The Effect And Mechanism Of Indole Propionic Acid（IPA） On Carbon Tetrachloride（CCl₄）-induced Liver Fibrosis In Mice

Background and Objective:Liver fibrosis is a common pathological process of various chronic liver diseases（CLD）,which can further develop into cirrhosis and even liver cancer,and is a key factor influencing clinical prognosis.It is currently believed that the key to the formation of liver fibrosis is the continuous activation of hepatic stellate cells（HSCs）,which causes excessive deposition of extracellular matrix（ECM）.Inflammation,oxidative stress,apoptosis,etc.are all important factors for activating HSCs,which indicates that reducing or inactivating HSCs in liver fibrosis through anti-inflammatory and anti-oxidation is the main goal of anti-fibrosis.Transforming growth factor（TGF-β）is one of the key activators of the positive feedback loop that promotes tissue repair.It mainly acts by regulating downstream Smads proteins.Smad2/Smad3 is considered to be a key inducer of fibrotic procedures.Smad7 has an anti-fibrosis effect.Therefore,terminating the feedback loop induced by TGF-βis essential to prevent the occurrence and development of fibrosis.The gut-liver axis also plays an important role in CLD.The liver not only obtains nutrients from the intestines but also the first target of intestinal microbes attack.After the intestinal barrier is destroyed,bacterial metabolites can directly act on liver cells or immune cells to induce inflammatory signals and oxidative stress damage and affect cell apoptosis.A large number of documents have confirmed the occurrence of intestinal leakage in CLD patients.Therefore,the microbiota-gut-liver axis is a potential target for the prevention and treatment of the progression of chronic liver disease.Indole propionic acid（IPA）is a tryptophan metabolic derivative produced by intestinal symbiotic flora（such as Clostridium spores）.It has been found to have anti-inflammatory,anti-cancer,anti-oxidant.Besides,it can maintain intestinal homeostasis.The latest research shows that IPA can effectively alleviate the progression of liver fibrosis in NAFLD rats by down-regulating the expression of liver fibrosis-related genes.Therefore,IPA seems to be an ideal natural anti-fibrosis compound,but the effect and mechanism of IPA on liver fibrosis have not been studied in other liver fibrosis models.The mouse liver fibrosis model induced by carbon tetrachloride（CCl₄）is the most widely used animal model of liver fibrosis.This study is the first to explore the effect and mechanism of IPA on liver fibrosis in this model.Methods:Taking male C57BL/6 mice as the research object,a liver fibrosis model was constructed by intraperitoneal injection of CCl₄.IPA solution was used for daily intragastric intervention.The mice were divided into 4 groups:normal control group,IPA control group,CCl₄ model group,and IPA intervention group.Serum level ALT,AST,total bilirubin（TBIL）and total bile acid（TBA）were used to assess liver injury,whlie H&E and Masson staining were used to assess liver pathological changes and liver fibrosis.ELISA and quantitative real-time polymerase chain reaction（q PCR）were used to detect inflammatory cytokines such as TNF-α,IL-1β,IL-6,IL-8,IL-10 in the liver to assess inflammation,and MDA,GSH,GSH-PX,SOD,and CAT kits were used to detect the level of oxidative stress in the liver.TUNEL analysis detects liver cell apoptosis.The m RNA expression levels ofα-SMA,collagen-I,TIMP1,MMP2,TGF-β1,and Smad3 in liver tissues were detected by q PCR.Western blot（WB）and immunohistochemical staining were used to detect the protein expression levels of TIMP1,MMP2,TGF-β1,and P-Smad2/3 in liver tissues.16s RNA sequencing was used to analyze the structure of intestinal flora.SPSS25.0 software was used for statistical analysis of the data,and the statistical methods were t-test and analysis of variance.Results:1.IPA gavage daily could effectively increase the concentration of IPA in the serum.2.The activity of ALT,AST and the content of bile acid and total bilirubin in the serum were increased more obvious in the mice treated by IPA plus CCl₄ than CCl₄alone.However,intragastric administration of IPA would not cause liver damage,which indicated that IPA solution had no hepatotoxicity,but it would exacerbate the liver damage induced by CCl₄ in mice.3.IPA intervention significantly increased CCl₄-induced hepatocyte necrosis,inflammatory and ECM deposition,indicating that IPA aggravated the formation of liver fibrosis induced by CCl₄.4.IPA intervention could ameliorate hypertriglyceridemia in mice with liver fibrosis induced by CCl_4.5.IPA intervention significantly increased the expression levels of pro-inflammatory factors in liver tissues,including TNF-α,IL-1β,IL-6,and IL-8,and decreased IL-10,an anti-inflammatory cytokine levels.This shows that IPA intervention will aggravate the inflammatory response induced by CCl₄.6.Compared with the CCl₄ model group,the IPA intervention group showed higher activity of CAT and lower level of GSH and GSH-PX,and did not affect the SOD activity and MDA content.The above results indicate that IPA intervention cannot alleviate the oxidative stress damage induced by CCl₄.7.The m RNA and protein levels ofα-SMA,Collagen-I,TIMP-1,and MMP-2 in the liver in the IPA intervention group were increased more obvious than the CCl₄ model group,suggesting that IPA aggravated CCl₄-induced liver fibrosis by promoting the activation of HSCs.8.The level of TGF-β1 and P-Smad2/3 in the IPA intervention group was increased more obvious than CCl₄ model group,while the m RNA and protein expression of Smad7 was inhibited in the IPA intervention group,indicating that IPA aggravated CCl₄-induced liver fibrosis is related to the activation of TGF-β1/Smads signaling pathway.9.IPA intervention could increase intestinal microbial diversity and Firmicutes/Bacteroides ratio,but it also increased the abundance of pathogenic bacteria.This shows that IPA intervention could affect the microbial structure in the mouse intestine.Conclusion:In conclusion,our data indicated that IPA may over-activate HSCs through the TGF-β1/Smad signaling pathway to exacerbate CCl₄-induced liver fibrosis.IPA aggravated the liver inflammation and cell apoptosis induced by CCl₄,but could not alleviate the liver lipid peroxidation damage caused by CCl₄.IPA intervention would affect the structure of the intestinal flora.