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Objective: To explore the key factors affecting plasma clot retraction and optimize the experimental method of plasma clot retraction, in order to study the regulation of platelet function and evaluate the modulatory effects of drugs on plasma clot retraction., Methods: The effects of different concentrations of thrombin, Ca2 + and platelets on plasma clot retraction were studied, and the detection system of plasma clot retraction was optimized. The availability of the detection system was then validated by analyzing the regulatory effects of multiple signaling pathway inhibitors on plasma clot retraction., Results: Through the optimization study of multiple factors, platelet rich plasma (PRP) containing 0.5 mmol/L Ca2 + and 40×10 9 /L platelets was treated with 0.2 U/ml thrombin to perform plasma clot retraction analysis. After treatment with thrombin for 15 min, plasma clot retracted significantly. After treatment with thrombin for 30 min, the percentage of plasma clot retraction was more than 50%. The regulatory effects of multiple signaling pathway inhibitors on plasma clot retraction were studied in this detection system. PKC inhibitor Go 6983 exhibited a significant inhibitory effect on plasma clot retraction, while PI3K inhibitor Ly294002 and p38 MAPK inhibitor SB203580 slightly suppressed plasma clot retraction., Conclusion: PRP containing 0.5 mmol/L Ca2 + and 40×10 9 /L platelets can be induced with 0.2 U/ml thrombin to conduct plasma clot retraction analysis, which can be used to study the regulation of platelet function and evaluate the modulatory effects of drugs on plasma clot retraction.

Objective: To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line., Methods: The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC 50 was computed by SPSS., Results: The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin., Conclusion: The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

Objective: To investigate the role of p38 mitogen-activated protein kinase (MAPK) signaling pathway in autophagy of neurons in hippocampus of sepsis rats., Methods: A sepsis model was established by cecal ligation and puncture (CLP). SD rats were randomly divided into sham-operated group (sham group), model group (CLP group), vehicle-treated group (CLP+Veh group) and inhibitor-treated group (CLP+SB203580 group), and each group was divided into 3, 6, 12, 24 and 48 h subgroups. CLP+Veh group and CLP+SB203580 group were injected with 1% DMSO 5 μL and 0.1 mmol/L SB203580 5 μL respectively in the lateral ventricle, and CLP was established 30 min after injection. The sham group only turned over the cecum and closed the abdomen without other treatments. The vital signs of rats were monitored, including mean arterial pressure (MAP) and heart rate (HR). Neurobehavioral score was used to investigate the brain injury in rats. Histopathological changes in hippocampus of rats were observed by HE staining. The process of neuronal autophagy in hippocampal of rats was observed under transmission electron microscope (TEM). Western blot assay was performed to detect the expression of microtubule associated protein 1 light chain 3 (LC3)Ⅱ, LC3Ⅰ, selective autophagy adaptor protein p62/sequestosome-1 (p62/SQSTM1), MAPK-activated protein kinase 2 (MK-2) and phosphorylation MK-2 (p-MK-2) in the hippocampus. The expressions of LC3 and p62/SQSTM1 in hippocampal neurons of rats were observed by immunofluorescence., Results: At different time points, MAP of CLP group was lower than sham group, while HR was higher than sham group, the change was most obvious at 12 h after molding; the neurobehavioral score of CLP group was the lowest; the histopathological changes in the hippocampus were obvious; and many autophagy vacuoles were observed under transmission electron microscope; compared with CLP group, the neurobehavioral score of CLP+SB203580 group increased; the pathological changes in the hippocampus improved; the inclusions in autophagy vacuoles were degraded under transmission electron microscopy; Western blot results showed:compared with sham group, expression of-LC3Ⅱ/LC3Ⅰ, p-MK-2/MK-2 increased, and p62/SQSTM1 decreased in hippocampal tissue of CLP group in rat, the former reaches its peak at 12 h, the latter bottomed out at 12 h. Compared with the other groups, at 12 h of modeling, the expression of LC3Ⅱ/LC3Ⅰ, p-MK-2/MK-2 was further increased, the expression of p62/SQSTM1 decreased further in hippocampal tissue of CLP+SB203580 group in rat ( P < 0.05); immunofluorescence observation showed that localization and expression of LC3 and p62/SQSTM1 in NeuN were consistent with Western blot., Conclusion: Inhibition of p38 MAPK signaling pathway in sepsis rats can further activate autophagy and protect neurons in the hippocampus., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Objective To illuminate whether IL-17 regulates receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in human periodontal ligament fibroblasts (HPDLFs) via p38MAPK signaling pathway. Methods HPDLFs were incubated in the presence of 20 ng/mL IL-17 for 0, 20, 40, 60 and 80 minutes. HPDLFs were divided randomly into 6 groups: control group, dimethyl sulfoxide (DMSO) group, p38MAPK pathway inhibitor SB203580 group, IL-17 group, IL-17 combined with DMSO group and IL-17 combined with SB203580 group. SB203580 (10 μmol/L) and IL-17 (20 ng/mL) were added to the corresponding groups. Real-time quantitative PCR was used to detect the expression of RANKL and OPG mRNAs in HPDLFs. The levels of phospho-p38MAPK (p-p38MAPK) and RANKL protein were measured using Western blot analysis. The protein level of OPG was detected by ELISA. Results After IL-17 stimulation, the expression level of p-p38MAPK protein gradually increased starting from 0 minute and reached its highest level at 60 minutes. It started to decline at 80 minutes. Stimulation with IL-17 could increase the mRNA and protein expression level of RANKL but decrease the mRNA and protein expression level of OPG. Nevertheless, unlike the IL-17 group, IL-17 combined with inhibitor SB203580 decreased the expression of RANKL mRNA and protein and increased OPG mRNA. Conclusion IL-17 can enhance the expression of RANKL in human periodontal fibroblasts and inhibit the expression of OPG mRNA through the p38MAPK signal transduction pathways.

Objective: To investigate the role of p38MAPK signal pathway in spinal cord and dorsal root ganglion (DRG) in rats with phantom limb pain and the effects of specific inhibitors.
 Methods: Healthy adult male SD rats (n=48) were cut off one side of the sciatic under anesthesia to establish a model of phantom limb pain. In addition, the healthy rats were taken as a sham group (group S, n=24). The animals were scored by observing the action of chewing (0=no chewing, 13=the worst chewing) after the operation and were sacrificed on the following day after the operation. The successful model of phantom limb pain were randomly divided into 2 groups: a phantom limb pain group (group P, n=24) and a phantom limb pain plus inhibitor group (group P+I, n=24). SB203580 was given to the rat at 0.8 mg/kg on every Monday until the rats were sacrificed, the rest of the rats received an equal amount of saline. Eight rats from each group were randomly taken for the determination of levels of P-p38MAPK in spinal cord and DRG before administration and on the 4th, 6th, 8th weekend following the administration, respectively.
 Results: In the sham group, no animal developed chewing. Meanwhile, rats in successful model of phantom limb pain group began chewing from the 2nd day after operation with scores at eight to eleven. The chewing scores in the P+I group were reduced after the treatment. Compared with group S, P-p38MAPK levels were elevated in groups of P and P+I (P<0.05 or P<0.01). Compared with group P, P-p38MAPK level was decreased in the group P+I (P<0.05 or P<0.01).
 Conclusion: P38MAPK signal pathway involves in the development of phantom limb pain.

Objective: To investigate the role of allograft inflammatory factor-1 (AIF-1) in colorectal cancer (CRC) progression and explore the possible mechanism., Methods: The expression levels of AIF-1 in 70 CRC tissues and paired adjacent tissues were detected using immunohistochemistry and Western blotting, and the correlation of AIF-1 expression with the clinicopathological features of the patients was analyzed. In the CRC cell line SW480, the functional role of AIF-1 in regulating tumor progression was investigated by transfecting the cells with an AIF-1-overexpressing plasmid (AIF-1) and a negative control plasmid (NC). EdU proliferation assay and flow cytometry were used to assess the cell proliferation and cell cycle changes; Transwell migration assay and Annexin V-APC/7-AAD apoptosis assay kit were used to analyze the cell migration and apoptosis. The changes in the biological behaviors of the cells were observed after application of SB203580 to block the p38 MAPK pathway. The expression levels of CDK4, cyclin D1, P21, P27, MMP2, MMP9, Bax, Bcl2, Bcl-xl, p38 and p-p38 were detected using Western blotting., Results: AIF-1 was down-regulated in CRC tissues compared with the adjacent normal tissues, and its expression level was positively correlated with lymph node metastasis (P=0.008), TNM stage (P=0.003) and tumor size (P=0.023). Overexpression of AIF-1 in SW480 cells significantly reduced EdU-positive cells and caused obvious cell cycle arrest in G1 phase (P<0.05). AIF-1 overexpression resulted in significantly lowered protein expressions of CDK4 and cyclin D1, enhanced expressions of P21 and P27, attenuated cell migration ability (P<0.001), and decreased protein levels of MMP2 and MMP9. AIF-1 overexpression also induced obvious apoptosis of SW480 cells (P<0.01), significantly increased the protein levels of Bax and p-p38, and decreased the protein levels of Bcl-2 and Bcl-xl; SB203580 significantly attenuated the apoptosis-inducing effect of AIF-1 overexpression., Conclusion: AIF-1 plays the role of a tumor suppressor in CRC by inhibiting cell proliferation, suppressing cell migration and inducing cell apoptosis. AIF-1 overexpression promotes the apoptosis of CRC cells by activating the p38 MAPK pathway.

Objective To investigate the effect and molecular mechanism of β-lapachone combined with NVP-BEZ235 on the proliferation and migration of BGC-823 human gastric cancer cells. Methods BGC-823 cells were randomly divided into four groups: control group, 1 μmol/L β-lapachone group, 50 nmol/L NVP-BEZ235 group and 1 μmol/L β-lapachone combined with 50 nmol/L NVP-BEZ235 group. The proliferation of cells was determined using the MTT assay and colony formation assay. The expression levels of proliferation-related proteins phosphorylated AKT (p-AKT), phosphorylated NF-κB (p-NF-κB), phosphorylated extracellular signal-regulated kinase (p-ERK) and cyclin D1 were detected by Western blotting. The migration of cells was measured by wound healing assay and Transwell TM migration assay. The expression levels of epithelial-mesenchymal transition (EMT) markers E-cadherin, vimentin, Snail and β-catenin were detected by Western blotting. Results Compared with β-lapachone or NVP-BEZ235 treatment, the combination of β-lapachone and NVP-BEZ235 showed more prominent inhibitory effect on the proliferation and colony formation of BGC-823 cells.The most effective suppression on the expressions of p-AKT, p-NF-κB, p-ERK and cyclin D1 was observed in the combination therapy. Combined treatment also showed more evident inhibitory effect on the migration of BGC-823 cells as compared with β-lapachone or NVP-BEZ235 treatment. The expression of E-cadherin was significantly up-regulated, but the expressions of vimentin, Snail and β-catenin were significantly down-regulated by the combined treatment. Conclusion β-lapachone combined with NVP-BEZ235 can effectively inhibit the proliferation of BGC-823 cells, which may be related to the down-regulation of p-AKT, p-NF-κB, p-ERK and cyclin D1. Moreover, the combined treatment can effectively suppress the migration of BGC-823 cells via modulating the EMT process.

Objective To compare and characterize the Th1 immune responses induced by the most commonly used commercial Toll-like receptor (TLR) agonists through in vivo and ex vivo experiments. Methods The concentrations of IL-12 were tested by ELISA after mouse dendritic cells (DCs) in vitro were stimulated by one of tested TLR agonists, including poly(I:C), monophosphoryl lipid A (MPLA), resiquimod (R848), cytosine polyguanine-C (CpG-C). The changes in percentage and phenotype of DCs, NK cells and effector T cells in the draining lymph nodes were analyzed by flow cytometry after BALB/c mice were immunized with ovalbumin (OVA) mixed with selected TLR agonist. The serum concentrations of specific anti-OVA IgG2a in the immunized mice were determined by ELISA. Results Only CpG-C and R848 could significantly induce the production of IL-12 from bone marrow-derived DCs in vitro. Among the tested TLR agonists, R848 was the most effective adjuvant in recruiting DCs and NK cells into lymph nodes, inducing the proliferation of CD4 + and CD8 + effector T cells and the production of specific anti-OVA IgG2a in vivo. Conclusion R848 was the most potential Th1-promoting adjuvant among the tested TLR agonists.

Objective: To study the effects of p38 mitogen-activated protein kinase (p38MAPK) signal transduction pathway inhibitor SB203580 on the inflammatory reaction and lung water clearance, and to explore the role of p38MAPK in acute lung injury, to provide new way for p38MAPK inhibitor -SB203580 intervene fat embolism syndrome induced lung injury., Methods: Twenty-four adult male SD rats were randomly assigned to normal control group (OA group) ( n =8), oleic acid-induced lung injury group (OA group, n =8)and SB203580 pretreatment group ( n =8). OA-group was administered oleic acid (0.20 ml/kg) via right jugular vein; In SB203580-group, SB203580(5 mg/kg) was injected via jugular vein, followed 30 min before by OA infusion; At the 4 hours animals were sacrificed. Arterial blood gas, the wet/dry weight(W/D)of the right lower lung were examined, lung index(LI), pulmonary permeability index(PPI) and levels of tumor necrosis factor α(TNF-α) in bronchoalveolar lavage fluid(BALF) were examined. The expressions of p38MAPK and phospho-p38MAPK (p-p38MAPK) were determined by Western blot and immunohistochemical method. Pathological changes of the lung tissue were examined with light microscrope., Results: Compared to control group, arterial oxygen partial pressure (PaO 2 ) and PaO 2 /FiO 2 were decreased in the animals of OA-group, while right lower lung wet/dry ratio, lung index, PPI, levels of TNF-α in BALF and the protein expression of p-p38MAPK were increased significantly ( P <0.01). The pathological changes were observed significantly in injured lung tissue. Compared to OA-group, those indexes were improved in SB203580 pretreated group., Conclusions: p38MAPK signal transaction path mediated inflammatory response process and played an important role in acute lung injury. SB203580 could inhibit the expression of inflammatory cytokines, reduce lung edema, protect lung tissue of rats from OA-induced lung injury obviously. Therefore, inhibition of p38MAPK activity provides a new way for the clinical treatment of fat embolism syndrome induced lung injury.

Objective: To investigate the effect of zoledronate on protein interaction between Ca(2+)/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and calmodulin and protein expression of nuclear factor of activation of T cells-1 (NFATc1) and tartrate resistant acid phosphatase (TRAP) during osteoclast differentiation. Methods: Mouse RAW264.7 cells were divided into group A and B and were cultured. Group A was induced with 50 mg/L receptor activator of NF-κB ligand (RANKL) for osteoclastogenesis, and group B was treated with 1×10(-6) zoledronate for two days from day 2. Co-immunoprcipitation (Co-IP) and reverse Co-IP were used to detect the protein-binding between CaMKⅡ and calmodulin. Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc1 and TRAP in both groups. Osteoclast formation was also analyzed. Results: In group B, the number of osteoclasts, number and size of dentin resorption lacunaes were 11.3±1.5, 8.7±2.1 and (5 034.4±775.4) μm(2) respevtively, which were significantly lower than those (37.7±5.7, 23.0±4.0 and [15 042.7±1 906.0] μm(2)) in group A ( P< 0.01). Co-IP and reverse Co-IP examination indicated that protein-binding between CaMKⅡ and calmodulin significantly decreased by 59.8% and 50.9% in group B compared with group A ( P< 0.01). The protein level of calmodulin and CaMKⅡ in total cellular proteins also significantly decreased by 52.1% and 51.5% in group B compared with group A ( P< 0.01). NFATc1 and TRAP protein decreased by 52.4% and 38.9% in group B than in group A ( P< 0.01), respectively. Conclusions: Zoledronate could significantly inhibit protein-binding between CaMKⅡ and calmodulin and down-regulate protein level of NFATc1 and TRAP.

Objective: To investigate whether the p38 mitogen-activated protein kinase (MAPK) signaling pathway mediates advanced oxidation protein products (AOPPs)-induced epithelial-to-mesenchymal transition (EMT) in tubular cells., Methods: Human proximal tubular cells (HK-2 cells) exposed to AOPP-bovine serum albumin (BSA) were examined for expressions of p38 MAPK and phosphorylated p38 MAPK using Western blotting. Western blotting and quantitative RT-PCR were used to examine the protein and mRNA expressions of EMT markers E-cadherin and vimentin and endoplasmic reticulum stress marker glucose-regulated protein (GRP) 78 in cells treated with SB203580 (an inhibitor of the p38 MAPK signaling pathway) prior to AOPP exposure. The cells treated with AOPPs following pretreatment with salubrinal (an inhibitor of endoplasmic reticulum stress) were also examined for expressions of p38 MAPK and phosphorylated p38 MAPK., Results: AOPP treatment induced the phosphorylation of p38 MAPK in HK-2 cells. AOPP-induced decrease in E-cadherin expression and overexpression of vimentin and GRP78 were partly inhibited by pretreatment of the cells with SB203580. Salubrina partly suppressed AOPP-induced phosphorylation of p38 MAPK in the cells., Conclusion: p38 MAPK signaling pathway, which is regulated by endoplasmic reticulum stress, might mediate AOPP-induced EMT in HK-2 cells.

Vascular smooth muscle cells(VSMCs)phenotype switching plays an essential role in the pathogenesis of various vascular diseases.The present study aims to investigate the role of receptor-interacting protein kinases 1(RIPK1)in VSMCs phenotypic switching induced by AngiotensinⅡ(AngⅡ).Expression of mRNA and protein of RIPK1,markers of VSMCs phenotypic switching and secretion,phosphorylation of the P65 subunit of NF-κB were measured by real-time PCR and Western blot.Meanwhile,EdU incorporation assay and wound scratch assay were performed to determine the cell proliferation and migration respectively.At the same time,Necrostatin-1(Nec-1,an known RIPK1inhibitor)and RIPK1-specific small interference RNA(siRNA)were used to inhibit the expression of RIPK1.The experimental data demonstrated that the mRNA and protein levels of RIPK1 and P65phosphorylation were increased significantly in the process of VSMC phenotypic switching induced by Ang II.Moreover,the expression of RIPK1 and P65phosphorylation were significantly down-regulated in VSMCs pretreated with Nec-1or transfected with RIPK1-siRNA.Furthermore,the proliferation,secretion and migration of VSMCs were also markedly suppressed after inhibition of RIPK1 by Nec-1or its specific siRNA.The results suggested that RIPK1 might be involved in VSMC phenotypic switching induced by Ang II,which was possibly via up-regulating the NF-κB signaling pathway.

To study the synergistic anti-tumor effects of doxorubicin and axitinib in combination, and investigate the underlying mechanism, we performed the MTT cytotoxic assay and tumor spheroids inhibition to investigate in vitro using A549 cells. The cell internalization, cell cycle distribution and DNA ladder experiments were performed to study the synergistic mechanisms. A549 xenograft was established in nude mice and adopted to study the in vivo anti-tumor effect of doxorubicin and axitinib. Results showed that combination of doxorubicin and axitinib exerted significantly higher cytotoxicity than each single drug, and induced a synergistic effect on tumor spheroids growth inhibition. The combination achieved the highest tumor growth suppression in vivo in the A549 xenograft. The combination of axitinib and doxorubicin exhibited the best anti-tumor effects both in vitro and in vivo than each single drug.

Objective: To investigate the role of p53 on ran transcription in myeloma cells., Methods: Using real-time fluorescence quantitative PCR, the ran transcription level was measured in 8 human myeloma cell lines such as OPM-2, RPMI-8226, U-266, KAS6/1, ANML-6, H-929, MM1.S and MOLP-8. The ran transcription level and P53 expression were detected by Q-PCR in MM1.S treated with Nutlin-3a for 24, 48 and 72 hours, respectively. The Western blot was used to detect the expression levels of ran and P53 proteins, and ran expression level after transfection of MM1.S cells using different concentration of plasmids which express the P53 luciferase reporter., Results: H-929 and MM1.S cells showed the highest ran transcription level among the above-mentioned 8 cell lines (P<0.05). After treatment with Nutlin-3a, ran transcription level in MM1.S cells decreased (P<0.05), (r=-1.00, P=0.04) and P53 expression increased (r=1.00, P=0.06) in time-dependence manner. The detection by p53 luciferase reporter showed that the ran transcription decreased and the plasmid increased to 25 ng (P<0.05)., Conclusion: This study demonstrated that ran is a target gene regulated by P53 in myeloma cells for the first time.

Objective: To investigate the effect of blocking p38 mitogen-activated protein kinase (p38MAPK) pathway on the proliferation of OT-II cells mediated by splenic dendritic cells (DCs)., Methods: Splenic DCs of C57BL/6 mice were purified with anti-CD11c immunomagnetic beads, and OT-II cells were isolated from the splenic of CD4(+)-ovalbumin transgenic mice (OT-IItransgenic mice) by mouse CD4 T cell isolation kits. After being pretreated with SB203580, an inhibitor of p38MAPK, DCs were stimulated with lipopolysaccharides (LPS). Then the expression levels of co-stimulatory molecules (CD80, CD86) and MHCII in DCs, and antigen-presenting ability of DCs treated with the 52-68 fragment of the E alpha-chain of I-E class II molecules (Eα52-68 peptide) were detected by flow cytometry. The protein levels of tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 6 (IL-6) and transforming growth factor β (TGF-β) in the culture supernatants were measured by ELISA. The proliferation of OT-II cells which were co-cultured with OVA323-339-treated DCs was analyzed by flow cytometry., Results: The purity of both DCs and OT-II cells reached over 90% after isolation. SB203580 downregulated the expressions of CD80, CD86 and MHC II, and suppressed the antigen-presenting ability of DCs. The expressions of TNF-α, IL-1α and IL-6 were downregulated, while the expression of TGF-β was raised. Finally, SB203580 inhibited DCs-mediated proliferation of OT-II cells., Conclusion: Blocking p38MAPK pathway with SB203580 could inhibit DCs-mediated proliferation of OT-II cells, which might be involved in modulating the expressions of CD80, CD86 and MHC II, the antigen-presenting ability, as well as the expressions of pro-inflammatory and anti-inflammatory cytokines.

Objective: To investigate the effect of necrostatin-1 (Nec-1) on the expression of liver monocyte chemotactic protein-1 (MCP-1) in septic rats and its mechanism., Methods: Forty-eight male Sprague-Dawley (SD) rats were randomly divided into sham group, model group, and Nec-1 group by randomized digital number method, with 16 rats in each group. The model of sepsis was reproduced by cecal ligation and puncture (CLP). Rats in sham group received anesthesia, and flipping the cecum followed by closure of the abdomen without ligation of the cecum. Rats in Nec-1 group were given 1 mg/kg Nec-1 [25 mg Nec-1 solution dissolved in 2.5 mL of dimethyl sulfoxide (DMSO)] through caudal vein 30 minutes before operation, while the rats in model group were given 0.1 mL/kg of DMSO only. Blood from abdominal aorta and liver tissue in each group were collected at 0 hour and 8 hours after operation. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined with automatic biochemistry analyzer. The pathological changes in liver were observed under light microscope using hematoxylin-eosin (HE) staining. The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined by enzyme linked immunosorbent assay (ELISA). The MCP-1 mRNA expression in the liver was determined by reverse transcription-polymerase chain reaction (RT-PCR)., Results: There was no significant differences in the levels of serum ALT, AST, TNF-α, IL-6 and expressions of liver MCP-1 mRNA at 0 hour among three groups, and the liver cellular structure was normal. At 8 hours, compared with sham group, the expressions of serum ALT, AST, TNF-α, IL-6 and liver MCP-1 mRNA were significantly increased in model group and Nec-1 group [ALT (U/L): 172.35±21.88, 129.67±18.20 vs. 60.04±11.74, AST (U/L): 511.03±34.92, 363.51±25.25 vs. 254.83±31.04, TNF-α(ng/L): 603.96±24.18, 483.87±26.60 vs. 265.74±15.14, IL-6 (ng/L): 975.62±65.37, 712.09±45.47 vs. 310.42±13.88, MCP-1 mRNA (2-ΔΔCt): 7.09±0.18, 5.51±0.45 vs. 0.99±0.06, all P < 0.05]. Levels of the above parameters in Nec-1 group at 8 hours were significantly decreased compared with those of model group (all P < 0.05). Under light microscopy, it was noted that the structure of hepatic lobules was destroyed, with exacerbation of immunocyte infiltration at 8 hours in model group. At 8 hours, it was found that Nec-1 alleviated the pathological damage in Nec-1 group., Conclusion: Nec-1 can protect the liver of rats with sepsis, lower the expression of serum TNF-α and serum IL-6 and liver MCP-1 mRNA, and obviously reduce the damage of inflammation.

Objective: To observe the effect of different concentrations of zoledronic acid on human gingival fibroblast (HGF) cells proliferation and apoptosis, and to investigate the mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) caused by zoledronic acid., Methods: Different concentrations (0, 0.5, l.0, 5.0, 10.0 µmol/L) of zoledronic acid acted on the HGF. After 24 hours, flow cytometry was used to detect the rate of apoptosis and methyl thiazolyl terazolium (MTT) assay used to observe the proliferation of HGF. The effect of different concentrations of zoledronic acid on cell proliferation was examined by proliferation test on day 2, 4 and 7., Results: Flow cytometry showed that when the concentration of zoledronic acid was 0, 0.5, 1.0, 5.0, 10.0 µmol/L, the apoptosis rate was (0.67 ± 0.50)%, (2.13 ± 0.21)%, (3.27 ± 0.23)%, (4.17 ± 0.35)%, (9.87 ± 1.79)% respectively. The difference in HGF cells between control group and zoledronic acid groups was significant (P < 0.05) at three concentrations of zoledronic acid (1.0, 5.0, 10.0 µmol/L). The cell proliferation test showed an concentration-time dependent effect. With increase of concentration and time, the cell proliferation was significantly inhibited. There was significant difference in absorbance value among the different concentration groups (P < 0.05)., Conclusions: Zoledronic acid can inhibit HGF proliferation and promote its apoptosis.

Objective: To investigate the effects and mechanisms of high glucose on the phenotype transformation of rat vascular smooth muscle cells (VSMCs)., Methods: VSMCs ere isolated from rat thoracic aorta and the 3rd-5th VSMCs were incubated with normal glucose (5.5 mmol/L), high glucose (25 mmol/L), or high glucose (25 mmol/L) + P38 inhibitor (25 mmol/L +SB203580) for another 24 hours. Then the gene expression of osteopontin (OPN), alpha smooth-actin (alpha-SMA), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9(MMP-9) were assayed by real time RT-PCR, the protein expression of P38 were assayed by Western blot., Results: (1) High glucose promoted the phenotype transformation of VSMCs and up-regulated the expression of MMP-2 and MMP-9. (2) High glucose promoted the phosphorylation of P38. (3) SB203580, the inhibitor of P38/MAPK signal pathway, inhibited the effects of high glucose on phenotype transformation and expression of MMP-2 and MMP-9., Conclusion: High glucose may promote phenotype transformation of VSMCs via the signal pathway of P38/MAPK.

Objective: To determine the effect of p38 MAPK inhibitor (SB203580) on TNF-α -induced high mobility group protein B1 (HMGB1) expression in microglial cells.
, Methods: Microglial cells were treated with TNF-α (25 ng/mL, TNF-α group), TNF-α plus SB203580 (10 μmol/L, TNF-α+SB203580 group), SB203580 (SB203580 group) or serum-free medium (control group). After 16 h of incubation, the protein levels of p-p38 MAPK and HMGB1, and mRNA levels of HMGB1 were examined by ELISA, Western Blot and RT-PCR, respectively.
, Results: There was a significant increase in p-p38 MAPK and HMGB1 levels in TNF-α-treated microglia cells (P<0.01). The TNF-α-induced HMGB1 protein and mRNA expression was suppressed by SB203580.
, Conclusion: TNF-α up-regulates HMGB1 expression in microglial cells through activation of the p38 MAPK pathway.

Objective: To investigate the influence of zoledronic acid on vascular endothelial cells., Methods: The influence of zoledronic acid on proliferation, migration and adhesion of vascular endothelial cells were tested with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell migration assay and cell adhesion assay. The results of each experimental group were compared with the control group and the data statistically analyzed., Results: In a concentration of 0-0.5 mmol/L, the absorbance value decreased from 0.09 to 0.34 as the drug concentration increased. Scratch test showed that the change of width of scratch before and after 24 hours in control, low, medium and high concentration groups were (38.7 ± 0.42), (35.8 ± 4.17), (19.9 ± 0.57) mm (P < 0.001), (12.5 ± 3.89) mm (P < 0.05). Adhesion test showed that the absorbance value in control, low, medium and high concentration groups were 1.14 ± 0.18, 0.95 ± 0.13, 0.81 ± 0.11 (P < 0.01), 0.67 ± 0.19 (P < 0.001). Comparisons between control and experimental groups were analyzed by t-test and P values < 0.05 were considered statistically significant., Conclusions: Zoledronic acid inhibits the proliferation, migration and adhesion of vascular endothelial cells.

Objective: To explore whether edaravone protects cells damage via mitogen-activated protein kinases (MAPKs) signal pathway, and which procedure of p38 be affected so as to add theories for AD pathogenesis and treatments., Methods: According to different drugs treated, PC12 cells in vitro were divided into four groups. Negative control group: cells were treated with media alone. AD model group: cells were treated with 30 pmol/L Abeta(25-35). Inhibitor control group: cells were treated with 10 micromol/L SB203580 Cp38 mitogen-activated protein kinase (p38) inhibitor], 10 micromol/L SP600125 [c-Jun NH2 terminal kinase (JNK) inhibitor], or 10 micromol/L PD98059 extracelular signal regulated kinase (ERK) inhibitor]. Low-dose, middle-dose and high-dose edaravone group: cells plated for 24 hours treated with 30 micromol/L Abeta(25-35) and co-treated with 20, 40, 80 micromol/L edaravone 3 hours, respectively. The morphology of the treated cells were observed, the p-p38, p-JNK and p-ERK proteins in each group were tested by the Western blot. The p38 mRNA were tested in each group above (only add SB203580 10 micromol/L in third group) by the real time PCR., Results: (1) The p-p38 protein was significantly increased in model control group compared with that in negative control group (P<0.05). The p-p38 protein in the inhibitor group and edaravone groups was decreased significantly (P<0.05) when compared with that in model control group. The p-p38 proteins were significantly increased in the three edaravone groups compared with that in inhibiter control group (P<0.05). The p-p38 protein in middle-dose edaravone group was decreased compared with that in low-dose edaravone group (P<0.05). There was no relationship in dose-dependent manner about edaravone. Compared with three edaravone groups, the p-p38 protein was lower than it in high-dose edaravone & inhibiter group (P<0.05). (2) The p-JNK protein was significantly increased in model control group compared with that in negative control group (P<0.05). The p-JNK protein in the inhibitor group was decreased compared with that in model control group (P<0.05). (3) No significantly difference of p-ERK protein concentration was observed in other groups when compared with that in negative control group (P>0.05 each). (4) Compared with negative control group, the p38 mRNA in model control group was significantly increased, and it was significantly decreased in inhibitor control group (P<0.05 each). In 40 micromol/L and 80 micromol/L edaravone groups, the p38 mRNA was significantly decreased compared with that in model control group, and it still was decreased compared with that in inhibitor control group (P<0.05). The p38 mRNA in 40 micromol/L edaravone group was the lowest among three edaravone groups, and it was obviously different from that in 20 micromol/L and 80 micromol/L edaravone groups (P<0.05)., Conclusion: Abeta(25-35) could increase the p-p38 and p-JNK protein expression in cultured PC12 cells, but there was no obviously expression of p-ERK protein. These indicated that Abeta(25-35) might activate MAPKs signal pathway, especially p38 and JNK, and lead to PC12 cell damage. Edaravone could decrease p38 mRNA induced-Abeta(25-35), which indicated edaravone could protect PC12 cell damage via blocking p38 signal pathway in mRNA stage and protein stage simultaneously. Hence, it is promising that edaravone would be a new medicine for AD.

The rice stripe disease is a viral disease transmitted by small brown planthopper, Laodelphax striatellus, which outbroke a few years ago in the Yangtze River basin, especially Jiangsu region, China. To study the effects of imidacloprid stress on rice stripe virus (RSV) in rice plants, the rice seedlings were treated with imidacloprid 1, 2, 3 and 4 times (B1, B2, B3 and B4), respectively, after artificial inoculation by L. striatellus for 48 h, and the expression levels of relative genes including RSV NS3, CP, SP and NSvc4, as well as the protein concentrations of CP and SP were detected at different stages by real-time PCR and Western blotting. The results showed that the effects of imidacloprid treatment on the expression levels of four genes were gene-specific and correlated with application frequencies of imidacloprid. The expression levels of NS3 gene were upregulated in three treatments, and the highest expression level (10.86) was observed 16 days after inoculation in B4 treatment, but a significant down-regulation of NS3 gene was found in all other treatments. The expression levels of CP, SP and NSvc4 genes were down-regulated significantly (0-0.74) in almost all B2 and B3 treatments, while a significant up-regulation was found in half of B1 and B4 treatments, and the highest expression levels of SP gene were observed 16 days after inoculation in B1 (258.89) and B4 (730.54) treatment, respectively. On the other hand, the effects of imidacloprid stress on the expression patterns of CP and SP genes were different from those of CP and SP proteins. For example, the expression level of CP gene was almost no expression (0) 19 days after inoculation in B1 treatment, while significantly up-regulated (23.08) was observdd for CP protein.

Objective: To explore the role of MAPK signaling pathway in apoptosis and necroptosis induced by aluminum in SH-SY5Y cells., Methods: To imitate neural cell death induced by aluminium, AlCl3 x 6H2O (4 mmol/L) was used to treat SH-SY5Y cells. Necrostatin-1 (Nec-1,60 μmol/L), the specific inhibitor for necroptosis, and zVAD-fmk (20 μmol/L), the specific inhibitor for apoptosis, were added into cultures for inhibiting the occurrence of necroptosis and apoptosis. CCK-8 was performed to measure cell viability, flow cytometry was used to test the difference of apoptosis rate and necrosis rate between groups, and western-blot was used to detect the change of MAPK protein., Results: Compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group, cell viabiligy of Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group decreaced (P < 0.05). Compared with Al(3+) exposed group, cell viability of Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group increased (P < 0.05). Necrotic rate and apoptotic rate in Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group obviously increased compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group (P < 0.05). Compared with Al(3+) exposed group, necrotic and apaptotic rate of Al(3+) plus zVAD-fmk group and Al(3+) plus Nec-1 group were statistically significant decreased (P < 0.05). Compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group, expression of p-p38 in Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group increased obviously (P < 0.05), and expression of p-ERK decreased significantly (P < 0.05). Compared with Al(3+) exposed group, expression of p-p38 decreased (P < 0.05), but p-ERK increased in Al(3+) plus Nec-1 group (P < 0.05)., Conclusion: The ERK and p38 MAPK signaling pathways are involved in aluminum-induced necroptosis in SH-SY5Y cells, but only ERK signaling pathway is involved in aluminum-induced apoptosis, and JNK signaling pathway is not involved in aluminum-induced cell death.

Objective: To explore the effect of zoledronate (ZOL) on osteoclast differentiation and expressions of transient receptor potential vanilloid 5 channel (TRPV5) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1)., Methods: RAW264.7 cells were divided into two groups for treatment with RANKL for 5 days (group A) or with additional ZOL treatment in the last 2 days of RANKL treatment (group B). Osteoclastogenesis of the cells and the mRNA and protein expressions of TRPV5 and NFATc1 after the treatments were examined., Results: In group B, the number of newly generated osteoclasts (≥ 3 nuclei), number and size of dentin resorption lacunaes were 29.0 ± 2.4, 24.8 ± 1.1, and 2 030.0 ± 165.7 µm², respectively, which were significantly lower than those in group A (56.5 ± 4.5, 49.3 ± 0.9, and 3 946.7 ± 367.5 µm², respectively, P<0.01). Fluorescent intensity of TRPV5 and NFATc1 were also significantly decreased in group B (P<0.01). Compared with those in group A, TRPV5 mRNA and protein expressions in group B were down-regulated by 50.4% and 37.8%, and those of NFATc1 by 68.0% and 48.4%, respectively (P<0.01)., Conclusion: ZOL can significantly inhibit osteoclastogenesis and bone resorption, which may be attributed, at least partly, to ZOL-induced inhibition of TRPV5 and NFATc1 expressions.

The aim of the present study is to investigate the expressions of ATP-sensitive K(+) channels (KATP) in pulmonary artery smooth muscle cells (PASMCs) and the relationship with p38 MAPK signal pathway in rats. Male SD rat PASMCs were cultured in vitro, and a model of hypoxia and hypercapnia was reconstructed. PASMCs were divided to normal (N), hypoxia-hypercapnia (H), hypoxia-hypercapnia+DMSO incubation (HD), hypoxia-hypercapnia+SB203580 (inhibitor of p38 MAPK pathway) incubation (HS) and hypoxia-hypercapnia+Anisomycin (agonist of p38 MAPK pathway) incubation (HA) groups. Western blot was used to detect the protein expression of SUR2B and Kir6.1; semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of SUR2B and Kir6.1. The results demonstrated that: (1) Compared with N, H, HD and HS groups, the expressions of Kir6.1 mRNA and protein in PASMCs of HA group were decreased significantly (P < 0.01), but there were no differences among N, H, HD and HS groups (P > 0.05); (2) Compared with N group, the expressions of SUR2B mRNA and protein in H, HD, HS and HA groups were increased significantly (P < 0.05), but there were no differences among H, HD, HS and HA groups (P > 0.05). The results imply that: (1) Hypoxia-hypercapnia, SB203580 didn't change the expressions of Kir6.1 mRNA and protein in PASMCs, but Anisomycin decreased the expressions of Kir6.1 mRNA and protein, so Kir6.1 may be regulated by the other subfamily of MAPK pathway; (2) Hypoxia-hypercapnia raised SUR2B mRNA and protein expressions in PASMCs, but SB203580 and Anisomycin did not affect the changes, so the increasing of SUR2B mRNA and protein induced by hypoxia-hypercapnia may be not depend on p38 MAPK pathway.

To investigate the murine double minute 2 (MDM2) localization and expression pattern in brain, immunohistochemistry, immunofluorescent staining and immunoblotting methods were used to analyze it in brains of Kunming mice during postnatal development, in brains of adult SD rats and in primarily cultured neurons. The distribution of MDM2 and markers of axon initial segment (AIS) was analyzed by double immunolabeling. In addition, Nutlin-3, a MDM2 antagonist, was injected into hippocampus to analyze the effect on the distribution of MDM2 and AIS protein Nav1.6 in AIS. The results showed that the dynamic expression patterns of MDM2 protein in cerebral cortex and hippocampus of Kunming mice after birth were different. However, it was similar that MDM2 was gradually enriched to AIS during postnatal development, especially after postnatal day 7. The MDM2 in AIS was also observed in different brain regions of adult SD rat brain and in primarily cultured neurons, where MDM2 was colocalized with AIS markers such as AnkG and Nav1.6. In addition, hippocampal injection of Nutlin-3 could induce the loss of the characteristic distribution of MDM2 in AIS. Moreover, Nutlin-3 not only caused a decrease of Nav1.6 distributing in AIS, but also disrupted the polarized distribution of MAP2 in neurons. These results indicate that MDM2 can be enriched at the AIS of adult rodent brain, which might play a role in regulation of the maintenance of AIS function and neuronal polarity.

In this study, the rescue effect of receptor activator for nuclear factor-kappaB ligand (RANKL) on zoledronate acid (ZOL) induced inhibition of osteoclastogenesis and gene expression of NF-kappaB p50 and c-Jun was investigated. Mice calvarial osteoblasts (OBs) were harvested and co-cultured with RAW264.7 cells and the cells were divided into 4 groups and received treatment with ZOL and RANKL, either single or combined. The formation of multi-nucleated osteoclast (OC) was examined and gene expression of NF-kappaB p50 and c-Jun was detected. Group B (ZOL) showed least multi-nucleated OC and resorption lacunae among the 4 groups (P < 0.05 or P < 0.01) and it was followed by group C (ZOL+RANKL). Group D (RANKL) showed highest OC and resorption lacunae while it was similar to Group A (control) (P > 0.05). Gene expression of NF-kappaB p50 and c-Jun was the lowest in group B (P < 0.05 or P < 0.01) among the four groups and was significantly increased in group C when compared with group B (P < 0.05). Group A and D showed highest gene expression and they were similar to each other (P > 0.05). This study suggest that RANKL might partly rescue ZOL induced inhibition of osteoclastogenesis, and the effect of RANKL and ZOL on osteoclastogenesis may be mediated by NF-kappaB p50 and c-Jun.

Objective: To investigate the effects of necrostatin-1( Nec-1) on the liver of rats with trauma induced hemorrhagic shock., Methods: Trauma induced hemorrhagic shock model was produced by adopting the left femur, tibia fracture and soft tissue injury, bleeding and reperfusion in male Sprague-Dawley (SD) rats. A total of 22 rats were divided into model group and Nec-1 group with 11 rats in each group by randomized digital number method and the 72-hour mortality was observed. In addition, 72 rats were randomly divided into sham group, model group, Nec-1 group with 24 rats in each group. Rats in sham group were only received anesthesia, separating a nd ligating blood vessels, without trauma induced hemorrhagic and reperfusion, and the rats in Nec-1 group were received 1 mg/kg Nec-1 through femoral vein 5 minutes before reperfusion, and the rats in Nec-1 group were received the same amount of solvent. The serum and liver tissues of each group were collected at 2, 4, 8 hours after reperfusion. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by automatic biochemistry analyzer. The pathology changes in liver were observed by hematoxylin-eosin (HE) staining. The mRNA expressions of tumor necrosis factor-α (TNF-α) and interleukin -1β (IL-1β) in the liver were determined by reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of receptor interaction of protease 1/3( RIP1/RIP3) were also assessed by Western blot analysis., Results: Compared to the model group, Nec-1 significantly reduced the 72 hour mortality [18.18% (2/11) vs. 63.64% (7/11), P = 0.040]. Two hours after trauma induced hemorrhagic shock and reperfusion, the expressions of ALT and AST in model group were significantly increased compared with those in sham group. [ ALT (U/L): 110.21 ± 22.32 vs. 80.98 ± 19.94, AST (U/L): 364.29 ± 64.83 vs. 279.76 ± 70.64, both P<0.05], and reached the peak at 8 hours [ALT (U/L): 387.41± 47.11 vs. 82.76 ±22.44, AST ( U/L): 973.35 ±77.51 vs.261.49 ± 52.03, both P <0.01]. Levels of serum ALT and AST in NEc-1 group were significantly decreased compared with model group [ALT (U/L) 4 hours: 144.64 ± 33.79 vs. 213.96 ± 36.21, 8 hours: 159.48 ± 43.57 vs. 387.41>11; AST (U/L): 4 hours: 398.78 ± 59.48 vs. 630.61 ± 59.93, 8 hours: 427.38 ± 80.75 vs. 973.35 ± 77.51, all P < 0.01] Under light microscopy, it was noted that the hepatic sinus expansion, liver cells degeneration, necrosis, as well as infiltration of abundant inflammatory cells were observed. But the pathology changes in hepatic tissues were significantly mitigated in Nec-1 group. Along with the time extension, the mRNA expressions of TNF-α and IL-β and the protein expressions of RIP1 and RIP3 were markedly up-regulated. Compared with model group, difference in the mRNA expressions of TNF-α and IL-β in hepatic tissues in Nec-1 group were statistically significant, and the most obvious difference was at 8 hours [TNF-α mRNA: 1.457 ± 0.081 vs. 2.317 ± 0.062, IL-β mRNA: 0.690 ± 0.087 vs. 1.812 ± 0.112, both P<0.01]. But there was no statistically significant difference in RIP1 and RIP3 between Nec-1 group and model group [RIP1 protein 8 hours: 0.561 ± 0.033 vs. 0.587 ± 0.036, RIP3 protein 8 hours: 0.976 ± 0.040 vs. 1.044 ± 0.115, both P > 0.05]., Conclusion: Nec-1 may be remarkable protect effect on the liver of rats with trauma induced hemorrhage shock and reperfusion, and the intrinsic mechanisms need further investigation.

Objective: To investigate the effect of zoledronate acid on osteoclast differentiation and gene expression of calmodulin (CAM) and calmodulin-dependent protein kinase (CAMK)II., Methods: Receptor activation of nuclear factor κB ligand (RANKL) was used to induce differentiation of RAW264.7 cells into osteoclasts in vitro. The cells were divided into two groups, group A and group B. Both groups were treated with RANKL for 5 days, whereas group B was also treated with zoledronate for the last 2 days.Osteoclastogenesis and gene expression of CAM and CAMK II were examined., Results: In group B, the number of new-generated osteoclasts (≥3 nuclei), number and size of dentin resorption lacunaes were (23 ± 3) , (19 ± 2) and (4951 ± 223) µm(2) respectively, which were significantly lower than those [(44 ± 3) , (46 ± 1) and (13 331 ± 248) µm(2)] in group A (P < 0.01).mRNA and protein level of CAM and CAMK II were also significantly down-regulated in group B when compared with group A (P < 0.01) and the decrease was 26.7% and 37.2% respectively for CAM, 57.0% and 76.1% respectively for CAMK II., Conclusions: Zoledronate acid could significantly inhibit formation and resorption function of osteoclasts. CAM and CAMKII may be involved in the inhibition process.

Objective: To examine the effects of niacin on lysophosphatidylcholine (LPC)-induced intercellular adhesion molecule-1 (ICAM-1), and gained insight to the mechanisms., Method: Human umbilical vein endothelial cell line was cultured using Medium 200 medium in incubator at 37 °C and 5% CO2 condition.Experimental groups:(1) the negative control group:medium; (2) LPC different time groups:the medium added with 20 µmol/L final concentration of LPC, were cultured for 10 min and 8 h, 24 h; (3) LPC+ p38-mitogen-activated protein kinase (p38MAPK) inhibitor (SB203580) group:the medium added with 10 µmol/L p38MAPK inhibitor (SB203580) was cultured for 1 h, then human umbilical vein endothelial cells (HUVECs) added with the LPC were cultured for 10 min, 8 h and 24 h.(4) LPC+different niacin dose group:after separately adding with 0.25, 0.5, 1 mmol/L niacin, the cells were cultured for 18 h, then HUVECs added with the LPC were cultured for 10 min, 8 h and 24 h. Cell concentration in each group was 5×10(5)/ml, inoculated in 6-well plates, each well 1 ml. Detected by Western blot analysis of pp38MAPK, ICAM-1 protein content, real-time quantitative PCR to detect endothelial cell ICAM-1 mRNA expression, cell immunofluorescence to detect LPC-induced ICAM-1 protein expression., Result: In LPC 24 h group, the expression of ICAM-1 protein was significantly increased 0.786 ± 0.02, the LPC+niacin group, ICAM-1 protein levels (0.487 ± 0.015) was significantly lower than the LPC 24 h group (P < 0.01), in LPC+SB203580 intervention group, ICAM-1 protein levels (0.461 ± 0.011) was significantly lower than that of the LPC 24 h group (P < 0.01), but did not reach the level of the control group. Adding LPC to culture for 10 min, phosphorylation of p38MAPK (pp38MAPK) reached its peak (0.47 ± 0.02), niacin could reduce the pp38MAPK (0.07 ± 0.02), SB203580 could also reduce its activity (0.11 ± 0.02). Adding LPC to culture for 8 h, ICAM-1 mRNA expression (8.16 ± 0.15) compared with the control group (1.00 ± 0.02) had a significant increase (t = 24.34, P < 0.01). Compared with the LPC 8 h, niacin reduced LPC-induced ICAM-1 mRNA expression (3.85 ± 0.14), and showed a dose-dependent manner (F = 8.06, P < 0.01), while SB203580 could not effectively reduce the ICAM-1 mRNA (8.09 ± 0.11)., Conclusion: Niacin prevented LPC-induced endothelial dysfunction by reducing expression of ICAM-1. These mechanisms appeared to be at least partly mediated by suppression of the pp38MAPK in endothelial cells. These pleiotropic effects of niacin may potentially contribute to the beneficial effects of risk reduction for atherosclerotic disease.

The present study was to investigate the effects of exogenous insulin-like growth factor binding protein 7 (IGFBP7) on the proliferation of human breast cancer cell line MDA-MB-453 and its possible mechanism. By means of MTT method in vitro, the results showed exogenous IGFBP7 inhibited the growth of MDA-MB-453 cells (IC50 of IGFBP7 = 8.49 μg/mL) in time- and concentration-dependent manner. SB203580, p38(MAPK) inhibitor, blocked the anti-proliferative effect of exogenous IGFBP7. The flow cytometry assay showed that exogenous IGFBP7 remarkably induced G0/G1 arrest in MDA-MB-453 cells. The Western blot showed that exogenous IGFBP7 promoted phosphorylation of p38(MAPK), up-regulated expression of p21(CIP1/WAF1), and inhibited phosphorylation of Rb. SB203580 restrained exogenous IGFBP7-induced regulation of p21(CIP1/WAF1) and p-Rb in MDA-MB-453 cells. In conclusion, the present study suggests that exogenous IGFBP7 could activate the p38(MAPK) signaling pathway, upregulate p21(CIP1/WAF1) expression, inhibit phosphorylation of Rb, and finally induce G0/G1 arrest in MDA-MB-453 cells.

Objective: To study the inhibitory effect of the dual usage of BEZ235 and U0126, the inhibitor of phosphatidyl inositol-3-kinase/protein kinase B pathway and extracellular regulated proteinkinase/mitogen-activated protein kinase pathway, respectively, on cell proliferation., Methods: Phosphatase and tensin homolog knockout mouse embryonic fibroblast (PTEN-/-MEF) cell lines were used as the cellular model for malignant tumors. BEZ235, the dual inhibitor of phosphatidyl inositol-3-kinase and mammalian target of rapamycin, and U0126, the inhibitor of mitogen-activated protein kinase were used to treat the cells individually and in a combination manner. The inhibitory effects to cell proliferation were monitored by MTT., Results: Both BEZ235 and U0126 suppressed PTEN knockout cell proliferation, and their half inhibitory concentrations were 6.257 nmol/L and 22.85 μmol/L, respectively. However, the combination treatment of the two drugs showed antagonistic rather than synergistic effect on cell proliferation., Conclusion: BEZ235 and U0126 are not suitable for a combined target therapy regimen.

Objective: To explore the signal transduction mechanism of p38 mitogen activated protein kinase (p38MAPK) in human facial hypertrophic scar fibroblast (FB) differentiation into myofibroblasts (MFB)., Methods: Fibroblasts of primary culture were simple randomly assigned into two groups: cyclic stretch (control group) and cyclic stretch pre-treated with SB203580(experimental group). Expression of P-p38MAPK and α-smooth muscle actin (α-SMA) protein were examined using Western blotting and expression of transforming growth factor β1 (TGF-β1) mRNA and α-SMA mRNA were examined using reverse transcription PCR (RT-PCR)., Results: In control group, the expressions of α-SMA, p38MAPK, TGF-β1 mRNA and α-SMA mRNA (0 h: 0.134 ± 0.011, 0.239 ± 0.015, 0.214 ± 0.018, 0.252 ± 0.010; 6 h: 0.152 ± 0.014, 0.287 ± 0.016, 0.288 ± 0.011, 0.277 ± 0.013; 12 h: 0.172 ± 0.017, 0.320 ± 0.017, 0.335 ± 0.013, 0.297 ± 0.006) , were significantly increased with loading time (6 h>0 h; 12 h>0 and 6 h). In experimental group (pre-treated with SB203580), the expressions of α-SMA, p38MAPK, TGF-β1 mRNA,α-SMA mRNA (6 h: 0.116 ± 0.017,0.128 ± 0.016,0.134 ± 0.014,0.163 ± 0.009; 12 h: 0.149 ± 0.013,0.136 ± 0.018,0.144 ± 0.013,0.187 ± 0.010) on corresponding time decreased sharply compared with those in control groups (6, 12 h)., Conclusions: The human facial hypertrophic scar fibroblasts differentiation in response to cyclic stretch was mediated by p38MAPK phosporylation.

Objective: To explore the effects of p38MAPK inhibitor SB203580 (SB) on the occurrence of acute GVHD and intestine damage after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in mice., Methods: Sixty BALB/c mice, as recipients, were randomized to control group, irradiation group, model group and intervention group. C57BL/6 mice, as donors, were raised to prepare the bone marrow cells (BMCs) and spleen cells (SCs), which were injected into irradiated recipients mice by tail vein. Except control group, other groups accepted 7.5Gy total body irradiation. Model group and intervention group were infused with BMCs 5×10⁶ and SCs 5×10⁵ by less than 4 h after irradiation. SB was injected into intervention group by intraperitoneally, but only DMSO for model group. The general status and survival rate of each group were evaluated. The expression of p-p38MAPK, Fas and FasL in intestine were determined by RT-PCR, Western blot and immunohistochemistry (IHC)., Results: The weight changes of intervention group (13.00±0.50)% was significantly lighter than that of model group (25.00±0.75)% (P<0.05). The clinical score of acute GVHD in the intervention group (3.33±0.82) was significantly lower than that of model group (6.33±1.36) (P<0.05). The expression levels of p-p38MAPK, Fas and FasL in small intestine of intervention group (1.43±0.02, 0.81±0.03, 0.97±0.03) were lower than those of model group (1.76±0.05, 1.52±0.04, 1.48±0.04)., Conclusion: SB inhibited the activation of p38MAPK and Fas/ FasL signal pathway and alleviated the apoptosis of small intestine. And SB could relieve small intestine damages induced by allogeneic T lymphocytes.

Objective: To investigate the changes of cyclooxygenase-2 (COX-2) expression in the spinal cord dorsal horn after intrathecal a specific p38MAPK inhibitor-SB203580 on neuropathic pain in rats induced by chronic constrictive injury (CCI) to the sciatic nerve., Methods: Twenty-four male SD rats after intrathecal catheter placement were randomly divided into 4 groups: a sham group with sham surgery, the neuropathic pain model of a NS group, a DMSO group and an SB group were established by CCI to sciatic nerve. NS or DMSO or SB203580 was injected IT NS or 2%DMSO or SB203580 twice a day for 5 consecutive days starting at 6th day when the model of chronic constrictive injury was established. Mechanical stimuli were measured before the surgery and on 1st, 3rd, 5th, 7th, 9th, and 11th day after the surgery. Then all rats were sacrificed and the lumbar segment of spinal cord was removed to determine the COX-2 expression in the dorsal horn by immunocytochemistry., Results: Day 1 to 11 after the surgery, the threshold to mechanical on the surgery side was significantly lower in the NS group and the DMSO group than in the sham group. Day 7 to 11 after the sugery, the threshold to mechanical on the surgery side was significantly lower in the SB group than in the NS group and the DMSO group. The expression of spinal COX-2 was higher in the NS group and the DMSO group than in the sham group, but lower in the SB group than in NS group and the DMSO group., Conclusion: Intrathecal administration of SB203580 has significant analgesic effect in the CCI rat model. Expression of COX-2 is significantly reduced when p38MAPK is inhibited by intrathecal SB203580, and p38MAPK stimulation is essential for COX-2 expression.

Background: It has been proven that cobrotoxin has anti-tumor effect while its role in lung cancer is rarely studied. The aim of this study is to assay the anti-tumor effect of cobrotoxin in cell line A549, and also to explore its possible mechanism related to autophagy and P38-MARK pathway., Methods: Using MTT method to observe the inhibition effect of cobrotoxin on the growth of adenocarcinoma cell A549 and human lung fibroblast cell HFL1, as well as on that of A549 pretreated with 3-MA and SB203580, which are the inhibitor of autophagy and P38-MARK pathway respectively. Cell colony tablet cloning experiment was executed to detect the effect of cobrotoxin on colony formation of A549. Determining the protein levels of beclin-1, LC3, p38 and pP38 in A549 by Western blot after cells were exposed to cobrotoxin, or to cobrotoxin combined with either 3-MA or SB203580., Results: All of different concentrations of cobrotoxin inhibited the growth of A549, but had no obvious effect on that of HFL1. After treating with 3-MA or SB203580, the suppress effect of cobrotoxin on A549 reduced. What's more, different concentrations of cobrotoxin all significantly suppressed the colony formation of A549. The expression of beclin-1 and pP38 in A549 increased obviously after exposure to cobrotoxin, and also the ratio of LC3 II to LC3 I amplified with a dose-dependant manner, but P62 decreased. The protein level of beclin-1 and the ratio of LC3 II to LC3 I in cells pretreated with 3-MA were reduced, while that of p62 was increased. Also, in cells that treated with SB203580 before exposed to cobrotoxin, the expression of beclin-1, pP38 and the ratio of of LC3 II to LC3 I were reduced, but the expression of P62 increased., Conclusions: Cobrotoxin can suppress the growth of A549 in vitro. And the activating of P38-MARK pathway, then upregulating autophagy, was involved in cobrotoxin induced anti-tumor process.

Objective: To investigate the effects of phosphorylated mitogen-activated protein kinases (MAPK), including the phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), the phosphorylated protein p38 (p-p38), the phosphorylated c-Jun N-terminal kinase (p-JNK), on phosgene inhalation-induced lung injury and its relationship with matrix metalloproteinase 9 (MMP-9)., Methods: According to the random number table, 30 male Wistar rats were divided into air control group (C), phosgene inhalation group (P), PD98059 (specific inhibitor of ERK1/2) group, SB203580 (specific inhibitor of p38) group, and SP600125 (specific inhibitor of JNK) group, with 6 rats in each group. The number of neutrophils in the bronchoalveolar lavage fluid (BALF) was counted and the lung wet-dry ratio (W/D) was examined. The serum levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 were determined with ELISA. The protein expressions of p-ERK1/2, p-p38, p-JNK, and MMP-9 in lung tissue were detected with Western blotting. The mRNA level of MMP-9 in lung tissue was detected with real-time fluorescence quantitative PCR. Data were processed with one-way analysis of variance (among groups) and SNK method (paired comparison)., Results: Compared with those of group C [respectively (2.0 ± 0.7)×10(4) /mL and 3.7 ± 0.6], the number of neutrophils and W/D of group P [respectively (10.7 ± 1.4)×10(4) /mL and 7.6 ± 0.4] were increased. The number of neutrophils in group SB203580 and group SP600125 was respectively (8.3 ± 1.1)×10(4), (7.9 ± 1.3)×10(4)/mL, with W/D respectively 6.1 ± 1.4, 6.1 ± 0.9, all of which decreased as compared with those of group P (with P values all below 0.01). Compared with those of group C, the levels of TNF-a, IL-1β, IL-6, and IL-8 of group P were increased, but decreased in group SB203580 and group SP600125 compared with that of group P, though still higher than those of group C, and the differences were statistically significant (P < 0.05 or P<0.01). Protein quantities of p-p38 and p-JNK were higher in group P (respectively 1.19 ± 0.22 and 1.43 ± 0.14) than in group C (respectively 0.76 ± 0.06 and 0.74 ± 0.05). Compared with those of group P, the protein levels of p-ERK1/2 (0.47 ± 0.05) in group PD98059, p-p38 (0.88 ± 0.07) in group SB203580, and p-JNK (0.91 ± 0.07) in group SP600125 were significantly reduced (P < 0.05 or P < 0.01). The protein and mRNA levels of MMP-9 were higher in group P (respectively 2.23 ± 0.18 and 4.93 ± 0.12) than in group C (respectively 1.26 ± 0.14 and 1.80 ± 0.03). The protein and mRNA levels of MMP-9 in group SB203580 (respectively 1.58 ± 0.14 and 2.96 ± 0.09) and group SP600125 (respectively 1.55 ± 0.30 and 3.00 ± 0.13) were lower than those in group P (P < 0.05 or P < 0.01)., Conclusions: The phosgene inhalation can activate the MAPK signaling protein pathway by increasing expressions of p-p38 and p-JNK, which lead to an up-regulation of MMP-9, and this may contribute to the phosgene inhalation-induced lung injury.

Objective: To investigate the role of p38 mitogen-activated protein kinases (MAPKs) in the apoptosis of human bronchial epithelial cells (BEAS-2B) induced by refractory ceramic fibers (RCFs)., Methods: BEAS-2B cells were exposed to 10, 20, 40, 80, and 160 µg/cm(2) RCF1, RCF2, and RCF3 for 24 h, and the cell viability was measured by CCK-8 assay. BEAS-2B cells were exposed to 20, 40, and 100 µg/cm(2) RCF1, RCF2, and RCF3 for 24 h, and the cell apoptosis rate was measured by flow cytometry. BEAS-2B cells were exposed to 40 µg/cm(2) RCF1, RCF2, and RCF3, and the expression levels of phospho-p38 MAPK and caspase-3 were measured by Western blot. In each of the above treatments, the BEAS-2B cells were divided into positive control, p38 inhibitor SB203580 intervention, and normal groups., Results: As the concentration of RCFs rose, the RCF exposure groups showed decreased cell viability and increased cell apoptosis rate. After SB203580 intervention, the intervention groups (all concentrations of asbestos + SB, 20, 40, 80, and 160 µg/cm(2)RCF1+SB, and 40, 80, and 160 µg/cm(2) RCF2 and RCF3+SB) had significantly increased cell viabilities (P < 0.05), and the intervention groups (asbestos + SB and 20, 40, and 100 µg/cm(2) RCF1, RCF2, and RCF3 + SB) had significantly decreased cell apoptosis rates (P < 0.05). Compared with the normal group, the RCF (40 µg/cm(2)) exposure and positive control groups had significantly increased expression of phospho-p38 MAPK (P < 0.05), and the RCF (40 µg/cm(2)) exposure group had significantly increased expression of caspase-3 (P < 0.05). The intervention groups (asbestos + SB and 40 µg/cm(2) RCF1, RCF2, and RCF3 + SB) had significantly decreased expression of caspase-3 after SB203580 intervention., Conclusion: p38 MAPKs play an important role in RCF-induced apoptosis of BEAS-2B cells.

Objective: To elucidate the role of p38 mitogen-activated protein kinase (p38MAPK) in the pathogenesis of ulcerative colitis (UC) and DSS-induced colitis in mice., Methods: (1) 27 Balb/c mice were divided randomly into three groups: DSS-induced colitis group, normal control group and SB203580 treatment group. In DSS-induced colitis group, mice were feed with 5% DSS solution. In SB203580 treatment group, mice were feed with 5%DSS solution for 72 hours, then treated with intraperitoneal injection of SB203580 (1 mg/kg) once daily. Disease activity index (DAI) was record to evaluate the severity of colitis. The mice were executed after 7 days. The levels of TNF-alpha and IL-1beta were measured by ELISA. (2) A total of 54 cases were included in the study. 36 cases were patients with active ulcerative colitis, 18 cases were normal mucosa from 18 colon cancer cases served as control. Effects of SB203580 (a selective p38MAPK inhibitor) on expression of TNF-alpha and IL-1beta in intestinal mucosal biopsy specimens from patients with ulcerative colitis were determined on condition of tissue culture., Results: (1) The DAI scores, the levels of TNF-alpha and IL-1beta in SB203580 group were lower significantly compared with DSS group (P < 0.05), and were increased significantly compared with normal control group (P < 0.05). (2) The levels of TNF-alpha and IL-1beta in intestinal mucosal biopsy specimens in SB203580 treatment group were lower significantly than those in UC group (P < 0.05)., Conclusion: SB203580 can inhibit p38MAPK signal transduction pathway, then reduce the expression of pro-inflammatory cytokine TNF-alpha and IL-1beta.

This study was aimed to explore the effect of NVP-BEZ235, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on proliferation, cell cycle and colony forming capability of CD34(+)CD38(-) human acute myeloid leukemia (AML) KG1a cells. Flow cytometry was used to detect expression of CD34 and CD38 on the surface of human AML KG1a cells; Trypan blue assay was used to analyze the effect of NVP-BEZ235 at various concentrations on proliferation of KG1a cells; flow cytometry was performed to examine the cell cycle of KG1a cells after NVP-BEZ235 treatment; Soft agar colony-forming experiment was used to detect the colony forming ability of KG1a cells treated with NVP-BEZ235 at various concentrations. The results indicated that the percentage of CD34(+)CD38(-) AML KG1a cells was (98.02 ± 0.72)%. NVP-BEZ235 (0.125 - 1 µmol/L) inhibited the proliferation of KG1a cells in a time-and dose-dependent manner (P < 0.05) and the 50% inhibition concentrations (IC50) at 24 h and 48 h were 0.597 µmol/L and 0.102 µmol/L, respectively. KG1a cells were arrested at G0/G1 phase after treating with 0.5 µmol/L NVP-BEZ235 for 24 h, it was significantly higher than that of control group (83.2 ± 3.80)% vs (43.47 ± 9.60)% (P < 0.05). KG1a cells treated with NVP-BEZ235 (0 - 1 µmol/L) for 14 d and 21 d, the number of colony decreased respectively from (375.67 ± 21.46) per 2500 KG1a cells and (706.33 ± 87.31) per 2500 KG1a cells to 0, with statistical significance (P < 0.05). It is concluded that NVP-BEZ235 can inhibit proliferation and colony-forming capability of CD34(+)CD38(-) human AML KG1a cells.

Objective: By analyzing the function and mechanism of nitric oxide in initiating producing lignin peroxidases by phanerochaete chrysosporium, we studied the regulation mechanism triggering the secondary metabolism of white-rot fungi., Methods: Mutant (pcR5305) and wild-type (pc530) strains of phanerochaete chrysosporium were respectively cultured under both the conditions of nitrogen limitation and nitrogen sufficiency. To compare their lignin peroxidases (LiP)-production and nitric oxide(NO)-production kinetics and their different influences on producing LiP after the NO donor Sodium Nitroprusside (SNP) and scavenger cPTIO were respectively added to the nitrogen limitation or sufficiency culture medium to show the function and mechanism of nitric oxide in initiating production of lignin peroxidases by white-rot fungi., Results: Both strains produced nitric oxide (NO) under the two opposite nutritional conditions, but the levels of NO produced were related with the type of strain and the nutritional conditions. Strain pc530 produced NO requiring nutrition depletion and producing of NO was strongly delayed and reduced when it was cultured under nitrogen sufficiency condition. On the contrary, pcR5305 did not require nitrogen depletion to trigger and the levels of NO were higher than that of pc530. The results indicate that LiP content had positive correlation with NO value except the occurrence time of LiP peak value was later than that of NO. The ability of producing LiP was promoted after the NO donor SNP added, but SNP affected more on pc530 than pcR5305 in promoting producing LiP. 15mM cPTIO would greatly repress producing LiP, but could not completely restrain the synthesis of LiP for both strains., Conclusion: By producing NO, Phanerochaete chrysosporium triggers LiP synthesis. However, the evidences do not indicate that NO participates or effect directly in LiP synthesis. It is more likely that NO is reacting as an upstream signal molecule. Besides NO, there are other signal molecules that have a positive effect on NO levels also involving in the regulation producing LiP. The mechanism of the resistance to nutritional repression of pcR5305 in synthesizing lignin degrading peroxidases may be the answer to the different NO production mechanism of pcR5305 from pc530.

Objective: To investigate the regulatory effect of erythropoietin-producing hepatocellular receptor (EphA2) on the expression of VEGF protein, a pro-angiogenic factor, via p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in squamous cell carcinoma of the head and neck(SCCHN) in vitro., Methods: SCCHN Tu686 cells were transfected with EphA2 overexpression vector pEGFP-N1-EphA2. Western blot was used to detect the expression of p38 MAPK and enzyme-linked immunosorbent assay (ELISA) was applied to assay of VEGF. SB203580 as a inhibitor of p38 MAPK signaling pathway was used., Results: The expression of VEGF protein was significantly up-regulated in Tu686 cells transfected with EphA2 overexpression vector (535.31 ± 45.71) pg/ml, when compared with Tu686 cells transfected with empty vector (400.99 ± 33.50) pg/ml and Tu686 cells with no transfection (385.30 ± 33.50) pg/ml (F = 17.091, P < 0.01). The expression of phosphorylated p38 MAPK was obviously increased in Tu686 cells with EphA2 overexpression. SB203580 inhibited the expressions of VEGF and phosphorylated p38 MAPK proteins in Tu686 cells with EphA2 overexpression., Conclusion: EphA2 can regulate the expression of VEGF protein and stimulate p38 MAPK signaling pathway.

Objective: To detect the inhibitory effect of a p38MAPK inhibitor SB203580 in combination with gefitinib on lung adenocarcinoma cell line PC-9 cells and A549 cells, and its cellular and molecular mechanisms of action., Methods: MTT test was used to detect the growth inhibition of PC-9 and A549 cells by SB203580 alone and in combination with gefitinib. Cell apoptosis and cell cycles were determined by flow cytometry. The expressions of p38 and phosphorylated -p38 proteins in the two cell lines were analyzed by immunofluorescence microscopy. The associated protein expression was determined by Western-blot., Results: Compared with the SB203580 group and gefitinib group, the growth inhibition and cell apoptosis of PC-9 cells in the SB203580 + gefitinib group were significantly increased (P < 0.05). The inhibition rate of PC-9 cells of 2 µmol/L SB203580 + 0.01 µmol/L gefitinib group was (46.6 ± 2.4)%, significantly higher than that induced by 0.01 µmol/L gefitinib (12.7 ± 1.5%) (P < 0.05). Immunofluorescence microscopy showed a low expression of phosphorylated-p38 protein in A549 cells and high expression in PC-9 cells. Flow cytometry showed that PC-9 cells in the SB203580 + gefitinib group were (77.35 ± 2.83)% at G0/G1 phase, (3.38 ± 0.84)% at S phase, and (19.56 ± 1.99)% at G2/M phase. Western-blotting showed that compared with the control group, the expression of phosphorylated Akt and phospho-p38 proteins in PC-9 cells of the SB203580 + gefitinib group was almost completely suppressed., Conclusions: The results indicate that the small molecular inhibitor SB203580 can effectively enhance the inhibitory effect of gefitinib on lung adenocarcinoma PC-9 cells. The enhanced inhibitory effect of SB203580 may be correlated with the blockage of p38MAPK signal transduction pathway.

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.

Objective To investigate the effect of zoledronate (Zol) on the cytotoxicity of γδ T cells from peripheral blood mononuclear cells (PBMCs) of osteosarcoma (OS) patients against OS. Methods γδ T cells, derived from PBMCs of patients with primary, recurrent and metastatic OS, were ex vivo expanded by Zol and IL-2. γδ T cell cytotoxicity against target cells was analyzed by a standard lactate dehydrogenase (LDH) release assay. IFN-γ secreted from γδ T cells was determined by ELISA kits. γδ T cells were incubated with different blocking Abs in order to investigate the mechanisms of recognition and killing of OS cells. Nude mice were inoculated with human OS cell line (HOS) by subcutaneous injection to create in vivo OS tumor models, and received normal saline, Zol, γδ T cells, or γδ T cells + Zol by tail vein injection, respectively. The inhibition of tumor growth by the different methods was observed and compared. Results γδ T cells from PBMCs of patients with OS were selectively expanded by Zol+IL-2 in vitro, and showed cytotoxicity against OS. In addition, γδ T cells showed significantly higher cytotoxicity against OS cells after treatment with Zol, and the mechanism of cytotoxicity was largely dependent on TCR pathway and perforin/plasmin pathway, partly on TRAIL pathway and NKG2D pathway. Combination treatment of γδ T cells and Zol had stronger and longer-lasting inhibition on tumor growth in vivo, compared with either drug alone. Conclusion Zol can promote γδ T cells' proliferation from PBMCs of OS patients and enhance γδ T cells' cytotoxicity against OS.

Objective: To find out the more efficient induction method through investigating the expansion efficiencies of HIV-infected patients' Vdelta2 T cells induced by zoledronic acid (Zol) or gammadelta TCR monoclonal antibody (mAb)., Methods: 38 healthy control subjects (HC group) and 65 HIV infected patients (HIV group) were enrolled in this research. Peripheral blood mononuclear cells (PBMCs) of individuals were stimulated by Zol or gammadelta TCR mAb respectively for 14 days at 2.0 x 10(7) cells/ well, and then gammadelta T cells and Vdelta2 subsets frequencies were measured by flow cytometry (FCM) on 0, 7 and 14 day. The absolute numbers of Vdelta2 T cells were calculated and the Vdelta2 T cell expansion efficiencies by these two methods were compared., Results: The absolute numbers and frequencies of Vdelta2 T cell of HIV groups were lower than those of HC groups significantly on 0 day. After 14 days, the frequencies of Vdelta2 T cell of HIV group and HC group were(17.6 +/- 19.8)% and(64.3 +/- 4.5)% respectively, and the expansion indexes of Vdelta2 T cell were 54 +/- 40 and 74 +/- 29 respectively by induction of gammadelta TCR mAb. However, the frequencies of Vgammadelta2 T cell of HIV group and HC group were (69.6 +/- 21.2)% and (97.3 +/- 1.7)% respectively, and the Vgammadelta2 T cell expansion indexes were 538 +/- 11 and 5984 +/- 721 respectively by induction of Zol., Conclusion: Zol could induce the vast expansion of Vgammadelta2 T cells of HIV infected patients. The expansion efficiency by Zol was better than that by the gammadelta TCR mAb.

This study was aimed to investigate whether the inhibition of NHE1 activity and intracellular acidification can reverse resistance of leukemia cells to the imatinib and to explore downstream signal molecule networks of BCR/ABL in the cells of chronic myelocytic leukemia (CML) patients. The mRNA and protein expression of P-glycoprotein (Pgp) and the drug accumulation were assayed after acidifying the primary leukemia cells of patients or K562/DOX and K562/G01 cells. The effects of intracellular acidification of primary leukemia cells on the phosphorylation level changes of ERK1/2 and p38 MAPK were analyzed by Western blot. The results showed that the intracellular concentration of drugs in the advanced patients increased and the sensitivity of K562/DOX and K562/G01 cells to imatinib was enhanced after intracellular acidification or treatment with NHE1 inhibitor cariporide. With downregulation of intracellular pH, the phosphorylation of p38 MAPK decreased in advanced patients and the phosphorylation of ERK1/2 increased within 3 min and then decreased after 30 min. SB203580, the specific inhibitor of p38 MAPK, displayed a synergistic effect with the inhibitor of NHE1 to downregulate the mRNA and protein expression of Pgp. It is concluded that the inhibiton of NHE1 can significantly decrease the protein expression of Pgp in K562/DOX and K562/G01 cells, increase the accumulation of Rhodamine123 and doxorubicin in the cells of advanced patients and enhance the sensitivity of cells to imatinib in which the p38 MAPK signal transduction pathways involves.

In order to effectively control the damage of Frankliniella occidentalis (Pergande), Phaseolus vuglaris was dipped with imidacloprid, phoxim, and emamectin benzoate, respectively to select the resistance populations of F. occidentalis from its susceptible population, and the resistance inheritance and resistance risk were analyzed with the resistance reality heredity. After 32, 32, and 24 generations' selection, the F. occidentalis populations obtained 13.8-fold, 29.4-fold and 39.0-fold resistance to imidacloprid, phoxim, and emamectin benzoate, respectively. The resistance reality heritability to imidacloprid, phoxim, and emamectin benzoate was 0.112, 0.166, and 0.259, respectively. The resistance development rate to emamectin benzoate was the fastest, followed by to phoxim, and to imidacloprid. The higher the resistance levels of the selected populations, the lower the differences between the larva and adult susceptibility to imidacloprid, phoxim, and emamectin benzoate. Stopping selection for 12 continuous generations, the resistance level of the selected resistance populations to imidacloprid, phoxim, and emamectin benzoate had definite decline, but it was difficult to regain the original susceptibility. F. occidentalis had a greater potential to gain high level resistance to imidacloprid, phoxim, and emamectin benzoate. Compared with the resistance of F. occidentalis to phoxim and emamectin benzoate, the resistance to imidacloprid increased slower and decreased faster, and thus, imidacloprid was more appropriate to control F. occidentalis in practice.

The study is to investigate the effect of angiotensin II (Ang II) and its receptor blockers on migration and endothelin-1 (ET-1) expression of rat vascular adventitial fibroblast subpopulations. Vascular adventitial fibroblasts were individually expanded by using cloning rings, and the effects of Ang II on the migration of adventitial fibroblast subpopulations were evaluated by Transwell. Fluorescence quantitative-PCR detected the expression of preproET-1 mRNA induced by Ang II, and its receptor antagonists losartan and PD-123319. The concentration of ET-1 was determined by ELISA. It showed that spindle shaped and epithelioid shaped cells were isolated by using cloning rings, named as spindle cells and round cells. RT-PCR showed that fibroblast subpopulations did not have leukocytes, endothelial cells and smooth muscle cells, namely pure cell lines. Compared with respective control cells, two subpopulations had transferring ability. Ang II significantly improved round cells migration in a concentration-dependent manner, and had no obvious influence on spindle cells migration. Ang II (1 x 10(-8) - 1 x 10(-6) mol x L(-1)) significantly increased the expression of preproET-1 mRNA in round cells (P < 0.01), and had no significant effect on the expression of preproET-1 mRNA in spindle cells. Losartan blocked the expression of preproET-1 mRNA induced by Ang II in round cells, and had no significant effect on the expression of preproET-1 mRNA in spindle cells. The effects of Ang II and ET-1 receptor inhibitors on the release of ET-1 were similar to the expression of preproET-1 mRNA. The results indicate that there are two cell subpopulations: round cells and spindle cells in rat vascular adventitial fibroblasts. Ang II significantly improved cells migration, and increased the expression of ET-1 in round cell subpopulation. It suggested that there may be different migratory mechanisms in two cell subpopulations, and the two subpopulations may play a different role in vascular remodeling and reparative process.

Objective: To establish a model of cigarette smoke exposure to asthmatic rats and glucocorticoid resistance induced by nicotine in alveolar epithelioid cells A549 and study the mechanism for the change of glucocorticoid sensitivity induced by p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580., Methods: Sixty Wistar rats were randomly divided into 4 groups: normal group, asthmatic group, cigarette smoke exposure to asthmatic group and SB203580 group. The mRNA expressions of glucocorticoid receptor (GR), heat shock protein 90 (HSP90) and p38 MAPK were detected by real time-polymerase chain reaction (RT-PCR) while their protein expressions detected by Western blot in vivo. A549 cells were divided averagely into 4 groups: group A: normal; group B: 1 µmol/L dexamethasone (DEX); group C: 1 µmol/L DEX +1 µmol/L nicotine; group D: 1 µmol/L DEX +1 µmol/L nicotine+1 µmol/L SB203580. Immunofluorescence staining was used to study the in vitro colocalization of glucocorticoid receptor (GR) in A549 cells., Results: The mRNA expression of GR was 0.671 ± 0.002 in cigarette smoke exposure to asthmatic group and 0.595 ± 0.061 in SB203580 group (P = 0.065). The protein expression of GR was 0.700 ± 0.033 in cigarette smoke exposure to asthmatic group and 0.628 ± 0.091 in SB203580 group (P = 0.148). The mRNA expression of HSP90 was 0.558 ± 0.009 in cigarette smoke exposure to asthmatic group and 0.377 ± 0.046 in SB203580 group (P = 0.000). The protein expression of HSP90 was 0.507 ± 0.030 in cigarette smoke exposure to asthmatic group and 0.402 ± 0.050 in SB203580 group (P = 0.005). The mRNA expression of p38 MAPK was 0.971 ± 0.012 in cigarette smoke exposure to asthmatic group and 0.278 ± 0.049 in SB203580 group (P = 0.000). The protein expression of p38 MAPK was 0.982 ± 0.038 in cigarette smoke exposure to asthmatic group and 0.338 ± 0.042 in SB203580 group (P = 0.000). The ratio of GR amount within A549 nucleus versus that in cytoplasm was 0.077 ± 0.047 in group C and 0.592 ± 0.249 in group D (P = 0.000)., Conclusion: The mechanism of SB203580 enhancing the corticosteroid sensitivity may be improving nuclear translocation of GR to elevate corticosteroid sensitivity.