Effect And Mechanism Of Zishen Pills On Insulin Resistance In Db/db Mice With Type 2 Diabetes Mellitu

Objective:To evaluate the effect of Zishen Pill(ZSP)on glucolipid metabolism and insulin resistance of genetic type 2 diabetic model db/db mice,explore the underlying mechanisms for its antidiabetic effects based on transcriptome analyses,and provide evidence for clinical use.Methods:1.ZSP was prepared with the formular of Rhizoma Anemarrhenae:Cortex Phellodendri:Cinnamomum cassia=10:10:1.Liquid chromatography(LC)-mass spectrometry(MS)/MS analysis was used to detect the compounds in ZSP.Extracts were dissolved by double distilled water before the LC-MS/MS analysis.2.A total number of 14 six-week-old male C57BL/KsJ-db/db mice and 7 C57BL/KsJ-wt/wt mice of the same age and gender were used for animal experiments.Db/db mice were divided into two groups(ZSP group,receiving 3.3g/kg ZSP by gavage;model group,receiving distilled water by gavage).Wt/wt mice were used as normal group(receiving distilled water by gavage).The treatment lasted for 40 days.The body weight,food intake and fast glucose levels were recorded.Oral glucose tolerance test(OGTT)was conducted on mice after over-night fasting at day 35.After 40 days of treatment,mice were sacrificed after anesthesia by isoflurane.Blood was drawn and centrifuged to obtain the serum.Liver and adipose tissues were taken out and weighed.Hematoxylin&eosin(H&E)staining was conducted for morphological observation,oil red O staining and periodic acid-Schiff(PAS)staining was performed to examine the lipid accumulation and glycogen content,respectively.Serum lipids including total cholesterol(TC),triglyceride(TG),high-density-lipoprotein(HDL)and low-density-lipoprotein(LDL),as well as hepatic function indicator alanine transaminase(ALT)and aspartate aminotransferase(AST)levels were measured.Fast serum insulin levels were measured by enzyme-linked immunosorbent assay(ELISA).Homeostatic model assessment for insulin resistance(HOMAIR)was calculated,in order to observe the effect of ZSP on the glucolipid metabolic process of db/db mice.3.Transcriptome analyses were performed on the livers,adipose tissues and skeletal muscles of mice,threshold for differentially expressed genes(DEGs)were defined as P value<0.05 and foldchange>1.5.For functional enrichment,Gene Ontology(GO)database and Kyoto Encyclopedia of Genes and Genomes(KEGG)database were used to annotate and analyze identified DEGs.4.Experiments were performed to confirm the mechanisms identified from results of transcriptome analyses.The mRNA expression of PI3K encoding genes Pik3ca,Pik3cb,Pikscd,Pik3cg and AKT encoding genes Akt1.Akt2 of liver and adipose tissues were tested by qPCR.Hk2,Hk3,Gck,Pfkb1,G6pase,Pepck,Gys,Insr,Irs1,Irs2,FltS,Csf1,Hgf,Vegfd,Pdgfc,Angpt2 mRNA expression in the liver were also detected by qPCR.P21,Vegfa,Pdgfd,Irs1,Irs2 mRNA expression in the adipose tissues were also detected by qPCR.Western blotting analyses were performed to examine the protein expression of PI3K,P-PI3K,AKT,P-AKT in the liver and P21 in the adipose tissues.Immunohistochemistry was used to analyze the protein expression of GYS in the liver.Results:1.Compounds from Rhizoma Anemarrhenae including mangiferin,isomangiferin,neomangiferin,timosaponin A Ⅲ and timosaponin BⅡ;compounds from Cortex Phellodendri including jatrorrhizine,berberrubine,berberine,epiberberine,tetrahydroberberine THB,demethyleneberberine and phellodendrine chloride;compounds from Cinnamomum cassia including cinnamic acid,cinnamaldehyde and trans-cinnamic acid were detected form ZSP solution.2.Compared with wt/wt mice in normal group,db/db mice in model group showed significant obesity and hyperglycemia.ZSP administration significantly reduced the fasting blood glucose levels and body weight of db/db mice.ZSP treatment significantly reduced weight of liver and epididymal adipose,improved glucose tolerance and HOMA-IR,decreased serum TC levels,ameliorated hepatic lipid accumulation and pathological changes in liver,adipose tissues and skeletal muscle,as well as increased the glycogen content in mice livers.Moreover,ZSP treatment had no detectable impact on the body length,food intake,water intake and serum AST and ALT levels of db/db mice.3.The data of transcriptomic analyses showed no significant abnormity in the quality assessment,indicating the reliability of transcriptomic data.ZSP showed substantial regulatory effects on the liver and adipose transcriptome of db/db mice.In the liver,DEGs were mainly enriched in glucokinase activity,hexokinase activity and fructokinase activity terms,and gene expression in PI3K/AKT pathway were significantly altered.Furthermore,ZSP also upregulated some genes encoding growth factors and receptor tyrosine kinases(RTK).In adipose tissue,the DEGs were mainly enriched in cholesterol metabolism processes.ZSP also upregulated several genes encoding the upstream factors of PI3K/AKT pathway and downregulated the transcription of P21.4.In the livers,compared with model group,ZSP significantly upregulated the mRNA expression of PI3K encoding genes Pik3ca,Pik3cb,Pikscd,Pik3cg and AKT encoding genes Akt1.Akt2,and induced the protein expression of PI3K,P-PI3K,AKT,P-AKT and GYS.It significantly upregulated the transcription of Hk2,Hk3,Gck,Pfkb1,G6pase,Gys,Flt3,Csf1;downregulated the mRNA expression of G6pase.In adipose tissues,compared with model group.ZSP significantly upregulated the mRNA expression of PI3K encoding genes Pik3ch,Pikscd.Pik3cg and AKT encoding genes Akt2,inhibited the mRNA and protein expression of P21.Meanwhile,it also induced the mRNA transcription of Vegfa,Pdgfd,Irs1,Irs2.Conclusions:1.ZSP had positive effects on the glucolipid metabolism of db/db mice and improved the insulin sensitivity.ZSP at dose of 3.3 g/kg showed no toxic or side effect on db/db mice.2.ZSP had substantial impact on the liver and adipose transcriptome of db/db mice but did not significantly alter the skeletal muscle transcriptome.In the liver,ZSP activated the PI3K/AKT signaling pathway via upregulating growth factors and RTKs.This further promoted glycogen synthesis and glycolysis,inhibited gluconeogenesis and glycogenolysis and improved glucose metabolism of db/db mice.3.In adipose tissues.ZSP activated PI3K/AKT signaling pathway by upregulating multiple upstream factors including growth factors and IRS.This further inhibited the transcription and translation of P21 and improved insulin sensitivity and reduced obesity.