基于“肝脾同调”研究蓝布正的补血效应及作用机制

Geum japonicum Thunb. var. chinense F.Bolle (Chinese name “lanbuzheng”, LBZ) is a special ethnomedicine in Guizhou province, which is abundant and validated to show blood-enriching effect in minority nationality areas. However, the effect and mechanism of enriching blood of LBZ have not been completely elucidated yet because of little basic research. It is necessary to deeply explore the LBZ. Our recent clinical and animal studies demonstrated that LBZ had the most effective activity on recovering the peripheral hemogram of people and mice, liver and spleen indexes of mice. Under the “Qi and Blood” theory of traditional Chinese medicine, the property and our previous experiment results of LBZ, we propose that we could investigate the effect and mechanism of enriching blood of LBZ based on “regulation of liver with spleen”. This project intends to adopt a mice model of acetylphenylhydrazine cyclophosphamide complex-induced blood deficient (BD) to comprehensively study the enriching blood effects of LBZ in terms of the change of peripheral hemogram, bone marrow, immune organs and energy metabolism; Moreover, the protective effects of LBZ on tissue pathology, apoptosis and oxidative damage of liver and spleen are respectively studied; the regulatory effects on liver (anabolism and release of hematopoietic factors) and spleen function (immune cell count of peripheral blood and secretion of hematopoietic factors) are explored; And then the molecular mechanism of double effects will be further investigated in vivo by detecting the gene and protein expression of factors in the liver and spleen tissue. Finally, the models of liver and spleen cells of “BD state” will be established, and then the further enriching blood effect and mechanism of LBZ based on “regulation of liver with spleen” are analyzed. The present project is aimed to explain the enriching blood effect and the mechanism of LBZ and to provide theoretical and experimental evidence for the scientific and rational use and development of LBZ.

蓝布正(LBZ)为贵州民族特色药，民间补血功效明确且资源丰富，然而基础研究的薄弱使其补血效应和作用机制尚未阐明。我们最近在临床实践和动物研究中发现LBZ显著恢复了血虚后的外周血象及小鼠肝脾指数。据此，本研究结合中医理论、LBZ归经主治及现代药理结果，拟基于肝脾同调探讨LBZ的补血效应及作用机制。采用化学损伤致小鼠血虚模型，在外周血象、骨髓象等环节研究LBZ的补血效应；在此基础上，探索LBZ对肝脾组织形态、凋亡及氧化损伤的保护效应，同时研究LBZ对肝（合成代谢与释放造血相关因子）及脾功能（调节免疫和分泌造血相关因子）的调节效应，并通过检测肝脾组织中目的因子的mRNA和蛋白水平表达分析其作用机制；最后通过构建“血虚态”肝、脾细胞模型，深入研究LBZ肝脾同调的补血效应及作用机制。本研究旨在从对肝脾保护及功能调节的双重作用上揭示LBZ的补血效应及作用机制，为其科学合理使用和开发提供理论和实验依据。

本研究拟通过建立的公认的乙酰苯肼和环磷酰胺联用致小鼠血虚的复合模型，多指标综合评价蓝布正的补血效应；同时在补血效应基础上，明确蓝布正对肝脾的保护及功能调节效应；在明确蓝布正对肝脾的双重效应基础上，体内外揭示其肝脾同调补血的作用机制。研究结果显示，蓝布正水提物可增加血虚小鼠骨髓有核细胞数，增加骨髓有核细胞数中造血干细胞的比例，改善外周血常规指标，降低肝脾脏指数。更有意义的是，蓝布正可改善小鼠肝细胞气球样变减少且炎细胞浸润现象减弱，减轻肝脏的内质网扩张；可降低AST和ALT水平，对IL-1、IL-3和TNF-α及EPO水平也均有显著回调作用；RT-PCR结果也显示蓝布正可显著回调肝脏组织中造血因子COX-2、Hepc及TNF-α的mRNA水平。与此同时，蓝布正可抑制环磷酰胺诱导的脾脏萎缩及病理学损伤，表现为脾脏指数的升高，脾脏红髓和白髓分界清晰，红髓淤血减少，淋巴细胞数量有所增加；LBZ能刺激血虚小鼠脾组织分泌造血因子IL-6、GM-CSF、M-CSF，上调G-CSF、M-CSF、GM-CSF的mRNA表达水平的同时抑制TNF-α的mRNA表达。代谢组学研究发现，蓝布正可纠正21个肝脏差异代谢物的水平，其可能参与的代谢通路有：脂肪酸代谢、氨基酸代谢和胆汁酸代谢等。蓝布正干预后，21个代谢物出现不同程度的回调。进一步的通路分析发现，蓝布正对血虚小鼠代谢紊乱的调控可能与泛酸盐和CoA生物合成、肌醇代谢、脂肪酸氧化等途径密切相关相关。.此外，在基金的支持下，我们还作了一些其他方面的研究。如对蓝布正作用血虚小鼠后血浆代谢组学进行研究，给予蓝布正后有14代谢物向正常小鼠回调，其作用机制主要与影响嘧啶代谢、长链脂肪酸的β氧化、亚麻酸和亚油酸代谢、磷脂生物合成、鞘脂类代谢相关。本课题组还对蓝布正对血虚小鼠脾脏的抗凋亡作用进行了探索，结果发现蓝布正的作用机制可能与激活G-CSF/JAK2/STAT3信号通路以及抑制线粒体凋亡途径有关，发现了新的作用途径，有进一步深入研究的价值。