The Hujin Formula Recipe On Lipid Synthesis In Nonalcoholic Fatty Liver Was Investigated Based On SIRT1 Pathway

Objective:Nonalcoholic fatty liver disease(NAFLD)is a clinicopathological syndrome characterized by diffuse hepatocellular bullae steatosis,which is caused by alcohol and other specific liver damage factors.NAFLD is a metabolic stress liver disease closely related to insulin resistance and genetic susceptibility.[1]adenylate activated protein kinase(AMPK)plays an important role in bioenergy metabolism,and AMPK and its downstream pathways play an important role in the pathogenesis of nonalcoholic fatty liver disease(NAFLD).The silencing information regulator 1(Sirt1)itself is a bidirectional regulatory protein.Studies have shown that inhibiting the expression of Sirt1 and Ampk by means of interference can reduce steatosis.In this study,SIRT1/AMPK signaling pathway was used to investigate the positive effects of SIRT1 inhibition of AMPK on the treatment of NAFLD.From the perspective of normal physiological functions,the liver is an organ with mainly metabolic and chemical functions,including deoxidation,storage of liver sugars,secreted protein synthesis,etc.,which is related to the physiological functions of the spleen system and the liver system in the theory of traditional Chinese medicine.Therefore,in the treatment of NAFLD from the perspective of traditional Chinese medicine,the principles of soothing the liver and strengthening the spleen,strengthening the body,removing blood stasis,and removing toxins through dampness were adopted to simulate the clinical intervention of hujinfang.Preliminary studies have shown that hujinfang can improve the liver function and blood lipid of NAFLD model mice,as well as improve the pathological condition of liver tissue and the ultrastructure of liver cells.In this study,by establishing NAFLD mice and cell models and taking the SIRT1 lipid synthesis pathway as the entry point,the influence mechanism of hujin recipe on both in vivo and in vitro models was investigated to provide scientific basis for its therapeutic target of NAFLD.Methods1.50 C57BL/6 male mice,of which 10 were given maintenance feed and 40 were given high-fat feed for 14 weeks to establish NAFLD model,and the maintenance feed was divided into normal group.The high-fat feed was divided into model group,low dose group,medium dose group and high dose group.At week 15-24,the feeding mode remained unchanged.Normal group and model group were given normal saline once a week according to body weight ratio of 0.01ml/g.The rest groups were given different concentrations of hujin decoction water extract,while the observation group was given medium dose concentration of hujin decoction,and the dosage was gavage according to body weight ratio of 0.01ml/g.Liver tissues were frozen in the refrigerator at--80℃ for subsequent pathway detection:Sirtl,Ampk,srebp-1c and Fas mRNA expression were detected by rt-pcr.The expressions of Sirtl,Ampk,srebp-lc and Fas were detected by Western blot.2.HepG2 cells were selected for study.The appropriate concentration of tiger gold was determined by MTT method,and the final high,medium and low doses of tiger gold were set as 160ng/ml,80ng/ml and 40ng/ml,respectively.Oleic acid and palmitic acid were used to induce lipid deposition in HepG2 cells.Then,tiger gold formula extract solution was used to intervene the steatotic liver cells to detect the reduction of lipid droplets in HepG2 cells after drug intervention.The levels of ALT,AST,TG,FFAs and TG in the supernatant were also detected.SIRT1,SREBP-1C and FAS were measured by ELISA for quantitative analysis.The expressions of SIRT1,AMPK,SREBP-1C and FAS were detected by Western blot.Results:1)after 3 weeks of feeding,the weight of mice in the high-fat feed group was significantly different from that in the normal group,and the weight gain was faster in the model group;The mice in each group began to lose their hair at the 5th week,while those in the high-fat diet group lost their hair seriously and showed alopecia areata.According to relevant literatures,it was found to be caused by C57BL/6 mouse gene defect.At week 14,the high-fat feed group showed sluggish action,large abdomen and reduced food intake.After the administration of the drug at week 15,the weight of the mice in the administration group showed a downward trend,but the mice continued to rise slowly from week 16,possibly due to the continuation of high-fat diet,but the rate of weight gain was slower than that of the model group.After dissection of mice at week 24,liver wet weight was observed.The liver wet weight in the model group was higher than that in the normal group,and the liver wet weight in the drug administration group was decreased.The high-dose group was the closest to the normal group.2)level of animals:tiger jw gold label scotch on Sirtl and downstream Ampk pathway factor,the influence of Srebp,Fas:through the observation of the nucleic acid and protein expression,model group compared with normal group decreased Sirtl expression,tiger jw gold label scotch every dose group can promote the expression of Sirtl,we then by comparing the downstream genes Ampk,Srebp,Fas expression found that expression of model group were increased,the tiger jw gold label scotch each downstream gene dosage group were decreased.3)in vitro cell level:the appropriate dose concentration of hujinfang was determined by MTT method,and the high,medium and low dose of hujinfang were set as:160ng/ml,80ng/ml and 40ng/ml,respectively.ELISA kits on cells in albumin SIRT1,the expression of FAS and SREBP,results show that the model group SIRT1 expression quantity is on the decline in the normal group,the tiger jw gold label scotch to medicine group SIRT1 expression quantity higher than model group,SIRT1 gene FAS and downstream SREBP model group expression quantity has a rising trend in the normal group,the tiger jw gold label scotch to medicine group expression was compared with model group decreased;Meanwhile,western-bloting method was used to further detect the SIRT1,FAS,SREBP and AMPK proteins.The SIRT1 protein expression in the model group was lower than that in the normal group,but the SIRT1 protein expression in the low dose group was lower than that in the model group.The SIRT1 protein expression in the medium and high dose group was significantly higher than that in the model group.The expression of the downstream target AMPK protein in the model group was increased compared with the normal group,and the expression of the drug administration group was decreased compared with the model group.There was no significant difference between the normal group and the model group of the FAS and SREBP targets,but the expression of the high dose group and the model group showed a certain decreasing trend.Conclusion:Experimental results in vivo indicate that hujinfang may regulate the target gene of downstream lipid synthesis through Sirtl target to reduce the synthesis of fat,so as to achieve the purpose of treating fatty liver.But in the cell in vitro experiments:FAS and SREBP target is not obvious difference of the normal group and model group,to illustrate the two targets may exist the possibility of gene regulation by other targets,but tiger jw gold label scotch compared with model group,high dose group still makes two targets downward trend,this suggests that the tiger jw gold label scotch may other pathways through factor to adjust the two targets,follow-up needs further research.