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“863”课题子课题负责人陈子珺免疫学分析

文字:[大][中][小] 手机页面二维码 2017/6/12     浏览次数:    

博士学位论文

 

 

王枣子免疫活性成分的筛选及

免疫抑制成分IG-1的机理研究

 

Study on screening immunocompetent component in Rabdosia amethystoieds (Benth.) Hara and the immunosuppressive action of IG-1

 

 

 

 

生: 陈子珺

    师: 李仪奎

    业: 中药学

 

 

 

上海中医药大学

 

00四年五月


 

   

目的:选择作用可靠、化学结构明确的中药提取物,在分离、纯化及确定其化学结构的基础上,开展活性及作用机制的研究,不仅能在分子水平上揭示这些天然产物的药理作用、阐明活性与功能的关系有重要意义,而且对于创制具有我国特色、并能符合国际规范的新药也有重要的指导意义。王枣子系唇形科香茶菜属植物香茶菜Rabdosia amethystoieds (Benth.) Hara的全草,为安徽省宿州地区民间习用草药。民间主要用于治疗系统性红斑狼疮,类风湿性关节炎,肺脓疡及老年性慢性支气管炎等。王枣子在安徽省宿州的药材种植基地,已有近十年的栽培经验,对资源持续性开发提供了保证为促进王枣子的进一步的研究开发,我们应用药理与化学密切合作的研究方法,以活性评价为导向,从水煎剂出发,对王枣子影响免疫功能作用的活性物质进行追踪分离,获得了具有免疫抑制活性的化学单体IG-1,并对其进行了作用机理研究,以期为王枣子的应用开发奠定基础。

方法:

(1) 采用2,4-二硝基氟苯所致的小鼠迟发型超敏反应模型和绵羊红细胞所致小鼠溶血素抗体生成模型,观察王枣子水煎剂对整体动物细胞免疫和体液免疫的影响。

(2) 用有丝分裂原伴刀豆蛋白A(ConA)活化小鼠脾脏T淋巴细胞,采用噻唑盐(MTT)实验方法,体外检测王枣子提取物对淋巴细胞增殖反应的影响,筛选具有免疫活性的有效成分IG-1

(3) 采用MTT法观察IG-1的细胞毒性及对ConA诱导的小鼠脾脏T淋巴细胞增殖、脂多糖(LPS)诱导的小鼠脾脏B淋巴细胞增殖和混合淋巴细胞培养反应的影响。

(4) 采用Cytometric Bead Array (CBA)法检测IG-1对小鼠脾脏淋巴细胞Th1/Th25种细胞因子(IL-2IFN-γTNF-α、IL-4IL-5)产生的影响。

(5) 采用吖啶橙/溴化乙锭(AO/EB)荧光染色法及流式细胞分析术观察IG-1对小鼠脾脏淋巴细胞凋亡的作用。

(6) 采用流式细胞分析术观察IG-1对小鼠脾脏淋巴细胞增殖周期的影响。

(7) 采用Fura-2/AM双波长荧光法,观察IG-1对小鼠脾脏淋巴细胞胞浆内游离钙离子浓度的影响。

结果:

(1) 王枣子水煎剂(10g.kg-15g.kg-12。5g.kg-1)24-二硝基氟苯(2,4-DNFB)所致小鼠迟发型超敏反应有显著的抑制作用,对胸腺、脾脏指数无明显影响;王枣子水煎剂(10g.kg-15g.kg-12.5g.kg-1)对绵羊红细胞所致小鼠特异性抗体生成(小鼠血清溶血素水平)有显著的增强作用。

(2) ConA诱导小鼠脾脏T淋巴细胞增殖,王枣子不同的提取部位FAFBFCFD作用不同,而同一部位在不同浓度下作用也不一样。其中FAFC呈增强作用,FBFD则在不同浓度下有增强与抑制的不同作用,提示王枣子中含有不同性质的免疫活性成分。推测FB中可能含有较强免疫抑制活性的成分,进而对FB进行分离提取,得到4个部分FB-1FB-2FB-3FB-4。同法进行体外筛选,结果表明:FB-1FB-2以增强作用为主;而FB-3FB-4则以抑制作用为主。推测FB-4可能是免疫抑制活性成分集中部位,进而对FB-4进行了深入的化学研究,最终从FB-4中获得了单体化合物IG-1

(3) IG-1主要具有免疫抑制作用,在体外它能显著抑制ConALPS诱导的小鼠TB淋巴细胞增殖及混合淋巴细胞培养反应;细胞毒性实验结果表明:IG-1与小鼠脾细胞共同培养24h48h72h未表现与药物相关的细胞毒性作用。提示IG-1的免疫抑制作用与细胞毒性无关。

(4) IG-1ConA诱导的Th1细胞来源的细胞因子(IL-2IFN-γTNF-α) Th2细胞来源的细胞因子(IL-4IL-5)的产生均有显著的抑制作用,且IG-1Th1细胞来源的细胞因子的抑制作用要强于Th2细胞来源的;对未经ConA活化的小鼠脾细胞, IG-1能抑制IL-2IFN-γTNF-α的产生,对于细胞因子IL-4IL-5IG-1则有促进作用。

(5) IG-1能诱导小鼠静止的脾细胞凋亡;对活化的小鼠脾脏淋巴细胞,IG-1作用不明显。

(6) 对于静止的小鼠脾细胞,IG-1可以抑制细胞从S期进入G2/M

(7) IG-1可降低ConA活化所致的小鼠脾脏淋巴细胞胞浆内[Ca2+i升高。

结论:

(1) 王枣子水煎剂的整体实验结果初步显示:它细胞介导的免疫反应有抑制作用,对体液介导的免疫反应有增强作用。王枣子具有显著的免疫活性。

  (2) 应用药理与化学密切合作的研究方法,以ConA诱导的小鼠脾脏T淋巴细胞增殖为活性指标,从水煎剂出发,对王枣子影响免疫功能作用的活性物质进行追踪分离,最终获得了化学单体IG-1

  (3) IG-1在体外能显著抑制ConALPS诱导的小鼠TB淋巴细胞增殖和混合淋巴细胞培养反应,它对抗原激活的淋巴细胞增殖的抑制作用与细胞毒作用无关。IG-1具有免疫抑制活性。

  (4) IG-1免疫抑制作用的机理主要是:IG-1对抗原激活的淋巴细胞增殖具有的抑制作用;IG-1Th1/Th2细胞因子产生的抑制以及在抑制状态下对Th1/Th2细胞因子相对值的影响; IG-1抑制细胞胞浆内游离钙的释放进而可能影响细胞信号的转导。

  (5) IG-1能诱导小鼠静止的脾细胞凋亡。对于静止的小鼠脾细胞,IG-1还能对细胞周期产生影响。这些实验结果提示IG-1可能还具有其它作用,尚待进一步研究。

 

关键词:王枣子;免疫抑制;细胞因子;细胞凋亡;细胞周期;钙离子


Abstract

  Objective: In order to promote research and development of Rabdosia amethystoieds (Benth。) Hara (RAB), the method of close collaboration between chemical and pharmacological studies was employed in discovering immunocompetent component from Rabdosia amethystoieds (Benth。) Hara under guidance of activity evaluation and IG-1 was isolated in the end. The immunosuppressive mechanism of action of IG-1 was studied further.

  Methods:

  (1) We used two methods to observe the action of RAB decoction on cell-mediated Immunity and humoral Immunity in vivo. One is the mouse delayed-type hypersensitivity (DTH) model induced by 2,4-DNFB and delayed hypersensitivity reflected by ear skin response. The other is the production of specific haemolysin antibody against sheep red blood cell (SRBC) in serum.

  (2) Spleen cells of mice were stimulated by mitogen concannavalin A (ConA), and proliferation of lymphocytes were measured with tetrazolium salts (MTT) in vitro, which was employed as the activity evaluating parameter to guide stepwise fraction evaluation. Immunocompetent component IG-1 was separated under guidance of activity evaluation in the end.

  (3) MTT colorimetric assay was applied to study the cytotoxicity of IG-1 and the effect of IG-1 on mixed lymphocyte reaction (MLR) and the proliferation of lymphocytes stimulated by ConA and lipopolysaccharide (LPS)。

  (4) The effects of IG-1 on murine lymphocytes cytokine production was assessed by a Cytometric Bead Array (CBA) method.

  (5)Cellular morphaological observation, DNA agarose gel electrophoresis and flow cytometry analysis were used to study the effects of IG-1 on apoptosis of murine splenocyte lymphocytes。

  (6) The influence of IG-1 on cell proliferation cycle was observed with flow cytometry。

  (7) Intracellular free calcium concentration was tested by spectrofluorophotometry using fura-2 as the calcium probe。

  Results:

  (1) RAB decoction could markedly inhibit delayed hypersensi- tivity induced by 2,4-DNFB in mice, but had no significant effects on the index of thymus and spleen. It also could increase the level of hemolysin induced by sheeperythrocyte immunization in the serum.

  (2) FA, FB, FCand FD are the extractions from RAB. To murine lymphocyte proliferation induced by ConA in vitro, their effects are different. FA and FC could promote proliferation but FB and FD had stimulative and inhibitory effect respectively at different concentration. FB showed stronger immuno- competent activity. We carry out further research around FB and got four fraction FB-1, FB-2, FB-3 and FB-4 from it. To the proliferation of murine lymphocytes, FB-1 and FB-2 had promotive effect but EB-3 and FB-4 had suppressive effect mainly. In the end we isolated IG-1 from FB-4.

  (3) IG-1 had showed primarily immunosuppressive activity。 It could inhibited murine T and B lymphocyte proliferation induced by ConA and LPS and lympho- cyte proliferation stimulated in MLR。 It’s immunosuppressive effect is not due to cytotoxicity.

  (4) IG-1could inhibited the production of Th1 and Th2 type cytokine (IL-2, IFN-γ,TNF-α,IL-4 and IL-5) induced by ConA. Moreover, IG-1 was found to inhibit the production of IL-2, IFN-γand TNF-αwhile enhancing the production of IL-4, IL-5 to the rest spleen cells.

  (5) IG-1 could induce apoptosis of resting spleen lymphocytes but had no effect on activated lymphocytes。    (6) IG-1 could affect cell cycle obviously, mainly causing the percentage of G2/M phase cells decreased and lympho- cyte blocked at S cycle stage。

  (7) The concentration of free calcium increased on murine spleenocyte activated by ConA. IG-1 could reduce spleenocyte intracellular calcium delivering.

   Conclusion:

  (1) RAB decoction could inhibit the function of cell- mediated Immunity (CMI) and promote the function of humoral Immunity in vivo.

  (2) The method of close collaboration between chemical and pharma- cological studies was employed in discovering the immunosuppressors from RAB under guidance of activity evaluation and IG-1 was separated in the end.    

  (3) IG-1 could inhibit murine T and B lymphocyte proliferation induced by ConA and LPS and lymphocyte proliferation stimulated in MLR. It’s immunosuppressive effect is not due to cytoxicity. IG-1 mainly showed immunosuppressive activity.

  (4) IG-1 could inhibit lymphocyte proliferation induced by antigen, inhibit the production of Th1 and Th2 type cytokine (IL-2, IFN-γ,TNF-α,IL-4 and IL-5) induced by ConA and affect on the relative value of Th1 and Th2 type cytokine, reduce spleenocyte intracellular calcium delivering, which may be the important mechanism of immunosuppressive action of IG-1.

  (5) IG-1 could induce apoptosis of resting spleen lymphocytes but had no effect on activated lymphocytes. IG-1 could affect cell cycle obviously, mainly causing the percentage of G2/M phase cells decreased and lymphocyte blocked at S cycle stage. These were related to other effect of IG-1.

  Key words: Rabdosia amethystoieds (Benth.) Hara;  immunosuppression;  cytokine;  apoptosis;  cell cycle;  intracellular calcium

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