Effect Of GCL And Proteins Related To Mitochondrion On The Oxidative Injury Of Glial Cell Line

Objective:Oxidative stress has an important effect in the injury of glial cell line , however the molecular mechanism is not very clear. H2O2 is intermediate products in oxidation metabolism , easily penetrate cellular membrane and generate cytotoxicity. anti-inflammatory agent SAS can induce cell injury at a high dose. GSH is one of very important index in the oxidative stress , participate in protein and DNA biosynthesis , also regulates enzymatic active , moreover antioxygen aspect takes a key effect , its biosynthesis is an important cell protect pathway. GCL is a key enzyme in the GSH synthesis , its activity have a direct impact on GSH levers of cells. in the process cell injure induced by oxidative stress , chloride channel act a elector who will be research penetratingly , in order to consummate the molecular mechanism of cell injury in the oxidative stress. reseach object in this experiment is rat glioma C6 cells , and observe H2O2 or SAS , influence on cell apoptosis , approach GSH synthetic influencein the cell injury that induced by oxidative stress. The expressions of proteins related to mitochondria , Bcl-2,Bax,Cyt-C,CLIC4 , and the effect or mechanism in the injury process , in order to provide new idea and rationale for developing and consummating the molecular mechanism of oxidative stress.Method:Cell culture ; The viability of cells was measured by MTT assay ; Observe cell morphology change in the administration process ; Determine LDH activity;determine GSH levels ; mRNA levels detectting of Bcl-2,Bax,GCLC,GCLM,CLIC4 by RT-PCR ; Protein levels detectting of Bcl-2,Bax,GCLC,GCLM,CLIC4 by Western Blotting.Result:1. MTT assay showed that Both H2O2 or SAS can decease the survival rate of C6 cells in a dosage dependent mode . When H2O2 or SAS and NPPB or NFA joint acted cells , the cell survival rate did not change obviously than H2O2 or SAS group .2. LDH activity displayed that both H2O2 or SAS lead an inceasing of LDH release rate of C6 cells after treated for 6 hours . LDH release rate decreased obviously than H2O2 or SAS group treated alone when H2O2 or SAS and NPPB or NFA joint acted cells .3. GSH level detectting manifested that GSH contents lowerobviously affer H2O2 or SAS treated C6 cells for 24 hours , but there is not conspicuous discrepancy between H2O2 or SAS group treated alone and H2O2 or SAS and NPPB or NFA joint action groups.4. RT-PCR results indicated , Bcl-2 mRNA level decreased , Bax mRNA level changeless and Bax/Bcl-2 increase obviously when H2O2 or SAS treated C6 cells for 24h . CLIC4,GCLC,GCLM mRNA level decrease in H2O2 group , Bcl-2,Bax,CLIC4,GCLC,GCLM mRNA level are not distinction between H2O2 group and H2O2,NPPB or NFA joint action groups .5. Western Blotting results manifested , Bcl-2 level lower , Bax level enhanced , Bax/Bcl-2 ratio increased significantly when H2O2 or SAS treated C6 cells for 24h . CLIC4 level enhanced significantly in H2O2 group , and also higher than H2O2 and NPPB or NFA joint action groups . CLIC4 level is not distinction obviously between SAS and SAS NFA treated C6 cells groups. Mitochondria Cyt-C decreased and cytoplasmic Cyt-C increased in SAS treated group , but 100μmol/L NFA can inhibited the Cyt-C release prosess which induced by SAS .Discussion:H2O2 is an oxidizer which can exchanged membrane easily , meanwhile , also is active oxygen , often was used to making mode of cellular oxidation injury . High dose SAS can induced cell injury too , so this experiment acted H2O2 and SAS as cell injury mode .MTT results showed , H2O2 and SAS induced C6 cell injury by dose dependent mode . Determined LDH results also exposed that H2O2 and SAS induced C6 cell injury . GSH also is an important agent in the process . So , the research provoked oxidative stress is an important cause in the cell injury process .Cell damage induced by oxidative stress is complex. Not only can the short of anti-oxidatant ability lead cell apoptosis , but also ralative with Bcl-2 family gene expression . In our research , as different concerntration H2O2 and SAS treated , Bcl-2 expression down regulated , and Bax expression up regulated , and the cells terated by SAS group , mitochondrial Cyt-C down regulated , cytoplasmic Cyt-C up regulated. Experiment results indicated that when oxidative stress , Bax/Bcl-2 ratio step up , can intensify ROS generation , meanwhile provoke Caspase actived and the mitochondrial Cyt-C release , induce cell death . So, we concluded that anti-apoptotic gene down regulated and the pro-apoptotic gene up regulated is a major mechanism of H2O2 and SAS induce cell injury .Chloride channel have close relations with the regulation of cell function , and also can regulate cell inner pH . But the effect of chloride channel in cell apoptosis is not very clear . This research indicated chloride channel blockers can not inhibit cell death induced by Bcl-2,Bax changing and GSH exhausted , GSH exhaust is important in the oxidative damage , cell antioxidant abilityinsufficient directly influence on DNA , Protein , plasma membrane function , and result in cell death . Chloride channel blockers lower LDH release rate , especially depress mitochondria Cyt-C release . Experimental results indicated , To some extent , chloride channel blockers can protect cell membrane .CLIC4 acts as a cell intracellular chloride channel who participates regulations of the cytoplasmic membrane and mitochondria membrane stability also can maintains membrane potential . This research indicated that H2O2 can induce CLIC4 strengthened but chloride channel blocking agent make CLIC4 approach control group . Cell death or apoptosis is participated by lots of signals . Chloride channel blocking agent inhibited chloride channel function and activity , regulated stability of cytoplasmic membrane and mitochondria membrane , meanwhile , repressed mitochondria Cyt-C release , but it could not deteriorate cell death induced by Bcl-2,Bax changed and GSH exhausted .Conclusion:H2O2 or SAS induced injury of C6 cells which mechanism as follow. Firstly , inhibited the synthetase of GSH , GCL activity , that resulted in GCLC,GCLM level decreased , GSH synthesis was inhibited , meanwhile cellular oxidation consume GSH directly lead deficiency of anti-oxidant ability of cells . Secondly , oxidative stress case Bcl-2 deceased , Bax increased and mitochondria Cyt-Creleased . Resulted in cell apoptosis. Thirdly , with chloride channel changing in oxidative stress process , particularly CLIC4 level . Many signals participate in the cell death or cell apoptosis process , chloride channel blocker can inhibited chloride channel function and activity , regulated cellular membrane and mitochondria membrane functions, meanwhile , inhibited mitochondria Cyt-C release , but it could not inhibited cell apoptosis induced by Bcl-2,Bax changed and GSH exhausted . This research provided a new idea and rationale for developing and consummating molecular mechanism of oxidative stress.