宣白承气汤加味联合西药改善流感病毒与肺炎链球菌共感染小鼠肺炎作用机制

Mechanism of Modified Xuanbai Chengqi Decoction Combined with Western Medicine in Improving Pneumonia in Influenza Virus/Streptococcus Pneumoniae Co-infected Mice

目的 基于TMT(tandem mass tag)标记定量蛋白质组学技术探究流感病毒/肺炎链球菌(IV/Spn)共感染肺炎小鼠的发病机制及中药复方宣白承气汤加味对其的改善作用。方法 选用幼龄健康雄性SPF级Balb/c小鼠40只,分别设置正常组、模型组、中药组、西药组、中西医结合组,每组8只。造膜成功后,分别给予小鼠宣白承气汤加味(0.15 g/mL)、奥司他韦(0.25 mg/mL)/头孢克洛干(0.48 g/mL)及中西药联合干预治疗,正常组与模型组予以等体积的生理盐水灌胃,7天后进行取材,分离各组小鼠肺部组织样品。所有样本经TMT 10标试剂进行标记定量、蛋白酶解、TMT肽段标记与肽段分级、LC-MS/MS分析后,用数据库检索方法确定差异蛋白,并进行生信分析。结果 与正常组比较,模型组小鼠的肺指数明显升高(P<0.05);与模型组比较,各给药组小鼠肺指数明显降低(P<0.05,P<0.01),中药组、西药组及中西结合组的肺指数抑制率分别为68.66%、77.61%、85.07%。与正常组比较,IV/Spn共感染肺炎小鼠共导致259个蛋白的表达变化,涉及补体和凝血级联、氨基糖和核糖代谢、Fcγ介导的吞噬作用等信号通路;宣白承气汤加味干预后共涉及了13个蛋白的表达变化,主要富集于丙酸、丁酸、半乳糖代谢等通路;西药组干预后共涉97个蛋白的表达变化,主要富集于补体和凝血级联、氨基糖和核糖代谢、视黄酸诱导基因蛋白1样受体等通路;中西医结合组干预后共涉144个差异蛋白,主要富集于细胞外基质相互作用、小细胞肺癌、视黄酸诱导基因蛋白1样受体等通路。结论 小鼠肺脏组织中发生的免疫炎性反应是导致IV/Spn共感染肺炎发病的主要原因;宣白承气汤加味可以通过调节肠道菌群失调及物质能量代谢对IV/Spn共感染肺炎起到纠正作用;西药组可以通过免疫调控缓解IV/Spn共感染后的肺部炎症;中西医结合组可以在调节机体免疫、炎症反应的同时为机体提供物质能量。

Objective To explore the pathogenesis of co-infection with influenza virus/Streptococcus pneumoniae(IV/Spn) in mice and the therapeutic effects of Chinese medicine formula Modified Xuanbai Chengqi Decoction using the tandem mass tag(TMT) labelled quantitative proteomics technology. Methods Forty young healthy male SPF grade Balb/c mice were divided into normal group,model group,Chinese medicine group,Western medicine group,and integrated Chinese and Western medicine group,8 mice in each group. After successful modelling,the mice were treated with Modified Xuanbai Chengqi Decoction(0.15 g/mL),oseltamivir(0.25 mg/mL)/ceftriaxone(0.48 g/mL),and combined Chinese and Western medicine intervention,respectively,while the normal group and model group were given equal volumes of normal saline by gavage.Lung tissue samples were collected after 7 days,and the samples from each group of mice were isolated. All samples were labelled and quantified using reagents TMT 10,followed by protein digestion,TMT peptide labelling and peptide fractionation,and LC-MS/MS analysis. Differential proteins were identified using a database search method and subjected to bioinformatics analysis. Results Compared with normal group,the lung index of mice in model group increased(P<0.05). Compared with the model group,the lung index of mice in all administration groups decreased(P<0.05,P<0.01),and the inhibition rate of lung index in Chinese medicine group,Western medicine group and integrated Chinese and Western combination group was 68.66%,77.61%and 85.07%, respectively. Compared with the normal group of mice,co-infection with IV/Spn pneumonia in mice led to changes in the expression of 259 proteins,involving complement and coagulation cascades,amino sugar and nucleotide sugar metabolism, Fcγ-mediated phagocytosis,and other signalling pathways. After intervention with Modified Xuanbai Chengqi Decoction,changes in the expression of 13 proteins were observed,mainly enriched in pathways such as propionate,butyrate,and galactose metabolism. Intervention with Western medicine resulted in changes in the expression of 97 proteins,mainly enriched in pathways such as complement and coagulation cascades,amino sugar and nucleotide sugar metabolism,and retinoic acid-inducible gene 1(RIG-1)-like receptor signalling. After intervention with integrated Chinese and Western medicine,changes in the expression of 144 differential proteins were observed,mainly enriched in pathways such as extracellular matrix(ECM) interaction,small cell lung cancer,and retinoic acid-inducible gene 1 like receptor signalling.Conclusions The immune-inflammatory reaction occurring in the lung tissue of mice is the main cause of IV/Spn co-infection pneumonia. Modified Xuanbai Chengqi Decoction can correct IV/Spn co-infection pneumonia by regulating intestinal dysbiosis and substance energy metabolism. Western medicine can alleviate lung inflammation after IV/Spn co-infection by immune regulation. Integrated Chinese and Western medicine can regulate the body's immune and inflammatory responses while providing substance energy for the body.