Asiatic Acid Ameliorates Acute Hepatic Injury By Reducing Endoplasmic Reticulum Stress And Triggering Hepatocyte Autophagy

Objective:To investigate the effects of asiatic acid from potentilla chinensis(AAPC)on lipopolysaccharide/d-galn(LPS/D-GalN)-induced acute liver injury(ALI)and its regulatory mechanism based on the cross-talk relationship among er stress(ERS),autophagy and apoptosis.Methods:1.Preparation of ALI mouse modelA total of sixty 6-8 week male C57BL/6 mice were randomly divided into normal control group(saline),AAPC control group(8 mg/kg),model group(saline)and AAPC-treated groups(4,8 mg/kg),continuous oral administration for 15 days,once a day.One hour after the last administration,except the normal group and the AAPC negative control group,the mice in other group were injected intraperitoneally with LPS(50 μg/kg)/D-GalN(800 mg/kg)to establish the model of ALI in mice.The mice were sacrificed by cervical dislocation 6 h after injection.Blood samples and liver samples were taken for further examination.2.The effect of AAPC on hepatic injury in ALI miceThe activities of serum ALT,AST,ALB and GLB were measured by an automatic biochemical analyzer.In addition,the pathological changes and apoptosis of hepatocytes were detected by Hermatoxylin-eosin(H&E)and TUNEL staining.3.Establishment of ERS model in LO2 cellsCultured LO2 cells with 1 μmol/L of thapsigargin for 3,6,12,24 and 48 h.Western blot was used to detect the expression of ERS marker protein GRP78 and autophagy marker proteins LC3II/I,P62,and to screen the best time to prepare ERS cell model.4.The effect of AAPC on ERS-induced damage to LO2 cellsThe concentration of AAPC was set to 10 μmol/L,20 μmol/L,and 40μmol/L,according to the previous MTT results of our research group.Then cells were divided into 7 groups: normal control group,model group(1 μmol/L),3-MA group(5 mmol/L),4-PBA group(1 μmol/L),and AAPC-treated groups(10,20 and 40 μmol/L).The content of lactate dehydrogenase(LDH)was detected by the kit.The Annexin V-FITC/PI apoptosis assay kit was used to detect the effect of AAPC on apoptosis.The effects of apoptosis-related proteins Bax and Bcl-2 were detected by Western blot.5.Effect of AAPC on the ERS signaling pathway protein in vivo and in vitroThe expressions of GRP78,p-PERK/PERK,p-IRE1/IRE1 and ATF6 in liver tissues were detected by Western blot.Meanwhile,the expressions of GRP78,p-PERK/PERK,p-e IF2α/e IF2α,ATF4,CHOP,p-IRE1/IRE1,XBP-1,p-JNK/JNK and ATF6 in LO2 cells were also detected.6.The effect of AAPC on autophagy in vivo and in vitroWestern blot was used to detect the expression of LC3II/I,Beclin1,Atg5 and Atg7 proteins in liver tissues;the formation of autophagosomes and autolysosomes in LO2 cells were observed by transmission electron microscopy.Results:1.AAPC ameliorated liver injury in ALI miceThe results of the automatic biochemical analyzer showed that compared with the model group,AAPC pretreatment significantly reduced serum ALT and AST levels(p<0.05).In addition,the H&E and TUNEL staining results showed that AAPC pretreatment could significantly ameliorate the hepatocyte damage and reduce the occurrence of hepatocyte apoptosis.These results indicate that AAPC ameliorates hepatic damage in ALI mice.2.AAPC ameliorated thapsigargin-induced LO2 cell apoptosisThe result showed that compared with the model group,AAPC pretreatment could significantly reduce LDH levels(p<0.05).In addition,flow cytometry showed that compared with the model group,AAPC significantly inhibited the apoptosis of LO2 cells(p < 0.5),and it was concentration-dependent.The results suggest that the protective effect of AAPC on thapsigargin-induced hepatocyte injury may be mediated by its anti-apoptotic effect.3.AAPC inhibited ERS signaling pathway in vivo and in vitroIn vivo results showed that the protein levels of GRP78,ATF6,p-PERK,and p-IRE1 in the LPS/D-GalN model group were significantly increased,while AAPC pretreatment significantly down-regulated these protein expressions,indicating that AAPC pretreatment significantly reduced ERS in LPS/D-GalN-induced ALI mice.In addition,we further found that the protein levels of GRP78,p-PERK/PERK,p-e IF2α/e IF2α,ATF4,CHOP,p-IRE1/IRE1,XBP-1,p-JNK/JNK and ATF6 were significantly up-regulated after treated with thapsigargin in LO2 cells(p<0.05),and AAPC pretreatment could significantly reverse the expression of these abnormal proteins(p<0.05).Taken together,these results indicatethat AAPC may reduce ERS by inhibiting the proteins expression of ERS signaling pathway.4.AAPC induced autophagy in vivo and in vitroIn vivo researches showed that the expression of autophagy-related proteins LC3II/I,Beclin1,Atg5 and Atg7 increased in the LPS/D-GalN model group(p<0.05),and the expression of these proteins were further increased after AAPC pretreatment(p<0.05).In addition,in vitro experiments showed that compared with the model group,the number of autophagosomes and autosomes was increased and the process of autolysosome maturation was promoted in the AAPC group.These results indicate that AAPC induced autophagy in vivo and in vitro.Conclusion:AAPC can significantly alleviate LPS/D-GalN-induced ALI in mice,which may be attributed to its ability to inhibit the ERS signaling pathway thereby reducing hepatocyte damage and apoptosis,and promote hepatocyte survival by inducing induced autophagy.