六味小青龙汤抑制p38MA...喘大鼠慢性炎症作用机制研究_陈秀峰.pdf

41第 2 5 卷 第 2 期 2023 年 2 月辽 宁 中 医 药 大 学 学 报JOURNAL OF LIAONING UNIVERSITY OF TCMVol.25 No.2 Feb.，2023六味小青龙汤抑制p38MAPK/p22phox通路减轻哮喘大鼠慢性炎症作用机制研究陈秀峰，肖臻，姜之炎，姜永红，李文，刘秀秀，王明晶（上海中医药大学附属龙华医院，上海 200032）基金项目：国家自然科学基金青年科学基金项目（82104924）；上海市临床重点专科建设项目（shslczdzk04102）；上海市进一步加快中医药传承 创新发展三年行动计划项目（ZY-0206-01）作者简介：陈秀峰（1991-），女，浙江诸暨人，主治医师，硕士，研究方向：儿童呼吸系统疾病。通讯作者：肖臻（1964-），男，上海人，主任医师，硕士，研究方向：儿童呼吸系统疾病。摘要：目的 探讨六味小青龙汤抑制 p38MAPK/p22phox哮减轻喘大鼠慢性炎症的作用机制。方法 120只SD大鼠分为空白组，模型组，六味小青龙汤低、中、高剂量组，地塞米松组6组，酶联免疫吸附测定法（ELISA）检测各组大鼠干预后血清，支气管肺泡灌洗液（BALF）白细胞介素-4（IL-4）、白细胞介素-5（IL-5），肿瘤坏死因子-（TNF-）水平；苏木精-伊红（HE）染色观察各组肺组织病理改变，比较肺组织炎症细胞浸润；实时荧光定量聚合酶链式反应（Real-time PCR）检测各组肺组织p38MAPKmRNA、p22phoxmRNA表达；蛋白免疫印迹法（Western blot）检 测 各 组DUOX1、DUOX2、p38MAPK及HO-1蛋 白 表 达。结 果 模 型 组BALF IL-4、IL-5、TNF-水平均高于空白组（P0.05），地塞米松及六味小青龙汤能降低哮喘大鼠BALF中IL-4、IL-5含量（P0.05）。p22phoxmRNA、p38MAPKmRNA在模型组中表达升高（P0.05），中剂量六味小青龙汤能降低p22phoxmRNA表达（P0.05），低、中、高剂量六味小青龙汤组均能使p38MAPKmRNA表达降低（P0.05）。DUOX1在初期模型组中表达较空白组升高，六味小青龙汤高剂量能降低DUOX1的表达（P0.05）。DUOX2在模型组中较空白组表达升高，六味小青龙汤中、高剂量能降低DUOX2的表达（P0.05）。HO-1在模型组中较空白组表达升高，高剂量六味小青龙汤以及地塞米松能降低HO-1的表达（P0.05）。p38MAPK蛋白在模型组中较空白组表达升高，六味小青龙汤中、高剂量以及地塞米松均能降低p38MAPK蛋白表达（P0.05）。六味小青龙汤及地塞米松组肺组织炎症细胞浸润均较模型组改善。结论 六味小青龙汤可抑制p38MAPK/p22phox通路减轻哮喘大鼠肺组织病变和慢性炎症，下调p38MAPKmRNA、22phoxmRNA与DUOX1、DUOX2、HO-1、p38MAPK蛋白表达，降低IL-4、IL-5、TNF-水平。关键词：六味小青龙汤；p38MAPK/p22phox通路；哮喘；慢性炎症中图分类号：R285.5 文献标志码：A 文章编号：1673-842X (2023)02-0041-08Mechanism of Liuwei Xiaoqinglong Decoction（六味小青龙汤）Inhibiting p38MAPK/p22phox Pathway and Alleviating Chronic Inflammation in Asthmatic RatsCHEN Xiufeng，XIAO Zhen，JIANG Zhiyan，JIANG Yonghong，LI Wen，LIU Xiuxiu，WANG Mingjing（Longhua Hospital Shanghai University of Traditional Chinese Medicine，Shanghai 200032，China）Abstract：Objective To explore the mechanism of Liuwei Xiaoqinglong Decoction（六 味 小 青 9 HARRISON R.Structure and function of xanthine oxidoreductase：where are we now?J .Free Radic Biol Med，2002，33(6 )：774-797.10 JING L，GAO R，ZHANG J，et al.Norwogonin attenuates hypoxia-induced oxidative stress and apoptosis in PC12 cells J .BMC Complement Med Ther，2021，21(1 )：18.11 TSIKAS D.Assessment of lipid peroxidation by measuring malondialdehyde（MDA）and relatives in biological samples：Analytical and biological challenges J .Anal Biochem，2017，524：13-30.12 HAMID ERA，ALI WH，et al.Oxidative Stress and Anti-Oxidant Markers in Premature Infants with Respiratory Distress Syndrome J .Open Access Maced J Med Sci，2019，7(17 )：2858-2863.13 蔡亮，张炳东.PI3K/AKT信号通路在脑缺血再灌注损伤中的研究进展 J .广西医科大学学报，2021，38(10 )：2012-2016.14 ALI T，KIM T，REHMAN SU，et al.Natural Dietary Supplementation of Anthocyanins via PI3K/Akt/Nrf2/HO-1 Pathways Mitigate Oxidative Stress，Neurodegeneration，and Memory Impairment in a Mouse Model of Alzheimers Disease J .Mol Neurobiol，2018，55(7 )：6076-6093.15 PARK JH，LEE TK，KIM DW，et al.Neuroprotective Effects of Salicin in a Gerbil Model of Transient Forebrain Ischemia by Attenuating Oxidative Stress and Activating PI3K/Akt/GSK3 Pathway J .Antioxidants（Basel），2021，10(4 )：629.16 KALOGERIS T，BAO Y，KORTHUIS RJ.Mitochondrial reactive oxygen species：A double edged sword in ischemia/reperfusion vs preconditioning J .Redox Biology，2014(2 )：702-714.17 LIU H，JING X，DONG A，et al.Overexpression of TIMP3 Protects Against Cardiac Ischemia/Reperfusion Injury by Inhibiting Myocardial Apoptosis Through ROS/Mapks Pathway J .Cell Physiol Biochem，2017，44：1011-1023.18 CHEN X，ZHANG X，XUE L，et al.Treatment with Enriched Environment Reduces Neuronal Apoptosis in the Periinfarct Cortex after Cerebral Ischemia/Reperfusion Injury J .Cell Physiol Biochem，2017，41：1445-1456.19 YU H，ZHANG H，ZHAO W，et al.Gypenoside Protects against Myocardial Ischemia-Reperfusion Injury by Inhibiting Cardiomyocytes Apoptosis via Inhibition of CHOP Pathway and Activation of PI3K/Akt Pathway In vivo and In vitro J .Cell Physiol Biochem，2016，39：123-136.DOI：10.13194/j.issn.1673-842x.2023.02.01042 辽宁中医药大学学报 25 卷龙汤）inhibiting p38MAPK/p22phox asthma and alleviating chronic inflammation in asthmatic rats.Methods 120 SD rats were divided into blank group，model group，low-dose，medium-dose，and high-dose groups of Liuwei Xiaoqinglong Decoction and dexamethasone group.The interleukin-4（IL-4），interleukin-5（IL-5）and tumor necrosis factor-（TNF-）level in bronchoalveolar lavage fluid（BALF）were detected by enzyme-linked immunosorbent assay（ELISA）.Hematoxylin eosin（HE）staining was used to observe the pathological changes of lung tissue and compare the infiltration of inflammatory cells in lung tissue.The expressions of p38MAPK mRNA and p22phox mRNA in lung tissues of each group were detected by real-time fluorescence quantitative polymerase chain reaction（real-time PCR）.The protein expressions of duox1，DUOX2，p38MAPK and HO-1 were detected by Western blot.Results IL-4，IL-5 and TNF-in the model group were higher than those in the blank group（P0.05）.Dexamethasone and Liuwei Xiaoqinglong Decoction could reduce the contents of IL-4 and IL-5 in BALF of asthmatic rats（P0.05）.The expression of p22phox mRNA and p38MAPK mRNA increased in the model group（P0.05）.Me