Objectives: To explore the mechanism of moxibustion in improving synovitis inflammatory injury in rheumatoid arthritis (RA) model rats based on the ferroptosis-lipid metabolism pathway.
Methods: Forty-eight SD rats were randomly divided into normal, model, moxibustion and moxibustion + long-chain acyl-CoA synthetase 4 (ACSL4) inhibitor groups, with 12 rats in each group. The RA model was replicated using environmental factors of wind, cold, and dampness combined with Freund's complete adjuvant injection. The moxibustion group received moxibustion at "Shenshu" (BL23) and "Zusanli" (ST36) for 20 minutes per acupoint each time, once daily at a single acupoint (both sides) with alternating acupoints over 15 consecutive days. The moxibustion + ACSL4 inhibitor group received intraperitoneal injection of the ACSL4 inhibitor rosiglitazone (0.4 mg/kg) after moxibustion, once daily for 15 consecutive days. Histopathological changes in synovial tissue were observed using HE staining；serum contents of glutathione (GSH) and malondialdehyde (MDA) were detected using biochemical methods；reactive oxygen species (ROS) levels in synovial tissues were detected using flow cytometry；the expressions of glutathione peroxidase 4 (GPX4), ACSL4, and lysophosphatidylcholine acyltransferase 3 (LPCAT3) proteins in synovial tissues were detected using Western blot；and serum contents of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were detected using ELISA.
Results: Compared with the normal group, the serum contents of GSH decreased (P<0.01) while MDA, IL-6, and TNF-α contents increased (P<0.01)；the ROS levels, ACSL4 and LPCAT3 protein expressions increased (P<0.01) while GPX4 protein expression decreased (P<0.01) in synovial tissue in the model group. Compared with the model group, the serum contents of GSH increased (P<0.01) while MDA, IL-6, and TNF-α contents decreased (P<0.01)；the ROS levels, ACSL4 and LPCAT3 protein expressions decreased (P<0.01) while GPX4 protein expression increased (P<0.01) in synovial tissue in the moxibustion group and moxibustion + ACSL4 inhibitor group. Compared with the moxibustion group, the serum contents of GSH increased (P<0.01) while MDA, IL-6, and TNF-α contents decreased (P<0.01)；the ROS levels, ACSL4 and LPCAT3 protein expressions decreased (P<0.01, P<0.05) while GPX4 protein expression increased (P<0.05) in synovial tissue in the moxibustion + ACSL4 inhibitor group. HE staining showed that the model group had significantly increased synovial hyperplasia and inflammatory cell infiltration；the moxibustion group and moxibustion + ACSL4 inhibitor group showed varying degrees of alleviation in inflammatory cell infiltration and hyperplasia in synovial tissue；compared with the moxibustion group, the moxibustion + ACSL4 inhibitor group showed more significant improvements in inflammatory infiltration and hyperplasia of synovial cells, reduced layers of synovium.
Conclusions: Moxibustion at BL23 and ST36 can alleviate synovial inflammatory injury, and its mechanism may be related to reducing lipid peroxidation and ROS levels, and inhibiting the occurrence of ferroptosis.
目的: 基于铁死亡-脂质代谢通路探讨艾灸改善类风湿性关节炎（RA）模型大鼠滑膜炎性损伤的作用机制。方法: SD大鼠随机分为正常组、模型组、艾灸组、艾灸+长链脂酰辅酶A合成酶4（ACSL4）抑制剂组，每组12只。采用风、寒、湿环境结合弗氏完全佐剂注射复制RA模型。艾灸组予以艾灸“肾俞”“足三里”，每次每穴20 min，每日1穴（双侧），两穴交替选取，1次/d，连续15 d；艾灸+ACSL4抑制剂组在施灸后给予腹腔注射ACSL4抑制剂罗格列酮（0.4 mg/kg），1次/d，连续15 d。HE染色法观察各组大鼠滑膜组织病理形态学变化；生化法检测各组大鼠血清谷胱甘肽（GSH）和丙二醛（MDA）含量；流式细胞术检测各组大鼠滑膜组织活性氧（ROS）水平；Western blot法检测各组大鼠滑膜组织谷胱甘肽过氧化物酶4（GPX4）、ACSL4、溶血卵磷脂酰基转移酶3（LPCAT3）蛋白表达；ELISA法检测各组大鼠血清白细胞介素（IL）-6和肿瘤坏死因子-α（TNF-α）的含量。结果: 与正常组比较，模型组大鼠滑膜增生明显、大量炎性细胞浸润；血清中GSH含量显著降低（P<0.01），MDA、IL-6、TNF-α含量显著升高（P<0.01）；滑膜组织ROS水平、ACSL4和LPCAT3蛋白表达显著升高（P<0.01），GPX4蛋白表达显著降低（P<0.01）。与模型组比较，艾灸组、艾灸+ACSL4抑制剂组大鼠滑膜组织中的炎性细胞浸润和增生有不同程度的缓解；血清中GSH含量显著升高（P<0.01），MDA、IL-6、TNF-α含量显著降低（P<0.01）；滑膜组织ROS水平、ACSL4和LPCAT3蛋白表达显著降低（P<0.01），GPX4蛋白表达显著升高（P<0.01）。与艾灸组比较，艾灸+ACSL4抑制剂组大鼠滑膜组织炎性细胞浸润和滑膜细胞增生改善较为明显、滑膜层数减少；血清中GSH含量显著升高（P<0.01），MDA、IL-6、TNF-α含量显著降低（P<0.01）；滑膜组织ROS水平、ACSL4和LPCAT3蛋白表达显著降低（P<0.01，P<0.05），GPX4蛋白表达显著升高（P<0.05）。结论: 艾灸“肾俞”“足三里”能够减轻RA大鼠滑膜炎性损伤，其作用机制可能与降低脂质过氧化及ROS水平，抑制铁死亡的发生有关。.
Keywords: ACSL4; Ferroptosis; Lipid metabolism; Moxibustion; Rheumatoid arthritis.