解毒清肺合剂对肺炎支原体感染大鼠肺组织NF-κB和p38 MAPK通路的影响

Effects of Jiedu-Qingfei mixture on NF-κB and p38 MAPK pathways in lung tissues of rats with Mycoplasma pneumoniae infection

目的:观察解毒清肺合剂对肺炎支原体(MP)感染大鼠肺组织的治疗作用并探讨其作用机制。方法:SD大鼠40只,随机分为对照组、模型组、解毒清肺组和阳性对照组4组,每组10只。实验组大鼠鼻孔内缓慢滴入1×10~9CFU/L MP菌液连续4 d,各组在接种完成后第2天处死1只大鼠进行MP核酸检测,同时其余大鼠开始灌胃治疗,空白对照组和模型组给予等量生理盐水灌胃,解毒清肺组每天给予8 mL/kg的解毒清肺合剂,阳性对照组给予地塞米松磷酸钠(0.5 mg·kg·d^(-1)),连续治疗4周。实验结束后处死大鼠,收集血清和支气管肺泡灌洗液(BALF),ELISA法检测血清和BALF中白细胞介素12(IL-12)、IL-13和肿瘤坏死因子α(TNF-α)水平;取右肺组织用于观察病理形态和HE染色,左肺组织用于NF-κB p50、I-κBα和p38丝裂原活化蛋白激酶(p38 MAPK)的mRNA及蛋白水平的检测。结果:MP核酸检测结果显示,除空白对照组外其余大鼠均感染了MP,表明MP感染大鼠造模成功。治疗第1天模型组和解毒清肺组肺组织病理评分显著高于空白对照组(P<0.05),治疗结束后模型组的肺组织病理评分显著高于空白对照组、解毒清肺组和阳性对照组(P<0.01)。MP感染后模型组血清和BALF中IL-12水平显著低于空白对照组(P<0.05),解毒清肺组和阳性对照组血清和BALF中IL-12水平显著高于模型组(P<0.05);模型组大鼠血清和BALF中IL-13和TNF-α水平显著升高,解毒清肺组和阳性对照组IL-13和TNF-α水平显著低于模型组(P<0.05),解毒清肺组与阳性对照组无显著差异。RT-qPCR检测结果显示,模型组NF-κB p50和p38 MAPK的mRNA表达水平显著升高(P<0.01),解毒清肺组和阳性对照组NF-κB p50和p38 MAPK的mRNA表达水平与模型组比较显著降低(P<0.01);模型组I-κBα的mRNA表达水平较对照组显著降低,解毒清肺组和阳性对照组I-κBα的mRNA表达水平较模型组显著升高(P<0.05),解毒清肺组NF-κB p50、p38、MAPK和I-κBα与阳性对照组比较差异无统计学显著性。Western blot检测结果显示肺组织中模型组的NF-κB p50和p38 MAPK蛋白表达水平显著高于空白对照组,治疗后表达水平显著降低;模型组I-κBα蛋白表达水平显著低于空白对照组,解毒清肺组其蛋白表达水平显著升高,解毒清肺组与阳性对照组无显著差异。结论:解毒清肺合剂可能通过NF-κB和p38 MAPK通路减轻肺炎支原体引起的肺组织炎症。

AIM:To observe the therapeutic effect of Jiedu-Qingfei mixture on Mycoplasma pneumoniae(MP)-infected rat lung tissues and to explore its mechanism.METHODS:SD rats(n=40)were randomly divided into 4 groups:blank control group,model group,Jiedu-Qingfei group and positive control group,with 10 rats in each group.The rats in experimental groups were slowly dripped with 1×10^9 CFU/L MP solution into their nostrils for 4 d.One rat in each group was sacrificed for MP nucleic acid detection at the second day after inoculation,and the other rats were given gavage therapy.The rats in blank control group and model group were intragastrically given the same volume of normal saline,the rats in Jiedu-Qingfei group were given 8 mL/kg Jiedu-Qingfei mixture daily for 4 weeks,and the rats in psoitive control group were given dexmethasone sodium phosphate(0.5 mg·kg^-1·d^-1).After the experiment,the rats were killed.The serum and bronchoalveolar lavage fluid(BALF)were collected for detecting the levels of interleukin-12(IL-12),IL-13 and TNF-αby ELISA.The right lung tissues were used for pathological observation and HE staining,while the left lung tissues were used to detect the expression of NF-κB p50,I-κBαand p38 mitogen-activated protein kinase(p38 MAPK)at mRNA and protein levels.RESULTS:The results of MP nucleic acid detection showed that all the rats except blank control group were MP nucleic acid positive,indicating that the rat model of MP infection was successfully established.On the 1st day of the treatment,the pathological scores of the lung tissues in model group and Jiedu-Qingfei group were significantly higher than those in blank control group(P<0.05).After treatment,the pathological scores of the lung tissues in mo-del group were significantly higher than those in blank control group and Jiedu-Qingfei group.The levels of IL-12 in the serum and BALF in model group were significantly lower than those in blank control group after MP infection(P<0.05),while those after treatment with Jiedu-Qingfei mixture were significantly higher than those in model group(P<0.05).The levels of IL-13 and TNF-αin the serum and BALF of MP-infected rats were increased significantly,while those after treatment with Jiedu-Qingfei mixture were significantly lower than those in model group(P<0.05).The mRNA expression levels of NF-κB p50 and p38 MAPK in model group were increased significantly(P<0.01).After treatment,the mRNA expression levels of NF-κB p50 and p38 MAPK were decreased significantly compared with model group(P<0.01).The mRNA expression level of I-κBαin model group was significantly lower than that in control group.After treatment,the mRNA expression of I-κBαin Jiedu-Qingfei group was significantly higher than that in model group(P<0.05).The protein levels of NF-κB p50 and p38 MAPK in the lung tissues of model group were significantly higher than those of blank control group.After treatment,the protein expression of NF-κB p50 and p38 MAPK was decreased significantly.The protein level of I-κBαin model group was significantly lower than that in blank control group,and after treatment with Jiedu-Qingfei mixture,the protein expression level of I-κBαwas increased significantly(P<0.05).CONCLUSION:Jiedu-Qingfei mixture may attenuate lung tissue inflammation caused by MP through NF-κB and p38 MAPK pathways.