大黄-桃仁介导AhR通路影响Th17/Treg和肠道菌群平衡改善肠粘膜屏障功能防治粘连性肠梗阻的机制研究

Intestinal mucosal barrier is the trigger point of the formation of adhesive intestinal obstruction (AIO). Intestinal flora plays an important role in maintaining the stability of function of intestinal mucosal barrier. AhR pathway is involved in the synthesis and secretion of intestinal mucosal barrier. The previous work of our research group confirmed that many prescriptions with herb pairs such as Rhei Radix Et Rhizoma and Persicae Semen (RRER-PS) were clinically effective in the treatment of AIO. Based on the network pharmacology, the core target network of the RRER-PS treated AIO was screened. Accordingly, we made a hypothesis: the mechanism of RRER-PS in the treatment of AIO may be related to remodeling intestinal flora to affect intestinal mucosal barrier and the regulation of AhR signaling pathway. In this study, Caco-2 cells were used as the research objects to study the effect of RRER-PS on Caco-2 cell activity and adhesion protein, as well as the pathway by applying small interfering RNA and overexpression, cell culture and molecular biology techniques. In addition, with AIO rats as the research objects, hematoxylin-eosin staining, 16s rRNA sequencing analysis, immunohistochemistry, Western blotting were applied to discuss the expression of AHR pathway related index, confirm the molecular mechanism of RRER-PS in the prevention and treatment of AIO to mediate intestinal mucosa Th17/Treg balance remodeling intestinal flora.

肠粘膜屏障功能失调是粘连性肠梗阻（AIO）形成的重要因素，肠道菌群可维持肠粘膜屏障结构稳定，AhR通路参与肠粘膜屏障功能的调控。课题组前期工作证实以大黄-桃仁为药对的诸多方剂在外科临床治疗AIO临床疗效显著，并结合网络药理学筛选出其效应的核心靶点网络，为此提出科学假说：大黄-桃仁药对防治AIO的机制可能与调控AhR信号通路影响Th17/Treg稳态重塑肠道菌群作用肠粘膜屏障有关。本研究拟①以Caco-2细胞为研究对象，采用siRNA干扰和过表达观察大黄-桃仁对LPS刺激Caco-2细胞活性及AhR信号通路的影响及其作用途径；②以AIO大鼠为研究对象，采用16s rRNA测序、RT-PCR、WB等分子生物技术，从肠道菌群变化、AhR通路相关指标表达方面探讨大黄-桃仁干预AhR通路介导肠粘膜固有层Th17/Treg平衡重塑肠道菌群修复粘膜从而实现其防治AIO的作用的具体机制。

目的：阐释大黄-桃仁及其有效成分大黄酸介导AhR信号通路调控肠粘膜稳态影响肠道菌群分布改善肠粘膜屏障功能防治AIO的具体机制。方法：建立大鼠肠梗阻模型，应用HE染色、组织学评分，观察肠粘膜损伤情况；通过血清ELISA和流式细胞术分别检测炎性细胞因子水平和Th17、Treg 细胞水平，16s rRNA测序分析肠道菌群的群落结构和数量变化，免疫组化观察各组肠黏膜SIgA 、CD4+T、CD8+T和AhR细胞的表达情况及活性的变化；通过蛋白质免疫印迹观察AhR、p-ERK、XRE、p-JNK、p-P38, ERK、JNK和P38的蛋白水平。方法：利用100ng/ml TNF-α刺激Caco-2细胞 24h构建肠道屏障障碍细胞模型。通过CCK-8法检测不同浓度TNF-α刺激Caco-2细胞后活力变化及不同浓度大黄酸对细胞模型活力的影响，WB检测TLR4、MyD88、NF-kB、p-NF-kB、MLCK的蛋白表达；荧光定量检测NF-kB，IL-8，IL-1β，MLCK等细胞屏障 mRNA表达；结合透射电镜下观察大黄酸对TNF-α刺激Caco-2细胞的影响；同时确定TNF-α的造模浓度和大黄酸的干预浓度。荧光定量检测AhR，FOXP3，RORyt，STAT3，ZO-1等肠道屏障 mRNA表达；WB检测AhR、STAT3、p-STAT3、ZO-1等肠道屏障的蛋白表达。结果：与模型组相比，大黄-桃仁水煎剂低、中、高剂量可不同程度降低血清IL-1β、D-LA、ET、DAO、MCP-1和IL-17表达水平，同时升高IL-10表达水平（P＜0.01）。流式细胞检测显示大黄-桃仁高剂量可降低AIO模型大鼠血清中 Th17、Treg 细胞含量（P＜0.05）。Western blot 结果显示不同剂量大黄-桃仁组的AhR、p-ERK、XRE、p-JNK、和p-P38蛋白水平降低（P <0. 05）。大黄酸干预后Foxp３和RORγt的表达增加；给药组AhR、ZO-1等肠道屏障蛋白表达水平较模型组均显著升高，而p-STAT3蛋白表达水平较模型组显著降低。结论：大黄-桃仁及其有效成分大黄酸可能通过改变肠道菌群的变化和肠黏膜免疫环境，修复肠道黏膜屏障功能起到防治AIO的作用。