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Objective: To test the mechanism and upstream pathway of outer hair cell apoptosis in Cadherin 23 (Cdh23) gene mutant mice. Method: The mutant Cdh 23 (erl/erl) ( erl ) mice were collected as the study group, while the C 57 BL /6 J ( B 6) mice were chosen as the control group . A total of 70 mice per group were used in this study . The study group and control group underwent auditory - evoked brainstem response ( ABR ) tests at the same age . The terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) assay was performed to detect outer hair cell ( OHC ) apoptosis . The qRT - PCR was conducted to test the expression of ER stress markers immunoglobulin - binding protein ( BiP ) and C / EBP homologous protein ( CHOP ) mRNA . The expression and location of BiP and CHOP protein in OHC were detected by immunostaining . The expression of BiP protein in cochleae was identified by Western blot . The expression and location of CDH 23 protein in OHC were discovered by immunostaining . Results: The ABR thresholds in erl mice were significantly higher than those in B6 mice at the age of 1 and 3 months (both P <0.05). The surface preparation with TUNEL staining confirmed OHC apoptosis in erl mouse cochleae which showed a higher TUNEL positive cell ratio than B6 mouse( t =11.291, P <0.01). The ER stress marker Bip and Chop mRNA were upregulated in the erl mouse inner ear, when compared with those in the B6 mouse(both P <0.05). The BiP protein extracted from the erl mouse cochleae was significantly higher than that of B6 mouse measured by Western blot ( t =3.66, P =0.02). Immunostaining showed that BiP and CHOP were highly detected in the OHC in erl mouse cochleae, and was mainly detected in the perinuclear region of OHC. However, a bare BiP and CHOP signal were shown in B6 mouse cochleae. The CDH23 protein was specifically localized at the top of the OHC in B6 mice, indicating the localization of the tip links in hair bundle stereocilia. On the contrary, the CDH23 (erl) protein was found to be localized from the top to the nuclei of the OHC in erl mice. Portions of the CDH23 (erl) proteins failed to reach the top of the hair bundles and remained in the OHC cytoplasm. Conclusion: As the downstream response of the Cdh23 gene mutation, portions of the mutant CDH23 (erl) protein was accumulated in ER lumen resulting in the increase of ER loading and ultimately triggered ER stress and hair cell apoptosis in erl mouse cochleae.

Objective: To investigate the mechanisms of stem cell-derived extracellular vesicles (EVs) alleviating acute oxidative stress injury to Leydig cells., Methods: The model of testicular ischemia-reperfusion was established by unilateral testicular resection in 10 SD rats followed by injection of EVs into the tail vein in 5 of the animals (the experimental group) and that of isotonic PBS in the other 5(the control group).At 2 hours after reperfusion, the infiltration of neutrophilsin the testicular tissue was detected by HE staining, the apoptosis of Leydig cells measured by TUNEL and Caspase-3 immunohistochemistry, and the proliferation of Leydig cellsdetermined by Ki-67 immunohistochemical staining., Results: Compared with the control, the experimental group showed significantlymilderinfiltration of neutrophils in the interstitial tissueof the testis and lower apoptosis and higher proliferation of Leydig cells., Conclusions: Stem cell EVs can reduce the adhesion of neutrophils in the acute phase and alleviate acute oxidative stress injury to Leydig cells by decreasing the production of reactive oxygen free radicals.

Objective: To evaluate the feasibility and therapeutic efficacy of transhepatic arterial embolization with superparamagnetic iron oxide (SPIO) and lipiodol (LIP) for the treatment of VX2 tumor in rabbits.
 Methods: Twenty-four rabbits with hepatic VX2 tumors by surgical implantation were randomly divided into 4 groups and treated with transhepatic arterial embolization of 4 different agents as follows (n=6 each): doxorubicin (DOX) group, DOX-LIP group, SPIO-DOX group, and SPIO-DOX-LIP group. Liver function (AST and ALT) was measured at 0, 1, 3, 5 and 7 d after transhepatic arterial embolization. The serum DOX level was measured at 0, 5, 15, 30, 60, and 120 minutes after transhepatic arterial embolization. MRI was performed at 7 d after the treatment to assess the distribution of SPIO in the SPIO-DOX group and SPIO-DOX-LIP group, while CT was performed to assess the distribution of LIP in the DOX-LIP group and SPIO-DOX-LIP group. All the rabbits were sacrificed and their livers were removed at 7 d after treatment for the detection of tissue DOX level. The histopathologic examinations were performed including HE staining, Prussian blue staining and TUNEL assay, and then the tumor necrosis percentage and apoptosis index were calculated.
 Results: Compared to the DOX group, the levels of AST and ALT in other 3 groups were significantly elevated at 1 and 3 d after embolization (P<0.05). The levels of ALT and AST in the DOX group, DOX-LIP group or SPIO-DOX-LIP group returned to the baseline at day 7, there were no significant differences (P>0.05). The SPIO-DOX-LIP group exhibited the lowest serum DOX level at all time points up to 120 minutes after embolization (P<0.05). However, the tissue DOX level in the SPIO-DOX-LIP group was the highest among all groups at day 7 (P<0.05). The SPIO-DOX group and SPIO-DOX-LIP group showed significantly lower MRI signal intensity of tumors in T2 weighted imaging (T2WI) at day 7. Meanwhile DOX-LIP group and SPIO-DOX-LIP group showed that high-density lipiodol was deposited in the tumors in CT images. Histopathologic findings showed an almost complete central necrosis coagulation of tumors in the SPIO-DOX-LIP group, and the tumor necrosis percentage and tumor apoptosis index were significantly increased in the SPIO-DOX-LIP group compared to those in other 3 groups (P<0.05).
 Conclusion: This novel drug-delivery system of SPIO nano-drug carrier together with LIP is safe and feasible when it is used for transhepatic arterial embolization for liver tumor. It provides an excellent MR and CT visualization and improves the therapeutic efficacy for the treatment of rabbit VX2 liver tumor.

Objective: To investigate the role of pH2AX in the reversibility of mouse testicular reproductive function impaired by single heat stress., Methods: Twenty-four C57 male mice were randomly divided into heat stress and control groups and immersed in water at 43℃ and 25℃, respectively, for 15 minutes. At 1, 7, and 14 days of heat exposure, all the mice were sacrificed and their testis tissues collected for determining the apoptosis of the germ cells by TUNEL and measuring the expression level of the pH2AX protein by immunohistochemistry and Western blot., Results: The highest percentage of apoptotic cells were found in the seminiferous tubules of the mice in the heat stress group on the 1st day of the exposure and almost no apoptosis was observed at 7 and 14 days. The pH2AX protein was expressed in the nuclei of the basement membrane of adjacent seminiferous tubules. Compared with the control group, the expression of pH2AX was significantly increased on the 1st day of exposure (0.47 ± 0.02 vs 1.61 ± 0.04, P <0.01), then decreased at 7 days (0.85 ± 0.03) in comparison with that on the 1st day (P <0.01), and again elevated at 14 days (1.72 ± 0.02) as compared with either those at 1 and 7 days (P <0.01) or that of the control (P <0.01)., Conclusions: Heat stress causes dynamic changes of the pH2AX expression in the testis of the mouse, which are associated with heat stress-induced proliferation and division of the testicular spermatogenic cells.

To investigate the effects of dexmedetomidine (DEX) on hypoxia/reoxygenation (H/R) injury-induced cell apoptosis and caspase-12 expression, A549 cells were randomly divided into 4 groups: control group, DEX group, H/R group and DEX+H/R group. Cells of control and DEX groups were cultured in the normoxic incubator for 30 h. Cells of H/R and DEX+ H/R groups were incubated in the anoxic cultivation for 6 h, followed by normoxic culture for 24 h, and DEX (1 nmol/L) was added into the culture medium in DEX and DEX+H/R groups. Morphological changes were observed under the inverted microscope. Cell viability was detected by CCK-8. The apoptosis index (AI) of A549 cells was detected by TUNEL method. The activity of caspase-3 enzyme in cells was detected by using caspase-3 kit. The expressions of GRP78, caspase-12 protein and mRNA were determined by Western blot and RT-PCR respectively. Compared with control group, the morphological changes of the cultured cells were observed: some of the cell fusion occurred and the shape of the cells was multilateral; the cell viability was decreased significantly (P < 0.01), the number of apoptotic cells and the AI value, caspase-3 activity, and the expressions of GRP78, caspase-12 protein/mRNA were significantly increased (P < 0.01) in H/R group. While the administration of DEX alleviated the H/R injury-induced cell damage, obviously increased the cell viability (P < 0.01), significantly decreased the increment of apoptotic cells and the AI value induced by H/R injury (P < 0.01), and also dramatically decreased the H/R injury-induced high level of caspase-3 activity (P < 0.01) as well as high expression of caspase-12 protein and mRNA (P < 0.01). Taken together, the results suggest that DEX can effectively protect A549 cells from the H/R injury, which may be mediated by down-regulating the expression of caspase-12 and inhibiting cell apoptosis.

Objective: To explore the effect and mechanism of over-expression of miR-17-92 gene cluster on the biological characteristics of prostate cancer cells. Methods: DU145 cells were transfected with miR-17-92 gene expression plasmid and clones with stable ectopic miR-17-92 overexpression were established. The cell viabilities of DU145-17-92 and DU145-control cells were monitored by xCELLigence system. Cell proliferation and apoptosis were analyzed by Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cell cycle was detected by flow cytometry. Expression levels of proteins involved in apoptosis and Akt pathway were determined by western blotting. Results: xCELLigence RTCA array data showed that the growth rate of DU145-17-92 cells was significantly higher than that of DU145-control cells after 24 h of seeding ( P <0.01). The Ki-67-positive rates of the DU145-control group at 24, 48 and 72 hours were (56.57±1.68)%, (85.48±0.26)% and (90.85±2.08)%, respectively. While the Ki-67 positive rates of the DU145-17-92 group at the desired time points were (73.64±0.68)%, (93.43±1.23)% and (97.36±0.86)%, respectively, with a statistically significant difference at 24 hours ( P <0.01). The percentages of apoptotic cells of the DU145-control group at 24, 48 and 72 hours were (6.76±0.09)%, (14.51±0.86)% and (20.73±1.64)%, respectively, while the apoptotic percentages of the DU145-17-92 group were (1.86±0.15)%, (7.90±0.40)% and (4.92±0.48)%, respectively. The percentages of apoptotic cells of the DU145-control group at different time were significantly higher than those of DU145-17-92 group ( P <0.01 for all). The result of western blotting showed that the protein expression levels of Bcl-2 interacting mediator of cell death (BIM) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in DU145-control cells were 0.83±0.00 and 0.91±0.00, respectively, significantly higher than 0.16±0.00 and 0.13±0.00 of DU145-17-92 cells (both P <0.01). Overexpression of miR17-92 induced the phosphorylation of protein kinase B (Akt) at Ser473 while no appreciable effect on the phosphorylation of Akt at Thr308. The phosphorylated level of extracellular regulated protein kinases (ERK) in DU145-control cells was 0.21±0.01, significantly lower than 0.72±0.01 of DU145-17-92 cells ( P <0.01). Conclusions: Overexpression of miR-17-92 gene plays a pivotal role in growth, proliferation, apoptosis and cell cycle of DU145 cells through down-regulation of apoptotic protein BIM and tumor suppressor PTEN and activation of Akt and ERK signaling pathway.

Objective: To explore the effects of Kudzu Root plus Cinnamon Granules (KR+C) on prostatic hyperplasia (PH) in mice., Methods: Sixty 4-week-old Kunming male mice were randomly divided into six groups: blank control, PH model, high-, medium- and low-dose KR+C, and finasteride control. All the mice except those in the blank control group were subcutaneously injected with testosterone propionate (5 mg / ［kg·d］) at 7 days after surgical castration. The animals of different groups were treated intragastrically with different doses of KR+C, finasteride, and normal saline respectively for 3 weeks and then sacrificed for weighing of the prostate, calculation of the prostatic index, observation of the morphological changes in the prostate after HE staining, determination of the expressions of FGF2, Ki67 and TGF-β1 by immunohistochemistry, detection of 5α-reductase activity by ELISA, and measurement of the apoptosis index of the prostatic cells by TUNEL., Results: Compared with the model controls, the mice of the other groups showed significantly reduced prostatic volume (P <0.05), prostatic index (P <0.05), expressions of FGF2, Ki67 and TGF-β1, and activity of 5 α-reductase (P <0.05), but remarkably increased apoptosis index of the prostatic cells (P <0.05). However, no statistically significant differences were observed in the above parameters between the finasteride control and the three KR+C groups (P>0.05)., Conclusions: KR+C can reduce the prostatic volume of PH mice by decreasing the activity of 5α- reductase, inhibiting the expressions of FGF2, Ki67 and TGF-β1, and promoting the apoptosis of prostatic cells.

Objective: To explore the effect of delphinidin on breast cancer and the underlying mechanisms.
 Methods: Human epidermal growth factor receptor-2 (HER-2) positive breast cancer cells MDA-MB-453 were treated by delphinidin. Proliferation of MDA-MB-453 cells was detected by CCK-8 after 48 h. TdT-mediated dUTP nick end labeling (TUNEL) assay and Western blot were used to explore apoptotic status for MDA-MB-453 cells. Fluorescence dot assay, immunofluorescence, and Western blot were used to identify autophagy in breast cancer cells.
 Results: Delphinidin suppressed proliferation of MDA-MB-453 cells. Delphinidin increased the number of TUNEL positive cells. Delphinidin downregulated the expression of caspase-3 and caspase-9, while upregulated the expression of cleaved caspase-3 and cleaved caspase-9 in a dose-dependent manner. Delphinidin enhanced the number of GFP-LC3 punctate dots, LC3 immunofluorescence dots and the expression of LC3-II and ATG5. Delphinidin inhibited the expression of proteins in mTOR signaling pathway, including AKT, mTOR, eIF4E and p70s6k.
 Conclusion: Delphinidin induced apoptosis and autophagy by inhibition of AKT/mTOR pathway in HER positive breast cancer cells.

Objective: To investigate the effect of nucleolin on cardiac cell apoptosis in Type 2 diabetic cardiomyopathy mice.
 Methods: Mice were fed with high-fat and high-sugar food for 20 weeks (mice were injected intraperitoneally with 60 mg/kg streptozotocin in the 5th and 6th weeks) to establish a mouse model of Type 2 diabetes. The mice were divided into 4 groups: a wild type (WT) control group, a nucleolin transgenic (TG) control group, a WT diabetic group, a TG diabetic group. Diabetes-related indicators were detected at the end of the 8th week. At the end of the 20th week, HE staining was used to observe myocardial morphological changes; TUNEL staining and caspase-3 activity were used to detect the extent of apoptosis of cardiac myocytes.
 Results: The level of fasting blood glucose was significantly increased in the diabetic group than that in the control group. In WT diabetic group, myocardial disarrangement, fragmentation and dissolution were observed (determined by HE staining); cellular apoptosis (determined by TUNEL staining and caspase-3 activity) also increased markedly in the WT diabetic group. Compared with the wild mice in the diabetic group, myocardial morphological changes and cardiac myocytes apoptosis were alleviated significantly. 
 Conclusion: Nucleolin overexpression affectes the occurrence and development of diabetic cardiomyopathy through inhibition of cardiac myocyte apoptosis.

The present study was aimed to investigate the underlying mechanisms of the protective effect of proanthocyanidin (Pro) against hypoxia/reoxygenation (H/R) injury in H9C2 cells with a focus on Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway. H9C2 cells were randomly assigned to 5 groups, including the control group (Con), the H/R-injured group (H/R), the Pro-treated group (H/R+Pro), the JAK2 siRNA-treated group (H/R+Pro+JAK2 siRNA) and the JAK2 siRNA control group (H/R+JAK2 siRNA). The cells were pretreated with Pro (40 µmol/L) for 8 h before 2 h of hypoxia and 4 h of reoxygenation. Cellular viability and apoptosis rate were detected by MTT and TUNEL methods, and superoxide generation was measured. JAK2/STAT3 signaling, oxidative stress markers and endoplasmic reticulum stress markers were also detected by Western blot. We found that Pro treatment significantly improved cellular viability and reduced apoptosis rate in H/R-treated H9C2 cells. In addition, Pro treatment significantly up-regulated the phosphorylation levels of JAK2 and STAT3, down-regulated the superoxide generation, gp91 phox , glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and caspase-12 expression. However, these protective effects of Pro were all attenuated by JAK2 siRNA administration. Taken together, we demonstrated that Pro protects H9C2 cells against H/R-induced oxidative stress and endoplasmic reticulum stress injury via JAK2/STAT3 signaling pathway.

Objective: To explore the effects of ischemic preconditioning on the level of serum testosterone (T) and apoptosis of spermatogenic cells in rabbits with testicular ischemia-reperfusion injury induced by testicular torsion., Methods: A total of 15 New Zealand male rabbits were randomly divided into groups A (control), B (ischemia-reperfusion), and C (ischemic preconditioning). The animals of group A were subjected to exposure of the right spermatic cord without ischemia, those of group B received 60-minute non-invasive occlusion of the right spermatic cord followed by 3 days of reperfusion, and those of group C underwent 5-minute occlusion plus 5-minute reperfusion of the right spermatic cord followed by the same procedure as that for group B. Then the rabbits were narcotized with 3% barbital sodium, the whole blood collected for examination of the serum T content and the testis tissues obtained from both the ischemic and healthy sides for HE and TUNEL staining., Results: After operation, the body weight was significantly increased as compared with the baseline in groups A (［2.65±0.07］ vs ［2.45±0.07］ kg, P<0.05) and C (［3.03±0.11］ vs ［2.92±0.07］ kg, P<0.05), but not in group B (［3.05±0.07］ vs ［3.05±0.07］ kg, P>0.05). The serum T level showed no statistically significant difference in group A before and after operation (［139.59±9.39］ vs ［140.19±9.47］ ng/L, P>0.05), but was remarkably lower after operation than the baseline in groups B ［148.06±3.31］ vs ［74.12±4.00］ ng/L, P<0.01) and C (［133.75±6.48］ vs［94.76±3.13］ ng/L, P<0.01) as well as than the postoperative index in group A (P<0.01). In comparison with group A and the healthy side of group B, the testis tissue of the ischemic side in group B exhibited structural damage of most of the seminiferous tubules with disappearance of spermatogenic cell structures, apoptosis of spermatogenic cells, and exudation of light-eosin edema fluid in the mesenchyme and lumen, with a markedly increased apoptosis index (P<0.01) and a significantly decreased Johsen's score (P<0.01). Compared with ischemic side of group B, The testis tissue of the ischemic side in group C was restored to normal as compared with that in group B, with a dramatically decreased apoptosis index (P<0.01) and a remarkably increased Johnsen's score (P<0.01)., Conclusions: Ischemic preconditioning can raise the decreased serum T level and reduce the apoptosis of spermatogenic cells in rabbits with testicular ischemia-reperfusion injury, which could be applied as a potential option for the clinical treatment of testicular ischemia-reperfusion injury.

Objective: To study the protective effect of Salubrinal on human lens epithelial cells and its mechanism in endoplasmic reticulum stress (ERS)., Methods: Hydrogen peroxide (H2O2 200 μmol/L) was used to intervene in the cultured human lens epithelial cells B3 (HLE-B3) so as to create an oxidative stress model and induce ERS in the model. Different concentration of Salubrinal (10, 15, 20, 25, 30 and 35 μmol/L) were added to the cultured HLE-B3 with or without H2O2 intervention. Then the cells were cultured for 24 hours. The cell counting kit (CCK-8) assay was used to test the viability of HLE-B3. The HLE-B3 cells were divided into three groups: Group A (normal control group), Group B (H2O2 200 μmol/L group), and Group C (H2O2 200 μmol/L+ Salubrinal 25 μmol/L group). After 48 h, TUNEL and flow cytometry assay (FCM) were used to examine the effect of Salubrinal on HLE-B3 apoptosis. The expression of glucose-regulated protein 78(GRP78), C/EBP homologous protein (CHOP), cysteinyl aspartate specific proteinase 12 (Caspase-12) and phosphorylation eukaryotic translation initiation factor 2α (p-elF2α) were tested by western blot at different points in time. Data from different groups was analyzed by one-way analysis of variance (ANOVA) while Dunnett t test was used under an equal condition, Dunnett's T3 for the unequal variances., Results: CCK-8 results showed that without the intervention of H2O2, different concentrations of Salubrinal had no inhibitive effect on HLE-B3 viability, and that survival rates were (98.6±3.3) %, (98.7±2.6) %, (99.4±3.2) %, (98.6±1.9) %, (98.8±2.5) %, (99.3±3.2) % and (99.5±2.4) %. There was no statistically significant difference between them (F=0.09, P=0.10). With the increasing of Salubrinal concentration, the survival rates of HLE-B3 in the presence of H2O2 intervention were (52.9±4.7) %, (65.0±3.6) %, (72.9±3.8) %, (84.5±3.6) %, (91.6±2.1) %, (93.1±2.9) %, (92.0±3.3) %. There was statistically significant difference from the control group (all P<0.01). However, the survival rates no longer increased (P=0.56, 0.88) if the Salubrinal concentration was greater than 25 μmol/L. FCM results indicated that apoptosis rates of Group A, B and C were (1.9±0.7) %, (8.8±0.5) %, (4.3±0.3) %, respectively and the differences were statistically significant (F=396.26, P<0.01, comparing with Group A, all P<0.01). TUNEL results showed that apoptosis indexes of Group A, B, and C were (7.7±1.0) %, (36.9±1.0) %, (16.7±2.2) %, respectively and the differences were statistically significant. (F=618.39, P<0.01, comparing with Group A, all P<0.01). RESULTS of western blotting in group B at different points in time (0, 12, 24, 36, 48 h) showed that p-elF2α had increased by (2.16±0.38) times at 6 h; GRP78 had increased by (2.56±0.15) times at 12 h; CHOP started to rise after 12 h until it dropped after 24 h, and its amount had increased by (2.49±0.23) times at 48 h; Caspase-12 had increased significantly by (3.53±0.30) times at 48 h. The expression of GRP78, CHOP and p-elF2α in group C was greater than that in Group B, but the expression of Caspase-12 in Group C was lower than that in Group B (GRP78: F=37.85, P<0.01; CHOP: F=61.09, P<0.01; Caspse-12: F=22.27, P<0.01; p-eIF2α: F=15.11, P<0.01)., Conclusion: Salubrinal might protect HLE-B3 against H2O2-induced apoptosis by inhibiting ERS related apoptosis pathways.(Chin J Ophthalmol, 2016, 52: 437-443).

Objective: To study the impact of left varicocele (VC) and varicocelectomy (VCT) on the apoptosis of spermatogenic cells and the levels of nitrogen monoxidum (NO) and interleukin 1 (IL-1) in the rat testis., Methods: We randomly divided 60 adolescent male SD rats into four groups of equal number: sham operation control, VC model 1 (VC1), VC model 2 (VC2), and VCT. We determined the semen quality and levels of NO and IL-1 in the testis tissue, detected the apoptosis of spermatogenic cells by TUNEL, and compared the indexes obtained among different groups., Results: An experimental VC model was successfully established by partially ligating the left renal vein of the rats. Sperm concentration and motility were significantly decreased in the VC1 ([1.54 ± 1.16] x 10⁶/ml and [44.23 ± 15.46]%) as compared with those in the sham operation group ([2.80 ± 1.62] x 10⁶/ml and [72.34 ± 12.62]%) (P < 0.05), but remarkably higher in the VCT ([1.82 ± 1.34] x 10⁶/mI and [51.21 ± 12.62]%) than in the VC2 group ([1.04 ± 1.21] x 10⁶/ml and [39.23 ± 13.21]%) (P < 0.05). The levels of NO and IL-1 in the left testes were markedly elevated in the VC1 ([0.172 ± 0.030] ng/ml and [1.468 ± 0.080 ] mg/ml) in comparison with those in the sham operation group ([0.134 ± 0.021] ng/ml and [0.782 ± 0.079 ] mg/ml) (P < 0.05), and significantly higher in the VC2 ([0.198 ± 0.020] ng/ml and [1.994 ± 0.090] mg/ml) than in the VCT group ([0.141 ± 0.010] ng/ml and [0.781 ± 0.036] mg/ml) (P < 0.05). However, the NO and IL-1 levels in the right testis showed no statistically significant differences between the two groups, and the two levels were positively correlated (r = 0.492, P < 0.01). The rats of the VC1 group exhibited remarkable apoptosis of spermatogenic cells in the bilateral testes, with significant differences in the apoptosis index ( AL) between the two sides (P < 0.05) as well as in the same side in comparison with the sham operation group (P < 0.01). The Als of spermatogenic cells in the bilateral testes showed statistically significant differences in the VCT (P < 0.05) but not in the VC2 group (P > 0.05), and those in the same side manifested dramatic differences between the VCT and VC2 groups (P < 0.01)., Conclusion: Varicocele induces changes of the NO and IL-1 levels in the testis tissue and increases the apoptosis of spermatogenic cells, which might be one of the causes of testis damage and spermatogenic dysfunction.

Objective: To investigate the impact of in utero exposure to di-n-butyl phthalate (DBP) on the apoptosis of testicular cells in the pubertal male rat offspring., Methods: Ten pregnant SD rats were randomly divided into a control and an experimental group to be treated intragastrically with olive oil (1 ml per day) and DBP (500 mg per kg of body weight per day) respectively between gestation days 12 and 19. At the pubertal age (postnatal day 45, PND 45), the testes of the male rat offspring were removed for observation of the cell structure under the transmission electron microscope and the development of different spermatogenetic cells by HE staining. The apoptosis of testicular cells was detected by the TUNEL method, the expressions of the apoptosis-regulating proteins Bcl-2, Bcl-XL, Bax and p53 were determined by immunohistochemistry and Western blot, and the data obtained were compared between the two groups by t-test., Results: Transmission electron microscopy revealed increased apoptosis and vacuolization of testicular cells in the PND-45 rat offspring, HE staining showed markedly decreased numbers of different spermatogenetic cells, TUNEL manifested significantly increased apoptosis of testicular cells in the experimental group as compared with the control (12.00 ± 5. 22 vs 3.17 ± 1.47, P < 0.01), and immunohistochemistry and Western blot exhibited remarkably higher expressions of Bax and p53 in the former than in the latter group (P < 0.05)., Conclusion: In utero exposure to DBP can increase the apoptosis of germ cells and Sertoli cells, induce the vacuolization of testicular cells, and significantly elevate the expressions of the apoptosis-promoting proteins Bax and p53 in the pubertal male rat offspring.

Objective: To investgate the effects of rapamycin(RPM)and RPM-loaded poly(lactic-co-glycolic)acid(PLGA)nanoparticles(NPs)on the apoptosis of human umbilical arterial vascular smooth muscle cells(HUASMCs)in vitro and expression of bcl-2 and p27(kip1) protein., Methods: HUASMCs were cultured in vitro and divided to RPM and RPM-PLGA-NPs groups treated at 3 different concentration by 12 and 24 hours,with M231-smooth muscle growth supplements medium and null-PLGA-NPs treated groups as controlled. The apoptosis of HUASMCs was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining and flow cytometry. The expressions of bcl-2 and p27(kip1) were detected by streptacidin/peroxidase immunohistochemical method. The effect on cellular proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidecolorimetry., Results: The proliferation of HUASMCs was inhibited by RPM and RPM-PLGA-NPs in a dose-dependent manner. DNA electrophoresis showed DNA ladder in RPM and RPM-PLGA-NPs groups and classical scalar strips in control groups. The apoptotic indexes of RPM 100 ng/ml group and RPM-PLGA-NPs 500 ng/ml group detected by flow cytometry were(45.45<2.36)% and(35.04<5.64)%,respectively,which were significantly higher than that of M231-smooth muscle growth supplements control group [(2.60<0.95)%,all P<0.01]. The apoptotic indexes of groups incubated with RPM and RPM-PLGA-NPs for 24 hours were significantly higher than those of groups which incubated for 12 hours(P<0.05,P<0.01). The positive expression indexes(PEI)of p27(kip1) and bcl-2 protein were higher in RPM and RPM-PLGA-NPs groups than that of control groups. The Spearman's rank correlation coefficient test showed that there was no significant correlation between the PEI of p27(kip1) and the apoptotic indexes in the RPM group and RPM-PLGA-NPs group(P>0.05)., Conclusions: Rapamycin-loaded PLGA nanoparticles and rapamycin have similar effects in inhibiting proliferation and inducing apoptosis;meanwhile,they upregulate the expression of p27(kip1) protein without downregulating the expression of bcl-2 protein in HUASMCs in vitro. RPM-PLGA-NPs has more potent pro-apoptotic effect than equivalent dose of RPM but is not linearly correlated with the p27(kip1) expression level.

Objective: To observe the pathological changes in rabbits with spinal cord injury induced by decompression sickness (DCS), and to investigate the role of tumor necrosis factor-alpha (TNF-α) in spinal cord injury induced by DCS., Methods: Rabbits were randomly divided into normal control group, DCS group, and safe decompression group. The rabbit model of DCS was established. Light microscopy, real-time PCR, and immunohistochemical method were used to observe the pathomorphological changes in the thoracolumbar spinal cord and the mRNA and protein expression of TNF-α, respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to observe the apoptosis in the spinal cord., Results: In the DCS group, cavities formed in the white matter of spinal cord and gliosis occurred around necrotic areas. Moreover, the mRNA and protein expression of TNF-α was significantly higher in the DCS group than in the normal control group and the safe decompression group (P<0.01). The results of TUNEL showed that the number of positive apoptotic cells was significantly larger in the DCS group than in the normal control group and the safe decompression group (P<0.05)., Conclusion: Apoptosis plays an important role in spinal cord injury induced by DCS. In the early stage of DCS, the massive release of TNF-α initiates apoptosis and contributes to the pathological changes in spinal cord injury induced by DCS.

Objective: To observe the change of quantitative distribution,apoptosis and proliferation of T and B cells in the skin of KM mutant mice., Methods: We chose 1-,3-,6-,9-,22-day,3-,6-month-old KM mutant and wild-type mice to detect the changes of T and B lymphocytes using blood routine tests and immunohistochemical staining. Apoptosis was detected by TUNEL staining and proliferation by proliferating cell nuclear antigen (PCNA) staining., Results: T cells on KM mutant mice skin were mainly seen in epidermis and dermis. They increased on the first day to 6(th) day after birth and decreased on the 9(th) and 22(nd) day,but after 3-month-old,their number began to increase;at the time of 6 months,the number of B cells also increased. The apoptosis of the skin hair follicle and sebaceous gland cells were more obvious in KM mutant mice than in wild-type mice,with the maximal apoptosis occurred at the age of 22-day-old in both groups. The proliferation of epidermal basal cells in KM mutant mice between 1 to 9-day-old was not significantly different from that in the wild-type mice,but decreasing on the 22(nd) day and 3(rd) month and increasing in the 6(th) month. The proliferation in hair follicle and sebaceous glands decreased on 9(th) day,increased on 22(nd) day,and deceased on the 3(rd) month again., Conclusions: The quantitative distribution,apoptosis,and proliferation of T and B lymphocytes abnormally change in the skin tissue of KM spontaneous mutant mice. They may lead to immune and hair growth disorders and promote the inflammatory responses.

Objective: To investigate the effects of crypotanshinone (CPT) on the proliferation and apoptosis of DU145 prostate cancer cells as well as on the metadherin expression and the downstream PI3K/AKT signaling pathway in the DU145 cells., Methods: We treated DU145 prostate cancer cells with different concentrations of CPT for 24, 48, and 72 hours followed by evaluation of the proliferation and apoptosis of the cells by MTT assay and TUNEL, respectively. We determined the expressions of metadherin protein and mRNA in the DU145 cells by Western blot and RT-PCR respectively at different time points after CPT treatment. We also detected the expressions of the proteins metadherin, AKT, p-AKT, and Bcl-2 in the CPT-treated DU145 cells at 48 hours., Results: CPT significantly inhibited the proliferation of the DU145 cells in a dose- and time-dependent manner (P < 0.05). After treatment with 10 µmol/L CPT for 24, 48, and 72 hours, the apoptosis rates of the DU145 cells were (29.42 ± 4.51), (55.07 ± 5.67) and (70.84 ± 4.66)%, respectively, significantly higher than (3.1 ± 2.48)% in the control group (P < 0.05). The expression of metadherin was remarkably downregulated at the transcription and translation levels (P < 0.05) and the expressions of the AKT signaling pathway and the Bcl-2 protein were markedly inhibited in the DU145 cells after treated with 10 µmol/L CPT for 48 hours (P < 0.05)., Conclusion: CPT can inhibit the proliferation and induce the apoptosis of DU145 prostate cancer cells, which may be associated with its suppression of the downstream PI3K/AKT signaling pathway by reducing the expression of metadherin in the DU145 cells.

Objective: To establish a model of hypoxic postconditioning of dermal microvascular endothelial cells., Methods: During reoxygenation after hypoxia, cells received three times of hypoxic/ reoxygenation alternate treatment for a certain time. The cells were seeded on 6-well plates, with one plate for one group. They were divided into 6 groups as group 1 ( Control), group 2 (8h hypoxia + 24h reoxygenation), group 3 (8h hypoxia + 2 min x 3 times post-hypoxia treatment) , group 4 (8h hypoxia + 5 min x 3 times post-hypoxia treatment), group 5 (8h hypoxia + 10 min x 3 times post-hypoxia treatment), 6 group (8h hypoxia + 20 min x 3 times post-hypoxia treatment). Each group underwent 8 h hypoxia + 24 h hypoxia Buffer and reoxygenation. Lactate dehydrogenase (LDH) was detected during the process. Apoptosis rate was calculated by staining Tunel method. Bcl-2, Bax and activated caspase-3 protein were detected by Western Blot., Results: In the continuous hypoxia process, the LDH was (1563 ± 83.35) IU/L at 8h and (582.85 ± 58.25 ) IU/L at 0h, showing a statistical difference (P = 0.0001). Western blotting results showed that the expression of Bax/Bcl-2 in group 2 was 0.38 ± 0.02, showing a significant difference when compared with that in group 3 (0.23 ± 0.01) and group 4 (0.22 ± 0.02) (P = 0.012, P = 0.005), while not when compared with that in group 5 (0.33 ± 0.02) and 6 groups (0.34 ± 0.01) P > 0.05). The ratio of Activated caspase-3/caspase-3 in group 2 (6.30 ± 1.50) was significantly higher than that in group 3 (2.17 ± 0.26) and group 4 (2.63 ± 0.31) (P = 0.008, P = 0.019); while not in group 5 (4.36 ± 0.29) and group 6 (4.97 ± 0.51) (P > 0.05)., Conclusions: The model of hypoxic postconditioning of human dermal microvascular endothelial cells is successfully established.

Objective: To explore the effect of electro-acupuncture to improve the bladder function after acute spinal cord injury in rats and its possible mechanism., Methods: Sixty healthy adult male SD rats of SPF grade, with body weight of 220 to 250 g, one week after feeding adaptation, were randomly divided into sham operation group, model group, electro-acupuncture group, electro-acupuncture control group with 15 rats in each group. Sham operation group underwent no stimulation, and the moderate damage model of spinal cord injury were made in other three groups according to modified Allens method. The model group were not treated, electro-acupuncture group were treated with electro-acupuncture on Zhibianxue and Shuidaoxue, and electro-acupuncture control group were treated with electro-acupuncture on 0.5 inch next to Zhibianxue and Shuidaoxue. The frequency of 2/100 Hz, current of 1 mA, stimulation time of 15 min, once a day, left and right alternately stimulate every time, for a total of 7 times. The changes of residual urine volume and urine output in rats at the 1st and the 7th days after operation were observed. And 7 d later, the rats were sacrificed and the injured spinal cord were taken out to observe the apoptosis, and to detect the changes of Bcl-2, Bax, Bad content., Results: After modeling,the rats of three groups showed different bladder dysfunction. In electro-acupuncture group and electro-acupuncture control group, the residual urine volume of the 7th day after operation was significant lower than the 1st day after operation (P < 0.001), and there was statistically significant difference on the 7th day after operation between two groups (P < 0.001). Compared with model group, the urine output of electro-acupuncture group and electro-acupuncture control group was significantly increased on the 7th day after operation, and there was sig- nificant difference between electro-acupuncture group and electro-acupuncture control group (P < 0.001). Electro-acupuncture can inhibit apoptosis of spinal cord neurons by TUNEL detection. Postoperative at 7 d, the rate of nerve cell apoptosis in electro -acupuncture group and electro-acupuncture control group was significant increased than model group (P < 0.01, P < 0.05), and there was significant difference between electro-acupuncture group and electro-acupuncture control group (P < 0.005). Compared with model group, the positive expression rate of Bax, Bad decreased (P < 0.01, P < 0.05), and Bcl-2 increased (P < 0.01) in electro-acupuncture group and electro-acupuncture control group,there was significant difference between electro-acupuncture group and electro-acupuncture control group (P < 0.01)., Conclusion: Electro-acupuncture can obviously promote the repair of acute spinal cord injury,its mechanism may be through increasing Bcl-2, inhibiting the expression of Bax, Bad, which inhibits the apoptosis of spinal cord neurons.

Objective: To explore the relationship between the expression of interferon-induced protein with tetratricopetide repeats 1 (IFIT1) and liver cell apoptosis in the acute stress period after severe burns.
, Methods: A total of 25 C57/129 adult mice were randomly divided into the normal control group (0 h) and the groups at 1, 6, 12 or 24 after severe burns (n=5 per group). A model with third degree (20% of the total body surface area) burn injury was established and then liver tissues were taken. IFIT1 expression was examined by Western blot. The expression of caspase-3 and -8 was measured by immunohistochemistry. Liver cell apoptosis was detected by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL).
, Results: After burns, IFIT1 expression was increased at 1 h, which reached the highest level at 
6 h followed by a decrease at 12 h, which reached minimum level at 24 h. The differences between groups were significant (P<0.01). The caspase-3 and -8 levels significantly increased after burns in a time-dependent manner (P<0.01). Although at 0 h and 1 h there was no significant increase in liver cell apoptosis, the increase reached significance from 6 h to 24 h (P<0.01).
, Conclusion: The increase in IFIT1 expression after severe burns promotes liver cell apoptosis.

Objective: To investigate the role of endoplasmic reticulum stress in the apoptosis of testicular germ cells in hyperlipidemic rats., Methods: We randomly assigned 42 four-week-old male Wistar rats into a normal control group (n = 12) and a high-fat group (n = 30) to be fed on a normal diet and a high-fat diet, respectively, for 10 weeks. Then we measured the concentrations of triglyceride (TG) and total cholesterol (TC) in the serum using an automatic biochemistry analyzer, detected the apoptosis of testicular germ cells by TUNEL staining, and determined the protein and mRNA expressions of GRP78 and. caspase-12 in the testis tissue by immunohistochemistry and RT-PCR, respectively., Results: The concentrations of TG and TC were significantly increased in the animals of the high-fat group ([3.00 ± 0.92] and [3.04 ± 0.39] mmol/L) as compared with the control rats ([1.43 ± 0.41] and [1.55 ± 0.23] mmol/L) (P < 0.01), and so was the apoptosis index of the testicular germ cells ([37.17 ± 2.74]% vs [5.16 ± 0.81]%, P < 0.01). The high-fat group, in comparison with the control, also showed remarkably upregulated protein and mRNA expressions of GRP78 (0.32 ± 0.03 and 0.86 ± 0.05 vs 0.19 ± 0.01 and 0.37 ± 0.03, P < 0.01) and caspase-12 (0.34 ± 0.02 and 0.87 ± 0.01 vs 0.12 ± 0.01 and 0.34 ± 0.03, P < 0.01) in the testis tissue., Conclusion: The apoptosis of testicular germ cells is increased in hyperlipidemic rats, which may be attributed to endoplasmic reticulum stress.

Objective: To explore the status of apoptosis in human striatum derived neural stem cells (NSCs) aft er accutase dissociation and passage., Methods: The NSCs were isolated from fetuses obtained through spontaneous abortion at 13- 18 weeks of pregnancy, which formed neurospheres in vitro. At passages of 3-5, the neurospheres were disassociated into single cell by accutase digestion and then passaged. At 1, 24 and 72 h after passage, the apoptosis of NSCs was measured by several methods, including active caspase-3 or TUNEL staining for fixed cells, Annexin V, Hoechst or PI staining for live cells., Results: At all of the 3 time points, the staining of TUNEL and active caspase-3 overlapped perfectly. The apoptosis rate of NSCs increased significantly from 20%-25% at 1 h to 75%-80% at 24 h after passage (P<0.01). At 72 h, the apoptosis rate was significantly decreased as compared to that at 24 h time point because of the self-renewal and proliferation of survived NSCs (P<0.01)., Conclusion: Many cells in the neurospheres formed by human striatum-derived NSCs underwent apoptosis soon after accutase disassociation. For NSCs cultured in vitro, anti-apoptosis treatments might be a good method to increase the self-renewal and the proliferation of NSCs.

Objective: To observe the expression pattern of caspase-3 and HCLS1-associated protein X-1 (HAX-1) at different time after cerebral contusion in rat, and explore the new method for estimating the injury interval., Methods: The cerebral contusion model was established using adult SD male rats. Then the rats were randomly allocated into 8 groups: 2 h, 6 h, 12 h, 1 d, 3 d, and 7 d after cerebral contusion, sham-operation and normal control. Expression of caspase-3 and HAX-1 protein after cerebral contusion in rat was detected by Western blotting. Laser scanning confocal microscope was used to observe the number of HAX-1 positive cells and TUNEL-stained cells after cerebral contusion., Results: The expression of caspase-3 increased parallelly with the time after cerebral contusion and reached the peak value on 3 d. The expression of caspase-3 decreased gradually and still maintained a high level expression on 7 d (P < 0.05). The expression of HAX-1 positive cell went up after injury, and reached the peak value at 6 h (P < 0.05), then turned down gradually after 12 h and went out of detection after 3 d. The number of TUNEL-stained cells increased obviously at 2 h and reached the peak value on 3 d. The number of TUNEL-stained apoptotic cells decreased gradually and still maintained a high level expression on 7 d (P < 0.05)., Conclusion: The expression of caspase-3 and HAX-1 after cerebral contusion has time sequential regularity, which may provide new evidence for forensic diagnosis of cerebral contusion interval.

Objective: To explore the effect of high glucose and lipid intervention on islet cell apoptosis through the inhibition of prostate apoptosis response factor-4 (Par-4) expression and the underlying mechanisms., Methods: The mice islet β cells (NIT-1 cells) were randomly divided into a control group, a Par-4 inhibited group, a glucose and fatty acid intervented group and a glucose and fatty acid intervented+Par-4 inhibited group. Cell apoptosis was detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), and the protein expression levels of Par-4 and glucose regulated protein 78 (GRP78) were detected by Western blot., Results: Compared with the control group [(3.14 ± 1.08)%], the apoptosis rate of islet beta cell [(33.82 ± 3.15)%] in the glucose and fatty acid intervented group was significantly increased accompanied by the dramatically elevated Par-4 and GRP78 expression (both P<0.05). Compared with the glucose and fatty acid intervented group, the apoptosis rate in glucose and fatty acid intervented+Par-4 inhibit group [(18.3 4 ± 2.11)%] was significantly decreased concomitant with the significantly decreased Par-4 and GRP78 expression (both P<0.05)., Conclusion: The glucose and fatty acid-induced apoptosis of mice islet β cells could be improved through the inhibition of Par-4 expression, which might be related to reduction of endoplasmic reticulum stress.

Objective: To explore the protective mechanism of simvastatin on retina ischemia-reperfusion injury in a rat model., Methods: It was a experiment study.One hundred and sixty-five adult male SD rats were randomly divided into three groups using digital table method, normal control group (CON, 55 rats), ischemia-reperfusion model group (MOD, 55 rats) and the medicine of simvastatin group (SIM, 55 rats).Each group was divided into five points in time of 4 hours, 8 hours, 16 hours, 24 hours and 48 hours, and there were 11 rats in each point. The right cephalic artery of each rat was clipped in model group and simvastatin group, but it was exposed in control group. Expression of Bcl-2 and Bax protein were determined by the immunohistochemical method, the number of cell apoptosis in retina were examined by the TUNEL method and express of Bcl-2 and Bax mRNA were measured by the real-time PCR method. The expression of proteins and mRNAs of Bcl-2 and Bax and also apoptosis of the rat retinas in each group at corresponding time point are compared using single factor analysis of variance and LSD-t test., Results: Expression of Bcl-2 protein in model group began to decline at 4 h, reached the lowest at 24 h, with the data of (0.192 ± 0.011), (0.192 ± 0.015) , (0.189 ± 0.015), (0.183 ± 0.012) and (0.187 ± 0.010) .Expression of Bcl-2 protein in simvastatin group were higher than model group at each time point, with the information of (0.208 ± 0.011), (0.220 ± 0.011) , (0.221 ± 0.014), (0.228 ± 0.007) and (0.206 ± 0.015). The numbers were statistically significant at corresponding time point in each group (F(4, 8, 16, 24, 48) = 8.079, 9.005, 9.904, 35.563, 8.810, P < 0.05). Expression of Bax protein in model group began to increased at 4 h, reached the highest at 24 h, with the data of (0.255 ± 0.010), (0.261 ± 0.033), (0.276 ± 0.025), (0.324 ± 0.037) and (0.234 ± 0.018). Expression of Bax protein in simvastatin group were lower than model group at each time point, with the information of (0.222 ± 0.012), (0.219 ± 0.017), (0.223 ± 0.008), (0.232 ± 0.021) and (0.214 ± 0.008). The numbers were statistically significant at corresponding time point in each group (F(4, 8, 16, 24, 48) = 16.601, 13.525, 10.303, 25.849, 13.805, P < 0.05). AI of retina in model group began to increased at 4 h, reached the highest at 24 h, with the data of (11.771 ± 0.722), (13.705 ± 0.512), (15.533 ± 0.370), (21.210 ± 1.221) and (17.660 ± 0.414). AI of retina in simvastatin group were lower than model group at each time point, with the information of (9.061 ± 0.289), (11.717 ± 0.565), (13.506 ± 0.541), (16.586 ± 0.488) and (14.428 ± 0.559). The numbers were statistically significant at corresponding time point in each group (F(4, 8, 16, 24, 48) = 87.096, 232.245, 575.564, 389.205, 771.658, P < 0.05). Bcl-2 mRNA in model group were lower than in control group at each time point, with the data of (0.360 ± 0.017), (0.232 ± 0.066), (0.314 ± 0.012), (0.179 ± 0.019) and (0.357 ± 0.084). But Bcl-2 mRNA in simvastatin group were higher than those in model group, with the information of (1.718 ± 0.247), (1.981 ± 0.317), (1.309 ± 0.031), (1.289 ± 0.209) and (0.684 ± 0.071). The differences had statistical significance at corresponding time point in each group (F(4, 8, 16, 24, 48) = 112.934, 109.750, 3534.800, 112.428, 128.140, P < 0.05). And Bax mRNA in the model group were higher than those in control group at each time point, with the data of (2.140 ± 0.288), (3.189 ± 0.492), (2.896 ± 0.466), (2.392 ± 0.119) and (1.789 ± 0.169). However, Bax mRNA in simvastatin group were lower than those in model group, with the information of (0.658 ± 0.197), (1.746 ± 0.315), (0.670 ± 0.221), (0.952 ± 0.164) and (0.575 ± 0.174). The differences had statistical significance at corresponding time point in each group (F(4, 8, 16, 24, 48) = 74.115, 54.504, 81.271, 243.743, 97.163, P < 0.05)., Conclusions: Simvastatin has protective effects on retinal ischemia reperfusion injury, and the mechanism is closely related to inhibiting retinal cell apoptosis by adjusting the express of Bcl-2 and Bax.

Objective: To investigate if stimulating Toll-like receptor 2 (TLR2) specifically induces the cell apoptosis in mouse lung epithelial cells (MLE-12) and related potential mechanism., Methods: TLR2 was stimulated by 0, 1, 3.3, 10, 33 ng/mL PAM3CYS4 for 24 hours, or 33 ng/mL PAM3CYS4 for 0, 8, 16, 24, 36, 48 hours, respectively. The expressions of neuronal cell death inducible putative kinase (NIPK) and cleaved caspase-3 were detected by Western blotting; the number of apoptotic cells was counted by TUNEL staining in MLE-12 lung epithelial cells., Results: Responding to TLR2-specific stimulation, the expressions of NIPK and cleaved caspase-3 were significantly elevated, and the number of apoptotic cells increased. Inhibiting NIPK by siRNA transfection decreased the expression of cleaved caspase-3 and the number of apoptotic cells., Conclusion: Stimulating TLR2 signaling using TLR2-specific ligands PAM3CYS4 promotes the cell apoptosis through increasing the expression of NIPK.

Objective: To intervene the expression of CD59 on cervical cancer HeLa cells by RNA interference and phage display random peptide library, respectively, and detect the effect of down-regulated CD59 expression on proliferation and apoptosis of the cervical cancer cells., Methods: HeLa cells were divided into normal control group, CD59 peptide seal group, pSUPER transfected group and pSUPER-siCD59 transfected group. The CD59 peptide seal group was treated with 10 μg/mL CD59 peptide seal for 8 hours. The pSUPER and pSUPER-siCD59 transfected groups were transfected with empty plasmid pSUPER and recombinant plasmid pSUPER-siCD59, respectively. MTT assay was used for the detection of cell proliferation. TUNEL, annexin V-PE/7-AAD staining combined with flow cytometry were adopted for the detection of cell apoptosis., Results: Compared with the control groups, both CD59 peptide seal group and pSUPER-siCD59 transfected group presented a diminished cell proliferation activity and a strengthened apoptosis. What's more, the effect of peptide seal on CD59 expression inhibition was better than that of pSUPER-siCD59 plasmid., Conclusion: Down-regulation of CD59 expression could inhibit HeLa cells' proliferation and promote apoptosis, and the inhibitory effect of peptide seal was better than that of CD59 interference plasmid.

Objective: To infect lung adenocarcinoma A549 cells with recombinant LaSota strain Newcastle disease virus (NDV) vaccine expressing the rabies virus glycoprotein (rL-RVG), and explore the effect of rL-RVG on proliferation and apoptosis of lung cancer cells., Methods: A549 cells were infected with the rL-RVG and then detected for the expressions of RVG and NDV proteins by Western blotting. The cell proliferation was examined by MTT assay and apoptosis index and cell early apoptosis were respectively detected by TUNEL and annexin V-FITC/PI staining combined with flow cytometry. The expression of pro-apoptotic protein caspase-3 was observed by Western blotting. The LaSota strain of NDV was used as control group and PBS was the blank control., Results: Both RVG and NDV proteins were stably expressed in A549 cells infected with rL-RVG. MTT assay results showed that cell proliferation was significantly inhibited, and the inhibition rate of the rL-RVG group was higher than that of LaSota group. The apoptosis of A549 cells were promoted by rL-RVG infection. Flow cytometry revealed that the early apoptotic cells of the rL-RVG group increased as compared with the other two groups (P<0.05). Consistently, TUNEL assay showed the apoptotic index increased in the rL-RVG group as compared with the other two groups (P<0.05). Western blotting demonstrated that the expression of the pro-apoptotic protein caspase-3 was up-regulated. However, when we added specific broad-spectrum caspase inhibitor Z-VAD-FMK, the expression of the caspase-3 protein was obviously reduced., Conclusion: The rL-RVG is stably expressed in the infected A549 cells. The rL-RVG inhibits lung cancer cell growth and promote cell apoptosis, and the effect of rL-RVG is better than the wild LaSota strain.

Objective: To observe the intervention of Dachengqi Granule (DG) on the apoptosis of small intestine smooth muscle cells (SMCs) in rats with multiple organ dysfunction syndrome (MODS) and its mechanisms., Methods: Healthy 100 adult Wistar rats were randomly divided into the control group (n =20), the MODS model group (n =40), and the DG group (n =40).E. coli suspension was peritoneally injected to rats in the model group and the DG group to establish bacterial peritonitis induced MODS model. DG at 1 mL/100 g was administered by gastrogavage to rats of the DG group, twice daily for 3 successive days. Twenty-four hours after modeling, the proximal segment of intestine was taken and stained by using terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and immunohistochemistry. Changes of apoptosis quantity of SMCs and the expression of Bcl-2 associated X protein (Bax), B cell lymphoma/leukemia-2 (Bcl-2) and cytochrome c protein (Cyt c) in mitochondrial apoptotic signaling pathway were observed., Results: Compared with the control group, the apoptosis quantity of SMCs and the expression of Bax and Cyt c protein significantly increased, and the expression of Bcl-2 protein significantly decreased in the MODS model group (P <0.01). Compared with the MODS model group, the apoptosis quantity of SMCs and the expression of Bax and Cyt c proteins significantly decreased, and the expression of Bcl-2 protein significantly increased in the DG group (allP <0.01)., Conclusion: DG could inhibit apoptosis of SMCs through suppressing activation of mitochondrial apoptotic signaling pathway in intestinal SMCs, thus promoting the recovery of the gastrointestinal motility function in rats with MODS.

Objective: To investigate the effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the proliferation of ovarian carcinoma 3AO cells in vitro and its molecular mechanism., Methods: Ovarian carcinoma 3AO cells were incubated with 12.5, 25.0, 50.0 ng/mL TRAIL proteins for 72 hours, respectively. At 24, 48 and 72 hours, the cells were collected to observe the morphological change under an inverted microscope, detect the cell proliferation using methyl thiazolyl tetrazolium (MTT) assay, the apoptosis rate and cell cycle using flow cytometry (FCM), the morphological features of apoptotic cells using TdT-mediated-dUTP nick end labeling (TUNEL) and the expression of caspase-3 protein using Western blotting., Results: The growth of 3AO cells was inhibited by different concentrations of TRAIL (P<0.05). Morphological change of 3AO cells was clearly observed. TRAIL protein at 25 and 50 ng/mL significantly induced 3AO cell apoptosis and cell cycle arrest. With the treatment time went by, the percentage of cells at G1 phase increased and cells at S and G2/M phase decreased. The expression of caspase-3 protein was raised by TRAIL. No significant differences were noted in the apoptosis rate and the expression of caspase-3 protein between the 25 and 50 ng/mL TRAIL groups (P>0.05)., Conclusion: TRAIL protein could promote the apoptosis of 3AO cells through inhibiting cell growth cycle, blocking DNA synthesis and activating caspase-3.

Objective: To study the effect of chronic poisoning of ketamine on brain cell apoptosis in adult mouse under different duration and doses., Methods: The mouse model of chronic poisoning of ketamine was established on adult mouse by tail vein injection of ketamine twice every week with different doses (4, 10, 20 and 30 mg/kg). The mice were sacrificed after continuous injection of ketamine of 1, 2, 4, 8 and 12 weeks. The qualitative assessment of apoptosis was made by transmission electron microscope and the quantitative assessment was made by Caspase-3 immumofluorescence staining method and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to estimate the time point of apoptosis. All the experimental results were statistically analyzed., Results: The neuron apoptosis was observed in hippocampus and corpus striatum by transmission electron microscope one week after administration, and continued for eight weeks. High level of Caspase-3 expression was observed one week after administration, but with a low level expression after 4 weeks. The number of TUNEL positive cells obviously increased one week after administration and maintained in a high number at 4 weeks., Conclusion: Ketamine by tail vein injection could induce neuron apoptosis in adult mouse.

Objective: To observe the effect of apoptosis of human umbilical vein endothelial cells (HUVEC) induced by IgA1 from Henoch-Schönlein purpura (HSP) patients., Method: HUVEC were cultured in 3 different conditional media with IgA1 from HSP patients, normal healthy children and simply the cell culture medium. Serum IgA1 was purified by jacalin affinity chromatography, rates of apoptosis in HUVEC cells at different concentration and different times after incubation with IgA1 were determined by TUNEL method and flow cytometry. Real-time PCR and Western blot methods were used to detect the expression of caspase-3 and Fas, respectively., Result: Apoptosis rate of HUVEC by IgA1 isolated from HSP patients were significantly higher than that of the blank control [(14.77 ± 2.23)% vs. (2.25 ± 0.77)%, P < 0.01] and the apoptosis rate of HUVEC induced by IgA1 from normal healthy children was higher than that of blank control [(7.97 ± 1.48)% vs. (2.25 ± 0.77)%, P < 0.01]. The apoptosis rate of HUVEC induced by IgA1 from HSP was time and concentration-dependent. Moreover IgA1 isolated from HSP patients could significantly increase the caspase-3 and Fas expression (P < 0.01)., Conclusion: The IgA1 from HSP patients could induce the apoptosis of HUVEC, which might be related to the progression of HSP.

Objective: To explore the effects of marrow mesenchymal stem cell (BMSC) transplantation on retinal cells apoptosis and changes to neurotrophin-3 (NT-3 and ciliary neurotrophic factor (CNTF) in rats with retinopathy of prematurity (ROP)., Methods: Seven-day-old Sprague-Dawley rats were randomly divided into normal control (CON), ROP, BMSC transplantation (BMSCs were transplanted 5 days after oxygen conditioning) and phosphate buffered saline (PBS) groups. The ROP model was prepared according to the classic hyperoxygen method. Seven days after transplantation, TUNEL/DAPI, NT-3/API and CNTF/DAPI double-labeled immunofluorescence were used to examine the effects of BMSC transplantation on both the apoptosis of retinal cells and the expression of NT-3 and CNTF protein in the retinal cells of the ROP rats., Results: Seven days after BMSC transplantation, there were few TUNEL+ DAPI+ cells observed in the CON group. There were fewer TUNEL+DAPI+ cells observed in the BMSC group than in the ROP group (P<0.01), but there was no significant difference between the ROP and PBS groups (P>0.05). There were few NT-3+DAPI+ cells and CNTF+DAPI+ cells in the CON group. There were more NT-3+DAPI+ and CNTF+DAPI+ cells in the ROP group than in the CON group, but there was no significant difference between the ROP and CON groups (P>0.05). More NT-3+DAPI+ and CNTF+DAPI+ cells were observed in the BMSC group compared with the ROP group (P<0.01), and there was no significant difference in either NT-3+DAPI+ or CNTF+DAPI+ cells between the ROP and PBS groups (P>0.05)., Conclusions: BMSC transplantation therapy could alleviate the apoptosis of retinal cells in ROP rats, and its mechanisms might be associated with promoting the expression of NT-3 and CNTF protein in retinal cells.

Objective: To study the therapeutic efficacy of baicalin and its effect on apoptosis of inflammatory cells in spinal cords in Wistar rats with autoimmune encephalomyelitis (EAE)., Methods: Forty-four rats were randomly divided into four groups: normal control group (control, n=10), EAE group (n=12), and two intervention groups with dexamethasone (DXM) or baicalin. Seven days after immunization, the two intervention groups were injected intraperitoneally with DXM (1 mg/kg) and baicalin (200 mg/kg) for 1 week, respectively. The spinal cords were removed 14 days after immunization, and stained with hematoxylin and eosin. MBP expression in spinal cords was detected by immunohistochemistry. The apoptosis of inflammatory cells in spinal cords was detected by TUNEL., Results: The weight gain rate in the untreated EAE and the DXM or baicalin intervention groups were significantly lower than that in the control group (P<0.05). The weight gain rate in the baicalin intervention group was significantly higher than that in the untreated EAE and the DXM intervention groups (P<0.05). The scores of neurological function in the two intervention groups were significantly higher than that in the untreated EAE group (P<0.05). DXM or baicalin treatment significantly increased the MBP expression compared with the untreated EAE group (P<0.05). The apoptosis of inflammatory cells increased more in the DXM and the baicalin intervention groups compared with the untreated EAE groups (P<0.05)., Conclusions: Baicalin has protective effects against EAE in rats. It can promote the apoptosis of inflammatory cells in spinal cords.

Objective: To observe the effect of Ciclosporin (CsP) on apoptosis and expression of the associated protein Bcl-2, Caspase-3 in gingival epithelium of rats in order to approach the mechanism of CsP-induced gingival epithelium overgrowth., Methods: Eighty SPF grade male 7-week-old Wistar rats were randomly divided into experimental group and control group, and each group was divided into 4 subgroups according to the duration of treatment (10, 20, 30 and 40 days). The experimental objects were given fresh milk including CsP intragastrically and the control ones were given only fresh milk. After perfusion of 4% paraform for internal fixation, the specimens' bucco-lingual paraffin sections at lower first molar were made. Apoptosis was detected using TdT-mediated dUT nick end labeling (TUNEL) and the expression of Bcl-2 and Caspase-3 using immunohistochemisty of PV. The apoptotic index, positive cell rate of Caspase-3 and average gray scale of Bcl-2 was measured with an image analysis system. Data were analyzed by two-way analysis of variance of factorial design., Results: The apoptosis index and positive cell rate of Caspase-3 were down-regulated in the experimental group, and were significant difference from the control group (P < 0.05). The average gray scale of Bcl-2 was up-regulated in the experimental group, and was significant difference from the control group (P < 0.05)., Conclusion: CsP-induced gingival epithelial overgrowth is likely to associated with interference to the path of mitochondrial apoptosis and inhibition apoptosis.

To explore gambogenic acid (GNA)-induced apoptosis and underlying mechanism in vivo. A549 nude mice xenografts were used as in vivo model to study anticancer effect of GNA by observing tumor growth curve and weight of the tumor. Ultrastructure of A549 cells treated by GNA was observed by TE. Expression of COX-2 and VEGF were detected by immunohistochemistry. TUNEL assay was applied in examining apoptosis index of tumor cells. The tumor isolated from mice treated by GNA (8, 16 mg kg(-1)) took on a slow growth condition compared with control group. The results suggested that weight and volume of the tumor from experimental groups were remarkably decreased compared with control group (P < 0.05). Ultrastructure change of the tumor, such as vacuolization, abnormal distribution of the heterochromosome, volume of the tumor cells, even apoptotic bodies, were observed in GNA-treated group. While no apparent morphological change was observed in the normal group. Typical apoptotic characteristics could be distinctly observed in the mouse treated by GNA for 20 days and apoptosis index in GNA-treated group was significantly higher than model group. Expression of COX-2 and VEGF were significantly down-regulated in GNA-treated groups in comparison with control group (P < 0.01). These results indicate that GNA could affect the development and progression of A549 cells through inducing apoptosis, mediating the expression of VEGF in vascular cells and COX-2 in tumor cells.

Objective: Inducible nitric oxide synthase (iNOS) is a main rate-limiting enzyme resulting in over-production of nitric oxide following hypoxia-ischemia (HI). The aim of this study was to observe the expression of iNOS protein and gliacyte apoptosis in the brains of premature rats after HI, in order to explore possible relationships of iNOS with white matter damage (WMD)., Methods: One hundred and twelve 2-day-old premature rats were randomly subjected to right carotid ligation followed by 4 hrs hypoxic stress (WMD group) or sham operation (control group). The pups were sacrificed at 1, 3, 6, 12 hrs, and 1, 3 and 7 days after HI. Immunohistochemical technique was applied to determine the iNOS expression in periventricular white matter tissues. Gliacyte apoptosis was detected in these tissues by TUNEL., Results: Compared with the control group, iNOS expression began to increase 1 hr after HI and reached the peak 1 day after HI in the WMD group. Gliacyte apoptosis increased 1 hr after HI and peaked 1 day after HI in the WMD group compared with the control group., Conclusions: In the neonatal rats with WMD, the expression of iNOS may be involved in the ischemic cellular events including apoptosis, and plays a role in the pathophysiological process of WMD.

Objective: To study the effects of edaravone on glial fibrillary acidic protein (GFAP) and interleukin-1β (IL-lβ) expression and neuronal apoptosis in the juvenile rat hippocampus after status convulsion (SC)., Methods: One hundred and ninety-five juvenile male Sprague-Dawley (SD) rats were randomly divided into 3 groups: normal saline control and SC with and without edaravone treatment. Each of the 3 groups was further subdivided into subgroups sacrificed at 4, 12, 24, 48 and 72 hrs after SC (n=15). The SC model was prepared using lithium-pilocarpine. The expression of GFAP and IL-lβ protein was detected with immunohistochemistry methods. The neuronal apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL). The hippocampal GFAP mRNA expression was detected by RT-PCR., Results: The value of IOD of GFAP and IL-lβ positive cells measured by immunohistochemistry in the untreated SC group increased compared with the control group. Expression of GFAP and IL-lβ protein was significantly reduced in the edaravone treated SC group compared with the untreated SC group. RT-PCR showed the expression trend of GFAP mRNA was similar to that of protein. The TUNEL positive cells in the hippocampus CA1 in the untreated SC group increased significantly 12 hrs after SC and reached a peak at 48 hrs compared with the control group. The intervention with edaravone decreased significantly TUNEL positive cells between 12-48 hrs after SC, but the number of TUNEL positive cells in the intervention group remained significantly greater than in the control group., Conclusions: The expression of GFAP and IL-lβ in the hippocampus increases after SC in rats. Edaravone may decrease the expression of GFAP and IL-1β and reduce the number of neuronal apoptosis. These results suggest that edaravone may have protective effects against brain damage caused by SC.

Objective: To investigate the effects of the calmodulin inhibitor W-7 on the expression of the key marker of ERS GRP78 and neuronal apoptosis in the immature rat hippocampus after status convulsion (SC)., Methods: One hundred and seventeen male Sprague-Dawley rats aged 19-21 days were randomly divided into three groups: normal saline control (control), SC with and without W-7 pretreatment. Each of the 3 groups was further subdivided into subgroups sacrificed at 4, 24 and 48 hrs. SC model was prepared using lithium-pilocarpine. GRP78 mRNA expression in the hippocampus was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). GRP78 protein was ascertained by immunohistochemistry. Neuronal apoptosis was observed with TdT-mediated dUTP nick end labeling (TUNEL)., Results: The expression of GRP78 mRNA was significantly increased in the non-pretreated SC group compared with the control group 24 hrs after injection of saline or lithium-pilocarpine (P<0.01), and the expression of GRP78 protein also increased markedly in the seizure group compared with the control group 24 and 48 hrs after the injection (P<0.01). The expression of GRP78 mRNA and protein in the W-7 pretreatment group was significantly higher than both the control and the non-pretreated seizure groups 24 and 48 hrs after injection. The TUNEL positive cells in the hippocampus CA1 in the non-pretreated SC group 24 and 48 hrs after injection (21.0 ± 2.5 and 29.4 ± 2.8, respectively) were increased compared to the control group (7.1 ± 1.4 and 7.3 ± 1.6, respectively; P<0.01). W-7 pretreatment decreased TUNEL positive cells to 15.0 ± 2.5 and 20.0 ± 2.9 at 24 and 48 hrs after injection compared to the non-pretreated seizure group (P<0.01), but the number of TUNEL positive cells in the W-7 pretreatment group remained significantly greater than in the control group (P<0.01)., Conclusions: W-7 may up-regulate the expression of GRP78 and reduce the number of apoptotic neurons, thus provides a neuroprotective effect against brain damage following SC.

Objective: The aim of the present study is to investigate the effect of rhein treatment on the first-phase insulin secretory function in db/db mice., Method: Twenty 4-week-old male db/db mice were randomized to treatment with rhein (120 mg x kg(-1), n = 10) and placebo respectively (1% natrium cellulose solution, n = 10) by gavage for 8 weeks respectively. Ten age-matched non-diabetic male littermates db/m mice treated with placebo were studied as non-diabetic control. Body weight and fasting blood glucose level were measured before and after medication. The islets were isolated after 8 weeks' gavage. Islet perifusion system was set up, and all columns were perfused in parallel at a flow rate of 0.5 mL x min(-1) with KRB (2.8 mmol L(-1) glucose) at 37 degrees C. After 60-min static incubation with KRB (2.8 mmol x L(-1) glucose), the islets were stimulated in the continuous presence of a high concentration of 16.7 mmol x L(-1) glucose. Samples were collected every 20-second until 2-min, every 1-min until 5-min, thereafter every 5-min until 30-min. Samples were immediately stocked at -80 degrees C until further analysis., Result: Compared with the db/db control group, the fasting glucose concentration was significantly decreased in the rhein treatment group. The first-phase insulin secretory function was impaired significantly in db/db mice, while the first-phase insulin secretory peak was obvious in the rhein treatment mice., Conclusion: Rhein treatment significantly improved glucose tolerance, restored the first-phase insulin secretion and protected the islets function.

Aim: By observing the rats with PDTC after the intervention, myocardial ischemic reperfusion injury (MIRI) during apoptosis, NF-κB and iNOS expression. Analysis of NF-κB and iNOS expression in the rat heart ischemic-reperfusion injury, apoptosis in the process of regulation and its mechanism., Methods: Manufacture of myocardial ischemic-reperfusion model. Using transmission electron microscope methods and TUNEL method to observe the apoptosis myocardial cells and calculated the apoptotic index; by immunohistochemistry (SP method), the detection of myocardial cell NF-κB p65 expression, and the use of immunofluorescence to detect iNOS expression in myocardial cells., Results: IR group and PDTC +IR group, apoptosis and NF-κB and iNOS expression was significantly stronger than that of Sham group (P<0.05). PDTC +IR group, apoptosis, NF-κB and iNOS expression was significantly weaker than the IR group, the difference was significant (P<0.05)., Conclusion: Myocardial ischemia reperfusion injury, there is significant apoptosis. IR can induce NF-κB activation and iNOS formation. The NF-κB activation and iNOS formation, apoptosis of cardiac muscle cells are a catalyst. PDTC can effectively reduce the expression of NF-κB, to improve the oxygen scavenging cell function, decreased cell apoptosis, reduce the MIRI injury.

Objective: To investigate the expression of serine protease Omi/HtrA2 in kidneys of postasphyxial neonatal rats, and to study the effects of Ucf-101 on apoptosis and the expression of Omi/HtrA2 in these rats., Methods: Seventy-two neonatal Wistar rats of 7-10 days old were randomly divided into 3 groups: control, postasphyxial model, Ucf-101-treated postasphyxialThe postasphyxial model was established by normobaric asphyxiaExpression of Omi/HtrA2 was determined with streptavidin-peroxidase immunohistochemistry 2, 24 and 48 hrs after asphyxia. Terminal deoxynuleotidyl-mediated nick end labeling (TUNEL) was used to ascertain the apoptosis of renal cells., Results: Compared with the control group, OmiHtrA2 expression in renal cells began to increase 2 hrs after asphyxia and peaked at 24 hrs. The expression of Omi/HtrA2 in the Ucf-101-treated postasphyxial group was significantly lower than that in the postasphyxial model group (P<0.01). TUNEL-positive cells began to increase 2 hrs after asphyxia and peaked at 24 hrs in the postasphyxial model group when compared with the control group. The number of TUNEL-positive cells in the Ucf-101-treated postasphyxial group was significantly lower than that in the postasphyxial model group at all time points (P<0.01)., Conclusions: The expression of Omi/HtrA2 in kidneys is increased in postasphyxial neonatal rats. The increased Omi/HtrA2 expression may play an important role in the development of postasphyxial renal injury. Treatment with Ucf-101 can reduce the expression of Omi/HtrA2 in kidneys of postasphyxial neonatal rats and thus reduce renal tububar epithelial cell apoptosis.

Objective: To investigate the influence of di-(2-ethylexyl) phthalate (DEHP) on cell apoptosis and expression of Bcl-2 and bax in cultured human first trimester cytotrophoblasts., Methods: Human first trimester cytotrophoblasts were cultured with DEHP at concentration of 0, 25, 50, 100 µmol/L for 24 hours. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and flow cytometer method. The expression of apoptosis-associated genes, including Bcl-2 and bax, were detected by reverse transcription (RT)-PCR in cultured cytotrophoblast cells. The protein expression of Bcl-2 and bax in cytotrophoblast cells was measured by western blot., Results: (1) The expression of Bcl-2: when incubated with DEHP at concentration of 0, 25, 50 and 100 µmol/L, the expression of Bcl-2 were 1.00 ± 0.05, 1.03 ± 0.04, 1.04 ± 0.03, 1.04 ± 0.04, which did not show statistical difference (P > 0.05). The expression of Bcl-2 protein were 0.11 ± 0.02, 0.11 ± 0.04, 0.12 ± 0.02, 0.12 ± 0.03, which also didn't reach statistical difference (P > 0.05). (2) The expression of bax: when incubated with DEHP at concentration of 50 and 100 µmol/L, the expression of bax protein were 0.63 ± 0.04 and 0.81 ± 0.04, which were significantly higher than 0.23 ± 0.05 with DEHP at 0 µmol/L (P < 0.05). The expression of bax mRNA were 0.96 ± 0.04 and 1.02 ± 0.04, which was significantly higher than 0.81 ± 0.05 with DEHP at 0 µmol/L (P < 0.05). (3) Apoptosis: when incubated with DEHP at concentration of 50 and 100 µmol/L for 24 hours, the apoptotic cell ratio were (18.8 ± 2.6) % and (20.3 ± 2.0) % by annexin V-FITC/PI staining, which were significantly higher than (10.6 ± 1.4) % at 0 µmol/L and (18.1 ± 4.6) % and (19.5 ± 1.2) % by TUNEL staining, which were significantly higher than (11.2 ± 3.1) % at 0 µmol/L of DEHP (P < 0.05)., Conclusion: DEHP could induce apoptosis of cytotrophoblast cells by increasing bax gene expression, but had no effect on Bcl-2 expression.

Objective: To study the changes of brain-derived neurotrophic factor (BDNF) following repeated febrile seizures in rats and its possible correlation with neurocyte apoptosis., Methods: Fifty-one male Sprague-Dawley (SD) rats were randomly assigned to three groups: normal control (n=14), febrile seizure (FS, n=18), hyperthermia alone (n=19). Febrile seizures were induced by hot water bath. The level of BDNF in the hippocampal homogenate was measured using ELISA and the expression of BDNF in various brain regions was measured by immunohistochemistry. The neurocyte apoptosis of the brain was determined by TdT-mediated biotinylated-dUTP nick end labling (TUNEL)., Results: The level of BDNF in the hippocampus in the FS group(89.9+/-12.5 ng/g)was higher than that in the normal control group(54.4+/-18.9 ng/g)and in the hyperthermia alone group (64.1+/-15.0 ng/g) (P<0.01). The OD value of BDNF positive neurons in various brain regions of the FS group was significantly higher than that of the normal control group (P<0.01) and the hyperthermia alone group (P<0.01). The FS group had significantly higher apoptotic index in various brain regions than the normal control and the hyperthermia alone groups (P<0.01). There was a positive correlation between the expression of BDNF and the apoptotic index in various brain regions (r=0.332, P<0.05)., Conclusions: BDNF expression in the brain increases following repeated febrile seizures in rats, and the increased BDNF expression is correlated with neurocyte apoptosis.

Objective: To investigate the anti-apoptosis effects of Rubus alceaefolius total alkaloids in rats with hepatic injury., Method: The hepatic injury model of rat was induced by intraperitoneal injection with CCl4. Sixty SD rats were randomly divided into the normal group, the model group, the R. alceaefolius total alkaloids intervened group, and the bifendate intervened group. The expressions of the levels of liver cell apoptosis were determined by TUNEL. Ultrastructures of the liver cells were observed with transmission electron microscope., Result: Compared with the model group, the degree of hepatic injury and the positive expressions of apoptosis in liver tissues in the R. alceaefolius total alkaloids intervened groups and the bifendate intervened group were significantly lower., Conclusion: R. alceaefolius total alkaloids could reduce the pathological changes and degree of hepatic injury in rats, which may be partially through inhibiting the expressions of apoptosis in liver tissue.

Objective: To examine the expression of Fas and Fas ligand (FasL) in T lymphocytes of oral lichen planus (OLP) and the effects of Fas, FasL and activation-induced cell death (AICD) on OLP., Methods: The oral mucosa samples from patients with OLP and normal oral mucosa were assessed by in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine-5'-triphosphate (dUTP)-biotin nick end-labelling (TUNEL) assay for nucleosomal DNA fragmentation of apoptotic cells in infiltrating lymphocytes. Immunohistochemical technique was used for detection of expression level of Fas and FasL. Immunohistochemical double labeling was performed to examine the expression of Fas and FasL in CD8+ T cells and CD4+ T cells., Results: The rate of apoptosis in infiltrating lymphocytes of OLP was (1.9 +/- 1.8)%, which was lower than that of normal oral mucosa (P = 0.013). The expression of Fas and FasL were increased in lymphocytes of OLP (P = 0.005 and 0.000 respectively). The positive rate of double labeled cells of CD8+ and Fas+ was not increased in OLP (P = 0.313), and of CD8+ and FasL+ in OLP was higher than that of normal oral mucosa (P = 0.002). The expression of double labeled cells of CD4+ and Fas+ in OLP was higher than that of control group (P = 0.031). The expression of CD4+ and Fas+ T cells in reticular OLP were increased (P = 0.019), and of CD4+ and FasL+ did not show remarkable change (P = 0.097)., Conclusions: There was a low frequency of lymphocytic apoptosis. T cells, especially CD8+ T cells in OLP and CD4+T cells in atrophic-erosive OLP appeared to escape from AICD, which may account for the persistence of inflammation. Some CD4+ T cells in reticular OLP may go through AICD.

Objective: To study the possible protection of edaravone on neurons of the hippocampus after status convulsion (SC) and its effects on the expression of interleukin-1beta (IL-lbeta) in juvenile rats., Methods: One hundred and ninety-five juvenile male Sprague-Dawley rats were randomly divided into three groups: SC, edaravone pretreatment and normal saline control (control group). Each group was subdivided into five groups sacrificed at 4, 12, 24, 48 and 72 hrs after SC induction. SC model was prepared using lithium-pilocarpine. The edaravone pretreatment group received edaravone by intraperitoneal injection once daily three days before convulsion induction. Histopathologic changes in the hippocampus were viewed under a light microscope and an electron microscope. Expression of apoptosis cells was observed by TdT-mediated dUTP nick end labeling (TUNEL). Expression of IL-lbeta protein was determined by immunohistochemistry., Results: Under the electron microscrope, a small quantity of neurons showed karyopycnosis and endocytoplasmic reticulum (ER) expanded remarkably 24 hrs after SC induction; at 48 hrs the ER expanding was alleviated somewhat but mitochomdria swelling was more severe. The edaravone pretreatment group showed less severe neuronal changes compared with the SC group under the microscopes. The TUNEL positive cells in the hippocampus of the SC group were significantly more than those of the control group 12 hrs, and peaked at 48 hrs after SC induction. The edaravone pretreatment group showed decreased TUNEL positive cells in the hippocampus compared with the SC group, although the positive cells were more than those in the control group between 12 and 48 hrs after SC induction. The immunohistochemistry assay demonstrated that the expression of IL-lbeta in the hippocampus of the SC group increased significantly compared with that of the control group 12, 24, 48 and 72 hrs after SC induction. Edaravone pretreatment resulted in a significantly decreased IL-lbeta expression in the hippocampus as compared with the SC group., Conclusions: Edaravone pretreatment may decrease the IL-1beta expression and neuronal apoptosis in the hippocampus. This suggests that edaravone may have protective effects against the hippocampal damage caused by SC.

Objective: To observe the effect of the extract of Ginkgo biloba (EGb761) and the components isolated from the extract named ginkgolide B (GB) against damage of glutamate in pretreatment modes so that determine their application value and approach., Method: Based on glutamate-induced excitotoxicity to primary cultures from neonatal Sprague-Dawley (SD) rat hippocampal neuron, our experiment utilized trypan blue, TUNEL and LDH to study the effect of EGb761 and GB on neuron in different doses pretreatment modes, as well as to compare with the NMDA receptor uncompetitive antagonist-MK-801., Result: EGb761 and GB can recrease cell viability, reduce apoptosis rate and decrease LDH leakage in different degree and depended on dose in certain range. The maximal protection was achieved at a concentration of 100 mg x L(-1), 100 micromol x L(-1), but inferior to MK-801 (10 micromol x L(-1)). The protective effect of GB is superior to EGb761., Conclusion: Treatment with EGb761 and GB could protect the neurons against glutamate-induced injury. The maximal protection of GB was achieved by pretreatment is superior to EGb761, so its precautionanary intervention to high-risk population could have more value.

Objective: Castrated rats exhibit significant shrinkage of the ventral prostate and apoptosis of prostatic cells, which can be attributed to the reduced blood supply to the prostate. But what causes the blood decrease in the prostate remains unknown. This study aims to explore the molecular mechanism of the changes in the microcirculation of the ventral prostate of rats following castration., Methods: We randomized 24 male adult rats into 6 groups of equal number, and collected their ventral prostates at 0, 1/2, 1, 2, 3 and 7 d, respectively, after castration. Then we observed the changes of the microvessels under the transmission electron microscope, detected the apoptosis of endothelial cells by TUNEL, and determined the expressions of VEGF, endostatin, angiostatin and angiopoietin-2 by Western blot., Results: The castrated rats showed dramatic changes in the microvessels of the ventral prostate, obvious apoptosis of the endothelial cells, down-regulated expression of VEGF, and up-regulated expressions of endostatin and angiostatin, while angiopoietin-2 remained unchanged., Conclusion: The decreased level of VEGF and increased levels of endostatin and angiostatin might underlie the mechanism of the changes in the microcirculation of the ventral prostate of rats following castration.