清金颗粒防治多重耐药细菌感染的免疫机制研究

The incidence of multidrug-resistant bacterial infections (MDRO), especially the incidence of MRSA increased year by year(Representing approximately 50% of S. aureus infections),which leads to high mortality, high economic burden. “ Qingjin particles” is the valid prescription that is often used by the famous Chinese medicine professor Shao changrong to treat MDRO infection such as MRSA、MDR.TB and so on . In addition, “ Qingjin particles” can help to make the expression of TLR2 In the vitro macrophages with M.tb infection increased . We also carried out a study to optimize the prescription for MRSA before. This study will carry out a control experiment about the MRSA-infected littermate wild-type mice intervened with“ Qingjin particles” and TLR2-/ - knockout mice. The research aims to study the affect of “ Qingjin particles” for the blood mononuclear cells,the peritoneal macrophage activation ,the expression of the mode receptor (TLR1, TLR2, TLR6 and TLR4) and the secretion of TNF-ɑ, IL-6 and IL10 in blood in the infected host on the 1 \ 3 \ 7d respectively. And at the same time, We need to observe the relationship between TLR2 and the acquired immune. So that,We will culture macrophages in vitro to study the regulatory mechanisms of Serum containing “ Qingjin particles” for the expression of TLR2 in macrophages ,the Intracellular signal transduction pathways (MyD88-NF-κB), NF-KB p65 nuclear translocation and the secretion of TNF-ɑ, IL-6 and IL10 in blood .So that , we can use these findings guide clinical treatment better.

多重耐药细菌感染（MDRO），尤其MRSA发病率逐年上升（约占金葡菌感染50%），高病死率、高经济负担。“清金颗粒”是全国名老中医邵长荣治疗MRSA、MDR.TB等MDRO感染的验方，体外能使M.tb感染巨噬细胞TLR2表达上调，且针对MRSA感染的进行了组方优化实验研究。本研究设计清金颗粒干预MRSA感染同窝野生小鼠与TLR2-/-基因敲除小鼠对照实验，研究其对感染宿主第1\3\7d时间点血单核细胞、腹腔巨噬细胞的活化、模式受体表达（TLR1、TLR2、TLR6和TLR4）及血TNF-ɑ、IL-6和IL10分泌的调控效应；观察TLR2与获得性免疫的可能关联；同时，体外培养巨噬细胞，研究“清金颗粒”含药血清对巨噬细胞TLR2表达、胞内信号转导通路（MyD88-NF-κB）、NFκB p65入核及TNF-ɑ、IL-6和IL10分泌等MyD88依赖通道内的调控机制，指导临床更加有针对性的用药。

随着抗菌药物的迅猛发展和普遍应用，给人类带来高效解决病痛的同时，也不断催生新的多重耐药病原菌( Multidru g-resistant organism ,MDRO)。TLR2活化被认为是宿主固有免疫清除MRSA等MRDO病原菌的主要路径。药物对 TLR 免疫应答的抑制可能是治疗宿主炎症性疾病的一种重要途径。“清金颗粒”是在“芩部丹”基础上加减而成的中药复方，且临床疗效优于“芩部丹”，结合其临床针对多种MDRO 感染广泛应用，及中医“异病同治”理论，本研究设计清金颗粒干预MRSA感染C57小鼠与TLR2-/-小鼠对照实验，研究其对感染宿主第1\3\7d时间点肝脏载菌、病理，脾脏流式、炎症因子表达，腹腔巨噬细胞的活化及极化、模式受体表达（TLR1、TLR2和TLR4）及腹腔灌洗液TNF-ɑ、IL-6和IL10分泌的调控效应；同时，体外培养巨噬细胞，研究“清金颗粒”对巨噬细胞TLR2表达、胞内信号转导通路（MyD88-NF-κB）及TNF-ɑ、IL-6和IL10分泌等MyD88依赖通道内的调控机制。结果发现：⑴ 清金颗粒显著降低MRSA感染C57小鼠第3天、7天肝脏载菌量，显著降低TLR2-/-小鼠第3天肝脏载菌量；⑵ 清金颗粒清除脏器MRSA载菌量并不是通过TLR2信号转导通路，且不存在TLR1/TLR4的协同表达，其对TLR2的表达、活化及下游TAK1/P38的磷酸化没有影响；对脾脏、腹腔巨噬细胞炎症因子、趋化因子的表达没有影响；对腹腔灌洗液炎症因子及抗体的含量没有影响。⑶ 清金颗粒显著增加MRSA感染C57小鼠及TLR2-/-小鼠第3天脾脏CTL细胞及B细胞数量；⑷ MRSA在体内可能通过巨噬细胞发生逃逸，并定植到脏器组织，清金颗粒可能是通过上调CTL细胞分化，明显减少MRSA感染胞内免疫逃逸，但值得进一步深入研究；⑸ 清金颗粒可以通过免疫调控作用从而发挥对MRSA感染的治疗作用。本研究最终明确了清金颗粒治疗MRSA感染的免疫机制，为临床上应用清金颗粒治疗耐多药菌（MRSA/MDR-TB）及胞内菌提供了基础依据及支撑。将不同MDRO 感染具有共同宿主免疫应答机制与中医“异病同治”理论相结合，能够进一步指导更多中药验方用于MRSA 等MDRO 感染性疾病。