痰瘀同治法调控JNK信号通路对糖尿病大鼠心肌保护的作用机制

Mechanism of phlegm-stasis co-treatment for myocardial protection in rats with diabetes mellitus by regulating JNK signaling pathway

目的观察痰瘀同治法对糖尿病模型大鼠心肌c-Jun氨基末端激酶(JNK)信号通路的干预机制。方法选取健康SPF级雄性SD大鼠50只,采用单次腹腔注射链脲佐菌素(STZ)溶液55 mg/kg建立糖尿病模型。继续喂养3周后,将造模成功大鼠按随机数字表法分为正常组、模型组、阿拉氯胺组、化瘀组、化痰组、痰瘀同治组,每组6只。化痰组、化瘀组、痰瘀同治组分别灌胃小陷胸汤(4.05 g/kg)、血府逐瘀汤(7.02 g/kg)、抵当陷胸汤(8.10 g/kg),阿拉氯胺组灌胃阿拉氯胺(3 mg/kg)。连续灌药8周,采用HE染色观察各组大鼠心肌组织形态学改变;Masson染色观察大鼠心肌间质胶原沉积情况;免疫组化染色检测心肌组织中JNK1蛋白表达;实时荧光定量PCR法检测大鼠心肌组织中JNK1、胰岛素受体底物-1(IRS-1)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)mRNA水平;Western blot法检测IRS-1、p-Akt、NLRP3蛋白表达。结果模型组大鼠心肌细胞排列混乱,细胞肥大,纹理模糊,间质有炎性浸润,胶原纤维增多,出现局灶性坏死;各给药组均可不同程度地改善纤维化、炎性浸润状况,减少心肌胶原沉积。与模型组比较,痰瘀同治组JNK1、NLRP3 mRNA及蛋白表达降低(P<0.01),IRS-1 mRNA水平及蛋白表达升高(P<0.01),p-Akt蛋白表达升高(P<0.01)。结论痰瘀同治法可有效改善糖尿病大鼠心肌病变,且效果较单纯使用化痰法或化瘀法更佳,其机制可能与抑制JNK信号通路激活,下调JNK1、NLRP3蛋白,上调IRS-1、Akt蛋白表达有关。

Objective To observe the intervention mechanism of phlegm-stasis co-treatment for the JNK signaling pathway in the myocardium of diabetes rats.Methods Totally 50 male SD rats of SPF grade were selected.Diabetes model was established by single intraperitoneal injection of 55 mg/kg streptozotocin(STZ)solution.After continued feeding for 3 weeks,the rats were divided into normal group,model group,alachloramine group,blood stasis removing group,phlegm removing group and phlegm-blood stasis co-treatment group according to random number table method,with 6 rats in each group.Xiaoxianxiong Decoction(4.05 g/kg),Xuefu Zhuyu Decoction(7.02 g/kg),Didang Xianxiong Decoction(8.10 g/kg)were administered to the stomach respectively in the phlegm removing group,the blood stasis removing group and the phlegm-blood stasis co-treatment group.Alachloramine(3 mg/kg)was administered to the stomach by gavage in the alachloramine group.After 8 weeks,HE staining was used to observe the morphological changes of myocardial tissue in diabetic rats.Masson staining was used to observe the deposition of collagen fibers in the myocardial interstitium in rats.The expression of JNK1 protein was determined by immunohistochemistry.JNK1 mRNA,IRS1 mRNA and NLRP3 expression levels were detected by Real-time PCR.Western blot was used to detect the protein expressions of IRS-1,p-Akt and NLRP3.Results The myocardial cells in the model group were disorganized,with hypertrophy,blurred texture,inflammatory infiltration of interstitium,increased collagen fibers,and focal necrosis.All treatment groups could improve fibrosis,inflammatory infiltration and reduce myocardial collagen deposition in different degrees.Compared with the model group,the mRNA and protein expressions of JNK1 and NLRP3 bodies decreased(P<0.01),the IRS-1 mRNA and protein increased(P<0.01),and p-Akt protein expression increased(P<0.01).Conclusions The phlegm and stasis co-treatment can effectively improve the cardiomyopathy of diabetes rats,and the effect is better than the phlegm-resolving method or the stasis resolving method alone.The mechanism may be related to the inhibition of JNK signaling pathway activation,reduce the expressions of JNK1 and NLRP3,and increase the IRS-1 and Akt.