【作者】 孙丽娟；

【导师】 刘建文；

【作者基本信息】 华东理工大学 ， 生物化工， 2011， 博士

【摘要】 Withanolide类型化合物是一类具有28个碳原子的麦角甾烷型的化合物,其特征性结构是22位与26位碳连接形成一个δ内酯环的侧链。Withanolide不仅可以用来表示麦角甾烷型的22,26-内酯,也包括与之有生源关系的化合物。这类化合物主要分布在茄科植物中,具有抗炎,抗菌,抗病毒,抗微生物,抗氧化,抗肿瘤,抗惊厥,以及抑制乙酰胆碱酯酶的活性。本课题首先对茄科植物的Withanolide部位进行了活性检测,结果显示该部位具有较强的免疫抑制生物活性。继续采用生物追踪法,由合作实验室对该Withanolide部位进一步的分离纯化,得到一系列Withanolide单体化合物。采用体外细胞增殖模型,通过对这些化合物进行免疫抑制活性的筛选。结果发现,Withangulatin A(WA), Physagulin L, Physalin H等都具有显著的免疫抑制活性。根据筛选结果,初步总结出Withanolide骨架化合物免疫抑制活性的构效关系。本论文在此基础上以WA为代表,深入探讨其抗自身免疫性疾病的具体作用机理。本论文研究了WA对正常的淋巴细胞、异常增殖的淋巴细胞和混合淋巴细胞的增殖抑制活性,与阳性药物环胞菌素A (CsA)进行比较,结果显示,WA具有较CsA毒性更低,免疫抑制活性更强的潜在作用。小鼠耳廓肿胀实验结果显示,WA同样具有比CsA作用更强的抑制小鼠耳廓肿胀的抗炎作用,且对小鼠的体重影响更小。这为深入探索WA的免疫抑制活性提供了重要的理论基础。本论文分别从调节淋巴细胞周期,细胞亚型和Th1/Th2型细胞因子平衡等方面对WA免疫抑制活性机理进行了初步的探索。流式细胞技术结果表明WA通过阻滞细胞从G1期到S期的转换,从而阻滞T淋巴细胞有丝分裂的完成；抑制CD4+/CD8+和CD25+细胞亚型,提高了抑制性T淋巴细胞相对数量来抑制淋巴细胞的异常增殖。体内外ELISA结果显示WA调节Th1/Th2型细胞因子平衡是其免疫抑制活性的作用机理之一。本论文研究了WA对保护性基因HO-1的调节作用。HO-1具有调节免疫,保护细胞损伤等作用,因此诱导HO-1表达在免疫调节方面将成为一种新型的治疗方法。本研究选择HO-1作为分子靶点,对WA免疫抑制的作用机理进行探讨。Western blot和RT-PCR结果显示,WA通过ERK/MAPK途径,呈浓度和时间依赖性地促进小鼠淋巴细胞中HO-1蛋白和mRNA的表达。采用Western blot方法,继续分析了NF-κB p65核内外的表达水平,结果表明WA抑制NF-κB p65核转位效应,与HO-1表达具有直接或间接联系,同时可能参与WA上调过氧化物酶体增殖物激活受体-γ(PPAR-γ),下调环氧合酶-2(COX-2)表达的作用。本论文建立了基于双荧光素酶报告基因法的特异性抑制COX-2表达的体外分子筛选模型。荧光素酶报告基因法是检测基因启动子活性的一种有效的方法,具有检测速度快、灵敏度高、费用低、不需使用放射性同位素等优点。该模型通过分别构建含有COX-1和COX-2基因启动子的荧光素酶报告基因载体,并同海肾荧光素酶报告基因载体共同组成双荧光素酶报告基因系统,采用脂质体法将两个载体共转染到细胞中,利用化学发光仪测定荧光强度变化,从而分别反映出COX-1和COX-2基因启动子的活性。本研究利用COX-2特异性抑制剂的阳性药物Celecoxib对该分子模型进行了验证,结果显示该模型可信度高、灵敏度高及适于大规模筛选。由于该模型中荧光强度仅仅由COX-2启动子的活性调控,由于该启动子区域中包含特定的已知作用位点,因此还适用于特异性抑制COX-2表达的分子机理探讨。基于双荧光素酶报告基因的分析结果,本研究进一步对WA特异性抑制COX-2的分子机理进行了探索。Western Blot和PCR等实验结果表明WA通过抑制ERK/MAPK、p38/MAPK途径和抑制NF-κB p65核转位来特异性地抑制COX-2的蛋白和基因表达,进而降低其PGE2产量,而对COX-1表达几乎没有影响。MAPK的三种抑制剂参与NF-κB核转位的结果表明,MAPK可能参与COX-2启动子上除NF-κB以外的其他作用位点来调控COX-2的表达。本论文利用成熟的MRL/lpr红斑狼疮小鼠,初步探讨了WA对系统性红斑狼疮疾病的治疗作用。结果表明WA可能通过BAFF/BAFF-R通路,有效改善免疫状况,减轻脾脏及淋巴结肿大,同时显著改善MRL/lpr小鼠的肾脏和肺功能,减轻蛋白尿症状和降低血清中抗ds-DNA水平,延长小鼠平均生存期,进一步验证了WA用于治疗系统性红斑狼疮的潜力。本论文分别从体外的细胞和分子水平,体内炎症和MRL/lpr红斑狼疮鼠模型等几个方面对WA的免疫抑制活性这一新用途进行了多方面、多角度的研究。结果表明WA通过阻滞小鼠T淋巴细胞有丝分裂的完成,抑制CD4+/CD8+和CD25+细胞亚型和调节Th1/Th2型细胞因子的平衡来抑制淋巴细胞的异常增殖；同时上调保护性基因HO-1的表达,特异性地抑制炎症因子COX-2表达来发挥其免疫抑制活性。此外WA对炎症小鼠和红斑狼疮小鼠均有很好的治疗作用。以上实验结果为进一步开发和利用WA以及Withanolide类成分的新的免疫抑制活性提供了科学依据。更多还原

【Abstract】 Withanolides are structurally diverse steroids with an ergostane skeleton in which C22 and C26 are oxidized to form aδ-lactone. Withanolides represent not only aδ-lactone with C22 and C26 oxidized, but also all biosynthectic pathways related compounds. These compounds show anti-inflammatory, antibacterial, antiviral, anti-microbial, anti-oxidation, anti-tumor, anti-convulsions, as well as inhibition activity of acetylcholinesterase. In this study, the immunosuppressive fraction of withanolides from Solanaceae plant were screened, the result demonstrated that this fraction showed significant immunosuppression activity. In order to clarify its effective chemicals relation to immunosuppression, the thirty compounds were isolated out from the effective fractions by our cooperative Lab. We found that Withangulatin A (WA), Physagulin L, Physalin H, and et al, exhibited significant immunosuppression activity. According to the results, the structure-immunosuppression activity relationship was summarized. In order to further explore its immunosuppressive mechanisms, WA was selected as typical compounds for its significance activity.In this study, we evaluated the cytotoxicity and immunosuppressive activity of WA in normal lymphocyte, ab-normal proliferation and mixed lymphocyte. The results illustrated that WA showed more effective and lower cytotoxicty than positive drug of cyclosporine A (CsA). At the same time, we measured immunosuppressive activity of WA in Xylene-induced ear tumefaction mice, the result also showed WA was more effective than CsA in inhibiting xylene-induced ear tumefaction while the weight of mice was not significantly different. These results provide an important scientific evidence of WA for further research.In this study, we investigated the effect on mice T lymphocytes function of WA. FCM data documented that WA obstructed DNA replication in G1-S1 stages to regulate cell cycle and decreased CD4+/CD8+ and CD25+ to inhibit the overproduced lymphocyte. In addition, the results of ELISA showed that WA regulated Thl/Th2 balance in vitro and in vivo to show immunosuppressive activity.In this study, further immunosuppression mechanism of WA was studied through a molecular target of protective gene Heme oxygenase-1(HO-1), which plays immune regulation and protects cell from injury. So induction of HO-1 expression would become a new type of treatment in the immune regulation. The results of Western blot and RT-PCR displayed that WA induced the expression of HO-1 in the LPS-induced mice T lymphocytes through ERK/MAPK pathway in a dose- and time-dependent manner. Furthermore, WA inhibited LPS-induced nuclear factorκB (NF-κB p65) translocation directly or indirectly mediated by HO-1, which probably involved in increasing PPAR-y while decreasing COX-2 expression.In this study, we established a model for screening COX-2 specific inhibitor on account of dual-luciferase report gene system. Dual-luciferase report gene is an effective method in promoter activity assay, and this method is rapid, sensitive, economic and without pollution of isotope. We constructed recombination luciferase report gene vector dependent COX-1 or COX-2 gene promoter, which the recombination vector and control vector were co-transfected into cells and analyzed activity luciferase. The activity luciferase was index of activity of COX-1 and COX-2 gene promoter. In addition, we used COX-2 specific inhibitor Celecoxib to confirm this model for its regulation in COX-2 gene expression, and the results showed that this model was precise and sensitive. Therefore, this model could be used to screen COX-2 specific inhibitor, and also used to probe mechanism of targeted inhibiting COX-2 expression.Based on dual-luciferase reporter gene analysis, our studies further explore the molecular mechanism of targeted inhibiting COX-2. The results of Western blot and RT-PCR displayed that WA significantly inhibited COX-2 expression and PGE2 production through ERK/MAPK, p38/MAPK pathways and NF-κB nuclear translocation, while the expression level of COX-1 was not influenced markedly. The results showed that MAPK participated in the translocation of NF-κB, which possiblely regulated some other transcription factors.In order to further verify the immunosuppressive activity of WA, the present studies preliminary investigate the therapeutic effect of WA on the disease systemic lupus erythematosus with the MRL/lpr lupus mice model. The results showed that WA effectively improved immune status and reduced the splenomegaly and lymphadenopathy possibly through BAFF/BAFF-R pathway. Moreover, WA significantly improved kidneys and lung function of MRL/lpr mice, reduced proteinuria symptoms and anti-dsDNA level of serum, as well as to improve their lives function, which might provide new clues for the immunosuppressive activity of WA in vivo.In this paper, we investigated the immunosuppressive activity of WA based on a series of cellular, molecular level and mice experiments, The results showed that WA suppressed the over-proliferation of mice T lymphocytes through blocking cell mitosis and suppressing CD4+/CD8+ and CD25+ cell subsets, as well as regulated Th1/Th2 balance. WA might play immune suppression activity by increasing protective genes HO-1 expression and targeted inhibiting the expression of COX-2. At the same time, the results of inflammation mice and MRL/lpr lupus mice model showed a very good therapeutic effect of WA. The above results in vitro and in vivo further provide scientific evidences for the potential of new immunosuppressive activity of WA and Withanolide compound.更多还原