杯苋甾酮通过激活自噬并抑制TLR4/MyD88/NF-κB通路改善小鼠胶原诱导性关节炎

doi:10.5246/jcps.2026.05.031

中国药学(英文版) ›› 2026, Vol. 35 ›› Issue (5): 438-453.DOI: 10.5246/jcps.2026.05.031

• 【研究论文】 • 上一篇

杯苋甾酮通过激活自噬并抑制TLR4/MyD88/NF-κB通路改善小鼠胶原诱导性关节炎

谭新访¹^,^#, 刘源²^,^#, 史鹏博³, 马江涛², 黄成曦⁴, 吴志方⁵^,^*(), 胡笑燊⁶^,^*(), 徐亮亮¹^,^*()

^1. 广州中医药大学第一附属医院，岭南医学研究中心骨科与创伤实验室，广东广州 510006
^2. 河南省洛阳正骨医院河南省骨科医院，河南郑州 450016
^3. 河南中医药大学第一附属医院骨伤科，河南郑州 450099
^4. 科尔沃学院，美国印第安纳州 46511
^5. 广州中医药大学第三附属医院骨伤科，广东广州 510006
^6. 成都中医药大学康复医学院，四川成都 610075

收稿日期:2026-02-16 修回日期:2026-03-21 接受日期:2026-04-05 出版日期:2026-05-31 发布日期:2026-05-31
通讯作者: 吴志方, 胡笑燊, 徐亮亮

Cyasterone alleviates collagen-induced arthritis in mice by activating autophagy and inhibiting inflammation via the TLR4/MyD88/NF-κB signaling pathway

Xinfang Tan¹^,^#, Yuan Liu²^,^#, Pengbo Shi³, Jiangtao Ma², Chengxi Huang⁴, Zhifang Wu⁵^,^*(), Xiaoshen Hu⁶^,^*(), Liangliang Xu¹^,^*()

^1. Laboratory of Orthopedics & Traumatology, Lingnan Medical Research Center, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, China
^2. Luoyang Orthopedic Hospital of Henan Province & Orthopedic Hospital of Henan Province, Zhengzhou 450016, Henan, China
^3. Department of Orthopedics and Traumatology, the First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450099, Henan, China
^4. Culver Academies, 1300 Academy Rd, Culver 46511, Indiana, United States of America
^5. Laboratory of Orthopaedics & Traumatology, The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, China
^6. School of Health Preservation and Rehabilitation, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, Sichuan, China

Received:2026-02-16 Revised:2026-03-21 Accepted:2026-04-05 Online:2026-05-31 Published:2026-05-31
Contact: Zhifang Wu, Xiaoshen Hu, Liangliang Xu
About author:
^# Xinfang Tan and Yuan Liu contributed equally to this work.
Supported by:
The National Natural Science Foundation of China (Grant No. 82205239) and the Natural Science Foundation of Sichuan Province (Grant No. 2025ZNSFSC0636).

摘要/Abstract

摘要：

类风湿性关节炎是一种慢性自身免疫性疾病，杯苋甾酮作为一种植物蜕皮激素，在既往研究中虽已显示出抗炎潜力，但其在类风湿性关节炎中的具体作用机制尚不清楚。本研究旨在通过建立牛Ⅱ型胶原尾静脉注射诱导的小鼠胶原诱导性关节炎(CIA)模型，评估杯苋甾酮的治疗效果。通过关节炎指数、爪肿胀度等指标评价CIA进展；采用酶联免疫吸附法测定炎症因子水平；通过实时荧光定量PCR和Western blot检测基因及蛋白表达；利用Transwell实验、TUNEL染色及流式细胞术分别检测滑膜成纤维细胞(RA-FLS)的迁移、侵袭及凋亡情况。结果显示，杯苋甾酮以剂量依赖的方式抑制RA-FLS的增殖、迁移与侵袭能力，同时促进细胞凋亡并减少炎症因子释放，表明其对RA具有保护作用。在机制上，杯苋甾酮能抑制TLR4/MyD88/NF-κB信号通路并激活自噬，具体表现为降低TLR4、MyD88、磷酸化p65及磷酸化mTOR蛋白水平，同时提高Beclin 1表达及LC3 II/I比值。体内实验进一步证实，杯苋甾酮可以显著降低CIA小鼠的关节炎指数与足爪肿胀程度，降低血清炎症因子水平，减轻滑膜增生及软骨结构破坏，表现出抗软骨损伤的效果。综上所述，杯苋甾酮通过调控TLR4/MyD88/NF-κB信号通路并激活自噬，对CIA小鼠发挥治疗作用，有望成为关节炎治疗的潜在候选药物。

关键词: 关节炎, 杯苋甾酮, 滑膜成纤维细胞, TLR4/MyD88/NF-κB信号通路, 炎症

Abstract:

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Cyasterone, a phytoecdysteroid, demonstrates anti-inflammatory potential, but its mechanism in RA remains unclear. This study aims to investigate Cyasterone’s therapeutic effect and underlying mechanisms in collagen-induced arthritis (CIA) mice. Cell migration and cell invasion capabilities were measured by the transwell assay. Apoptosis was detected by TUNEL or flow cytometry. Tail vein injection of bovine type II collagen was used to establish the mouse CIA model. The enzyme-linked immunoassay was performed to measure inflammatory factor concentrations. Quantitative real-time PCR or WB analysis was performed to detect gene or protein expression. CIA was assessed by the arthritis index and claw thickness curve, etc. The results demonstrated that Cyasterone had dose-dependent inhibitory effects on the proliferation, migration, and invasion of rheumatoid arthritis-fibroblast like synoviocytes (RA-FLS). Cyasterone increased apoptosis and reduced inflammation in RA-FLS, indicating its protective effects in RA. Cyasterone inhibited TLR4/MyD88/NF-κB pathway and increased autophagy in RA-FLS, as demonstrated by reduced TLR4, MyD88, phosphorylated p65, and p-mTOR levels while increasing Beclin 1 expression and light chain 3 II/I ratio. The in vivo results further confirmed that Cyasterone reduced the arthritis index and paw swelling, significantly reduced the serum levels of inflammatory factors, alleviated synovial hyperplasia and destruction of cartilage structure, demonstrating an anti-cartilage damage effect. In conclusion, Cyasterone protected against CIA through the TLR4/MyD88/NF-κB signaling pathway, which might be a promising candidate for arthritis treatment.

Key words: Arthritis, Cyasterone, Fibroblast-like synoviocytes, TLR4/MyD88/NF-κB signaling pathway, Inflammation

Supporting: /attached/file/20260529/20260529192238_479.pdf

谭新访, 刘源, 史鹏博, 马江涛, 黄成曦, 吴志方, 胡笑燊, 徐亮亮. 杯苋甾酮通过激活自噬并抑制TLR4/MyD88/NF-κB通路改善小鼠胶原诱导性关节炎[J]. 中国药学(英文版), 2026, 35(5): 438-453.

Xinfang Tan, Yuan Liu, Pengbo Shi, Jiangtao Ma, Chengxi Huang, Zhifang Wu, Xiaoshen Hu, Liangliang Xu. Cyasterone alleviates collagen-induced arthritis in mice by activating autophagy and inhibiting inflammation via the TLR4/MyD88/NF-κB signaling pathway[J]. Journal of Chinese Pharmaceutical Sciences, 2026, 35(5): 438-453.

图/表 6

Figure 1. Cyasterone inhibited RA-FLS proliferation, migration, and invasion. (A) The effect of Cyasterone on normal FLS cell viability. (B–D) The effect of Cyasterone on RA-FLS cell proliferation (B), cell migration (C) and cell invasion ability (D). One or two-way ANOVA was used for multiple group statistical analyses and comparisons. The data were expressed as mean ± SD (n = 6). *P < 0.05 vs. FLS, #P < 0.05 vs. RA-FLS.

Figure 2. Cyasterone promoted apoptosis and reduced inflammation in RA-FLS. (A) Flow cytometry assay was performed to measure apoptosis; (B) IL-10, TNF-α, IL-6, and IL-17 expression. One-way ANOVA was used for multiple-group statistical analyses and comparisons. The data were expressed as mean ± SD (n = 6). *P < 0.05 vs. FLS, # P < 0.05 vs. RA-FLS.

Figure 3. Cyasterone inhibited TLR4/MyD88/NF-κB pathway and increased autophagy in RA-FLS. (A) TLR4 and MyD88 expression was determined by Qpcr; (B) TLR4, MyD88, NF-κB, IkBα, p-p65, and p-IkBα expression; (C) LC3II/I, Beclin1, p62, mTOR, and p-mTOR levels in RS-FLS. One-way ANOVA was used for multiple-group statistical analyses and comparisons. The data were expressed as mean ± SD (n = 6). *P < 0.05 vs. FLS, #P < 0.05 vs. RA-FLS.

Figure 4. Cyasterone attenuates arthritis progression in CIA mice. (A) Animal images; (B) Arthritis assessment (arthritis index and paw thickness); (C) H&E staining was performed to observe knee joint injury; (D) The injury in the knee cartilage; (E) The knee joint tissue apoptosis was assessed by TUNEL fluorescence.

Figure 5. The effect of Cyasterone on serum IL-1β, IL-6, IL-10, IL-17, MMP-3, TNF-α, 5-LOX, and COX-2 in the serum of rats in different groups. The indicated factor was detected by ELISA assay. The data were expressed as mean ± SD (n = 6). *P < 0.05, compared with the Sham group; #P < 0.05, compared with the CIA model group.

Figure 6. Cyasterone inhibited TLR4/MyD88/NF-κB pathway and induced autophagy in vivo. (A) TLR4 and MyD88 expression were detected by qPCR; (B) TLR4, MyD88, NF-κB, IkBα, p-p65, and p-IkBα expression were detected by Western blot; (C) Western blot was applied to assess LC3II/I, Beclin1, p62, mTOR, and p-mTOR levels. One or two-way ANOVA was used for multiple group statistical analyses and comparisons. The data was expressed as mean ± SD (n = 6). *P < 0.05 vs. sham, #P < 0.05 vs. model.

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杯苋甾酮通过激活自噬并抑制TLR4/MyD88/NF-κB通路改善小鼠胶原诱导性关节炎

Cyasterone alleviates collagen-induced arthritis in mice by activating autophagy and inhibiting inflammation via the TLR4/MyD88/NF-κB signaling pathway

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