芩百清肺浓缩丸治疗肺炎支原体感染大鼠模型细胞因子表达水平变化

Effect of Qinbai Qingfei concentrated pills on expression levels of inflammatory cytokines of rat models with Mycoplasma pneumoniae infection

目的分析芩百清肺浓缩丸治疗肺炎支原体(MP)感染大鼠细胞因子表达水平变化,进一步探究芩百清肺浓缩丸对其免疫机制及炎症反应的调控。方法 SPF级成年雄性Wistar大鼠(90~110 g)60只随机分为4组:正常对照组、肺炎支原体肺炎模型(MP组)、芩百清肺浓缩丸组及阿奇霉素组。通过经鼻反复感染MP构建感染模型,对照组鼻内滴入等体积支原体改良培养基。芩百清肺浓缩丸组及阿奇霉素组从MP感染日起连续4天分别给与芩百清肺浓缩丸3.2 g/kg及阿奇霉素25 mg/kg,每日1次,模型组给予等量生理盐水灌胃。给药后第5天抽取血样、分离血清检测各组大鼠血清中肿瘤坏死因子-α(TNF-α)及γ干扰素(IFN-γ)水平,留取大鼠肺组织检测Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核转录因子-κB(NF-κB)蛋白表达水平。结果与对照组比较,MP组、芩百清肺浓缩丸组及阿奇霉素组大鼠血清中TNF-α含量显著升高(均P<0.05),与MP组比较,芩百清肺浓缩丸组及阿奇霉素组TNF-α水平显著降低(均P<0.05),但芩百清肺浓缩丸组与阿奇霉素组TNF-α水平无显著性差异;与对照组比较,MP组、芩百清肺浓缩丸组及阿奇霉素组IFN-γ含量均呈不同程度的降低(均P<0.05),与MP组比较,芩百清肺浓缩丸组及阿奇霉素组的IFN-γ水平均升高(均P<0.05),但芩百清肺浓缩丸组与阿奇霉素组IFN-γ水平无显著性差异;肺组织实时荧光PCR(qPCR)结果显示,与对照组比较,MP组、芩百清肺浓缩丸组及阿奇霉素组的TLR4、MyD88、 NF-κB mRNA表达水平均呈不同程度的升高(均P<0.05),与MP组比较,芩百清肺浓缩丸组和阿奇霉素组上述指标水平显著降低(均P<0.05),但芩百清肺浓缩丸组和阿奇霉素组上述指标水平无显著性差异。免疫印迹(WB)结果示TLR4、MyD88、 NF-κB的蛋白表达变化趋势与mRNA一致。结论芩百清肺浓缩丸可降低MP感染大鼠TLR4、MyD88、NF-κB、TNF-α的表达水平,增加IFN-γ的水平,从而减轻MP肺炎的炎症反应。

OBJECTIVE To investigate the effect of Qinbai qingfei concentrated pills on expression levels of cytokines of the rats with Mycoplasma pneumoniae infection and further explore the regulation of Qinbai qingfei concentrated pills on its immune mechanism and inflammatory response. METHODS Totally 60 SPF adult male Wistar rats(90-110 g) were randomly divided into 4 groups: the control group, the Mycoplasma pneumoniae pneumonia group(the MP group),the Qinbai qingfei concentrated pills group and the azithromycin group.The infection models were constructed through repeated nasal infection with MP, the control group was treated with intranasal drip of isopyknic MP to modify medium, the Qinbai qingfei concentrated pills group and the azithromycin group were respectively treated with 3.2 g/kg of Qinbai qingfei concentrated pills and 25 mg/kg of azithromycin for 4 consecutive days from the start of MP infection, once a day, and the model group was given the same amount of normal saline for gavage.The blood specimens were drawn for separation of serum after the drug therapies for 5 days so as to detect the expression levels of serum tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ), and the lung tissues were collected to detect the expression levels of toll like receptor 4(TLR4), myeloid differentiation factor 88(MyD88) and nuclear factor kappa-B(NF-κB). RESULTS The serum TNF-α level was significantly higher in the MP group, the Qinbai qingfei concentrated pills group and the azithromycin group than in the control group(all P<0.05);the TNF-α level was significantly lower in the Qinbai qingfei concentrated pills group and the azithromycin group than in the MP group(all P<0.05);there was no significant difference in the IFN-γ level between the Qinbai qingfei concentrated pills group and the azithromycin group.The result of qPCR of lung tissues showed that the levels of TLR4, MyD88 and NF-κB mRNA were significantly higher in the MP group, the Qinbai qingfei concentrated pills group and the azithromycin group than in the control group(all P<0.05), the levels of above indexes were significantly lower in the Qinbai qingfei concentrated pills group and the azithromycin group than in the MP group(all P<0.05), however, there were no significant differences in the levels of above indexes between the Qinbai qingfei concentrated pills group and the azithromycin group.The result of Western blotting indicated that the changing trends of TLR4, MyD88 and NF-κB were consistent with the mRNA. CONCLUSION Qinbai qingfei concentrated pills can reduce the levels of TLR4, MyD88, NF-κB and TNF-α of the rats with MP infection and raise the IFN-γ level so as to alleviate the inflammatory response of the MP pneumonia.