中等强度有氧运动调节PPARγ/PI3K/AKT通路改善2型糖尿病大鼠肝脏糖、脂代谢紊乱及炎症

The Moderate-intensity Aerobic Exercise Relieves Hepatic Glucose and Lipid Metabolism Disorder and Inflammation of Type 2 Diabetic Rats by Modulating PPARγ/PI3K/AKT Pathway

目的:探讨中等强度跑台运动对2型糖尿病(T2DM)大鼠肝脏糖、脂代谢紊乱和炎症的调节效果及作用机制。方法:将31只SPF级雄性SD大鼠随机选取8只作为正常对照组(NC组),剩余大鼠经链脲佐菌素(STZ)进行造模,将造模成功的大鼠随机分为2型糖尿病模型组(DC组)、有氧运动干预组(DE组),每组8只。NC组和DC组大鼠正常饲养,DE组大鼠采用水平动物跑台进行运动,训练强度为15.2 m/min,持续运动60 min/d,5 d/w,共干预8周。实验期间检测各组大鼠体重、血糖、摄食量和饮水量。干预结束后,取材肝脏计算大鼠肝脏指数;采用ELISA法检测血清胰岛素、血脂四项水平;通过苏木素-伊红(HE)染色、油红O染色和PAS染色观察肝脏组织形态学和脂肪变性病理变化;采用RT-qPCR检测各组大鼠肝脏肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)mRNA表达水平;运用Western Blot方法检测肝脏过氧化物酶体增殖物激活受体γ(PPARγ)、磷酸化磷酯酰肌醇3激酶(P-PI3K)、磷脂酰肌醇3激酶(PI3K)、磷酸化蛋白激酶B(P-AKT)、蛋白激酶B(AKT)、核转录因子-κB(NF-κB)蛋白的表达。结果:(1)运动干预8周后,与NC组相比,DC组大鼠体重显著降低(P<0.01),血糖、饮水量、摄食量、肝脏指数均显著升高(P<0.01);DE组大鼠体重显著高于DC组(P<0.05),血糖、饮水量、摄食量、肝脏指数均显著低于DC组(P<0.05,P<0.05,P<0.01,P<0.05)。(2)与NC组相比,DC组大鼠血清胰岛素(insulin)水平、高密度脂蛋白胆固醇(HDL-C)水平均显著降低(P<0.01,P<0.05),血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)水平均显著升高(P<0.01);DE组大鼠血清Insulin水平、HDL-C水平均显著高于DC组(P<0.05),TC、TG、LDL-C水平均显著低于DC组(P<0.05,P<0.01,P<0.05)。(3)与NC组相比,DC组大鼠肝脏组织HE染色结果显示肝脏组织中肝细胞排列紊乱,出现脂肪变性和炎性细胞浸润,油红O染色结果显示阳性区域面积显著增加(P<0.01),PAS染色结果显示,肝糖原染色阳性区域面积显著降低(P<0.01);与DC组相比,DE组大鼠HE染色结果显示肝细胞排列相对整齐,肝细胞内脂肪空泡和脂滴明显减少,油红O染色结果显示阳性区域面积显著减少(P<0.01),PAS染色结果显示肝糖原染色阳性区域面积显著增加(P<0.01)。(4)RT-qPCR结果显示,与NC组相比,DC组大鼠TNF-α、IL-6、IL-1βmRNA表达水平均显著升高(P<0.01);DE组大鼠TNF-α、IL-6、IL-1βm RNA表达水平均显著低于DC组(P<0.05,P<0.01,P<0.01)。(5)Western Blot结果显示,与NC组相比,DC组大鼠PPARγ、P-PI3K/PI3K、P-AKT/AKT蛋白表达均显著降低(P<0.05,P<0.01,P<0.01),NF-κB蛋白表达显著升高(P<0.05);DE组PPARγ、P-PI3K/PI3K、P-AKT/AKT蛋白表达均显著高于DC组(P<0.05,P<0.05,P<0.01),NF-κB蛋白表达显著低于DC组(P<0.05)。结论:8周中等强度跑台运动可改善T2DM大鼠的糖、脂代谢紊乱和炎症反应,其作用机制可能与PPARγ/PI3K/AKT信号通路的激活有关。

Objective To explore the regulatory effect and mechanism of moderate-intensity treadmill exercise on hepatic glucose and lipid metabolism disorder,and inflammation in type 2 diabetes mellitus(T2DM)rats.Methods Eight of 31 SPF male Sprague-Dawley rats were randomly selected into a normal control group(NC group),while the remaining rats were modelled with streptozotocin(STZ).Then those modelled successfully were randomly divided into a T2DM model group(DC group)and an aerobic exercise intervention group(DE group),each of 8.Both the NC and DC groups were fed normally,while the DE group underwent a daily 60-minute running on a horizontal treadmill at 15.2 m/min,5 days a week for 8 weeks.During the experiment,the body weight,blood sugar,food intake and water intake of all groups were detected.After the intervention,the livers were collected to calculate the liver indexes,while the levels of serum insulin and blood lipids were measured by using the enzyme-linked immunosorbent assay.Moreover,the histomorphology and steatosis pathology of livers were observed by hematoxylin-eosin(HE)staining,oil red O staining and PAS staining.Meanwhile,the mRNA expression of the tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in livers were detected by using the RT-qPCR and the expressions of peroxisome proliferatoractivated receptorγ(PPARγ),phosphorylated phosphatidylinositol 3-kinase(P-PI3K),phosphatidylinositol 3-kinase(PI3K),phosphorylated protein kinase B(P-AKT),protein kinase B(AKT)and nuclear transcription factor-κB(NF-κB)protein in livers were measured by using Western blotting.Results(1)After 8 weeks of exercise intervention,compared with the NC group,the body weight of the DC group decreased significantly(P<0.01),but the blood glucose,water intake,food intake,and liver indexes increased significantly(P<0.01).Meanwhile,the body weight of the DE group was significantly higher than the DC group(P<0.05),while the blood glucose,water intake,food intake,and liver indexes were significantly lower than the DC group(P<0.05,P<0.05,P<0.01,and P<0.05).(2)Compared with the NC group,the serum insulin level and high density lipoprotein cholesterol(HDL-C)level of the DC group were significantly lower(P<0.01,P<0.05),but the serum total cholesterol(TC),triglyceride(TG),and low density lipoprotein cholesterol(LDL-C)levels increased significantly(P<0.01).Moreover,the serum insulin and HDL-C of the DE group were significantly higher than the DC group(P<0.05),while the levels of TC,TG and LDL-C were significantly lower than the latter(P<0.05,P<0.01 and P<0.05).(3)According to the HE staining,compared with the NC group,the liver tissues of the DC group were in disorder,with fatty degeneration and inflammatory cell infiltration.Moreover,the oil red O staining found a significant increase in the positive area(P<0.01),but PAS staining showed a significant decrease in the positive area of liver glycogen staining(P<0.01).Meanwhile,compared with the DC group,the hepatocytes of the DE group were arranged more neatly,with a significant decrease in the fat vacuoles and lipid droplets in the liver cells,a significant decrease in the positive area according to the oil red O staining,but a significant increase in the positive area of liver glycogen staining according to the PAS staining(P<0.01).(4)The results of RT-qPCR showed that compared with the NC group,the expression levels of TNF-α,IL-6,and IL-1βm RNA in the DC group increased significantly(P<0.01),while those of TNF-α,IL-6 and IL-1βm RNA in DE group were significantly lower than the DC group(P<0.05,P<0.01,and P<0.01).(5)Western blotting results showed that compared with the NC group,the protein expressions of PPARγ,P-PI3K/PI3K,and P-AKT/AKT decreased significantly in the DC group(P<0.05,P<0.01 and P<0.01),but those of NF-κB increased significantly(P<0.05).Moreover,the protein expression of PPARγ,P-PI3K/PI3K,and PAKT/AKT of the DE group were significantly higher than the DC group(P<0.05,P<0.05,P<0.01),while that of NF-κB was significantly lower than the latter(P<0.05).Conclusion Eight-week moderateintensity treadmill exercise can relieve glucose and lipid metabolism disorders and inflammatory responses in T2DM rats,and its mechanism may be related to the activation of the PPARγ/PI3K/AKT signaling pathway.