Caveolin-1 Protects Against Liver Damage Exacerbated By Acetaminophen In Non-alcoholic Fatty Liver Disease By Inhibiting The ERK/HIF-1α Pathway

Acetaminophen(APAP)is a commonly used antipyretic and analgesic drug,but its use in large amounts or for long periods of time can lead to liver damage.The use of APAP in non-alcoholic fatty liver disease(NAFLD)exacerbates liver injury,and the level of hypoxia-inducible factor 1-alpha(HIF-1α)is significantly elevated.However,the underlying mechanism remains unclear.Therefore,it is necessary to discuss the specific pathogenesis and possible therapeutic strategies.Caveolin-1(CAV1)has been shown to have a protective effect against NAFLD.Aims:The aim of this study was to investigate whether CAV1 can exert a protective effect against exacerbation of liver injury with APAP in NAFLD status by affecting HIF-1α or its related targets.Methods:1.In vivo experimentSeven-week-old C57BL/6 male mice(18-20 g)were randomly divided into six groups,(1)normal group(N),(2)high-fat diet group(HFD),(3)acetaminophen group(APAP,300 mg/kg),(4)HFD and APAP group(HFD+APAP),(5)HFD+APAP+Adenoassociatedvirus-Control group(HFD+APAP+AAV-Control),and(6)HFD+APAP+Adeno-associatedvirus-Caveolin-1group(HFD+APAP+AAV-Caveolin-1).In vivo experiments were performed using adenovirus overexpressing CAV1(Adeno-associatedvirus-Caveolin-1,AAV-CAV1)injected tail vein into mice to overexpress CAV1,while an equal amount of control adenovirus(Adeno-associatedvirus-Control,AAV-Control)as a control.The mice were administered with APAP(300 mg/kg)by gavage 24 h before the completion of modeling,and the other groups were given an equal amount of distilled water by gavage with no food or water.H&E staining and Oil Red O staining were used to detect the pathological changes of liver tissues;kits were used to detect the changes of ALT,AST,TG,GSH,MDA and SOD activities in serum;immunohistochemistry was used to detect the expression of HIF-1α.The DHE probe was used to detect ROS expression.Western Blot was used to detect the expression of CAV1 and SREBP-1c p-ERK,HIF-1α protein.2.In vitro experiment2.1 The hepatocyte lipid deposition model was established in vitro by culturing a mouse normal hepatocyte line(AML12)with a mixture of palmitic acid and oleic acid(PA/OA)for 48 h.The effect of APAP on hepatocyte lipid deposition model cells was observed by adding 4 m M of APAP at 24 h before the end of modeling and continuing the culture until the end of modeling.2.2 The effects of APAP on lipid deposition,oxidative stress and ERK/HIF-1αpathway protein expression in a cellular model of hepatocyte lipid deposition were observed by overexpression of CAV1 and silencing of CAV1 by SiRNA technology through GV146-CAV1 transfection of AML12 cells,respectively.2.3 An ERK pathway inhibitor——PD98059,was used in a hepatocyte lipid deposition cell model to observe its effects on oxidative stress and ERK/HIF-1αpathway proteins in NAFLD exacerbated by APAP.Results:1.In vivo resultsThe H&E and Oil Red O staining results revealed that the number of fat vacuoles and orange-red lipid droplets in the HFD group’s liver tissue was dramatically higher than in the N group,and these phenomena were aggravated in the HFD+APAP group.The Western Blot results revealed that CAV1 protein expression decreased in the HFD group compared to the normal group,while SREBP-1c,HIF-1,and p-ERK1/2 protein expression increased in the HFD group.These changes were more obvious in the HFD+APAP group.In contrast,mice in the HFD+APAP group with tail vein injection of AAV-CAV1 showed a significant reduction in the index of liver injury and lipid accumulation,a significant increase in GSH and SOD activities,and a significant decrease in MDA levels,while CAV1,SREBP-1c,HIF-1α,and p-ERK1/2 protein expression was reversed.The above results indicated that CAV1 could reduce the lipid accumulation and oxidative stress exacerbated by APAP in the non-alcoholic fatty liver state.Meanwhile,Western Blot results showed that CAV1 significantly inhibited the elevated level of ERK1/2 phosphorylation,suggesting that CAV1 may play a protective role by alleviating oxidative stress through inhibiting the ERK/HIF-1α pathway.2.In vitro resultsThe results of Oil Red O staining and TG activity detection showed that the orange-red lipid droplets and TG levels in P/O group were significantly increased compared with those in N group.The above phenomena were more serious in P/O+APAP group.Meanwhile,Western Blot results showed that compared with normal group,CAV1 protein expression in P/O group was significantly decreased,while SREBP-1c,HIF-1α and p-ERK1/2 protein expression levels were increased.The addition of APAP will aggravate these phenomena.Compared with the corresponding negative control group,overexpression of CAV1 resulted in significantly better lipid deposition in the P/O+CAV1 group,significantly lower ROS levels,and reversal of CAV1,SREBP-1c and HIF-1α protein expression,as well as significantly lower phosphorylation levels of ERK1/2,which is consistent with the in vivo results.In contrast,silencing of CAV1 yielded the opposite results.The above results further suggest that CAV1 may play a protective role by alleviating oxidative stress through inhibition of ERK/HIF-1α pathway.In the P/O+APAP+CAV1-SiRNA group,after using the ERK pathway inhibitor PD98059,the levels of cellular lipid deposition、oxidative stress were all significantly reduced and the expression of cellular phosphorylated ERK was inhibited.This confirms that the protective effect of CAV1 on liver injury exacerbated by APAP in the fatty liver state is achieved by attenuating oxidative stress through inhibition of the ERK/HIF-1α pathway.Conclusion:1.The use of APAP in the NAFLD state exacerbates lipid deposition and oxidative stress.2.The protective effect of CAV1 on liver injury exacerbated by APAP in NAFLD state may be related to its inhibition of ERK/HIF-1α pathway to attenuate oxidative stress.