葛根素对淋巴细胞P-糖蛋白表达的影响及降低激素耐药的作用

目的：探讨葛根素对淋巴细胞P-糖蛋白（P-glycoprotein,P-gp）表达的影响及降低糖皮质激素耐药的作用,为提高糖皮质激素耐药患者的临床疗效提供新的思路和方法。方法：取健康志愿者静脉血10ml,密度梯度离心法分离淋巴细胞,贴壁培养去除单核细胞。予小剂量甲基泼尼松龙（2μg/ml）诱导细胞耐药,用非细胞毒浓度（80μg/ml）的葛根素干预,MTT试验检测激素耐药倍数和葛根素逆转倍数,流式细胞术检测淋巴细胞P-gp和200μg/ml甲基泼尼松龙刺激24h后AnnexinⅤ。结果：小剂量甲基泼尼松龙刺激72h可诱导淋巴细胞P-gp显著升高,甲基泼尼松龙耐药倍数为2.45±0.42。葛根素干预可显著降低耐药细胞P-gp,使甲基泼尼松龙对细胞的半数抑制率IC50显著降低,对激素耐药的逆转倍数为1.55±0.30;葛根素干预可使甲基泼尼松龙诱导细胞的AnnexinⅤ显著增高。葛根素独立干预无显著诱导细胞凋亡作用。结论：葛根素干预可显著降低淋巴细胞糖皮质激素耐药性,显著增强激素诱导淋巴细胞凋亡的作用。葛根素显著下调P-gp表达的作用可能是其降低激素耐药的重要机制之一。

Effects of Puerarin on Expression of P-glycoprotein and Glucocorticoid Resistance in Lymphocytes

Objective：To investigate the effects of Puerarin on the expression of P-glycoprotein and lessening of glucocorticoid resistance in lymphocytes.Methods：Ten ml Venous blood from each healthy volunteer was obtained.The lymphocytes were collected and purified using density gradient centrifugation,and the Monocytes were removed by in vitro adherence method.Glucocorticoid resistance were induced by the low dose of Methylprednisolone（2 μg/ml） for 72 hours in lymphocytes which were treated with Puerarin at the same time.The glucocorticoid resistance multiple and the multiple of reversing glucocorticoid resistant of Puerarin in lymphocytes were detected by MTT method.The level of AnnexinⅤand the expression of P-gp in the lymphocytes were detected by flow cytometry after being stimulated with 200 μg/ml of Methylprednisolone for 24 hours.Results：The expression of P-gp in peripheral blood lymphocytes induced by the low dose Methylprednisolone was up-regulated significantly compared with that in the control cells.The glucocorticoid resistance multiple was 2.45±0.42 in the lymphocytes with P-gp upregulated.In lymphocytes interfered with Puerarin,the expression of P-gp was down-regulated and the multiple of reversing glucocorticoid resistant was 1.55±0.30.In lymphocytes stimulated with Methylprednisolone of 200 μg/ml for 24 h,the level of AnnexinⅤof glucocorticoid resistance decreased significantly compared with that in the control group.But the level of AnnexinⅤ increased significantly in the lymphocytes stimulated with Puerarin at the same time.Conclusion：Puerarin can reduce glucocorticoid resistance,and increase the effects of Methylprednisolone induced lymphocytes apoptosis.The role of down-regulating the expression P-gp could be one of important explanations of Puerarin to reduce glucocorticoid resistant.