[1]赵 帆,李佳钰,陆麒瑾,等.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[J].南方医科大学学报,2020,(08):1119-1126.
 Danggui Niantong decoction induces apoptosis by activating Fas/caspase-8 pathway inrheumatoid arthritis fibroblast-like synoviocytes[J].Journal of Southern Medical University,2020,(08):1119-1126.
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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()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2020年08期
页码:
1119-1126
栏目:
出版日期:
2020-07-30

文章信息/Info

Title:
Danggui Niantong decoction induces apoptosis by activating Fas/caspase-8 pathway in rheumatoid arthritis fibroblast-like synoviocytes
作者:
赵 帆李佳钰陆麒瑾陈恩生袁立霞
关键词:
当归拈痛汤Fas/caspase-8通路类风湿关节炎滑膜成纤维细胞细胞凋亡
Keywords:
Danggui Niantong decoction Fas/caspase-8 signal pathway rheumatoid arthritis fibroblast-like synoviocytes apoptosis
文献标志码:
A
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.
更新日期/Last Update: 2020-07-29