炙甘草汤对异丙肾上腺素诱导的心肌纤维化大鼠心肌NF-κB信号通路的调节作用研究

Study on regulation of Zhigancao Decoction on myocardial NF-κB signal pathway in rats with myocardial fibrosis induced by isoproterenol

目的研究炙甘草汤对异丙肾上腺素诱导的心肌纤维化大鼠NF-κB信号通路的调节作用。方法将72只健康雄性Wistar大鼠随机分为正常对照组、心肌纤维化组、炙甘草汤低剂量组、炙甘草汤中剂量组、炙甘草汤高剂量组及吡咯二硫氨基甲酸酯组,每组12只。除正常对照组外,其余组大鼠均采用腹腔注射异丙肾上腺素的方法建立心肌纤维化模型。造模成功后,炙甘草汤低、中、高剂量组分别给予2 mL/kg、4 mL/kg、8 mL/kg的炙甘草汤灌胃,吡咯二硫氨基甲酸酯组腹腔注射200 mg/kg的吡咯二硫氨基甲酸酯,均1次/d。连续干预21 d后,利用小动物超声仪测定各组大鼠左心室射血分数(LVEF)、心室短轴缩短率(LVFS),经颈动脉心室内插管法测定左室收缩压(LVSP)及左室舒张末压(LVEDP),使用酶联免疫吸附法(ELISA)试剂盒检测心肌组织中肿瘤坏死因子-α(TNF-α)、转化生长因子-β_(1)(TGF-β_(1))、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白水平,HE及Masson染色法观察心肌病理学改变,Western blot法检测心肌组织中NF-κB p65、p-NF-κB p65蛋白表达水平,RTq-PCR法测定心肌组织中NF-κB p65 mRNA表达量。结果与正常对照组比较,心肌纤维化组大鼠LVEF、LVFS及LVSP均显著降低(P均<0.05),LVEDP及心肌组织中TNF-α、TGF-β_(1)、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、NF-κB p65蛋白、p-NF-κB p65蛋白表达水平及NF-κB p65 mRNA表达量均显著升高(P均<0.05);与心肌纤维化组比较,炙甘草汤各剂量组大鼠LVEF、LVFS及LVSP均显著升高(P均<0.05),LVEDP及心肌组织中TNF-α、TGF-β_(1)、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、NF-κB p65蛋白、p-NF-κB p65蛋白表达水平及NF-κB p65 mRNA表达量均显著降低(P均<0.05),且上述指标随炙甘草汤的剂量增加变化越明显,组间两两比较差异均有统计学意义(P均<0.05)。结论炙甘草汤能够改善异丙肾上腺素诱导的心肌纤维化大鼠心功能,抑制大鼠心肌纤维化,其机制可能与调节NF-κB信号通路有关。

Objective It is to study the regulatory effect of Zhigancao Decoction on NF-κB signaling pathway in rats with myocardial fibrosis induced by isoproterenol.Methods 72 healthy male Wistar rats were randomly divided into normal control group,myocardial fibrosis group,low-dose Zhigancao Decoction group,medium-dose Zhigancao Decoction group,high-dose Zhigancao Decoction group and pyrrole dithiocarbamate group,each group had 12 animals.Except for the normal control group,the rats in the other groups were injected with isoproterenol intraperitoneally to establish myocardial fibrosis models.After successful modeling,the low,medium and high dose Zhigancao Decoction groups were given 2 mL/kg,4 mL/kg,8 mL/kg Zhigancao Decoction,respectively,and the pyrrole dithiocarbamate group was intraperitoneally injected with 200 mg/kg pyrrole Dithiocarbamate,all once per day.After 21 days of continuous intervention,the left ventricular ejection fraction(LVEF)and left ventricularshort-axis fractional shortening(LVFS)of each group were measured by a small animal ultrasound system,and left ventricular systolic blood pressure(LVSP)and left ventricular end diastolic pressure(LVEDP)were measured by intracardiac intubation via carotid artery,the levels of tumor necrosis factor-α(TNF-α),transforming growth factor-β_(1)(TGF-β_(1))and type I collagen,Ⅲcollagen in myocardial tissue were detected by enzyme-linked immunosorbent assay(ELISA),the myocardial pathological changes were observed by HE and Masson staining method,the protein expression levels of NF-κB p65,p-NF-κB p65 in myocardial tissue were detected by Western blot method,the expression levels of NF-κB p65 mRNA in myocardial tissue were detected by RTq-PCR method.Results Compared with the normal control group,the LVEF,LVFS and LVSP of rats in the myocardial fibrosis group were significantly reduced(all P<0.05),LVEDP and the expression levels of TNF-α,TGF-β_(1),typeⅠcollagen and typeⅢcollagen protein,NF-κB p65 protein,p-NF-κB p65 protein,and NF-κB p65 mRNA expression in myocardial tissue were significantly increased(all P<0.05);compared with the myocardial fibrosis group,LVEF,LVFS and LVSP of the rats in every dose Zhigancao Decoction group were significantly increased(all P<0.05),LVEDP and the expression levels of TNF-α,TGF-β_(1),typeⅠcollagen,typeⅢcollagen,NF-κB p65 protein,p-NF-κB p65 protein and the expression of NF-κB p65 mRNA in myocardial tissue were significantly reduced(all P<0.05),and the above indicators changed more significantly with the increase of the dose of Zhigancao Decoction,and the differences between every two groups were statistically significant(all P<0.05).Conclusion Zhigancao Decoction can improve the cardiac function and inhibit myocardial fibrosis of rats with myocardial fibrosis induced by isoproterenol.The mechanism may be related to the regulation of NF-κB signaling pathway.