AGE-LDL对人主动脉瓣间质细胞钙化和炎症反应的诱导作用及其机制

AGE-LDL induces calcification and inflammatory response of human aortic valve interstitial cells

目的观察糖基化终产物修饰的低密度脂蛋白(AGE-LDL)对人主动脉瓣膜间质细胞(HAVICs)钙化和炎症反应的诱导作用,并探讨其机制。方法非CAVD瓣膜组织(non-CAVD组)取自手术治疗的主动脉瓣脱垂患者,CAVD瓣膜组织(CAVD组)取自因钙化性主动脉瓣狭窄而进行主动脉瓣膜置换术的患者。采用免疫组化法检测non-CAVD组和CAVD组中的AGE。取non-CAVD组的主动脉瓣膜组织,分离HAVICs,分为对照组、LDL组、AGE-HSA组和AGE-LDL组。对照组给予M199培养液,LDL组给予100μg/mL的LDL,AGE-HSA组给予100μg/mL的AGE-HSA,AGE-LDL组分别给予50、100、200μg/mL的AGE-LDL。取对照组和AGE-LDL组细胞培养21 d后进行茜红素染色,镜下观察细胞钙化情况。给药48 h后检测各组细胞中的细胞间黏附分子1(ICAM-1)、IL-6蛋白及细胞上清液中的IL-6、IL-8。用100μg/mL的AGE-LDL分别刺激HAVICs 0.5、1、2、4、8 h,以只加DMSO的细胞作为对照,检测HAVICs中NF-κB磷酸化水平。将HAVICs随机分为5个组,对照组给予M199培养液;LDL组给予100μg/mL的LDL;DMSO组给予含有1‰DMSO的M199培养液;AGE-LDL组给予100μg/mL的AGE-LDL;Bay11-7082组予2.5μmol/L的Bay11-7082干预30 min,然后给予100μg/mL的AGE-LDL;给药48 h后检测各组细胞中的ICAM、IL-6蛋白。结果与对照组相比,AGE-LDL组(100μg/mL亚组)细胞中钙盐沉积明显,形成钙结节。AGE-LDL组细胞中ICAM-1、IL-6蛋白相对表达量及培养液上清中IL-6、IL-8水平均高于对照组、LDL组、AGE-HSA组,且AGE-LDL组的50μg/mL亚组、100μg/mL亚组、200μg/mL亚组ICAM-1、IL-6蛋白相对表达量及IL-6、IL-8水平也依次增高(P均<0.05)。AGE-LDL可呈时间依赖性方式刺激HAVICs中的NF-κB/p65磷酸化,于作用2 h后达到峰值,4 h后开始减弱。AGE-LDL组、Bay11-7082组细胞中ICAM、IL-6蛋白相对表达量高于对照组、LDL组、DMSO组,且Bay11-7082组ICAM、IL-6蛋白相对表达量低于AGE-LDL组(P均<0.05)。结论 AGE-LDL可诱导HAVICs发生钙化及炎症反应,其机制可能与NF-κB信号通路活化有关。

Objective To observe the calcification and inflammatory response induced by advanced glycation end products of low-density lipoprotein（ AGE-LDL） in human aortic valve interstitial cells（ HAVICs） and to explore its mechanism. Methods The non-CAVD valvular tissues（ non-CAVD group） were taken from patients with surgical treatment of aortic valve prolapse,the CAVD valvular tissues（ CAVD group） were taken from patients undergoing aortic valve replacement because of calcified aortic valve stenosis. The expression levels of AGE in the non-CAVD group and CAVD group were tested by immunohistochemistry. HAVICs were separated from aortic valve tissues in the non-CAVD group,and then were divided into the control group,LDL group,AGE-HSA group,and AGE-LDL group. The control group was given M199 medium,the LDL group was given 100 μg/mL LDL,the AGE-HSA group was given 100 μg/mL AGE-HSA,and the AGE-LDL group was given 50,100 and 200 μg/mL AGE-LDL,respectively. After 21 days of cell culture in the control group and AGE-LDL group,the cells were stained with alizarin red to observe cell calcification by microscope. Intercellular adhesion molecule-1（ ICAM-1）,interleukin-6（ IL-6） and interleukin-8（ IL-8） in the cell supernatant and cell lysate were detected in each group after stimulation. We stimulated HAVICs with AGE-LDL（ 100 μg/mL） for 0. 5,1,2,4,and8 h,respectively; DMSO group was taken as the control. We detected the phosphorylation level of NF-κB. HAVICs were randomly divided into the five groups; the control group was given M199 medium; the LDL group was given 100 μg/mL LDL; the DMSO group was given M199 medium containing 1‰ DMSO; the AGE-LDL group was given 100 μg/mL AGELDL; the BAY11-7082 group was given 2. 5 μmol/L BAY 11-7082 before stimulation with AGE-LDL（ 100 μg/mL） for 30 min. ICAM and IL-6 proteins were detected in each group after 4 8 h. Results Compared with the control group,calcium salt deposits significantly increased in the AGE-LDL group（ the 100 μg/mL subgroup）,forming calcium nodules. The expression levels of ICAM-1 and IL-6 proteins in the cells,and the levels of IL-6 and IL-8 in the supernatant were were higher than those of the control group,LDL group,and AGE-HSA group; the expression levels of ICAM-1 and IL-6 proteins in the cells,and the levels of IL-6 and IL-8 in the supernatant of the AGE-LDL groups（ including 50 μg/mL,100 μg/mL,and 200 μg/mL subgroups） increased gradually（ all P 0. 05）. AGE-LDL stimulated NF-κB/p65 phosphorylation in a time-dependent manner,it reached the peak after 2 h and started to decinle at 4 h. The expression levels of ICAM and IL-6 in the AGE-LDL group and the BAY11-7082 group were higher than those in the control group,the LDL group,and the DMSO group; the expression levels of ICAM and IL-6 in the BAY11-7082 group were lower than those in the AGE-LDL group（ both P 0. 05）. Conclusion AGE-LDL induced calcification and inflammatory response of HAVICs,and its mechanism may be related to the activation of NF-κB signaling pathway.