The Effects Of Neu-p11 And Danggui-shaoyao-san On Glucose And Lipid Metabolism And The Mechanism Mediated By HPA Axis In Chronic Stress Rats With Hight-fat Diet

ObjectiveTo investigate the effects and HPA axis related mechanisms of novel melatonin agonist Neu-p11 and Danggui-Shaoyao-San on glucose and lipid metabolism in chronic stress rats fed with high-fat diet, providing new drugs on new target and theoretical basis for the treatment of type 2 diabetes mellitus. MethodThe experiments were divided into five batches successively, each batch contains 60 male Wistar rats, were randomly divided into normal control group(Control), model group(Model), melatonin group(MLT) and three other groups treated with different drugs. Normal control group and model group were treated with equal amount saline, melatonin group were treated with 20 mg·kg-1MLT. Three drug-treated groups were treated with: the 1st batch were treated with Neu-p11(dose were respectively 5mg·kg-1,10mg·kg-1 and 20mg·kg-1),the 2nd batch were treated with Neu-p11(dose were respectively 10mg·kg-1,20mg·kg-1 and 40 mg·kg-1),the 3rd batch were treated with Neu-p11(doses were 5mg·kg-1,10mg·kg-1 and 20mg·kg-1 respectively), the 4th batch were treated with three kinds of traditional Chinese medicine( 445mg·kg-1 alcohol extract of Kaixin-San, 3360 mg·kg-1 aqueous extract of Danggui-Shaoyao-San and 300 mg·kg-1 extract of St. John’s wort).The 5th batch were treated with aqueous extract of Danggui-Shaoyao-San(doses were 840 mg·kg-1,1680 mg·kg-1and 3360 mg·kg-1 respectively). All the groups were given intragastric administration with the volume of 1ml·200g-1. In the 1st batch, rats were injected intraperitoneally 2.5 g·kg-1 glucose and 300mg·kg-1 epinephrine to establish the acute hyperglycemia model, and blood glucose levels of 0, 30, 60, 120 min was determined, and the blood glucose curve area was calculated. In the rest of four batches, control rats was given daily normal diet, and any stimulation was avoided. The other five groups rats were fed with high fat-diet, while randomly chronic stress was given once a day, the experiment duration lasted for 4~6weeks, daily water and food consumption were recorded, body weight and fasting blood glucose(FBG) levels of rats were measured weekly. At different time points, the intraperitoneal glucose tolerance test(IPGTT) and insulin tolerance test(ITT) were carried out. The levels of plasm lipids [contains: triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)], plasma insulin(INS), corticotropin releasing hormone(CRH), adrenocorticotropic hormone(ACTH), corticosterone(CORT) and melatonin(MLT) were detected. The 24 h rat urine was collected to measure the level of CORT. At the end of the experiment, rats were decapitated, the liver tissue and muscle were removed to measure the glycogen levels, bilateral adrenal glands and kidney, spleen, liver were weighed, the organ index was calculated. The activities of liver glucokinase(GK), glucose-6-phosphatase(G-6-Pase), MLT content in pineal gland were measured. The expression of MT1, MT2 m RNA in adrenal gland; GR, 11β-HSD1 and TNF-α m RNA in hippocampus; GR, MT1, MT2, GLUT-4, PPAR-γ m RNA in liver; GR, GLUT-4, PPAR-γ, Adipo, Leptin, TNF-α and IL-6 m RNA levels in adipose tissue were detected by RT-PCR technique. Protein expression of GR in liver, as well as MT1, MT2 in hippocampus and adrenal glands were detected by Western Blot technique. ResultsExperimental results of The 1st batch showed: Compared with the model group, Neu-p11(10 mg·kg-1) significantly reduced the 30min’s blood glucose by 11.83%(P<0.05). The experimental results of the other four batches showed: Compared with the normal group, weight gain was lowered in model group, the relative food intake was reduced significantly(P<0.05), FBG was significantly increased(P<0.05).The level of IPGTT and ITT was significantly lowered(P<0.05); plasma TC, TG were significantly increased(P<0.05, P<0.01), and HDL-C was decreased, LDL-C was increased. Liver glycogen and muscle glycogen content was significantly decreased(P<0.05). HPA hormone(including CRH, ACTH and CORT) and INS in plasma was increased, and CORT level was significantly increased in urine(P<0.05).The experimental results of the 2nd batche showed: Compared with the model group, the body weight, relative food intake, glucose tolerance were significantly increased in Neu-p11 treated group, the FBG was reduced(P<0.05), lipid levels of model rats were close to normal. Liver glycogen was reduced further, INS content was increased in prior period, but reduced in later period, HPA hormone levels was tended to normal. The results of the 3rd batche showed: Compared with the model group, rat body weight of Neu-p11(10 mg·kg-1)group was increased and relative food intake in the second week significantly increased by 9.38%(P<0.05), FBG was significantly reduced by 20.98%(P<0.01). The glucose intolerance were improved, TC and TG was significantly decreased(P<0.05), HDL-C had an increasing tendency, LDL-C tended to be decreased. Liver glycogen was significantly reduced by 53.66%(P<0.01).The 4th batches showed: Kaixin-San, Danggui-Shaoyao-San and St.John’s wort had a significant improvement on glucose lipid disorders and HPA axis hormone levels. Compared with the model group, the glucose intolerance was improved in Danggui-Shaoyao-San group,and FBG was significantly reduced by 8.45%(P<0.05), TG was significantly reduced by 40.00%(P<0.05), LDL-C was significantly reduced by 56.41%(P<0.05), the level of serum CRH, ACTH, CORT and INS decreased,the content of serum MLT, hepatic glycogen and muscle glycogen increased, although there is no significant differences. The 5th batche results showed: Compared with the model group, FBG of Danggui-Shaoyao-San(1680 mg·kg-1) group was significantly reduced by 15.69%(P<0.01), glucose intolerance and insulin intolerance was improved(P<0.05), TG was significantly reduced by 45.22%(P<0.05),HDL-C was significantly increased by 20.93%(P<0.05)on the 42 th day. The glycogen content in liver and muscle tended to increase.The related mechanism research showed: Compared with the control group, hepatic G-6-Pase activity was significantly reduced in model group(P<0.05), the level of CRH、ACTH、CORT and INS was decreased,and the MLT content of plasma and pineal gland were tended to reduce, expression level of gene and protein related to HPA axis and glucose lipid metabolism were abnormal. Compared with the model group, Neu-p11 have a regulatory effect on above indicators, and upregulated MT1 and MT2 m RNA in adrenal glands and liver, GR m RNA in hippocampus, liver and adipose significantly(P<0.01), upregulated GLUT-4 and PPAR-γ m RNA in adipose tissue(P<0.01). 11β-HSD1 and TNF-α m RNA in hippocampus were downregulated(P<0.01); adipo m RNA was upregulated(P<0.01); leptin m RNA and IL-6 m RNA was significantly reduced in adipose tissue(P<0.01); GR protein in liver and MT2 protein in hippocampus was significantly increased(P<0.01).Compared with the model group, in Danggui-Shaoyao-San treated groups, G-6-Pase activity activity was decreased(P<0.05); the levels of CRH, ACTH, CORT and INS were reduced(P<0.05), and the MLT content of plasma and pineal gland were tended to increase, the expression of MT2 in adrenal gland and GR in liver tissue was increased(P<0.05). ConclusionNeu-p11 has an improvment effect on glycolipid metabolism in acute hyperglycemic rats induced by glucose and in chronic stress rats fed with high-fat diet, the probalble mechanism is through inhibiting HPA hyperfunction by regulating related genes and proteins in adrenal gland, hippocampus, liver and adipose tissue.Kaixin-San, Danggui-Shaoyao-San and St.John’s Wort can improve glucose and lipid metabolism disorders in chronic stress rats fed with high-fat diet. Above all, the effect of Danggui-Shaoyao-San is the best, the mechanism may be mediated through regulating HPA axis activity by stimulating the secretion of MLT and upregulating the expressing of receptors.