游泳训练对小鼠心肌PKCδ/P66shc蛋白表达的影响

Effect of swimming training on the expression of PKCδ/p66Shc protein in mouse myocardium

目的:探究不同强度的游泳训练对小鼠心肌P66shc蛋白的影响。方法:将50只昆明小鼠随机分为对照组(C组)、负重游泳组(E组)、负重游泳+药物组(ER组)、非负重游泳组(P组)、非负重游泳+药物组(PR组),10只/组。C组不运动,E组、ER组、P组、PR组进行4周游泳训练,其中E组、ER组以体重3%负荷进行负重游泳,P组、PR组无负重游泳,60 min/d,每周6次。ER组、PR组小鼠在最后2次运动前腹腔注射PKCδ抑制剂Rottlerin(0.3 mg/kg),C组、E组、P组注射同等剂量生理盐水。在训练结束24 h后取样,Western blot测定小鼠心肌PKCδ、P-PKCδ、P66shc、P-P66shc、NOX2蛋白表达;免疫共沉淀测PKCδ和P66shc;生化分析心肌及血清丙二醛(MDA)、心肌活性氧(ROS)、超氧化物歧化酶(SOD)。结果:与C组比较,E组的PKCδ、P-PKCδ、P66shc、P-P66shc、NOX2蛋白表达均明显增加(P<0.01),血清和心肌MDA水平、心肌ROS明显增加(P<0.05或P<0.01),心肌SOD活性降低(P<0.01),P组的PKCδ、P-PKCδ、P-P66shc和NOX2明显增加(P<0.05或P<0.01),心肌SOD活性增强(P<0.05);与E组比较,ER组PKCδ(P<0.01)、P-PKCδ(P<0.01)、P66shc(P<0.05)、P-P66shc(P<0.01)、NOX2(P<0.05)蛋白表达明显减少,P组P66shc蛋白表达显著减少(P<0.05),心肌MDA(P<0.01)和ROS(P<0.05)减少,SOD活性增强(P<0.01);与P组比较,PR组的PKCδ、P-PKCδ、P-P66shc蛋白表达明显减少(P<0.01),NOX2增加(P<0.05)。结论:两种强度的游泳训练均促使小鼠心肌细胞内PKCδ蛋白及其磷酸化增加;高强度游泳训练可显著增强P66shc蛋白表达及磷酸化水平,导致ROS大量生成,抗氧化酶活性下降;低强度游泳训练增强P66shc磷酸化但不促进其蛋白表达,心肌抗氧化能力增强,产生运动适应。

Objective:To investigate the effects of different intensity of swimming training on p66Shc protein in mouse myocardium.Methods:Fifty Kunming mice were randomly divided into control group(Group C),weight-bearing swimming group(Group E),weight-bearing swimming+drug group(Group ER),non weight-bearing swimming group(Group P),non weight-bearing swimming+drug group(Group PR),with 10 mice in each group.Group C did not exercise.Groups E,ER,P,and PR received swimming training for 4 weeks.Groups E and ER performed weight-bearing swimming with a 3%body weight,and Group P and Group PR were swimming without weight-bearing,60 min/d,6 times/w.Mice in ER and PR groups were injected intraperitoneally with Rottlerin(0.3 mg/kg),a PKCδinhibitor,before the last two exercises.Groups C,E,and P were injected with the same dose of normal saline.Samples were collected after training finished for 24 hours.The protein expressions of PKCδ,P-PKCδ,P66Shc,P-P66shc and NOX2 were detected by Western blot;PKCδand P66Shc were detected by immunoprecipitation;malondialdehyde(MDA),reactive oxygen species(ROS)and superoxide dismutase(SOD)in myocardium and serum were analyzed by biochemistry.Results:Compared with Group C,the protein expressions of PKCδ,P-PKCδ,P66Shc,P-P66shc and NOX2 in Group E were increased significantly(P<0.01),the serum and myocardial MDA levels,myocardial ROS were increased significantly(P<0.05 or P<0.01),and the myocardial SOD activity was decreased(P<0.01),the PKCδ,P-PKCδ,P-P66shc and NOX2 in Group P were increased significantly(P<0.05 or P<0.01),and the myocardial SOD activity was enhanced(P<0.05).Compared with Group E,the protein expressionS of PKCδ(P<0.01),P-PKCδ(P<0.01),P66Shc(P<0.05),P-P66shc(P<0.01),NOX2(P<0.05)in Group ER was decreased significantly,the protein expression of P66Shc in Group P was decreased significantly(P<0.05),the myocardial MDA(P<0.01)and ROS(P<0.05)were decreased,and the activity of SOD was enhanced(P<0.01).Compared with Group P,the protein expressions of PKCδ,P-PKCδand P-P66shc in Group PR were decreased significantly(P<0.01),while the expression of NOX2 was increased(P<0.05).Conclusion:Both swimming training of two intensities promoted the increase of PKCδprotein and its phosphorylation in mouse cardiomyocytes.High-intensity swimming training could significantly enhance the expression and phosphorylation level of p66Shc protein,resulting in the production of ROS and the decrease of antioxidant enzyme activity.Low-intensity swimming training enhanced the phosphorylation of p66Shc,but did not promote its protein expression,resulting in the enhancement of myocardial antioxidant capacity and exercise adaptation.