Huang-Qi-San Improves Obesity And Nonalcoholic Fatty Liver By Regulating FABP4 In A High-fat Diet-induced Obese Rats

Nonalcoholic fatty liver disease(nonalcoholic fatty liver diseases,NAFLD)is a common cause of chronic liver disease among people who are with no history of heavy drinking,and the main pathological features are in hepatic lobule with fatty degeneration of hepatocytes and accumulation of lipids.The current assumption is that NAFLD is not an independent disease,the etiology is associated with many metabolic disorders such as obesity,hyperlipemia and diabetes.At present,the understanding of NAFLD pathogenesis is the two hit hypothesis.The first hit hypothesis is that insulin resistance is the key factor in the pathogenesis of NAFLD.And the second hit hypothesis is involved in the participation of free fatty acid,adipokines,cytokine secreted by adipose tissue,stress response and other multiple factors.So to say,there are many associated pathophysiological basis in the development of obesity-specific NAFLD in humans.Lots of researches indicate that obesity is a common soil of various diseases,which is also the important embodiment of adipose tissue dyfunction.Adipose tissue is one of the largest endocrine and paracrine organ in the body,and the secretion of Free fatty acid(FFA),as a kind of signal molecule,can disrupt the body sugar and lipid metabolic disorder through activation of inflammation and stress,which in turn caused the metabolism of other organs such as liver,heart,skeletal muscle lesions.FABP4(Adipocyte fatty acid binding proteins,also called aP2)is a member of the family of fatty acid binding protein,which can combine with fatty acid to promote the transportation of FFA as a molecular chaperone,and in turn involved in metabolism and signal transduction process.There have been reports that the FABP4 and FFA levels in the blood in patients of obesity,fatty liver disease and diabetes were higher than normal people.FABP4 was abundantly expressed in the adipose tissue,but the expression and significance in the liver has not been reported.Emerging evidence shows that endoplasmic reticulum stress(endoplasmic reticulum stress,ERs)may be the core mechanism of linking obesity(FABP4)and nonalcoholic fatty liver disease.Endoplasmic reticulum(endoplasmic reticulum,ER)is a critical organelle involved in synthesis of cellular proteins and lipid,and participate in calcium saved and its signal transduction.When nutrition surplus,higher fatty acids,calcium balance disorders,glucose deprivation and pathogenic microorganism stimulation,the metastability of endoplasmic reticulum will will be upset,leading to endoplasmic reticulum stress.ERs mainly includes three signaling pathways:1)unfolded protein response(UPR);2)endoplasmic reticulum overload response(EOR);3)sterol regulatory cascade.The UPR is considered the main pathway of endoplasmic reticulum stress,which is mediated by three transmembrane proteins that are PER-like ER kinase(PERK),Inositol requiring-1(IRE1)and Activating transcription factor-6(ATF6).Sterol regulatory cascade is mainly SREBP1c,and it is as a main regulatory factor of hepatic fatty acid synthesis.When the expression of SREBPlc increased,fatty liver would happen.Huang-Qi-San is the an ancient prescriptions for the treatment of dabetes.The formula is composed with Radix Astragali,Radix Pueraria and Cortex Mori,of which these three herbs are commonly used Chinese medicine in the treatment of various diseases for thousands of years.In our previous research,Huang-Qi-San significantly decreased the body weight and FFA level in diabetic rat,and improved insulin sensitivity and fatty liver.However,the mechanism of its effects of weight loss and liver protection is not clear yet.To sum up,the main concent of this pape is as follows:ObjectiveIn this study,we will propose the high fat diet-induced obese and NAFLD rat model and cell model,using Elisa,RT-PCR and WB methods,and on the whole animal and cellular levels,to observe the improvement effect of Huang-Qi-San on obesity and fatty liver and how to effect FABP4 and ERs.The participating role of FABP4 and ERs in nonalcoholic fatty liver disease was confirmed by using transcriptome sequencing(RNA-Seq)method.The preliminary mechanism of FABP4 effecting ERs was in FABP4 overexpression way.Finally,this paper disscusses Huang-Qi-San how to improve obesity and nonalcoholic fatty liver by regulating FABP4.In addition,due to the mechanisms of multi-targets effects of traditional Chinese medicine,we need to expand the mechanism of Huang-Qi-San on metabolic diseases and to furnish basis for clinical applecation.Methods1.Experimental study on preventive and therapeutic effects of Huang-Qi-San in a high-fat diet-induced obese and NAFLD ratsThe experiment includes two parts,preventive and therapeutic effects of Huang-Qi-San.72 healthy male SD rats were randomly divided into normal group(SD,n=12)and building-model group(n=60).The rats in SD group were fed with basic diet and in building-model group with high fat diet,in which to choose 12 rats for model group(HFD).The preventive effect of Huang-Qi-San:the rats in building-model group were randomly divided into HQS-L low dose group(1.2 g/kg.d),HQS-H high dose group(2.4 g/kg.d),8 in each group,building and administration were at the same time for 15 weeks.The therapeutic effect of Huang-Qi-San:the rats in building-model group were fed with high fat diet for 7 weeks.After this,24 obese rats were choosed to randomly divide into HQS-L low dose group(1.2 g/kg.d),HQS-H high dose group(2.4 g/kg.d),Lipitor positive medicine group(Lipitor,2 mg/kg.d),8 in each group,beginning drugs for 15 weeks.To investigate the effects of Huang-Qi-San on body weight,glucolipid metabolism,insulin,glucose tolerance,liver lipid(TC,TG)and homocysteine(Hcy),glutamyl transpeptidase(GGT1)levels,and epididymal fat and hepatic histopathology,hepatic steatosis(HE and Oil red 0 stainings).At the same time,to obeserve the effect of Huang-Qi-San on fat rate,subcut-aneous fat,visceral fat and liver fat rate using micro-CT method.Finally,the study evaluates the preventive and therapeutic effects of Huang-Qi-San in a high-fat diet-induced obese and NAFLD rats.2.Effect of Huang-Qi-San on FABP4 nad ERs in a high-fat diet-induced obese ratsTo further investigate the mechanism in effect of Huang-Qi-San in a high-fat diet-induced obese rats.Mmunohistochemistry was used to evaluste FABP4 expression in epididymal fat and liver.Elisa and ACS-ACOD methods were used to determine the levels of FABP4 and FFA.The method of RT-PCR was used to measure the mRNAs of FABP4 and FAS in epididymal fat,and the mRNAs of FABP4,FAS,ApoB100,PPAR y and SREBP-lc,ATF6,PERK,JNK in liver.The method of WB was used to measure the proteins of FABP4 in epididymal fat and liver,and the proteins of FAS,SphK2、p-AKT、SREBP-lc、PERK、IRE-1 a/p-IRE-1 a in liver.Finally,this study disscusses Huang-Qi-San how to improve obesity and nonalcoholic fatty liver by regulating FABP4.3.RNA-Seq study of Huang-Qi-San in a high-fat diet-induced obese ratsThere were 3 cases respectively epididymal fat and liver from control group,model group,lipitor group and HQS-H groups of preventive and therapeutic were gained for extracting total RNA.After the quality and quantitative to mRN enrichment using oligo(dT)mRNA magnetic beads,construct RNA sequencing library,library quantitative by RT-PCR method.Using the Illumina Hiseq 4000 sequencing machine to sequence,and the sequencing results are standardized and screening differentially expressed genes,GO and Pathway function analysis.Through the observation of the differences in gene expression in liver and adipose tissue before and after medicine,and combined with the function analysis,to explore the participating role of FABP4 and ERs in nonalcoholic fatty liver disease in prevention and treatment of Huang-Qi-San.4.Effect of FABP4 on ERs and intervention study of Huang-Qi-San① To obeserve the content of FABP4 in supernatant fluid,3T3-L1 preadipocyte was induced mature adipocyte using the classic“cocktail”induction method.Then to obeserve the effect of Huang-Qi-San on 3T3-L1 cell’s proliferation and FABP4 and PPAR y.② The L02 liver cells of fatty degeneration of hepatocytes model were induced with different concentration of palmitic acid(PA).According to MTT and Oil red 0,the optimal concentration of FA was determined.In L02 fatty degeneration of hepatocytes model,the FABP4 overexpression added,the exprement was divided into 7 groups,control group,PA group,FABP4 group,PA+FABP4 group,PA+HQS group,FABP4+HQS group,PA+FABP4+HQS group.The method of RT-PCR was used to measure the mRNAs of FABP4 PERK,ATF6,IRE-1 a in L02 cells.The test was preliminary evaluated the effects of FABP4 and Huang-Qi-San on endoplasmic reticulum stress.Results1.Compared with normal group,the body weight of rats in model group were increased significantly,and the liver tissue pathologic and lesions were also significant obvious,the obese and NAFLD ras model was successfully made.From two parts of preventive and therapeutic effects of Huang-Qi-San,Huang-Qi-San could significantly decrease the body weight,FPG,TC,TG,LDL-C levels,improve glucose tolerance and decrease FINS level;Huang-Qi-San could significantly decrease TC,TG,Hcy,GGT1 levels in liver,and protect the liver tissue pathologic and lesions;Huang-Qi-San could significantly decrease fat rat,subcutaneous fat,visceral fat and liver fat rate.The above results suggest that Huang-Qi-San has better improve obesity and fatty liver function,preventive superior to therapeutic effects,and superior to lipitor.2.Immunohistochemical results showed that FABP4 was expressed in the cytoplasm and nucleus in the fat cells of endothelial cells in adipose tissue and hepatic vein endothelial cells.Huang-Qi-San could significantly decrease FABP4 and FFA levels in the blood.RT-PCR and WB test results showed that Huang-Qi-San could significantly decrease the mRNAs and protein of FABP4 in adipose tissue and liver,and also decreased the mRNAs or proteins of FAS,PPARγ,ATF6,SREBP-lc,PERK,IRE-la/p-IRE-la,and increased the proteins of SphK2 and p-AKT.There is a difference between preventive and therapeutic effects.The above results suggest that Huang-Qi-San can regulate FABP4 and endoplasmic reticulum stress.3.The gene sequencing results were analysed,and the differential expression gene were compared among each group.FABP4,as one of the differential expression genes,was increased in the model group,and decreased in HQS-H groups of preventive and therapeutic,but no significantly decrease in lipitor group.At the same time,through function analysis,the many GO terms related to stress were effected by Huang-Qi-San.And we also find more differentially expressed genes,which were decreased in HQS group.4.① In the process of adipogenic differentiation,the content of FABP4 in the supernatant fluid was gradually increased.Huang-Qi-San could significantly decrease the mRNA of FABP4 and PPAR y.② The resluts of various concentrations of PA on L02 cells found that PA at the concentration of 0.5 mM could obviously increase the hepatic steatosis and successfully make the fatty degeneration of hepatocytes model.Compared with the PA group,after FABP4 expression,the mRNAs of FABP4,PERK,ATF6,IRE-la in FABP4 group and PA+FABP4 group were significantly increased,and the bigger rise in PA+FABP4 group;Compared with PA group,PA+HQS group,FABP4+HQS and PA+HQS+FABP4 group can significantly reduce the mRNAs of FABP4 and PERK,ATF6,IRE-la.And in FABP4 expression,the improvement of endoplasmic reticulum stress in PA+FABP4+HQS was lower than that in the PA+HQS and FABP4+HQS group.The above results suggest that Huang-Qi-San can inhibit the expression of FABP4,alleviate liver cells endoplasmic reticulum stress.Conclusion1.Both preventive and therapeutic of Huang-Qi-San can significantly reduce body weight,glucoselipid and subcutaneous and visceral fat,and improve the effects of liver tissue pathological changes and steatosis in obese rat,with weight loss and protecting liver function.2.FABP4 was expressed in adipose tissue and liver.Huang-Qi-San can significantly decrease FABP4,FFA levels in the blood and the mRNA and protein of FABP4 in the adipose tissue and liver,improve the endoplasmic reticulum stress in liver tissue.This suggests Huang-Qi-San can improve obesity and nonalcoholic fatty liver by regulating FABP4 and endoplasmic reticulum stress.3.At the cellular level,Huang-Qi-San also can obviously reduce the FABP4 level in the mature fat cells fluid,improve the PPAR gamma expression,to suggest it has good effect reducing weight.Huang-Qi-San can significantly improve the endoplasmic reticulum stress in liver cells.After FABP4 expression,the effect of Huang-Qi-San on the improvement of the endoplasmic reticulum stress is abate.Huang-Qi-San may inhibit FABP4 expression,alleviate the liver endoplasmic reticulum stress,improve hepatocyte fatty degeneration.4.The transcriptome sequencing results also confirmed that FABP4 can be significantly decreased under the effect of Huang-Qi-San.The other targeted gene also needs to continue to expand.