山东大学学报 (医学版) ›› 2023, Vol. 61 ›› Issue (9): 84-93.doi: 10.6040/j.issn.1671-7554.0.2023.0460
刘金波,刘凯文,向崇鑫,程雷
LIU Jinbo, LIU Kaiwen, XIANG Chongxin, CHENG Lei
摘要: 目的 探讨西红花苷对椎间盘退变的治疗效果和作用机制。 方法 将收集的人类髓核组织Pfirrmann II级样本分成两部分,一部分提取髓核细胞并培养,根据培养基的成分,将髓核细胞分为4组,包括空白对照组、肿瘤坏死因子α(TNF-α)刺激组、西红花苷低浓度治疗组(10 μg/mL)以及西红花苷高浓度治疗组(40 μg/mL)。培养24 h后,采用实时定量 PCR检测4组髓核细胞中炎症因子和代谢标志物的mRNA表达水平。培养0、15、30 min和48 h后,采用Western blotting 检测4组髓核细胞中炎症因子、代谢标志物、凋亡标志物以及核因子活化B细胞κ轻链增强子(NF-κB)信号通路关键蛋白p-P65的蛋白表达水平。采用双荧光素酶报告基因检测西红花苷对NF-κB信号通路的作用效果。另一部分髓核组织进行离体培养,根据培养基不同,分为空白对照组、TNF-α刺激组以及西红花苷治疗组(40 μg/mL)。使用免疫组化检测各组髓核组织中炎症因子以及细胞代谢标物的蛋白表达水平。 结果 实时定量 PCR检测与Western blotting 检测结果显示,与空白对照组相比,TNF-α刺激组髓核细胞的炎症反应及细胞外基质降解加剧,促炎因子诱导型一氧化氮合酶、环氧化酶-2、去整合素和金属蛋白酶-5及基质金属蛋白酶13的基因转录与蛋白表达水平明显升高(P<0.05),而软骨可聚蛋白多糖与II型胶原蛋白的基因转录与蛋白表达水平明显下降(P<0.05)。同时Western blotting 检测结果也显示,与空白对照组相比,TNF-α刺激组髓核细胞的抗凋亡因子B淋巴细胞瘤-2基因(Bcl-2)的蛋白表达水平明显下降(P<0.001),而促凋亡因子中的Bcl-2相关X蛋白(P=0.004)和活化的天冬氨酸特异性半胱氨酸蛋白酶的蛋白表达水平明显升高(P=0.005)。而在10 μg/mL和40 μg/mL的西红花苷治疗组中上述由TNF-α所诱导的变化均被明显抑制(P<0.05)。此外,在NF-κB信号通路的实验中,与空白对照组相比,TNF-α刺激组中p-P65的蛋白表达水平明显升高(P<0.05),而在10 μg/mL和40 μg/mL的西红花苷治疗组中p-P65的蛋白表达水平均明显下降(P<0.05),并且在治疗时长15、30 min的西红花苷组中p-P65的蛋白表达水平均明显下降(P<0.05)。同时在双荧光素酶报告基因检测中,相较于空白组,TNF-α刺激组的荧光强度增加(P<0.001),而经过西红花苷治疗后,荧光强度明显下降(P=0.006)。 结论 西红花苷可抑制髓核细胞的炎症反应、细胞外基质降解及细胞凋亡, NF-κB信号通路在西红花苷抑制椎间盘退变中发挥重要作用。
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