电针预处理对脓毒症小鼠肠黏膜屏障功能保护作用及MLCK/MLC信号通路的调节作用

Protective effect of electroacupuncture pretreatment on intestinal mucosal barrier function and its regulatory effect on MLCK/MLC signaling pathway in sepsis mice

目的观察电针足三里穴预处理对脂多糖(lipopolysaccharide,LPS)诱导的脓毒症小鼠肠黏膜功能损伤的保护作用及其机制研究。方法将40只美国癌症研究所(Institute of Cancer Research,ICR)小鼠随机分为正常组、模型组、电针组和非经非穴组,电针组小鼠选取足三里穴予以电针预处理,每日1次,每次30 min,持续5 d;第5天时采用腹腔注射LPS构建脓毒症肠黏膜损伤小鼠模型。通过苏木精-伊红(hematoxylin-eosin,HE)染色法观察小鼠回肠组织病理学改变并评分;采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测小鼠血清中C-反应蛋白(C-reactive protein,CRP)、降钙素原(procalcitonin,PCT)和结肠组织中白细胞介素-1(interleukin-1,IL-1)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达水平;蛋白印迹(Western blot,WB)法测定结肠组织中IL-1、IL-6、TNF-α和闭合蛋白(occludin)表达的水平;免疫荧光法检测胞质紧密粘连蛋白-1(zonula occludens-1,ZO-1)和连接蛋白-5(claudin-5)在回肠组织的表达水平;逆转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)检测肌球蛋白轻链激酶(myosin light chain kinase,MLCK)、肌球蛋白轻链(myosin light chain,MLC)、ZO-1、claudin-5、occludin在结肠组织中的含量。结果电针组中因LPS引起细胞排列紊乱与缺失、结构破坏、腺体变形等病理损伤均有改善,电针组小鼠回肠组织病理评分明显低于模型组(P<0.05),电针组小鼠结肠组织中IL-1、IL-6和TNF-α的含量低于模型组(P<0.05)。与模型组比较,电针组小鼠血清中CRP和PCT含量较低(P<0.05),小鼠结肠组织中occludin蛋白、occludin mRNA、claudin-5 mRNA、ZO-1 mRNA的相对表达量较高(P<0.05),小鼠结肠组织中MLCK和MLC mRNA相对表达量较低(P<0.05)。结论电针足三里预处理可通过抑制炎症反应和MLCK/MLC信号通路上调紧密连接蛋白的表达,发挥对LPS诱导的脓毒症肠黏膜屏障功能损伤的保护作用。

Objective To observe the protective effect of electroacupuncture pretreatment at Zusanli(ST36)on intestinal mucosal function in sepsis mice induced by lipopolysaccharide(LPS)and explore the mechanism.Method Forty Institute of Cancer Research(ICR)mice were randomly divided into a normal group,a model group,an electroacupuncture group,and a non-acupoint group.Mice in the electroacupuncture group received electroacupuncture pretreatment at Zusanli,30 min each time,once daily for 5 d;on the 5th day,intraperitoneal injection of LPS was performed to develop sepsis models with intestinal mucosal injuries.Hematoxylin-eosin(HE)staining was used to observe and score the pathological changes in the ileum tissues.Enzyme-linked immunosorbent assay(ELISA)was adopted to determine the expression levels of C-reactive protein(CRP)and procalcitonin(PCT)in the serum and interleukin-1(IL-1),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in the colon tissues.The expression levels of IL-1,IL-6,TNF-α,and occludin proteins in the colon tissues were detected by Western blot(WB).The immunofluorescence method was used to determine the expression of zonula occludens-1(ZO-1)and claudin-5 in the ileum tissues.The contents of myosin light chain kinase(MLCK),myosin light chain(MLC),ZO-1,claudin-5,and occludin in the colon tissues were detected by the reverse transcription polymerase chain reaction(RT-PCR).Result The electroacupuncture group demonstrated improvements in pathological injuries caused by LPS,such as disordered cells or cell loss,damaged structure,and deformed glands;the histopathological score of the ileum tissues was notably lower in the electroacupuncture group than in the model group(P<0.05).The contents of IL-1,IL-6,and TNF-αin the colon tissues were lower in the electroacupuncture group than in the model group(P<0.05).Compared with the model group,the serum contents of CRP and PCT were smaller in the electroacupuncture group(P<0.05);the relative expression levels of occludin protein,occludin mRNA,claudin-5 mRNA,and ZO-1 mRNA in the colon tissues were higher(P<0.05),and the relative expression levels of MLCK and MLC mRNA in the colon tissues were lower in the electroacupuncture group(P<0.05).Conclusion By inhibiting inflammatory reactions and elevating the expression of zonula occludens in the MLCK/MLC signaling pathway,electroacupuncture pretreatment at Zusanli produces protective effects on the damaged intestinal mucosal barrier function in sepsis induced by LPS.