椒蒿挥发油对病毒性心肌炎小鼠心肌损伤的影响

Effects of volatile oil from artemisia dracunculus for treatment of mice with myocardial injury caused by viral myocarditis

目的观察椒蒿挥发油对病毒性心肌炎小鼠心肌损伤的影响及作用机制。方法选取160只BALB／c雄性小鼠，取10只作为正常对照组，余150只按计算机产生的随机数字分为模型组、黄芪组和低、中、高剂量椒蒿挥发油组。采用腹腔接种柯萨奇B3亲心肌病毒株建立病毒性心肌炎（VMC）小鼠模型。病毒感染1h后，正常对照组和模型组均给予生理盐水灌胃；黄芪组腹腔注射黄芪注射液0．1mL；椒蒿挥发油低、中、高剂量组给予2％、5％、10％的椒蒿挥发油0．3mL灌胃。各组均每日1次，持续给药1周。观察各组小鼠死亡率、心脏／体质量比值、自然杀伤细胞（NK细胞）活性、心肌组织匀浆中病毒滴度、血清心肌肌钙蛋白I（cTnI）含量及心肌组织病理学变化。结果①死亡率：模型组死亡率高于正常对照组、黄芪组和椒蒿挥发油低、中剂量组（60．0％比0％、23．3％、20．0％、28．7％），模型组与椒蒿挥发油高剂量组比较差异无统计学意义（60．0％比46．7％，P〉0．05）；黄芪组死亡率明显低于椒蒿挥发油高剂量组（P〈0．01），黄芪组与椒蒿挥发油中、低剂量组比较差异无统计学意义（P〉0．05）；椒蒿挥发油低剂量与中剂量组死亡率比较差异亦无统计学意义（P〉0．05）。②免疫指标：制模后8d，模型组NK细胞活性较正常对照组明显降低[（15．91±3．87）％比（38．50±2．32）％]，黄芪组和椒蒿挥发油低、中剂量组NK细胞活性较模型组明显升高[（19．38±3．27）％、（18．54±3．09）％、（18．36±2．64）％比（15．91±3．87）％，均P〈0．05]。正常对照组心肌组织匀浆中未检出病毒，黄芪组和椒蒿挥发油低、中剂量组心肌组织匀浆病毒滴度较模型组明显下降（10^-9／mL：1．96±0．44、1．95±0．46、1．95±0．48比2．41±0．51，均JP〈0．01）。③肌损伤指标：正常对照组cTnI水平〈0．1μg/L，模型组[（15．84±3．89）μg/L]较正常对照组明显升高，心脏／体质量比值亦较正常对照组明显升高（×10^-4：8．3±1．3比4．6±0．1），黄芪组和椒蒿挥发油低、中剂量组cTnI、心脏／体质量比值均较模型组明显降低[cTnI（mg／L）：10．03±2．35、10．81±2．56、11．10±1．89比15．84±3．89，心脏／体质量比值（×10^-4）：7．2±0．8、7．3±1．0、7．3±0．6比8．3±1．3]。正常对照组心肌组织无炎性浸润和坏死，心肌病理变化积分均为0，模型组炎性浸润积分（分：3．25±0．45）和坏死积分（分：2．91±0．51）均较正常对照组明显升高，黄芪组和椒蒿挥发油低、中剂量组浸润积分和坏死积分均较模型组明显降低[浸润积分（分）：2．92±0．39、2．95±0．35、2．95±0．37比3．25±0．45，坏死积分（分）：2．46±0．50、2．50±0．51、2．54±0．50比2．91±0．51，均P〈0．05]。结论椒蒿挥发油可清除体内病毒、调节免疫功能等从而起到保护心肌细胞的作用，但椒蒿挥发油的作用与剂量有关，低、中剂量较高剂量效果更好。

Objective To investigate the effects of volatile oil from artemisia dracunculus on myocardial injury caused by viral myocarditis in mice and explore its possible mechanism. Methods Totally 160 adult male BALB/c mice were randomly divided into normal control group （10） and viral myocarditis group （150）. Viral myocarditis mice models were reproduced by intraperitoneal inoculation with a solution of coxsackievirus B3 （CVB3）, a viral strain with affinity to myocardium, and then randomly divided into model, astragalus group, and low-, medium-, and high-dose volatile oil from artemisia dracunculus groups. After 1 hour of viral infection, normal control group and model group mice were given normal saline by intragastric administration, astragalus group mice were injected with astragalus 0.1 mL in each mouse by intraperitoneal injection, and the mice in other three groups were given low, medium and high dose （2%, 5%, 10%） 0.3 mL volatile oil from artemisia dracunculus in each mouse by intragastric administration, respectively, once a day for one week consecutively. The mortality, heart/body weight ratio, the activity of natural killer cells （NK cell）, virus titer in myocardial homogenate, serum cardiac troponin I （cTnI） level and myocardial pathological changes were observed. Results （1） Mortality： the mortality of model group was higher than that of the normal control group, astragalus group, low and medium dose volatile oil from artemisia dracunculus groups （60.0% vs. 0%, 23.3%, 20.0%, 28.7%）, and the difference in the mortality being of no statistical significance between model group and that of high-dose volatile oil from artemisia dracunculus group （60.0% vs. 47.6%, P 〉 0.05）; the mortality of astragalus group was obviously lower than that of high-dose volatile oil from artemisia dracunculus group （P 〈 0.01）, and the differences in comparisons between the mortalities of astragalus intervention group, and medium- and low-dose volatile oil groups were not statistically significant （all P 〉 0.05）, and the comparison of mortality between low- and medium-dose volatile oil groups were also not statistically significant （P 〉 0.05）. （2） Immunization parameters： on the 8th day after modeling, the activity of NK cells in the model group was significantly lower than that in the normal control group [（15.91 ± 3.87）% vs. （38.50 ± 2.32）%], the activities of NK cells in astragalus group, medium- and low-dose volatile oil from artemisia dracunculus groups were significantly higher than that in model group [（19.38 ± 3.27）%, （18.54 ± 3.09）%, （18.36 ± 2.64）% vs. （15.91 ± 3.87）%, all P 〈 0.05]. None of virus was detected in the myocardial homogenate in the normal control group, and the virus titers in astragalus group, low and medium dose volatile oil from artemisia dracunculus groups were significantly lower than the titer of the model group （10^-9/mL： 1.96 ± 0.44, 1.95 ± 0.46, 1.95 ± 0.48 vs. 2.41 ± 0.51, all P 〈 0.01）. （3） Myocardial injury parameters： the level of cTnI in the normal control group was less than 0.1 μg/L, obviously lower than that in the model group [（15.84 ± 3.89） μg/L], as well as the ratio of heart/body weight in model group was also significantly higher than that in normal control group （ ×10^-4：8.3 ± 1.3 vs. 4.6 ± 0.1）, and the cTnI and the ratio of heart/body weight of astragalus intervention group, low and medium dose volatile oil from artemisia dracunculus groups were markedly lower than those of model group [cTnI （mg/L）： 10.03 ± 2.35, 10.81 ± 2.56, 11.10 ± 1.89 vs. 15.84 ± 3.89, ratio of heart/body weight （ ×10^-4）： 7.2 ± 0.8, 7.3 ± 1.0, 7.3 ± 0.6 vs. 8.3 ± 1.3]. In the normal control group, there were no inflammatory cell infiltration and necrosis in myocardial tissue, the scores of myocardial pathological changes were 0. In the model group, the scores of inflammatory cell infiltration （3.25 ± 0.45） and of necrosis （2.91 ± 0.51） were markedly higher than those in the normal control group. And the above scores in astragalus group, low and medium dose volatile oil from artemisia dracunculus groups were significantly lower than those of the model group （infiltration score： 2.92 ± 0.39, 2.95 ± 0.35, 2.95 ± 0.37 vs. 3.25 ± 0.45, necrosis score： 2.46 ± 0.50, 2.50 ± 0.51, 2.54 ± 0.50 vs. 2.91 ± 0.51, all P 〈 0.05）. Condusions Volatile oil from artemisia dracunculus can protect cardiomyocytes by removing the virus and regulating the immune function in the body. But the protective effects of volatile oil from artemisia dracunculus is related to the dosage, and the effects of low and medium dose are better.