桂枝芍药知母汤抑制cGAS/STING/NF-κB通路对类风湿关节炎大鼠炎症活动的影响

Guishao Zhimu Decoction alleviates inflammatory response in rheumatoid arthritis rats via inhibiting cGAS-STING-NFκB axis

目的基于环状GMP-AMP合成酶(cGAS)/干扰素基因刺激因子(STING)/核因子-κB(NF-κB)通路探讨桂枝芍药知母汤对类风湿关节炎大鼠炎症活动的影响及机制。方法取70只Wistar大鼠,随机选择10只作为正常组,其余60只大鼠制备胶原诱导性关节炎模型。将造模成功大鼠随机分为模型组、甲氨蝶呤组、雷公藤组及桂芍知母汤低、中、高剂量组,每组10只。甲氨蝶呤组给予0.104 mg/mL的甲氨蝶呤片混悬液灌胃,1次/周,其余时间每天给予蒸馏水灌胃1次;雷公藤组给予0.625 mg/mL的雷公藤多甙片混悬液灌胃,桂芍知母汤低、中、高剂量组分别给予0.958 g/mL、1.916 g/mL、2.874 g/mL的桂枝芍药知母汤灌胃,正常组、模型组给予蒸馏水灌胃,均1次/d。各组均干预4周。测量各组大鼠干预1,2,3,4周末足趾肿胀程度,并计算关节炎指数(AI);末次干预结束后,ELISA法检测血清白细胞介素-1β(IL-1β)、白细胞介素-17(IL-17)、肿瘤坏死因子-α(TNF-α)水平,Western blot法检测滑膜组织中cGAS、STING、NF-κB p65、IκBα蛋白表达情况。结果干预1,2,3,4周末,模型组大鼠的关节肿胀度均明显高于正常组(P均<0.05);随时间推移,模型组大鼠的关节肿胀度逐渐增加,各药物干预组大鼠的关节肿胀度有不同程度的降低,其中桂芍知母汤中剂量组干预4周末的关节肿胀度明显低于模型组(P<0.05)。干预1周末,各组间AI评分比较差异均无统计学意义(P均>0.05);干预2,3,4周末,桂芍知母汤高剂量组AI评分均明显低于模型组和桂芍知母汤低剂量组(P均<0.05),且桂芍知母汤高剂量组干预2周末的AI评分明显低于雷公藤组(P<0.05);干预3周末,甲氨蝶呤组、桂芍知母汤中剂量组AI评分均明显低于模型组(P均<0.05);干预4周末,甲氨蝶呤组、雷公藤组、桂芍知母汤中剂量组、桂芍知母汤低剂量组AI评分均明显低于模型组(P均<0.05)。与正常组比较,模型组血清IL-1β、IL-17、TNF-α水平及滑膜组织中cGAS、STING、NF-κB p65蛋白相对表达量均明显升高(P均<0.05),滑膜组织中IκBα蛋白相对表达量明显降低(P<0.05);与模型组比较,各药物干预组血清IL-1β、IL-17、TNF-α水平及滑膜组织中cGAS、STING、NF-κB p65蛋白相对表达量均明显降低(P均<0.05),滑膜组织中IκBα蛋白相对表达量均明显升高(P均<0.05)。桂芍知母汤高剂量组和甲氨蝶呤组血清IL-1β、IL-17和TNF-α水平及滑膜组织中cGAS、STING、NF-κB p65蛋白相对表达量均明显低于桂芍知母汤低剂量组(P均<0.05),滑膜组织中IκBα蛋白相对表达量均明显高于桂芍知母汤低剂量组(P均<0.05)。结论桂枝芍药知母汤可明显减轻类风湿关节炎大鼠关节肿胀和关节炎症,机制可能与抑制cGAS/STING/NF-κB通路,降低炎症因子水平有关。

Objective It is to investigate the effect and mechanism of Guishao Zhimu Decoction(GSZD)on inflammatory activity in rheumatoid arthritis rats via inhibiting cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)-nuclear factor kappa-B(NF-κB)axis.Methods Seventy Wistar rats were taken,in which 10 rats were randomly selected as the normal group,and the remaining 60 rats were prepared to establish models of collagen-induced arthritis.The successfully modeled rats were randomly divided into model group,meth otrexate(MTX)group,tripterysium glycosides(TG)group and low,medium and high dose groups of GSZD,with 10 rats in each group.The MTX group was given 0.104 mg/mL MTX tablet suspension by gavage once per week,and given distilled water by gavage once per day for the rest of the week;the TG group was given 0.625 mg/mL TG tablet suspension by gavage,and the low,medium,and high dosage groups of GSZD were respectively given 0.958 g/mL,1.916 g/mL,and 2.874 g/mL GSZD by gavage,and the normal group and model group were given distilled water by gavage,all once daily.All the groups were treated for 4 weeks.The degree of toe swelling of rats in each group was measured after 1,2,3,and 4 weeks of treatment,and the arthritis index(AI)was calculated;at the end of the final intervention,the serum levels of interleukin-1β(IL-1β),interleukin-17(IL-17)and tumor necrosis factor-α(TNF-α)were detected by ELISA,and the expressions of cGAS,STING,NF-κB p65,and IκBαproteins in synovial tissue were detected by Western blot.Results At the end of 1,2,3 and 4 weekends of the intervention,the joint swelling of rats of the model group was significantly higher than that of the normal group(all P<0.05);with the increase in time,the joint swelling of rats in the model group were gradually increased,and that of rats in every treatment group were decreased to different degrees,the joint swelling of GSZD medium dose group was significantly lower than that of the model group at the end of 4 weekends of the intervention(P<0.05).At the end of one week of intervention,the differences in AI scores among each group were not statistically significant(all P>0.05);at the end of 2,3,and 4 weeks of intervention,the AI scores of the GSZD high-dose group were significantly lower than those of the model group and GSZD low-dose group(all P<0.05),and the AI scores of the GSZD high-dose group were significantly lower than those of the TG group at the end of 2 weeks of intervention(all P<0.05);at the end of 3 weeks of intervention,the AI scores of the MTX group and the GSZD medium-dose group were significantly lower than those of the model group(all P<0.05);at the end of 4 weeks of intervention,the AI scores of the MTX group,GSZD medium-dose group and GSZD low-dose group were all significantly lower than those of the model group(all P<0.05).Compared with the normal group,the serum levels of IL-1β,IL-17,TNF-αand relative expressions of cGAS,STING and NF-κB p65 proteins in synovial tissues of the model group were significantly increased(all P<0.05),and the relative expression of IκBαprotein in synovial tissues was significantly decreased(P<0.05);compared with the model group,the serum levels of IL-1β,IL-17 and TNF-αand relative expressions of cGAS,STING,NF-κB p65 proteins in synovial tissues of each treatment group were significantly reduced(all P<0.05),and relative expression of IκBαprotein in synovial tissues was significantly increased(all P<0.05).The serum levels of IL-1β,IL-17 and TNF-αand relative expressions of cGAS,STING,NF-κB p65 proteins in synovial tissues of the MTX group and GSZD high dose group were significantly lower,while the relative expression of IκBαprotein in synovial tissues was significantly higher than those of the GSZD low dose group(all P<0.05).Conclusion Guishao Zhimu Decoction can significantly alleviate joint swelling and joint inflammation in rats with rheumatoid arthritis,and the mechanism may be related to inhibit cGAS/STING/NF-κB pathway,thus to reduce the levels of inflammatory factors.