Functional Role And Regulatory Mechanism Of NLRC5 In Alcoholic Fatty Liver

Alcoholic liver disease(ALD)is a systemic disease which is caused by continuously excessive alcohol consumption.The consequences of alcohol consumption on human health vary according to the personal drinking pattern(excessive or not,acute or chronic),environmental(diet)and individual factors(gene polymorphism).Excess alcohol consumption once became one of the primary causes of liver-related mortality in Western countries.With recent changes in the economic development and increases in average incomes in Asia,there has been a rapid rise in individual alcohol consumption.The WHO International Cancer Research Institute classified ethanol in alcoholic beverages into Ⅰclass of carcinogens in 2017.Now,ALD has became a global problem of human health.ALD comprises a complex spectrum,ranging from alcoholic steatosis to alcoholic hepatitis(AH),alcoholic liver fibrosis and alcoholic cirrhosis.Histologically ALD is characterized by hepatocyte steatosis,ballooning and apoptosis,lobular inflammation of liver,deposition of extracellular matrix(ECM)and formation of regenerative nodules.It has been recognized that once alcoholic steatosis was considered benign,it will act as a condition that may result in advanced liver diseases,such as cirrhosis and hepatocellular carcinoma(HCC).Alcoholic cirrhosis has been estimated to account for about 50% of all deaths from cirrhosis.However,once alcoholic cirrhosis develops,it will hard to be reversed.Hence,it is significant to control the deterioration of alcoholic steatosis and even achieve the reversion.Alcoholic steatosis is a systemic disease characterized by hepatocytes damage,deposition of intracellular fat droplets,and infiltration of liver tissue by inflammatory cells.Acetaldehyde,the metabolites of alcohol in the body,has direct toxic effects on hepatocytes,leading to larger cell gap,increased vacuoles,resulting in disorder of hepatic cord and the destroyed normal liver structure.Simultaneously,alcohol stimulation can increase steroid regulatory element-binding protein-1c(SREBP-1c)and promote lipid synthesis.And also decreases peroxisome proliferator-activating receptor α(PPAR-α)to reduce fatty acid oxidation.Two-pronged approach,leading to excessive deposition of lipid droplets within the liver cells,finally liver steatosis.This study will try to find the research targets which are abnormally expressed in alcoholic fatty liver and participate in the regulation of disease progression,trying to provide new ideas for the prevention and treatment of ALD.Recent studies have found that members of the NOD-like receptor family,involved in innate immunity,play important role in many non-immune response.The largest member of the family,NLRC5,has been shown to promote the development of hepatocellular carcinoma and liver fibrosis through multiple signaling pathways.Our research group established the Binge model of alcoholic fatty liver and constructed NLRC5 knockout mice to further explore the possible abnormal changes,biological functions and molecular mechanism of NLRC5 in early stage of ALD.NLRC5 expression was firstly measured in the liver of Et OH-fed mice in the Binge model.Immunohistochemistry and Western Blot experiments confirmed that NLRC5 gene was highly expressed in the steatosis liver of mice.Further,the Binge model was established in wild-type mice and NLRC5 knockout mice,respectively.After the modeling period,the serum of mice was used for biochemical detection,and liver tissues were collected for pathological observation and immunohistochemical detection of related genes.Then,perfusion technique was used to isolate the liver hepatocytes and Kupffer cells for Realtime-q PCR and Western Blot detection.The results showed that compared with wild-type mice,serum concentrations of triglyceride(TG),total cholesterol(T-CHO),alanine aminotransferase ALT)and aspartate transaminase(AST)in NLRC5 gene knockout mice were reduced.HE and Oil-Red O staining showed that the normal structure of liver in alcohol-fed wild-type mice was destroyed,present more vacuoles and obvious lipid droplets.However,compared with those of wild-type mice,livers of NLRC5 knockout mice experienced less liver damage and steatosis after Et OH-feeding.Moreover,alleviated alcohol-induced abnormal expression of SREBP-1c and PPAR-α was measured in NLRC5 gene knockout mice by Realtime-q PCR and Western Blot detection.These result suggest that the NLRC5 gene knockout mice has a certain tolerance to alcohol,leading to mice liver suffer from less alcohol-induced liver damage and steatosis.Mouse hepatocytes and Kupffer cells were separated by perfusion,and abnormal expression of NLRC5 gene were mainly observed in hepatocytes.Therefore,immortalized mouse AML-12 hepatocytes were used for silencing and over-expression of NLRC5 gene in vitro.Consistent with in vivo results,silencing NLRC5 has a protective effect on the liver,whereas over-expression of NLRC5 aggravates the toxic effects of alcohol.Further study found that with the same Et OH-fed conditions,amount of autophagosome in livers of NLRC5 knockout mice were more than those of wild-type mice.In general,increased expression of microtubule-associated light chain 3-II(LC3-II)and decreased expression of the multifunctional protein sequestome 1(p62 / SQSTM1,p62)are considered to be signs of autophagy.The results of Western Blot confirmed these changes of LC3-Ⅱ and p62 protein expression in the liver of NLRC5 knockout mice.Interestingly,autophagy as a highly conserved intracellular process of catabolism is thought to play a protective role in the development of alcoholic fatty liver.In this study,3-methyladenine(3-MA),an autophagy inhibitor,and autophagy activator rapamycin were co-administered with Et OH-feeding respectively.After 3-MA inhibit autophagy in mice,alcohol-induced hepatic injury and steatosis were aggravated,while the hepatic toxicity of alcohol was attenuated by rapamycin-induced autophagy.This result confirms the role of autophagy in relieving alcohol damage to the liver.Previous results confirmed that knockout NLRC5 can induce autophagy,so NLRC5 knockout mice were given intraperitoneal injection of 3-MA to observe whether inhibition of autophagy influence the biological function of NLRC5 in ALD.Consistent with experimental hypotheses,administration of 3-MA caused elevated levels of TG,T-CHO,ALT and AST in serum of Et OH-fed NLRC5-knockout mice,mice compared to those injected with control solvent.And lipid droplets deposition was also enhanced,accompanied by disorder of fat biosynthesis and metabolism genes.These results suggested that 3-MA may partially block the protective effect of NLRC5 knockout in liver through inhibition of autophagy.These above studies showed that,NLRC5 was abnormally increased after alcohol consumption and it may be involved in the pathogenesis of alcoholic fatty liver disease.Whereas,alcohol-induced liver damage and steatosis were alleviated after NLRC5 knockout,and this biological function may be associated with autophagy.