基于TGF-β1/Smads信号通路探讨鹿红益心颗粒对心力衰竭大鼠心肌纤维化的影响

Effects of Luhong Yixin Granules on myocardial fibrosis in heart failure rats based on TGF-β1/Smads signaling pathway

旨在探讨鹿红益心颗粒对心力衰竭大鼠心肌纤维化的影响及可能的作用机制。将60只雄性Wistar大鼠随机分为对照组、模型组以及鹿红益心颗粒低、中、高剂量组,每组12只。除对照组外,其余各组大鼠通过腹腔注射阿霉素(DOX)诱导心力衰竭模型。将鹿红益心颗粒溶于等量生理盐水,分别予以低、中、高剂量(2.8、5.6、11.2 g·kg^(-1)·d^(-1))进行灌胃,对照组和模型组均给予等量生理盐水,持续灌胃40 d。给药结束后,超声心动图测量左心室射血分数(LVEF)和左心室缩短分数(LVFS);测量大鼠体质量和心脏体质量,计算心脏体质量比;酶联免疫吸附法测定血清中白细胞介素-6(IL-6)、白细胞介素-17(IL-17)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β1(TGF-β1)、生长刺激表达基因2蛋白(ST2)、N末端B型利钠肽原(NT-proBNP)、半乳糖凝集素-3(Gal-3)和肌酸激酶同工酶(CK-MB)水平;苏木素-伊红(HE)染色和Masson染色观察心肌组织病理形态;蛋白免疫印迹法和实时荧光定量聚合酶链式反应分别检测心肌组织中IL-6、IL-17、TNF-α、TGF-β1、Smad3、Smad7、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(COL-Ⅰ)蛋白及其mRNA表达水平。结果显示,与对照组相比,模型组大鼠LVEF、LVFS、心脏体质量比值均显著下降(P<0.05),IL-6、IL-17、TNF-α、TGF-β1、ST2、NT-proBNP、Gal-3、CK-MB水平均显著上升(P<0.05);HE染色可见心肌组织炎性改变;Masson染色显示心脏横截面面积及室腔面积减小且出现不同程度的心肌纤维化(P<0.05);心肌组织中IL-6、IL-17、TNF-α、TGF-β1、Smad3、α-SMA、COL-Ⅰ蛋白及其mRNA表达均显著升高(P<0.05),Smad7蛋白及其mRNA表达显著降低(P<0.01)。与模型组相比,鹿红益心颗粒低、中、高剂量组大鼠LVEF、LVFS及心脏体质量比值均显著上升(P<0.05),IL-6、IL-17、TNF-α、TGF-β1、ST2、NT-proBNP、Gal-3、CK-MB水平均下显著下降(P<0.05);HE染色显示心肌组织炎性改变逐渐减轻;Masson染色显示心脏横截面面积及室腔面积增大,心肌纤维化面积减少(P<0.05);心肌组织中IL-6、IL-17、TNF-α、TGF-β1、Smad3、α-SMA、COL-Ⅰ蛋白及其mRNA表达均显著降低(P<0.05),Smad7蛋白及其mRNA表达均显著升高(P<0.05)。综上所述,鹿红益心颗粒可能通过调节TGF-β1/Smads信号通路,抑制炎症相关蛋白表达,减少细胞外基质的沉积,缓解心肌纤维化,对心力衰竭的治疗具有重要价值。

To investigate the effects of Luhong Yixin Granules on myocardial fibrosis in rats with heart failure and its possible mechanism,a total of 60 male Wistar rats were randomly divided into the control group,model group,and low-,medium-and high-dose Luhong Yixin Granules groups,with 12 rats in each group.Except for those in the control group,rats in the other groups were induced by intraperitoneal injection of doxorubicin(DOX)into a rat model.After the Luhong Yixin Granules were dissolved in the same amount of normal saline,they were given by gavage at low,medium and high doses(2.8,5.6,11.2 g·kg^(-1)·d^(-1)),and the control group and the model group were given the same amount of normal saline by gavage for 40 days.After the end of dosing,echocardiography was used to measure left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS).Rat body weight(BW)and heart weight(HW)were calculated as HW/BW.Enzyme-linked immunosorbent assay was used to measure the levels of interleukin-6(IL-6),interleukin-17(IL-17),tumor necrosis factor-α(TNF-α),transforming growth factor-β1(TGF-β1),growth stimulation expressed gene 2 protein(ST2),N-terminal pro-B-type natriuretic peptide(NT-proBNP),galectin-3(Gal-3)and creatine kinase isoenzyme(CK-MB)in serum.Hematoxylin-eosin(HE)staining and Masson staining were used to observe the pathological morphology of myocardial tissue.Western blot and quantitative real-time polymerase chain reaction were used to detect the protein and mRNA expression levels of IL-6,IL-17,TNF-α,TGF-β1,Smad3,Smad7,α-smooth muscle actin(α-SMA),and collagenⅠ(COL-Ⅰ),respectively.Results showed that compared with those in the control group,LVEF,LVFS,and HW/BW in the model group were decreased(P<0.05),and the levels of IL-6,IL-17,TNF-α,TGF-β1,ST2,NT-proBNP,Gal-3,and CK-MB were increased(P<0.05).HE staining showed inflammatory changes in myocardial tissue;Masson staining showed decreases in the cross-sectional area and ventricular cavity area of the heart,and myocardial fibrosis of varying degrees(P<0.05).The protein and mRNA expression of IL-6,IL-17,TNF-α,TGF-β1,Smad3,α-SMA,and COL-Ⅰwere increased(P<0.05),and the protein and mRNA expression of Smad7 protein was decreased(P<0.01).Compared with those in the model group,LVEF,LVFS and HW/BW of the low-,medium-and high-dose Luhong Yixin Granules groups were increased(P<0.05),and the levels of IL-6,IL-17,TNF-α,TGF-β1,ST2,NT-proBNP,Gal-3 and CK-MB were decreased(P<0.05).HE staining showed gradually reduced inflammatory changes of myocardial tissue,and Masson staining showed increased cross-sectional area and ventricular cavity area of the heart and decreased area of myocardial fibrosis(P<0.05).The protein and mRNA expression levels of IL-6,IL-17,TNF-α,TGF-β1,Smad3,α-SMA,and COL-Ⅰwere decreased(P<0.05),while the protein and mRNA expression levels of Smad7 were increased(P<0.05).Luhong Yixin Granules may be of great value in the treatment of heart failure by regulating the TGF-β1/Smads signaling pathway,inhibiting the expression of inflammation-related proteins,reducing the deposition of extracellular matrix,and alleviating myocardial fibrosis.