“863”課題子課題負責人陳子珺免疫學分析

博士學位論文

王棗子免疫活性成分的篩選及

免疫抑制成分IG-1的機理研究

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

博 士 生： 陳子珺

導    師： 李儀奎

專    業： 中藥學

上海中醫藥大學

二00四年五月

摘    要

目的：選擇作用可靠、化學結構明確的中藥提取物，在分離、純化及確定其化學結構的基礎上，開展活性及作用機制的研究，不僅能在分子水平上揭示這些天然產物的藥理作用、闡明活性與功能的關系有重要意義，而且對于創制具有我國特色、并能符合國際規范的新藥也有重要的指導意義。王棗子系唇形科香茶菜屬植物香茶菜Rabdosia amethystoieds (Benth.) Hara的全草，為安徽省宿州地區民間習用草藥。民間主要用于治療系統性紅斑狼瘡，類風濕性關節炎，肺膿瘍及老年性慢性支氣管炎等。王棗子在安徽省宿州的藥材種植基地，已有近十年的栽培經驗，對資源持續性開發提供了保證。為促進王棗子的進一步的研究開發，我們應用藥理與化學密切合作的研究方法，以活性評價為導向，從水煎劑出發，對王棗子影響免疫功能作用的活性物質進行追蹤分離，獲得了具有免疫抑制活性的化學單體IG-1，并對其進行了作用機理研究，以期為王棗子的應用開發奠定基礎。

方法：

(1) 采用2,4-二硝基氟苯所致的小鼠遲發型超敏反應模型和綿羊紅細胞所致小鼠溶血素抗體生成模型，觀察王棗子水煎劑對整體動物細胞免疫和體液免疫的影響。

(2) 用有絲分裂原伴刀豆蛋白A(ConA)活化小鼠脾臟T淋巴細胞，采用噻唑鹽(MTT)實驗方法，體外檢測王棗子提取物對淋巴細胞增殖反應的影響，篩選具有免疫活性的有效成分IG-1。

(3) 采用MTT法觀察IG-1的細胞毒性及對ConA誘導的小鼠脾臟T淋巴細胞增殖、脂多糖(LPS)誘導的小鼠脾臟B淋巴細胞增殖和混合淋巴細胞培養反應的影響。

(4) 采用Cytometric Bead Array (CBA)法檢測IG-1對小鼠脾臟淋巴細胞Th1/Th2型5種細胞因子(IL-2、IFN-γ、TNF-α、IL-4、IL-5)產生的影響。

(5) 采用吖啶橙/溴化乙錠(AO/EB)熒光染色法及流式細胞分析術觀察IG-1對小鼠脾臟淋巴細胞凋亡的作用。

(6) 采用流式細胞分析術觀察IG-1對小鼠脾臟淋巴細胞增殖周期的影響。

(7) 采用Fura-2/AM雙波長熒光法，觀察IG-1對小鼠脾臟淋巴細胞胞漿內游離鈣離子濃度的影響。

結果：

(1) 王棗子水煎劑(10g.kg^-1，5g.kg^-1，2.5g.kg^-1)對2，4-二硝基氟苯（2,4-DNFB）所致小鼠遲發型超敏反應有顯著的抑制作用，對胸腺、脾臟指數無明顯影響；王棗子水煎劑(10g.kg^-1，5g.kg^-1，2.5g.kg^-1)對綿羊紅細胞所致小鼠特異性抗體生成(小鼠血清溶血素水平)有顯著的增強作用。

(2) 對ConA誘導小鼠脾臟T淋巴細胞增殖，王棗子不同的提取部位FA、FB、FC和FD作用不同，而同一部位在不同濃度下作用也不一樣。其中FA、FC呈增強作用,FB和FD則在不同濃度下有增強與抑制的不同作用，提示王棗子中含有不同性質的免疫活性成分。推測FB中可能含有較強免疫抑制活性的成分，進而對FB進行分離提取，得到4個部分FB-1、FB-2、FB-3和FB-4。同法進行體外篩選，結果表明：FB-1和FB-2以增強作用為主；而FB-3和FB-4則以抑制作用為主。推測FB-4可能是免疫抑制活性成分集中部位，進而對FB-4進行了深入的化學研究，最終從FB-4中獲得了單體化合物IG-1。

(3) IG-1主要具有免疫抑制作用，在體外它能顯著抑制ConA、LPS誘導的小鼠T、B淋巴細胞增殖及混合淋巴細胞培養反應；細胞毒性實驗結果表明：IG-1與小鼠脾細胞共同培養24h、48h、72h，未表現與藥物相關的細胞毒性作用。提示IG-1的免疫抑制作用與細胞毒性無關。

(4) IG-1對ConA誘導的Th1細胞來源的細胞因子(IL-2、IFN-γ、TNF-α) 和Th2細胞來源的細胞因子(IL-4、IL-5)的產生均有顯著的抑制作用，且IG-1對Th1細胞來源的細胞因子的抑制作用要強于Th2細胞來源的；對未經ConA活化的小鼠脾細胞， IG-1能抑制IL-2、IFN-γ和TNF-α的產生，對于細胞因子IL-4、IL-5，IG-1則有促進作用。

(5) IG-1能誘導小鼠靜止的脾細胞凋亡；對活化的小鼠脾臟淋巴細胞，IG-1作用不明顯。

(6) 對于靜止的小鼠脾細胞，IG-1可以抑制細胞從S期進入G₂/M期。

(7) IG-1可降低ConA活化所致的小鼠脾臟淋巴細胞胞漿內［Ca²⁺］i升高。

結論：

(1) 王棗子水煎劑的整體實驗結果初步顯示：它細胞介導的免疫反應有抑制作用，對體液介導的免疫反應有增強作用。王棗子具有顯著的免疫活性。

　　(2) 應用藥理與化學密切合作的研究方法，以ConA誘導的小鼠脾臟T淋巴細胞增殖為活性指標，從水煎劑出發，對王棗子影響免疫功能作用的活性物質進行追蹤分離，最終獲得了化學單體IG-1。

　　(3) IG-1在體外能顯著抑制ConA、LPS誘導的小鼠T、B淋巴細胞增殖和混合淋巴細胞培養反應，它對抗原激活的淋巴細胞增殖的抑制作用與細胞毒作用無關。IG-1具有免疫抑制活性。

　　(4) IG-1免疫抑制作用的機理主要是：IG-1對抗原激活的淋巴細胞增殖具有的抑制作用；IG-1對Th1/Th2細胞因子產生的抑制以及在抑制狀態下對Th1/Th2細胞因子相對值的影響； IG-1抑制細胞胞漿內游離鈣的釋放進而可能影響細胞信號的轉導。

　　(5) IG-1能誘導小鼠靜止的脾細胞凋亡。對于靜止的小鼠脾細胞，IG-1還能對細胞周期產生影響。這些實驗結果提示IG-1可能還具有其它作用，尚待進一步研究。

關鍵詞：王棗子；免疫抑制；細胞因子；細胞凋亡；細胞周期；鈣離子

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