DSS-induced Chronic Experimental Colitis Enhances Hepatic Fibrogenesis In Mice With CCl₄-induced Liver Fibrosis

Hepatic fibrosis (HF) is a repair response to virus, alcohol liver, toxicmetabolism. Liver fibrosis is reversible, while it difficult to reverse cirrhosisof the liver. It is important to study the pathogenesis and early intervene oreven reverse hepatic fibrosis. In recent years, it found that induction ofintestinal inflammation in experimental non-alcoholic steatohepatitis (NASH)promotes liver inflammation and fibrogenesis, so the intestinal mucosal barrierinjured which may play a role in the progression of liver diseases.Inflammatory bowel disease (IBD) is found to be associated with severalkinds of liver diseases. The hepatobiliary system diseases is IBD commonextraintestinal manifestation. At the onset of IBD, the inflammatory cytokinesproduced, which caused colonic mucosal barrier damaged, imbalance ofintestinal flora, bacterial translocation secondarily, resulting in endotoxemia.In1998, Marshall proposed the concept of the “gut-liver axis”. He pointed outthat the immune defense function between the intestine and liver is mutualinfluence, mutual adjustment and inseparable. It was one of the hotspots ofcurrent research that intestinal mucosal barrier function, intestinal flora andendotoxin in portal system and then induced liver disease. It Oftenaccompanied by intestinal endotoxemia in the development of various liverdiseases. The intestinal absorption of endotoxin increased, which was one ofthe main mechanisms of endotoxemia, which was further cause secondaryhepatic injury.It is the central link that the activation and proliferation of hepatic stellatecells (HSCs) which was in the course of HF. Liver macrophages (KCs)secretion of inflammatory and fibrogenic cytokine activation in the inductionof HSCs, control the development of hepatic fibrosis also play a major role.The activation of hepatic macrophages (Kupffer cell, KCs) aggregation at sites of injury, caused by a variety of inflammatory factors, such as TNF-α, IL-1β,IL-6, TGF-β secretion, thus promote the occurrence of liver inflammation andfibrosis. It induces the secretion of chemokines from activated HSCs andchemotaxis of Kupffer cells which secrete the profibrogenic cytokinetransforming growth factor beta (TGF-β). LPS-induced TLR4-dependentsignals augment TGF-β signaling on an intracellular level via down-regulationof the TGF-β pseudoreceptor which is mediated by a TLR4-MyD88-NF-κBdependent pathway.In recent years, lipopolysaccharide (LPS)/Toll like receptor4(TLR4)signal transduction pathway plays a pivotal role in the pathogenesis of variouschronic liver diseases. Gut-derived LPS, a gramnegative bacterial cell wallcomponent, released after bacterial dissociated or adhered to other cells. TLRsare a kind of innate immune receptors that identify pathogens. Within thisfamily of TLRs proteins, TLR4plays a critical role in immunity system. TLR4are cell surface receptors that can swtich LPS into the cells, through itscytoplasmic domain Myeloid differentiation protein88(MyD-88) furtherinfluence on its downstream gene tumor necrosis receptor-associatedfactor(TRAF6), followed by the activation of NF-κB, resulting in a largenumber of proinflammatory cytokine production which mediated liverdiseases. Gabele E who showed that induction of intestinal inflammation inNASH promotes liver inflammation and fibrogenesis, which underscore thepathophysiological role of the gut-liver axis in liver diseases. Therefore,Combined with intraperitoneal injection of CCl4and drinking DSS inducedliver fibrosis with established intestinal damage model, we intend to researchthe effects of colitis on liver inflammation and fibrosis; at the same time toobserve the chronic experimental colitis associated with liver disease, toexplore the role of endotoxin and gut-liver axis. which provided a theoreticalfoundation for delaying the progress of liver fibrosis and the comprehensivetreatment of IBD.Objective: To intvestigate the role of combination with dextran sodiumsulfate (DSS) in hepatitis and fibrosis in mice treated by with CCl4. Methods:50male C57BL/6mice were randomly divided into fivegroups. Control group (n=10), DSS group (n=10), Olive oil group (n=10),CCl4group (n=10) and CCl4+DSS group (n=10). Chronic colitis was inducedby administration of2%DSS drinking water intermittently for28days, liverfibrosis was induced by intraperitoneal injection of5%CCl4olive oil10μL/gintermittently for28days, and the overlapping group was induced by drinking2%DSS combined with intraperitoneal injection of5%CCl4olive oil10μL/gIntermittently for28days. Severity of colitis was evaluated by disease activityindex (DAI), gross score, myeloperoxidase (MPO) and histology. Bacterialtranslocation and serum LPS levels were detected. Pro-inflammatorycytokines including TNF-α, IFN-γ, IL-17A and tight junction (TJ) proteins inthe colon mucosa were also checked by immunohistochemistry, western blotand real-time Q-PCR respectively. Haematoxylin and eosin staining (H&Estaining), Sirius red staining and Masson’s trichrome staining (MT staining)were used to evaluate liver histopathology, serological tests were used toobserve liver function. The protein and mRNA expressions of TNF-α, IFN-γ,IL-17A, TGF-β1, α-SMA, collagen type I and III, MMP-2, TIMP-2, TLR4,TRAF6and NF-κB in liver tissues were observed by immunohistochemistry,western blot and real-time Q-PCR, respectively.Results:(1) The results of chronic colitis showed that BW hadsignificantly greater loss in the DSS group and CCl4+DSS group comparedwith that in the control group. DAI and pathology scores in the DSS group andCCl4+DSS group were significantly higher, and colon length was shorter(P<0.05); the degree of congestion and edema of the colon wall, infiltrationinto the mucosa superficial layers of inflammatory cells and multifocalshallow ulcers were much severer (P<0.05). Mice in DSS group andCCl4+DSS group had much higher MPO (P<0.05). The results ofimmunohistochemical staining, Western blot and real-time Q-PCR showedthat the expressions of the TNF-α, IFN-γ and IL-17A in colon tissuessignificantly increased in DSS group and CCl4+DSS group (P<0.05),whilethere is no significant differences between the two groups (P>0.05).(2) Liver tissue pathology change of H&E shows that DSS group inflammatory cellinfiltration periportal, liver cell edema, loosening of cytoplasm; while in CCl4group and CCl4+DSS group, it shows that liver plate arranged disordered, livercell edema, loosening of cytoplasm, cell vacuolar degeneration, necrosis ofliver cells, periportal infiltration of inflammatory cells. Which was obviousespecially in CCl4+DSS group. Inflammation scores show that: compared withthe control group, DSS group, CCl4group, CCl4+DSS group weresignificantly increased, especially increased obviously in the overlappinggroup (P<0.05).(3) Liver tissue pathology change of MT and Sirius reddyeing shows that in DSS group, there was central veins of hepatic lobuleswall fiber, a little fibrous tissue deposition to the portal area and interlobularsepta, which was similar to the control group. While in CCl4group andCCl4+DSS group it shows with the modeling time prolonged, the visiblestructure disorder of hepatic lobules, fibrous tissue hyperplasia formationaround the small bile ducts, deposition of fibrous tissue increased around thecentral vein and portal tracts. Which induces liver fibrosis, Especially inCCl4+DSS group. The degree of fibrosis grading show that: compared withthe control group, DSS group had little difference (P>0.05), while in CCl4group and CCl4+DSS group were significantly increased, especially inCCl4+DSS group, the difference was statistically significant (P<0.05).(4)Hydroxyproline test results show that, compared with the control group, thecontent of hydroxyproline slightly increased in DSS group (P>0.05), whilethat increased significantly in CCl4and CCl4+DSS group, but thatsignificantly higher in CCl4+DSS group (P<0.05);(5) The measurement ofliver function shows, compared with the control group, alanineaminotransferase (ALT) and aspartate aminotransferase (AST) levels in theDSS group elevated, while the albumin levels decreased (P<0.05), but totalbilirubin(TBIL) and direct bilirubin (DBIL) levels increased indistinctively(P>0.05), that changed obviously in CCl4and CCl4+DSS group, which wassignificantly changed in CCl4+DSS group (P<0.05);(6) The results ofimmunohistochemical staining, Western blot and real-time Q-PCR revealed that the expressions of the TNF-α, IFN-γ, IL-17A in liver tissues significantlyincreased in DSS group, CCl4group and CCl4+DSS group compared withthose of the control group, while that in CCl4+DSS group more significantlythan others (P<0.05);(7) The results of immunohistochemical staining,Western blot and real-time Q-PCR revealed that the expressions of theCollagen typeⅠ, Collagen type Ⅲ slightly increased in DSS group (P>0.05),but that increased obviously in CCl4and CCl4+DSS group, while that inCCl4+DSS group more significantly than other (P<0.05);(8) The results ofimmunohistochemical staining, Western blot and real-time Q-PCR revealedthat the expressions of the α-SMA, TGF-β1, MMP-2and TIMP-2slightlyincreased in DSS group (P>0.05), but that increased distinctly in CCl4andCCl4+DSS group, while that in CCl4+DSS group more significantly than other(P<0.05);(9)The level of serum LPS in DSS group, CCl4group andCCl4+DSS group significantly increased compared with that of the controlgroup, while the LPS level increased distinctly in CCl4+DSS group (P<0.05);Bacterial translocation remarkably increased in DSS group(80%), CCl4group(60%), CCl4+DSS group(90%) compared with that of the control group(0%), Olive oil group(10%). The results of immunohistochemical staining,Western blot and real-time Q-PCR showed that the expressions of the occludin,claudin-1in colon remarkably decreased in DSS group and CCl4+DSS group(P<0.05)，while there were no significant differences between the two groups(P>0.05);(10) The results of immunohistochemical staining, Western blot andreal-time Q-PCR revealed that the expressions of the TLR4、TRAF6andNF-κB in liver tissues increased obviously in DSS group, CCl4group andCCl4+DSS group compared with those of the control group, while that inCCl4+DSS group more significantly than others (P<0.05).Conclusion: Induction of intestinal inflammation in hepatic fibrosispromotes liver inflammation and fibrogenesis probably through LPS/TLR4signaling pathway. The scavenging capacity of liver to intestinal endotoxemiadecreased, and the presence of intestinal inflammation, that can significantlypromote the translocation of LPS, and upregulate LPS/TLR4signal transduction pathway which mediated liver diseases.