Role Of Indoleamine 2,3-dioxygenase In Hepatic Failure Complicated With Hepatic Encephalopathy Through Tryptophan Pathway

Research background:Liver failure complicated by hepatic encephalopathy(HE)is associated with severe metabolic disturbances and high mortality,and the mechanisms of this complex metabolic disorder are still unclear.Previous studies have shown that the important metabolite of tryptophan,N-methyl derivative,is significantly elevated in the exhaled gas of patients with liver failure and that the tryptophan-kynurenine metabolic pathway is associated with abnormal brain function metabolism,but its role and key regulatory sites in liver failure complicated by hepatic encephalopathy have not been elucidated.Objectives:Through clinical cases and animal experiments,we analyzed the upstream and downstream molecules of tryptophan-kynurenine metabolic pathway and the changes of key rate-limiting enzyme indoleamine 2,3-dioxygenase 1(IDO1)to elucidate the mechanism of tryptophan-kynurenine metabolic pathway in liver failure complicated by hepatic encephalopathy.The results of the study will provide new ideas for the early diagnosis and treatment of liver failure complicated by hepatic encephalopathy.Methods:1.In vitro human testing:Thirty patients with liver failure with hepatic encephalopathy and 30 patients with liver failure without hepatic encephalopathy who visited the Department of Infection of the First Affiliated Hospital of Nanchang University from December2021 to April 2022 were selected,as well as 30 healthy individuals who visited our physical examination center during the same period.Blood was collected from the study subjects,and the levels of tryptophan(TRP),kynurenine(KYN),kynurenine quinolinic acid(KYNA)and 5-hydroxytryptamine(5-HT),which are key molecules in the tryptophan metabolic pathway,were measured by high performance liquid chromatography(HPLC)technique to analyze the changes in tryptophan metabolism and the causes of the patients.2.Animal experiments:Six-week-old C57BL/6 male mice were used,and according to the experimental design,the experimental animals were divided into 6 groups according to the principle of random grouping method: 9 healthy control,9 liver failure,9 liver failure combined with hepatic encephalopathy,9 healthy control + IDO1 inhibitor,9 liver failure + IDO1 inhibitor;9 liver failure combined with hepatic encephalopathy +IDO1 inhibitor group.Lipopolysaccharide/D-aminogalactose(LPS/D-Gal N)was used to construct the animal model of liver failure.Thioacetamide(TAA)was used to prepare the model of liver failure combined with hepatic encephalopathy.The IDO1 inhibitor 1-methyltryptophan(1-MT)was used to treat the above mice separately.The levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and blood ammonia were measured in each group of mice;hematoxylin-eosin(H&E)staining was used to observe liver histopathology,RT-q PCR to detect liver inflammatory factors [interleukin-6(IL-6),tumor necrosis factor alpha(TNF-α)],IDO1 transcript levels,Western-blot,immunohistochemistry to detect IDO1 expression in liver tissues,to clarify the expression of IDO1 in liver failure and hepatic encephalopathy disease models and the effect of IDO1 inhibition on the progression of liver failure and hepatic encephalopathy.Results:1.HPLC chromatography showed that 5-HT levels decreased in the liver failure and hepatic encephalopathy groups compared with the healthy control group(P<0.05),while KYNA levels increased significantly in both the liver failure and hepatic encephalopathy groups compared with the healthy control group(P<0.05).TRP content and KYN content were not statistically different among the three groups,but KYN/TRP was significantly higher in the liver failure and hepatic encephalopathy groups compared to the healthy control group(P<0.05);2.liver IDO1 expression was upregulated in the liver failure and hepatic encephalopathy groups(P<0.05);3.IDO1 inhibitor 1-MT significantly improved serum transaminases and liver pathological damage in mice in liver failure and hepatic encephalopathy groups,and IDO1 inhibitor 1-MT improved the symptoms of hepatic encephalopathy in mice;4.The expression of IL-6 and TNF-α in liver tissues of mice in liver failure group and hepatic encephalopathy group were significantly up-regulated(P<0.05).And IDO1 inhibitor 1-MT significantly reduced the levels of the above pro-inflammatory factors(P<0.05).Conclusion:1.In patients with liver failure and liver failure complicated by hepatic encephalopathy,there is a severe disorder of tryptophan metabolism,with a weakened5-hydroxytryptamine(5-HT)metabolic pathway,an enhanced kynurenine(KYN)metabolic pathway,and enhanced IDO1 activity(KYN/TRP);IDO1 expression levels are also significantly elevated in mice with liver failure and hepatic encephalopathy,which may cause an enhanced tryptophan-kynurenine metabolic pathway and thus promote the progression of liver failure and hepatic encephalopathy.2.IDO1 inhibitor 1-MT can reduce serum transaminases,liver pathological damage,and liver pro-inflammatory factor levels in mice with liver failure and hepatic encephalopathy.It is suggested that IDO1,the only rate-limiting enzyme for tryptophan metabolism along the kynurenine pathway,attenuated the disease progression of liver failure and hepatic encephalopathy after inhibition.These results provide new insights into the future clinical treatment of liver failure or hepatic encephalopathy.