扶正透邪解毒方调控NLRP3/Caspase-1/GSDMD通路治疗多重耐药菌感染肺炎的药效与作用机制

Efficacy and mechanism of action of Fuzheng Touxie Jiedu Prescription in modulating NLRP3/Caspase-1/GSDMD pathways in rats with pneumonia caused by multi-drug resistant organisms

关键词:  多重耐药菌感染肺炎  扶正透邪解毒方  NLRP3/Caspase-1/GSDMD信号通路  液相色谱-四极杆-飞行时间质谱  网络药理学

Key words:  pneumonia caused by multi-drug resistant organisms  Fuzheng Touxie Jiedu Prescription  NLRP3/Caspase-1/GSDMD signaling pathway  LC-Q-TOF-MS  network pharmacology

摘要:

目的 基于液相色谱-四极杆-飞行时间质谱（LC-Q-TOF-MS）、网络药理学、分子对接及动物实验探讨扶正透邪解毒方对多重耐药菌感染肺炎大鼠的药效影响及作用机制。方法 采用多重耐药铜绿假单胞菌气管滴注法构建多重耐药菌感染肺炎大鼠模型，将30只大鼠随机分为空白组、模型组、中药组、西药组和中药+西药联合治疗组5组，另取6只大鼠分为正常组和给药组，用于含药血清制备。采用LC-Q-TOF-MS技术对大鼠进行含药血清化学分析，结合网络药理学对扶正透邪解毒方活性成分、核心靶点及信号通路进行预测，采用CB-Dock2平台进行分子对接。采用HE染色观察大鼠肺组织病理变化，ELISA法检测大鼠血清白细胞介素（IL）-18、IL-1β、肿瘤坏死因子（TNF）-α及IL-6的表达水平，荧光定量PCR和Western blot法检测大鼠肺组织中核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）、凋亡相关斑点样蛋白（ASC）、半胱氨酸天冬氨酸特异性蛋白酶-1（Caspase-1）及消皮素D（GSDMD）mRNA和蛋白表达水平。结果 筛选出34种活性成分，包括176个疾病和中药复方的交集靶点和38个核心靶点。扶正透邪解毒方治疗多重耐药菌感染肺炎的核心成分包括：7-羟基香豆素、Salvianono、槲皮素、甘草苷等，核心靶点包括核因子（NF）KB1、TNF、IL6、蛋白激酶Bα（AKT1）等，主要涉及丝裂原活化蛋白激酶（MAPK）信号通路、磷脂酰肌醇3-激酶-蛋白激酶B（PI3K-Akt）信号通路、叉头框O（FoxO）信号通路等炎症与免疫相关通路，分子对接结果显示，关键成分与核心靶点结合活性较强。动物实验结果表明，扶正透邪解毒方可减轻多重耐药菌感染肺炎大鼠肺组织病理改变，抑制血清IL-18、IL-1β、TNF-α和IL-6炎症因子表达，下调大鼠肺组织NLRP3、ASC、Caspase-1、GSDMD mRNA和蛋白表达水平。结论 扶正透邪解毒方对多重耐药菌感染肺炎大鼠具有治疗作用，其具体机制可能与调节NLRP3/Caspase-1/GSDMD信号通路，抑制炎症和免疫反应有关。

Abstract:

Objective To explore the pharmacodynamic effects and mechanisms of Fuzheng Touxie Jiedu Prescription on rats with pneumonia caused by multi-drug resistant organisms based on liquid chromatography-quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS), network pharmacology, molecular docking, and animal experiments. Methods A rat model of pneumonia caused by multi-drug resistant Pseudomonas aeruginosa was established via tracheal instillation. Thirty rats were randomly divided into 5 groups: blank control group, model group, traditional Chinese medicine (TCM) group (Fuzheng Touxie Jiedu Prescription), Western medicine group, and integrated Chinese-Western medicine group. Additionally, another 6 rats were divided into the normal group and the drug-administered group for the preparation of drug-containing serum. LC-Q-TOF-MS was used for chemical analysis of drug-containing serum. Network pharmacology predicted the active components, core targets, and signaling pathways of the prescription. Molecular docking was performed using the CB-Dock2 platform. Hematoxylin and eosin (HE) staining assessed lung tissue pathology. ELISA was used to detect the expression olevels of interleukin (IL) -18, IL-1β, tumor necrosis factor (TNF) -α, and IL-6 in rat serum. Quantitative real-time PCR (qPCR) and Western blot measured mRNA and protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), Caspase-1, and gasdermin D (GSDMD) in rat lung tissue. Results Thirty-four active components were identified. Analysis revealed 176 intersecting targets related to both the disease and the prescription, including 38 core targets (NFKB1, TNF, IL6, AKT1, etc.). Core active components included 7-Hydroxycoumarin, Salvianono, Quercetin, Liquiritin, etc. Key signaling pathway involved MAPK, PI3K-Akt, FoxO signaling pathways, primaily related to inflammation and immunity. Molecular docking demonstrated strong binding activity between key components and core targets. Animal experiments showed that the Fuzheng Touxie Jiedu Prescription alleviated pathological lung tissue damage, and inhibited the expressions of inflammatory factors including IL-18, IL-1β, TNF-α, and IL-6 in serum, downregulated mRNA and protein expression of NLRP3, ASC, Caspase-1, and GSDMD in lung tissue. Conclusion The Fuzheng Touxie Jiedu Prescription exhibits therapeutic effects against multi-drug resistant organisms pneumonia in rats. Its mechanisms likely involves regulating the NLRP3/Caspase-1/GSDMD signaling pathway to inhibit excessive inflammation and immune responses.