The Effect Of Bicyclol On Proliferation, Apoptosis And The Expression Of Caspase-3,Bcl-2Protein Of Rat Hepatic Stellate Cell In Vitro

Chronic liver disease is a kind of serious harm to human health worldwide, Hepatic fibrosis is middle and key element of this process, Though the pathogenesis of diverse hepatic diseases is different,The ultimate approach to liver fibrosis is similar.It is well known that the critical event of liver fibrosis is that hepatic stellate cells （HSCs） become active, The increasing of production and secretion of extracellular matrix （ECM） and the decreasing of degradation of them result in the accumulation of over ECM in the live.So inducing the apoptosis of the activated hepatic stellate cells can reverse the process of hepatic fibrosis.Bicyclol is the Chinese academy of medical sciences institute of drug resistance hepatitis drug development,The chemical name for a₁,5a carbonyl group, Which was a2,3,2,3pair of methylene2oxygen radicals4,4Dimeth yl oxygen radicals.Research shows, many experiments bicylol on acute and chronic liver injury were significantly protective effect. Clinical treatments for chronic hepatitis B, C viral hepatitis have achieved good effec.Bicyclol still can make liver fibrosis rats procollage peptide （PIIIP） level decreased significantly,Albumin levels significantly increased.Therefore, The purpose of our present study was to investigate the effect of bicyclol on proliferation, Apoptosis and the expression of bcl-2,caspase-3protein of hepatic stellate cells and the possible mechanisim.Objective:The experiment mainly through different concentration of bicylol intervention hepatic stellate cells in vitro to investigate the effect on proliferation, apoptosis, and the expression of caspase-3、bcl-2protein.Methods:1、Rat HSC was cultured in vitro and divided into4groups,â‘ blank control group;â‘¡bicylol group（0.25mmol/）l;â‘¢bicylol group（0.5mmol/l）;â‘£bicylol group（lmmol/1）,⑤without DMSO group.2、Proliferation effects of bicylol on activated hepatic stellate cell were measured by MTT test;3、With Hoechst3325staining by fluorescence microscope to detect cell apoptosis rate; Cell apoptosis rate and cell cycle were analyzed using Annexin â…¤-FITC/PI double-labled kit and propduim kit by flow cytometry.4、The protein expression levels of capase-3in hepatic stellate cells were measured by immuno-cytochemcal staining.5、The protein expression levels of bcl-2in hepatic stellate cells were measured by Western blotting.Results:1、The effect of bicyclol on HSCs proliferation:After different concentrations bicyclol（0.25mmol/、0.5mmol/l、1mmol/l） for12h, compared control group（3.056±0.110）,the overall mean of A490value of concentration bicyclol groups （2.365±0.210、1.480±0.2460.797±0.120） had significantly difference（P<0.05）; after different concentrations bicyclol for24h, compared control group（2.796±0.098）,the overall mean of A490value of concentration bicyclol groups （1.960±0.152、1.160±0.143、0.655±0.064） had significantly difference（P<0.05）,after different concentrations bicyclol for48h,compared control group（3.279±0.172）, the overall mean of A490value of concentration bicyclol groups（2.599±0.180、1.672±0.117、0.895±0.105） had significantly difference（P<0.05）;no significant difference was found between the A490value of the control group and the group without DMSO （P>0.05）.After0.25mmol/l bicyclol treatment for12h,24h,36h, A490value（2.365±0.210、1.960±0.152、2.599±0.180）,The difference of overall mean of A490value of each group was statistically significant （p<0.05）,After0.5mmol/l bicyclol treatment for12h,24h,36h, A490value（1.480±0.246、1.160±0.143、1.672±0.117）,The difference of overall mean of A490value of each group was statistically significant （p<0.05）,After lmmol/1bicyclol treatment for12h,24h,36h, A490value（0.797±0.120、0.655±0.0640.895±0.105）,The difference of overall mean of A490value of each group was statistically significant （p<0.05）.2、The effect of bicyclol on HSC apoptosis:â‘ The effect of different concentrations bicyclol （0.25mmol/1,0.5mmol/1, lmmol/l）on HSCs for24h,We found that the condensed nuclei and the formation of apoptotic body in HSCs after different concentrations bicyclol treatment, Staining with Hoechest33342by fluorescence microscopy. The results suggested that bicyclol had effect on the rate of HSCs apoptosis. Compared control group （3.963%±1.242%） the overall mean of apoptosis rates of different concentrations bicyclol group （7.763%±1.476%11.267%±1.340%、23.256%±1.936%） had significantly difference（P<0.05）;except the control group between without DMSO group have no difference （P>0.05）.â‘¡Different concentrations bicyclol effection on cell cycle and apoptosis rate of HSCs by flow cytometry detecting:The cell cycle results indicated that an increase in G2/M phase cells and a decrease in S phase cells after bicyclol treatment for24h, with dose-dependent a decrease in S phase cells （31.12%±0.83%、29.29%±1.07%、28.87%±1.10%vs43.32%±1.00%,P<0.05） and an increase in G2/M phase cells （20.10%±0.76%、23.12%±0.67%、33.17%±0.72%vs15.79%±0.68%,P<0.05）,Bicyclol induced cell cycle arrest in G2/M phase. Meanwhile, With the same concentration groups and the same time, the apoptotic rates was also increasing in a dose-dependent maner.The overall mean of the apoptotic rates with the different concentrations bicyclol （7.12%±0.78%、14.76%±1.04%,24.37%±1.05%）have significantly elevated, Which have significantly difference compared with the control group （4.41%±0.91%）,（P<0.05）; no significant difference was found between the control group and the group without DMSO （P>0.05）.3, After treated with different concentrations of bicyclol （0.25mmol/1,0.5mmol/l,1mmol/l）for24h, The expressional levels of caspase-3in hepatic stellate cells with immunocytochemisty:Caspase-3was dispersively expressed in the cytoplasm of hepatic stellate cells,While with the raising of concentrations of bicyclol,Caspase-3were significantly upper than that in control group,Compared control group（0.306±0.024）,Different concentrations bicyclol group（0.362±0.017、0.439±0.037、0.542±0.021）had significantly difference（P<0.05）; no significant difference was found between the control group and the group without DMSO （P>0.05）.4、After treated with bicyclol24h, The expressional levels of bcl-2in hepatic stellate cells were significantly lower than those in control group,Compared control group（0.657±0.028） the overall mean of the expressional levels of different concentrations bicyclol group （0.475±0.037）、（0.381±0.035）、（0.238±0.023） had significantly difference（P<0.05）; no significant difference was found between the control group and the group without DMSO （P>0.05）.Conclusion:1、Cell cultured in vitro studies have shown that, Bicyclol can inhibiteHSCs proliferation, The inhibitory effect on HSCs was significantly in dose-interrelated manner.2、Bicyclol could induced the activated HSCs apoptosis.3、Bicyclol can induce apoptosis in HSC-T6and may be related with some apoptosis-related gene such as bcl-2, and caspase-3.