白皮杉醇对糖尿病心肌病模型大鼠的治疗机制研究

Therapeutic effect and underlying mechanism of picatanol on diabetic cardiomyopathy in a rat model

目的探讨白皮杉醇(Pic)对糖尿病心肌病(DCM)模型大鼠的治疗效果及作用机制。方法H9c2细胞分为三组:正常组、高糖(HG)组和HG+Pic组,CCK-8检测各组细胞活性,免疫荧光技术测定H9c2细胞中核因子-κB(NF-κB)p65的入核率。腹腔注射链脲佐菌素建立大鼠Ⅰ型糖尿病动物模型。48只SD大鼠按照随机数字表法分为正常组、DCM组、白皮杉醇低剂量(Pic-L)治疗组[5 mg/(kg·d)]、白皮杉醇高剂量(Pic-H)治疗组[10 mg/(kg·d)],每组12只。给药12周后,应用超声心功能检测在体评价各组大鼠心功能,检测各组大鼠血糖、心脏质量/体质量比(HW/BW),HE染色和天狼猩红染色观察各组大鼠心肌组织病理情况。采用酶联免疫吸附试验(ELISA)检测氧化应激反应相关指标超氧化物歧化酶(SOD)和丙二醛(MDA)水平。TUNEL染色观察心肌细胞凋亡情况。结果CCK-8结果显示,Pic可以提高HG下H9c2的细胞活性[(102.00±5.57)%比(84.67±4.93)%;P<0.05]。细胞免疫荧光染色结果显示,Pic降低HG下H9c2细胞中NF-κB p65的入核率。与DCM组比较,Pic-L治疗组、Pic-H治疗组大鼠心肌左心室舒张末期直径(LVIDd)和左心室收缩率(LVFS)提高[LVIDd:(6.50±0.16)mm、(7.41±0.09)mm比(5.64±0.13)mm,P<0.01;LVFS:(44.83±1.36)%、(46.77±0.99)%比(36.10±0.40)%,P<0.01],左心室后壁下降[(1.59±0.05)mm、(1.52±0.02)mm比(1.69±0.05)mm,P<0.01],同时心肌细胞形态及排列状态有所改善。与DCM组大鼠比较,Pic-H治疗组血糖[(13.70±2.18)mmol/L比(25.13±1.74)mmol/L,P<0.01]、HW/BW[(3.70±0.46)mg/g比(5.20±0.46)mg/g,P<0.05]下降。ELISA结果显示,与DCM组比较,Pic-L治疗组和Pic-H治疗组的SOD水平提高[(59.41±1.98)U/mg protein、(72.30±3.99)U/mg protein比(45.33±3.96)U/mg protein,P<0.01],MDA水平下降[(4.19±0.18)nmol/mg protein、(3.55±0.13)nmol/mg protein比(5.05±0.26)nmol/mg protein,P<0.01]。TUNEL染色结果表明,Pic治疗组能显著降低大鼠心肌细胞凋亡。结论Pic可能通过抑制心肌氧化应激和炎症反应,减轻心肌细胞凋亡和心肌组织纤维化,预防DCM大鼠心肌损伤。

Objective To assess the therapeutic effect and the underlying molecular events of piceatannol(Pic)on diabetes cardiomyopathy(DCM)in a rat model.Methods H9c2 cells were grown in normal,high-glucose(HG),and HG+Pic intervention(HG+Pic)culture media and then subjected to CCK-8 assay for detection of cell viability and immunofluorescence for detection of NF-κB p65 protein nuclear translocation.After that,a rat model of type I diabetes was established by the intraperitoneal injection of streptozotocin,i.e.,48 rats were randomly divided into normal,DCM,DCM+Pic low[Pic-L;5 mg/(kg·d)]and DCM+Pic-H[10 mg/(kg·d)]treatment groups(n=12 per group)for 12 weeks of Pic administration.The heart functions of each animal was evaluated using the ultrasonic cardiac function detection device,while the blood glucose and heart weight/body weight ratio(HW/BW)of each animal were also analyzed and histology of rat myocardial tissues was evaluated after H&E and Picrosirius red staining.ELISA was used to assay levels of oxidative stress related indicators superoxide dismutase(SOD)and malondialdehyde(MDA),while TUNEL assay was used to observe level of myocardial cell apoptosis.Results The CCK-8 results showed that Pic could improve H9c2 cell viability under the HG growth condition(102.00±5.57 vs.84.67±4.93%;P<0.05)and the immunofluorescence data showed that Pic blocked the NF-κB p65 protein nuclear translocation.Moreover,compared with the DCM group,the rat left ventricular end-diastolic diameter(6.50±0.16 and 7.41±0.09 vs.5.64±0.13 mm,respectively;P<0.01)and left ventricular systolic rate(44.83±1.36 and 46.77±0.99 vs.36.10±0.40%,respectively;P<0.01)in Pic-L and Pic-H treatment groups were all increased,whereas the left ventricular posterior wall(1.59±0.05 and 1.52±0.02 vs.1.69±0.05 mm,respectively;P<0.01)was decreased.Meanwhile,the myocardial cell morphology and arrangement were improved after the treatments.In addition,compared with DCM rats,the blood glucose(13.70±2.18 vs.25.13±1.74 mmol/L;P<0.01)and the HW/BW(3.70±0.46 vs.5.20±0.46 mg/g;P<0.05)were reduced after Pic-H treatment.The ELISA results showed that compared with DCM group,level of SOD in Pic-L and Pic-H treatment groups increased(59.41±1.98 and 72.30±3.99 vs.45.33±3.96 U/mg protein,respectively;P<0.01),whereas MDA level was decreased(4.19±0.18 and 3.55±0.13 vs.5.05±0.26 nmol/mg protein,respectively;P<0.01).The TUNEL data revealed that Pic treatment significantly reduced myocardial cell apoptosis compared to that of the model group of rats.Conclusion Pic treatment was able to reduce the myocardial oxidative stress and inflammatory reaction;thus,to reduce myocardial cell apoptosis and myocardial fibrosis and prevent myocardial injury in a rat model of diabetes cardiomyopathy.