FBXW7和RhoA/Rho激酶蛋白表达参与糖尿病大鼠心肌纤维化

Protein expressions of FBXW7 and RhoA/Rho kinase play role in myocardial fibrosis in diabetic rats

目的探究F框/含蛋白7 WD-40域蛋白(FBXW7)、Ras同源基因家族成员A(RhoA)、Rho相关卷曲螺旋蛋白激酶(ROCK)的表达与糖尿病心肌纤维化的关系。方法将SD大鼠随机分为正常对照组和糖尿病模型组,采用一次性ip链脲佐菌素60 mg·kg^(-1)建立1型糖尿病大鼠模型,分别于2,4,8,12和16周进行检测。HE染色观察大鼠心脏组织形态变化;Masson染色和测定心肌羟脯氨酸(HYP)含量观察心肌胶原沉积程度;ELISA法检测血清中FBXW7,转化生长因子β_1(TGF-β_1)和结缔组织生长因子(CTGF)的含量;Western蛋白印迹和免疫组化法检测心肌中FBXW7,RhoA和ROCK1蛋白的表达。结果显微镜下可见糖尿病模型组大鼠心肌不同程度局灶性心肌细胞变性、坏死和纤维组织增生等。与正常对照组相比,4～16周时,模型组大鼠心肌组织中胶原沉积面积和胶原含量显著升高(P<0.05,P<0.01),呈递增趋势;2～4周时,大鼠血清中FBXW7含量显著增加(P<0.01),第8周时降低,但仍显著高于正常对照组(P<0.01);8～16周时,大鼠血清中TGF-β_1和CTGF含量逐渐升高(P<0.01)。Western蛋白印迹和免疫组化结果显示,模型组大鼠心肌RhoA和ROCK1蛋白表达在各时间点均显著高于正常对照组(P<0.05,P<0.01);2～12周模型组大鼠心肌FBXW7表达较正常对照组显著升高(P<0.01,P<0.01),第4周表达最高,8～16周表达下降,与时间呈负相关(r=-0.988,P<0.05),但仍高于正常对照组(P<0.01)。结论 FBXW7和RhoA/Rho激酶参与糖尿病大鼠心肌纤维化的发生发展,可能成为治疗糖尿病心肌病的靶点。

OBJECTIVE To investigate the relationships between F-box/WD-40 domain-containing protein 7(FBXW7),member A of Ras homologous gene family(RhoA),Rho-related coiled-coil kinase(ROCK)and diabetic myocardial fibrosis.METHODS SD rats were one-time ip administered with streptozotocin 60 mg·kg-1to establish a type 1 diabetic rat model.Body mass,cardiac index,left ventricular index and blood glucose levels were detected on the 2nd,4th,8th,12th and 16thweeks,respectively.The morphology of rat heart tissue was observed by HE staining.The degree of myocardial collagen deposition was detected by Masson staining and content of hydroxyproline(HYP).ELISA was used to detect the contents of FBXW7,transforming growth factor-β1(TGF-β1)and connective tissue growth factor(CTGF)in serum.The expression of FBXW7,RhoA and ROCK1 in the myocardium were determined by immunohistochemistry and Western blotting.RESULTS The myocardial cells of the model group were degenerated and necrotic,and fibrous tissue hyperplasia was abserved under the microscope.Compared with normal control group,the collagen deposition area and collagen content in the myocardial tissue of the model group increased significantly from the 4thto 16thweeks(P<0.05,P<0.01),and kept increasing.The serum FBXW7 content increased in the first 2-4 weeks(P<0.01),decreased at the 8thweek,but remained higher than that of normal control group(P<0.01).The serum levels of TGF-β1 and CTGF increased from the 8th to 16thweeks(P<0.01).Western blotting and immunohistochemistry showed that the expressions of RhoA and ROCK1 protein in the myocardium of the model group were significantly higher than those of normal control group at each time point(P<0.05,P<0.01).The expression of FBXW7 in the myocardium of the 2nd-12thweeks model group was significantly increased compared with normal control group(P<0.01),and reached the peak in the 4thweek,but decreased in the 8th-16th weeks(P<0.01).CONCLUSION FBXW7 and RhoA/Rho kinase are involved in the occurrence and development of myocardial fibrosis in diabetic rats and may be a target for the treatment of diabetic cardiomyopathy.