| Objective:The aim of this study was to investigate the effect of Asiatic acid from Potentilla Chinensis(AAPC)on lipid deposition,oxidative stress,inflammation and Endoplasmic reticulum stress(ERS)in rats with non-alcoholic fatty liver disease(NAFLD),exploring the underlying mechanism of AAPC on lipid metabolism.Method:1.Preparation of NAFLD rat modelThe NAFLD was induced by high fat diet(HFD)in male Sprague-Dawley(SD)rats.A total of 80 male rats were divided into two groups:I group(28 animals)and II group(52 animals).The rats in the I group were fed with basal feed,and the animals in the II group were fed with HFD for 8 weeks.Then,the I group was divided into two groups including the normal control group and AAPC control group.In addition,the rats in the ? groups were randomly divided into four groups including the HFD model group,positive control group(Silibinin-treated group),low-and high-dosages of AAPC-treated groups.Next,in addition to HFD,the rats in the Silibinin-treated group received Silibinin 26.25 mg/kg/d intragastrically;animals in the low-and high-dosages of AAPC-treated groups were intragastric administrated AAPC 4,8mg/kg/d,respectively;rats in the AAPC control group were received AAPC 8 mg/kg;and the rats in the model control group and the normal control group were given intragastrically an equivalent normal saline.After treatment for 10 weeks,all animals were sacrificed,and the blood and liver tissue were obtained for further examination.2.The effect of AAPC on lipid deposition in NAFLD ratsThe activities of serum TC,TG,LDL and HDL were measured by an automatic biochemical analyzer.Serum FFA was detected using a commercially available kit according to the manufacturer's instruction.Furthermore,the Lipid droplets were observed by Oil red O staining.3.Effect of AAPC on lipogenesis related proteins in NAFLD ratsThe expressions of SREBP-1c,LXR?,FAS,ACC1,AMPK and p-AMPK were detected by western blot.4.The effect of AAPC on hepatic injury in NAFLD ratsThe activities of serum AST,ALT,ALB and GLB were detected by an automatic biochemical analyzer.Moreover,the pathological changes of liver were observed by H&E staining and Masson staining.5.The effect of AAPC on oxidative stress and lipid peroxidation in NAFLD ratsThe levels of serum SOD,MDA,as well as hepatic SOD,MDA,GSH-Rd,GSH-Px and MPO were assayed using commercially available kits according to the manufacturer's instruction.6.The effect of AAPC on inflammatory response in NAFLD ratsThe inflammation cytokines including TNF-? and IL-6 were detected using ELISA kits according to the manufacturer's instructions.And the protein expressions of IL-6,TNF-?,NF-?B p65,p-NF-?B p65,I?B-? and p-I?B-?were detected by western blot.7.Effect of AAPC on the ERS signaling pathway related gene and protein of in NAFLD ratsThe mRNA expressions of GRP78,PERK,eIF2a,CHOP and ATF6 in liver were analyzed by RT-PCR.And the protein expressions of GRP78,PERK,p-PERK,eIF2?,p-eIF2?,ATF4,CHOP,IRE1,p-IRE1,XBP-1,JNK,p-JNK,ATF6 and Caspacel2 were detected by western blot.Result:1.AAPC alleviated lipid deposition in NAFLD ratsIn this study,the NAFLD was induced by HFD for 8 weeks in SD rats.The results revealed that the liver index(liver weight/body weight)was higher in the model control group than the normal control group.The serum levels of TC,TG,LDL,FFA,AST and ALT were increased in the model group compared with the normal control group,while the level of HDL was decreased.Moreover,severe changes were observed in liver morphology in the model control group,including liver steatosis,accumulation of collagen and accumulation of lipid droplets.These results indicated that the NAFLD model in rats was successfully induced.After treatment with AAPC,the results showed that AAPC significantly decreased the serum levels of TC,TG,LDL and FFA,and increased the level of HDL(p<0.01).Moreover,the Oil Red O staining showed that a striking increase in the hepatic lipid droplets was observed in the HFD model group,and AAPC treatment obviously reduced the lipid droplets accumulation.These data suggest that AAPC can attenuate steatosis and lipid accumulation.2.AAPC regulated the expressions of lipogenesis-related proteins in NAFLD rat liverWestern blot results showed that the protein expressions of AMPK and p-AMPK were significantly down-regulated in the model group,while the expressions of SREBP-1c,ACC1,FAS and LXRa were significantly up-regulated.However,AAPC treatment could significantly reverse the abnormal expressions of lipogenic proteins in NAFLD rats(p<0.01),suggesting that AAPC may improve dyslipidemia by regulating lipogenesis-related proteins expression.3.AAPC ameliorated hepatic injury in NAFLD ratsThe results showed that AAPC treatment could markedly reduce the liver index and decreased the activities of serum ALT and AST(p<0.05 or p<0.01).Furthermore,the H&E and Masson staining showed that AAPC could significantly ameliorate liver cell injury and 'collagen deposition.These results indicate that AAPC ameliorates hepatic damage in rats with NAFLD.4.AAPC alleviated oxidative stress and lipid peroxidation in NAFLD ratsCompared with the normal control group,the level of SOD in both the serum and liver tissue was significantly decreased in the model group,while MDA level was significantly increased.However,AAPC treatment significantly increased the SOD level,while decreased the MDA level(p<0.05 or p<0.01).Moreover,AAPC could markedly reduce the MPO content and significantly increased the GSH-Px and GSH-Rd levels(p<0.05 or p<0.01).The results suggest that AAPC can significantly reduce the oxidative damage induced by NAFLD.5.AAPC alleviated inflammatory response in NAFLD ratsThe results of ELISA showed that the productions of TNF-? and IL-6 were higher in the HFD group than those in the normal control group,while the increased levels of both inflammatory cytokines were reversed by AAPC treatment(p<0.05 or p<0.01).Similarly,AAPC treatment significantly decreased the protein levels of TNF-? and IL-6.In addition,the Western blot results showed that AAPC treatment significantly increased the protein expressions of P-NF-?B and p-I?B?(p<0.01).These results indicate that AAPC inhibits the inflammatory response via inhibition of the NF-?B signaling pathway.6.AAPC inhibited the ERS signaling pathway related gene and protein in NAFLD ratsRT-PCR and Western.blot results showed that the expression of GRP78 was up-regulated in model group and evidently down-regulated by AAPC treatment(p<0.01).Furthermore,the protein expression levels of p-PERK,PERK,p-eIF2?,eIF2a,ATF4 and CHOP were significantly increased in the model group,and AAPC treatment markedly reversed the abnormal expression of these proteins Additionally,the results also showed that AAPC significantly inhibited the expressions of IRE 1,p-IRE1,XBP-1,JNK,p-JNK,ATF6 and Caspase12(p<0.01).Taken together,these results indicate that AAPC significantly reduces ERS by regulating the three canonical branches of the UPR.Conclusion:AAPC can significantly alleviate the hepatic steatosis and hepatocyte damage in rats with NAFLD,which may be attributed to its ability to alleviate oxidative stress,recruit the anti-oxidative defense system,inhibit the NF-?B pathway thereby reducing inflammatory cytokines,and alleviate hepatocyte apoptosis and lipid metabolism disorder by inhibiting ERS response. |
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