基于NF-κB/NLRP3炎症小体通路通督调神灸减轻脊髓损伤大鼠肠道炎症及肠屏障损伤

Tongdu Tiaoshen moxibustion alleviates intestinal inflammation and barrier injury in spinal cord injured rats based on the NF-κB/NLRP3 inflammasome pathway

关键词:  通督调神灸  脊髓损伤  NF-κB/NLRP3通路  肠道炎症  肠道屏障损伤

Key words:  Tongdu Tiaoshen moxibustion  spinal cord injury  NF-κB / NLRP3 inflammasome pathway  intestinal inflammation  intestinal barrier injury

摘要:

[目的]探讨通督调神灸通过调控核因子κB（NF-κB）/NOD样受体热蛋白结构域相关蛋白3（NLRP3）通路减轻脊髓损伤大鼠肠道炎症及屏障损伤的作用机制。[方法]将45只SD大鼠随机分为假手术组、模型组、艾灸组，每组15只，使用改良的Allen’s法制备脊髓损伤大鼠模型，艾灸组选取“百会”“大椎”“命门”督脉要穴进行悬灸，经14 d干预后，取结肠组织，采用苏木精-伊红（HE）染色观察病理形态，酶联免疫吸实验（ELISA）检测肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-23（IL-23）、白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）含量，蛋白免疫印迹法（Western blot）检测核因子-κB p65亚基（NF-κB p65）、CC趋化因子配体2（CCL2）、芳香烃受体（Ahr）、闭合蛋白-1（Claudin-1）、闭锁小带蛋白-1（ZO-1）蛋白表达，RT-qPCR检测NF-κB、NLRP3、半胱氨酸的天冬氨酸蛋白水解酶-1（Caspase-1）mRNA水平。[结果]模型组结肠黏膜结构破坏，炎性细胞浸润，TNF-α、IL-6、IL-23含量显著升高（P<0.05），Claudin-1、ZO-1蛋白表达降低（P<0.05），结肠出现炎症反应，肠屏障受损；模型组NF-κB p65、CCL2蛋白及NF-κB、NLRP3、Caspase-1 mRNA表达显著升高（P<0.05），Ahr蛋白降低（P<0.05）；经通督调神灸干预后，艾灸组中上述炎症因子含量降低，屏障蛋白（Claudin-1、ZO-1）及Ahr表达显著回升（P<0.05），NF-κB/NLRP3通路相关分子表达下调（P<0.05）。[结论]通督调神灸可通过抑制NF-κB/NLRP3炎症小体通路活化，减少促炎因子释放，上调紧密连接蛋白及Ahr表达，进而减轻SCI后肠道炎症并修复屏障损伤。

Abstract:

[Objective] To investigate the mechanism by which governor vessel-regulating and spirit-nourishing moxibustion alleviates intestinal inflammation and barrier damage in rats with spinal cord injury（SCI） through modulation of the NF-κB / NLRP3 inflammasome pathway. [Methods] Forty-five SD rats were randomly divided into sham-operated，model，and moxibustion groups，with 15 rats in each group. SCI was induced using the modified Allen’s method. The moxibustion group received suspended moxibustion at the Governor Vessel acupoints“Baihui”（GV20），“Dazhui”（GV14）and“Mingmen”（GV4）. After 14 days of intervention，colon tissues were collected；pathological morphology was examined by HE staining；ELISA was used to measure the contents of tumor necrosis factor-α（TNF-α），interleukin-6（IL-6），interleukin-23（IL-23），interleukin-1β（IL-1β）and interleukin-18（IL-18）. Western blot was performed to detect the protein expression of nuclear factor-κB p65 subunit（NF-κB p65），CC chemokine ligand 2（CCL2）， aryl hydrocarbon receptor（Ahr），Claudin-1 and zonula occludens-1（ZO-1）. RT-qPCR was conducted to quantify the mRNA levels of NF-κB，NLRP3 and Caspase-1. [Results] In the model group，the colonic mucosal structure was disrupted，inflammatory cells infiltrated，the contents of TNF-α，IL-6，IL-1β，IL-18 and IL-23 were significantly elevated（P<0.05），and the protein expression of Claudin-1 and ZO-1 was decreased（P<0.05），indicating colonic inflammation and impaired intestinal barrier；the expression of NF-κB p65 and CCL2 proteins and the mRNA levels of NFκB，NLRP3 and Caspase1 were markedly increased（P<0.05），whereas Ahr protein was reduced（P<0.05）. After Tongdu Tiaoshen moxibustion，the contents of the above inflammatory factors decreased，the expression of barrier proteins（Claudin-1 and ZO-1）and Ahr was significantly restored（P<0.05），and the expression of molecules associated with the NF-κB/NLRP3 pathway was down-regulated（P<0.05）. [Conclusion] Tongdu Tiaoshen moxibustion can attenuate spinal cord injury-induced intestinal inflammation and repair barrier damage by inhibiting NF-κB/NLRP3 inflammasome activation， reducing pro-inflammatory cytokine release and up-regulating the expression of tight-junction proteins and Ahr.