Study On The Effect And Mechanism Of Fufang Lvliu Granules On Improving Insulin Resistance In Type 2 Diabetic ZDF Rats

ObjectiveLLKL consists of Edgeworthia gardneri(Wall.)Meisn.,Sibiraea angustata and Crocus sativus L.(saffron).This study was conducted to evaluate the effects of LLKL on insulin resistance in ZDF(Zucker Diabetic Fatty)rats and whether LLKL had a better effect than individual herbs in LLKL,and explore the underlying mechanism providing evidence for it’s use in treating T2DM patients.MethodsAfter being induced-feeding with pumina#5008 diet for 4 weeks,male ZDF(fa/fa)rats with the FBG(Fasting blood glucose)higher than 7.8 mmol/L were consider as diabetic rats.According to the FBG and body weight(BW),64 diabetic rats were randomly assigned to the following 8 groups:diabetic model group(MOD,n=8),LLKL high dose group(LLKL_H,n=8),LLKL middle dose group(LLKL_M,n=8),LLKL low dose group(LLKL_L,n=8),Edgeworthia gardneri(Wall.)Meisn.group(n=8),Sibiraea angustata group(n=8),Crocus sativus L.(saffron)group(n=8)and Metformin group(MET,n=8).8 age-matched ZLN(Lean Zucker Lean Normoglycemic,+/fa)rats were used as normal control group(NC,n=8).Each group orally administered for 6 weeks(once a day)as follow:LLKL_H:4.68 g/kg/d,LLKL_M:2.34 g/kg/d,LLKL_L:1.17 g/kg/d,Edgeworthia gardneri(Wall.)Meisn.:1.35 g/kg/d,Sibiraea angustata:0.9 g/kg/d,Crocus sativus L.(saffron):0.09 g/kg/d.Metformin at a dose of 0.135 g/kg/d was used as the positive control to evaluate the hypoglycemic effectiveness in this study.NC group and MOD group were administered with lm/100g deionized water.BW and FBG were measured once per week.After 6 week-treatment,food intake was recorded and OGTT and ITT were performed.Then,after 12 h-fasting,all rats were anaesthetized with 1%sodium pentobarbital(45 mg/kg),and the blood samples were collected and centrifuged at 3000 g,4℃ for 15 min to obtain serum.The liver was separated and weighed,and the liver weight index was calculated(Liver weight/BW).Faeces,liver and intestinal tissues were quickly collected and fixed with 4%paraformaldehyde or frozen in liquid nitrogen and then stored at-80℃ until use.The FINS(Fasting insulin),FFA(Free fatty acids),LPS(Lipopolysaccharide),IL-6(Interleukin-6)and TNF-α(Tumor necrosis factor)were measured.Liver tissue was measured by Haematoxylin and Eosin(HE staining),PAS and oil red O staining.TG(Triglyceride)and TC(Total cholesterol)of liver were measured.HE staining and Occludin IHC(immunohistochemical)staining were performed to observe the effect of LLKL on intestinal tissue.Liver transcriptome sequencing and bioinformatics analysis were performed to explore the effect of LLKL on liver in ZDF rats.16S rDNA sequencing were carried out to explore the effect of LLKL on the gut microbiota in ZDF rats.Results1 Effect of LLKL on insulin resistance of ZDF rats①The effect of LLKL on insulin resistance:compared to the NC group,BW,food intake,FBG,OGTT-AUC,ITT-AUC,FINS and HOMA-IR in MOD group were significantly increased(P<0.001).Compared with the MOD group,the levels of FBG,OGTT-AUC,ITT-AUC.FINS,HOMA-IR in LLKL_H,LLKL_M,LLKL_L and MET groups were decreased(P<0.05,P<0.01 or P<0.001).After 6-week treatment,compared to the MOD group,there was no significant change of BW and food intake in LLKL_H,LLKL_M,LLKL_L and MET groups(P>0.05).These results provide evidence to suggest that LLKL treatment improves glycemic control and insulin resistance in ZDF rats,and LLKL_H shows the best effect,which was better than the MET(0.135 g/kg/d).②Comparison of the effects of LLKL and individual herbs in LLKL on insulin resistance:after 6-week treatment,compared to the MOD group,Edgeworthia gardneri(Wall.)Meisn.,Sibiraea angustata and Crocus sativus L.(saffron)groups decreased the FBG,OGTT-AUC,ITT-AUC,FINS and HOMA-IR(P<0.05,P<0.01 or P<0.001),which were higher than the LLKL_M group.Compared to the MOD and LLKL_M groups,there was no significant change of BW and food intake in Edgeworthia gardneri(Wall.)Meisn.,Sibiraea angustata and Crocus sativus L.(saffron)groups(P>0.05).Taken together,these data provide evidence to suggest that Edgeworthia gardneri(Wall.)Meisn..Sibiraea angustata and Crocus sativus L.(saffron)treatment also improve glycemic control and insulin resistance in ZDF rats,while LLKL has a better effect than the individual herbs.2 Effects of LLKL on liver glucose and lipid metabolism and transcriptome in ZDF ratsAfter 6-week treatment,compared with NC group,the HE and Oil Red O staining revealed an obviously increase in the number and size of the lipid droplets,and morphology injury in MOD group which were reversed by LLKL_H,LLKL_M and LLKL L treatments.In addition.the PAS staining of liver showed a significantly lower glycogen deposition in MOD group compared to the NC group,while administration with LLKL promoted the glycogen accumulation.Consistently,the liver weight index was enhanced in MOD group,while LLKL_H,LLKL M and LLKL L treatments decreased the liver weight index.Moreover.compared to the NC group,the levels of serum FFA,liver TC and liver TG in MOD group were notably evaluated(P<0.001),and these effects were reversed by LLKL_H,LLKL_M and LLKL_L treatments(P<0.05,P<0.01 or P<0.001).In conclusion,these results reveal an effectively protective role of LLKL on hepatic morphology recovery and lipid metabolism in ZDF rats.The results of liver transcriptome analysis:we identified 1,495 differentially expressed genes(DEGs)in the comparison of MOD group and NC group,and 740 DEGs were detected in the LLKL_H versus MOD groups.The results of functional annotation analysis revealed that:DEGs in MOD vs.NC group were significantly associated with the biological processes such as go:0055114～oxidation reduction process,go:0006468～protein phosphorylation,and were involved in the metabolic pathways(rno01100:metabolic pathways)and oxidative phosphorylation(rno00190:oxidative)KEGG signaling pathways.DEGs in LLKL_H vs.MOD were significantly associated with protein translation(GO:0006412～translation),cellular response to DNA damage stimulus and other biological processes and Metabolic pathways(RNO01100:Metabolic pathways)and Toll-like receptor signaling pathway(RNO04620:Toll-like receptor signaling pathway)KEGG pathways.RT-qPCR verified that TLR4,CTSK and MyD88 were highly expressed in the MOD group(P<0.001)and decreased in the LLKL_H group(P<0.001),indicating that the toll-like receptor signaling pathway might play an important role in the treatment of LLKL.3 Effect of LLKL on small intestine and gut microbiota of ZDF ratsAfter 6-week treatment,MOD group displayed abnormal morphological alterations characterized by loss of normal villus structure of the small intestine epithelium,including disorganized,collapsed villi and lower villus height and crypt depth while these disorders were restored by LKL_H.LLKL_M and LLKL_L treatments,suggesting that LLKL could diminish the intestinal epithelial villus damage.Occludin exhibited lower expression in MOD group compared to the NC group(P<0.001),whereas LKL_H,LLKL_M and LLKL_L administration increased Occludin expression level(P<0.001).The serum LPS and inflammatory cytokines,including TNF-α and IL-6 were detected,compared to the NC group,elevated LPS,IL-6 and TNF-α levels were observed in the MOD group(P<0.001),while LKL_H,LLKL_M and LLKL_L treatment significantly reduced LPS,IL-6 and TNF-α levels(P<0.01 or P<0.001).These results support the idea that intestinal epithelial barrier damage is diminished by administration of LLKL in ZDF rats,resulting in a reduction in LPS and inflammatory cytokines release from the gut into the bloodstream.The results of 16S rDNA sequencing:the a diversity and β diversity of the MOD group were significantly lower than that of the NC group,and the a diversity and β diversity of LLKL_H and LLKL_M groups were significantly higher than that of the model group.At taxonomic level,the top 5 phyla in 4 groups are Firmicutes,Actinobacteria,Proteobacteria,Bacteroidetes and Verrucomicrobia.Compared to the NC group,the abundance of Bacteroidetes in the MOD group was lower,Firmicutes was higher and Bacteroidetes/Firmicutes was significantly lower.Compared with the MOD group,the abundance of Bacteroidetes in LLKL_H and LLKL_M administration groups increased,Firmicutes decreased and Bacteroidetes/Firmicutes increased significantly.The results of GSEA analysis:high expression of MyD88 was positively correlated with glycerolipid metabolism(NES=1.347158,P=0.05814,FDR q=1)and negative correlated with insulin signaling pathway(NES=-1.0536,P=0.388614,FDR q=1),while high expression of CTSK was positively correlated with fatty acid metabolism(NES=1.626464,P=0.008032,FDR q=0.469346)and glycerolipid metabolism(NES=1.515651,P=0.071567,FDR q=0.602147)and negative correlated with insulin signaling pathway(NES=-1.3491,P=0.044922,FDR q=1).These results suggested that the insulin signaling pathway was enriched and upregulated in LLKL_H treatment group,while glycerolipid metabolism and fatty acid metabolism were implicated and downregulated in LLKL_H treatment group.Conclusion1 LLKL administration improves glycemic control and insulin resistance in ZDF rats,which display a better effect than individual herbs involved in LLKL2 LLKL administration improves the glucose and lipid metabolism of the liver in ZDF rats,and the mechanism may be related to its regulation of cellular response to DNA damage stimulus,Metabolic pathways,toll-like receptor signaling pathway and other biological processes.RT-qPCR verified that TLR4,CTSK and MyD88 were highly expressed in the MOD group and decreased in the LLKL_H group,indicating that the toll-like receptor signaling pathway might play an important role in the treatment of LLKL3 LLKL ameliorates hyperglycemia,modulates the gut microbiota in ZDF rats,increases gut microbiota diversity,increases Bacteroidetes,reduces Firmicutes which results of a increased proportion of Bacteroidetes/Firmicutes.Moreover,LLKL protects the intestinal,increases intestinal barrier function and reduces LPS and inflammatory to regulate the toll-like receptor signaling pathway.LLKL decreases the expression of CTSK and MyD88 to inhibit the fatty acid metabolism and glycerolipid metabolism and enhance insulin signaling pathway which contribute to the glycemic control and insulin resistance.