糖尿病大鼠心肌纤维化的发生及基质金属蛋白酶-14的表达

目的:观察糖尿病(DM)大鼠不同病程阶段是否出现心肌纤维化,并检测心肌基质金属蛋白酶(MMP)-14表达,分析DM引起心肌纤维化的可能机制.方法:雄性SD大鼠30只,随机分为对照组、高糖高脂组(HC组)和DM 4周、8周、12周组,各6只.对照组正常饮食,HC组和DM各组给予高糖高脂饮食;喂养4周后,DM组腹腔注射链脲佐菌素建模,对照组及HC组腹腔注射等剂量柠檬酸钠缓冲液;持续喂养至12周.比较各组大鼠空腹血糖(FBG)和血浆三酰甘油(TG)、总胆固醇(TC)水平及体质量(BW)、左心室质量指数(LVWI)、心脏指数(H/B);采用HE和Masson染色,比较各组大鼠心肌组织形态学改变和纤维化情况;采用ELISA法测定大鼠心肌组织Ⅰ型胶原(colⅠ)、Ⅲ型胶原(colⅢ)含量,并计算colⅠ/colⅢ比值;Western blot法检测大鼠心肌组织colⅠ和MMP-14蛋白表达量.结果:DM各组大鼠FBG均显著高于HC组和对照组(P<0.01);且随病程延长,DM大鼠FBG逐渐升高(P<0.01).DM组和HC组大鼠TG、TC水平均明显高于对照组(P<0.01),其中DM 8周组和12周组TG、TC水平亦均明显高于HC组(P<0.01);随病程延长,DM大鼠TG、TC水平均逐渐升高(P<0.05～P<0.01).各组大鼠BW间差异无统计学意义(P>0.05).DM 8周和12周组大鼠H/B和LVWI均明显高于对照组、HC组和DM 4周组(P<0.01),DM 12周组大鼠H/B亦明显高于DM 8周组(P<0.01).HE染色结果显示,DM组大鼠心肌细胞增生肥大,纤维排列紊乱、断裂不连续;且随病程延长,病理改变更加明显.Masson染色显示,DM组胶原纤维粗大,排布不均,可见明显断裂,大量胶原沉积;且随时间延长,胶原沉积更加明显,以DM 12周组变化最为显著.ELISA结果显示,HC组和对照组大鼠colⅠ、colⅢ含量差异均无统计学意义(P>0.05);DM组大鼠心肌组织colⅠ、colⅢ含量均明显高于对照组和HC组(P<0.01);且随病程延长,DM组大鼠colⅠ、colⅢ含量均逐渐升高(P<0.01);DM 8周和12周组大鼠colⅠ/colⅢ比值均明显高于对照组、HC组和DM 4周组(P<0.01).Western blot结果显示,DM 8周、12周组大鼠colⅠ、MMP-14蛋白表达量均明显高于对照组、HC组和DM 4周组(P<0.01),其中DM 12周组colⅠ、MMP-14表达亦均高于DM 8周组(P<0.01);而DM 4周组大鼠colⅠ、MMP-14蛋白表达量与对照组和HC组差异均无统计学意义(P>0.05).结论:DM可引起大鼠心肌纤维化,并随病程延长逐渐加重,其机制可能与心肌MMP-14表达量持续升高有关.

The occurrence of myocardial fibrosis and the expression of MMP-14 in diabetic rats

Objective:To observe the occurrence of myocardial fibrosis and the change of MMP-14 in different courses of diabetic rats and analyze the possible mechanism. Methods:All male Sprague Dawley(SD) rats were randomly divided into five groups(n=6):control group(CON),high glucose and high caloric group(HC) group and type 2 diabetes mellitus groups at 4-week(DM4w),8-week (DM8w) and 12-week(DM12w). Plasma triglyceride (TG),total cholesterol(TC) and fasting blood glucose (FBG) levels were measured,body weight ( BW ) , the ratio of heart weight/body weight ( H/B ) , left ventricular weight index ( LVWI ) levels were calculated. The myocardial morphological and fibrotic changes were discovered by HE and Masson staining methods. The contents of colⅠ and colⅢin heart tissue were evaluated by ELISA and the ratio of colⅠ/colⅢwas calculated. The protein expressions of MMP-14 and col Ⅰwere measured by Western blotting. Results:Compared with CON group,plasma TG and TC levels increased significantly in HC and DM groups. The FBG level increased in DM groups. With the course of diabetes extended, FBG, H/B, LVWI and plasma TG and TC levels further increased. HE staining showed cardiac cells were arranged irregularly and fractures were not continuous in DM groups,Masson staining showed that a large amount of interstitial collagen fiber hyperplasia, collagen accumulation or uneven distribution appeared in DM groups. The contents of col Ⅰ,col Ⅲ,the ratio of col Ⅰ/colⅢ and the protein expressions of col Ⅰ and MMP-14 in myocardial tissue were further increased with the extension of diabetes course. Conclusions:Type 2 diabetes can induced the happening of myocardial fibrosis,the fibrosis degree will gradually aggravated with the course of diabetes extended. The mechanism may be related to the continuously increase of the expression of MMP-14 in myocardium.