Objective: To observe the effect of moxibustion on expression of autophagy related gene(Atg), serine/threonine protein kinase-uncoordinated 51 like kinase-1 (ULK1), Beclin1 and microtubule associated proteins light chain 3 (LC3) and ultrastructure of synovium in rheumatoid arthritis (RA) rats, so as to explore its mechanisms underlying improvement of RA.
Methods: Forty SD rats were randomly divided into normal control, RA model, moxibustion, cigarette-roasting and medication groups (n=8 rats in each group). The RA model was established by keeping the rats in wind, cold and wet environment for 12 h, once a day for 20 days and subcutaneous injection of Freund's adjuvant complete into the sole of the left hind paw. Moxibustion was applied to the left "Zusanli" (ST36) for 20 min, once a day for 15 days. Rats of the cigarette-roasting group was treated by ignited cigarettes instead of moxa strips. Rats of the medication group was treated by gavage of Tripterygium wilfordii polyglycoside tablet suspension (0.8 mg/100 g) once a day for 15 days. The rats' paw volume of the left hindlimb was measured by using a water-based paw plethysmometer. The synovial tissue of the left plantar joint was harvested at the end of experiments for observing changes of the ultrastructure with transmission electron microscope, and the expression of ULK1, Atg3, Atg5, and Atg12 mRNAs was detected with quantitative real-time PCR and the expression of LC3-Ⅱ and Beclin-1 proteins were detected with Western blot.
Results: Following modeling, the paw volume of the left hindlimb was significantly increased (P<0.01), while the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins of the synovial tissue were notably down-regulated in the model group relevant to the normal control group (P<0.01). The increase of the paw volume in the moxibustion and medication groups and the down-regulation of synovial Atg3, Atg12 and ULK1 mRNAs in the 3 intervention groups, and Atg5 mRNA , and LC3-Ⅱ and Beclin-1 proteins in both moxibustion and medication groups were considerably suppressed (P<0.01, P<0.05). The therapeutic effect of moxibustion was apparently superior to that of cigarette-roasting in down-regulating the paw volume, and up-regulating the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins (P<0.05, P<0.01), and notably weaker than that of medication in up-regulating Atg3 and ULK1 mRNAs (P<0.01), but was comparable to that of medication in up-regulating the expression levels of Atg5 and Atg12 mRNAs, LC3-Ⅱand Beclin-1 proteins (P＞0.05). Results of the ultrastructural observation showed an obvious injury of synovial cells, such as unclear and incomplete nuclear membrane, chromatin condensation, swollen mitochondria with broken crests, cavitation-like degeneration of cytoplasma, and appearance of autophagosomes and lysosomes in the model group, which was relatively milder in the 3 intervention groups.
Conclusion: Moxibustion can reduce the paw edema and inflammatory injury of the plantar synovial tissue in RA rats, which may be related to its effects in up-regulating Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins to enhance the cellular autophagy. The therapeutic effect of moxibustion is obviously superior to that of cigarette-roasting and medication in relieving swelling.
目的：观察艾灸对类风湿性关节炎(RA)大鼠滑膜组织中自噬相关基因(Atg)、丝氨酸/苏氨酸蛋白激酶-失调51样激酶1(ULK1)、Beclin-1和微管相关蛋白轻链3(LC3)表达及滑膜细胞超微结构的影响，探讨艾灸通过细胞自噬途径发挥抗炎和调节细胞增殖作用的机制。方法：40只SD大鼠随机分为正常组、模型组、艾灸组、香烟灸组和药物组，每组8只。采用风、寒、湿环境因素结合左后足跖皮下注射完全弗氏佐剂建立RA模型。艾灸组使用直径0.9 cm的艾条对“足三里”(左侧)施灸，施灸距离皮肤2 cm，每日1次，每穴每次灸20 min，共灸15 d；香烟灸组处理方法同艾灸组，用直径0.8 cm普通卷烟代替艾条；药物组予以雷公藤多苷片混悬液灌胃(0.8 mg/100 g)，每日1次，连续给药15 d。用足跖容积测量仪测量各组大鼠左后肢足跖容积，透射电镜观察滑膜细胞超微结构，荧光定量PCR法检测滑膜组织Atg3、Atg5、Atg12、ULK1 mRNA的表达，Western blot法检测滑膜组织LC3-Ⅱ、Beclin-1蛋白的表达。结果：与正常组比较，模型组大鼠左后肢足跖容积增加(P<0.01)，滑膜组织Atg3、Atg5、Atg12、ULK1 mRNA及LC3-Ⅱ、Beclin-1蛋白表达均显著降低(P<0.01)。与模型组比较，艾灸组和药物组大鼠足跖关节容积均显著降低(P<0.01)，Atg3、Atg5、Atg12、ULK1 mRNA及LC3-Ⅱ、Beclin-1蛋白表达均显著升高(P<0.01)；香烟灸组Atg3、Atg12、ULK1 mRNA表达显著增加(P<0.05，P<0.01)。与艾灸组比较，药物组、香烟灸组大鼠左后肢足跖关节容积均增加(P<0.05，P<0.01)，香烟灸组Atg3、Atg5、Atg12、ULK1 mRNA及LC3-Ⅱ、Beclin-1蛋白表达均降低(P<0.01，P<0.05)，药物组Atg3、ULK1 mRNA表达显著升高(P<0.01)。与香烟灸组比较，药物组左后肢足跖关节容积显著降低(P<0.01)，Atg3、Atg5、Atg12、ULK1 mRNA及LC3-Ⅱ、Beclin-1蛋白表达均显著升高(P<0.01)。与正常组比较，模型组滑膜细胞损伤明显，出现病理性改变；与模型组比较，艾灸组、药物组和香烟灸组均有不同程度缓解。结论：RA模型大鼠关节滑膜细胞的自噬水平低于正常大鼠，艾灸和雷公藤多苷灌胃干预能改善关节肿胀和滑膜细胞损伤，增强其自噬水平，且艾灸的消肿作用强于雷公藤多苷。艾灸可能通过提高自噬水平发挥修复RA滑膜细胞损伤的作用。.
Keywords: Autophagy-related molecules; Cellular autophagy; Cigarette-roasting; Moxibustion; Rheumatoid arthritis; Synoviocytes; Ultrastructure.