Expression Of PI3K/AKT Signaling Pathway In Tibetan Minpig Model With Insulin Resistance And The Effects Of Guanxinning Tablets

Objective To explore the pathogenesis mechanism of insulin resistance(IR)cardiovascular diseases and the effects of Guanxining tablets(GXNT).In this study,the Tibetan minipigs model with IR was induced by high fat diet,and to investigate the experession of PI3K/AKT signaling pathway and its related signal molecules in the Tibetan minipigs model with IR and the effects of GXNT.Methods 4-5 months old male Tibetan minipigs,after adaptive feeding for 4 weeks,were randomly divided into normal control group(n=4)and IR model group(n=12)according to their weight,the normal control group was fed with normal diet,IR model group was fed with high fat diet.After being model for 8 weeks,the IR model group was divided into model control group(n=6)and GXNT group(n=6)according to the fasting blood glucose level.The minipigs of GXNT group were daily mixed in diet administered 81mg/kg GXNT for 8 weeks.The body weight,fasting blood glucose(FBG),blood lipid,haematological parameters(WBC、MONO、NEUT),fasting insulin(FINS),leptin,hs-CRP,blood pressure and glucose tolerance test were detected every 4 weeks,and the area under blood glucose curve(AUC),Artherosclerosis index(AI),insulin resistance index(HOMA-IR)and insulin sensitivity index(HOMA-IS)were calculated.After administrated for 8 weeks,the wet weight of heart,liver,kidney and pancreas were detected,and calculated their organ coefficients.Meanwhile,the changes of mRNA expression of IRS-1,IRS-2,GLUT4,FOXO1 and mTOR in liver,muscle and adipose tissues were detected by RT-PCR methods,and the expression changes of IRS-1,IRS-2,PI3K,p-AKT and AKT protein in liver and muscle tissues were also detected by Western Blot methods,while the expression changes of IRS-1,mTOR and p-AKT protein were detected by immunohistochemistry method.In addition,the pathological changes of liver,pancreas and abdominal aorta were staining with H&E dying,while the pathological change of coronary artery was stainging with oil red dying.Results(1)Compared with normal control group,the body weight of IR model group was significantly increased at the beginning of 8th week(P<0.05),and the levels of TC,HDL-C,LDL-C and AI index in IR model group were significantly increased after 4 weeks(P<0.01).The SBP,DBP,MAP,AUC,FINS,HOMA-IR,WBC,MONO and NEUT in IR model group were also markedly increased(P<0.05,P<0.01),while HOMA-IS was significantly decreased(P<0.01).(2)Compared with model control group,the blood glucose and blood lipid tended to decrease in different degrees(P>0.05),the hs-CRP,WBC,MONO,MAP,SBP,DBP,FINS and HOMA-IR in GXNT group were significantly lowered,and HOMA-IS was significantly increased(P<0.01).Meanwhile,the weight of heart,liver and spleen were increased at different degrees in IR model group,and the spleen weight and spleen coefficient was significantly increased(P<0.01).Spleen weight in GXNT group were significantly lowered(P<0.01).(3)Pathological observation showed that there was no obvious abnormal changes in the liver and pancreas tissues of normal control group,and the structure of the abdominal aorta and coronary artery was clear and no lipid was found,while the liver tissues of model control group had local hepatocellular edema,derangement and accompanied by lymphocyte infiltration,pancreatic acinar and some islet cells appear individual vacuolar degeneration and part of the β cell swelling.At the same time,abdominal aorta had obvious inflammatory cell infiltration,plaque lipid,calcification and atherosclerosis;the intima of coronary artery had visible red dye droplets;and given Guanxin Ning tablets can significantly improve the above lesions.(4)Compared with the normal control group,the expression of IRS-1 mRNA and protein in the liver tissue of model control group were significantly decreased(P<0.01),while the expression of IRS-1 protein in the muscle tissue was significantly decreased(P<0.01)and IRS-2 mRNA expression was decreased in liver,muscle and adipose tissue,and decreased significantly in the liver and adipose tissues(P<0.01),Meanwhile,the IRS-2 protein was also decreased in liver and muscle tissues.The expression of PI3K in the liver and muscle tissues was significantly drecreased(P<0.05),the expression of GLUT4 mRNA in the liver,muscle and adipose tissues was decreased,especially in the liver and muscle tissues(P<0.05,P<0.01),the expression of p-AKT protein was decreased in muscle tissue,and the FOXO1 mRNA increased in liver and adipose tissues and the expression of mTOR mRNA in muscle tissue was tendency to increase.The results of immunohistochemistry showed that the expression of IRS-1 protein was significantly dercreased(P<0.05)while mTOR protein was significantly increased in adipose tissue(P<0.01).In addition,the expression of IRS-1,IRS-2 and GLUT4 mRNA in the liver tissue of GXNT group were significantly higher than those of model control group(P<0.01),the expression of IRS-2 and GLUT-4 mRNA in the muscle tissue as well as IRS-2 mRNA in adipose tissue were increased,while the expression of FOXO1 mRNA in adipose tissue was tendency to decrease,and the expression of IRS-1 protein was significantly increased,while the expression of mTOR protein was significantly reduced in adipose tissues(P<0.05,P<0.01).Conclusions High fat diet can induce the Tibetan minipig appeared obvious hypertension,hyperlipidemia,impaired glucose tolerance and insulin resistance,and GXNT intervention can obviously improve the symptom of IR,and the pathological changes of abdominal aorta has a certain improving effect.The related factors of PI3K/AKT signaling pathway in different tissues has different expression and together regulation of the IR.After GXNT intervention,the expressions of related genes of PI3K/AKT signaling pathway in liver tissue has siginificantly increased,and the related genes and proteins in muscle and adipose tissues have also corresponding effects,which may be one of the mechanisms of GXNT in the treatment of insulin resistance cardiovascular diseases.