二苯乙烯苷对C反应蛋白诱导的小鼠巨噬细胞明胶酶A和B表达的影响

Depressive effects of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside on expressions of gelatinases A and B induced by C-reactive protein in mouse peritoneal macrophages

目的为探讨二苯乙烯苷(TSG)预防动脉粥样硬化斑块不稳定的可能机制,观察TSG对C反应蛋白(CRP)诱导的巨噬细胞表达明胶酶A和B的影响。方法体外培养的小鼠腹腔巨噬细胞,分为空白对照组、模型组(给CRP20mg·L-1)、模型+辛伐他汀100μg·L-1组、模型+TSG120及60μg·L-1组。给予CRP12h后加入干预药物,共同培养24h后进行指标的检测。用蛋白免疫印迹法观察各组细胞明胶酶A和B蛋白表达的差异;用逆转录聚合酶链反应法(RT-PCR)观察各组细胞明胶酶A和B mRNA表达的差异;ELISA法测定细胞培养液中白细胞介素6(IL-6)和肿瘤坏死因子α(TNFα)含量。结果蛋白免疫印迹分析显示,模型组明胶酶A和B蛋白的表达较空白对照组明显增加;与模型组(明胶酶A:1.14±0.26,明胶酶B:1.26±0.24)相比,辛伐他汀(明胶酶A:0.71±0.12,明胶酶B:0.73±0.15)及TSG(120μg·L-1组:明胶酶A,0.74±0.11,明胶酶B,0.88±0.13;60μg.±0.18,明胶酶B,1.12±0.18)均能降低明胶酶A和B蛋白的表达,并随TSG处理浓度的增加有下降趋势。RT-PCR显示,模型组明胶酶A和B的mRNA表达较空白对照组明显增加;与模型组(明胶酶A:2.45±0.18,明胶酶B:2.59±0.19)相比,辛伐他汀(明胶酶A:0.86±0.06,明胶酶B:0.98±0.10)及TSG(120μg·L-1组:明胶酶A,0.98±0.09,明胶酶B,1.24±0.13;60μg·L-1组:明胶酶A,1.32±0.12,明胶酶B,1.80±0.15)均能降低明胶酶A和B的mRNA表达,并随TSG处理浓度的增加有下降趋势。ELISA结果显示,与空白对照组比较,模型组IL-6和TNF-α水平明显升高;与模型组IL-6(614±52)ng·L-1,TNFα(82.5±4.7)mg·L-1相比,辛伐他汀IL-6(290±32)ng·L-1,TNFα(36.3±2.7)mg·L-1及TSG120μg·L-1组:IL-6(310±28)ng·L-1,TNFα(42.1±3.1)mg·L-1;60μg·L-1组:IL-6(498±46)ng·L-1,TNFα(58.6±3.4)mg·L-1能明显降低细胞培养液中IL-6和TNFα含量。结论TSG可通过抑制上调的明胶酶A和B的表达、IL-6及TNFα的分泌以抑制斑块不稳定。

AIM To investigate the mecha- nism of 2, 3, 5, 4＇-tetrahydroxystilbene-2-O- β-D-glucoside （TSG） on matrix remodeling in atherogenesis, the expressions of gelatinases A and B which were induced by C-reactive protein in macrophages were studied. METHODS Mouse peritoneal macrophages were cultured in vitro and intervened by different concentrations of TSG. There were 5 groups： normal control group, model group（ recombinant human CRP, rhCRP 20 mg·L^-1） , model ＋ simvastatin 100 μg·L^-1 group, model ＋ TSG 120 and 60 μg·L^-1 groups. After 24 h coincubation with drugs, gelatinases A and B proteins were deter- mined by Western blot, and gelatinase A mRNA and gelatinase B mRNA were measured by reverse transcriptase-polymerase chain reaction （RT-PCR）. The level of IL-6 and TNFα were determined by ELISA. RESULTS Compared with the normal control, the expression of proteins of gelatinases A and B significantly increased in model group （gelatinase A： 1.14 ±0.26,and gelatinase B ： 1.26 ± 0.24 ）. Compared with model group, simvastatin （gelatinase A, 0.71 ±0.12, and gelatinase B, 0.73 ± 0.15 ） and TSG（ 120 μg·L^-1 group ： gelatinase A 0.74± 0.11, and gelatinase B 0.88± 0.13 ; 60 μg·L^-1 group： gelatinase A 0.92± 0.18, and gelatinase B 1.12 ±0.18） significantly decreased protein expressions. The inhibitory effect of TSG was increased with the concentration increased. Compared with normal control group, the expressions of mRNA of gelatinases A and B also significantly increased in model group （gelatinase A mRNA, 2.45± 0.18, and gelatinase B mRNA, 2.59 ± 0. 19）. The expressions of mRNA of gelatinases A and B were reduced markedly by simvastatin （gelatinase A： 0. 86 ±0.06, and gelatinase B： 0. 98± 0.10） and TSG（ 120μg·L^-1 group： gelatinase A, 0.98 ±0.09, and gelatinase B： 1.24±0.13; 60μg·L^-1 group：gelatinase A, 1.32± 0. 12, and gelainase B, 1. 80 ± 0.15） in mouse peritoneal macrophages. The inhibitory effect of TSG was increased with the concentration increased. Compared with the normal control, the level of IL-6 and TNFα remarkably increased in model group [ IL-6 （ 614 ± 52） ng·L^-1 , and TNFα （ 82.5 ± 4.7 ）mg·L^-1 The levels of IL-6 and TNF-α were decreased markedly by simvastatin [ IL-6 （ 290± 32 ） ng·L^-1, and TNFet （36. 3 ± 2.7）mg·L^-1 3 and TSG [ 120μg·L^-1 group I IL-6 （ 310± 28 ） ng·L^-1 , and TNFet （42.1 ±3.1 ） mg. L - 1 ; 60 μg·L^-1 group ： IL-6 （498 ± 46） ng·L^-1 , and TNFα （58.6± 3.4） mg·L^-1 in mouse peritoneal macrophages. CONCLUSION TSG can prevent atherosclerosis plaques formation by downregulating expressions of gelatinases A and B, and inhibit the levels of IL-6 and TNFα.