Study On The Effect And Mechanism Of Combined Administration Of Zhujiangtang Granules And Sitagliptin On Insulin Resistance In ZDF Rats

ObjectiveTo obersve the effect of Sitagliptin phosphate(SP)combined with Fuzhujiangtang granule(FJG)on insulin resisitance and metabolism in type 2 diabetes mellitus ZDF rats,to explore the mechanism of using SP-FJG for treating insulin resistance and to provide the evidenct of using SP-FJG in combination for clinical use.MethodsForty eight-week-old ZDF rats were feeding rodent chow(Purina#5008,Harlan Teklad,Indianapolis,IN)for 4 weeks.40 rats which the blood glucose>11.1mM were identified as successful diabetes mellitus models.A normal control group(N)of age-matched Zucker lean control rats were also included in the study and fed a standard laboratory chow diet throughout the study.All rats were housed under controlled conditions(12:12-h light-dark cycle,24℃,and 50%relative humidity)with free access to water and food.40 successful diabetes mellitus model rats were randomly divided into the following groups and treated as indicated:diabetes mellitus group(DM)group(deionized water),SP group(SP[9mg/Kg·d-1]),FJG group(FJG[0.64g/Kg·d-1]);SP-FJG group(SP[9mg/Kg ·d-1]and FJG[0.64g/Kg·d-1]).Besides,NC group was intragastrically administered with the same volume of deionized water.All treatments were given via oral gavage once a day,while the SP-FJG group were given FJG at 9am and SP at9pm a day.The drugs were orally administered for 6 weeks in different groups.The activity,spirit,diet,body posture of rats were recorded.Weekly monitor body weight,FBG(fasting blood-glucose)and RBG(random blood glucose)at 7am to 9am.ITT,OGTT was measured at sixth weeks of administration and the area under the curve(AUC)was calculated.The fast serum insulin levels,Leptin,Adiponectin,tumor necrosis factor a,interleukin-6,high density lipoprotein,low density lipoprotein,total cholesterol,triglyceride,superoxide dismutase,malondialdehyde,Glutamic oxaloacetic transaminase,UREA,Creatinine were ananlyzed by kits and performed according to the manufacturers’recommendations.Total RNA was extracted from liver and sequenced in a single lane of the Illumina HiSeqTM 2500,analyced with bioinformatic methords.Results1.SP-FJG had a better improvements in regulating blood glucose,lipid metabolism,glucose tolerance and improving insulin resistance than using FJG or SP separately.ZDF rats showed obvious type 2 diabetes syndromes after fed with induction rodent chow.SP-FJG had a better improvements in decreasing body weight,while SP could decrease body weight but not as much as SP-FJG.The weight of liver in DM rats were larger than NC group.SP-FJG reduced the weight increase of liver significantly.As for decreasing blood glucose,SP-FJG reduced blood glucose at 120 min in OGTT test.Besides,SP-FJG had a better effect in regulating fast serum insulin levels,TNF-α,IL-6compared with using SP individually.As for lipid metabolism,the increased LDL,TC caused by using SP was attenuated by using FJG in combination.2.SP-FJG regulates several metabolism pathways and interfering with biological progress as metabolism,transport and inflammatory response.SP-FJG improved insulin resistance by down regulating the expression of pik3rl,up regulate Irak3 and FGF21.Compared with the DM group,1781 different expressed genes(DEGs)(include 1248 mRNA,211 ncRNA,202 cirRNA and 120 miRNA)were enriched in 58 pathways.The trend genes of traditional Chinese medicine are mainly associated with sulfation,cellular detoxification of nitrogen compound etc.The efficacy genes of SP group mainly focus on the biological process,for example purine nucleotide biosynthetic process,innate immune response,cellular response to jasmonic acid stimulus.The trend gene of drug combination group are mainly involved in biological processes,flavonoid glucuronidation,xenobiotic glucuronidation,response to organic cyclic compound.Both SP group and combined drug effect genes regulate the pocess in response to nutrient.The biological processes of fructose transport is regulated by both the combination of drugs and the pharmacophore of Chinese Medicine.According to the biological pathway of pharmacophore regulation,the pathway of TNF sigling pathway,Hematopoietic cell lineagethe pharmacophore are regulated by both Chinese medicine and Western Medicine.The trend genes of combination and Chinese medicine are both regulated Metabolism of xenobiotics by cytochrome P450,Metabolic pathways,Maturity onset diabetes of the young,drug metabolism-cytochrome P450 and Chemical carcinogenesis pathway.The pharmacophore of Western medicine(SP)is specially regulate Toll-like receptor sigling pathway and other pathways.Pharmacophore of Chinese medicine specifically regulates Alanine aspartate and glutamate metabolism,Apoptosis pathway.Ascorbate and aldarate metabolism and other pathways are regulated by the pharmacophore of the combined drug.Through analysis of ceRNA networks,we found that rno-miR-326-3p,rno-miR-423-5p,rno-miR-15b-5p,rno-let-7c-5p,rno-let-7b-5p were related to pharmacodynamics in different groups.By analyze the protein-protein interaction(PPI)and co-expression networks of the transcriptomes of different groups,it’s inferred that Irak3 may be pharmacodynamic gen es for Type 2 Diabetes Mellitus(T2DM).Our research compared the treatment of SP3FJG and SP-FJG,and acquainted the PPI network,co-expression network,mutations and pharmacodynamics genes,which reveals the new mechanisms of pathogenesis of T2DM.By using the PPI and co-expression networks of the transcriptomes in different treatments groups,we generated highly connected modules were used to enrich the gene mutations in ZDF rats using WGCNA algorithm.Using string database,PPI network analysis found that Lrrk2,Irak3,and other18genes.RT-PCR was used to verify the expression of Irak3,Pik3r1,FGF21 etc.and confirmed that the sequencing was reliable.Besides,5SP-FJG improved insulin resistance by down regulating the expression of pik3rl,up regulate Irak3 and FGF21.ConclusionThe effect of SP-FJG in improving insulin resistance is better than using FJG or SP separately.The mechanism relies on its effect on interfering with multiple metabolism pathways and biological progress as metabolism,transport and inflammatory response.SP-FJG improved insulin resistance by down regulating the expression of pik3rl,up regulate Irak3 and FGF21.