Study on the Effect and Mechanism of Wheat-Grain Moxibustion on Adjuvant Arthritis Rats Based on ROS-NLRP3 Pathway
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摘要:
目的 探讨麦粒灸通过调控ROS-NLRP3炎性通路从而对类风湿关节炎(Rheumatoid arthritis, RA)发挥抗炎镇痛效应的分子机制。 方法 将47只雄性SD大鼠通过SPSS软件随机程序分为空白组、模型组、模型艾灸组、ROS过表达组、ROS过表达艾灸组。其中空白组7只, 其余4组每组10只。对空白组大鼠从右后足垫进行皮内注射无菌生理盐水, 其余通过右后足垫皮内注射弗氏完全佐剂(Freund's complete adjuvant, FCA)诱导关节炎(Adjuvant arthritis, AA)大鼠模型。FCA造模后, ROS过表达组和ROS过表达艾灸组于造模后第2天皮下注射鱼藤酮油溶液(1.5 mg·kg-1)制备ROS过表达模型。模型艾灸组和ROS过表达艾灸组造模后7 d取大鼠足三里穴、肾俞穴进行麦粒灸治疗, 各穴5壮, 两侧交替。艾灸治疗6天为1个疗程, 疗程之间休息1 d, 共治疗3个疗程。空白组、模型组及ROS过表达组按同法固定, 不予艾灸治疗。造模、治疗前后观察大鼠足跖肿胀度、热痛阈及屈腿嘶鸣评分变化; 治疗结束后取材, 采用ELISA法检测大鼠血清中IL-1β、IL-18炎症因子变化; Western blot法检测大鼠踝关节炎性组织NLRP3蛋白、ROS相关蛋白活性氧调节因子1(ROMO1)表达量; qPCR法检测下丘脑ROS相关ROMO1 mRNA的表达量。 结果 ROS过表达组体质量在实验周期内无明显增加, 其余各组均有增加(P < 0.05)。与空白组相比, 模型组右足肿胀度、热痛阈提高百分率、屈腿嘶鸣评分均有显著升高(P < 0.01), 而艾灸后模型艾灸组右足肿胀度、热痛阈提高百分率、屈腿嘶鸣评分则显著下降(P < 0.01)。同时, 与空白组相比, 模型组踝关节炎性组织NLRP3、ROMO1表达量及下丘脑ROMO1 mRNA表达量、IL-1β、IL-18水平显著上升(P < 0.01), 而模型艾灸组艾灸后以上炎症通路及炎性因子表达量均显著下降(P < 0.01);与ROS过表达组相比, ROS过表达艾灸组行为学表现(足肿胀度、痛阈提高百分率、屈腿嘶鸣评分)均有显著改善(P < 0.01), 踝关节炎性组织NLRP3、ROMO1表达量及下丘脑ROMO1 mRNA表达量、IL-1β、IL-18水平显著下降(P < 0.01)。 结论 麦粒灸可能通过抑制AA模型大鼠ROS-NLRP3-IL-1β炎性通路从而发挥抗炎、镇痛作用。 Abstract:OBJECTIVE To explore the molecular mechanism by which wheat-grain moxibustion exerts anti-inflammatory and analgesic effects on rheumatoid arthritis (RA) by regulating the ROS-NLRP3 inflammatory pathway. METHODS 47 male SD rats were randomly divided into control group, model group, model moxibustion group, ROS overexpression group and ROS overexpression moxibustion group by SPSS random program. There were 7 rats in the control group, and 10 rats in each of the other 4 groups. Rats in the control group were intradermally injected with sterile normal saline from the right posterior foot pad, while the rest were intradermally injected with Freund's complete adjuvant (FCA) Adjuvant to induce adjuvant arthritis (AA). After FCA modeling, ROS overexpression group and ROS overexpression moxibustion group were injected subcutaneously with rotenone oil solution (1.5 mg·kg-1) on the second day after modeling to establish ROS overexpression model. The model moxibustion group and ROS overexpression moxibustion group were treated with wheat-grain moxibustion at Zusanli point and Shenshu point 7 days after modeling, with 5 grains on each point, alternating on both sides. Moxibustion treatment lasts for 6 days as a course of treatment, with a day off between the courses. There is a total of 3 courses of treatment. Control group and model group were fixed by the same method without moxibustion treatment. The swelling degree of the foot plantar, thermal pain threshold and arthritic flexion pain test score were observed before and after modeling and treatment. After treatment, samples were collected and ELISA was used to detect the changes of inflammatory factors IL-1β and IL-18 in serum of rats. The expressions of NLRP3 protein and ROS-related protein reactive oxygen regulatory Factor 1 (ROMO1) in ankle inflammatory tissue were detected by Western blot. The expression level of ROS-related ROMO1 mRNA in hypothalamus was detected by qPCR. RESULTS The body mass of the ROS overexpression group did not significantly increase during the experimental period, while all other groups increased (P < 0.05). Compared with control group, the swelling degree of the right foot, the percentage increase of thermal pain threshold and the score of arthritic flexion pain test were significantly increased in the model group (P < 0.01), while the swelling degree, the percentage increase of thermal pain threshold and the score of arthritic flexion pain test were significantly decreased in the model moxibustion group after moxibustion (P < 0.01). Meanwhile, compared with control group, the expressions of NLRP3 and ROMO1 in inflammatory ankle tissue and the expressions of ROMO1 mRNA, IL-1β and IL-18 in hypothalamus in model group were significantly increased (P < 0.01), while the expressions of inflammatory pathways and inflammatory factors above in model moxibustion group were significantly decreased after moxibustion (P < 0.01). Compared with the ROS overexpression group, the ROS overexpression moxibustion group showed significant improvement in behavioral performance (degree of foot swelling, percentage increase of thermal pain threshold and score of arthritic flexion pain test) (P < 0.01). The expressions of NLRP3 and ROMO1 in the inflammatory tissue of ankle joint and the ROMO1 mRNA expression, IL-1β and IL-18 in hypothalamus were significantly decreased (P < 0.01). CONCLUSION Wheat-grain moxibustion may inhibit ROS-NLRP3-IL-1β inflammatory pathways in AA model rats, thereby exerting anti-inflammatory and analgesic effects. -
Key words:
- moxibustion /
- rheumatoid arthritis /
- IL-1β /
- IL-18 /
- NLRP3 /
- ROS /
- pain /
- anti-inflammatory
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图 2 艾灸对不同ROS水平下AA模型大鼠足肿胀度的影响
注: 与空白组比较, **P < 0.01;与模型组比较, ΔΔP < 0.01;与ROS过表达组比较, ▲▲P < 0.01;与模型艾灸组比较, ##P < 0.01。
Figure 2. Effect of moxibustion on the degree of foot swelling in AA model rats with different ROS levels
图 3 艾灸对不同ROS水平下AA模型大鼠右足屈腿嘶鸣评分的影响
注: 与空白组比较, **P < 0.01;与模型组比较, ΔΔP < 0.01;与ROS过表达组比较, ▲▲P < 0.01;与模型艾灸组比较, ##P < 0.01。
Figure 3. Effect of moxibustion on the score of right foot flexion and neigh in AA model rats with different ROS levels
图 4 艾灸对不同ROS水平下AA模型大鼠热痛阈提高百分率的影响
注: 与空白组比较, **P < 0.01;与模型组比较, ΔΔP < 0.01;与ROS过表达组比较, ▲▲P < 0.01;与模型艾灸组比较, ##P < 0.01。
Figure 4. Effect of moxibustion on the percentage increase of heat pain threshold in AA model rats with different ROS levels
图 5 艾灸对不同ROS水平下AA模型大鼠踝关节炎性组织中ROS相关蛋白ROMO1、NLRP3表达水平的影响
注: 与空白组比较, **P < 0.01;与模型组比较, ΔΔP < 0.01;与ROS过表达组比较, ▲▲P < 0.01;与模型艾灸组比较, ##P < 0.01。x±s, n=3。
Figure 5. Effect of moxibustion on the expression levels of ROMO1 and NLRP3 proteins in ankle inflammatory tissue of AA model rats under different ROS levels
图 6 艾灸对不同ROS水平下AA模型大鼠下丘脑ROMO1 mRNA表达水平的影响
注: 与空白组比较, **P < 0.01;与模型组比较, ΔΔP < 0.01;与ROS过表达组比较, ▲▲P < 0.01;与模型艾灸组比较, ##P < 0.01。x±s, n=3。
Figure 6. Effect of moxibustion on the expression level of ROMO1 mRNA in hypothalamus of AA model rats with different ROS levels
图 7 各组大鼠治疗结束后(Day28)IL-1β、IL-18表达水平比较
注: 与空白组比较, **P < 0.01;与模型组比较, ΔΔP < 0.01;与ROS过表达组比较, ▲▲P < 0.01;与模型艾灸组比较, ##P < 0.01。x±s, n=3。
Figure 7. Effect of moxibustion on serum levels of IL-1β and IL-18 in AA model rats with different ROS levels
表 1 各组大鼠体质量在造模与艾灸治疗干预前后变化(x±s, g)
Table 1. Effects of moxibustion on body weight changes in AA model rats under different ROS levels (x±s, g)
分组 n 造模前(Day1) 造模后(Day9) 艾灸治疗后(Day28) 空白组 7 228.71±8.55 229.42±14.54 261.85±7.83 模型组 10 226.00±20.04 224.30±38.02 257.80±46.03 模型艾灸组 10 231.60±9.89 228.00±16.65 274.70±14.72 ROS过表达组 10 229.80±11.76 210.70±17.10** 237.70±29.34 ROS过表达艾灸组 10 234.60±24.28 212.20±18.77** 265.80±19.10Δ 注: 与模型组比较, **P < 0.01;与ROS过表达组比较, ΔP < 0.05。 
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