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目的:ERK1/ET1在慢性心肌炎病毒持续感染与纤维化表达及清心Ⅱ号干预作用。方法:150只清洁级体重14~16 g健康雄性Balb/c小鼠,采用随机数字表法分出10只作为正常组,其余140只采用腹腔注射含不同浓度柯莎奇病毒B3半数组织培养感染剂量=10-5(coxsackie virus B3tissue culture infective dose50=10-5,CVB3 TCID50=10-5)的病毒液3次,每次每只2 m L,首次1:2000浓度CVB3病毒液,2周后腹腔接种1∶1600浓度CVB3病毒液,4周后腹腔注射1∶800浓度CVB3病毒液,60天后模型构建成功,存活小鼠94只。随机选取90只,分为清心II号高、中、低剂量组各20只,卡托普利组20只,模型组10只。正常组和模型组给予生理盐水灌胃,治疗组分别给予卡托普利和清心II号高中低剂量灌胃。实验结束,摘眼球取血后颈椎脱臼法处死动物,采集心脏组织液氮速冻。用放射免疫法测各组血浆内皮素1(endothelium 1,ET1)含量;RT-PCR病毒、P-ERK1基因检测;MASSON染色观察各组纤维化形成情况,并检测胶原阳性面积占测量的面积的百分比的即容积分数。结果:造模后ET1、ERK1、CVB3RNA模型组及各治疗组均高于正常组(P<0.05);MASSON染色后正常组含少量胶原纤维,分布于血管周围;模型组心肌细胞排列紊乱,胶原纤维与正常组比较增生明显(P<0.05);治疗后各治疗组ERK1、ET1、CVB3RNA与模型组比较相应均减低(P<0.05),P-ERK1各治疗组均接近正常组(P>0.05),各治疗组胶原纤维较模型组减少(P<0.05),清心Ⅱ号各治疗组随清心Ⅱ号剂量增加ERK1、ET1、CVB3RNA逐渐减少,高剂量组胶原含量接近于正常组(P>0.05),清心Ⅱ号高中低剂量组与卡托普利组疗效相当(P>0.05)。结论:在慢性病毒性心肌炎心肌细胞中CVB3RNA持续存在,ET1与ERK1在心肌病毒持续感染及心肌纤维化形成过程中发挥重要作用,清心Ⅱ号可显著减少ET1、ERK1在心肌的表达,且具有清除柯莎奇病毒及显著抗心肌纤维化作用。
OBJECTIVE: To investigate the effect of ERK1 / ET1 on persistent infection and fibrosis of chronic myocarditis virus and the intervention of Qingxin Ⅱ. Methods: One hundred and fifty healthy male Balb / c mice weighing 14-16 g were divided into normal group by random number table and the other 140 were intraperitoneally injected with half of tissue culture inoculation with different concentration of coxsackievirus B3 The virus solution of the dose = 10-5 (coxsackie virus B3 tissue culture infective dose 50 = 10-5, CVB3 TCID50 = 10-5) was administered 3 times for 2 mL each for the first time at a concentration of 1: 2000 CVB3 virus, and after 2 weeks The CVB3 virus solution was inoculated intraperitoneally at the concentration of 1: 1600 and the CVB3 virus solution was injected intraperitoneally at a concentration of 1: 800 after 4 weeks. After 60 days, the model was successfully constructed and 94 survived mice. Ninety rabbits were randomly selected and divided into two groups: 20 in the high, medium and low dose group of Qingxin II, 20 in the captopril group and 10 in the model group. Normal group and model group were given normal saline, the treatment group were given Captopril and Qingxin II high school low dose gavage. At the end of the experiment, the animals were sacrificed by cervical dislocation after blood was taken from the eyeball, and frozen in liquid nitrogen of the heart tissue. The contents of endothelium 1 (ET1), RT-PCR and P-ERK1 in each group were detected by radioimmunoassay. The formation of fibrosis was observed by MASSON staining, and the area of the positive area of collagen The percentage is the volume fraction. Results: After modeling, ET1, ERK1, CVB3RNA model group and each treatment group were higher than the normal group (P <0.05); in the normal group, a small amount of collagen fibers were found in the normal group, distributed in the perivascular area; the myocardial cells in the model group were disordered, collagen Compared with the normal group, the proliferation of fibroblasts was significant (P <0.05). After treatment, ERK1, ET1 and CVB3RNA in each treatment group were significantly decreased compared with the model group (P <0.05), P-ERK1 treatment groups were close to the normal group ), The collagen fibers in each treatment group decreased compared with the model group (P <0.05). The levels of ERK1, ET1 and CVB3RNA decreased with the increase of the dosage of Qingxin II in each treatment group, and the collagen content of the high-dose group was close to that of the normal group ), Qingxin Ⅱ high school low dose group and captopril group (P> 0.05). CONCLUSION: CVB3RNA persists in cardiomyocytes of chronic viral myocarditis. ET1 and ERK1 play an important role in the persistent infection of myocarditis virus and the formation of myocardial fibrosis. Qingxin II can significantly reduce the expression of ET1 and ERK1 in myocardium, Saki virus and significant anti-myocardial fibrosis.