基于NF-kB信号通路探讨芍药苷对LPS诱导的人软骨细胞炎症及退变的作用机制

Mechanism of paeoniflorin on LPS-induced human chondrocyte inflammation and degeneration based on NF-kB signaling pathway

目的观察芍药苷(PF)对于脂多糖(LPS)诱导的人软骨细胞炎症及退变的影响,并初步探索其作用机制。方法取2019年04月—2019年10月于北京中医药大学第三附属医院行全膝关节置换术的原发性膝关节骨性关节炎(KOA)患者术中废弃的软骨组织,用胰酶和胶原酶消化成软骨细胞。用CCK-8法检测不同浓度PF对软骨细胞活力的影响,选取合适浓度用于后续实验。将软骨细胞随机分为5组,即空白组,模型组以及PF低、中、高浓度组,除空白组外,其余4组均予以LPS诱导模拟关节炎环境,PF低、中、高浓度组分别给予10、20、50μmol/L的PF进行干预。使用qPCR法检测各组细胞中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶13(MMP13)及聚蛋白多糖酶5(ADAMTS5)含量;Western-blot法检测各组细胞中核转录因子(NF-kB p65)、核转录因子kB抑制剂激活酶(IkK)及核转录因子kB抑制剂(IkB)表达水平。结果CCK-8结果显示10、20、50μmol/L的PF可以显著提高人软骨细胞增殖活力,而过高浓度(200μmol/L)的PF会抑制软骨细胞活力。qPCR结果显示,PF各组与模型组比较IL-1β、IL-6、TNF-α含量及MMP13、ADAMTS5表达水平均降低(均P<0.05);Western-blot结果显示,PF各组与模型组比较NF-kB p65及IkK表达水平明显降低,而IkB表达明显升高(均P<0.05)。结论芍药苷可以阻止NF-kB通路激活,抑制软骨细胞炎症及延缓软骨退变,是一种潜在的治疗KOA的药物。

Objective To observe the effects and explore the mechanism of paeoniflorin(PF)on the inflammation and degeneration of LPS-induced human chondrocytes.Methods Discarded cartilage tissues from primary knee osteoarthritis(KOA)patients undergoing total knee arthroplasty at The Third Affiliated Hospital of Beijing University of Chinese Medicine from April 2019 to October 2019 were collected and digested into chondrocytes using pancreatin and collagenase.The effect of different concentrations of PF on the viability of chondrocytes was detected using the CCK-8 assay,and appropriate concentrations were selected for subsequent experiments.Chondrocytes were randomly divided into five groups,namely the blank group,the model group,and the low,medium,and high concentration PF intervention groups.Except for the blank group,chondrocytes in the other four groups were induced by LPS to simulate an arthritis environment.In the low,medium,and high concentration PF intervention groups,10,20,and 50μMol/L PF were given respectively.The expressions of IL-1β,IL-6,TNF-α,MMP13 and ADAMTS5 in each group were detected by qPCR assay;and the expression levels of NF-kB p65,IKB and IkK were detected by Western-blot.Results The results of CCK-8 assay showed that 10,20,and 50μMol/L PF could significantly increase the level of proliferation and viability of human chondrocyte;while excessively high concentration(200μM)PF could inhibit chondrocyte viability.Further qPCR results showed that PF could significantly inhibit the expression levels of inflammatory factors IL-1β,IL-6,TNF-αand cartilage degradation factors MMP13 and ADAMTS5 in human chondrocytes after LPS intervention,compared with the model group(P<0.05);WB results showed that PF could significantly inhibit NF-kB p65 and IkK expresion and promotes IkB expression(P<0.05).Conclusion Paeoniflorin seems to inhibit LPS-induced human chondrocyte inflammation and delay cartilage degeneration by inhibiting the activation of NF-kB signaling pathway.