The Role And Mechanism Of Gut Inflammation In Non-alcoholic Steatohepatitis

Background and aimsAlthough multiple ‘hits’ have been reported to contribute to development and progression of non‐alcoholic fatty liver disease(NAFLD),mechanisms associated with the progression of NAFLD to non-alcoholic steatohepatitis(NASH)are not yet fully understood.In recent years,an increasing body of evidence indicates that gut microbiome and intestinal barrier integrity are associated with the development of NAFLD.Gut inflammation has been put forward to be associated with hepatic injury in some cases in the clinical practice,such as the coexistence of inflammatory bowel disease(IBD)and NAFLD.Intestinal dysbiosis could induce intestinal inflammation and further disrupt intestinal barrier,and a subsequent translocation of bacteria,bacterial products,toxins and inflammatory mediators promotes hepatic injury,suggesting a key role of intestinal barrier dysfunction in the development and progression of NAFLD.Thus,intestinal barrier disruption is the key mechanism responsible for the inflammatory transport through gut-liver axis and sustained immune activation in the development of NAFLD/NASH.As we all known,intestinal mucosal barrier plays an important role by acting as a first line of defense against microbial intrusion.Recent study showed that a second line of defense against microbiota exists beyond the well-known mucosal barrier.Gut–vascular barrier(GVB)can control the passage of antigens and prevent bacterial translocation and systemic dissemination of bacteria.Moreover,they found that GVB impairment could be responsible for liver damage in the patients with celiac disease.However,no patient data or experimental studies exist that have assessed the function of GVB in NAFLD/NASH.In the present study,we established a NAFLD model with intestinal inflammation to study the effect and mechanism of gut inflammation on hepatic injury in high-fat diet induced NAFLD model,and then we observed the intestinal barrier disruption and bacterial translocation and demonstrated the role of gut-liver axis derangement in progression of NAFLD,especially attention to the GVB dysfunction.Methods1.To establish a NAFLD model with intestinal inflammation,male C57bl/6 mice were fed with a high-fat diet(HFD)and 1% DSS for 12 weeks.Mice were randomly divided into four groups: normal chow diet(NCD)group,high-fat diet(HFD)group,DSS-induced colitis and DSS-induced colitis with high-fat diet(DSS+HFD)group.Colonic injury and inflammation were assessed by the disease activity index(DAI)score,colonic morphological changes and histological activity index(HAI)score of colon tissues.The mRNA expression levels of inflammatory factors IL-1 and TNF-α in colonic tissues were detected by RT-PCR.The grade of hepatic steatosis was observed by Oil Red O staining;the pathological changes and fibrosis in liver were observed by hematoxylin eosin(HE)staining and Masson staining.The mRNA expression levels of inflammatory factors IL-1,IL-6,TNF-α and MCP-1 in liver tissues were detected by RT-PCR.Moreover,the mRNA expression levels of profibrotic factors Collagen1,transforming growth factor β(TGF-β),Actin α2(ACTA2),tissue inhibitor of metalloproteinase-1(TIMP-1)and plasminogen activator inhibitor-1(PAI-1)in liver tissues were detected by PCR and the expression of Collagen 1 protein in liver tissues was detected by western blot.2.Endotoxin(LPS)levels were detected through an End Point Chromogenic Endotoxin Detection LAL Kit.Furthermore,the gene expression levels of pattern recognition receptor for bacterial components in the liver,such as Toll like receptor(TLR4)and Toll like receptor(TLR9)were detected by RT-PCR.The protein expression levels of tight junction-associated proteins,ZO-1 and claudin 1 in colonic tissues were assessed by Western blot.To assess the GVB permeability,2 mg of 70 KDa fluorescein isothiocyanate(FITC)-dextran(FD70)were injected in the colonic loop after laparotomy incision under anesthesia,the density of fluorescent dye FD70 was measured in the liver and spleen by immunofluorescence and the presence of serum FD70 levels were measured by a fluorescence microplate reader.In addition,the expression of plasmalemma vesicle-associated protein-1(PV1),which is a marker of endothelial cells permeability,was detected by Western blot.Furthermore,immunofluorescence confocal microscopy analysis was used to further detect the expression and distribution of PV-1 protein in colonic tissues.Results1.Compared to the control group,the DSS group and HFD+DSS group displayed body weight loss,significantly increased DAI scores and decreased length of colon.Additionally,DSS group and HFD+DSS group displayed marked mucosa destruction with a loss of crypts and goblet cells,crypt distortion and extensive submucosal edema accompanied by moderate inflammatory cell infiltration,which also shown higher histological damage score in colons.Furthermore,the proinflammatory cytokines IL-1β and TNF-α,were also increased in the colon of DSS and HFD+DSS group.The Oil Red O staining demonstrated evident lipid droplets in the liver of HFD mice,and much more serious in those with DSS-induced colitis.HE staining also showed a large amount of fat vacuoles and disorganized structures in the liver in HFD group and HFD+DSS group.Moreover,HFD+DSS group displayed evident inflammatory cell infiltration.In line with the pathological histology results,the mRNA expression levels of hepatic proinflammatory cytokines,IL-1,IL-6,TNF-α and MCP-1,were markedly increased in HFD+DSS group.Masson staining clearly showed significant branching fibrosis in HFD+DSS mice.Compared with HFD mice,the DSS-treated HFD mice showed evident increase of collagen I expression,a marker of hepatic fibrosis.Furthermore,HFD+DSS mice also presented upregulated mRNA expression of profibrogenic factors,such as Collagen 1,TGF-β,ACTA2,TIMP-1 and PAI-1,obviously higher than the simple HFD mice.In addition,DAI score and HAI score were positively correlated with severity of liver damage in NAFLD mice(r=0.813 and P<0.01;r=0.930 and P<0.01,respectively).2.Plasma endotoxin levels were detected in the portal vein blood by limulus amebocyte lysate(LAL)assay,which indicated a trend of elevation in circulatory endotoxin levels in HFD+DSS mice relative to the HFD or DSS treated mice,however,there was no significant difference.RT-PCR showed that the mRNA expression levels of TLR4 and TLR9 in HFD+DSS group were significantly higher than that in other three groups.Western blot analysis showed that the expression levels of tight junction proteins,ZO-1 and claudin1 in both DSS group and HFD+DSS group were significantly decreased relative to those without DSS-treatment.GVB permeability analysis showed that the FD70 density in the liver was markedly increased in HFD+DSS group relative to simple HFD or DSS group,as well as the FD70 distribution in the spleen and the FD70 levels in the serum.Additionally,Western blot analysis showed that the protein expression of PV-1 was significantly upregulated in DSS group and HFD+DSS group relative to relative to those without DSS-treatment.Confocal microscopy analysis showed that the expression of PV-1 was increased in DSS group and HFD+DSS group,even more evident in HFD+DSS mice compared to the HFD group,which was consistent with those of our western blot analysis.Additionally,the co-localisation of PV-1(red)and CD31(green)in colonic mucosa indicated that PV-1 was located in the colonic mucosal microvascular endothelial cells.Spearman correlation analysis showed that the expression level of PV-1 was positively correlated with severity of liver damage in NAFLD mice.ConclusionsGut inflammation exacerbates liver injury and fibrosis in the high-fat diet induced NAFLD mice,and contribute to the development of NASH.Apart from the damaged intestinal mucosal barrier,GVB disruption with bacterial translocation or endotoxin invading into bloodstream may play a key role in the pathogenesis of NASH.More importantly,these findings demonstrate a critical role for GVB in NASH pathogenesis and further underscore the complex interplay between diet,intestinal barriers and gut microbiota in driving NAFLD progression.Understanding the gut vascular barrier may provide new insights into the regulation of the gut-liver axis,thus giving a potential therapeutic target for the prevention and treatment of NASH.