羟基红花黄色素A延缓高磷诱导的血管平滑肌细胞钙化及其机制研究

Mechanism of hydroxysafflower yellow A in the regulation of vascular smooth muscle cell calcification

目的 探讨羟基红花黄色素A(Hydroxysafflor yellow A,HSYA)在β-甘油磷酸钠(β-GP)诱导的血管平滑肌细胞(VSMC)钙化过程中的作用及其机制.方法 VSMC用10%胎牛血清+1%双抗高糖DMEM培养液,于37℃、5%CO2培养箱中培养,根据细胞生长密度按1:4比例传代.实验分为3组:对照组(NC)、高磷诱导钙化组(HP)、HSYA干预组(HSYA).用茜素红染色和钙测定试剂盒(邻甲酚酞络合酮比色法)检测各组细胞钙沉积量;Western印迹法检测细胞钙化指标碱性磷酸酶(ALP),Runt相关转录因子2(RUNX2),核因子κB受体活化因子配体(RANKL),α平滑肌肌动蛋白(α-SMA),及TLR4/NF-κB通路和炎性反应相关指标Toll样受体4(TLR4),白细胞介素8(IL-8),肿瘤坏死因子α(TNF-α)的变化.抽提细胞核蛋白和细胞质蛋白,Western印迹法检测细胞核NF-κB p65和细胞质p65,及细胞总蛋白中p65和磷酸化p65的改变.超氧化物歧化酶(SOD)、丙二醛(MDA)试剂盒法检测细胞抗氧化酶和氧化终产物的改变.结果 Western印迹结果显示,HSYA组细胞钙化指标ALP、RUNX2、RANKL的表达量较HP组明显减少;α-SMA表达较HP组增加(均P<0.01).HSYA组TLR4、TNF-α、IL-8和p-p65/p65的表达量较HP组减少,p65入核减少,细胞质内p65的表达增多(均P<0.05).HSYA组细胞SOD含量较HP组明显升高,MDA含量较HP组明显降低(均P<0.01).结论羟基红花黄色素A可减轻高磷诱导的血管平滑肌细胞的钙化,减少钙沉积.其机制与羟基红花黄色素A抑制了TLR4/NF-κB通路的激活和氧化应激反应有关.

Objective To investigate the role and mechanism of Hydroxysafflor yellow A(HSYA)in the calcification of vascular smooth muscle cells(VSMC)induced byβ-glycerol phosphate(β-GP).Methods VSMC were cultured with 10%fetal bovine serum+1%double anti-high glucose DMEM medium at 37℃and 5%CO2 incubator,and were subcultured according to cell growth density at 1:4 ratio.The cells were divided into three groups:control group(NC),high-phosphate-induced calcification(HP)group,and HSYA intervention(HSYA)group.The Calcium deposition amount was measured by alizarin red staining and calcium determination kit.The expressions of ALP,RUNX2,RANKL,α-SMA and inflammation indicators TLR4,TNF-α,IL-8 were detected by Western blotting method;Western blotting was also used to detect calcification index alkaline phosphatase(ALP)and Runt-related transcription factor 2(RUNX2).Nuclear factor kappa B receptor activating factor ligand(RANKL),α-smooth muscle actin(α-SMA),and the expressions of TLR4/NF-κB pathway and inflammatory response-related indicators Toll-like receptor 4(TLR4),interleukin-8(IL-8)and tumor necrosis factor alpha(TNF-α).The nuclear protein and cytoplasmic proteins were respectively extracted.The expressions of p65 in nucleus and cytoplasm,as well as the expressions of p65 and phosphorylated p65 in total proteins were detected by Western blotting method.Superoxide dismutase(SOD)and malondialdehyde(MDA)kit were used to detect the content of antioxidant enzymes and oxidation end products in cells.Results Western blotting showed that the expressions of ALP,RUNX2 and RANKL in HSYA group were significantly lower than that in HP group.The expression ofα-SMA was increased than that of HP group(all P<0.01).The expression levels of TLR4,TNF-α,IL-8 and p-NF-κB/p65 in HSYA group were decreased compared with that in the HP group,and p65 was decreased in nucleus and increased in cytoplasm(all P<0.05).SOD content in HSYA group was significantly higher than that in HP group,and MDA content was significantly lower than that in HP group(all P<0.01).Conclusions HSYA can reduce calcification and calcium deposition of vascular smooth muscle cells induced by high phosphorus.The mechanism is related to the inhibition of the activation of TLR4/NF-κB pathway and the inhibition of oxidative stress.