人硫氧还蛋白基因表达对病毒性心肌炎小鼠心肌炎症反应的影响及其机制

Effects and related mechanism of overexpression of human thioredoxin on the inflammatory response in mice with viral myocarditis

目的探讨携带人硫氧还蛋白（hTRX）的重组腺病毒对柯萨奇B3（CVB3）病毒性心肌炎小鼠心肌炎症反应的影响及其机制。方法雄性Balb/c小鼠60只，4周龄，体重l2-14 g，分为3组，即对照组、心肌炎组和hTRX组，每组20只。hTRX组及心肌炎组小鼠腹腔注射100 TCID50的CVB3病毒0.1 ml，对照组同时给予等量的生理盐水。接种病毒前2 d，hTRX组小鼠心包腔穿刺注射滴度为2.5×108 pfu/ml的TRX重组腺病毒30 μl，心肌炎组小鼠心包腔穿刺注射滴度为2.5×108 pfu/ml的空载体重组腺病毒30 μl。于接种病毒的第7天将小鼠处死取心脏。心脏病理切片苏木素伊红染色观察各组心肌病理改变，电镜观察心肌超微结构改变，Western blot法检测心肌组织核因子κB（NF-κB）蛋白含量，ELISA法检测心肌组织肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）蛋白含量。结果（1）各组小鼠心肌组织光镜观察结果：心肌炎组小鼠心肌炎病变检出14只，hTRX组检出5只，对照组未检出。光镜下观察可见，对照组小鼠心肌组织未见炎性细胞浸润及坏死灶，细胞排列整齐，呈圆柱状，细胞质丰富红染，胞核卵圆，位于细胞中央；心肌炎组小鼠可见局灶性心肌坏死和炎性细胞浸润；hTRX组小鼠可见点状心肌坏死和炎性细胞浸润。（2）各组小鼠心肌组织透射电镜观察结果：心肌炎组小鼠心肌细胞肿胀、大量肌丝溶解、线粒体肿胀空化。而与心肌炎组相比，hTRX组小鼠心肌细胞肿胀较轻、肌丝溶解较少、线粒体肿胀空化较少。（3）各组小鼠心肌细胞核中NF-κB的蛋白含量：心肌炎组小鼠心肌组织细胞核NF-κB蛋白含量高于对照组（F=6.237，P〈0.01）, hTRX组则低于心肌炎组（F=6.009，P〈0.01）。（4）各组小鼠心肌组织中TNF-α和IL-1β的蛋白含量：心肌炎组小鼠心肌组织TNF-α和IL-1β蛋白含量均高于对照组（F值分别为8.670和8.462，P均〈0.01），而hTRX组则均低于心肌炎组（F值分别为7.542和7.219，P均〈0.01）。结论hTRX可减轻病毒性心肌炎小鼠的心肌损伤和炎症反应，其机制可能与hTRX可下调NF-κB、TNF-α和IL-1β蛋白表达密切相关。

Objective To observe the effects of recombinant adenovirus with human thioredoxin （hTRX） on the inflammatory response in mice with viral myocarditis and explore the related mechanism. Methods Sixty Balb/e male mice were randomly divided into control group, myocarditis group, and hTRX group according to the random number table （n=20 each group）. The myocarditis group and hTRX group were injected with 100 TCID50 Coxackie virus B3 （0.1 ml） in the abdomen and control group were injected with saline. Two days before the viral injection, the hTRX group were injected with recombinant adenovirus vector coding the human thioredoxin gene by pericardial puncture and the control group and myoearditis group were injected with recombinant adenovirus vector without coding gene by pericardial puncture, all these mice were killed and hearts were removed 7 days later. The morphology of myocardial tissue in each group was detected by HE staining and the ultrastrueture changes by electron microscope. The protein expressions of tumor necrosis factor （TNF）-α, interleukin （IL）-1β and NF-κB were detected by ELISA and Western blot. Immunohistochemical staining was performed to observe the protein expression levels of thioredoxin. Results Necrosis of myocardial cells and a small amount of cell infiltration were found in the myocarditis group and necrosis and cell infiltration were significantly reduced in the hTRX group and no myocardial lesion was found in control group on HE stained sections. Electron microscope examination evidenced ceil swelling and dissolved myofilament, vacuoles degeneration in mitochondria in the myocarditis group. These changes were significantly reduced in the hTRX group. There was no myocardial lesion in control group. The protein expression of TNF-α, IL-1β and NF-κB were significantly upregulated in myocarditis group than in control group （all P〈0.01）. The protein expression of TNF-α, IL-1β and NF-KB were significantly downregulated in hTRX group than in myocarditis group （all P〈0.01）. lmmunohistochemical staining showed that protein expression of hTRX was higher in hTRX group than in myocarditis group （P〈0.01）. Conclusion Recombinant adenovirus hTRX can attenuate cardiac injury in mice with acute myocarditis via inhibiting the inflammatory response and downregulating the expression of TNF-α, IL-1β and NF-κB.