Preliminary Study On Hypoglycemic Effect And Mechanism Of Extract Of Pressed Degreased Walnut Meal In T2DM Rats

Objective:Extract of pressed degreased walnut meal from Yunnan province(WMP)was used as candidate material.The type 2 diabetes rat model induced by high-fat diet combined with low-dose STZ was used to study the mechanism of WMP on the prevention and treatment of type 2 diabetes.Methods:1.The preventive effect of pressed degreased walnut meal extract(WMP)on type 2diabetes(T2DM): 43 SD male rats were randomly divided into normal control group(CD),model group(DM),low dose WMP group(WMP-L),and high dose WMP group(WMP-H).The CD group was fed with basic feed,the DM group fed with high-fat diet,WMP-L and WMP-H groups fed with high-fat feed and also supplemented with 500 and1000 mg/kg/d WMP,an equal amount of normal saline was given to CD and DM rats.After continuous feeding for 6 weeks,the CD group was intraperitoneally injected with citric acid buffer,and the remaining groups intraperitoneal injected with STZ(35mg/kg)for modeling.After a week later,the fasting blood glucose(FBG)of rats was determined,and the rats were considered to be T2 DM model when the FBG level of DM group exceeded 11.1 mmol/L.2.The therapeutic effect of WMP on T2 DM rats: 85 SD male rats were fed with a basic feed,after a week of adaptive feeding,10 rats were randomly selected as normal control group(CD)fed with normal feed,and the remaining 75 rats were fed with high-fat feed.After continuous feeding for 4 weeks,STZ(35mg/kg)was intraperitoneally injected for 75 rats to induce T2 DM rat model.Rats with successful modeling were randomly divided into model group(DM),positive group(MET,150mg/kg/d),low dose WMP group(WMP-L,125mg/kg/d),medium dose WMP group(WMP-M,250mg/kg/d),and high dose WMP group(WMP-H,500mg/kg/d),continuous intragastric administration for 5 weeks.Measured FBG,body weight,OGTT,TG,TC,LDL-C,HDL-C,ALT,AST,MDA,SOD,CAT,GSH,NO,FINS,GSP,IL-6,LPS,TNF-? and other indicators of rats;the morphological changes of liver and pancreas were observed by HE staining;rat feces were collected for high-throughput sequencing of bacterial 16 S rRNA gene to analyze the effects of WMP on the intestinal flora of T2 DM rats;Sequence the transcriptome of liver tissues in all rats so as to analyze the possible metabolic pathways and targets regulated by WMP.Results:1.The results of the preventive effect of WMP on T2 DM rats showed that: compared with the normal group,the weight of the model rats gradually decreased(p<0.01),oral glucose tolerance level was impaired,the AUC,GSP,FBG,FINS,HOMA-IR,and QUICK index increased significantly(p<0.001);TC,TG,LDL-C,ALT,AST,LPS,IL-6,TNF-?levels of serum and liver significantly increased(p<0.01),liver HDL-C levels significantly reduced(p<0.01);serum MDA(p<0.01)and NO(p<0.001)levels increased,SOD and CAT activity decreased(p<0.001);Hepatocytes was severely steatotic and swollen in volume;the number of islet cells was little,the volume was increased,and the shape was extremely irregular.Compared with the model group,WMP-L and WMP-H groups could significantly delay the weight loss of T2 DM rats(p<0.05,p<0.01),reduced the AUC(p<0.01,p<0.001),GSP(p<0.01),FBG(p<0.01),FINS(p<0.001),HOMA-IR(p<0.001),QUICK index(p<0.01),and TC,TG,LDL-C,ALT,AST,MDA,NO,LPS,IL-6,TNF-?levels of serum and liver(p<0.05,p<0.01,p<0.001);increased SOD and CAT activity(p<0.05,p<0.01,p<0.01);reduce the pathological changes of rat liver and pancreas,increase the number of islet cells.Compared with the normal group,the abundance and diversity of intestinal microflora in the model group decreased,the Firmicutes was increased,Bacteroidetes was decreased,the F/B ratios was significantly increased;compared with the model group,the abundance and species diversity of intestinal flora in the WMP group were increased,and the abundance of some beneficial bacteria butyrate-producing bacteria,such as Bacteroidaless 247 group,Lachnospiraceae,and Ruminococcaceae were increased.2.The results of the therapeutic effect of WMP on T2 DM rats showed that: compared with the model group,after 5 weeks of WMP treatment,the body weight of WMP-M and WMP-H rats significantly increased(p<0.05,p<0.01);the water intake and excretionvolume decreased significantly(p<0.01);the WMP-L,WMP-M,and WMP-H groups all reduced FBG levels(p<0.01,p<0.001),AUC level(p<0.001),FINS(p<0.05,p<0.01,p<0.001),HOMA-IR(p<0.001),HOMA-?(p<0.001),and GSP(p<0.001);increased ISI(p<0.01,p<0.001),reduced TC,TG,LDL-C,AST,ALT,MDA,LPS,IL-6,and TNF-?levels in serum and liver of rats(p<0.05,p<0.01,p<0.001),increased SOD(p<0.01,p<0.001)and CAT activity(p<0.05);the number of islet cells in the three WMP groups increased,the number of hepatic cell steatosis decreased,and pancreatic and liver tissue lesions were alleviated.The results of the therapeutic effect of WMP on gut bacteria flora in T2 DM rats showed that after WMP treatment,the diversity of intestinal microbes in T2 DM rats was significantly improved,and the compositional similarity of OTU in CD group increased;WMP could increase the relative abundance of Prevotellaceae,Lactobacillaceae,Ruminococcaceae,Lactobacillus,Prevotella-9,Romboutsia,unclassified_f_Prevotellaceae,and reduced the relative abundance of Firmicutes,norank_f_Muribaculaceae,Turicibacter,Anaerovibrio,Fusobacterium,Megamonas,Marvinbryantia in T2 DM rats.3.The results of rat liver RNA transcriptome showed that the number of genes commonly expressed in the CD,DM and WMP groups was 10600,the number of genes expressed in the WMP group and the CD group(115)was more than the number of genes expressed in the DM group and the CD group(88).The GO analysis found that genes regulated by WMP,and whose expression changes significantly were mainly focused on four processes: cell part,binding,cellular process,and metabolic process.Comparison and analysis with the KEGG database found that the differentially expressed genes mainly involved AMPK signaling pathway,PPAR signaling pathway,steroid biosynthesis,terpenoid backbone biosynthesis,cholesterol metabolism,insulin secretion,insulin signaling pathway,alanine,aspartate and glutamate metabolism.Key genes with significant expression included Gck,RT1-Ba,Fasn,Got1,Myh7 b.ConclusionWMP could significantly prevent and improve abnormal glycolipid metabolism,slow down weight loss,improve insulin resistance,reduce the abnormally elevated levels of FBG,blood lipids,liver lipids,and oxidative stress levels,the number of liver damage and hepatic cell steatosis,body inflammation level,the pathological changes of pancreatictissue,improve the diversity of intestinal microbes in T2 DM rats,and inhibit the growth of harmful bacteria.WMP mainly regulated the expression of energy metabolism,amino acid metabolism,glycolipid metabolism and insulin secretion and regulated the key genes expression of Gck,RT1-Ba,Fasn,Got1,Myh7 b to play an important role in abnormal glycolipid metabolism of T2 DM rats.