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Objective To explore whether the environmental pollutant triclosan (TCS) has negative effects on the various biological characteristics of dental pulp stem cells (DPSCs), as well as the distribution and hazards of TCS in rat dental pulp tissue in vivo, which will provide a basis for the clinical application of DPSCs and the safety of TCS. Methods Tooth collection was approved by the Ethics Committee of Tianyou Hospital Affiliated to Wuhan University of Science and Technology. Human DPSCs were extracted, cultured, and identified. Up to 0.08 mmol/L of TCS was added to the in vitro culture medium of DPSCs. The proliferation ability of DPSCs was detected by CCK-8. The migration ability of DPSCs was detected via scratch assay. The differentiation ability of DPSCs was detected by inducing trilineage differentiation. The gene or protein expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), inducible nitric oxide synthase (iNOS), and transforming growth factor-β (TGF-β) in DPSCs were detected. The level of reactive oxygen species (ROS) generated by DPSCs was analyzed using fluorescence staining. Changes in mitochondrial membrane potential of DPSCs were detected using a fluorescent probe. The activity of PI3K/Akt/mTOR, p38, and JNK pathways of DPSCs were detected. Animal experiments were approved by the Animal Ethics Committee of Wuhan University of Science and Technology. A rat model of short-term oral exposure to 50 mg/kg/d of TCS for 2 months was established, and the TCS concentration in the liver, brain, and dental pulp tissues of rats was detected through liquid chromatography-mass spectrometry. Results TCS at 0.02 mmol/L, 0.04 mmol/L, and 0.08 mmol/L significantly inhibited the proliferation ability of human-derived DPSCs on the 5th and 7th days of contact. TCS at 0.04 mmol/L and 0.08 mmol/L significantly inhibited the migration ability and tri-lineage differentiation ability of DPSCs on the 3rd day of contact. TCS induced the gene or protein expression of proinflammatory factors including TNF-α, IL-1β, IL-6, and iNOS, induced the gene or protein expression of TGF-β, and inhibited the protein expression of anti-inflammatory factor IL-10. On day 1, TCS at 0.04 mmol/L and 0.08 mmol/L induced the production of ROS in DPSCs and reduced the mitochondrial membrane potential of DPSCs. On day 3, TCS at these levels inhibited PI3K/Akt/mTOR pathway activity and enhanced p38 pathway activity of DPSCs, without affecting the pathway activity of JNK. After short-term intragastric exposure of rats to TCS, TCS was detected in liver (430 ng/mL) and brain (41.4 ng/mL) tissues but not in the dental pulp. The TCS concentration was highest in the liver, but no obvious histopathological changes were observed. Conclusion TCS inhibits a variety of biological characteristics of DPSCs and poses a potential risk to the organism. No TCS exists in the dental pulp tissue of rats exposed to TCS for a brief period of time, and the health of the rats is not damaged.

Objective: To investigate effects of adiponectin on survival, oxidative stress, inflammation and mitochondrial function of endothelial cell HUVEC induced by high glucose, and its potential mechanism. Methods: HUVEC cells were cultured with 5, 25 mmol/L glucose and divided into normal (NC) group and high glucose (HG) group. Adiponectin (1, 2, 4 µg/ml) treatment of high glucose induced endothelial cells were divided into low dose (L), medium dose (M) and high dose (H) groups. HUVEC cells treated with PBS, LPS and 4 µg/ml adiponectin were labeled as H+PBS group and H+LPS group. CCK8 and Annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis. Western blot was used to detect protein expressions of Cleaved caspase-3, NLPR3, amino acid transporter (ASC), Caspase-1, p-nuclear factor κB inhibitor protein (p-I-κBα) . ELISA was used to detect concentrations of lactate dehydrogenase (LDH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), IL-4, IL-6 and IL-1β. ROS fluorescence activity was detected by immunofluorescence. Mitochondrial membrane potential was detected by JC-1 staining. NLRP3 expression was detected by RT-qPCR. Results: Compared with NC group, 48 h, 72 h and 96 h survival rates of endothelial cells were significantly reduced, apoptosis rate was significantly increased, and levels of LDH, SOD, MDA, mitochondrial membrane potential, IL-4, IL-6 and IL-1β in cell supernatant were significantly increased, concentrations of ROS and GSH-Px were significantly decreased, and protein expressions of Cleared caspase-3, NLRP3, Caspase-1 and p-NF-κB were significantly increased, p-I-κBα expression was decreased significantly (P<0.05) . Adiponectin inhibited above changes of endothelial cells induced by high glucose in a concentration dependent manner. LPS attenuated above protective effects of adiponectin on HG induced endothelial cells. Conclusion: Adiponectin can reduce survival inhibition, oxidative stress, inflammatory response and mitochondrial dysfunction of endothelial cells induced by high glucose, whose protecting mechanism may be related to NF-κB pathway. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Science & Biotechnology is the property of Journal of Food Science & Biotechnology Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective To observe whether spleen aminopeptide oral solution has any effect on the proliferation, apoptosis and migration of human lung cancer cell line A549, and to further explore the possible mechanism of action of spleen aminopeptide oral solution. Methods A549 cells in the logarithmic growth phase were randomly divided into four groups： groups I, II, III, and IV. Cells in the groups I, II and III were added with 0. 5, 1. 0 and 2. 0 mg/mL spleen aminopeptide oral solution in DMEM medium containing 10% fetal bovine serum, and cells in the group IV were not treated. We observed the proliferation, apoptosis, and migration of cells and detected the mitochondrial membrane potential and reactive oxygen species (ROS) in cells of each group. Results Compared with the group IV, the cell viability of cells in the groups I, II and III at 24 h of culture was lower, the cell confluence of cells in the groups II and III at all time points was lower, and the density of relative scratch area was smaller; the apoptosis rates of the groups I, II and III at 24 h of culture were higher, the mitochondrial membrane potential was lower, and the relative expression levels of ROS in cells in the groups II and III at 24 h of culture were higher (all P<0. 05). With the increase of the concentration of splenic aminopeptide oral olutionn, the viability of A549 cells gradually decreased, the cell confluence decreased, the apoptosis rate increased, the density of relative scratch area decreased, the mitochondrial membrane potential decreased, and the relative expression of ROS increased, and it was in a dose-dependent manner (all P<0. 05). Conclusion Spleen aminopeptide oral solution could inhibit the proliferation and migration of A549 cells and promote the apoptosis of these cells, with the most obvious effect of spleen aminopeptide oral solution at a concentration of 2. 0 mg/mL. The spleen aminopeptide oral solution might exert the above effects by decreasing the mitochondrial membrane potential and promoting the expression of cellular ROS. [ABSTRACT FROM AUTHOR]

Objective To evaluate the effects of semen storage temperature and storage duration on mitochondrial membrane potential(MMP). Methods Thirty semen samples from healthy men were collected, and divided into 14 parts respectively, one part was immediately determined (within 1 h), two parts were stored at 4 T;, four parts were stored at room temperature, four parts were stored at 37 T, and three parts were immediately frozen at 一 80 戏.Semen stored at room temperature and 37 七 for 2, 4, 8, 24 h, refrigerated at 4 for 1, 3 d, and frozen at -80 for 1, 3, 7 d, respectively. Sperm mitochondrial membrane potential (MMP) was analyzed immediately after the storage time was reached. Results At room temperature and 37 兀,speim MMP decreased gradually with the extension of storage time. Compared with the real-time measurement, the MMP of sperm stored for 8, 24 h was significantly decreased (PvO.Ol). At 4, -80, the MMP of sperm stored for 1, 3, 7 d decreased significantly (P <0・ 01)・ Conclusion Storage conditions and storage time of semen in vitro are the important factors affecting mitochondrial fimction of sperm. At room temperature and 37 °C, semen analysis should be completed within 4 hours, and internal quality control before analysis is crucial to MMP test. [ABSTRACT FROM AUTHOR]

Objective: To discuss the inhibitory effect of gallic acid-lecithin complex (GA-LC) on the proliferation of the lung cancer A549 cells induced by X-ray irradiation, and to clarify its possible mechanism. Methods: The A549 cells were divided into different concentrations (0, 0. 05, 0. 10, 0. 15, 0. 20, 0. 25, 0. 30, and 0. 35 g·L-1) of GA-LC groups. CCK-8 assay was used to detect the proliferation activities of A549 cells in various groups, and the half-inhibitory concentration (IC50) value of GA-LC was determined. The A549 cells were divided into control group, GA-LC group, 4 Gy X-ray irradiation group, and GA-LC+4 Gy X-ray irradiation group. CCK-8 assay was used to detect the proliferation activities of cells in various groups; flow cytometry was used to detect the reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP) levels and apoptotic rates of cells in various groups; the mitochondrial green fluorescence probe Mito-tracker was used to detect the permeability of mitochondrial membrane. Results: Compared with 0 g·L-1 GA-LC group, the proliferation activities of the cells in 0. 15, 0. 20, 0. 25, 0. 30 and 0. 35 g·L-1 GA-LC groups were significantly decreased (P< 0. 05), and the IC50 value of GA-LC to the A549 cells was 0. 171 1 g·L-1 . Compared with control group, the proliferation activities and MMP levels of the cells in GA-LC group, 4 Gy X-ray irradiation group, and GA-LC+4 Gy X-ray irradiation group were significantly decreased (P<0. 05 or P<0. 01), while the ROS levels and apoptotic rates of the cells in GA-LC group and GA-LC+4 Gy X-ray irradiation group were significantly increased (P<0. 05 or P<0. 01). Compared with GA-LC group, the MMP level of the cells in GA-LC+4 Gy X-ray irradiation group was decreased (P<0. 01), while the ROS level and apoptotic rate of the cells in GA-LC+4 Gy X-ray irradiation group were significantly increased (P<0. 05); compared with 4 Gy X-ray irradiation group, the proliferation activity and MMP level of the cells in GA-LC+4 Gy X-ray irradiation group were decreased (P<0. 05), while the ROS level and apoptotic rate of the cells in GA-LC+4 Gy X-ray irradiation group were significantly increased (P<0. 05 or P<0. 01). The mitochondrial fluorescence probe Mito-tracker staining results showed that the green fluorescence could be observed under fluorescence microscope; compared with control group, the green fluorescence intensities in the cells in GA-LC group, 4 Gy X-ray irradiation group, and GA-LC+4 Gy X irradiation group were increased, and the green fluorescence intensity in the cells in GA-LC+4 Gy X-ray irradiation group was the most obvious, demonstrating that the mitochondrial membrane permeability was increased. Conclusion: GA-LC can enhance the inhibitory effect of X-ray irradiation on the proliferation activity of the lung cancer A549 cells, and its mechanism is related to the induction of oxidative stress, thereby increasing the mitochondrial permeability, decreasing the MMP and inducing the apoptosis. [ABSTRACT FROM AUTHOR]

Objective: To discuss the effect of baicalin on the proliferation of the human tongue squamous cell carcinoma (tongue squamous cell carcinoma) CAL27 cells, and to clarify its potential mechanism. Methods: The CAL27 cells at logarithmic growth phase were divided into control group and different concentrations (12. 5, 25. 0, 50. 0, 100. 0, and 200. 0 μmol·L-1)of baicalein groups, the clone formation of cells in various groups was observed by crystal violet staining; the proliferation rates of cells in various groups were detected by CCK-8 method; the levels of reactive oxygen species (ROS) in cells in vaious groups were detected by 2', 7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence probe; the mitochondrial membrane potential (MMP) of cells in various groups were detected by Rhodamine 123 fluorescence probe. The CAL27 cells at logarithmic growth phase were divided into control group and different concentrations (50, 100, and 200 μmol·L-1)of baicalin groups. Flow cytometry was used to detect the percentages of the cells at different cell cycles and the apoptotic rates of cells in various groups. The CAL27 cells at logarithmic growth phase were divided into control group and different concentrations (50 and 100 μmol·L-1)of baicalein groups, N-neneneba acetylcysteine (NAC) group, 50 μmol·L-1 baicalin+NAC group, and 100 μmol·L-1 baicalin+NAC group. The levels of ROS and MMP of cells in vraious groups were detected by DCFH-DA fluorescence probe and Rhodamine 123 fluorescence probe. Results: The crystal violet staining results showed that compared with control group, the numbers of clone formation of the cells in different concentrations of baicalin groups were decreased in a concentrationdependent manner, and there was almost no clone formation of the cells in 200 μmol·L-1 baicalin group. The CCK-8 assay results showed that compared with control group, the proliferation rates of the cells in different concentrations of baicalin groups were significantly decreased in a concentration-dependent manner (P<0. 05 or P<0. 01). Compared with control group, the ROS levels of the cells in different concentrations of baicalin groups were significantly increased (P<0. 05) and the MMP levels were significantly decreased (P<0. 05). Compared with control group, the percentages of the cells at S phase in different concentrations of baicalin groups were significantly increased (P<0. 05), the percentages of the cells at G0/G1 phase were significantly decreased (P<0. 05), and the apoptotic rates were significantly increased (P<0. 05). After the combination of baicalein and NAC, compared with control group, the ROS levels of the cells in 50 and 100 μmol·L-1 baicalein groups were significantly increased (P<0. 05) and the MMP levels were significantly decreased (P<0. 05); compared with 50 and 100 μmol·L-1 baicalein groups, the ROS levels of the cells in 50 μmol·L-1 baicalein+NAC group and 100 μmol·L-1 baicalein+ NAC group were significantly decreased (P<0. 05), and the MMP levels were significantly increased (P< 0. 05). Conclusion: Baicalein can inhibit the proliferation of the CAL27 cells by activating the mitochondrial oxidative stress pathway. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Chinese Institute of Food Science & Technology / Zhongguo Shipin Xuebao is the property of Journal of Chinese Institute of Food Science & Technology Periodical Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective: The protective effect of Polygonatum odoratum polysaccharides (POP) on D-galactose (D-gal) induced senescence in rat aortic smooth muscle cells (A7r5) was investigated. Methods: Firstly, the POP was extracted by the water extraction and alcohol precipitation methods. Next, the phenol concentrated sulfuric acid method was applied to further measure the total sugar content of POP. In addition, the A7r5-senescent cell model was established by the D-galactose treatment. Finally, the MTT assay, β-galactosidase staining, reactive oxygen species (ROS) staining as well as JC-1 staining were applied to further detect the protective effect of POP in A7r5-senescent cells induced by D-galactose. Results: This study found an yield of POP was 8.23% and its total sugar content was 76.48%±0.036%. As a result, the MTT assay showed the viability of the A7r5 cells was increased to 174.89%±3.30% after 24 h treatment by 400 μg/mL of POP, while it was decreased to 65.93%±1.63% after 48 h treatment by 40 mg/mL of D-galactose. Interestingly, when a concentration of POP was 100 μg/mL, this study had found the most significant protective effect in A7r5-senescent cells induced by D-galactose, which increased its cell viability to 226.87%±12.58% as compared with the control group (P

Objective To study the effect and mechanism of total astragalosides(ASTs) on myocardial cell apoptosis and mitochondrial membrane potential in rats with heart failure. Methods Rats were divided into control group(normal), heart failure group(model), AST low, medium and high intervention group (10, 20 and 40 mg/kg intragastric administration) and 12 rats in each group. Cardiac function was measured in rats. Myocardial tissue was taken from rats, apoptosis was detected by TUNEL assay, the activity of SOD was detected by xanthine oxidze method, the activity of GSH-PX was determined by dithio-dinitromethylbenzene method, MDA content was determined by thiobarbituric acid method, the mitochondrial membrane potential was measured by JC-1 method. Western blot was used to detect the expression levels of c-caspase-3, c-caspase-9, p-AKT, p-p38MAPK and Cyt-C in mitochondria and cytoplasm. Results Compared with normal group, LVSP,+dp/dtmax,-dp/dtmax in model group decreased(P＜0.05),the apoptotic rate and the level of c-caspase-3 and c-caspase-9 protein increased(P＜0.05), the activities of SOD and GSH-PX decreased(P＜0.05), the content of MDA increased(P＜0.05), the mitochondrial membrane potential decreased(P＜0.05), the level of Cyt-C protein in mitochondria decreased(P＜0.05), Cyt-c protein level in cytoplasm increased(P＜0.05), the level of p-AKT decreased, the level of p-p38MAPK increased(P＜0.05). Compared with model group, LVSP, +dp/dtmax,-dp/dtmax increased in AST-L, AST-M and AST-H groups, the apoptotic rate and c-caspase-3,c-caspase-9 protein levels decreased, the activities of SOD and GSH-PX increased, the content of MDA decreased, the mitochondrial membrane potential increased, the level of Cyt-C protein in mitochondria increased, Cyt-c protein level in cytoplasm decreased, the level of p-AKT increased, the level of p-p38 MAPK decreased, the higher the concentration of total astragalosides is, the greater the effect on heart failure rat model. Conclusions Total astragalosides inhibit cardiomyocyte apoptosis in rats with heart failure. The mechanism may be related to the increase of mitochondrial membrane potential and the influence of AKT and p38MAPK signaling pathways.

Objective To investigate the effect of down-regulation of Notch1 expression mediated by lentivirus on the activation of caspase-9 and mitochondrial membrane potential of fibroblast from pathological scars(PSF). Methods PSFs were isolated and cultured and then infected by Notch1 siRNA lentivirus and negative control lentivirus respectively.The level of Notch1 mRNA and protein was detected by real-time PCR and Western blot,respectively.MTT assay was used to detect cell proliferation; PI monochrome staining was used to detect cell cycle changes. Annexin V-FITC/PI assay was used to detect apoptotic changes; Western blot was used to detect the level of cyclin D1, CDK4, c-caspase-3, c-caspase-9 and cytochrome C protein levels in cytoplasm and mitochondria; JC-1 method was used to detect the mitochondrial membrane potential. Results The expression of Notch1 in PSFs transfected with Notch1 siRNA lentivirus was significantly lower than that in the negative control lentiviruses and the non-transfected PSFs(P＜0.05).The proliferation of PSFs decreased after Notch1 was down-regulated (P＜0.05);the percen-tage of G0/G1 phase increased(P＜0.05); the apoptotic rate increased(P＜0.05); the expression of c-caspase-3 and c-caspase-9 protein increased(P＜0.05); the mitochondrial membrane potential of the cells decreased significantly(P＜0.05); the level of cyclin D1 and CDK4 protein in cells decreased(P＜0.05); cytochrome C protein increased in cytoplasm and decreased in mitochondria(P＜0.05). Conclusions Down-regulation of Notch1 inhibits proliferation and induces apoptosis of PSF.

This study investigated the effects of ergosterol peroxide(EP) on the proliferation and apoptosis of MCF-7 breast cancer cells, explored its possible mechanisms of action, and verified the effects and mechanisms by in vitro experiments. Network pharmaco-logy was used to screen the target proteins of EP and construct target networks and protein-protein interaction(PPI) networks to predict the potential target proteins and related pathways involved in EP anti-breast cancer effects. The MTT assay was performed to measure the inhibitory effect of EP on MCF-7 cell proliferation, and the colony formation assay was used to assess the cell cloning ability. Flow cytometry and laser confocal microscopy were employed to evaluate cell apoptosis, mitochondrial membrane potential and reactive oxygen species(ROS) levels. Western blot analysis was conducted to examine the expression levels of B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), cytochrome C(Cyt C), caspase-7, cleaved caspase-7, phosphatidylinositol 3-kinase(PI3K), and se-rine/threonine kinase B(AKT) in MCF-7 cells treated with EP. The results of network pharmacology prediction yielded 173 common targets between EP and breast cancer; the results of Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis showed that EP treatment for breast cancer mainly affected the signaling pathways such as cancer pathway, PI3K-AKT signaling pathway, cellular senescence signaling pathway, and viral carcinogenesis pathway; and the MTT assay results showed that the viability of MCF-7 cells in the EP group was significantly lower than that in the control group, exhibiting a time-and concentration-dependent trend, and EP can inhibit colony formation of MCF-7 breast cancer cells. Treatment with 10, 20, and 40 μmol·L~(-1) EP for 24 h resulted in a significant increase in the total apoptosis rate of MCF-7 cells, a significant decrease in mitochondrial membrane potential, and a significant increase in ROS levels. In addition, treatment with EP led to an upregulation of Cyt C, Bax, and cleaved caspase-7 protein expression, and a downregulation of p-PI3K, p-AKT, and Bcl-2 protein expression in MCF-7 cells. Studies have shown that EP inhibits MCF-7 breast cancer cell proliferation and reduces colony formation by a mechanism that may be related to the PI3K-AKT pathway mediating the mitochondrial apoptotic pathway.

This study aims to investigate the effect of ergosterol peroxide(EP) on the apoptosis of human hepatocellular carcinoma and its mechanism of action. The cell viability of HepG2 and SK-Hep-1 cells with 0(blank control), 2.5, 5, 10, 20, 40, and 80 μmol·L~(-1) of EP after 24, 48, and 72 h of action was detected by using CCK-8 assay, and the half inhibitory concentrations(IC&#95;(50)) at 24, 48, and 72 h were calculated. Formal experiments were performed to detect the effect of EP on intracellular reactive oxygen species(ROS) using DCFH-DA staining, the effect of EP on intracellular mitochondrial membrane potential using JC-1 staining, the number of apoptotic cells using Annexin V-FITC/PI double-staining after HepG2 cells were co-cultured with 0(blank control), 10, 20, 40 μmol·L~(-1) EP for 48 h. The effects of EP at different concentrations on apoptotic morphology were detected using AO/EB staining. The effects of different concentrations of EP on the protein expression of mitochondrial apoptosis pathway-related proteins B cell lymphoma 2(Bcl-2), cytochrome C(Cyt-C), Bcl-2-related X protein(Bax), caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9 were examined by using Western blot. The results showed that different concentrations of EP could inhibit the proliferation of hepatocellular carcinoma with concentration-and time-dependent trends. Compared with the blank control group, the ROS level in the EP-treated group increased significantly(P&lt;0.05). The mitochondrial membrane potential decreased significantly(P&lt;0.05). The total apoptosis rate increased significantly(P&lt;0.05). The expression of Bcl-2 protein was significantly down-regulated, and the expression of Cyt-C, Bax, cleaved caspase-9, and cleaved caspase-3 were significantly up-regulated(P&lt;0.05). In summary, EP may inhibit the proliferation of hepatocellular carcinoma by modulating the mitochondria-mediated apoptosis pathway and induce apoptosis.

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective To explore the effects of C-reactive protein(CRP) on myocardial ischemia reperfusion injury in rat cardiomyocytes and its mechanisms. Methods We simulated ischemia reperfusion using oxygen-glucose deprivation/reoxygenation(OGD/R) in neonatal Sprague-Dawley rat cardiomyocytes. Cardiomyocytes were treated with CRP, Cyclosporine A(CsA), diazoxide(DZ) and divided into the control group, OGD/R group, CRP + OGD/R group, CRP + CsA +OGD/R group and CRP + DZ + OGD/R group. The myocardial cell viability,Lactic acid dehydrogenase(LDH) level in cell culture medium, mitochondrial membrane permeability transition pore(mPTP) opening level and mitochondrial membrane potential level were measured respectively. Cell viability was calculated as the percentage of the control group. Results Compared with the simple OGD/R group, the cell viability and mitochondrial membrane potential level decreased markedly, and the LDH leakage and mPTP opening level increased significantly in CRP + OGD/R group. All the differences were statistically significant(P < 0. 05). Compared with CRP + OGD/R group, in CRP + CsA + OGD/R group and CRP + DZ + OGD/R group, both cell viability and mitochondrial membrane potential level increased observably, and both LDH leakage and mPTP opening level reduced markedly. Conclusion Our results suggested that CRP directly aggravates myocardial ischemia reperfusion injury in myocardial cells and that this effect is primarily mediated by inhibiting mito KATP channels and promoting mPTP opening. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of lidamycin on the proliferation of human cervical cancer Caski cells and its underlying molecular mechanisms. Methods Human cervical cancer Caski cells were randomly divided the experimental groups (including the low-dose, medium-dose and high-dose groups) which were separately treated with 1 , 2.5 and 5 ng/mL lidamycin ( 100 μL) for 24 h, and the control group which was added with the same volume of RPM1-1640. MTT assay was used to detect the inhibition rate of proliferation. The cell cycle and mitochondrial membrane potential of CasKi cells were measured by flow cytometry. Transmission electronic microscope was used to observe the morphological changes of Caski cells. We calculated tlie apoptosis rate and mitotic catastrophe rate. Results The proliferation rate and survival rate of the experimental groups were significantly lower than those of the control group, and the decrease in the high-dose group was more significant ( all P <0. 05 ). flic percentage of cells in the S phase ol the control group and the medium-dose group was higher than those in the low-dose and high-dose groups (all P <0.05). The percentage of cells in the G0/G1 phase of the experimental groups was lower than that of the control group and the percentage of cells in the G2/M phase was higher than that of the control group, especially in the medium-dose and high-dose groups ( all P <0. 05). The ratios of mitochondrial membrane potential decrease in the low-dose, medium-dose and high-dose groups and control group were 34.77% ±5.33% , 37.87% ±5.73% , 18.37% ±3.91% , and 4. 17% ±0.65% , respectively. The mitochondrial membrane potential decreased ratios of the experimental groups were higher than that in the control group, especially in the low-dose and medium-dose groups (all P <0.05). Transmission electronic microscopy showed that the control group had normal morphology while the experimental groups had many forms of death cells, including apoptosis, necrosis and mitotic catastrophe. The apoptosis rate and mitotic catastrophe rate of the experimental groups were both higher than those of control group (both P <0.05). Conclusion Lidamvcin can significantly inhibit the proliferation of Caski cells with an dose-de-pendent manner by inducing the apoptosis and mitotic catastrophe of cells. [ABSTRACT FROM AUTHOR]

Objective To observe the effects of dichloroacetate (DCA)on the proliferation and apoptosis of bladder cancer cells T24 in vitro and to study the underlying mechanism. Methods The bladder cancer cells T24 were randomly divided into four groups: experimental group 1, experimental group 2, experimental group 3, and the control group. Cells in the experimental groups 1, 2, and 3 were added with 5, 10, and 20 mmol/ L DCA, respectively; cells in the control group were added with 0. 1% dimethyl sulfoxide (DMSO). The cell proliferation of T24 cells were detected by MTT assay at 24, 48, and 72 h after DCA treatment. At 48 h, the apoptosis was examined by Hoechst33258 staining,the mitochon-drial membrane potential was detected by using JC-1 staining,and the expression levels of Bax protein, Bcl-2 protein and Caspase-3 protein were determined by Western blotting. Results The cell proliferation in the experimental groups 1, 2, and 3 was lower than that of the the control group at 24, 48 and 72 h, and with the time and increasing concentrations of DCA, the inhibition of cell proliferation was more significant (all P < 0. 05). The number of apoptosis cells in the experimental groups 1,2 and 3 was higher than that of the control group, and the mitochondrial membrane potential was lower than that of the control group (all P < 0. 05). Compared with the control group, the expression of Bax protein and Caspase-3 protein significantly increased, while the expression of Bcl-2 protein significantly decreased in the experimental groups 1, 2, and 3 (all P < 0. 05). Conclusions DCA can inhibit the proliferation and induce the apoptosis of bladder cancer cell line T24, which suggests that the mechanism may be related to the decrease of mitochondrial membrane potential level,up-regulation of Bax protein and Caspase-3 protein, and down-regulation of Bcl-2 protein. [ABSTRACT FROM AUTHOR]

Background and objective The use of platinum-based combination chemotherpy remains the standard treatment for non-small cell lung cancer. However, the resistance to platinum limits further treatment clinically. Trichostatin A (TSA) is one of histone deacetylase (HDAC) inhibitors. It inhibits tumor cell proliferation and acts as a chemosensitizer. The aim of this study is to investigate the action mechanism of TSA on cisplatin-resistant human lung adenocarcinoma cell line A549/CDDP. Methods Cytotoxicity and cell viability was assayed by Neutral Red method. Morphologic assessment of apoptosis was determined by fluorescence microscope; cell cycle and mitochondrial membrane potential were detected by flow cytometry. In addition, A549/CDDP cells were transfected with Bcl-2 expression Vector and siRNA-bcl-2. Results A549/CDDP cells treated with TSA showed apparently cytotoxicity, IC50 of TSA was (446.59±27.32) nmol/L. The growth curve showed the ratio of growth decreased with the increase of concentration of TSA. The apoptosis appeared 24 hours after treated by (125-500) nmol/L TSA, morphologic changes including nuclear chromatin condensation. Fluorescence strength was observed with fluorescence microscope. Treated by TSA, mitochondrial membrane potential was decreased and cells were arrested at S phase. Western blotting analyses showed that the levels of Bcl-2 decreased, while expression of Bax increased. Simultaneously caspase-3 was activated. Over expression of Bcl-2 can inhibit TSA-induced A549/CDDP cell apoptosis, while the decrease of Bcl-2 enhanced the sensitivity of A549/CDDP cell to TSA. Conclusion TSA induce A549/CDDP cell apoptosis by mitochondria pathway.

Objective To investigate the influence of ischemia reperfusion （IR） on cell viability and apoptosis of rat myocardial cells H9c2 as well as the potential molecular mechanism. Methods H9c2 cells were divided into groups A （control） and B （IR） and were seeded in 96-well plates by 1 × 104/well. After adhering for 24 h, the culture medium of group B was replaced by 100 μL hypoxia buffer and the cells were incubated in 94% N2, 1% O2, and 5% CO2 for 90min. Then we discarded the hypoxia buffer, and incubated them in normal environment with 100 μL/well DMEM of high glucose medium for another 90 min. The cell viability was detected by MTT, apoptosis by TUNEL, mitochondrial membrane potential （MMP） and reactive oxygen species （ROS） by laser scanning confocal microscope. Furthermore, heat shock protein 60 （HSP60）, peroxiredoxin （Prx）, thioredoxin （Trx） and mitochondria fission protein （Fis1） were detected by Western blotting. Results The cell viability of groups A and B was 0. 77 ± 0. 01 and 0. 40 ± 0. 02, respectively （P< 0. 05）. The apoptosis rates in groups A and B were 2. 24% ± 0. 12% and 41. 78% ± 1. 43% （P < 0. 05）. Intracellular ROS and mitochondrial membrane potential of group A was 0. 58% ± 0. 02% and 1. 12% ± 0. 05%, and was 1. 13% ±0. 05% and 0. 76% ± 0. 01% in the group B （all P < 0. 05）. Compared with group A, the expression levels of HSP60 and Fis1 were increased, and the levels of Prx2 and Trx1 were decreased （all P < 0. 05）. Conclusion After ischemia reperfusion, the cell viability decreases and the apoptosis increases in H9c2 cells, and its mechanism may be that ischemia reperfusion promotes the expression of HSP60 and Fis1 protein and inhibits the expression of Prx2 and Trx1 protein. [ABSTRACT FROM AUTHOR]

Objective To investigate the protective effects of dexmedetomidine on bupivacaine-induced neurotoxicity. Methods Mouse neuroblastoma cell line N2a cells were divided into four groups. The cells in the control group were incubated with no drug adding while the cells in bupivacaine group were treated with 1 000 μmol/L bupivacaine for 24 h. The cells in the group Dex1 and Dex2 were incubated with 1 000 μmol/L bupivacaine and 50 μmol/L, 200 μmol/L dexmedetomidine for 24 h respectively. MTT assay was used to evaluate the cell viability. The reactive oxygen species (ROS) activity, mitochondrial membrane potential (MMP), the expression of Caspase-3 and apoptotic rate of N2a cells were detected by flow cytometry. Results The cell viabilities were significantly decreased after being treated with 1 000 μmol/L bupivacaine, MMP was also significantly decreased, and apoptotic rates, levels of ROS and Caspase-3 were significantly increased. The bupivacaine-induced cytotoxicity was inhibited by dexmedetomidine (50 and 200 μmol/L), which resulted in the increase in the cell viability and MMP, but decrease in apoptotic rate and levels of ROS and Caspase-3. These effects were more significant in 200 μmol/L dexmedetomidine group than those of 50 μmol/L dexmedetomidine group. Conclusion Dexmedetomidine attenuates bupivacaine-induced cytotoxicity of N2a cells, which may be related with the inhibition of ROS, the decrease in MMP and Caspase-3, and inhibiting appotosis in N2a cells. [ABSTRACT FROM AUTHOR]

Objective: This study aims to investigate the mechanism of Salidroside(Sal) protecting mitochondrial morphology and function in MPP+induced SH-SY5 Y cells. Methods: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay was used to test the cell activity; Mito Tracker Red CMXRos was used to test the mitochondrial morphology; Tetramethylrhodamine ethyl ester(TMRE) was used to test the mitochondrial membrane potential(MMP) and Western blot method was used to detect the protein level of PINK1 and Parkin. Results: Sal treatment of 24 h had no effect on cell activity, mitochondrial morphology and MMP(P>0.05). When compared with the control group, the cell activity and MMP levels were statistically decreased respectively(P<0.01), and the length of mitochondrial was statistically shortened(P<0.01) and in fragmentation after MPP+(500 μM) treated 24 h in SH-SY5 Y cells. However,pretreatment with Sal(25 μM) 24 h could significantly inhibit MPP+induced the decrease of cellular activity(P<0.01), and maintain mitochondrial length and increase the level of MMP(P<0.01). Moreover, pretreatment with Sal(25 μM) 24 h could significantly inhibit the MPP+(500 μM) induced the decrease of protein level of PINK1 and Parkin(P<0.01). Conclusion: Our vitro experiments showed that Sal could protect the MPP+induced the decrease of SH-SY5 Y cell activity and shortened the length of mitochondrial and in fragmentation.PINK1-Parkin pathway might be one of the mechanisms. It was provided experimental basis for Sal as a new drug for the treatment of PD. [ABSTRACT FROM AUTHOR]

Objective To observe the antioxidant effect on the proliferation of acute monocytic leukemia cells by establishing its reactive oxygen species (ROS) cell model. Methods U937 cell treated with different concentrations of hydrogen peroxide (H2O2) was used to establish ROS cell model. The proliferative cells were treated with various concentrations of antioxidant superoxide dismutase (SOD). We detected cell proliferation, intracellular content of ROS and mitochondrial membrane potential changes. Results (1) Low concentration of H2O2 (50μmol/L) stimulated the proliferation of U937 cells while high concentration of H2O2 (500μmol/L) produced a toxic effect on the cells. U937 cells were treated with 50μmol/L of H2O2 for 48 hours to establish the experimental model. (2) U937 cells contained a small amount of ROS and apoptotic cells; after 50μmol/L H2O2 treatment, the content of intracellular ROS, the mitochondrial membrane potential, cell proliferation and the newly synthesized DNA were increased, but the apoptosis rate declined. After treatment with various concentrations of antioxidants superoxide dismutase (SOD), the content of intracellular ROS, the mitochondrial membrane potential and the newly synthesized DNA decreased, and cell proliferation slowed down while the apoptosis rate was increased, which showed a dose-dependent manner. Conclusion Antioxidants can inhibit the proliferation of acute monocytic leukemia cells in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of rhein on proliferation and apoptosis of gastric cancer cell line HGC-27 and its related mechanisms., Methods: Human gastric cancer cells HGC-27 were treated with 0, 5, 10 or 20 mg/L rhein respectively for 24, 48 and 72 h in vitro, three duplicate wells were set in each group. The proliferation activity of HGC-27 cells was detected with CCK-8 method, the growth status of HGC-27 cells was observed by small high-content microscope, hoechst staining was used to analyze the karyotype of HGC-27 cells. Mitochondrial membrane potential was detected by JC-1 staining and flow cytometry, cell cycle was analyzed with flow cytometry, the levels of mRNA transcribing of bcl-2, bax, caspase-3, jak1, jak2, stat3 and notch genes were investigated with RT-qPCR method. Protein expressions were determined by Western blot., Results: Compared with HGC-27 cells treated with 0 mg/L rhein, HGC-27 cells treated with 5, 10 and 20 mg/L rhein for 24 h showed decreased mitochondrial membrane potential ( P ＜0.01), the cell proliferation activity was inhibited and apoptosis was induced. The effects were enhanced with the increase of rhein concentration and the extension of treatment time, but the cell cycle did not change significantly, and the expressions of bcl-2, jak1, jak2, stat3 and notch genes were down-regulated. The expression levels of bax and caspase-3 genes were increased significantly ( P ＜0.01)., Conclusion: Rhein can induce apoptosis of HGC-27 cells by influencing NOTCH/JAK/STAT signaling pathway, and has anti-gastric cancer effect.

AbstractObjective: To explore the effect and mechanism of curcumin on human T-cell acute lymphoblastic leukemia (T-ALL) cell apoptosis induced by Mcl-1 small molecule inhibitors UMI-77., Methods: T-ALL cell line Molt-4 was cultured, and the cells were treated with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77 for 24 h. The MTT method was used to detect the cell survival rate after different treatment; According to the results of curcumin and UMI-77, the experimental settings were divided into control group, curcumin group (20 μmol/L curcumin treated cells), UMI-77 group (15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells) and curcumin+ UMI-77 group (20 μmol/L curcumin and 15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells), MTT method was used to detect cell proliferation inhibition rate, Annexin V-FITC/PI double staining method and TUNEL staining were used to detect cell apoptosis, DCFH-DA probe was used to detect cell reactive oxygen species, JC-1 fluorescent probe was used to detect mitochondrial membrane potential, Western blot was used to detect the expression levels of apoptosis-related proteins and Notch1 signaling pathway-related proteins., Results: After the treatment of Molt-4 cells with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77, the cell survival rate was decreased (P<0.05); Compared with the control group, the cell proliferation inhibition rate of the curcumin group and the UMI-77 group were increased, the apoptosis rate of cell was increased, the level of ROS was increased, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, and the protein expression of Bcl-2 was reduced (P<0.05); Compared with the curcumin group or UMI-77 group, the cell proliferation inhibition rate and apoptosis rate of the curcumin+UMI-77 group were further increased, and the level of ROS was increased. At the same time, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, the protein expression of Bcl-2 was reduced (P<0.05); In addition, the mitochondrial membrane potential of the cells after curcumin treatment was decreased, and the proteins expression of Notch1 and HES1 were reduced (P<0.05)., Conclusion: Curcumin can enhance the apoptosis of T-ALL cells induced by Mcl-1 small molecule inhibitor UMI-77 by reducing the mitochondrial membrane potential, the mechanism may be related to the inhibition of Notch1 signaling pathway.

Objective To investigative the effects of dianhydrogalacitol(DAG)on cell proliferation and apoptosis of human glioma BT325 cells and its potential mechanism.Methods BT325 cells were divided into the control group and DAG-treated groups at various oncentrations.CCK-8 assay and colony formation assay were performed to detect the cell via-bility of BT325 cells.Hoechst33342 was employed to observe nucleus morphological changes and apoptotic rate.The mito-chondrial membrane potential (MMP)was detected by using Rhodamine 123 staining.Results With the increasing DAG concentration, the survival rate of BT325 cells decreased gradually (P <0.05).The survival rate of BT325 cells decreased with the prolonged incubation time in DAG-treated groups (all P <0.05).The IC50 of BT325 cells treated by DAG were 108.81, 26.61, and 11.38 μg/mL at 24, 48, and 72 h, respectively.The result of colony formation assay showed that DAG inhibited clones of BT325 cells and the colony formation rate in the control group was 16.78% ±1.50% (all P <0.01).The apoptotic rates of DAG-treated groups treated with 5, 10, 20, 40 and 80 μg/mL DAG were 11.79% ± 2.91%, 15.42% ±3.06%, 38.70% ±1.12%, 60.78% ±1.30%, and 80.35% ±3.22%, respectively, which were in an dose-dependent manner and were significantly increased as compared with that (4.27% ±1.72%)of the control group (all P <0.01).The MMP of control group and DAG-treated groups(5, 10, 20, and 40 μg/mL)were 0.193 ±0.014, 0.174 ±0.038, 0.142 ±0.037, 0.138 ±0.033, and 0.119 ±0.013, respectively.Significant difference was found in the MMP between the DAG-treated groups (20 and 40 μg/mL)and the control group (P <0.05 and P <0.01).Conclusions DAG inhibits the proliferation and induces apoptosis of BT325 cells.It is suggested that decreased MMP levels might in-volve in the mechanism. [ABSTRACT FROM AUTHOR]

Objective: To explore the mechanism of acupuncture in improving cognitive ability by regulating hippocampal phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in vascular dementia (VD) rats., Methods: A total of 80 male SD rats were randomized into sham operation, model, non-acupoint and acupoint groups ( n =18 per group). The VD model was established by ligation of the bilateral common carotid arteries. For rats of the acupoint group, "Baihui" (GV20) and bilateral "Zusanli "(ST36) were needled and stimulated by twirling the needles with reinforcing method, and for rats of the non-acupoint group, the bilateral subcostal spots (about 10 mm superior to the iliac cresta) were needled and stimulated by twirling the needles with uniform reinforcing and reducing method. The treatment was conducted once daily, 6 times a week for two weeks, beginning 3 days after successful modeling. Rats of the sham operation group and model group received grasps as those in the acupoint groups. Morris water maze test was used to detect the abilities of learning and spatial memory. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and the activities of acetylcholinesterase (AChE) and acetylcholine transferase (ChAT) in the hippocampus tissue were detected by using ELISA, changes of hippocampal mitochondrial membrane potential (MMP) detected using JC-1 fluorescence probe, and the expression levels of hippocampal phosphorylated (p)-PI3K, p-Akt and mTOR proteins measured using Western blot., Results: Compared with the sham operation group, the escape latency, contents of ROS and MDA, and AChE activity were significantly increased ( P <0.05), and the spatial memory ability, SOD activity, ChAT activity, MMP, p-PI3K, p-Akt and mTOR expression levels were significantly decreased in the model group ( P <0.05). Compared with the model group, carotid artery ligature-induced increase of the escape latency, contents of ROS and MDA, and AChE activity, and decrease of the spatial memory ability, SOD activity, ChAT activity, MMP, p-PI3K, p-Akt and mTOR expression levels were significantly reversed in the acupuncture group ( P < 0.05), but not in the non-acupoint group ( P >0.05). The therapeutic effects of acupoint needling were obviously superior to those of non-acupoint needling in decreasing the escape latency, contents of ROS and MDA, and AChE activity ( P <0.05), and in increasing the spatial memory ability, SOD activity, ChAT activity, MMP, p-PI3K, p-Akt and mTOR expre-ssion levels ( P <0.05)., Conclusion: Acupuncture can improve cognitive function of VD rats, which may be related with its functions in easing oxidative stress and MMP reduction by activating PI3K/Akt/mTOR signaling pathway in the hippocampus.

Objective: To investigate the protective effect of oleanolic acid(OA) on HgCl&#95;2 induced liver injury., Methods: L02 cells were divided into four groups according to different treatment, control group(Con), oleanolic acid group(OA, 10 μmol/L), HgCl&#95;2 group(HgCl&#95;2, 40 μmol/L) and oleanolic acid + HgCl&#95;2 group(OA + HgCl&#95;2). Cells of control group were given serum-free medium, cells of OA group were pretreated with OA solution for 8 hours, cells of HgCl&#95;2 group were exposed to HgCl&#95;2 solution for 6 hours, cells of OA + HgCl&#95;2 group were pretreated with OA solution for 8 hours, and then exposed to HgCl&#95;2 solution for 6 hours. MTT assay was used to detect cell viability. Laser confocal scanning was used to detect JC-1 probe fluorescence intensity to determine mitochondrial membrane potential. DCFH-DA fluorescence probe combined with flow cytometry was used to detect reactive oxygen species(ROS) level. Annexin V/PI double staining method combined with flow cytometry was used to determine cell apoptosis rate. Catalase(CAT), total superoxide dismutase(T-SOD), glutathione(GSH), malondialdehyde(MDA), Caspase 3 and Caspase 9 kits combined with enzyme labeled instrument were used to determine their activity or content respectively., Results: Compared with the control group, 40 μmol/L HgCl&#95;2 could significantly reduce cell viability, the level was 0.52±0.03(P&lt;0.05), OA pretreatment could significantly inhibit the decrease of cell viability induced by HgCl&#95;2, the level was 0.86±0.05(P&lt;0.05). The result of mitochondrial membrane potential detection showed that cell exposed to 40 μmol/L HgCl&#95;2 significantly reduced the intensity of red fluorescence, and the ratio of red to green fluorescence was 0.23±0.02(P&lt;0.05). OA pretreatment significantly increased red fluorescence, and the ratio of red fluorescence to green fluorescence was 1.32±0.08, which was significantly higher than that of HgCl&#95;2(P&lt;0.05). After exposure to 40 μmol/L HgCl&#95;2, the relative fluorescence intensity of ROS was 1.21±0.07, the apoptosis rate was about 8%, the activity levels of Casepase 3 and Casepase 9 were 3.11±0.20 and 2.94±0.17, respectively, which were all significantly higher than those in the control group(P&lt;0.05). OA pretreatment could significantly alleviate the changes of the above indexes, and the difference was statistically significant compared with HgCl&#95;2 group(P&lt;0.05). The level of T-SOD in HgCl&#95;2 group was(7.68±0.39)U/mL, which was significantly lower than that in control group(P&lt;0.05). Compared to the control group, the level of MDA was significantly increased to(4.99±0.26)nmol/mg(P&lt;0.05). OA pretreatment significantly increased level of T-SOD and decreased the level of MDA, the levels were(13.97±0.71)U/mL and(3.01±0.17)nmol/mg, respectively(P&lt;0.05)., Conclusion: A certain concentration of HgCl&#95;2 can induce hepatocyte damage. OA pretreatment may reduce cell damage by improving oxidative stress.

Objective: To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism., Methods: Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured in vitro . The cultured cells were treated with 0, 1, 10, 50, 100, 150, 200 and 500 μmol/L BPA, and the changes in cell proliferation, cell cycle, apoptosis and mitochondrial membrane potential were analyzed with CCK-8 method and flow cytometry. The protein expressions of Bcl-2, Bax, p53 and cyclin D1 in the treated cells were determined with Western blotting. Pregnant ICR mice were treated for a week with BPA at the concentration that produced significant effects on the preantral follicular granulosa cells, and the weight changes of the pregnant mice were recorded. The ovarian tissues of the offspring female mice were weighed at PND 10, 17, 21 and 42 followed by histological observation with HE staining and examination of Bcl-2 mRNA expression level with RT-qPCR., Results: Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 μmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation ( P < 0.05). Exposure to a higher BPA concentration at 200 μmol/L obviously enhanced cell apoptosis by reducing the mitochondrial membrane potential and repressed the cell proliferation ( P < 0.05). BPA exposure at 50 μmol/L and 200 μmol/L produced opposite effects on the protein expressions of Bcl-2 (P < 0.01), Bax ( P < 0.05) and p53 ( P < 0.05) in mouse ovarian preantral follicular granulosa cells. BPA exposure at the doses of 10 and 35 mg/kg caused rapid weight increment of the pregnant mice and changes in ovarian index of the offspring female mice. In the offspring female mice, the changes in Bcl-2 mRNA expression in the ovarian tissue showed a similar pattern to that of ovarian index. Exposure of the pregnant mice to a high BPA concentration at 35 mg/kg resulted in accelerated follicular development into antral follicular stage in PND 21 offspring female mice., Conclusions: BPA can concentration-dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.

Objective: To observe the effect of electroacupuncture (EA) on behavior, tyrosine hydroxylase (TH), mitochondrial complexes Ⅰ-Ⅳ, mitochondrial membrane potential and mitochondrial ultrastructure of Parkinson's disease (PD) mice, so as to explore its mechanism underlying improvement of PD., Methods: C57BL/6 mice were randomly divided into normal, model, medication (Madopar) and EA groups ( n =11 in each group). PD model was duplicated by intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP, 30 mg·kg -1 ·d -1 ) for consecutively 5 d. EA was performed on the chorea tremor areas on both sides of the head of mice for 15 min, once a day for 14 d. The behavioral changes of mice were observed. The expression of TH in substantia nigra compacta of midbrain was detected by immunohistochemistry. The activities of mitochondrial complexes Ⅰ-Ⅳ were measured. The changes of mitochondrial membrane potential were detected by JC-1 staining method. The ultrastructural changes of striatum mitochondria were observed by transmission electron microscope., Results: After modeling, the mice showed obvious behavioral abnormalities such as tremor, vertical hair and tail warping, and the pole test time in the model group was significantly longer than that in the normal group ( P <0.01). After 7 and 14 days of the treatment, the pole test time in the EA and medication groups was shorter than that in the model group ( P <0.05， P <0.01). Compared with the normal group, the number of TH positive cells of the substantia nigra, the mitochondrial membrane potential and the activity of mitochondrial complex I were decreased significantly in the model group ( P <0.01), and EA and medication intervention reversed these changes ( P <0.01). The mitochondrial structure of mice in the model group was obviously damaged, and the damage of mitochondrial structure was alleviated and the number of damaged mitochondria was decreased in the EA and medication groups., Conclusion: EA can protect and promote the recovery of mitochondrial structure and function in MPTP-induced PD mice, which may play a neuroprotective effect on PD mice by improving mitochondrial dysfunction, balancing cell homeostasis and reducing dopaminergic neuron damage.

Objective: To investigate the effects of hedyotis diffusa (injection) on mitochondrial membrane potential and expressions of apoptosis-related genes in human gastric cancer cell line MNK-45 cells. Methods: The human gastric cancer MNK-45 cells were divided into 4 groups, each group was set with 3 replicates. The control group was MNK-45 cells without added hedyotis diffusa; the 3 groups of experimental groups were treated with hedyotis diffusa at final concentrations of 20 , 30, 40 μg / ml respectively; each group was incubated for 48 h in a 5% carbon dioxide incubator, and the morphological changes of the cells were observed under a laser confocal microscope. Mitochondrial membrane potential was detected by flow cytometry. The expressions of Cytochrome C ( Cyt c ), caspase3 and caspase9 genes and proteins were detected by qRT-PCR and Western blot respectively. Results: Compared with the control group, the mitochondrial membrane potentials of MNK-45 cells were significantly reduced in the hedyotis diffusa treated groups at final concentrations of 20, 30, and 40 μg / ml ( P ＜0. 01). The gene expressions of Cyt c , caspase3 , and caspase9 were significantly up-regulated ( P ＜0. 01) and their protein expressions were also significantly increased ( P ＜0. 05 or P ＜0. 01). The 40 μg / ml hedyotis diffusa treatment group performed best. Conclusion: In the final concentration range of 20 ~ 40 μg / ml, hedyotis diffusa can reduce human gastric cancer MNK-45 cells mitochondrial membrane potential, induce apoptosis and up-regulate Cyt c, caspase3 and caspase9 gene expressions.

Objective: To study the impacts of aluminum chloride (AlCl3) on the sperm quality, sperm mitochondrial membrane potential (MMP) and sperm mitochondrial membrane permeability transition pore (MPTP) function of male rats, and the possible mechanisms of AlCl3 inducing the declination of sperm quality., Methods: According to the median lethal dose (LD50) of AlCl3・6H2O in drinking water, we randomly assigned 96 male Wistar rats weighing 180－200 g to four groups of equal number and fed them with AlCl3 aqueous drinking water at 256.72 mg/kg/d (1/5 LD50, high-dose group), 128.36 mg/kg/d (1/10 LD50, medium-dose group), 64.18 mg/kg/d (1/20 LD50, low-dose group) and 0 mg/kg/d (control group), respectively, all for 16 weeks. Then, we examined the quality of the epididymal sperm of the rats, observed the morphology of the sperm mitochondria under the transmission electron microscope, and determined the MMP level of the sperm mitochondria and the function of the MPTP by flow cytometry., Results: The percentage of progressively motile sperm was significantly decreased in the low-, medium- and high-dose AlCl3 groups as compared with that in the control group (［46.49 ± 5.37］%, ［33.50 ± 8.75］% and ［16.94 ± 5.00］% vs ［66.28 ± 5.68］%, P < 0.01), that of dead sperm was remarkably increased (［19.73 ± 5.57］%, ［35.80 ± 5.90］% and ［55.19 ± 4.97］% vs ［12.71 ± 4.84］%, P < 0.01), and so was that of morphologically abnormal sperm (［19.06 ± 2.44］%, ［23.78 ± 3.29］% and ［32.06 ± 4.65］% vs ［14.56 ± 1.62］%, P < 0.01). Sperm mitochondrial swelling was aggravated in the AlCl3-exposed rats in a dose-dependent manner. The sperm MMP level was significantly lower in the low-, medium- and high-dose AlCl3 groups than in the control (［60.88 ± 7.37］%, ［51.54 ± 6.12］% and ［37.70 ± 7.44］% vs ［74.35±4.67］%, P < 0.01), with a negative correlation to the dose of AlCl3 (rs = －0.819, P < 0.01), while the pathologically open MPTP was markedly higher in the former three than in the latter group (［27.80 ± 5.74］%, ［36.58 ± 6.67］% and ［64.95 ± 8.07］% vs ［15.37 ± 7.13］%, P < 0.01), with a positive correlation to the dose of AlCl3 (rs = 0.867, P < 0.01)., Conclusions: Exposure to aluminum can cause sperm mitochondrial swelling, decrease the sperm MMP level, induce pathological opening of the MPTP, and consequently reduce sperm quality in male rats.

Objective: To investigate the impact of Ureaplasma urealyticum (UU) infection (UUI) on the expression of the mitochondrial ribosomal protein S22 (MRPS22) in rat spermatogenic cells and the intervening effect of Zhibai Dihuang Decoction (ZBDH)., Methods: Forty-five SD male rats were randomly divided into four groups of equal number: normal control, UUI model control, ZBDH and azithromycin, and the UUI model was made by bladder injection of the standard UU strain in the latter three groups. After modeling, the rats in the ZBDH and azithromycin groups were treated intragastrically with ZBDH at 1 g/kg/d and azithromycin at 0.105 g/kg/d respectively, while those in the normal and UUI model control groups with normal saline at 1 ml/kg/d. At 21 days after intervention, all the animals were sacrificed and their testes harvested for observation of the apoptosis and mitochondrial ultrastructure of the spermatogenic cells, measurement of the mitochondrial membrane potential (MMP) by flow cytometry, and determination of the mRNA and protein expressions of MRPS22 by RT-PCR and Western blot, respectively., Results: The apoptotic index of the rat sperma-togenic cells was significantly higher in the UUI model control than in the ZBDH and azithromycin groups (［11.23 ± 1.65］ % vs ［6.62 ± 0.49］ % and ［7.82 ± 0.81］ %, P < 0.01), but lower in the ZBDH than in the azithromycin group (P < 0.05). The mitochondrial ultrastructure of the spermatogenic cells was markedly improved in the ZBDH and azithromycin groups as compared with that in the model control. The MMP level was remarkably lower in the model control than in the normal control (［8.77 ± 1.73］ % vs ［22.33 ± 1.66］ %, P < 0.01), but higher in the ZBDH (［18.26 ± 1.32］ %) than in the model control (P < 0.01) and the azithromycin group (［15.91 ± 1.69］ %) (P < 0.01). The mRNA and protein expressions of MRPS22 were significantly lower in the model control (8.02 ± 3.21 and 22.65 ± 5.31) than in the normal control (15.43 ± 2.54 and 33.31 ± 7.09), ZBDH (11.26 ± 3.82 and 33.35 ± 3.96), and azithromycin group (8.79 ± 2.03 and 28.11 ± 4.13) (all P < 0.01), but both higher in the ZBDH than in the azithromycin group (P < 0.01). There was a positive correlation between the MRPS22 protein expression and MMP (r = 0.639, P < 0.01)., Conclusions: UU infection induces the apoptosis of rat spermatogenic cells by inhibiting the mRNA and protein expressions of MRPS22 and reducing the mitochondrial membrane potential, while ZBDH can decrease the apoptosis of spermatogenic cells by improving the mRNA and protein expressions of MRPS22 and enhancing the mitochondrial membrane potential.

Objective: To study the effects of L-carnitine (LC) on cryopreserved human sperm., Methods: Ten semen samples were collected from normal sperm donors, each divided into six groups, fresh ejaculate (FE), non-LC cryopreservation (non-LC), and cryopreservation with LC at 1 mmol/L (LC-1), 2.5 mmol/L (LC-2), 5 mmol/L (LC-3) and 10 mmol/L (LC-4), respectively. The optimal concentration of LC was identified based on the motility and motion parameters of the post-thaw sperm. The plasma membrane integrity (PMI) of the sperm was assessed by eosin-nigrosin staining, their mitochondrial membrane potential (MMP) monitored by JC-1 assay, and the level of sperm ROS measured by the fluorescent probe DCFH-DA, followed by analysis of the mechanisms of LC protecting sperm against cryopreservation injury., Results: Compared with the sperm in the FE group, the post-thaw sperm in the non-LC and LC groups showed significantly decreased progressive motility, average path velocity (VAP), straight line velocity (VSP) and curvilinear velocity (VCP) (P < 0.05). In comparison with the non-LC group, the LC-3 group exhibited a remarkably higher percentage of progressively motile sperm (［41.9 ± 4.6］ vs ［47.0 ± 4.3］%, P = 0.0261) and VAP (［34.9 ± 2.6］ vs ［38.9 ± 4.2］ μm/s, P = 0.0152), indicating that the optimal concentration of LC was 5 mmol/L. Both PMI and MMP were significantly lower in the non-LC than in the FE group (［52.7 ± 5.7］ vs ［75.5 ± 5.4］%, P < 0.01 and ［44.5 ± 3.5］ vs ［57.3 ± 4.4］%, P < 0.01), but higher in the LC groups (［70.1 ± 8.2］% and ［50.3 ± 3.4］%) than in the non-LC group (P < 0.01 and P < 0.05). The level of sperm ROS, however, was markedly higher in the non-LC than in the FE group (［12.5 ± 3.9］ vs ［6.8 ± 2.4］, P < 0.01) but lower in the LC groups (［8.4 ± 5.3］%) than in the non-LC group (P = 0.05)., Conclusions: L-carnitine can improve the motility and motion parameters of cryopreserved human sperm by reducing sperm ROS, enhancing sperm mitochondrial membrane potential and protecting the sperm plasma membrane.

Objective: To explore the effects of the Chinese herbal compound Tongjingling (TJL) on the ROS level, mitochondrial membrane potential (MMP) and mitochondrial ultrastructure in spermatogenic cells in varicocele (VC) rats with liver-qi stagnation (LQS)., Methods: Totally, 72 male SPF Wistar rats were randomly divided into six groups of equal number, sham operation, VC+LQS model, low-, medium- and high-dose TJL, and L-carnitine (LC) control. The VC+LQS model was established by the Saypol method combined with tail-clamping. At 4 weeks after modeling, the rats in the low-, medium- and high-dose TJL groups were treated intragastrically with TJL at 0.6, 1.2 and 2.4 g/ml respectively, those in the sham operation and VC+LQS model groups with 0.9% saline, and those in the LC control group with LC oral liquid at 2.1 ml/kg, qd, for 8 weeks. Then the ROS level and MMP in the spermatogenic cells were measured by flow cytometry and the mitochondrial ultrastructure observed under the transmission electron microscope., Results: The ROS level in the spermatogenic cells was significantly higher in the VC+LQS model and low-dose TJL groups than in the sham operation group (［62.72 ± 9.90］% and ［52.25 ± 8.15］% vs ［36.53 ± 5.25］%, P < 0.01), but remarkably lower in the medium-dose TJL (［41.65 ± 4.89］%), high-dose TJL (［39.60 ± 5.19］%) and LC control (［38.33 ± 7.46］%) than in the VC+LQS model group (P < 0.01). MMP in the spermatogenic cells was significantly lower in the VC+LQS model and low-dose TJL than in the sham operation group (［34.80 ± 20.18］% and ［44.39 ± 19.60］% vs ［60.24 ± 22.25］%, P < 0.01 and P < 0.05), but markedly higher in the medium-dose TJL (［50.62 ± 14.83］%), high-dose TJL (［52.42 ± 7.55］%) and LC control (［50.75 ± 15.65］%) than in the VC+LQS model group (P < 0.05). The mitochondria in the spermatogenic cells of the VC+LQS model rats exhibited swelling, vacuolization, rupture or disappearance of the mitochondrial cristae, and breakdown of the internal structure. The pathological injury of the mitochondrial structure was improved in different degrees in the TJL and LC groups., Conclusions: TJL can reduce the ROS level, increase MMP and improve pathological injury of the mitochondrial ultrastructure in the spermatogenic cells of VC rats with LQS, which may be the underlying molecular mechanism of TJL improving fertility in infertile VC patients.

Objective: To investigate the changes of CD71(+) nucleated erythrocyte apoptosis, cytochrome C (Cyt-C) and mitochondrial membrane potential (MMP) in bone marrow of chronic mountain sickness (CMS). Methods: 14 patients with CMS and 15 patients with simple old fracture were divided into CMS group and control group, respectively.Bone marrow mononuclear cells (BMMNC) were separated, marked with CD71 monoclonal antibody and stained with Annexin V-FITC/PI.Then the apoptotic index of CD71(+) nucleated erythrocytes was determined by flow cytometry.CD71(+) nucleated erythrocytes were sorted out by magnetic column separation, and Cyt-C mRNA was detected by RT-qPCR, MMP was detected by JC-1 staining flow cytometry. Results: The apoptotic index of CD71(+) nucleated erythrocytes was (1.9±1.4)% in the CMS group, and was (3.2±1.5)% in the control group, with significant difference between the two groups ( P <0.05). The expression of Cyt-C mRNA was (0.72±0.14) in the CMS group, and was (1.00±0.15) in the control group, with significant difference between the two groups ( P <0.01). The MMP was (5.0±2.2) in the CMS group, and was (3.3±0.9) in the control group, with significant difference between the two groups ( P <0.05). The apoptotic index of CD71(+) nucleated erythrocytes was negatively correlated with hemoglobin in CMS group ( r =-0.569, P =0.034). But there was no significant correlation among apoptosis index, MMP and Cyt-C mRNA. Conclusions: The apoptosis index of CD71(+) nucleated erythrocytes decreased in CMS patients, which was negatively correlated with the level of hemoglobin, indicating that the decline of apoptosis index of CD71(+) nucleated erythrocytes may be related to the accumulation of red blood cells in CMS.The MMP increased and Cyt-C mRNA expression decreased in CD71(+) nucleated erythrocytes of CMS patients, which suggests that the change of mitochondrial pathway of apoptosis might be involved in the down-regulation of CD71(+) nucleated erythrocytes apoptosis in CMS patients.But there was no significant correlation among CD71(+) nucleated erythrocyte apoptosis index, MMP and Cyt-C mRNA levels, which indicates that the mechanism of CD71(+) nucleated erythrocytes apoptosis is complex in CMS.

Objective: To observe the effect of Huangjing Zanyu Capsule (HZC) on sperm mitochondrial membrane potential (MMP) in asthenozoospermia patients., Methods: We assigned 70 asthenozoospermia patients to a treatment group (n = 39) and a control group (n = 31), the former treated with oral HZC at the dose of 4 capsules tid for 3 months while the latter left untreated. We obtained semen parameters from the patients and detected their sperm mitochondrial membrane potentials (MMP) by JC-1 staining and flow cytometry before and after medication, followed by comparison between the two groups., Results: The total effectiveness rate was 71.05% in the treatment group and natural pregnancy was achieved in 3 cases during the medication. A total of 35 patients in the treatment group and 30 controls completed all the laboratory examinations after a 3-month observation. Compared with the controls, the patients treated with HZC exhibited significant improvement after medication in MMP (variation value: ［1.19 ± 10.36］% vs ［20.28 ± 14.21］%, P <0.01), total sperm motility (variation value: ［3.46 ± 8.67］% vs ［20.68 ± 14.12］%, P <0.01), the percentage of progressively motile sperm (variation value: ［2.26 ± 8.29］% vs ［17.58 ± 12.73］%, P <0.01), and the percentage of morphologically normal sperm (variation value: ［0.23 ± 3.48］% vs ［3.37 ± 3.99］%, P <0.01). MMP was significantly correlated with total sperm motility (r = 0.69, P <0.01), progressive sperm motility (r = 0.75, P <0.01) and normal sperm morphology (r = 0.26, P <0.01)., Conclusions: Huangjing Zanyu Capsule can enhance sperm mitochondrial membrane potential and sperm mitochondrial function, thus improving total sperm motility, progressive sperm motility and normal sperm morphology. It is safe and effective for the treatment of asthenospermia.

Objectives: To investigate the efficacy of three different digestion methods for the separation of neonatal rat cardiomyocytes., Methods: We employed three different digestion methods to separate neonatal rat cardiomyocytes. Group A was 0.08% trypsin digestion alone, group B was 0.08% trypsin+0.08% type 2 collagenase mixed digestion, group C was 0.08% trypsin and 0.08% type 2 collagenase isolated digestion. The number of cells and cell viability after differential adhesion were recorded. The purity of cardiomyocytes was evaluated by immunofluorescence staining. The cell vitality was assessed by the detection of mitochondrial membrane potential with JC-1 staining, and the ratio of red to green fluorescence intensity by laser scanning confocal microscope., Results: There was no statistically significant difference in the number of cells between three groups ( P >0.05).The rate of cell viability in group C was significantly higher than that in group A ( P <0.01). No statistically significant difference was found in the purity of cardiomyocytes between three groups ( P >0.05). The ratio of red to green fluorescence intensity in group A, B and C were 0.928±0.078, 0.943±0.099 and 1.160±0.089, respectively; the ratio in group C was significantly higher than that in group A and group B ( P <0.01)., Conclusion: The cell isolation method with 0.08% trypsin and 0.08% type 2 collagenase isolated digestion could be served as conventional method to separate neonatal rat cardiomyocytes.