Effects Of The Active Ingredient Of Jiaotaiwan On The Orexin A/HPA Axis And Skeletal Muscle GLUT4 In T2DM Rats

ObjectiveType 2 diabetes mellitus(T2DM)is a chronic disease characterized by elevated blood sugar and is a serious health hazard.Among them,insulin resistance(IR)is an important onset feature of T2DM.Pharmacological studies have shown that IR is closely related to the function of the orexin A/hypothalamus-pituitary-adrenal(HPA)axis pathway,and the Orexin A/HPA axis pathway is a potential target for improving IR.The neuroendocrine regulation of the Orexin A/HPA axis pathway in the pathogenesis of IR is similar to that of the Chinese medicine "heart and kidney intersection" theory.The Ming Dynasty "Han Shi Yi Tong" classic prescription Jiataiwan is made up of 10:1 compatibility of Rhizoma Coptidis and Cinnamon.It is a representative of the "traffic heart and kidney" water and fire treatment,the clinical efficacy of this side has been shown to improve IR and IR-induced glucose and lipid metabolism disorders,but its active ingredients and mechanism of action are still unclear.Previous studies have shown that Jiaotaiwan can regulate the Orexin A protein of Orexin A/HPA axis pathway.Therefore,the hypothesis is that the mechanism of action of Jiaotaiwan to improve IR may be related to the regulation of the Orexin A/HPA axis.Based on the above assumptions,this study established the fingerprint of Jiaotaiwan by HPLC method to further determine its main active components.On this basis,the effects of the main active components of Jiaotaiwan on the Orexin A/HPA axis pathway and skeletal muscle glucose transport 4 in T2DM rats were observed.To clarify the mechanism and material basis of Jiaotaiwan to improve IR,lay a foundation for the clinical application and development of this side,and further clarify the scientific connotation of TCM "heart and kidney intersection" theory.Methods1.Establishment of HPLC fingerprint of JiaotaiwanOptimize the extraction method of Jiaotaiwan by single factor investigation,and optimize different mobile phases,the pH of buffer salts,detection wavelength,flow rate,column and column temperature to establish chromatographic conditions.Furthermore,the samples of 15 batches of Jiaotaiwan were analyzed,and the HPLC chromatogram analysis and establishment of 15 batches of Jiaotaiwan were carried out using the TCM chromatographic fingerprint similarity evaluation system.2.Determination of multi-indicative components in JiaotaiwanHPLC method was used to determine the content of epiberberine,coptisine,berbericine,berberine hydrochloride and cinnamaldehyde in 15 batches of medicinal materials.Methodology examined linear relationships,precision,stability,repeatability,and average sample recovery.3.Effects of the active components of Jiaotaiwan in Orexin A/HPA-axis pathway and skeletal muscle GLUT4 in T2DM RatsIn this study,a high-sugar and high-fat diet plus a small dose of streptozotocin(STZ)was induced into a rat model of type 2 diabetes.To investigate the effects of the main active ingredients of Jiaotaiwan on the related indicators of Orexin A/HPA axis and the expression of GLUT4 in skeletal muscle of type 2 diabetic IR rats,and to explore the possible mechanism and effective components of Jiaotaiwan to improve IR.Resulis1.The HPLC fingerprint of Jiaotaiwan was established:Through the software analysis of the Chinese traditional medicine chromatographic feature atlas similarity evaluation system(2004 A Edition),five characteristic peaks and 16 common peaks were identified.The similarity of 15 batches of Jiaotaiwan was 0.910?0.997.2.HPLC method to determine the content of its five components:Its resolution is good,in the methodological investigation,the linear relationship is good(r>0.999 6),precision and reproducibility RSD are less than 3.0%,the average recovery rate is 95.24%?102.73%,RSD is 0.20%?2.49%.The average content of epiberberine,coptisine,berbericine,cerberine hydrochloride and cinnamaldehyde in the 15 batches was 1.87?2.96 mg/g,3.52?5.40 mg/g,3.80?5.79 mg/g,6.08?8.04 mg/g and 0.55?5.59 mg/g.3.Model group fasting blood glucose(FBG),fasting serum insulin(FINS),insulin resistance index(IRI),cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL-C)level,hypothalamic Orextotal in A,OX2R,corticotropin releasing hormone(CRH),pituitary and plasma adrenocorticotropic hormone(ACTH),serum and urinary corticosterone(CORT),and skeletal muscle 11?-HSD1 protein expression were increased;and insulin sensitivity index(ISI),high density lipoprotein(HDL-C),and the expression of GLUT4 protein in skeletal muscle were decreased(P<0.05 or P<0.01).Compared with the model group,the levels of FBG,FINS,IRI,TC,TG LDL-C in the combination of berberine hydrochloride and cinnamaldehyde in the main active ingredients of Jiaotaiwan were decreased;ISI and HDL-C levels were increased;hypothalamic Orexin A,OX2R,CRH,pituitary and plasma ACTH,serum and urine CORT levels were decreased;at the same time,the expression of 11?-HSD1 protein in skeletal muscle was down-regulated,and the expression of GLUT4 protein in skeletal muscle was up-regulated(P<0.05 or P<0.01).ConclusionThe active ingredients of Jiaotaiwan are mainly berberine hydrochloride(BBR)and cinnamaldehyde(CA),which are relatively high in content and stable.The active ingredient of Jiaotaiwan has improved IR and glycolipid metabolism in T2DM rat model,and its possible mechanism is related to the regulation of Orexin A/HPA axis pathway and up-regulation of skeletal muscle GLUT4 expression.