Improvement Effect Of 4-hydroxycinnamamide On Non-alcoholic Fatty Liver Disease And Its Mechanism

At present,non-alcoholic fatty liver disease(NAFLD)has become one of the main causes of chronic liver disease in Western countries.In recent years,the incidence of NAFLD in China has also shown a significant upward trend.Although researchers have a certain basis for research on the pathogenesis of NAFLD and the use of existing drugs for the treatment of NAFLD,the clinical research data on the effect of drug treatment are still incomplete.In addition,the toxic side effects caused by the long-term application of some drugs are obvious,which greatly limit the research progress and strategic application of NAFLD treatment drugs.4-hydroxycinnamamide is a natural small molecule compound extracted from barberry powder,which has the advantages of small molecular weight and low synthesis cost,and has broad development prospects.This study aims to preliminarily elucidate the improvement effect and mechanism of 4-hydroxycinnamamide on NAFLD based on in vitro and in vitro NAFLD models.At the cellular level,0.5 mM OA induced LO2 cells were used as an in vitro model of NAFLD,and the optimal concentration and time of action of 4-hydroxycinnamamide and OA were screened by MTT experiment.The effect of 4-hydroxycinnamamide on OAinduced lipid accumulation in LO2 cells was investigated by oil red O staining,intracellular TG,TC assay,PCR,and Western Blot technology.An animal model of NAFLD was established in male mice with C57BL/6 induced by a high-fat,high-cholesterol and highfructose diet for 24 weeks,and a control group,a model group,a positive drug group and a 4-hydroxycinnamamide group were set up to investigate the effect of 4hydroxycinnamamide on NAFLD mice.The effects of body weight,liver appearance and organ coefficients of mice in each group were observed.The oral glucose tolerance test and the expression of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglycerides(TG),total cholesterol(TC),and tumor necrosis factor-α were measured in each group of mice.The pathophysiological structure changes of liver tissues were judged by HE staining,oil red O staining,Masson staining and ELISA kit to detect triglycerides(TG),total cholesterol(TC)and free fatty acids(FFA)in the liver.Real-time PCR was used to determine the mRNA expression levels of lipid metabolism-related genes,inflammation-related genes,fibrosis-related genes,and endoplasmic reticulum stress-related genes.Western Blot detects the expression of lipid-related proteins and the expression of endoplasmic reticulumassociated proteins.The main findings are as follows:1.4-hydroxycinnamamide has no toxic effect on OA-induced LO2 cells at a certain concentration and time;2.4-hydroxycinnamamide significantly improved OA-induced intracellular lipid accumulation of LO2 and reduced intracellular TG and TC levels(P<0.01),which were dose-dependent.Compared with the OA group,the expression of SREBP-1C,FASN,PPARy,SCD-1,and ACC genes were significantly downregulated(P<0.01)after 4-hydroxycinnamamide intervention,and the trend was dose-dependent.At the same time,4-hydroxycinnamamide also reduced the expression of SREBP-1C,FASN,PPARy and ACC protein levels(P<0.01),and it was dose-dependent;3.The liver of the mice in the HFD group was enlarged,the color was slightly darker,the liver edge was passivated,the surface was rough and dull,and the cut surface was greasy;The symptoms of liver lesions in the simvastatin group and the 4hydroxycinnamamide group were improved,the liver enlargement was reduced,the liver edge was sharp,and the cut surface was not obviously greasy.Compared with mice in the HFD group,4-hydroxycinnamamide reduced liver weight(P<0.01)and liver index(P<0.001),but did not affect the weight,food intake and water intake of NAFLD mice;4.After 4-hydroxycinnamamide intervention in NAFLD mice,the fasting blood glucose(P<0.001),serum insulin level(P<0.05),insulin resistance index(P<0.001)and insulin sensitivity index(P<0.01)decreased,and oral glucose tolerance test results were also reduced,but there was no statistical significance;5.4-hydroxycinnamamide can reduce the expression of liver function(P<0.001),blood lipid four(P<0.05)and inflammatory factors TNF-α,IL-1β and IL-6 levels(P<0.05)in serum of the body.Compared with the HFD group,4hydroxycinnamamide could reduce the expression levels of TG(P<0.01),TC(P<0.01)and FFA(P<0.05)in the liver,and the expression of lipid metabolismrelated genes,inflammation and fibrosis-related genes in the liver of mice was down-regulated.The pathological section results showed that 4hydroxycinnamamide could reduce hepatic steatosis,liver lipid accumulation,and liver fibrosis in mice with NAFLD;6.4-hydroxycinnamamide regulates the expression levels of key proteins SREBP-1C,FASN,PPARy and ACC in the endoplasmic reticulum stress signaling pathway by downregulating the expression of eIF2α,ATF-4,CHOP and XBP-1 proteins.At the same time,the expression levels of CD36,LPL,ATGL and PPARα were also downregulated.In summary,4-hydroxycinnamamide can reduce OA-induced lipid deposition of LO2 cells,improve hepatic steatosis,liver lipid accumulation,inflammation and liver fibrosis in NAFLD mice,and improve liver lipid deposition in NAFLD mice by downregulating the expression of endoplasmic reticulum stress pathways eIF2α,ATF-4,CHOP and XBP-1.These studies provide new ideas for the development of therapeutic drugs for NAFLDrelated diseases.