| ObjectTo observe the effect of Fuzhu-Jiangtang Granule(FJG)combined with Metformin(Met)on insulin resistance(IR)in type 2 diabetes mellitus(T2DM),and to explore the mechanism with with high-throughput sequencing of microRNAs and molecular biology.MethodsMethods of animal experiment:28 spontaneous T2DM animal model,ZDF(fa/fa)rats,was used.After being induced-feedingwith pumina#5008 dietfor 4 weeks,the rats with random blood glucose higher than 11.1 mmol/L were used as diabetic rats.According to body weight and random blood glucose,ratswere randomly divided into model group,Metformin group(Met group),Fuzhu-Jiangtang Granule group(FJG group),Fuzhu-Jiangtang Granule combined with Metformin group(Combined group),and 7 rats were used in each group.7 normal ZDF(fa/+)rats were usedas normal group.All rats were treated for continuous 6 weeks.During the experiment,the general condition,body weight,fasting blood glucoseof rats wereobserved,OGTT and ITT were detected respectively at the sixth week.After 6 weeks,serum FBG,Fins,HOMA-IR,TG,TC,FFA,AST,Scr,BUN,MDA,SOD,CAT,TNF-alpha,Adiponectin,Resistin and hepatic glycogen content were measured,HE and PASstainingdetects the pathological changes of liver.The different expression microRNAs of liver were detected by high-throughput sequencing,and thefunction and pathway of different expresse-microRNAs were predicted with bioinformatics analysisof NCBI,KEGG,TIGR,Gene,ontology and miRBase.Phosphorylation level of IRSlser307/ser612/ser1101/Tyr989,PI3K,Akt,GSK-3 were measured by Westemblot,the expression InsR,G-6-P,PEPCK mRNA were measured byReal time-PCR,and all these were used for verifingthe results of microRNAs bioinformatics analysis.Methods of cellexperiment:50 male SD rats were randomly divided into normal group,Metformin group(Met group),Fuzhu-Jiangtang Granule group(FJG group)and Fuzhu-Jiang tang Granule combined with Metformin group(Combined group),10 rats in each group.All rats were administered 2 times each day for 7 times toget medicated serum.H4IIE liver cell was culturedconventionally,and induced bymedium containg 10-6mol/L insulin for 36 h to establish insulin resistance modelhepatic cell.After treated bymedicated serum,cell activity was detected by CCK-8,the gene and protein expression of key targets in insulin signaling way were detectedby Western blot and Real time-PCR.ResultsAnimal experiment results:1.serum detection:?the improvement of IR and lipid metabolism,comparing with normal rats,serum FBG,Fins and HOMA-IR of rats in model group increased significantly(P<0.01 or P<0.05),OGTT and ITT peak delayed(P<0.01 or P<0.05),weight,the liver weight and liver weight/body weight,TG,TC and FFA were significantly increased(P<0.01 or P<0.05),and hepatic glycogen content decreased significantly(P<0.01).Comparing withmodel group,the combined group differently decreased FBG,Fins,HOMA-IR,body weight,liver weight,liver weight/body weight and TG,TC and FFA(P<0.01 or P<0.05),significantly increased the content of hepatic glycogen(P<0.01),and the effects is better than Met group and FJG group(P<0.01 or P<0.05).?The improvementof oxidative stress and cytokines:comparing with normal rats,serum MDA,Resistin,TNF-alpha of rats in model groupwere significantly increased(P<0.01 or P<0.05),while SOD,CAT and Adiponectin were significantly decreased(P<0.01 or P<0.05).Comparing withmodel group,serum MDA,Resistin,TNF-alpha of combined group ratswas significantly decreased(P<0.01 or P<0.05),SOD,CAT and Adiponectin levels were significantly higher(P<0.01 or P<0.05).The effects of improving SOD,CAT,MDA,Adiponectin,TNF-alpha incombined groupis better than Met group,FJG group(P<0.01 or P<0.05).?The improvement of liver and kidney function:there is no difference of AST in the groups.Scr and BUN in model group ratswere significantly higher than those of the normal group(P<0.01),butMet group,FJG groupand combined group werelower than these in model group(P<0.01).Comparing with Met group,Scr incombined groupdecreased significantly(P<0.05).2.Morphological detection:HE staining showed that arrangement,fatty degeneration and infiltration of inflammatory cells of liver cells in the treated group were better than model group.PAS staining showed that the treated group in the cytoplasm of glycogen granules distribution increasedcompared with model group,and the effect in combined group was more obvious.3.Bioinformatic analysis of microRNAs:After 6 weeks of drug intervention,of liver microRNAs sequencing andgene difference trend wereanalyzed.Comparing with normal group and model group,the expression of rno-miR-182,rno-miR-33-3p,rno-miR-33-5p,rno-miR-3553,rno-miR-96-5p in Met group exhibiteddifferent trends;the expression ofrno-miR-122-3p,rno-miR-33-3p,rno-miR-33-5p and rno-miR-22-3p inFJG group exhibited different trends;the expression ofrno-miR-122-3p,rno-miR-33-3p,rno-miR-33-5p group,rno-miR-742-3p,rno-miR-22-3p incombined group exhibited different trends(P<0.01 or P<0.05).The target gene prediction,functional analysis and Pathway analysis of difference trend microRNAs showed that Met group,FJG group and combined group significantly affected insulin signal transduction,and combination group was significantly stronger than Met group,FJG group.4.Western blot and Real time-PCR detection:Compared with the model group,the phosphorylation level of IRS1 ser307/ser612/ser1101 in liverof combined group decreased significantly(P<0.01 or P<0.05),phosphorylation level ofIRS1 Tyr989,PI3K,Akt,GSK-3 protein was significantly increased(P<0.01 or P<0.05),the expression of mRNA InsR increased significantly(P<0.01),the expression of G-6-P and PEPCK mRNA decreased significantly(P<0.01).Compared with Met group and FJG group,the effects of combined group regulated the protein and gene expression in insulin signaling way is better significantly(P<0.01 or P<0.05).Cell experiment results:The activity of cells in model group was significantly lower than normal group(P<0.01);Met group and FJG groupand combined group increased significantly compared with model group(P<0.01).The activity of cells in combined group was significantly higher than that ofMet group and FJG group(P<0.01 or P<0.05).The phosphorylation of IRS1ser307/ser612/ser1101 protein was significantly decreased(P<0.01 or P<0.05),phosphorylation level of IRS1 Tyr989,PI3K,Akt,GSK-3 protein was significantly increased(P<0.01 or P<0.05),the expression of InsR mRNA increased significantly(P<0.01),and the expression of G-6-P and PEPCK mRNA decreased significantly(P<0.01).Regulationeffects ofprotein and gene expression in insulin signaling way of combined group is better than Met group and FJG group(P<0.01 or P<0.05).ConclusionsFJG combined with Met significantly improved IR,disorders of glucose-lipid metabolism and glycogen synthesisin diabetic rats,and the effects is better than Met and FJG alone,the mechanism may influencethe expression of IRS1,PI3K,Akt,GSK-3,G-6-P,InsR,PEPCK in insulin signaling way by regulating the expression difference of microRNA.In addition,the combined applicationhas the effects of antioxidant,regulating cytokines Adiponectin,Resistin,TNF-alpha,which maybe also one of the possible mechanism. |
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