Research On The Improvement Of Ramulus Mori(Sangzhi)Alkaloids On Adipose Tissue Metabolism And Inflammation In High-fat Diet-Induced Obese Mice

Objective:Ramulus Mori(Sangzhi)alkaloids(SZ-A),derived from Morus alba L.were used to treat type 2 diabetes(T2DM).Furthermore,SZ-A has been found to have various pharmacological effects in its clinical application,such as weight reduction and blood lipid regulation.Based on these results,this study was conducted to assess the efficacy of SZ-A on weight loss in simple obese mice.At the same time,we also studied the pharmacological effects and possible mechanisms of SZ-A on adipose tissue.3T3-L1 preadipocytes were also used as a model to investigate whether SZ-A has direct regulatory effects on adipocytes in this study.Methods:1、6-week-old C57BL/6J male mice were fed a high-fat diet continuously for 12 weeks and the weights of both groups were weighed and recorded weekly.When the body weight of mice in the model group increased by 20%compared to the normal group,the obese mouse model was considered to be successful.2、As the obese mouse model was successfully constructed,the mice were randomly divided into 5 groups:normal group,model group,SZ-A 100,300,and 400 mg/kg group.The mice in each group were treated daily by gavage,and the normal and model group were given the same volume of sanitary saline.During the administration period,mice in all groups except the normal group were still fed a high-fat diet.3、After 6 weeks of drug intervention,the body weight of mice in each group was compared,and an in-depth study was conducted on mice in the 400mg/kg group.The levels of lipids in mice were detected by a fully automatic biochemical analyzer;the levels of serum adiponectin in each group of mice were detected by ELISA kit;the levels of leptin and inflammatory factors in mice serum were detected using a mouse multifactor test kit.4、The morphological and structural changes of adipose tissue in mice of all groups were observed;the molecular biology of indicators related to lipid metabolism and inflammation in epididymal adipose tissue were analyzed by Western-blot and qRT-PCR.Functional enrichment of differential genes from transcriptome gene sequencing of mouse epididymal fat was analyzed.Immunohistochemical analysis was performed with F4/80 and CD86-labeled macrophages.5、3T3-L1 preadipocytes were treated with different concentrations of SZ-A.And the supernatants and proteins were collected for adipokine and protein assays.Results:1、After 6 weeks of drug intervention,mice in the SZ-A treated group showed a significant decrease in body weight,with the greatest decrease in the SZ-A 400 mg/kg group.Compared with the model group,the blood lipid level of the mice in the SZ-A 400 mg/kg group was significantly improved and the lipid droplets in the adipose tissue were significantly reduced.2、Compared with the model group,the mice in the SZ-A 400 treated group,the Phosphorylated acetyl coenzyme A carboxylase,triglyceride dismutase,and hormone-sensitive triglyceride dismutase protein expression and gene transcript levels were significantly increased in the epididymal adipose tissue(eWAT).Meanwhile,the peroxisome proliferator-activated receptor alpha and carnitine ester acyltransferase 1 A transcripts were increased.3、In the SZ-A 400 group,the serum inflammatory factors level of mice was reduced while the adiponectin level was increased.Down-regulated gene function enrichment pathways in epididymal adipose tissue focused on inflammatory response pathways.The macrophages were reduced in the eWAT,while pro-inflammatory factor transcription was inhibited and anti-inflammatory factor transcription was promoted.4、SZ-A promoted adipocyte secretion of lipocalin while inhibiting leptin,and increased protein expression of peroxisome proliferator-activated receptor alpha and phosphorylation levels of acetyl coenzyme A carboxylase.Conclusion:This study demonstrated that SZ-A could regulate adipokine secretion and protein expression in adipocytes,inhibit the fatty acid synthase expression,and promote the level of triglyceride catabolic enzymes in the eWAT,and thus reduce fat accumulation and inhibit weight gain resulting from the high-fat diet.In addition,the effect of SZ-A on ameliorating chronic inflammation levels in obesity-induced adipose tissue,including macrophage infiltration,at least in part through inhibition of Toll-like receptor pathways.Therefore,SZ-A,a natural anti-diabetic drug,has been shown to be effective in improving lipid metabolism and adipose tissue inflammation in HFD-induced obese mice.