甘草附子汤调控NF-κB信号通路抑制胶原诱导型关节炎小鼠骨质破坏的作用

Inhibitory Effect of Gancao Fuzitang on Bone Destruction in Collagen-induced Arthritis Mice by Regulating NF-κB Signaling Pathway

目的:探讨甘草附子汤(GCFZ)抑制胶原诱导型关节炎(CIA)小鼠模型骨质破坏的作用机制。方法:将30只雄性DBa/1J小鼠随机分为5组,分别为正常组、CIA组、甘草附子汤低剂量组(GCFZ-L,2.4 g·kg^(-1))、甘草附子汤高剂量组(GCFZ-H,4.8 g·kg^(-1))和甲氨蝶呤组(MTX,1 mg·kg^(-1)),每组6只,二次免疫法诱导CIA模型;观察记录各组小鼠关节炎指数,苏木素-伊红(HE)染色观察小鼠踝关节组织病理学变化;番红-固绿染色检测小鼠踝关节软骨破坏情况;显微计算机断层扫描(Micro-CT)扫描检测小鼠踝关节骨质破坏的情况;免疫组化法观察踝关节核转录因子-κB p65(NF-κB p65)、磷酸化核转录因子-κB p65(p-NF-κB p65)、核转录因子-κB抑制蛋白激酶α/β(IKKα/β)、磷酸化核转录因子-κB抑制蛋白激酶α/β(p-IKKα/β)的表达情况。结果:与正常组比较,CIA组关节肿胀明显,关节炎指数评分显著升高(P<0.01);与CIA组比较,GCFZ-L组、GCFZ-H组和MTX组均能显著抑制关节肿胀,并且明显降低关节炎指数评分(P<0.05,P<0.01)。HE染色和番红-固绿染色显示,与CIA组比较,治疗组(GCFZ-L组、GCFZ-H组和MTX组)能明显抑制滑膜的侵袭,并且关节软骨破坏明显减轻。Micro-CT扫描分析显示,与CIA组比较,治疗组(GCFZ-H组和MTX组)骨破坏评分显著降低(P<0.01)。免疫组化结果显示,与正常组比较,CIA组NF-κB p65、p-NF-κB p65、IKKα/β和p-IKKα/β积分吸光度均显著升高(P<0.01);而与CIA组比较,治疗组(GCFZ-H组和MTX组)NF-κB p65、p-NF-κB p65、IKKα/β和p-IKKα/β积分吸光度均明显降低(P<0.05,P<0.01);而GCFZ-L组仅NF-κB p65积分吸光度显著降低(P<0.01)。结论:甘草附子汤可能通过调控NF-κB信号通路来抑制CIA小鼠的骨质破坏。

Objective:To explore the mechanism of Gancao Fuzitang(GCFZ)in inhibiting the bone destruction of collagen-induced arthritis(CIA)model in mice.Method:Thirty male DBa/1J mice were randomly divided into normal group,CIA group,low-dose GCFZ group(GCFZ-L,2.4 g·kg^(-1)),high-dose GCFZ group(GCFZ-H,4.8 g·kg^(-1)),and methotrexate group(MTX,1 mg·kg^(-1)),with six mice in each group.The CIA model was induced by secondary immunization method.The arthritis index of mice in each group was observed and recorded,and the histopathological changes in ankle joint were observed by hematoxylin-eosin(HE)staining.The damage to ankle cartilage was detected by safranin O-fast green staining.Micro-CT scanning was used to detect the bone destruction of ankle joint,and the expression of nuclear factor-κB p65(NF-κB p65),p-NF-κB p65,inhibitory-κB kinaseα/β(IKKα/β),and p-IKKα/βwas observed by immunohistochemical staining.Result:Compared with the normal group,the CIA group showed manifest joint swelling and increased arthritis index score(P<0.01).Compared with the CIA group,the groups with drug intervention could inhibit joint swelling and reduce arthritis index score(P<0.05,P<0.01).As revealed by HE staining and safranine O-green staining,compared with the CIA group,the groups with drug intervention could inhibit synovial invasion and reduce the destruction of articular cartilage.Micro-CT scanning analysis showed that compared with the CIA group,the GCFZ-H group and the MTX group showed reduced bone destruction scores(P<0.01).The immunohistochemical results showed that compared with the normal group,the CIA group showed increased optical density values of NF-κB p65,p-NF-κB p65,IKKα/β,and p-IKKα/β(P<0.01).Compared with the CIA group,the GCFZ-H group and the MTX group showed reduced optical density values of NF-κB p65,p-NF-κB p65,IKKα/β,and p-IKKα/β(P<0.05,P<0.01).In the GCFZ-L group,only the NF-κB p65 optical density value decreased(P<0.01).Conclusion:GCFZ may inhibit bone destruction in CIA mice by regulating the NF-κB signaling pathway.