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Objective To study the effect and mechanisms of chloride channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) on thansforming growth factor β1 (TGF-β1) induced human conjunctival fibroblasts (HConF) fibrosis.Methods Cell counting kit (CCK-8) was used to screen out the optimal TGF-β1 treatment time and the optimal NPPB concentration.The cells were divided into control group,TGF-β1 treatment group and TGF-β1+NPPB group.Cell proliferation and cell cycle were detected by CCK-8 and flow cytometer,respectively.Cell migration ability were observed by scratch and transwell migration assays.Western blot and Real time-PCR were used to detect the expression of collagen Ⅰ (COL-Ⅰ),fibronectin (FN) and α-smooth muscle actin (α-SMA).The phosphorylation level of PI3K and Akt were measured by Western blot.Results TGF-β1 promotes cell proliferation in a time-dependent manner.There was no statistically significant difference in A values between 48 hours and 72 hours after TGF-β1 treatment (P =0.064).Forty-eight hours was selected as the most appropriate time for TGF-β1 treatment.NPPB inhibited HConF cell proliferation in a concentration-dependent manner.Compared with the control group,the proliferation A values of cells in the 50 mol/L and 100 mol/L NPPB groups were significantly reduced (P =0.020,0.000),and 100 mol/L was selected as the optimal concentration of NPPB.The cell proliferation A value,migration area and migration cell number of TGF-β1 +NPPB group were significantly lower than those of TGF-β1 treatment group (all at P＜0.05).Compared with the control group and TGF-β1 +NPPB group,the proportion of G1 phase cells in the TGF-β1 treatment group was reduced,and the proportion of cells in the S phase and G2/M phase were increased,with statistically significant differences between them (all at P ＜ 0.05).The protein and mRNA expression of α-SMA,COL-Ⅰ and FN in the TGF-β1 treatment group were higher than those in the control group and TGF-β1+NPPB group,with statistically significant differences between them(all at P＜0.05);the ratios of p-PI3K/PI3K and p-Akt/Akt in the TGF-β1 treatment group were significantly higher than those in the control group and TGF-β1 +NPPB group,with statistically significant differences between them (all at P＜0.05).Conclusions NPPB may inhibit TGF-β1 induced HConF fibrosis process by inhibiting phosphorylation of PI3K and Akt.
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Aim To clarify the effect of Borneol on the chloride channels and cell volume in poorly differentia-ted nasopharyngeal carcinoma CNE-2Z cells.Methods The technique of whole-cell patch clamp was used to detect the chloride currents and analyze the character-istics of the currents in CNE-2Z cells.The volume changes caused by Borneol were measured by the meth-od of time-lapse live cell imaging.Results The chlo-ride currents were induced by extracellular application of Borneol (20 μmol·L -1 )isotonic condition.The currents showed a characteristic of outward rectification and did not show voltage-dependent or time-dependent inactivation.The reversal potential of the currents was close to the CI-equilibrium potential. The currents were inhibited by the chloride channel blocker tamox-ifen.The currents were also inhibited by 47% hyper-tonic solution.Borneol decreased the cell volume by 9.4% in 30 min.Tamoxifen completely inhibited the Borneol-induced cell volume decrease.Conclusion Borneol can activate volume-sensitive chloride channels and induce volume decrease in CNE-2Z cells.Chloride channels play a pivotal role in the process of volume decrease caused by Borneol.
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Aim To investigate the effects of chloride channel blockers on the apoptosis of RIN-m? cells of pancreatic islet induced by H2O2.Methods The apoptotic model was made by H2O2 exposed for six hours with a concentration of 500 ?mol?L-1.The chloride channel blockers:DIDS,NPPB and NFA were administered to pretreat the samples respectively.The cell viability,morphological changes,and apoptosis rate were observed.Results Chloride channel blockers alone have no marked effects on the cell viability of RIN-m? cell.However,they elevated the cell viability of RIN-m?cell disposed of by H2O2.Compared to H2O2 group,the groups of DIDS +H2O2,NPPB+ H2O2 and NFA+H2O2 have significant difference in cell viability and apoptosis rate(P
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Aim To observe the protective effects of SITS and DIDS,two kinds of chloride channel blockers,on hippocampal neuronal damage induced by NO in culture.Methods The cultures were divided into three groups:control group,NO treatment group,NO treatment plus chloride channel blocker group. The cultures were detected with the methods of morphological stain (Hoechst 33258),and the apoptotic neurons and neuronal viabilities were observed through MTT quantitative analysis. The activated caspase-3 was analyzed with western blot.Results There were significant protective effects of SITS and DIDS on neuronal damage with dose-dependence.Conclusions Chloride channel blockers have some protective effects against neuronal injury induced by NO.
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Aim To investigate the effects of chloride on the injury of the ventricular myocytes from anoxia-reoxygenation. Methods Under the conditions of anoxia-reoxygenation injury, primary cultured rat ventricular myocytes were treated with 4-acetanide- 4′-isothiocya- natostilbene -2,2′-disulfonic acid (SITS),4,4′,-dii sothiocya-nostilbene-2,2′-disulfonicacid (DIDS) or replaced Cl~- with equimolar gluconate, respectively. Then the cell viability and the contents of Lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD)and glutathione peroxidase (GSH-Px) in the media were measured. Results The release of LDH and MDA was significantly increased in the anoxia-reoxygenation group, while the cell viability and the activity of SOD, GSH-Px decreased significantly compared with those in the control group. In both Cl~--free+ A-R group and SITS+A-R group, LDH and MDA release was noticeably lower than those of the A-R group, while the cell viability and the activity of SOD, GSH-Px significantly increased compared with those in the anoxia-reoxygenation group. But the cell viability and the contents of LDH, MDA, SOD and GSH-Px in the DIDS+A-R group had no significant change compared with those in the anoxia-reoxygenation group.Conclusion Cl~- plays an important role in anoxia reoxygenation injury. SITS provides effective protection to the cardiac myocyte subjected to anoxia reoxygenation injury, while DIDS cannot provide such protection.