佛耳草总黄酮联合抗生素对大鼠慢性阻塞性肺疾病引起的气道炎症的缓解作用  

Alleviation of Airway Inflammation Induced by Chronic Obstructive Pulmonary Disease in Rats by Total Flavonoids of Gnaphalium affine Combination with Antibiotics

作　　者：陈巍[1] 陈晓兰[1] 周思杨 解肖羽婷 王豪 杨海峰[1] CHEN Wei;CHEN Xiaolan;ZHOU Siyang;XIE Xiaoyuting;WANG Hao;YANG Haifeng(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;School of Applied Technology,Changzhou University,Changzhou 213164,China;Jiangsu Key Laboratory of Chiral Pharmaceuticals Biosynthesis,Taizhou University,Taizhou 225300,China;Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,China)

机构地区：[1]江苏农牧科技职业学院,泰州225300 [2]常州大学应用技术学院,常州213164 [3]泰州学院,江苏省手性医药化学品生物制造重点实验室,泰州225300 [4]中国科学院大连化学物理研究所,大连116023

出　　处：《中国畜牧兽医》2024年第12期5518-5527,共10页China Animal Husbandry & Veterinary Medicine

基　　金：江苏省高等学校基础科学(自然科学)研究重大项目(24KJA230001);江苏农牧科技职业学院校级科研揭榜挂帅项目(NSF2024JB01);泰州市科技支撑计划农业项目(TN202218);江苏省高校“青蓝工程”资助项目(苏教师函〔2022〕29号)。

摘　　要：【目的】探讨佛耳草总黄酮联合抗生素对肺炎克雷伯菌和脂多糖(LPS)诱发的慢性阻塞性肺疾病(COPD)的治疗效果,并从相关蛋白和基因层面探究其相关抗炎机制,为该疾病的治疗提供一个新的思路。【方法】将54只Wistar大鼠随机分为6组:对照组、模型组、氨茶碱组、佛耳草总黄酮组、抗生素组、佛耳草总黄酮+抗生素组,每组9只。除对照组外,其余各组大鼠均采用气管滴注肺炎克雷伯菌和脂多糖的方法建立COPD模型,对照组以等体积的生理盐水代替肺炎克雷伯菌和脂多糖同法处理。COPD造模12周确认成功后,治疗组大鼠每天分别给予氨茶碱(50 mg/kg BW)、佛耳草总黄酮(50 mg/kg BW)灌胃处理,头孢他啶(200 mg/kg BW)肌内注射处理,佛耳草总黄酮+抗生素组大鼠同时给予佛耳草总黄酮(50 mg/kg BW)和头孢他啶(200 mg/kg BW)处理,对照组和模型组大鼠不做处理。连续治疗7 d后,采用动物肺功能检测仪检测大鼠呼气峰流量(PEF)、吸气峰流量(PIF)、0.3 s用力呼气容积与用力肺活量比值(FEV0.3/FVC)。分别采用HE染色和TUNEL染色检测大鼠肺脏组织病理学形态和组织细胞凋亡水平;采用Western blotting检测大鼠肺脏组织B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、活化的半胱氨酸蛋白酶-3(Cleaved Caspase-3)、基质金属蛋白酶2(MMP-2)、MMP-9、金属蛋白酶组织抑制因子-1(TIMP-1)蛋白表达水平;采用实时荧光定量PCR检测大鼠肺脏组织中MMP-2、MMP-9及TIMP-1基因mRNA表达水平;采用ELISA方法检测各组大鼠血清及肺泡灌洗液(BALF)中白细胞介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α)水平;采用流式细胞术检测大鼠肺脏组织Th17、Treg细胞比例。【结果】与对照组相比,模型组大鼠PEF、PIF、FEV0.3/FVC、Bcl-2、Treg细胞比例均显著降低(P<0.05),TUNEL阳性率,Bax、Cleaved Caspase-3蛋白表达水平,Th17细胞比例、Th17/Treg比值,MMP-2、MMP-9、TIMP-1及IL-【Objective】This study was aimed to investigate the therapeutic effect of total flavonoids of Gnaphalium affine(G.affine)combined with antibiotics on chronic obstructive pulmonary disease induced by Klebsiella pneumoniae and lipopolysaccharides(LPS),and to explore the related anti-inflammatory mechanisms at the level of related proteins and genes to provide a new idea for the treatment of this disease.【Method】54 Wistar rats were divided into 6 groups:Control group,model group,aminophylline(AM)group,total flavonoids of G.affine(TFG)group,antibiotic(AB)group,and total flavonoids of G.affine+antibiotic(TFG+AB)group,with 9 rats in each group.Among them,chronic obstructive pulmonary disease(COPD)was established in all groups except for control group by tracheal instillation of Klebsiella pneumoniae and lipopolysaccharide(LPS).Rats in control group was treated with equal volume of normal saline instead of Klebsiella pneumoniae and LPS.After successful modeling of COPD for 12 weeks,the rats in treatment groups were treated with aminophylline(50 mg/kg BW)and total flavonoids of G.affine(50 mg/kg BW)by gavage,and ceftazidime(200 mg/kg BW)by intramuscular injection every day.The rats in TFG+antibiotic group were treated with total flavonoids of G.affine(50 mg/kg BW)and ceftazidime(200 mg/kg BW)every day.Control and model groups were not treated.After 7 days of continuous treatment,peak expiratory flow(PEF),peak inspiratory flow(PIF),and the ratio of forced expiratory volume in 0.3 s to forced vital capacity(FEV0.3/FVC)were measured by pulmonary function tester.HE staining and TUNEL staining were used to detect the pathological morphology of rat lung tissue and the level of apoptosis in rat lung tissue,respectively.Western blotting was used to detect the protein expression of Bax,Bcl-2,Cleaved Caspase-3,MMP-2,MMP-9 and TIMP-1 in lung tissues of rats.Real-time quantitative PCR was used to detect the expression of MMP-2,MMP-9 and TIMP-1 genes mRNA in lung tissues of rats.ELISA was used to detect the levels of IL-1β,IL

关 键 词：佛耳草总黄酮 抗生素 慢性阻塞性肺疾病 气道炎症 细胞凋亡 基质金属蛋白酶 

分 类 号：S859.7[农业科学—临床兽医学]