摘要
该研究旨在探讨通腑泻肺灌肠液(TFXF)通过p38丝裂原活化蛋白激酶(p38 MAPK)/肌球蛋白轻链激酶(MLCK)信号通路改善肠道屏障及肠道菌群结构治疗急性肺损伤大鼠的作用机制。将60只SPF级Wistar大鼠随机分为对照(CON)组、脂多糖(LPS,7.5 mg·kg^(-1))组、LPS+地塞米松(DEX,3.5 mg·kg^(-1))组、LPS+高剂量(HD,14.74 g·kg^(-1))-TFXF组、LPS+中剂量(MD,7.37 g·kg^(-1))-TFXF组、LPS+低剂量(LD,3.69 g·kg^(-1))-TFXF组。腹腔注射LPS建立大鼠急性肺损伤(ALI)模型。测定大鼠支气管肺泡灌洗液(BALF)中总蛋白浓度和乳酸脱氢酶(LDH)活性;酶联免疫吸附测定(ELISA)法检测大鼠肺组织和结肠组织中肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)等炎症因子水平;苏木素-伊红(HE)染色法检测大鼠肺组织和结肠组织的病理表达;实时荧光定量聚合酶链式反应(real-time PCR)检测大鼠肺组织p38丝裂原活化蛋白激酶(p38 MAPK)、TNF-α和IL-1βmRNA表达;蛋白免疫印迹(Western blot)法检测大鼠结肠组织中p38 MAPK/MLCK信号通路相关蛋白表达;16S rRNA测序检测大鼠肠道菌群组成及含量变化,相关性分析探讨肠道菌群改善ALI大鼠的调节作用。结果显示,与LPS组比较,各给药组大鼠肺及结肠组织病理学评分、BALF中LDH活性和总蛋白浓度均明显降低;除LPS+LD-TFXF组外,其余给药均能显著降低大鼠肺组织及结肠组织中TNF-α和IL-1β含量;各给药组大鼠结肠组织磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)/p38、磷酸化肌球蛋白轻链(p-MLC)/肌球蛋白轻链2(MLC2)和MLCK蛋白表达明显降低;LPS+HD-TFXF组肺组织p38 MAPK、TNF-α和IL-1βmRNA表达水平显著降低。16S rRNA测序结果显示,LPS+HD-TFXF组肠道菌群丰度显著升高,肠道菌群Sobs、Shannon、Npshannon指数均显著升高,肠道微生物β多样性分布趋向于CON组,厚壁菌门显著升高、变形杆菌门显著降低,拟杆菌属丰度显著降低,瘤胃球菌属显著升高,物种差异主要为经黏液真杆菌属Blautia、罗氏菌499(Roseburia_sp_499)、丁酸球菌属Butyricicoccus等。大鼠肺组织TNF-α和IL-1β分别与Muri菌属、未分类的粪真杆菌群、瘤胃球菌属呈负相关,与拟杆菌属呈显著正相关;大鼠结肠组织TNF-α和IL-1β分别与未分类的粪真杆菌群、瘤胃球菌属呈负相关,与拟杆菌属呈显著正相关。预测菌群生物功能与次生代谢物的生物合成、氨基酸的生物合成、糖代谢和氧化磷酸化等相关。上述研究表明,TFXF可调节ALI大鼠肠道菌群物种的丰度和多样性,修复肺及结肠组织结构,调节炎性因子水平。其治疗ALI大鼠的作用机制可能与通过p38 MAPK/MLCK信号通路抑制炎症,改善肠黏膜通透性,维持肠道菌群稳态和屏障有关。
The study is designed to observe the mechanism of Tongfu Xiefei Guanchang Solution(TFXF)in the treatment of acute lung injury(ALI)in rats by improving intestinal barrier and intestinal flora structure via p38 mitogen-activated protein kinase(p38 MAPK)/myosin light chain kinase(MLCK)signaling pathway.Sixty SPF-grade Wistar rats were randomly divided into the control(CON)group,lipopolysaccharide(LPS)group(7.5 mg·kg^(-1)),LPS+dexamethasone(DEX)group(3.5 mg·kg^(-1)),LPS+high-dose(HD)-TFXF group(14.74 g·kg^(-1)),LPS+middle-dose(MD)-TFXF group(7.37 g·kg^(-1)),and LPS+low-dose(LD)-TFXF group(3.69 g·kg^(-1)).ALI model of the rat was established by intraperitoneal injection of LPS.The lactate dehydrogenase(LDH)activity and total protein concentration in the bronchoalveolar lavage fluid(BALF)were measured;tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)levels in lung and colon tissue of rats were detected by enzyme linked immunosorbent assay(ELISA).Hematoxylin-eosin(HE)staining was used to observe the pathological expression in the lung and colon tissue of rats.The mRNA expression of p38 MAPK,TNF-α,and IL-1βin rat lung tissue was determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR).Western blot was used to detect the protein expression related to the p38 MAPK/MLCK signaling pathway in the colon tissue of rats.16S rRNA sequencing was used to detect changes in the composition and content of intestinal flora in rats,and correlation analyses were performed to explore the regulatory role of intestinal flora in improving ALI in rats.The results showed that compared with those in the LPS group,the histopathological scores of lung and colon tissue,LDH activity,and total protein concentration in BALF were significantly reduced in rats in all groups after drug administration.Except for the LPS+LD-TFXF group,the remaining groups significantly reduced the levels of TNF-αand IL-1βin the lung and colon tissue of rats.The protein expressions of phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK)/p38,phosphorylated myosin light chain(p-MLC)/myosin light chain 2(MLC2),and MLCK in colon tissue of rats in each drug administration group were significantly decreased.The mRNA expression levels of p38 MAPK,TNF-α,and IL-1βwere significantly reduced in the LPS+HD-TFXF group.16S rRNA sequencing results showed that the abundance of intestinal flora was significantly higher in the LPS+HD-TFXF group,and intestinal floras including Sobs,Shannon,and Npshannon were significantly higher.Theβ-diversity distribution of intestinal flora tends toward the CON group,and the abundance of Firmicutes was significantly higher.The abundance of Proteobacteria was significantly reduced;the abundance of Bacteroides was significantly reduced,and the abundance of Ruminococcus was significantly higher.The main species differences were Blautia,Roseburia_sp_499,and Butyricicoccus.TNF-αand IL-1βof lung tissue were negatively correlated with Muribaculaceae,unclassified norank_f_Eubacterium_coprostanoligenes,and Ruminococcus and positively correlated with Bacteroides.Meanwhile,TNF-αand IL-1βof colon tissue were negatively correlated with unclassified norank_f_Eubacterium_coprostanoligenes and Ruminococcus and positively correlated with Bacteroides.The predicted biological function of the flora was related to the biosynthesis of secondary metabolites,amino acid biosynthesis,sugar metabolism,and oxidative phosphorylation.The above studies show that TFXF can repair lung and colon tissue structure and regulate inflammatory factor levels by modulating the abundance and diversity of intestinal flora species in ALI rats.Its mechanism of action in ameliorating ALI in rats may be related to the inhibition of inflammation,improvement of intestinal mucosal permeability,and maintenance of intestinal flora homeostasis and barrier through the p38 MAPK/MLCK signaling pathway.
作者
马鸣
汪坤
杨艳华
岳孟茹
任权娜
陈雨晗
宋永真
徐子福
赵旭
MA Ming;WANG Kun;YANG Yan-hua;YUE Meng-ru;REN Quan-na;CHEN Yu-han;SONG Yong-zhen;XU Zi-fu;ZHAO Xu(Henan Province Hospital of Traditional Chinese Medicine/the Second Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450002,China;Zhoukou Hospital of Traditional Chinese Medicine,Zhoukou 466099,China;Henan University of Chinese Medicine,Zhengzhou 450000,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第21期5919-5931,共13页
China Journal of Chinese Materia Medica
基金
河南省中医药科学研究专项(2022ZY1093,2023ZY1015,2023ZY2081)
河南省中医药拔尖人才培养项目(豫卫中医函[2021]15号)。
