扶正解毒方通过调控髓系细胞的吞噬能力治疗脓毒症的机理

Bacteria dissemination and excesive production of inflmmatory cytokines are two major factors of multi-organ failure and mortality during sepsis. However, impairment of inflammatory cytokines production failed to decrease the mortality rate of severe sepsis in the clinic. Granulocytes, monocytes and macrophages, which are the major phagocytes derived from myeloid progenitors, play an important role in eliminating bacteria by phagocytosis that is induced by activating the PI3K signalling pathway. Fu-Zheng-Jie-Du-Fang, a traditional chinese decoction, is used in clinical therapies of peritonitis caused by acute liver failture and sepsis. In the preliminary study, we observed that Fu-Zheng-Jie-Du-Fang increased the survival rates of mice with colon ascending stent peritonitis (CASP) by reducing bacteria translocation. Fu-Zheng-Jie-Du-Fang enhanced the phagocytic ability of peritoneal macrophages and granulocytes in mice by intraperitoneal injection of LPS-fluorescent beads. However, Fu-Zheng-Jie-Du-Fang did not inhibit inflammatory cytokines in endotoxin-challenged mice. Based on above observations, we proposed that Fu-Zheng-Jie-Du-Fang might increase phagocytosis ability of myeloid cells by regulating PI3K signalling pathway during sepsis. With CASP model and primary cultured myeloid cells, we intend to investigate the effects of Fu-Zheng-Jie-Du-Fang on phagocytosis and the PI3K signalling pathway, explore its pharmacological mechanisms in regulating innate immune responses, and provide experimental evidences for future clinical treatment.

细菌播散和炎性细胞因子造成的免疫损伤是细菌性脓毒症的两大致病因素。既往研究表明单纯拮抗炎性细胞因子尚不能降低患者死亡率。髓系来源的单核-巨噬细胞和粒细胞是机体主要的吞噬细胞，激活PI3K信号通路，增强其吞噬能力，是治疗细菌性脓毒症的重要突破口。扶正解毒方对肝衰竭自发性腹膜炎和脓毒症具有良好的临床疗效。我们前期发现：该方能显著降低腹腔持续置管引流腹膜炎（CASP）重症脓毒症模型小鼠的死亡率，减轻菌群移位；能显著提高腹腔髓系细胞吞噬能力；而不直接降低炎性细胞因子水平。由此提出“扶正解毒方可能通过调控PI3K信号通路，增强髓系细胞吞噬能力是其治疗重症细菌性脓毒症的关键环节”。我们拟以CASP模型为体内研究基础，原代分离培养的髓系细胞为体外研究对象，明确扶正解毒方对髓系细胞吞噬功能及其相关的信号通路的影响，探讨扶正解毒方纠正重症感染机体免疫紊乱的机制，为中医药治疗重症感染提供新的思路和方法。

细菌播散和炎性细胞因子造成的免疫损伤是细菌性脓毒症的两大致病因素。既往研究表明单纯拮抗炎性细胞因子尚不能降低患者死亡率。髓系来源的单核-巨噬细胞和粒细胞是机体主要的吞噬细胞，激活信号通路，增强其吞噬能力，是治疗细菌性脓毒症的重要突破口。扶正解毒方对肝衰竭自发性腹膜炎和脓毒症具有良好的临床疗效。我们研究发现：该方能显著降低腹腔持续置管引流腹膜炎（CASP）重症脓毒症模型小鼠的死亡率。研究扶正解毒方拆方研究，发现虫草菌丝显著提高CASP 模型小鼠生存率，进一步分析发现虫草菌丝起效的主要有效组分是虫草菌丝糖蛋白。机制研究发现：扶正解毒方主要有效组分虫草菌丝1）通过小鼠腹腔注射荧光微球，流式细胞仪检测到扶正解毒方主要有效组分虫草菌丝增多腹腔吞噬荧光微球的髓系细胞数量；通过小鼠腹腔注射GFP-E.coli，扶正解毒方主要有效组分虫草菌丝增强腹腔髓系细胞吞噬GFP-E.coli能力，腹腔内游离的GFP-E.coli减少；2）扶正解毒方主要有效组分虫草菌丝通过增加髓系细胞表面吞噬受体CD16/CD32和CD64表达，促进髓系细胞肌动蛋白聚合，促进髓系细胞吞噬细菌，减轻菌群移位，而且吞噬没有菌群特异性；3）扶正解毒方主要有效组分虫草菌丝通过髓系细胞吞噬细菌，降低髓系细胞表面负共刺激分子及其它活化标志PD-1、PD-L1、PD-L2和CD80表达，抑制炎性因子表达，减轻脏器损伤；4）RNA-seq结果证明扶正解毒方主要有效组分虫草菌丝通过调控多条吞噬相关信号通路，提高髓系细胞吞噬能力。5）采用腹腔注射构建的稳定表达荧光素酶的产碳青霉烯酶肺炎克雷伯杆菌脓毒症模型和二次打击模型盲肠结扎穿孔（CLP）7天后鼻饲绿脓杆菌脓毒症模型，虫草菌丝促进髓系细胞吞噬细菌，显著提高两种细菌性脓毒症模型小鼠生存率。这些结果证实扶正解毒方通过主要成分虫草菌丝调控吞噬相关信号通路，增强髓系细胞吞噬能力是其治疗重症细菌性脓毒症的关键环节。我们的研究为中医药治疗重症感染和抗菌素耐药治疗提供新的思路和方法。