南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (08): 1119-1126.doi: 10.12122/j.issn.1673-4254.2020.08.08

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当归拈痛汤调控Fas/caspase-8通路促进类风湿关节炎滑膜成纤 维细胞凋亡

赵 帆，李佳钰，陆麒瑾，陈恩生，袁立霞   

  • 出版日期:2020-08-20 发布日期:2020-08-20
  • 基金资助:

Danggui Niantong decoction induces apoptosis by activating Fas/caspase-8 pathway in rheumatoid arthritis fibroblast-like synoviocytes

  

  • Online:2020-08-20 Published:2020-08-20

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摘要： 目的 探究当归拈痛汤（DGNTD）对类风湿关节炎滑膜成纤维细胞（FLS）凋亡及TNF受体超族成员6（Fas）/半胱氨酸天冬氨酸蛋白酶-8（caspase-8）通路的影响。方法 收集临床RA患者的滑膜组织，无菌条件下采用酶解法分离并培养出FLS细胞。采用免疫荧光染色法对细胞进行鉴定。FLS细胞分为空白组（NC组）、DGNTD低剂量组、DGNTD中剂量组、DGNTD高剂量组、Fas抑制剂联合DGNTD组（KR-33493+DGNTD组）。NC组：10%空白血清干预FLS细胞；DGNTD低剂量组：10% DGNTD低剂量含药血清干预 FLS 细胞；DGNTD 中剂量组：10% DGNTD 中剂量含药血清干预 FLS 细胞；DGNTD 高剂量组：10% DGNTD高剂量含药血清干预FLS细胞；KR-33493+DGNTD组：20 μmol/mL KR-33493+10% DGNTD高剂量含药血清干预FLS细胞。通过MTT法检测不同剂量DGNTD对FLS细胞增殖的影响；Hoechst 33342法和Annexin V-FITC/PI流式细胞术分别检测FLS细胞凋亡特性和凋亡率；qPCR和Western blot分别检测FLS细胞Fas、Fas相关蛋白（FADD）、caspase-8、caspase-3的mRNA和蛋白表达量。结果 免疫荧光实验证明本实验提取出的细胞为FLS细胞；MTT结果显示DGNTD含药血清随着剂量和干预时间的增加可显著抑制FLS的增殖（P<0.05）；Hoechst33342和流式细胞术结果显示不同剂量的DGNTD含药血清可以显著促进FLS细胞凋亡（P<0.05）；qPCR和Western blot均显示不同剂量的DGNTD含药血清可以显著提高Fas、FADD、caspase-8、caspase-3 mRNA和蛋白的表达水平（P<0.05）；进一步发现，Fas蛋白抑制剂（KR-33493）可以逆转DGNTD对FLS细胞的上述作用（P<0.05）。结论 当归拈痛汤可能通过激活Fas/caspase-8通路来诱导FLS细胞凋亡。

Abstract: Objective To explore the effect of Danggui Niantong decoction (DGNTD) on cell apoptosis and TNF receptor super family 6 (Fas)/caspase-8 pathway in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Methods FLS isolated from the synovial tissue of RA patients were cultured and identified using immunofluorescence staining. The cells were treated with 10% blank serum (blank control group), 10% sera containing low, moderate or high doses of DGNTD, or 20 μmol/mL KR-33493 (a Fas inhibitor) combined with 10% serum containing high-dose DGNTD. MTT assay was used to detect the proliferation of the cells after the treatments. Apoptosis of the cells was detected at 48 h in each group using Hoechst 33342 staining and flow cytometry with annexin V-FITC/PI staining. The mRNA and protein expressions of Fas, FADD, caspase-8 and caspase-3 in the cells at 48 h were detected using qPCR and Western blotting. Results Immunofluorescence staining identified the cultured cells as FLS. Treatment with DGNTD-containing sera significantly inhibited the proliferation of FLS, and the inhibitory effects were enhanced as the dose and intervention time increased (P<0.05). Hoechst 33342 staining and flow cytometry showed that the sera containing different doses of DGNTD significantly promoted apoptosis of FLS (P<0.05). The expression levels of Fas, FADD, caspase-8, and caspase-3 at both mRNA and protein levels were significantly increased in the cells after treatment with different doses of DGNTD-containing sera (P<0.05). The application of KR-33493 obviously reversed the effects of DGNTD on the FLS (P<0.05). Conclusion DGNTD can induce apoptosis of the FLS by activating Fas/caspase-8 signaling pathway.