Caveolin-1 Ameliorates Acetaminophen-aggravated Inflammatory Damage And Lipid Deposition In Non-alcoholic Fatty Liver Disease Via The ROS/TXNIP/NLRP3 Pathway

The overuse of acetaminophen(APAP)in patients with non-alcoholic fatty liver disease(NAFLD)may lead to more serious hepatotoxicity.Caveolin-1(CAV1),is an essential regulator of metabolic function,which can alleviate liver damage by scavenging reactive oxygen species(ROS).Evidence suggests that the NOD-like receptor family pyrin domain-containing 3(NLRP3)-mediated pyroptosis is involved in the development of NAFLD.Moreover,thioredoxin-interactive protein(TXNIP)activation is a key event linking ROS to NLRP3 inflammasome.Aims:The purpose of this study is to investigate whether CAV1 alleviates the liver injury aggravated by APAP in NAFLD by influencing hepatocyte pyroptosis.And the role of the ROS/TXNIP/NLRP3 pathway in this process is further discussed.Methods:1.In vivo experimentIn this study,male C57BL/6 mice(18-20 g,8-9 weeks)were injected with CAV1 gene overexpressed adeno-associated virus(AAV-CAV1)and control virus(AAVControl)through caudal vein at the end of the acclimation period,and then fed with a high-fat diet for two months to prepare the fatty liver mouse model,and 300mg/kg APAP was given by gavage 24 h on the last day of the experiment.The main experimental groups were: 1)normal group(N);2)high-fat diet group(HFD);3)APAP group(300 mg/kg)(APAP);4)HFD with APAP group(HFD+APAP);5)N with AAVControl group(N+CON);6)N with AAV-CAV1 group(N+AAV-CAV1);7)HFD with APAP and AAV-Control group(HFD+APAP+CON);and 8)HFD with APAP and AAV-CAV1 group(HFD+APAP+AAV-CAV1).After the experiment,biological tests were performed on the collected blood and tissue samples,including: ALT,AST,TG,LDH,GSH,MDA levels in serum,and SOD levels in the liver were detected by the kit.The levels of inflammatory cytokines IL-18 and IL-1β were detected by ELISA kit.Hepatic steatosis and pathological changes were observed by oil red O staining and H&E staining.The ROS levels in the liver were detected by DHE staining.The expression of CAV1 in the liver and its co-localization with specific markers of hepatocytes were analyzed by immunofluorescence(IF).Finally,ROS/TXNIP/NLRP3axis-mediated pyroptosis-related proteins in mouse liver were assessed by immunohistochemistry(IHC)and western blotting(WB),including: CAV1,TXNIP,NLRP3,Caspase-1 P20,GSDMD-N,IL-18,IL-1β.2.In vitro experiment2.1 In vitro,0.5mM free fatty acids(palmitic acid:oleic acid =1:2,PA:OA=1:2,P/O)were used for 48 h to establish the hepatocyte steatosis model,and 4mM APAP was added at 24 h to observe the effects of APAP on the hepatocytes(lipid deposition,oxidative stress,inflammatory injury).The specific experimental groups were: 1)normal group(N),2)acetaminophen group(APAP,4mM),3)free fatty acid group(FFA,P/O),and 4)free fatty acid+acetaminophen group(P/O+APAP).Firstly,intracellular lipid droplets were observed by oil red O staining,and the levels of TG and SREBP-1c protein expression were measured to determine whether the steatosis model was successfully established.The MMP changes were observed by confocal laser microscopy.The intracellular ROS levels were determined by flow cytometry.Scanning electron microscopy(SEM)was used to observe the pyroptosis characteristics of hepatocytes.The mRNA levels of NLRP3,IL-18,and IL-1β were determined by q RTPCR.Finally,WB was used to analyze the ROS/TXNIP/NLRP3 axis-mediated pyroptosis-related protein levels,including: CAV1,TXNIP,NLRP3,Caspase-1 P20,GSDMD-N,IL-18,IL-1β,and SREBP-1c,which could determine whether excessive APAP intervention induced hepatocyte pyroptosis through activation of NLRP3 inflammasome,aggravating inflammatory damage and lipid deposition in fatty liver.2.2 Transfection of CAV1-Si RNA and GV146-CAV1 was performed to observe the protective effect of CAV1 on liver injury caused by APAP in NAFLD,and to determine whether it was related to the regulation of ROS/TXNIP/NLRP3 axismediated pyroptosis pathway.The detection method was the same as above.The specific groups were: 1)N group,2)P/O+APAP group,3)P/O+APAP+Con-siRNA group,4)P/O+APAP+CAV1-siRNA group,5)P/O+APAP+GV146-Con group,6)P/O+APAP+GV146-CAV1 group.2.3 Transfection of TXNIP-siRNA and the use of N-Acetylcysteine(NAC),a reactive oxygen species inhibitor,further determine whether the ROS/TXNIP/NLRP3 pathway is involved in the regulation of CAV1 on APAP-induced hepatotoxicity in NAFLD.Flow cytometry was used to detect intracellular ROS levels to verify the success of NAC intervention.Finally,WB was used to analyze the protein levels of TXNIP,NLRP3,Caspase-1 P20,GSDMD-N,and IL-1β.Results:1.In vivo results1.1 Kit test results showed that compared with N group,serum ALT,AST,TG,and MDA levels were increased,GSH levels were decreased and SOD levels in liver tissue were decreased in HFD or APAP alone treatment groups.HE staining results showed that,it was obvious that liver size was significantly increased,liver color was altered,fat vacuoles were greater in number,and nuclear pleomorphism and inflammatory cell infiltration were observed in the livers of the HFD or APAP alone treatment group,which were more obvious in the HFD+APAP group.More severe lipid deposition was also observed in the HFD+APAP group compared to the HFD group by oil red O staining.DHE staining also showed that the ROS fluorescence intensity in HFD+APAP group was the highest,followed by HFD and N groups.Notably,IF results showed that the CAV1 expression in liver tissue was lowest in HFD+APAP group,and there was a typical co-localization between CAV1 and hepatocyte-specific marker Albumin.However,compared with HFD+APAP group injected with AAV-Control,the liver damage and lipid deposition were significantly reduced,the activities of GSH and SOD were significantly increased,and the level of MDA was significantly decreased in HFD+APAP group injected with AAV-CAV1,along with the increase of CAV1 protein expression.These results preliminarily suggest that CAV1 reduces lipid deposition and oxidative stress aggravated by APAP in NAFLD,which is mainly dependent on its expression in hepatocytes.Finally,ELISA results showed that serum LDH,IL-18 and IL-1β levels in the HFD+APAP group were higher than those in the HFD group.WB and IHC results also showed that the protein levels of NLRP3,TXNIP,Caspase-1 P20,GSDMD-N,IL-18,and IL-1β in the HFD+APAP group were increased compared with those in the HFD group,suggesting that APAP can promote hepatocyte pyroptosis.After caudal vein injection of AAV-CAV1,the expression of pyroptosis-related proteins were decreased,suggesting that CAV1 could inhibit pyroptosis of hepatocyte via the ROS/TXNIP/NLRP3 pathway,and alleviate the inflammatory injury aggravated by APAP in NAFLD.2.In vitro results2.1 Oil red O staining,the detection of SREBP-1c protein and TG levels showed that compared with P/O group,lipid deposition was significantly aggravated in P/O+APAP group.JC-1 staining and flow cytometry results showed that P/O or APAP alone treatment lowered MMP,which was further decreased in the P/O+APAP group,along with the increase of ROS levels.These results preliminarily indicated that overuse of APAP on the basis of fatty liver could promote lipid deposition and oxidative stress.SEM showed that the cell surface of N group was intact,but some holes appeared on the surface of the cells exposed to P/O+APAP.In addition,P/O or APAP alone treatment enhanced NLRP3 and pyroptosis-related protein levels compared to the N group,such as TXNIP,GSDMD-N,Caspase-1 P20,IL-18,and IL-1β,which were further increased in the P/O+APAP group.The same trend was observed in the mRNA expression levels of NLRP3,IL-18,and IL-1β,suggesting that APAP intervention aggravates inflammatory damage by promoting hepatocyte pyroptosis.2.2 Compared with the corresponding negative control group,lipid deposition and ROS levels in the P/O+APAP group were significantly reduced after CAV1 overexpression.The protein expression changes of NLRP3,TXNIP,GSDMD-N,Caspase-1 P20,IL-18,and IL-1β were reversed by WB,and the mRNA levels of NLRP3,IL-18,and IL-1β were significantly decreased,which was consistent with the results in vivo.However,opposite results were found after silencing CAV1.These results further suggest that CAV1 may play a protective role in the liver via the ROS/TXNIP/NLRP3 pathway.2.3 The role of TXNIP in NLRP3 inflammasome activation was further investigated using the siRNA strategy.WB results showed that TXNIP-siRNA significantly reduced the high expression levels of NLRP3,GSDMD-N,Caspase-1 P20,IL-18,and IL-1β induced by P/O+APAP.Flow cytometry showed that NAC intervention reduced the high ROS levels in P/O+APAP group treated with CAV1-siRNA.Moreover,WB results indicated that the increased expression levels of TXNIP,NLRP3,and pyroptotic proteins in the P/O+APAP+CAV1-siRNA group were reversed by NAC.Taken together,these results demonstrate that there is an interaction between TXNIP and NLRP3 and that CAV1 alleviates APAP-aggravated inflammatory damage in the fatty liver state via regulation of the ROS/TXNIP/NLRP3 pathway.Conclusion:1.The overuse of APAP in NAFLD aggravated inflammatory damage and lipid deposition.2.CAV1 ameliorated APAP-aggravated hepatotoxicity in NAFLD via regulating the NLRP3-mediated pyroptosis pathway.3.The ROS/TXNIP axis participated in the regulatory effect of CAV1 on NLRP3-mediated pyroptosis.