四妙散通过调节AKT/ROS/自噬轴抑制实验性类风湿关节炎中的炎症反应

Suppression of Inflammation by Si Miao San in Experimental Rheumatoid Arthritis Through Modulation of the AKT/ROS/Autophagy Axis.

作者信息

Zhang Honglin, Jiang Haixu, Wei Qiuzhu, Xu Jie, Zhao Zihan, Sun Yuhe, Lu Qingyi

机构信息

School of Life Sciences, Beijing University of Chinese Medicine, Beijing, People's Republic of China.

School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, People's Republic of China.

出版信息

J Inflamm Res. 2025 Jul 17;18:9459-9476. doi: 10.2147/JIR.S524871. eCollection 2025.

OBJECTIVE

Si Miao San is effective in ameliorating rheumatoid arthritis (RA) both clinically and experimentally. NETs play a fundamental role in the onset and progression of RA. The goal of this study was to explore the therapeutic effects of Si Miao San (SMS) on adjuvant-induced arthritis in mice and the regulatory mechanisms of NETs both in vivo and in vitro.

METHODS

SMS decoctions were identified using LC-MS/MS analysis and TCMSP. In the adjuvant induced RA murine model, SMS decoction and methotrexate were administered orally. Disease progression was analysed by assessing arthritic scores and joint diameter, H&E staining, safranin O-fast green staining, toluidine blue staining and micro-CT analysis. The expression of NE, MPO, PAD4, LC3B, CitH3, p-AKT and p-PI3K and the production of ROS were detected using IHC, WB and IF analyses. Cytokines in the sera of the mice were detected using cytometric bead arrays. After the in vitro culture of neutrophils, NE, MPO, PAD4, LC3B, CitH3, ROS, p-PI3K and p-AKT were measured using IF and WB analyses. Autophagy was further observed with TEM.

RESULTS

SMS decoction compounds were first identified. Compared with the model group, SMS significantly inhibited joint swelling, inflammation progression and bone destruction. The levels of NE, MPO, PAD4, CitH3, LC3B, ROS production and relative expression of p-AKT and p-PI3K in joint tissues were significantly reduced in the SMS group compared to the model group ( < 0.05). In vitro culture, SMS-containing serum significantly reduced the LC3B-II/LC3B-I ratio and the relative expression levels of p-AKT and p-PI3K, as well as the levels of ROS, NE, MPO, PAD4, and CitH3 compared with those in the PMA-treated group ( < 0.05), which was abolished by the treatment with the AKT activator SC79.

CONCLUSION

The SMS-induced suppression of inflammation in experimental RA occurred through the modulation of the AKT/ROS/autophagy axis.

目的

四妙散在临床和实验中均对类风湿关节炎（RA）具有改善作用。中性粒细胞胞外陷阱（NETs）在RA的发病和进展中起重要作用。本研究旨在探讨四妙散（SMS）对佐剂诱导的小鼠关节炎的治疗作用以及NETs在体内和体外的调控机制。

方法

采用液相色谱-串联质谱（LC-MS/MS）分析和中药系统药理学数据库与分析平台（TCMSP）鉴定四妙散汤剂中的成分。在佐剂诱导的RA小鼠模型中，口服给予四妙散汤剂和甲氨蝶呤。通过评估关节炎评分、关节直径、苏木精-伊红（H&E）染色、番红O-固绿染色、甲苯胺蓝染色和显微计算机断层扫描（micro-CT）分析来分析疾病进展。采用免疫组化（IHC）、蛋白质免疫印迹（WB）和免疫荧光（IF）分析检测中性粒细胞弹性蛋白酶（NE）、髓过氧化物酶（MPO）、肽瓜氨酸化酶4（PAD4）、微管相关蛋白1轻链3（LC3B）、瓜氨酸化组蛋白H３（CitH3）、磷酸化蛋白激酶B（p-AKT）和磷酸化磷脂酰肌醇-3激酶（p-PI3K）的表达以及活性氧（ROS）的产生。使用细胞计数微珠阵列检测小鼠血清中的细胞因子。中性粒细胞体外培养后,采用IF和WB分析检测NE、MPO、PAD４、LC3B、CitH3、ROS、p-PI3K和p-AKT。通过透射电子显微镜（TEM）进一步观察自噬情况。

结果

首次鉴定出四妙散汤剂中的化合物。与模型组相比，四妙散显著抑制关节肿胀、炎症进展和骨质破坏。与模型组相比，四妙散组关节组织中NE、MPO、PAD4、CitH3、LC3B、ROS产生以及p-AKT和p-PI3K的相对表达水平显著降低（P<0.05）。体外培养时，与佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理组相比，含四妙散血清显著降低LC3B-II/LC3B-I比值以及p-AKT和p-PI3K的相对表达水平，以及ROS、NE、MPO、PAD4和CitH3的水平（P<0.05），而AKT激活剂SC79处理可消除这种作用。