红茂草异紫堇碱抗炎与促细胞免疫活化机制研究

The Dicranostigma Leptopodum (Maxim.) Fedde, which contains the effective components of alkaloids charactering as the highest level of isocorydine, was traditionally used to treat the open tuberculosis of lymph node and all kinds of traumatic inflammation. It has been demonstrated that alkaloid extraction from Dicranostigma Leptopodum (Maxim.) Fedde functioned in the effects on anti-microbial, anti-tumor, protection-liver and immune-enhanced. This study aims to use the isocorydine alkali extracted from the Dicranostigma Leptopodum (Maxim.) Fedde to test its effects on the activity of macrophage and T lymphocyte as well as the alteration of the secretion of cytokines using immunoblotting and immunofluorescence strategies. Moreover, the effects of isocorydine on the lymphocyte DNA will be investigated employing the molecular biology methods to pinpoint the mechanism of isocorydine promoting cell immunocompetence. Therefore, the primary cultured endothelial cells, fibroblasts, Vero and Hela cell lines will subject to the isocorydine to examine the effects of isocorydine on the selective cell mitogenic or inhibitory of the various cell lines. The detections of free radical and apoptosis will be performed in normal cells and cancer cells. The effects of isocorydine on the bacterial cell wall and nucleic acid as well as the gene expression of Escherichia coli will also be subsequently conducted. The mechanism of isocorydine for promoting wound healing and anti-inflammatory will also be explored. This study will offer a theoretical guide for the development and clinical application of the Dicranostigma Leptopodum (Maxim.) Fedde and serve as a fundamental work for the research and development of novel drugs.

红茂草民间多用于治疗开放性淋巴结核和各类创伤性炎症。有效成分为生物碱，其中以异紫堇碱的含量最高。已有的研究证明由红茂草提取的生物碱具有抑菌、保肝、抗肿瘤、增强免疫机能等功效。本项目拟采用红茂草中分离得到的异紫堇碱为材料，采用免疫印迹技术、免疫荧光标记技术等免疫学方法，检测其对巨噬细胞和T淋巴细胞活性的影响及对免疫细胞因子分泌的改变。采用分子生物学技术研究红茂草异紫堇碱对淋巴细胞DNA的影响机制，确定异紫堇碱的促细胞免疫活性功能。通过作用于原代培养的内皮细胞与成纤维细胞及Vero与 Hela细胞系，检测其对不同细胞选择性促细胞分裂或抑制效应；对正常细胞与癌细胞染色体的作用；进行细胞凋亡及自由基检测。红茂草异紫堇碱处理大肠杆菌，检测对细菌细胞壁及核酸的作用；并检测细菌基因表达变化。探讨异紫堇碱的促伤口愈合与抗炎机制。本项目的研究为红茂草的开发和临床应用提供理论参考，为新药研制做好基础工作。

红茂草在民间多用于治疗开放性淋巴结核和各类创伤性炎症，其有效成分主要为生物碱，其中以异紫堇碱的含量最高。本项目以从红茂草（Dicranostigma leptopodum）中分离到的异紫堇碱为材料，使用现代免疫技术检测了其对巨噬细胞免疫细胞因子分泌的改变和免疫活性功能的影响。与此同时，异紫堇碱也被分别作用于原代培养的正常细胞株（HA人星形胶质细胞）、人宫颈癌细胞Hela和Huh7、SMMC-7721、HCC-LY5细胞株，检测了其对不同细胞株的选择性促细胞分裂或抑制效应。最后，红茂草异紫堇碱也被用于处理大肠杆菌，检测了其对细菌细胞壁及胞内酶的变化的作用。研究结果表明：红茂草异紫堇碱对大肠杆菌等试验菌株有较好的抑菌、杀菌作用，对DPPH 自由基和•OH和O2-•自由基具有清除作用，其清除率分别为58.70%和49.26%，说明红茂草异紫堇碱能通过水解细菌细胞壁中的糖苷键，破坏细胞壁结构，影响BCA的合成能力，进而抑制细菌生长。此外，红茂草异紫堇碱还能有效缓解小鼠肝脾和胸腺的萎缩，并延缓血清中IL-2、TNF-α、IL-1β和IFN-γ的含量下降；降低血清中转氨酶的含量，显著提高肝组织中T-SOD的含量。LPS诱导RAW 264.7细胞释放炎性因子实验表明红茂草生物总碱能够抑制促炎因子IL-1β、IL-6、PEG-2和TNF-α的释放；高浓度异紫堇碱对HeLa细胞有抑制作用，但对HA细胞的毒性不显著，其对HeLa细胞的IC50为3.67µg/µL，证明了红茂草异紫堇碱对Hela细胞具杀伤作用。对肝癌细胞系的研究表明红茂草异紫堇碱衍生物（d-ICD）可抑制Huh7、SMMC-7721、HCC-LY5 肝癌细胞的体外增殖和体内肿瘤形成能力，其IC50 浓度在15-25 μg/mL之间；其可诱导肝细胞癌细胞周期G2 期阻滞；通过下调Bcl-2 蛋白表达、上调Bax 蛋白表达，促进肝癌细胞凋亡；通过诱导C/EBPβ，转录性地上调了GADDA45A表达，增强了细胞周期抑制剂p21的表达，导致G2/M细胞周期阻滞和细胞凋亡。这项研究表明红茂草异紫堇碱及其衍生物是一个很有前途的治疗肝细胞癌的治疗剂。本项目的研究在为红茂草的开发和临床应用提供理论依据的同时，也为新药的研制做好基础工作。