伸筋草总生物碱对类风湿关节炎大鼠脾脏淋巴细胞增殖及IL-17A、TGF-β水平的影响

Effect of the total alkaloids of lycopodium clavatum on the proliferation of splenic lymphocytes and the levels of IL-17A and TGF-βin rats with rheumatoid arthritis

目的探究伸筋草总生物碱对类风湿关节炎大鼠脾脏淋巴细胞增殖及白细胞介素-17A(IL-17A)、转化生长因子-β(TGF-β)水平的影响。方法取60只雄性SD大鼠,随机选取10只作为正常对照组,余大鼠采用皮下注射弗氏佐剂的方法复制类风湿关节炎模型。将造模成功大鼠随机分为模型组、伸筋草低剂量组、伸筋草高剂量组和甲氨蝶呤组,每组10只。伸筋草低、高剂量组大鼠每日分别灌胃4 mg/kg和8 mg/kg的伸筋草总生物碱,甲氨蝶呤组每日灌胃0.5 mg/kg的甲氨蝶呤溶液,正常对照组和模型组每日灌胃等体积的生理盐水。连续灌胃21 d后,取各组大鼠脾脏,HE染色后光镜下观察脾脏组织病理形态;采用细胞增殖法(体外分离模型组大鼠脾脏中淋巴细胞,设空白组、伴刀豆球蛋白组、伸筋草25μmol/L组和伸筋草12.5μmol/L组)测定大鼠脾脏淋巴细胞增殖情况,采用酶联免疫吸附法测定脾细胞上清液中IL-17A和TGF-β水平。结果与模型组比较,伸筋草高、低剂量组和甲氨蝶呤组大鼠脾脏组织结构轮廓清晰,红髓充血、白髓增生减少,炎性浸润减少。细胞增殖实验显示,伸筋草25μmol/L组和伸筋草12.5μmol/L组脾脏淋巴细胞增殖OD值均明显低于伴刀豆球蛋白组(P均<0.05)。与模型组比较,伸筋草高剂量组和甲氨蝶呤组IL-17A水平均明显降低(P均<0.05),伸筋草高、低剂量组和甲氨蝶呤组TGF-β水平均明显升高(P均<0.05)。结论伸筋草总生物碱可以明显抑制类风湿关节炎大鼠脾脏组织淋巴细胞增殖和炎性细胞浸润,其作用与部分恢复辅助性T细胞17(Th17)/调节性T细胞(Treg)的动态平衡密切相关。

Objective It is to explore the effects of the total alkaloids of lycopodium clavatum on the proliferation of splenic lymphocytes and the levels of interleukin-17A(IL-17A)and transforming growth factor-β(TGF-β)in rats with rheumatoid arthritis.Methods Sixty male SD rats were selected,in which 10 rats were randomly selected as normal control group,and the remaining rats were subcutaneously injected with Freund’s adjuvant to replicate the rheumatoid arthritis models.The successfully modeled rats were randomly divided into the model group,the low-dose lycopodium clavatum group,the high-dose lycopodium clavatum group and the methotrexate group,with 10 rats in each group.The rats in the low and high-dose groups of lycopodium clavatum were respectively given 4 mg/kg and 8 mg/kg of total alkaloids of lycopodium clavatum daily,and the methotrexate group was given 0.5 mg/kg of methotrexate solution daily,the normal control group and the model group were given an equal volume of normal saline daily.After continuous gavage for 21 days,the spleens of the rats in each group were taken,and the pathological morphology of the spleen tissue was observed under light microscope after HE staining;cell proliferation method(blank group,concanavalin group,25μmol/L lycopodium clavatum group and 12.5μmol/L lycopodium clavatum group in were divided after isolation of lymphocytes in vitro in the spleen of rats in the model group)were used to determine the spleen lymph proliferation,and the levels of IL-17A and TGF-βin the supernatant of spleen cells were determined by enzyme-linked immunosorbent assay.Results Compared with the model group,the spleen tissue structure of the rats in the high and low dose groups of lycopodium clavatum and methotrexate groups was clear,with red marrow congestion,reduction of white marrow hyperplasia and inflammatory infiltration.The cell proliferation experiment showed that the OD values of splenic lymphocyte proliferation in the 25μmol/L lycopodium clavatum group and the 12.5μmol/L lycopodium clavatum group were significantly lower than those of the concanavalin group(both P<0.05).Compared with the model group,the level of IL-17A in the high-dose lycopodium clavatum group and methotrexate group was significantly reduced(P<0.05),and the level of TGF-βin the high-dose and low-dose groups and methotrexate groups were significantly increased(all P<0.05).Conclusion The total alkaloids of lycopodium clavatum can obviously inhibit the proliferation of lymphocytes and the infiltration of inflammatory cells in the spleen tissue of rheumatoid arthritis rats,and its role is closely related to the partial restoration of helper T cell 17(Th17)/regulatory T cell(Treg)dynamic balance.