A Mechanism Research Of Electroacupuncture On Hypothalamus Insulin PI3K Signaling Pathway In Insulin Resistance Rats

Objective:This subject is intended under the guidance of Chineseacupuncture theory“prevent disease”, in order to study the fastingplasma insulin, fasting plasma glucose, body weight, free fattyacid, triglyceride and total cholesterol, regulatory mechanisms ofPI3K signaling pathway in hypothalamus tissure of insulinresistance rats from celluar and molecular levels; Try to exploreand analysis the effect and mechanism of electroacupuncture forinsulin resistance model ratsMethods:608-week-old male Wistar rats, weight about180±20g, wererandomly divided into6groups: normal group, model group,prevention group, electroacupuncture group, cerebrospinal fluidgroup and wortmannin group, with10rats in each group respectively.Normal rats were feed normal food (3.8kcal/g, containing70%carbohydrate,20%protein,10%fat), rats in other groups were feedwith high-fat diet daily (5.4kcal/g, containing38.5%carbohydrate,15%protein,46.5%fat), to prepare insulin resistance model.Feeding8weeks later, blood samples of the rats tail vein bloodto test FPG, Fins, and calculate the insulin sensitive index,Insulin Activity Index (Insulin Activity Index=1/[FPG (mmol/L)×FIN (mIU/L)), the compared with normal group, IAI of model group rats significantly decreased (P <0.05).Ventricle perfusion method:Rats of wortmannin group were weighed and to abdominalanesthesia with chloral hydrate (10%). Rat were fixed withstereotaxic instrument, local presved skin, disinfeced, inaccordance with the stereotaxic atlas of positioning; Drilled ahole2mm in diameter with dental drill in the median of calvariabone, back of the bregma0.8mm, catheterized with stainless steeltube (18.0mm long, diameter0.64mm,0.39mm canon; Stainlesssteel with cap (17.0mm long,0.33mm in diameter)were put into andclosed the stainless steel tube; Fixed the tube by dentalcement.Rats awoke after the operation six to eight hours, suturedthe skin, prevented infection, put rats in an empty cage for itsrevival.After2days, took out the stainless steel and cleaned withartificial cerebrospinal fluid. Injected wortmannin (50nmol/L,3mg/kg) into stainless steel tube through PVC tube (0.3mm indiameter), injection speed is maintained at1mu L/min; Retainedthe needle8to10min after the injection to prevent drugregurgitation. Ventricle perfusion method of cerebrospinal fluidgroup were same as the wortmannin group.Cerebrospinal fluid configuration method: Na+145.5ml/L,K+2.8mol/L, Ca2+2.3mol/L, Mg2+2.2mol, Cl-128.5mol/L, HCO3-23.lmol/L,H2Po4-l.lmol/L, glucose0.6lg/L, osmotic pressure289.omosM/L, PH7.3, amount of perfusion, the operation method is the same asblockers group.Acupuncture operating method:0.30×25mm acupuncture needle were operated at the acupoints:CV4、ST36、ST40and CV12,and stimulated with a Hans acupoint never stimulator (HANS) EA apparatus, the following stimulus oarameterswere selected: continuous-wave at1Ma,2Hz,10min a day for8weeks.Result:1. Effect of acupuncture on fasting plasma insulin, fastingplasma glucose, body weight in rats: at the eighth week, insulinresistance model rats were set up, compared with the normal rats,fasting plasma insulin, fasting plasma glucose, body weight of ratsin model group, prevention group, electroacupuncture group,cerebrospinal fluid group and wortmannin group were increasedsignificantly（p<0.01）; At the sixteenth week, fasting plasmainsulin, fasting plasma glucose, body weight of model rats wereincreased significantly while compared with normal rats （p<0.01）;fasting plasma insulin, fasting plasma glucose, body weight ofprevention group and electroacupuncture group were decreased whilecompared with model group（p<0.05）; Compared with wortmannin group,fasting plasma insulin, fasting plasma glucose, body weight ofcerebrospinal fluid group decreased（p<0.05）. Compared with normalgroup, insulin activity index of model rats decreased（p<0.01）;insulin activity index of prevention group and electroacupuncturegroup raised while compared with model group（p<0.05）; Comparedwith wortmannin group, insulin activity index of cerebrospinalfluid group increased （p<0.05）.2. Effect of acupuncture on free fatty acid, triglyceride andtotal cholesterol in rats.After established the insulin resistancemodel rats, free fatty acid, triglyceride and total cholesterol ofmodel rats increased significantly（p<0.01）; At the sixteenth week,after electroacupuncture treatment, free fatty acid, triglycerideand total cholesterol in prevention group and electroacupuncture group decreased while compared with model rats （p<0.05）; Comparedwith wortmannin group, free fatty acid, triglyceride ofcerebrospinal fluid group increased （p<0.05）, total cholesterolwith no significant difference（p>0.05）.3. Effect of acupuncture on IRS2IOD of hypothalamus tissurein each group. After immunohistochemical test, compared withnormal rats, IRS2IOD decreased in model group（p<0.01）; IRS2IODof prevention group and electroacupuncture group raised whilecompared with model group （p<0.05）; Compared with wortmanningroup, IRS2IOD of cerebrospinal fluid group increased （p<0.05）.4. Effect of acupuncture on PI3Kp110and p-PI3Kp110ofhypothalamus tissure in each group: at the sixteenth week, afterwestern-blot test, compared with normal group, expression ofPI3Kp110protein in hypothalamus of model group and wortmanningroup dreased（P<0.01）; Compared with model group, expression ofPI3Kp110protein in hypothalamus of prevention group andelectroacupuncture group raised （P<0.05）; Compared withwortmannin group, expression of PI3Kp110protein in hypothalamusof cerebrospinal fluid group（P<0.05）.In addition, expression ofp-PI3Kp110protein in hypothalamus of model group and wortmanningroup decreased while compared with normal group（P<0.01）;Compared with model group, expression of p-PI3Kp110protein inhypothalamus of prevention group, electroacupuncture group raised（P<0.05）; Expression of p-PI3Kp110protein in hypothalamus ofcerebrospinal fluid group increased while compared withwortmannin group（P<0.05）.5. Effect of acupuncture on Akt2mRNA expression of hypothalamustissure in each group: at the sixteenth week, after real-time PCR test, compared with normal group, Akt2mRNA expression inhypothalamus of model group and wortmannin group dreased（P<0.01）;Compared with model group, Akt2mRNA expression in hypothalamus ofprevention group, electroacupuncture group raised（P<0.05）;Compared with wortmannin group, Akt2mRNA expression inhypothalamus of cerebrospinal fluid group increased（p<0.05）.Conclusion:1. Electroacupuncture had significant protective effect onregulating fasting plasma insulin, fasting plasma glucose, bodyweight, free fatty acid, triglyceride and total cholesterol ininsulin resistance rats, and relieve abnormal lipid metabolicdisorder which caused by insulin resistance.3. Electroacupuncture had significant protective effect onregulating the expression of IRS-2, PI3Kp110, p-PI3Kp110andAkt2mRNA in hypothalamus tissure of resistance rats. It issuggested that the electroacupuncture has a protective effect oninsulin resistance through regulating PI3K signaling pathways andthe related signaling molecules.