小鼠腘窝淋巴结模型评价3种中药注射液的潜在免疫毒性

Potential immunotoxicity of three traditional Chinese medicines using the popliteal lymph node assay in mice

目的:利用小鼠直接腘窝淋巴结(D-PLNA)和报告抗原腘窝淋巴结(RA-PLNA)对脉络宁、生脉、丹香冠心3种中药注射液的免疫毒性进行评价。方法:D-PLNA模型中设脉络宁、生脉、丹香冠心3种中药注射液,生理盐水对照组和阳性药物苯妥英钠对照组;RA-PLNA模型中上述各组分别加入报告抗原三硝基苯基聚蔗糖(TNP-Ficoll)和三硝基苯基卵蛋白(TNP-OVA),每组8只BALB/c小鼠,于左足趾部给药50μL。第7天处死小鼠分离腘窝淋巴结,研磨制备淋巴单细胞悬液并进行细胞计数,计算细胞指数;将细胞悬液培养于96孔板中,加入刺激剂共孵育后,利用酶联免疫吸附试验(ELISA)测定细胞上清液中免疫球蛋白Ig E和单核细胞趋化蛋白-1(MCP-1)的含量。结果:与生理盐水对照组比较,小鼠D-PLNA模型中,仅丹香冠心注射液组腘窝淋巴结细胞数显著增加(P<0.05);小鼠RA-PLNA模型中,各给药组与TNP-Ficoll作用时,细胞数目和MCP-1浓度均显著升高(P<0.05),各给药组与TNP-OVA作用时,脉络宁注射液能显著刺激IgE分泌,同时丹香冠心注射液IgE和MCP-1含量均显著升高(P均<0.05)。结论:脉络宁和生脉注射液仅在加入报告抗原后引起小鼠淋巴结阳性反应,说明两者中可能存在作为半抗原的小分子物质诱发Ⅰ型超敏反应,而丹香冠心注射液成分复杂,可能直接诱导Ⅰ型超敏反应,也可能存在新的抗原或表位诱发免疫反应。

OBJECTIVE:To evaluate the potential immunotoxicity of three traditional Chinese medicines:Mailuoning,Shengmai and Danxiangguanxin,in mice using the direct popliteal lymph node assay(D-PLNA)and reporter antigen popliteal lymph node assay(RA-PLNA).METHODS:In D-PLNA,mice were randomly divided into five groups:Mailuoning,Shengmai and Danxiangguanxin,normal saline control and diphenylhydantoin(DPH)positive control.In RA-PLNA,the above groups were combined administration with reporter antigens TNP-Ficoll or TNP-OVA.Mice were randomly assigned to their respective treatment groups,8 in each group and received a subcutaneous injection into left hind footpad with or without RAs in a final volume of 50μL.At 7 days after injection,mice were sacrificed and popliteal lymph nodes were excised and then grinded to prepare single cell suspensions.Cell number was counted and cell index was calculated.Concentration of IgE and MCP-1 cell supernatant were measured by ELISA assay after cell suspension was cultured with stimulant in a 96-well plate.RESULTS:In D-PLNA,compared with the control group,Danxiangguanxin injection increased PLN cellularity significantly(P<0.05).In RA-PLNA,each treatment group significantly increased PLN cellularity and the concentration of MCP-1 when co-injected with TNP-Ficoll(P<0.05).When co-injected with TNP-OVA,Mailuoning stimulated significant secretion of IgE while Danxiangguanxin increased the concentration of IgE and MCP-1(P<0.05).CONCLUSION:Mailuoning and Shengmai injection only caused positive reaction after RAs were added.This suggests they may contain low molecular weight compounds as hapten to induce type I hypersensitivity reaction.However,the composition of Danxiangguanxin is complex and may directly induce type I hypersensitivity reaction,and may also act as a neo-antigen or neo-epitope to elicit immune response.