活血通腑方调控巨噬细胞极化表型重构免疫微环境防治腹腔粘连的实验研究

Postoperative peritoneal adhesion is closely associated with the changes of peritoneal immune microenvironment. The M1/M2 polarization of peritoneal macrophages plays a key role of the development trend of peritoneal inflammation and tissue fibrosis after peritoneal immune microenvironment change. The JAK/STAT/PPARγ signal pathway regulates the state of M1/M2 polarization. Our previous studies have demonstrated that Blood-activating and Organ-purging formula effectively prevented postoperative peritoneal adhesion in animal models by up-regulating CD40、CD40L and MCP-1 protein levels , and down-regulating IL-10 protein levels, indicating that the therapeutic mechanisms may be involved in mediating the JAK/STAT/PPARγ signal transduction to regulate the state of macrophage M1/M2 polarization. Therefore, in animal models of peritoneal adhesion and cell models of peritoneal macrophages, this project will analysis the potential target PPARγ nuclear translocation level and the mediation of the JAK/SATA pathway to macrophage M1/M2 polarization by multispectral imaging flow cytometry; as well as detect the local immune microenvironment state of peritoneal adhesion by using xMAP technology. This project will mainly investigate and explore the intervention and the underlying molecular mechanism of Blood-activating and Organ-purging formula on peritoneal macrophage polarization and its mediated immune microenvironment change. These results from this study will provide the evidences for further exploring the immune mechanism of postoperative peritoneal adhesion, and guiding the effective prevention and treatment of traditional Chinese medicine.

术后出现腹腔粘连与腹腔免疫微环境的改变密切相关。腹腔巨噬细胞M1/M2极化表型决定腹腔免疫微环境变化后的腹腔炎症及组织纤维化反应趋势，而JAK/STAT/PPARγ信号通路调控M1/M2极化表型。前期研究结果表明：活血通腑方可通过下调腹腔CD40、CD40L和MCP-1蛋白水平及上调IL-10水平等有效降低术后粘连程度，推测其效应机制可能与介导JAK/STAT/PPARγ信号转导进而调控腹腔巨噬细胞M1/M2极化表型有关。为此，拟在腹腔粘连模型动物及腹腔巨噬细胞上，采用高通量高速成像流式技术量化分析潜在靶点PPARγ核转位水平及其通过JAK/SATA通路对巨噬细胞M1/M2极化状态的影响；运用细胞因子液相芯片技术分析粘连局部免疫微环境状态；重点研究并阐明活血通腑方对腹腔巨噬细胞极化及其介导免疫微环境变化的干预作用和分子效应机制，为进一步探讨术后腹腔粘连免疫机制及指导中医药有效防治提供依据。

腹腔粘连是腹部外科手术中最具挑战性的问题之一，它的出现与腹腔免疫微环境密切相关。腹腔巨噬细胞极化决定腹腔免疫微环境变化后的腹腔炎症及组织纤维化反应趋势，密切参与术后腹腔粘连的生理病理过程。JAK/STAT通路及其上下游信号是巨噬细胞极化激活的主要途径，导致巨噬细胞极化为不同的表型，M1型巨噬细胞（M1）分泌促炎细胞因子，M2型巨噬细胞（M2）分泌抗炎细胞因子，M1/M2的功能转换影响腹腔粘连的严重程度。活血通腑方（HXTF）能够有效防治腹腔粘连，由大黄、延胡索、桃仁、莱菔子、红花和芒硝组成，临床已运用二十余年，然而其作用机制仍不完全清楚。因此，在本研究中，我们建立RAW264.7巨噬细胞炎症模型和大鼠腹腔粘连模型，给予活血通腑方干预，进行不同分组。流式细胞术检测M1/M2极化程度和PPAR-γ核转位程度，ELISA法检测M1/M2相关细胞因子水平，HE染色法和Masson染色法观察粘连局部炎症和胶原纤维沉积情况，Westernblot法和real-time PCR法检测巨噬细胞和粘连组织SOCS/JAK2/STAT/PPAR-γ通路相关因子表达。观察到术后粘连局部炎症和胶原纤维沉积减少，与IL-4类似，HXTF诱导巨噬细胞向M2极化，促进PPAR-γ核转位，HXTF和PPAR-γ激动剂下调巨噬细胞M1极化相关因子IL-1、IL-6和TNF-α，上调M2极化相关因子IL-4、IL-10和TGF-β1。同时，HXTF和PPAR-γ激动剂下调SOCS3/JAK2/STAT1通路，激活SOCS1/STAT6/PPAR-γ通路。本研究揭示和阐明了活血通腑方对腹腔巨噬细胞极化及其介导免疫微环境变化的干预作用和分子效应机制，为进一步探讨术后腹腔粘连免疫机制及指导中医药有效防治提供依据。