深绿卷柏总双黄酮对H9c2心肌细胞缺氧复氧的保护作用

Protective effect of total diflavonoids from Selaginella doederleinii on H9c2 cells with hypoxia/reoxygenation injury

目的探究深绿卷柏总双黄酮对缺氧复氧(HyPoxia/Reoxygenation,H/R)诱导的H9c2心肌细胞损伤的保护作用。方法采用95%乙醇回流提取深绿卷柏,通过D101大孔树脂富集得到深绿卷柏总双黄酮。采用MTT比色法筛选深绿卷柏总双黄酮最佳药物浓度、药物作用时间和细胞缺氧时间,再通过流式细胞术筛选最佳复氧时间。将H9c2心肌细胞分为对照组(Control组)、H/R模型组(Model组)和深绿卷柏总双黄酮25、50、100μg/mL浓度组,分别检测细胞中白细胞介素6(Interleukin-6,IL-6)、肿瘤坏死因子α(Tumor necrosis factor-α,TNF-α)、乳酸脱氢酶(Lumber disc herniation,LDH)、白细胞介素10(Interleukin-10,IL-10)以及肌酸激酶同工酶MB(Creatine kinase-MB,CK-MB)的水平变化。结果通过富集纯化得到纯度为56%的深绿卷柏总双黄酮7.85 g。通过MTT比色法和流式细胞术得到最佳条件为:深绿卷柏总双黄酮浓度为100μg/mL,药物作用时间12 h,细胞H/R时间为缺氧12 h复氧4 h(H12R4 h)。在最佳处理条件下,与Model组比较,100μg/mL浓度组细胞中的IL-6、TNF-α、LDH和CK-MB的含量显著降低(P<0.01),IL-10的含量显著升高(P<0.01)。结论深绿卷柏总双黄酮对H9c2心肌细胞缺氧复氧诱导的损伤具有良好的保护作用。

Objective To explore the protective effects of the total diflavonoids from Selaginella doederleinii on the damage of H9c2 cardiomyocytes induced by hypoxia/reoxygenation(H/R).Methods Reflux extraction with 95%ethanol was used to extract Selaginella doederleinii and then enriched by D101 macroporous resin to obtain total biflavonoids from Selaginella doederleinii.MTT colorimetry was used to select the best drug concentration,action time and cell hypoxia time.The optimal reoxygenation conditions were screened by FCM.Then,the H9c2 cardiomyocytes were randomly divided into control group,H/R model group,total biflavonoids from Selaginella doederleinii(25,50 and 100μg/ml)groups.The expressions of TNF-α,IL-6,IL-10,LDH and CK-MB were detected.Results Through enrichment and purification,total biflavonoids from Selaginella doederleinii(7.85 g)with a purity of 56%was obtained.The best conditions performed by MTT colorimetry and FCM were:total diflavonoids concentration was 100μg/ml,action time was 12 h,the cell H/R time was hypoxia 12 h,reoxygenation was 4 h(H12R4 h).Under the optimal conditions,compared with the Model group,the levels of IL-6,TNF-α,LDH and CK-MB in the cells of total biflavonoids from Selaginella doederleinii(100μg/ml)group were reduced,and the content of IL-10 was increased.Conclusion The total diflavonoids of Selaginella doederleinii have a good protective effect on H9c2 cardiomyocyte damage induced by H/R.