左旋紫草素通过减轻坏死性凋亡抑制高糖心肌细胞炎症的作用研究

Effect of L-shikonin on Inhibition of High Glucose Induced Cardiomyocytes Inflammation by Alleviating Necroptosis

目的研究左旋紫草素抑制高糖心肌细胞炎症的机制。方法培养H9C2心肌细胞株,分为正常糖对照组(5.5 mmol/L葡萄糖)、高糖组(30 mmol/L葡萄糖)和左旋紫草素低、中、高浓度(25、50、100μmol/L)分别预处理30 min的高糖组。CCK8测定各组心肌细胞活力;ELISA测定各组肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)表达;2,7-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针测定活性氧簇ROS水平;蛋白质印迹技术检测坏死性凋亡相关蛋白Cleaved Caspase-3、Cleaved PARP表达;Hoechst 33258核染色测定心肌凋亡细胞数量。结果高糖处理H9C2心肌细胞24 h能明显降低细胞存活率、增加ROS生成,升高TNF-α、IL-1β和IL-6表达水平,上调Cleaved Caspase-3及Cleaved PARP蛋白表达和增加凋亡细胞数量。左旋紫草素可抑制高糖诱导的心肌细胞损伤,升高细胞存活率,降低ROS生成,降低TNF-α、IL-1β和IL-6炎症因子水平,减轻凋亡细胞数量及坏死性凋亡蛋白Cleaved Caspase-3、Cleaved PARP表达,呈剂量依赖性。结论左旋紫草素抑制高糖心肌细胞炎症可能是通过降低坏死性凋亡实现。

Objective To investigate the mechanism of L-shikonin in inhibiting high glucose induced cardiomyocytes inflammation.Methods H9C2 cardiomyocytes were cultured and divided into normal glucose control group(5.5 mmol/L glucose),high glucose group(30 mmol/L glucose)and high glucose plus L-shikonin groups,which were pretreated for 30 min with low,medium and high concentration of L-shikonin(25,50,100μmol/L)before the treatment of high glucose.Cardiomyocytes vitality was measured by cell counting kit-8(CCK8)method.And the expression of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)were detected by enzyme-linked immunosorbent assay(ELISA).Levels of reactive oxygen species(ROS)was detected by 2,7-Dichlorofluorescein diacetate(DCFH-DA)fluorescent probe.Expression of necroptosis related proteins Cleaved Caspase-3 and Cleaved PARP were detected by Western blot.The number of apoptotic cardiomyocytes was measured by Hoechst 33258 staining.Results Treating H9C2 cardiomyocytes with high glucose for 24 h could significantly reduce cell survival rate,increase ROS production,increase the expression levels of TNF-α,IL-1βand IL-6,up-regulate the protein expression of Cleaved Caspase-3 and Cleaved PARP,and increase the number of apoptotic cardiomyocytes.L-shikoninin can inhibit high glucose induced cardiomyocyte injury,increase cell survival rate,reduce ROS production,reduce the level of inflammatory factors TNF-α,IL-1βand IL-6,reduce the number of apoptotic cardiomyocytes,and reduce the expression of necroptosis related proteins Cleaved Caspase-3 and Cleaved PARP in a dose-dependent manner.Conclusion The effect of L-shikonin on inhibition of high glucose Induced cardiomyocytes inflammation may be achieved by reducing necroptosis.