苦参素对晚期糖基化产物诱导的人血管平滑肌细胞表型转化的抑制作用及其机制

Suppressive effect of matrine on advanced glycation end products-induced contractile-synthetic phenotypic conversion of human vascular smooth muscle cells and its mechanism

目的探讨晚期糖基化产物(AGEs)诱导人血管平滑肌细胞(HVSMCs)发生收缩-合成表型转化的分子机制及苦参素的抑制作用。方法将HVSMCs分为对照组(control)、低剂量AGEs组(L-AGEs)、高剂量AGEs组(H-AGEs)、低剂量苦参素组(AGEs+L-Mat)、中剂量苦参素组(AGEs+M-Mat)以及高剂量苦参素组(AGEs+H-Mat)。免疫荧光法评估HVSMCs平滑肌肌球蛋白重链11(MYH11)表达水平。酶联免疫吸附试验(ELISA)检测细胞培养上清液白介素(IL)1β及肿瘤坏死因子(TNF)α浓度。Western blotting法检测HVSMCs内葡萄糖调节蛋白78(GRP78)、Notch受体细胞内结合域1(NICD1)、Split多毛增强子1(HES1)、发状分裂相关增强子2(HEY2)、δ样蛋白-4(Dll4)表达水平及蛋白激酶RNA样内质网激酶(PERK)磷酸化水平。结果与control组相比,L-AGEs组及H-AGEs组HVSMCs中MYH11表达水平显著降低(P<0.05),细胞内GRP78、Dll4、NICD1、HES1、HEY2表达水平及PERK磷酸化水平显著升高(均P<0.05),细胞培养上清液IL-1β及TNF-α浓度显著升高(均P<0.05)。与L-AGEs组相比,H-AGEs组细胞MYH11、GRP78、Dll4、NICD1、HES1、HEY2表达水平及PERK磷酸化水平均显著升高(均P<0.05),细胞培养上清液IL-1β及TNF-α浓度均显著升高(均P<0.05)。与H-AGEs组相比,AGEs+L-Mat组、AGEs+M-Mat组及AGEs+H-Mat组MYH11表达水平显著升高(P<0.05),细胞内GRP78、Dll4、NICD1、HES1、HEY2表达水平及PERK磷酸化水平显著降低(均P<0.05),细胞培养上清液IL-1β及TNF-α浓度显著降低(均P<0.05);AGEs+L-Mat组、AGEs+M-Mat组以及AGEs+H-Mat组细胞MYH11、GRP78、Dll4、NICD1、HES1、HEY2表达水平及PERK磷酸化水及细胞培养上清液IL-1β及TNF-α浓度变化具有显著的苦参素剂量依赖性(均P<0.05)。结论AGEs通过激活内质网应激PERK/Notch信号通路诱导HVSMCs发生收缩-合成表型转化,苦参素能够通过抑制该通路活化阻遏AGEs诱导的VSMCs表型转化。

Objective To investigate the mechanism of advanced glycation end products(AGEs) inducing the contractile-synthetic phenotypic conversion of human vascular smooth muscle cells(HVSMCs) and the inhibitory effect of matrine. Methods HVSMCs were divided into control group, low-dose AGEs group(L-AGEs), high-dose AGEs group(H-AGEs), low-dose matrine group(AGEs+L-Mat), moderate-dose matrine group(AGEs+M-Mat) and high-dose matrine group(AGEs+H-Mat). Immunofluorecent staining was used to evaluate the expression of smooth muscle myosin heavy chain 11(MYH11). ELISA was used to determine the concentrations of interleukin(IL)-1β and tumor necrosis factor(TNF)-α. Western blotting was used to evaluate the expression levels of glucose regulated protein 78(GRP78), Notch intracellular domain 1(NICD1), enhancer split protein 1(HES1), delta-like 4(Dll4) and the phosphory-lation level of protein RNA-like ER kinase(PERK) in HVSMCs. Results Compared with control group, MYH11 expression was significantly decreased in L-AGEs group and H-AGEs group(P<0.05), the expression levels of GRP78, Dll4, NICD1, HES1, HEY2 and the phosphorylation level of PERK were increased(P<0.05), and the concentrations of IL-1β and TNF-α in cell medium were also significantly increased(P<0.05). The expression levels of MYH11, GRP78, Dll4, NICD1, HES1, HEY2, the phosphorylation level of PERK and the concentrations of IL-1β and TNF-α in cell medium were higher in H-AGEs group than in L-AGEs group(P<0.05). Compared with H-AGEs group, MYH11 expression level was significantly increased in AGEs+L-Mat group, AGEs+M-Mat group and AGEs+H-Mat group(P<0.05). Compared with H-AGEs group, the expression levels of GRP78, Dll4, NICD1, HES1, HEY2 and the phosphorylation level of PERK were significantly reduced in AGEs+L-Mat group, AGEs+M-Mat group and AGEs+H-Mat group(P<0.05), while IL-1β and TNF-α concentrations in cell medium were significantly decreased(P<0.05). And the expression levels of MYH11, GRP78, Dll4, NICD1, HES1, HEY2, the phosphorylation level of PERK, and IL-1β and TNF-α concentrations in cell medium showed dosage-dependent changes by matrine(P<0.05). Conclusion AGEs can induce the contractile-synthetic phenotypic conversion of HVSMCs via PERK/Notch signaling pathway. Matrine could suppress the phenotypic conversion by inhibiting this pathway.