The Effects And Mechanism Studies Of Natural Compound Alisol B On Non-alcoholic Steatohepatitis

Aims:Non-alcoholic fatty liver disease（NAFLD）refers to a pathological syndrome characterized by ectopic fat deposition in hepatocytes.Non-alcoholic steatohepatitis（NASH）is a progressive form of NAFLD.However,the pathogenesis of NASH is complex and no effective therapies have been clinically approved for NASH until now.Thus,discovering anti-NASH lead compounds is of vital significance.Alisol B is a natural compound isolated from Alisma orientalis（Sam.）,and its effects against NASH haven’t been reported so far.In this study,we systematically explored the therapeutic effects of Alisol B against NASH in several widely-used NASH models and explored the underlying molecular mechanisms.This study mainly foucused on the underlying molecular mechanisms of Alisol B against NASH via normalizing disturbed retinol metabolism and RARα-PPAR-γ-CD36 cascade,which provided experimental and theoretical basis for the development of anti-NASH lead compounds.Experimental methods:The high-fat diet plus carbon tetrachloride（DIO+CCl₄）-induced murine NASH model,choline-deficient and amino acid-defined（CDA）diet-induced murine NASH model and high-fat plus high-cholesterol（HFHC）diet-induced golden hamster NASH model were used to explore the therapeutic effects of Alisol B.The efficacy evaluation included the determination of serum Alanine aminotransferase（ALT）and Aspartic transaminase（AST）,and the determination of hepatic triglyceride（TG）and total cholesterol（TC）level.The NAS score was calculated according to Hematoxylin-Eosin staining and the percentage of heaptic collagen deposition was analyzed by Sirius Red staining.RT-PCR and Western Blot were used to detect the hepatic expression of pro-inflammatory cytokines and fibrosis-related markers.To explore the underlying machanisms,RNA-seq analysis was conducted in liver tissues of vehicle and Alisol B treated DIO+CCl₄-induced NASH mice.Free fatty acids（FFAs）-induced mouse primary hepatocytes were used to explore Alisol B’s effects on lipid accumulation and inflammatory cytokines expression.The fluorescent-labeled fatty acid analogue BODIPY-C16 and reactive oxygen species probe DCFH-DA were conducted to detect Alisol B’s effects on FFA uptake and oxidative stress in mouse primary hepatocytes.CD36 si RNA and CD36 plasmid were employed to clarify the correlation between CD36 and the improving effects of Alisol B on lipid accumulation,oxidative stress and inflammation.RARαsi RNA and RARαplasmid were conducted to explore the relationship between RARαand CD36 suppression by Alisol B.RT-PCR was used to detect the m RNA expression of the main retinol-metabolizing enzymes in the liver of NASH mice and the level of all-trans retinoic acid（at RA）in the liver was measured.Results:The chronic oral administration of Alisol B significantly lowered the serumALT,AST level and hepatic TG content,attenuated oxidative stress and inflammatory cells infiltration,decreased the percentage of collagen deposition and expression ofα-Smooth muscle（α-SMA）and Collagen type 1a1（Col-1a1）in DIO+CCl₄-induced NASH mice.In CDA diet-induced NASH mice and HFHC diet-induced NASH golden Syrian hamsters,chronic oral administration of Alisol B also decreased the hepatic TG and TC contents,attenuated hepatic steatosis and fibrosis.RNA-seq analysis indicated that Alisol B significantly decreased CD36 expression and markedly regulated hepatic retinol metabolism in DIO+CCl₄-induced NASH mice.In mouse primary hepatocytes,Alisol B dose-dependently decreased palmitate（PA）-induced cellular TG accumulation,oxidative stress and inflammatory cytokines expression,lowered the phosphorylation of c-JUN N-terminal kinase（JNK1/2）and nuclear factor-kappa B（NF-κB）.These curable effects were dependent on the down-regulation of CD36.Further study revealed that Alisol B increased the m RNA expression of RARα,and decreased HNF4α,PPAR-γand CD36 expression.The suppressed HNF4α,PPAR-γand CD36 expression and decreased lipid accumulation and oxidative stress by Alisol B were fully blocked by RARαknockdown,suggested Alisol B down-regulated CD36 expression via regulating RARα-HNF4α-PPAR-γcascade.Moreover,we discovered that Alisol B didn’t have the direct agonistic activity against RARα.The up-regulation of RARαby Alisol B may due to the increased hepatic at RA level in NASH mice,which might be associated with the increased transcript expression of retinyl ester hydrolases and aldehyde dehydrogenase by Alisol B.Conclusions:This is the first study reported that Alisol B had therapeutic effects against NASH in several NASH models.We discovered the novel mechanism that Alisol B regulated hepatic retinol metabolism homostasis,normalized the at RA level,increased RARαexpression,and decreased CD36 expression via regulating RARα-HNF4α-PPAR-γcascade,which further alleviated lipid accumulation,oxidative stress and inflammation in the liver of NASH mice.In this study,Alisol B was used as a probe to explore the possibility of normalizing disturbed endogenous retinol metabolism or RARα-PPAR-γ-CD36 cascade as a new strategy for the treatment of NASH,and to provide experimental and theoretical basis for Alisol B as a lead compound for the treatment of NASH.