基于慢病毒介导的NLRP3过表达对艾灸调控实验性RA家兔关节滑膜液anti-CCP、RF水平的影响

Study on the Effect of Lentivirus-Mediated NLRP3 Overexpression on Levels of Anti-CCP and RF in Synovial Fluid of Experimental RA Rabbits Regulated by Moxibustion

目的观察艾灸对实验性类风湿性关节炎(RA)家兔的抗炎消肿作用,通过在RA家兔实验模型过表达由慢病毒载体介导的NOD样受体相关蛋白3(NLRP3),研究艾灸对其膝关节滑膜液中抗环瓜氨酸多肽抗体(anti-CCP)、类风湿因子(RF)、白细胞介素-18(IL-18)含量的影响及其NLRP3调控机制。方法日本大耳白兔30只,随机分为空白组、模型组、艾灸组、NLRP3过表达组、NLRP3阴性对照组,每组各6只动物。福氏完全佐剂(FCA)平均注入家兔双膝关节腔(0.5 mL·kg^(-1))。RA模型塑造后第3天及治疗后第10天,将NLRP3过表达慢病毒载体(Lv-NLRP3-GFP)10μL注入NLRP3过表达组兔双后膝关节腔,NLRP3阴性对照组家兔同法注入等量携带GFP的慢病毒载体(LvGFP)作对照。艾柱灸艾灸各组家兔双侧“肾俞”、“足三里”穴(5壮/穴/天,6天1疗程,疗程间间隔1天,3个疗程)。治疗后观测各家兔左、右后膝关节周长,ELISA法检测关节滑膜液中anti-CCP、RF、IL-18的含量。结果与空白组比较,模型组左、右膝关节周长增大,膝关节滑膜液anti-CCP、RF、IL-18含量显著升高(P<0.01);与模型组比较,艾灸组左、右膝关节周长缩小,膝关节滑膜液anti-CCP、RF、IL-18含量显著降低(P<0.01);与艾灸组相比,NLRP3过表达组左、右膝关节周长增大(P<0.05),膝关节滑膜液anti-CCP、RF、IL-18含量升高(P<0.05)。结论艾灸对实验性RA家兔具有抗炎效应,艾灸“肾俞”、“足三里”穴可显著降低RA家兔膝关节滑膜液中免疫效应因子anti-CCP、RF以及炎性细胞因子IL-18的含量,在NLRP3过表达的情况下其作用显著降低,提示艾灸抑制实验性RA家兔异常免疫功能,从而实现其抗炎调节作用可能与NLRP3的表达情况有密切关联。

Objective Observation of the anti-inflammatory and detumescence effects on experimental rheumatoid arthritis(RA)rabbits by moxibustion,With lentiviral vector mediated NLRP3 overexpression in RA animal models,to investigate the effect of moxibustion on anti-CCP/RF levels in synovial fluid of RA rabbit joints and its regulation mechanism of NLRP3.Methods Randomly divided thirty Japanese big-ear white rabbits into 5 groups:group of blank,group of model,group of moxibustion,NLRP3 overexpression group and NLRP3 overexpression negative group,with 6 rabbits in each group.Both joint of the model group,and the groups with moxibustion was injected into FCA(0.5 mL·kg^(-1)),the blank group used the same method to inject sterile saline.On the 3rd day and the 10th day after treatment,10μL of NLRP3 overexpression lentiviral vector was injected into the joint cavity of the NLRP3 overexpression group of rabbits.The rabbits in the NLRP3 overexpression negative group were injected with the same amount of LvGFP as Contrast.moxibustion bilateral"BL23","ST 36"of the groups with moxibustion(5 cones with one point in a day.6 days for 1 course,1 day for rest,3 courses of treatment in total.)After treatment,observe the circumference of each rabbit's left and right knee joints,the content of RF,anti-CCP,IL-18 in synovial fluid was detected by ELISA.Results Compared with the blank group,the left and right knee joint circumferences in the model group increased,and the anti-CCP,RF,and IL-18 contents in the synovial fluid of the knee joint increased significantly(P<0.01);compared with the modle group,The circumference of the knee joint decreased,and the content of anti-CCP,RF and IL-18 decreased significantly(P<0.01);compared with the moxibustion group,the circumference of the left and right knee joints in the NLRP3 overexpression group increased(P<0.05),the content of anti-CCP,RF and IL-18 increased(P<0.05).Conclusions Moxibustion has anti-inflammatory effects on experimental RA rabbits.Moxibustion at Shenshu(BL23)and Zusanli(ST36)can significantly reduce the level of anti-CCP,RF the immune effector,also IL-18,the in synovial fluid of RA rabbits knee joint,yet this effect can be declined significantly under NLRP3 overexpression,suggesting that moxibustion performed its anti-inflammatory regulation by suppressing abnormal immune function in experimental RA rabbits,which may be closely related to the NLRP3 expression.