运动和表没食子儿茶素没食子酸酯对糖尿病大鼠心肌间质蛋白表达的影响

Effects of swimming and epigallocatechin gallate on interstitial proteins expression of myocardium from type 2 diabetic rats

目的观察游泳训练和/或表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对2型糖尿病大鼠心肌间质蛋白表达的影响及其与心肌TGF-β/Smads信号通路的关系。方法6周龄SPF级雄性SD大鼠,采用6周高脂饲料喂养加腹腔注射链脲佐菌素(STZ,30 mg/kg)复制2型糖尿病大鼠模型,随机分为糖尿病模型组(D组)、糖尿病(EGCG)组(DG组)、糖尿病运动组(DE组)、糖尿病运动EGCG组(DEG组),每组8只,另增设同批次雄性SD大鼠6只为正常对照组(C组)。运动组大鼠进行无负重游泳训练,游泳时间为第1周15 min/d,时间以每周15 min递增延长,直到延长至90 min/d,每周运动5天,休息2天。EGCG给药组(100 mg/kg BW)大鼠每天灌胃,连续7天。游泳训练和/或EGCG干预共12周。干预实验结束48小时后,过夜禁食12 h后麻醉处死大鼠,取部分左心室采用免疫组化法检测心肌多克隆抗体Ⅰ型胶原(Col Ⅰ)、Ⅳ型胶原(ColⅣ)、纤维连接蛋白(FN)蛋白表达,另取部分左心室采用碱水解法检测心肌羟脯氨酸(Hyp)含量、免疫印迹检测转化生长因子-β1(TGF-β1)蛋白表达、RT-PCR检测Smad2和Smad7 mRNA基因表达。结果与C组相比,D组大鼠血糖、心肌TGF-β1、Smad2 mRNA表达和心肌Hyp含量、Col Ⅰ、Col Ⅳ、FN蛋白表达均显著升高(P<0.01),Smad7 mRNA表达显著下降(P<0.01)。与D组相比,DG组、DE组、DEG组大鼠血糖、心肌TGF-β1、Smad2 mRNA表达和心肌Hyp含量、Col Ⅰ、Col Ⅳ、FN蛋白表达均显著下降(P<0.05或P<0.01),DG组、DEG组大鼠心肌Smad7 mRNA表达显著上升(P<0.05或P<0.01)。与DG组比较,DEG组大鼠血糖、心肌Smad2 mRNA表达和心肌Hyp含量、Col Ⅰ、Col Ⅳ蛋白表达均显著下降(P<0.05或P<0.01),心肌Smad7 mRNA表达显著上升(P<0.05)。与DE组比较,DEG组大鼠血糖、心肌TGF-β1、Smad2 mRNA表达和心肌Hyp含量、Col Ⅰ、Col Ⅳ蛋白表达均显著下降(P<0.05或P<0.01),心肌Smad7 mRNA表达显著上升(P<0.05)。DEG组大鼠血糖、心肌Smad2 mRNA表达和心肌Hyp含量、Col Ⅰ、Col Ⅳ、FN蛋白表达仍显著高于C组(P<0.05或P<0.01)。结论12周的游泳训练和/或EGCG给药均不同程度的降低了2型糖尿病大鼠心肌间质蛋白的表达,且二者联合干预效果更佳。

OBJECTIVE To observe the effects of swimming training and/or epigallocatechin gallate(EGCG)on the expression of myocardial interstitial protein and its relationship with transforming growth factor-beta(TGF-β)/Smads signaling pathway in type 2 diabetic rats.METHODS Male SD rats(SPF grade,6 weeks old)were fed with high-fat diet for 6 weeks and intraperitoneal injection of streptozotocin(STZ,30 mg/kg).The rats were randomly divided into four groups:diabetic model group(D),diabetes EGCG group(DG),diabetes exercise group(DE),and diabetes exercise EGCG group(DEG),with 8 rats in each group,and 6 male SD rats in the same batch were added as the normal C group.The rats in the exercise group were given swimming training without load.The swimming time was 15 min/d in the first week,and the time was increased by 15 min a week until it was extended to 90 min/d.The rats in the exercise group were trained for 5 days a week.Rats in the EGCG group(100 mg/kg BW)were intragastrical administered for 7 days.Swimming training and/or EGCG intervention lasted for 12 weeks.48 hours after the end of the intervention experiment,the rats were fasted overnight for 12 hours.Myocardial collagen Ⅰ(Col Ⅰ),collagen Ⅳ(Col Ⅳ)and fibronectin(FN)protein expression were detected by immunohistochemistry in some left ventricles,hydroxyproline(Hyp)content was detected by colorimetry,TGF-β1 protein expression was detected by WB,Smad2 and Smad7 mRNA expression were detected by RT-PCR.RESULTS The levels of blood glucose,Hyp content,TGF-beta1,Smad2 mRNA,Col Ⅰ,Col Ⅳ,FN of the rats from D group were significantly higher than those of the rats from C group(P<0.01),but the level of Smad7 mRNA was significantly lower than it of the rats from C group(P<0.01).Compared with D group,blood glucose,TGF-β1,Smad2 mRNA expression,Hyp content,Col Ⅰ,Col Ⅳ,FN protein expression in DG group,DE group and DEG group were significantly decreased(P<0.05 or P<0.01),and the Smad7 mRNA expression increased significantly(P<0.05 or P<0.01).Compared with DG group,blood glucose,Smad2 mRNA expression,Hyp content,Col Ⅰ,Col Ⅳ protein expression in DEG group were significantly decreased(P<0.05 or P<0.01),and the myocardial Smad7 mRNA expression was significantly increased(P<0.05).Compared with DE group,the levels of blood glucose,TGF-β1,Smad2 mRNA,Hyp content,Col Ⅰ,Col Ⅳ protein expression were significantly decreased in the EGCG group(P<0.05 or P<0.01),and the expression of Smad7 mRNA was significantly increased(P<0.05).Blood glucose,Smad2 mRNA expression,Hyp content,Col Ⅰ,Col Ⅳ,FN protein expression in DEG group were significantly higher than those in C group(P<0.05 or P<0.01).CONCLUSION 12 weeks swimming training and/or EGCG administration can reduce the expression of myocardial interstitial protein in type 2 diabetic rats,and the combined intervention effect is better.