解毒凉血健脾方阻抑中性粒细胞定向迁移治疗急性肝衰竭的机制研究

The increase of neutrophil in peripheral blood was demonstrated to reflect circular amplification of immune injury, which is associated with the prognosis of patients with acute liver failure. In our clinical studies, therapy of Jiedu-Liangxue-Jianpi Decoction was effective to improve survival of patients with acute liver failure by tempering neutrophil. In our previous work, the acute liver failure mouse model established with administration of ConA performed elevated absolute numbers and percentages of hematopoietic stem cells and decreased granulocyte/monocyte progenitors in bone marrow, suggesting that numerous neutrophils were mobilized from bone marrow. In addition, the tissues distribution of neutrophils were analyzed and sorted by the flow cytometry which found that numerous neutrophils were recruited to the liver. Accordingly, we proposed that Jiedu-Liangxue-Jianpi Decoction could temper the liner inflammatory response and treat acute liver failure by inhibiting the neutrophil directional migration. The aim of this study is:1)to evaluate the curative effect of Jiedu-Liangxue-Jianpi Decoction in treating acute liver failure with ConA model and identify the major chemotatic factors;2)to confirm the key role of neutrophil in the treatment of acute liver failure through cell clearance and transplantation experiments;3)to define the regulating effect of the decotion on the polarization and chemotaxis in cytological level;4)to further reveal the molecular mechanism of neutrophil directional migration regulation,finally to provide experimental evidence for the treatment of acute liver failure with traditional Chinese medicine.

中性粒细胞介导的炎症损伤循环放大，与急性肝衰竭患者的病情和预后密切相关。我们临床研究发现：解毒凉血健脾方能够降低肝衰竭患者外周血中性粒细胞水平，改善生存率。我们前期应用ConA急性肝衰竭小鼠模型发现骨髓造血干细胞明显升高，粒/单核系祖细胞明显降低，提示中性粒细胞从骨髓中大量动员。同时，我们利用流式细胞术检测粒细胞在组织间的分布，发现中性粒细胞被大量募集至肝脏。据此我们提出假说：“解毒凉血健脾方通过阻抑中性粒细胞向肝脏定向迁移，减轻肝损伤，治疗急性肝衰竭”。本课题拟1）采用ConA模型评价该方疗效，筛选调节中性粒细胞定向迁移的关键趋化因子；2）结合细胞清除、移植实验确证中性粒细胞在治疗急性肝衰竭中的关键作用；3）在细胞水平应用Zigmond和Transwell技术，明确该方对中性粒细胞极性和趋化性的调节作用；4）在分子水平分析药物干预中性粒细胞定向迁移的作用机制，为中医药治疗肝衰竭提供依据。

中性粒细胞介导的炎症损伤循环放大，与急性肝衰竭患者的病情和预后密切相关。深入探讨中性粒细胞趋化过程中的信号机制，对寻找新的治疗和干预靶点，防止中性粒细胞异常激活导致的组织损伤具有重要意义。但是，目前有关急性肝衰竭发生发展过程中，中性粒细胞向肝脏定向迁移的相关机制还有待进一步探讨，并且当前还没有明确的治疗方案能够控制急性肝细胞坏死后，肝脏中性粒细胞的募集和激活。.本研究发现：1）解毒凉血健脾方具有明显的抵抗急性肝损伤和抗炎作用，主要表现在提高Con A急性肝衰竭模型小鼠的生存率，降低模型小鼠的血清转氨酶（ALT和AST）水平，抑制炎性因子TNF-α、IL-6、IL-1β、MCP-1的表达，减少肝细胞凋亡，改善模型小鼠的肝组织病理学损伤。2）Luminex和免疫组化结果显示CXCL2是解毒凉血健脾方阻抑中性粒细胞定向迁移治疗急性肝衰竭的关键趋化因子。3）通过阻断实验证实抑制中性粒细胞趋化因子受体CXCR2和FPR1能明显降低急性肝衰竭小鼠转氨酶和炎症因子水平，减轻肝损伤。并通过免疫荧光技术证实解毒凉血健脾方能够明显降低中性粒细胞趋化因子受体CXCR2和FPR1的表达。4）通过PCR方法发现抑制中粒细胞趋化因子受体CXCR2和FPR1能够上调PI3K和AKTmRNA表达。5）通过PCR、Western blot法发现解毒凉血健脾方通过激活PI3K/AKT/Gas-3β信号通路，下调Caspase3表达，发挥减少肝细胞凋亡，减轻肝损伤的作用。6）细胞学实验通过Transwell技术再次验证解毒凉血健脾方能够抑制中性粒细胞的趋化能力，并通过免疫荧光和流式细胞学技术证实解毒凉血健脾方含药血清能够改善ConA刺激的AML12细胞的凋亡。本研究结果不仅有助于充分揭示解毒凉血健脾方抗急性肝衰竭的科学内涵，也为中医药治疗急性、慢加急性及亚急性肝衰竭提供了新的思路和方法。