基于p38 MAPK/NF-κB信号通路探讨藏红花素对糖尿病心肌病大鼠的影响

Effect of Crocin on diabetic cardiomyopathy in rats via p38 MAPK/NF-κB signaling pathway

目的基于p38丝裂原活化蛋白激酶/核因子-κB(p38 MAPK/NF-κB)信号通路探讨藏红花素对糖尿病心肌病(DCM)大鼠的影响。方法取45只Wistar大鼠,随机选择8只作为正常组,其余大鼠采用高糖高脂饮食12周并负荷一次性30 mg/kg链脲佐菌素的方法构建DCM模型。将32只造模成功的大鼠随机分为模型组、藏红花素组、藏红花素+p38 MAPK抑制剂组和藏红花素+p38 MAPK激动剂组,每组8只。藏红花素组给予50 mg/kg藏红花素腹腔注射,藏红花素+p38 MAPK抑制剂组给予50 mg/kg藏红花素和5 mg/kg SB203580腹腔注射,藏红花素+p38 MAPK激动剂组给予50 mg/kg藏红花素和2 mg/kg ANS腹腔注射,正常组和模型组给予等体积生理盐水腹腔注射,均1次/d。连续注射12周后,心脏超声检测大鼠心功能指标[左心室收缩末期压(LVESD)、左心室舒张末期压(LVEDD)、左心室短轴缩短率(LVFS)],ELISA法检测血清心肌酶[谷草转氨酶(AST)、乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)]和炎症因子[白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)、肿瘤坏死因子-α(TNF-α)]水平,苏木精-伊红、马森染色观察心肌组织病变和纤维化情况,RT-PCR法、免疫组织化学法检测心肌组织中p38 MAPK、NF-κB p65、转化生长因子-β1(TGF-β1)、I型胶原蛋白(Col-I)mRNA和蛋白表达情况。结果与模型组比较,藏红花素组、藏红花素+p38 MAPK抑制剂组大鼠LVESD、LVEDD及血清AST、LDH、CK-MB、IL-1β、IL-18、TNF-α水平均明显降低(P均<0.05),LVFS明显升高(P<0.05);心肌组织病变和纤维化情况均明显改善,心肌组织损伤评分和胶原容积分数均明显降低(P均<0.05);心肌组织中p38 MAPK、NF-κB p65、TGF-β1、Col-I mRNA相对表达量和蛋白表达积分吸光度均明显降低(P均<0.05)。与藏红花素组比较,SB203580可显著增强藏红花素对DCM大鼠心功能指标、心肌酶和炎症因子水平、心肌组织病变和纤维化情况、p38 MAPK/NF-κB信号通路相关因子mRNA和蛋白表达的调控作用,ANS则明显逆转藏红花素对DCM大鼠的上述作用。结论藏红花素可能通过抑制p38 MAPK/NF-κB信号通路减轻炎症反应和组织纤维化,从而对DCM大鼠起到保护作用。

Objective It is to investigate the effect of Crocin on diabetic cardiomyopathy(DCM)in rats via p38 mitogen-activated protein kinase/nuclear factor-κB(p38 MAPK/NF-κB)signaling pathway.Methods Forty-five Wistar rats were taken,in which 8 rats were randomly selected as normal group,and the remaining rats were fed with high-sugar and high-fat diet for 12 weeks and loaded with 30 mg/kg streptozotocin to establish models of DCM.32 successfully modeled rats were randomly divided into model group,Crocin group,Crocin+p38 MAPK inhibitor group and Crocin+p38 MAPK agonist group,with 8 rats in each group.The Crocin group was given 50 mg/kg Crocin intraperitoneally,the Crocin+p38 MAPK inhibitor group was given 50 mg/kg Crocin and 5 mg/kg SB203580 intraperitoneally,the Crocin+p38 MAPK agonist group was given 50 mg/kg Crocin and 2 mg/kg ANS intraperitoneally,and the normal group and model group were given equal volumes of normal saline by intraperitoneal injection,all once daily.After 12 weeks of administration,the cardiac function indexes[left ventricular end-systolic diameter index(LVESD),left ventricular end diastolic diameter(LVEDD),left ventricular fractional shortening(LVFS)]of the rats were detected by cardiac ultrasound,the serum levels of myocardial enzyme[aspartate transaminase(AST),lactate dehydrogenase(LDH),creatine kinase isoenzyme MB(CK-MB)]and inflammatory factors[interleukin-1β(IL-1β),IL-18,tumor necrosis factor-α(TNF-α)]in myocardial tissue were detected by ELISA,the myocardial tissue pathological changes and fibrosis were observed by hematoxylin-eosin or masson staining,The mRNA and protein expressions of p38 MAPK,NF-κB p65,transforming growth faction-β1(TGF-β1),type I collagen(Col-I)were detected by RT-PCR or immunohistochemical(IHC)method.Results Compared with the model group,the LVESD,LVEDD,and serum levels of AST,LDH,CK-MB,IL-1β,IL-18,TNF-αin the Crocin group and Crocin+p38 MAPK inhibitor group were significantly decreased,and the LVFS was significantly increased(all P<0.05);the myocardial tissue pathological changes and fibrosis were significantly improved,the injury score and collagen volume fraction were significantly decreased(all P<0.05);the mRNA and protein expressions of p38 MAPK,NF-κB p65,TGF-β1,Col-I were significantly decreased(all P<0.05).Compared with the Crocin group,SB203580 could significantly enhance the regulatory effects of Crocin on cardiac function indexes and levels of myocardial enzyme and inflammatory factors,myocardial tissue pathological changes and fibrosis,p38 MAPK/NF-κB signaling pathway related mRNA and protein expression in DCM rats;while ANS could reverse these effects of Crocin on DCM rats.Conclusion Crocin may alleviate inflammation and tissue fibrosis via inhibiting the p38 MAPK/NF-κB signaling pathway,thereby protect DCM rats.