Caveolin-1 Alleviates Acetaminophen-Induced Vascular Oxidative Stress And Inflammation In Non-Alcoholic Fatty Liver Disease

Background:Acetaminophen(APAP)is one of the most widely used drugs in the world.The literature shows that excessive or long-term use of APAP can lead to increased cardiovascular dysfunction.An acute increase in angiotensin Ⅱ(Ang Ⅱ)caused by APAP use in fatty liver disease may increase the risk and severity of vascular injury.However,the underlying mechanism remains unclear.Caveolin-1(CAV1)is a broad-spectrum kinase inhibitor that significantly determines endothelial function.Aims: This study aimed to observe the effects of APAP on the vasculature in non-alcoholic fatty liver disease(NAFLD)and to determine whether CAV1 could alleviate vascular oxidative stress and inflammation by targeting Ang Ⅱ or its downstream pathways.Methods: Seven-week-old C57BL/6 male mice(18-20 g)were used.Mice were randomly divided into 12 groups(n=8 in each group):(1)normal group(N),(2)high-fat diet group(HFD),(3)APAP group(APAP),(4)HFD group plus APAP group(HFD+APAP),(5)N with adeno-associated virus control(AAV-Control)group(N+AAV-Control),(6)HFD with AAV-Control group(HFD+AAV-Control),(7)APAP with AAV-Control group(APAP+AAV-Control),(8)HFD with APAP and AAV-Control group(HFD+APAP+AAV-Control),(9)N with AAV-CAV1 group(N+AAV-CAV1),(10)HFD with AAV-CAV1 group(HFD+AAV-CAV1),(11)APAP with AAV-CAV1 group(APAP+AAV-CAV1),(12)HFD with APAP and AAV-CAV1 group(HFD+APAP+AAV-CAV1),followed by high-fat diet(Tp26300)for 56 days and intragastric administration of APAP(300 mg/kg)or an equivalent amount of PBS solution within the last 24 h.Mice were executed after 24 h and serum,liver and vascular tissues were collected as soon as possible.H&E staining was performed to detect pathological changes in liver and vascular tissues;SEM was performed to observe the status of thoracic aortic endothelium;kits were used to detect serum levels of ALT,AST,GSH,MDA and TG;ELISA was performed to detect inflammatory factors TNFα,IL-1β,IL-6 and Ang II;immunohistochemical staining was performed to observe liver and vascular 3-NT and vcam-1 indicators.Western blotting was performed to detect the expression of CAV1,ACE2,Ang II,3-NT,vcam-1,TNFα,IL-1β,IL-6,PKC,MAPK proteins.A vascular endothelial injury model was established in vitro using Ang II(300 n M)cultured human umbilical vein endothelial cells(HUVEC)for 48 h.The cell models were tested for inflammation and oxidative stress indexes with the help of Western blot,RT-q PCR,and flow assay.CAV1 overexpression plasmid,small interfering RNA and PKC inhibitor were used to treat Ang II-stimulated HUVEC cells to investigate whether CAV1 exerts protective effects through PKC/MAPK pathway,respectively.The localization of intracellular PKC and its content were further quantified by immunofluorescence.Results: In animal experiments,the kit results showed that ALT,AST,and TG activities were higher in the HFD group than in the N group.In addition,these parameters were significantly higher in the HFD+APAP group than in the APAP group.WB results showed increased expression of lipid synthesis-related protein SREBP-1c and decreased expression of lipid oxidation-related protein PPARα in the HFD+APAP group.Consistent with these results,severe lipid deposition,inflammatory cell infiltration and increased intrahepatic necrosis were observed in the liver tissue of HFD+APAP mice.Thoracic aortic sections and ELISA showed increased intima-media thickness and serum Ang II activity in the thoracic aorta of the HFD+APAP group compared with the APAP group.WB results showed increased Ang II expression and decreased CAV1 expression in the vascular tissues of the HFD+APAP group of mice.Scanning electron microscopy results showed that the integrity of vascular endothelium was better in the N and APAP groups.The endothelial surface was rougher in the HFD group compared with the N group,and the endothelial connections were loosened and multiple damage pores appeared in the HFD+APAP group.The detection of oxidative stress-related indexes in vascular tissues revealed that GSH and SOD activities were decreased and MDA levels were increased in the HFD and APAP groups compared with the N group;compared with the APAP group,GSH and SOD activities were further decreased and MDA levels were significantly increased in the HFD+APAP group.Immunohistochemical analysis of the thoracic aorta showed that 3-NT expression was increased in the HFD and APAP groups,with the greatest increase in the HFD+APAP group.We also observed increased levels of 3-NT in the vascular and sinusoidal endothelium within the liver tissue of the HFD+APAP group.ELISA results showed increased levels of inflammatory factors in the HFD and APAP groups,with higher levels of inflammatory factors in the HFD+APAP group than in the APAP group,and consistent with these results was the increased level of inflammatory factor-related protein expression in the thoracic aortic tissue of mice in the HFD+APAP group.Immunohistochemical results showed increased expression of vcam-1 in the thoracic aorta,liver tissue vessels,and hepatic sinusoidal endothelium of HFD+APAP mice.Indicators of liver injury and the extent of lipid accumulation were significantly reduced in mice injected with AAV-CAV1 compared with the HFD+APAP+Control group.The intima-media thickness,oxidative stress and inflammation indexes of blood vessels were significantly reduced.The above results suggest that CAV1 can reduce hepatic steatosis and endothelial oxidative stress as well as inflammation in mice aggravated by APAP.In addition,WB results showed that CAV1 inhibited the increase in phosphorylation levels of PKC and MAPK induced by APAP+HFD.The above results suggest that CAV1 may alleviate acetaminophen-induced endothelial inflammation and oxidative stress in NAFLD state by inhibiting PKC/MAPK pathway.In in vitro experiments,WB and PCR after incubation of human umbilical vein endothelial cells(HUVEC)with Ang II(300 n M)for 48 h showed a decrease in CAV1 expression and an increase in inflammatory factor expression levels.Flow assay showed increased levels of oxidative stress.Immunofluorescence results showed that PKC expression was increased and more aggregated in mitochondrial sites,and subsequently we found a decrease in mitochondrial membrane potential after flow assay.Compared to the negative control group,overexpression of CAV1 increased CAV1 expression while decreasing inflammatory factor expression levels and reducing oxidative stress.At the same time,immunofluorescence showed reduced levels of PKC expression.And the opposite result appeared after silencing CAV1.These results further suggest that CAV1 may play a protective role by inhibiting the PKC pathway.To further determine whether CAV1 exerts regulatory effects through PKC pathway we still used PKC inhibitors.h UVECs treated with PKC inhibitors showed no significant changes in CAV1 protein expression,but PKC and p-P38 protein levels decreased in Ang II+PKC412 and Ang II+PKC412+CAV1-sirna groups.Immunofluorescence results also showed that the fluorescence levels of PKC and PKC in mitochondria were reduced and mitochondrial membrane potential was restored after using PKC412.Meanwhile,Ang II or Ang II+CAV1-si RNA-induced oxidative stress and inflammation in HUVECs were also inhibited by PKC412.Conclusion:(1)APAP increase vascular oxidative stress and inflammation in NAFLD;(2)CAV1 has a protective effect against APAP-induced vascular injury in NAFLD and may be associated with its inhibition of the PKC/MAPK pathway.