法舒地尔调控巨噬细胞极化改善糖尿病小鼠心肌纤维化

目的:探讨盐酸法舒地尔(HF)对糖尿病(D)小鼠心肌纤维化及巨噬细胞极化的影响。方法:将60只C57BL/6小鼠随机分为正常组(NS组)、正常+法舒地尔组(N+HF组)、糖尿病组(D+NS组)、低剂量法舒地尔组(D+LHF组)、中剂量法舒地尔组(D+MHF组)及高剂量法舒地尔组(D+HHF组)。采用链脲佐菌素(STZ)连续腹腔注射建立1型糖尿病小鼠模型。干预8周后处死小鼠,观察法舒地尔对小鼠体重及血糖的影响;HE及Masson染色观察心脏组织形态改变,测量心肌胶原容积分数(collagen volume fraction,CVF);免疫组化观察心脏组织中巨噬细胞极化及白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和IL-10的蛋白水平;Western blot测定心脏组织中p-MYPT1 Thr853、诱导型一氧化氮合酶(iNOS)和精氨酸酶1(Arg-1)的蛋白水平。结果:与NS组相比,D+NS组小鼠成模后体重下降,血糖升高(P<0.05);与D+NS组相比,各治疗组血糖和体重差异无统计学显著性。与NS组相比,D+NS组CVF增大,M1型巨噬细胞数量增加,M2型巨噬细胞数量减少,IL-6、TNF-α、p-MYPT1 Thr853和iNOS的蛋白水平升高,IL-10和Arg-1的蛋白水平降低(P<0.05);与D+NS组相比,各治疗组CVF下降,M1型巨噬细胞数量减少,M2型巨噬细胞数量增加,IL-6和TNF-α的蛋白水平降低,IL-10的蛋白水平升高;与D+NS组相比,D+MHF和D+HHF组的p-MYPT1 Thr853和iNOS的蛋白水平下降,Arg-1的蛋白水平升高(P<0.05)。与NS组相比,N+HF组各检测指标差异无统计学显著性。结论:法舒地尔能改善糖尿病心肌病小鼠心肌纤维化,其可能的机制与诱导M2型巨噬细胞极化,减少M1型巨噬细胞极化和炎症反应有关。

Fasudil ameliorates myocardial fibrosis by regulating polarization of macrophages in diabetic mice

AIM:To investigate the effect of hydroxyl fasudil(HF)on myocardial fibrosis and macrophage polarization in the diabetic(D)mice.METHODS:C57BL/6 mice(n=60)were randomly divided into normal saline group(NS group),normal+hydroxyl fasudil group(N+HF group),diabetes group(D+NS group),diabetes+low dose of HF group(D+LHF group),diabetes+middle dose of HF group(D+MHF group)and diabetes+high dose of HF group(D+HHF group).A mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin(STZ).The mice in treatment groups received different doses of fasudil through intraperitoneal injection for 8 weeks.At the end of the study,the effects of fasudil at different doses on the body weight and blood glucose were observed.The histopathological changes of the cardiac tissues were observed by HE staining.The myocardial collagen volume fraction(CVF)was calculated by Masson staining.Immumohistochemical staining was used to test the macrophage polarization and protein expression of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and IL-10 and Western blot was applied to determine the protein levels of p-MYPT1 Thr853,inducible nitric oxide synthase(iNOS)and Arginase-1(Arg-1).RESULTS:Compared with NS group,the body weight of the mice in D+NS group was decreased and the blood glucose was increased significantly(P<0.05).However,no statistically difference of blood glucose and body weight between the treatment groups and D+NS group was observed.Compared with NS group,CVF,the number of M1-type macrophages and the protein levels of IL-6,TNF-α,p-MYPT1 Thr853 and iNOS were increased markedly,while M2-type macrophages and the expression of IL-10 and Arg-1 were decreased in D+NS group(P<0.05).Compared with D+NS group,CVF,the number of M1-type macrophages,and the protein levels of IL-6 and TNF-αwere relatively decreased,conversely the number of M2-type macrophages and the protein level of IL-10 was increased in treatment groups(P<0.05).Moreover,the protein levels of p-MYPT1 Thr853 and iNOS were reduced and the protein level of Arg-1 was increased in D+MHF and D+HHF group compared with D+NS group(P<0.05).No statistical difference in above mentioned indexes between NS group and N+HF group was observed.CONCLUSION:Fasudil significantly attenuates the myocardial fibrosis of diabetic cardiomyopathy in mice,which is possibly related to increased polarization of M2-type macrophages,decreased polarization of M1-type macrophages and inflammation.