清瘟解热方对湿热证型病毒性肺炎大鼠肺损伤及CCL3/CCR5信号通路的作用  

Effects of Qingwen Jiere Formula on lung injury and CCL3/CCR5 signaling pathway in rats with viral pneumonia of damp-heat pattern

作　　者：张琳曼 李钦 张庭瑞 温伟波 ZHANG Linman;LI Qin;ZHANG Tingrui;WEN Weibo(Yunnan University of Chinese Medicine,Kunming,Yunnan 650021,China;Yunnan Key Laboratory of Integrated Chinese and Western Medicine for Chronic Disease Prevention and Treatment,Kunming,Yunnan 650021,China)

机构地区：[1]云南中医药大学,云南昆明650021 [2]云南省中西医结合慢病防治重点实验室,云南昆明650021

出　　处：《湖南中医药大学学报》2024年第11期1951-1958,共8页Journal of Hunan University of Chinese Medicine

基　　金：国家自然科学基金地区基金项目(82260930,82060864);国家重点研发计划课题(2023YFF0724803);国家中医药管理局新型冠状病毒感染中医药应急专项课题(2023ZYLCYJ02-24);国家中医药多学科交叉创新团队项目(ZYYCXTD-D-202201);云南省科学技术厅中医联合专项项目(202101AZ070001-020);云南省科学技术厅重点研发计划项目(202103AC100005)。

摘　　要：目的探讨清瘟解热方(以下简称QWJR)对湿热证型病毒性肺炎大鼠肺损伤及CCL3/CCR5信号通路的作用。方法将48只大鼠随机分为正常组、湿热模型组、阳性药物组(甲泼尼龙)、清瘟解热低剂量组(QWJR-L)、清瘟解热中剂量组(QWJR-M)、清瘟解热高剂量组(QWJR-H),每组8只,雌雄各半,正常组自然环境饲养,其余各组湿热外因+内因饲养14 d。14 d后,除正常组气管注射50μL PBS外,其余组均运用Poly(I:C)气管注射,建立湿热证型病毒性肺炎大鼠模型。造模2 h后,正常组和湿热模型组灌胃生理盐水,其余各组给予相应药物干预,连续给药3 d。收集样本后检测相关指标。(1)湿热证评价指标:大鼠症状、证候积分、体质量、血清总胆固醇(cholesterol,CHOL)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)含量、血常规;(2)肺泡毛细血管屏障变化、肺损伤情况:肺组织的干湿比、支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)总细胞计数、BALF总蛋白浓度、HE染色观察大鼠肺组织病理变化;(3)趋化因子CCL3(CC chemokine Ligand 3,CCL3)/CC趋化因子受体-5(CC chemokine receptor 5,CCR5)通路及下游因子:qPCR法检测肺组织中CCL3、CCR5、磷脂酶C(phospholipase C,PLC)、蛋白激酶C(proteinkinase C,PKC)mRNA表达水平和Western-Blot法检测CCL3、CCR5、PLC、PKC蛋白表达水平。结果QWJR干预后大鼠大便黏滞或溏泄伴臭秽,肛门红肿污秽情况有所减轻,大鼠湿热证候积分降低,血脂CHOL、LDL-C水平降低(P<0.05);血常规中WBC及NE水平较模型组降低(P<0.05);QWJR-M组和QWJR-H组肺组织W/D值、BULF中总蛋白含量、总细胞数均降低,且以高剂量组治疗效果最显著(P<0.01),并有效改善大鼠肺组织病理损伤;与模型组相比,阳性药物组、QWJR-H组大鼠CCR5、PLC、PKC蛋白表达水平降低(P<0.05,P<0.01),CCL3、CCR5、PLC、PKC mRNA表达量降低(P<Objective To investigate the effects of Qingwen Jiere Formula(QWJR)on lung injury and CC chemokine Ligand 3(CCL3)/CC chemokine receptor 5(CCR5)signaling pathway in rats with viral pneumonia of damp-heat pattern.Methods Forty-eight rats were randomized into normal group,model group,positive drug group(methylprednisolone),as well as low-,medium-,and high-dose QWJR groups(i.e.QWJR-L,QWJR-M,and QWJR-H groups),with eight rats in each group,half male and half female.The normal group was raised in a natural environment,while the other groups were subjected to both exogenous and endogenous damp-heat conditions for 14 days.After 14 days,except for the normal group which received a tracheal injection of PBS 50μL,all other groups were administered with Poly(I∶C)via tracheal injection to establish a rat model of viral pneumonia with damp-heat pattern.Two hours after model establishment,the normal and model groups were gavaged with saline,while the other groups received corresponding drug interventions for 3 consecutive days.Samples were collected for the following measurements:(1)Evaluation indicators of damp-heat pattern:rat symptoms,pattern scores,body mass,serum levels of chol-esterol(CHOL),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C),and blood routine test;(2)Changes in alveolar-capillary barrier and lung injury:measurement of the lung wet-to-dry weight ratio(W/D),as well as the total cell count and total protein concentration in the bronchoalveolar lavage fluid(BALF),and observation of pathological changes in the lung tissue using HE staining;(3)CCL3/CCR5 pathway and downstream factors:determination of CCL3,CCR5,PLC,and PKC protein and mRNA expression levels using qPCR and Western blot,respectively.Results After QWJR intervention,symptoms such as sticky or loose stools with a foul odor,and redness,swelling,and soiling around the anus were alleviated in the rats;the pattern scores for damp-heat were reduced,along with lower levels of serum CHOL and LDL-C(P<0.05);the levels

关 键 词：病毒性肺炎 清瘟解热方 肺损伤 湿热证 湿热证动物模型 CCL3/CCR5信号通路 

分 类 号：R285.5[医药卫生—中药学]