高强度游泳训练后AngⅡ介导小鼠心肌细胞损伤中TLR4信号通路的作用

Study on Effect of TLR4 Signal Channel in Damaged Mice's Myocardial Cell by Inducing the Ang Ⅱ After High-intensity Swimming Training

目的:探讨高强度游泳训练后Ang II介导小鼠心肌细胞损伤中TLR4及My D88蛋白的作用。方法:选择清洁级昆明小鼠36只,分为3组,即正常对照组、普通强度训练组和高强度训练组,每组12只。运动负荷训练8周,普通强度训练组和高强度训练组每周训练6 d,每天训练1次。低强度训练组:游泳时间45 min/d;高强度训练组:用铅皮在小鼠尾部负重5%,游泳训练90 min/d;正常对照组正常饲养。游泳运动训练结束后,检测实验动物心脏指数、心肌组织Ang II含量以及血液中Ang II、c Tn I、CK-MB指标,制备心肌组织病理切片。培养乳鼠心肌细胞,分组为空白对照组、抑制剂组、Ang II组、Ang II+抑制剂组,加药24 h后流式细胞仪检测ROS。收集心肌组织和培养的心肌细胞,western blot法检测TLR4和My D88表达。结果:1)体内实验:与空白对照组相比较,低强度游泳训练组体重增加,心脏重量增加,心脏指数相差不大;心肌组织中Ang II升高,血液中Ang II、c Tn I、CK-MB均变化不大;心肌纤维略致密,纤维及间质变化不大。而与空白对照组比较,高强度游泳训练组出现体重下降、心脏重量增加的现象,心脏指数增大(P<0.05);心肌组织中Ang II升高,血液中Ang II、c Tn I、CK-MB均升高(P<0.01);心肌纤维结构紊乱,边界不清,细胞间隙大,出现心肌纤维增粗及肥大趋势,心肌横截面空白区域增多,间质间隙增大。2)体外实验:与对照组比较,Ang II组细胞内ROS增加,加入抑制剂后ROS水平有所下降。3)Western blot结果:随着游泳强度的增加,心肌组织中TLR4蛋白表达含量增加显著(P<0.01),My D88蛋白增加显著(P<0.01),但高强度游泳训练后出现下降。细胞实验显示,正常心肌组织加入抑制剂后,TLR4表达量显著下降(P<0.05),My D88表达显著增加(P<0.01);Ang II作用后诱导TLR4表达显著增加(P<0.01),My D88增加不显著,加入抑制剂后,TLR4表达显著下降,My D88表达显著增加(P<0.01)。结论:高强度游泳训练后,可引起实验动物心肌细胞的损伤;高强度游泳运动训练可导致实验动物心肌组织和血液中Ang II增加;高强度游泳训练运动训练对实验动物造成的心肌损伤有可能是通过Ang II增加所引起;Ang II可引起心肌细胞TLR4的表达增加和My D88蛋白表达降低。

Objective： This paper attempted to discuss the effect of Ang II on TLR4 and My D88 protein in mice with myocardial cell injury after high intensity swimming training. Methods： Thirty-six Kunming mice of clean grade were chose and divided into normal control group,normal intensity exercise group and high intensity exercise group with 12 mice respectively. They took training for 8 weeks. Normal and high intensity group took training 6days per week with once a day. For normal intensity group,the mice swam 45 minutes a day while high intensity group swam 90 minutes a day with 5% load lead sheath tied on tails. Normal control group were raised as usual.After the swimming training,cardiac index,Ang II content of myocardial tissue,Ang II,c Tn I,and CK-MB in blood were tested and pathological section of myocardium were made. The author also cultured myocardial cells of neonatal rats,and divided them into control group,inhibitor group,Ang II group and Ang II ＋ inhibitor group.Twenty-four hours after dosing,ROS were tested by flow cytometry. Myocardial tissue and cultured cardiac muscle cells were collected and the expression of their TLR4 and MyD 88 were tested by WB method. Results： In vivo experiment,normal intensity group＇s weight and heart weight increased compared with the control group,but has little difference on cardiac index; AngI I in myocardial tissue increased,but AngI I,cT nI and CK-MB in blood changed little; Myocardial fibers were slightly denser while fibers and mesenchyme changed little. Compared with the control group,high intensity group showed weight loss,heart weight and cardiac index increased（ P 0. 05）;AngI I in heart tissue and blood,cT nI and CK-MB in blood increased（ P 0. 01）; myocardial fiber structure appeared to be disordered,with unclear boundary and large intercellular space; myocardial fiber showed a trend of thickening and hypertrophy; myocardial cross section had more blank area and mesenchyme gap increased. In vitro experiment,compared with the control group,the intracellular ROS increased in the AngI I group,and the level of reactive oxygen species decreased after the addition of the inhibitor. WB test showed that with the increasing of swimming intensity,TLR4 and MyD 88 protein expressed in myocardial tissue increased significantly（ P 0. 01）,but decreased after high intensity swimming. In the cell experiment,after the addition of inhibitor,normal myocardial tissue had a significantly decrease of TLR4 protein expression（ P 0. 05） and a significantly increase of MyD 88 protein expression（ P 0. 01）; AngI I induced the TLR4 protein expression increase significantly（ P 0. 01） but not for MyD 88; After adding inhibitor,TLR4 protein expression decreased significantly while MyD 88increased（ P 0. 01）. Conclusions： High intensity swimming training can cause the myocardial injury of the experimental animal and the increase of AngI I in blood and myocardial tissue. The injury might be caused by the increase of AngI I. AngI I can increase the expression of TLR4 protein and decrease the expression of MyD 88 protein.