尼可地尔通过NLRP3/Caspase-1通路对糖尿病心肌病大鼠心脏损伤的影响

Effects of nicorandil on cardiac injury in rats with diabetic cardiomyopathy through NLRP3/Caspase-1 pathway

目的基于NOD样受体3(NLRP3)/半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)通路研究尼可地尔对糖尿病心肌病大鼠心脏损伤的影响。方法2020年10月在中国中医科学院西苑医院基础医学研究所进行实验。健康Wistar大鼠24只随机选取16只采用高糖高脂饲料联合腹腔注射链脲佐菌素建立糖尿病大鼠模型,余8只常规饲料喂养(正常组)。将成模大鼠随机分为模型组、尼可地尔组,各8只,尼可地尔组按照5 mg/kg剂量灌胃,正常组、模型组给予同体积的蒸馏水,每天1次,连续干预4周。实验结束后,高分辨率小动物超声仪检测大鼠心功能,HE染色观察心肌病理学改变,Masson染色观察心肌纤维化的改变,蛋白免疫印迹法(Western-blot)检测心肌组织NLRP3、Caspase-1的表达,免疫组化法(IHC)检测心肌组织消皮素D(GSDMD)的表达,酶联免疫吸附法(ELISA)检测血清中白介素-1β(IL-1β)、IL-18水平。结果与正常组比较,模型组大鼠左心室射血分数(LVEF)、左心室缩短率(FS)、每搏输出量(SV)显著降低(t/P=5.567/<0.001、3.192/0.012、4.205/0.002),心肌胶原容积分数升高(t/P=4.896/0.001);心肌组织NLRP3、Caspase-1、GSDMD蛋白表达水平明显升高(t/P=3.546/0.024、3.877/0.018、5.347/<0.001);血清IL-1β、IL-18水平明显升高(t/P=9.673/<0.001、7.972/<0.001)。与模型组比较,尼可地尔组大鼠LVEF、SV明显升高(t/P=2.080/0.048、3.171/0.010),心肌胶原容积分数明显降低(t/P=2.163/0.036);心肌组织NLRP3、Caspase-1、GSDMD蛋白表达水平显著降低(t/P=4.201/0.014、3.069/0.037、2.388/0.038);血清中IL-1β、IL-18水平明显降低(t/P=9.929/<0.001、5.473/<0.001)。结论尼可地尔可能通过调控NLRP3/Caspase-1信号通路抑制心肌细胞焦亡,减轻心肌纤维化,改善糖尿病大鼠心功能。

Objective To investigate the effect of nicorandil on cardiac injury in diabetic cardiomyopathy rats based on NOD-like receptor 3(NLRP3)/cysteine aspartate proteolytic enzyme 1(Caspase-1)pathway.Methods In October 2020,the experiment was conducted at the Institute of Basic Medicine,Xiyuan Hospital,Chinese Academy of Chinese Medical Sciences.From 24 healthy Wistar rats,16 rats were randomly selected with high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin to establish a diabetic rat model,and the remaining 8 rats were fed with conventional diet(normal group).The model rats were randomly divided into model group and nicorandil group,8 in each group.The nicorandil group was intragastrically administered at a dose of 5 mg/kg,and the normal group and model group were given the same volume of distilled water,once a day,for 4 weeks of continuous intervention.After the experiment,high-resolution small animal ultrasound was used to detect the cardiac function of the rats,and hematoxylin-eosin(HE)staining was used to observe the pathological changes of the myocardium.The changes of myocardial fibrosis were observed by Masson staining.The expression of NLRP3 and Caspase-1 in myocardial tissue was detected by Western blot.Immunohistochemical method(IHC)was used to detect the expression of desmoxidin D(GSDMD)in myocardial tissue.The levels of interleukin 1β(IL-1β)and IL-18 in serum were detected by enzyme-linked immunosorbent assay(ELISA).Results Compared with the normal group,the left ventricular ejection fraction(LVEF),left ventricular shortening rate(FS),and stroke volume(SV)in the model group were significantly decreased(t/P=5.567/<0.001,3.192/0.012,4.205/0.002).The myocardial collagen volume fraction increased(t/P=4.896/0.001).The protein expression levels of NLRP3,Caspase-1 and GSDMD in myocardial tissue were significantly increased(t/P=3.546/0.024,3.877/0.018,5.347/<0.001).Serum IL-1βand IL-18 levels were significantly increased(t/P=9.673/<0.001,7.972/<0.001).Compared with the model group,the LVEF and SV of the nicorandil group were significantly increased(t/P=2.080/0.048,3.171/0.010),and the myocardial collagen volume fraction was significantly decreased(t/P=2.163/0.036).The protein expression levels of NLRP3,Caspase-1 and GSDMD in myocardial tissue were significantly decreased(t/P=4.201/0.014,3.069/0.037,2.388/0.038).The levels of IL-1βand IL-18 in serum were significantly decreased(t/P=9.929/<0.001,5.473/<0.001).Conclusion Nicorandil may inhibit cardiomyocyte pyroptosis,alleviate myocardial fibrosis and improve cardiac function in diabetic rats by regulating the NLRP3/Caspase-1 signaling pathway.