基于内质网应激的丹参酮ⅡA磺酸钠调节主动脉瓣膜间质细胞钙化机制研究

Sodium Tanshinone ⅡA Sulfonate Attenuates Aortic Valve Calcification Via Regulating ER Stress Related Inflammatory Pathways

目的:研究丹参酮IIA磺酸钠对低密度脂蛋白(ox LDL)诱导的猪主动脉瓣膜间质细胞(porcine valvular interstitial cells,VICs)钙化模型内质网应激(ERS)因子作用的影响。方法:分离VICs免疫荧光法鉴定细胞,应用ox LDL建立细胞模型,MTT法筛选最佳丹参酮IIA磺酸钠干预浓度,茜素红染色测量钙化结节,免疫印记法及实时荧光定量PCR法检测ERS通路中BIP、Chop、Runx2及Osteocalcin蛋白及基因表达,ELISA法检测细胞培养上清液中炎症因子表达,应用MCP-1中和抗体观察MCP-1在成骨化中的作用。结果:在成功建立ox LDL诱导的VIC细胞模型基础上,茜素红染色显示丹参酮IIA磺酸钠组钙结节形成较ox LDL模型组差异有统计学意义;丹参酮IIA磺酸钠可抑制BIP、Chop细胞内基因的表达,抑制Runx2及Osteocalcin蛋白表达,同时可抑制IL-6、IL-8及MCP-1炎症因子的表达,其表达与模型组比较差异有统计学意义,但MCP-1中和抗体对成骨化因素影响较小。结论:丹参酮IIA磺酸钠可抑制ox LDL诱导的VIC炎症反应及成骨化,其具体机制可能与抑制ERS通路有关。

Objective： To study the effects of sodium tanshinone IIA sulfonate on low density lipoprotein（ox LDL）induced porcine aortic valve interstitial cells（VICs） calcification,and its mechanisms related to endoplasmic reticulum stress（ERS）. Methods： Isolated porcine aortic valve interstitial cells were indentified by optical microscope and fluorescence microscope,ox LDL were used to establish the cell model of calcification,sodium tanshinone IIA sulfonate were used as intervention after ascertain the optimal concentration by MTT,alizarin red staining,western blot and real-time fluorescence quantitative PCR assay were performed for detection of BIP,Chop,Runx2 and Osteocalcin protein,ELISA method was used to detect the expression of inflammatory cytokines in the supernatant. MCP-1 anntibodies were used to test its role in osteogenesis. Results： we successfully established the VIC model of calcification. Alizarin red staining showed that tanshinone IIA sulfonate decreased the formation of calcium nodules compared with ox LDL group. Sodium tanshinone IIA can inhibit the expression of BIP and Chop genes within the cell,and can inhibit the expression of Runx2 and Osteocalcin protein. ELISA results showed that the inhibition of IL-6,IL-8 and inflammatory factor MCP-1 were also decreased by tanshinone IIA sulfonate. However,MCP-1 antibodies had littile effect on osteogenesis. Conclusion： Sodium tanshinone IIA sulfonate can inhibit the inflammatory response and osteogenic activity VIC,and its mechanism may related to the inhibition of the ERS.