基于LPS/p38MAKP通路研究血府逐瘀汤治疗非酒精性脂肪肝的作用机制

Xuefu Zhuyu Tang, a classic formula, can promote blood circulation to remove stasis and move qi to relieve pain. It has been proven effective in treating non-alcoholic fatty liver disease (NAFLD) with obvious advantages. However, its action mechanism is unclear. LPS/p38MAKP pathway is crucial in the pathogenesis of NAFLD, which is marked by neutrophil chemotaxis and infiltration and subsequent microinflammation damage. Our previous study revealed that Xuefu Zhuyu Tang can effectively improve the liver steatosis of NAFLD mice model by alleviating inflammatory cell infiltration typically and meanwhile reducing chemokine MIP2 and KC expression; and different combinations of its guiding herbs affect the distribution of its effective constituents in the hepatic tissue. Hereby, we would like to propose the hypothesis that "Xuefu Zhuyu Tang may intervene LPS/p38MAKP pathway to reduce neutrophils infiltration, interdict subsequent inflammatory injury, and treat NAFLD effectively; and its role is associated with different combination of guiding herbs". This study will establish in vitro and in vivo models and apply such techniques as IF, FCM, qRT-PCR and WB to explore the rules and features of Xuefu Zhuyu Tang's effect of reducing neutrophils infiltration and to investigate the correlation between LPS/p38MAKP pathway and the formula's effect of relieving neutrophil infiltration when herbs are combined in various ways.

中医经典古方血府逐瘀汤具有活血祛瘀、行气止痛之功，临床用于治疗非酒精性脂肪肝(NAFLD)疗效确切，优势明显，但作用机制不清。LPS/p38MAKP通路在NAFLD的发病过程中发挥关键作用，其核心是趋化中性粒细胞浸润及后续微炎症损伤。前期研究发现，血府逐瘀汤可有效改善NAFLD模型小鼠肝脂肪变性，以减轻炎性细胞浸润为病理改善特点，同时可减少趋化因子MIP2和KC的表达；本方中引经药的不同配伍可影响其有效成分在肝组织中的分布。据此，提出“血府逐瘀汤可通过干预LPS/p38MAKP通路，对抗中性粒细胞浸润，阻断后续炎症损伤，发挥治疗NAFLD的作用，且该作用与引经药配伍有关”的研究假说。本研究拟通过建立体内外研究模型，采用IF、FCM、qRT-PCR、WB等技术，探索血府逐瘀汤对抗中性粒细胞浸润的作用规律和特点，探讨其不同配伍形式的抗中性粒细胞浸润作用与LPS/p38MAKP通路之间的相关性。

血府逐瘀汤治疗NAFLD疗效确切，LPS/p38MAKP通路在本病中发挥关键作用，其核心是趋化中性粒细胞浸润及后续微炎症损伤。本研究探索血府逐瘀汤及其不同引经药配伍形式对抗中性粒细胞浸润的作用规律和特点，以及与LPS/p38MAKP通路之间的相关性。. 主要研究内容：.所有实验分为四组进行：血府逐瘀汤全方组、全方减牛膝组、全方减桔梗组、全方减牛膝桔梗组。.1.体内实验：高脂饮食复合亚临床剂量LPS刺激建立C57BL/6JApoE-/-小鼠NAFLD模型，免疫荧光技术观察肝组织中中性粒细胞浸润情况，流式细胞技术检测外周血中性粒细胞活化程度。双色免疫组化TUNEL法检测肝细胞凋亡水平，HE&OilRedO染色计算NAFLD活动评分。.2.体外实验：C57BL/6J小鼠中性粒细胞体外培养，观察含药血清对中性粒细胞迁移能力的影响。.3.对LPS/p38MAKP通路的影响：qRT-PCR、WB、ELISA技术检测相关基因及蛋白的表达水平：通路调控分子p-p38MAPK(Thr180/Tyr182)、p38MAPK、MKP7、MPO；通路效应分子MIP2、KC、PARP、caspase-3、Bcl-2、Bax、Fas、FasL。. 重要结果：.1.血府逐瘀汤治疗NAFLD与其对抗中性粒细胞浸润、减轻炎症反应有关，其机制为抑制p38MAPK通路激活：预防肝组织脂肪变性和炎症，改善症状，降低p38MAPK的相对表达量，抑制LPS/p38MAPK信号通路，抑制中性粒细胞激活，降低KC和MIP-2的浓度，阻止活化的中性粒细胞向炎症部位募集，降低Caspase-3和Bax、Fas的表达，促进Bcl-2的表达。.2.血府逐瘀汤抗中性粒细胞浸润的作用规律和作用特点：抑制中性粒细胞激活，降低KC和MIP-2的浓度，阻止活化的中性粒细胞向炎症部位募集。.2.3引经药配伍在NAFLD治疗中的意义：牛膝、桔梗能加强抑制LPS/p38MAPK信号通路、减少炎症因子释放及抑制细胞凋亡；牛膝能促进MKP7蛋白的表达、抑制Caspase-3蛋白的表达。. 本研究证实了“血府逐瘀汤可通过干预LPS/p38MAKP通路，对抗中性粒细胞浸润，阻断后续炎症损伤，发挥治疗NAFLD的作用，且该作用与引经药配伍有关”的假说，对血府逐瘀汤的临床应用，以及方剂引经药配伍理论的发展具有积极的意义。