大承气汤通过内源性抗菌肽mCRAMP对脓毒症小鼠肠屏障的保护作用及机制  

Da Chengqitang Protects Intestinal Barrier in Septic Mice via Endogenous Antimicrobial Peptide mCRAMP

作　　者：李袁袁 钟旋[3] 刘磊[4] 孙淑亚 凌家俊[1] 林荣锋[1,2] LI Yuanyuan;ZHONG Xuan;LIU Lei;SUN Shuya;LING Jiajun;LIN Rongfeng(School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Mathematical Engineering Academy of Chinese Medicine,Dongguan 523808,China;Guangdong Women and Children Hospital,Guangzhou 511442,China;The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine,Shenzhen Traditional Chinese Medicine Hospital,Shenzhen 518033,China)

机构地区：[1]广州中医药大学中药学院,广州510006 [2]东莞广州中医药大学研究院,广东东莞523808 [3]广东省妇幼保健院,广州511442 [4]广州中医药大学第四临床医学院,深圳市中医院,广东深圳518033

出　　处：《中国实验方剂学杂志》2024年第6期20-28,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：广东省中医药局科研课题面上项目(20211045);东莞广州中医药大学研究院培育项目(2023PY0203)。

摘　　要：目的:采用分子对接和体内动物实验探讨大承气汤对脓毒症小鼠肠屏障的保护作用及分子机制。方法:采用文本挖掘方法筛选大承气汤的活性成分,利用AutoDock Tools和Discovery Studio分子对接软件研究其关键活性成分与脓毒症主要作用靶点紧密连接蛋白-1(Claudin-1)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、抗菌肽(mCRAMP)、Toll样受体4(TLR4)和髓样分化因子88(MyD88)的相互作用。动物实验采用C57BL/6小鼠50只,每组10只,随机分成5组,分别为假手术组、模型组、大承气汤低、高剂量(4、8 g·kg^(-1))组和乌司他丁(50000 U·kg^(-1))组。造模前、手术当天和术后,各组给予相应的药物进行干预。除假手术组外,其余各组小鼠均采用盲肠结扎穿刺法(CLP)制备脓毒症模型。采用酶联免疫吸附测定法(ELISA)检测小鼠血清D-乳酸水平,评估肠黏膜通透性;采用苏木素-伊红(HE)染色法观察回肠组织病理变化,评估肠黏膜损伤程度及炎症浸润;采用蛋白免疫印迹法(Western blot)检测小鼠回肠组织Claudin-1和Occludin蛋白表达水平,评估肠机械屏障功能;采用ELISA检测小鼠回肠组织TNF-α和IL-6水平,评估肠道炎症水平;采用免疫组化法(IHC)观察回肠组织mCRAMP蛋白表达;采用实时荧光定量聚合酶链式反应(Real-time PCR)检测小鼠回肠组织内mCRAMP、TLR4和MyD88的基因表达水平,探讨大承气汤对脓毒症小鼠肠屏障的保护机制。结果:分子对接结果表明,采用文本挖掘方法所筛选出的10个大承气汤活性成分中多数与脓毒症靶点具有结合活性,主要通过范德华力、氢键和其他共轭体系相连。动物实验结果表明,与模型组比较,大承气汤低、高剂量组小鼠血清D-乳酸水平显著降低(P<0.01);回肠组织损伤、黏膜水肿减少,小肠绒毛完整性较好,炎症细胞浸润减少;回肠组织Claudin-1蛋白表达水平显著升高(P<0.01);回肠组织IL-6水平显著降低(P<0.01);回Objective:Molecular docking and animal experiments were employed to explore the protective effect and mechanism of Da Chengqitang(DCQD)on intestinal barrier in septic mice.Method:Text mining method was used to screen the active ingredients in DCQD.AutoDock Tools and Discovery Studio were used to study the interactions of active components with the core target proteins[claudin-1,tumor necrosis factor(TNF)-α,interleukin(IL)-6,endogenous antimicrobial peptide mCRAMP,Toll-like receptor 4(TLR4),and myeloid differentiation primary response gene 88(MyD88)]in sepsis.Fifty C57BL/6 mice were randomized into sham,model,low-and high-dose(4 g·kg^(-1) and 8 g·kg^(-1))DCQD,and ulinastatin groups(n=10).Before,during,and after the day of modeling surgery,each group was administrated with corresponding drugs.The mice in other groups except the model group were subjected to modeling by cecal ligation and puncture.Enzymelinked immunosorbent assay(ELISA)was used measure the serum level of D-lactic acid to assess intestinal mucosa permeability.Hematoxylin-eosin staining was employed to observe the histopathological changes in the ileum and assess the intestinal mucosal damage and inflammatory infiltration.Western blotting was employed to determine the expression levels of tight junction proteins claudin-1 and occludin in the ileal tissue,which were indicative of the bowel barrier function.The TNF-α and IL-6 levels were measured by ELISA to assess the intestinal inflammation.The expression of mCRAMP in the ileal tissue was observed by immunohistochemistry.The mRNA levels of mCRAMP,TLR4,and MyD88 in mouse ileal tissue were determined by Real-time polymerase chain reaction,on the basis of which the mechanism of DCQD in protecting the intestinal barrier of septic mice was explored.Result:Molecular docking results showed that most of the 10 active ingredients of DCQD that were screened out by text mining could bind to sepsis targets by van der Waals force,hydrogen bonding,and other conjugated systems.The results of animal experiments s

关 键 词：大承气汤 脓毒症 肠屏障 抗菌肽 Toll样受体4(TLR4)/髓样分化因子88(MyD88) 

分 类 号：R284[医药卫生—中药学] R285[医药卫生—中医学] R289R287R22R2-031R33R24