| Hepatic fibrosis,characterized by accumulation of excessive extracellular matrix(ECM),is a pathophysiological process response to repeated damage caused by various pathogenic factors.Part of them will develop into cirrhosis or liver cancer which account for poor prognosis and high mortality.In normal liver quiescent HSCs serve as a major storage of vitamin A.During liver injury,quiescent HSCs lost vitamin A lipid droplets and convert to activated phenotype with strengthened ability of proliferation,migration and ECM production.This process plays a key role during liver fibrosis formation.Recent studies demonstrated that liver fibrosis can be reversed after removal of pathogenic factors.Increased degradation and decreased secretion of ECM are the necessary for the reversal of liver fibrosis.Activated HSCs are the main source of collagens.Two rodent studies demonstrated that approximately 50% of activated HSCs undergo apoptosis during fibrosis reversal,and the rest revert to a quiescent phenotype.Therefore,induction of apoptosis and suppression of proliferation and secretion can reverse fibrosis,ideally targeting activated HSCs.Due to lack of effective treatment of hepatic fibrosis,the research on the mechanism of apoptosis of activated HSCs is of great significance.Cell apoptosis includes extrinsic apoptotic pathway and intrinsic apoptotic pathway.Intrinsic apoptotic pathway,also called mitochondrial apoptotic pathway,is marked by one central event — mitochondrial outer membrane permeabilization(MOMP).BH3-only proteins generally stimulate MOMP by inducing the oligomerization of BCL-2-associated X protein(BAX)and/or BCL-2 antagonist or killer(BAK)in the outer mitochondrial membrane,thereby forming supramolecular channels that mediate the liberation of soluble proteins from the mitochondrial inter membrane space,such as cytochrome c which can trigger intrinsic apoptosis.Therefore,mitochondrial homeostasis plays a vital role in modulating cell apoptosis.Liver is a high energy consuming organ.Reactive oxygen species(ROS),produced by mitochondria during the energy generation,will damage the mitochondrial proteins,lipids and nucleic acids.The damage of functional proteins will increase the ROS production and aggravate mitochondrial damage.The cells will remove dysfunctional mitochondria through the selectively autophagy known as mitophagy,when the protein and DNA damage accumulated beyond its own repair capacity.Although it is believed that mitophagy coexists with cell apoptosis,there are no clear evidence to prove the relationship between them.The relationship between mitochondrial clearance in HSCs and hepatic fibrosis has not been reported.At present,views consider that Bcl-2 family protein involved in both mitophagy and cell apoptosis.The family encompasses six anti-apoptotic proteins(Bcl-2,Bcl-B,Bcl-w,Bcl-x L,Bfl-1 and Mcl-1),many pro-apoptotic proteins,BH3 domain-only proteins and the effector proteins(Bax and Bak).The anti-apoptotic Bcl-2 proteins generally localize to the mitochondrial membrane.Here they regulate MOMP and inhibit intrinsic apoptotic execution through blocking Bax/Bak oligomerization.The Bcl-2 can also block the formation of autophagosomes by binding to the autophagic factor beclin-1.The interaction between Bcl-2 and Beclin-1 is considered as a key of autophagy.Bcl-B(BCL-2-like protein-10),with typical structural characteristics of the family,is the most recently discovered and least studied anti-apoptotic Bcl-2 family protein.Bcl-B protein is widely expressed in tissues of humans and selectively binds Bax to suppress apoptosis in HEK293 T cells.Its physiological function is largely unknown.There are no literature indicate the role of Bcl-B in mitochondrial clearance and the expression and function during hepatic fibrosis formation,reversal or HSC apoptosis.This study is aim to observe the changes of apoptotic-related protein BCL-B expression and mitochondrial content during HSC apoptosis and hepatic fibrosis reversion,to elucidate the effect of Bcl-B on HSC apoptosis and its possible molecular mechanism.The experiment contains three parts,shown as follows:Part 1 During mice liver fibrosis reversal,the mitochondrial content and the expression of BCL-B reduced in primary HSCs.Objective: To establish liver fibrosis reversal mice model and observe the changes of mitochondrial content and BCL-B expression.Methods: Liver fibrosis mice model,which was established by intraperitoneal injection of carbon tetrachloride(CCl4),spontaneous reversed after stopping intervention.Mice were injected intraperitoneally with CCl4 twice a week over a period 6 weeks,olive oil(vehicle)was utilized as a control.Livers were excised 24 hours,1 week,2 weeks and 4 weeks after the final injection of CCl4.Livers were embedded in paraffin and sectioned.Sections were used for hematoxylin and eosin(H&E)and Masson staining to measure pathological changes.The serum ALT and AST were measured by Reitman-Frankel methods.Furthermore,the expression of collagen I and ?-SMA were examined by immunohistochemistry(IHC)and western blot.Primary mice HSCs were isolated by orthotopic perfusion digestion followed by density gradient centrifugation.The quiescent HSCs were tested immediately after isolated by fluorescence microscope and then cultured for 3 days.The expression of BCL-B and TOM20 protein were detected by western blot.Primary HSCs apoptosis was investigated by TUNEL and flow cytometry(FCM).Meanwhile,PicoGreen reflected the total amount of Mitochondrial DNA(mt DNA)in primary HSCs.Results:1 Liver fibrosis reversal mice model was established successfully.In order to establish the liver fibrosis reversal model,CCl4 intraperitoneal injection was used to induce liver fibrosis,and the fibrosis spontaneous reversed after stopping injection.The experiment was divided into 5 groups: control group(oil),fibrosis group(6w),recovery 1 week group(R1w),recovery 2 weeks group(R2w),recovery 4 weeks group(R4w).The livers were soft,smooth and brightly-colored in oil group mice.The livers were shrinking,firm and granular in 6w group,while restored smooth and soft gradually with prolonged recovery time.H&E and Masson staining results showed,neatly arranged liver plate,complete hepatic lobules and less collagen in oil group,and sever distortion of architecture,hemorrhagic necrosis,hepatocytes vacuolar degeneration,inflammatory cells and collagen deposition in 6w group.With prolonged of CCl4 withdrawal time,hepatic steatosis and collagen deposition decreased,followed by liver necrosis and liver plate gradually restored in R1 w,R2w and R4 w.As shown in IHC,collagen I and ?-SMA expression was significantly increased in 6w group,while little was detected in oil group.Compared with 6w group,there were no obviously changes in R1 w group,while obviously decreased in R4 w group.Western blot results showed an increase of collagen I and ?-SMA expression in 6w group compared with that in oil group,whereas decreased gradually in fibrosis reversed liver,and reached the lowest level in R4 w group.The detection of serum ALT and AST showed a high level in 6w group and decreased with the prolongation of withdrawal time.2 Primary mice HSCs were isolated successfully.The primary HSCs were isolated by sequential digestion of the liver with pronase and type IV collagenase,followed by single step density gradient centrifugation.HSCs show blue fluorescence under the inverted fluorescence microscope(L=328 nm).The primary HSCs is round and with bigger volume and high refraction under the microscope.Cell viability and cell purity were greater than 80%,with a yield ranging from 1×106 to 2×106 HSCs/mouse.The cells were all adherent after inoculation in plastic culture flask for 24 h.In this study,the primary HSCs cultured for 3 days were used for the experiment.At this time,the cells were still not activated,and were close to the state in vivo.3 HSCs apoptosis was increased during the process of fibrosis reversal.The results of TUNEL showed that the proportion of apoptosis HSCs was higher in oil group,and less was in apoptotic state in 6w group.During liver fibrosis reversal stage,the HSCs apoptosis significantly increased,but was no correlation with reversed time.The HSCs were stained with Annexin-V/PI,followed by flow cytometry to detect cell apoptosis.The results showed a lower apoptotic rate in 6w group compared with oil group.Addition of HSCs apoptosis was observed after CCl4 discontinuation.In R2 w group the apoptotic rate was the most significant.The major expression was early apoptosis in R2 w group,while late apoptosis in R4 w group.There was no significant correlation between total rate of HSCs apoptosis and withdrawal time.These results indicate that the HSCs apoptosis increased during the hepatic fibrosis reversal.4 The mitochondrial content and BCL-B expression of HSCs were down-regulated during the process of fibrosis reversal.Protein was extracted from primary HSCs and the mitochondrial membrane protein TOM20 was detected by western blot.Results showed that the expression of TOM20 was obviously increased in 6w group and decreased gradually with the withdrawal time from CCl4 prolonged.In R4 w group,the TOM20 expression is the lowest,but still higher than oil group.These results indicated that the mitochondrial content is abundant in HSCs in fibrotic liver,while increased mitochondrial clearence resulted in a lower content of mitochondria in HSCs during liver fibrosis reversal.The content of mtDNA in primary HSCs was observed by PicoGreen staining.The results showed higher brightness and agglomerate fluorescence in HSCs in 6w group compared with oil group,indicating that the mitochondria increased and fused.The fluorescence brightness decreased and volume reduced significantly in R1 w.There was no change in R2 w compared with R1 w.There was no significant change in fluorescence intensity in the R4 w group,but the fluorescence mass disappeared and the fluorescence in the cytoplasm was uniform.These results indicated that mitochondria number increased and fused in HSCs during liver fibrosis formation,but mitochondria number decreased and homogeneously distributed in the cytoplasm in HSCs during liver fibrosis reversal.Western blot results showed a lower expression of BCL-B in oil group and a significantly increased expression 6w group.As the withdrawal from CCl4 and fibrosis reversal,the expression of BCL-B decreased and tended to oil group,indicating a correlation between BCL-B expression and primary HSCs apoptosis.Conclusions: During liver fibrosis reversal,HSCs apoptosis and mitochondrial clearance increased,meanwhile,BCL-B expression down-regulated.Part 2 The effect of BCL-B on HSCs apoptosis and mitochondrial clearenceObjective: To induce HSC-LX2 apoptosis and verify the results obtained in primary HSCs.Inhibit mitochondrial clearence and investigate the effect of mitochondrial clearence on HSCs apoptosis.Knockdown and overexpress BCL-B in LX2 and observe its effect on LX2 apoptosis and mitochondrial clearence.Methods: To establish LX2 apoptosis cell model,mitophagy inhibitor CsA was used to inhibit mitochondrial clearence.Design and synthesize BCL-B targeted siRNA and adenovirus to knockdown and overexpress BCL-B in LX2.To indicate the level of cell apoptosis,mitochondrial clearence and BCL-B expression,western blot was used for detecting expression of BCL-B,mitochondrial protein,apoptotic-related protein and activation-related proteins,PicoGreen for mtDNA,TUNEL for cell apoptosis.Results:1 Induced apoptosis of HSCs accompanied with increased mitochondrial clearence and decreased expression of BCL-B.In order to simulate liver fibrosis reversal at the cellular level,the gliotoxin(GTX)was used to induce LX2 apoptosis.(1)The TUNEL assay showed a higher rate of apoptosis in GTX group compared with control group.The results of western blot showed that,compared with control group,the expression of apoptotic-related protein cleaved-caspase3 and activated intrinsic apoptotic-related protein cleaved-caspase9 were significantly increased,but the activation-related proteins collagen I and ?-SMA decreased in GTX group,indicating that the GTX induced LX2 apoptosis successfully.(2)The expression of mitochondrial protein TOM20 was significantly reduced in GTX group.PicoGreen showed that the green fluorescence was high intensity and granulated in control group,while the fluorescence intensity decreased and scattered after apoptosis induced.These results indicated that mitochondrial clearence increased and mitochondria content decreased in apoptotic LX2.(3)The western blot showed that the expression of BCL-B reduced after GTX treatment.2 HSCs apoptosis was inhibited and BCL-B expression up-regulated by blocking mitochondrial clearence.(1)Western blot analysis showed that the expression of TOM20 was increased after mitophagy inhibitor CsA treatment.PicoGreen results showed that the intensity and area of green fluorescence increased in GTX + CsA group indicating that the mitochondrial clearence was suppressed.(2)TUNEL results showed that there was a lower apoptosis rate in GTX + CsA compared with GTX group.Western blot showed a decreased expression of cleaved-caspase9 and cleaved-caspase3 and increased expression of collagen I and ?-SMA after CsA treatment.These results indicated that the LX2 apoptosis reduced after mitochondrial clearence inhibition.(3)Western blot results showed that BCL-B was significantly increased in GTX + CsA group than in GTX group,indicating that the inhibition of mitochondrial clearence up-regulated BCL-B expression.3 Knockdown of BCL-B promoted HSCs apoptosis and mitochondrial clearence.The BCL-B targeted si RNA(siBCL-B)and control siRNA(siCon)were transfected into LX2,and then extracted protein to detect.(1)Western blot test showed that the expression of BCL-B protein in siBCL-B group was reduced by 79% compared with siCon group,indicating that BCL-B expression was knockdown successfully.(2)TUNEL result showed a higher apoptosis rate in siBCL-B group compared with siCon group.Western blot showed that,in siBCL-B group,the expression levels of cleaved-caspase3 and cleaved-caspase9 were significantly higher than those in siCon group,while the expression of collagen I and ?-SMA was lower than that in siCon group.These results indicating that knockdown of BCL-B increased cell apoptosis.(3)Western blot analysis showed that the expression of TOM20 was obviously reduced in siBCL-B group than that in siCon group.PicoGreen showed that the intensity and area of fluorescence reduced in si BCL-B group indicating decreased mtDNA content.These results indicated that the mitochondrial clearence increased and mitochondria content reduced after BCL-B knockdown.4 Overexpression of BCL-B inhibited LX2 apoptosis and mitochondrial clearence.BCL-B targeted adenovirus(adBCL-B)and control adenovirus(adCon)were transfected into LX2 with infection efficiency up to 90%.(1)Western blot showed that BCL-B expression in adBCL-B group was about 4.7 times higher than that in adCon group,indicating that BCL-B could successfully overexpress through adenovirus transfection.(2)The results of TUNEL staining showed that the cell apoptosis rate was significantly decreased in adBCL-B group compared with adCon group.The western blot results showed that the expression of cleaved-caspase9 and cleaved-caspase3 in adBCL-B group was lower than that in adCon group,while the expression of collagen I and ?-SMA was higher than that in adCon group.These results indicated that the overexpression of BCL-B inhibited LX2 apoptosis and promoted its activation.(3)Overexpression of BCL-B inhibited mitochondrial clearence.Western blot analysis showed that the expression of TOM20 protein was up-regulated in adBCL-B group compared with ad Con group.The results indicated that mitochondrial clearence is inhibited and mitochondria content increased in BCL-B overexpressed LX2.Conclusion: Inhibition of mitochondrial clearence leads to a decreased apoptosis.BCL-B can inhibit LX2 apoptosis and mitochondrial clearence.Part 3 BCL-B inhibits HSC apoptosis by inhibiting cell mitochondrial clearenceObjective: To investigate whether BCL-B regulates HSC apoptosis by inhibiting mitochondrial clearance,and explore the molecular mechanism.Methods: Mitophagy inhibitor was used in LX2 that BCL-B was knockdown.mitochondrial clearence inhibition was demonstrated by Western blot detection for mitochondrial protein expression and PicoGreen for mt NDA content.Western blot was used for detecting apoptosis-related proteins expression;TUNEL for apoptosis rate.BCL-B was knockdown or overexpression to detect the changes of p-Parkin expression.The combination and colocalization of BCL-B and p-Parkin in LX2 cells was detected by Co-immunoprecipitation(Co-IP)and double-labeled fluorescent immunostaining.Results:1 Inhibition of mitochondrial clearence can block the effect of BCL-B knockdown on LX2.In order to determine whether BCL-B suppress LX2 apoptosis by inhibiting mitochondrial clearence,the mitphagy was blocked in LX2 after BCL-B knockdown.(1)CsA successfully inhibited mitochondrial clearence.Western blot analysis showed that the expression of TOM20 was increased in si BCL-B+CsA group compared with siBCL-B group.PicoGreen showed lighter and gathered fluorescence in siBCL-B+CsA group,indicating that the mitochondrial clearence was inhibited by CsA.(2)TUNEL showed that the rate of LX2 apoptosis was obviously reduced in siBCL-B + CsA group compared with siBCL-B group.The expression of cleaved-caspase3 and cleaved-caspase9 in siBCL-B + Cs A group was significantly lower than that in siBCL-B group,while the expression of collagen I and ?-SMA was higher than that in siBCL-B group.These results indicated that inhibition of mitochondrial clearence reduced LX2 apoptosis caused by BCL-B knockdown.2 BCL-B down-regulated the level of p-Parkin.The phosphorylation of Parkin is the key to initiate mitophagy.Western blot analysis showed that the expression of p-Parkin was increased by knockdown of BCL-B and was significantly decreased by overexpression of BCL-B,indicating that BCL-B can inhibit the activation of mitophagy key protein Parkin.3 BCL-B interacts with p-Parkin in LX2.(1)Co-IP was conduced by immunoprecipitation with BCL-B or p-Parkin in experimental group,Ig G in control group.BCL-B and p-Parkin was detected respectively in the p-Parkin group and BCL-B group,indicating that BCL-B combined with p-Parkin in LX2 cell.(2)in double-labeled fluorescent immunostaining,BCL-B,p-Parkin and nuclei were marked by red fluorescence,green fluorescence and blue fluorescence,respectively.Results revealed that BCL-B marked by red fluorescence was diffusely distributed in cytoplasm,p-Parkin marked by green fluorescence was mainly located in cytoplasm especially around nuclear,and there are partial overlaps shown as yellow fluorescence.Above results suggested that there is colocalization between these two proteins.Conclusions: BCL-B suppresses LX2 apoptosis by inhibiting mitochondrial clearence.BCL-B interacts with p-Parkin in HSCs.BCL-B may inhibit phosphorylation of Parkin by binding to p-Parkin,thereby inhibiting mitochondrial clearence. |
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