透骨血竭散含药血清通过阻断PI3K/AKT信号抑制人类风湿关节炎滑膜成纤维细胞的恶性行为

Medicinal serum of Tougu Xuejie Powder inhibiting the malignant behavior of human rheumatoid arthritis synovial fibroblasts by blocking the PI3K/AKT signaling pathway

关键词:  透骨血竭散  PI3K/AKT信号通路  类风湿关节炎  滑膜成纤维细胞  恶性行为

Key words:  Tougu Xuejie Powder  PI3K/AKT signaling pathway  rheumatoid arthritis  synovial fibroblasts  malignant behavior

摘要:

[目的] 探究透骨血竭散含药血清是否通过阻断磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）信号抑制人类风湿关节炎滑膜成纤维（RA-FLS）细胞的恶性行为。[方法] 体外培养人RA-FLS细胞，细胞共分为以下4组：A组（正常组）、B组[模型组，脂多糖（LPS）诱导炎症微环境]、C组（LPS+透骨血竭散组）和D组（LPS+透骨血竭散+PI3K激活剂组）。通过CCK-8检测细胞活力，流式细胞术检测细胞凋亡，划痕实验检测细胞迁移，Transwell检测细胞侵袭，酶联免疫吸附测定（ELISA）检测细胞上清液炎性因子水平[肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、白细胞介素-10（IL-10）]，蛋白免疫印迹法（Western blot）检测PI3K/AKT信号通路相关蛋白表达水平。[结果] CCK-8实验发现，在干预24和48 h后，15%和20%的透骨血竭散含药血清均能显著降低RA-FLS的细胞活力（P<0.05）。与A组相比，B组细胞增殖、迁移、侵袭数量及细胞上清液TNF-α、IL-1β、IL-6水平显著升高，细胞凋亡率及细胞上清液IL-10有所降低，且p-PI3K/PI3K、pAKT/AKT、p-mTOR/mTOR和p-GSK-3β/GSK-3β比值升高（均P<0.05）。这表示LPS刺激促进RA-FLS细胞的恶性行为，激活PI3K/AKT信号通路，促进下游关键分子（如mTOR、GSK-3β）磷酸化水平。而经透骨血竭散含药血清处理24 h后，RA-FLS细胞增殖、迁移、侵袭数量、细胞上清液TNF-α、IL-1β、IL-6水平及p-PI3K/PI3K、pAKT/AKT、p-mTOR/mTOR和p-糖原合成酶激酶（GSK）-3β/GSK-3β比值降低，细胞凋亡率及细胞上清液IL-10水平升高。激活PI3K/AKT信号部分逆转透骨血竭散含药血清对RA-FLS细胞增殖、迁移、侵袭能力的抑制作用，及对RA-FLS细胞炎症反应的改善作用。[结论] 透骨血竭散含药血清通过阻断PI3K/AKT信号抑制人类风湿关节炎滑膜成纤维细胞的恶性行为。

Abstract:

[Objective] To investigate whether the drug-containing serum of Tonggu Xuejie Powder can inhibit the malignant behavior of human rheumatoid arthritis synovial fibroblasts（RA-FLS） by blocking the phosphoinositide 3-kinase（PI3K）/protein kenase B（AKT） signaling pathway. [Methods] Human RA-FLS cells were cultured in vitro. The cells were divided into the following 4 groups：Group A（normal group），Group B [model group，induced inflammatory microenvironment by lipopolysaccharide（LPS）]，Group C（LPS+Tougu Xuejie Powder group），and Group D（LPS+Tougu Xuejie Powder+PI3K activator group）. Cell viability was detected by CCK-8，apoptosis was detected by flow cytometry，cell migration was detected by scratch assay，cell invasion was detected by Transwell，and the levels of inflammatory factors in cell supernatants[Tumor necrosis factor-α（TNF-α），interleukin-1β（IL-1β），interleukin-6（IL-6），interleukin-10（IL-10）] were detected by Enzyme-linked immunosorbent assay（ELISA），and the expression levels of PI3K/AKT signaling pathway-related proteins were detected by western blot. [Results] The CCK8 experiment results showed that 15% and 20% of the drug-containing serum of Tougu Xuejie Powder significantly reduced the cell viability of RA-FLS（both P<0.05） at 24 hours and 48 hours of intervention. Compared with group A，the cell proliferation，migration，invasion quantity，and levels of TNF-α，IL-1β，and IL-6 in the supernatant of cells in group B were significantly increased，while the apoptosis rate and the level of IL-10 in the supernatant of cells decreased，and the ratios of p-PI3K/PI3K，pAKT/AKT，p-mTOR/mTOR and p-GSK-3β/GSK-3β increased（all P<0.05）. This indicates that LPS stimulation promotes the malignant behavior of RA-FLS cells，activates the PI3K/AKT signaling pathway，and enhances the phosphorylation levels of downstream key molecules（such as mTOR and GSK-3β）.After 24 hours of treatment with the drug-containing serum of Tougu Xuejie Powder，the proliferation，migration，invasion quantity，levels of TNF-α，IL-1β，and IL-6 in the supernatant of RA-FLS cells，and the ratios of p-PI3K/PI3K，pAKT/AKT，p-mTOR/mTOR and p-GSK-3β/GSK-3β decreased，while the apoptosis rate and the level of IL-10 in the supernatant of cells increased. Activation of the PI3K/AKT signaling partially reversed the inhibitory effect of the drug-containing serum of Tougu Xuejie Powder on the proliferation，migration，and invasion abilities of RA-FLS cells，as well as the improvement effect on the inflammatory response of RA-FLS cells. [Conclusion] The medicinal serum of Tougu Xuejie Powder inhibits the malignant behavior of human rheumatoid arthritis synovial fibroblasts by blocking the PI3K/AKT signaling pathway.