尪痹片调控cGAS-STING信号通路改善肾虚证胶原诱导性关节炎大鼠炎症水平机制研究

Wangbi Tablets reduce inflammation in rat model of collagen-induced arthritis with syndrome of kidney deficiency by regulating cGAS-STING signaling pathway

探究尪痹片(Wangbi Tablets,WBT)通过调控cGAS-STING信号通路改善肾虚证胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠炎症水平的作用机制。24只SPF级SD大鼠,随机选取18只构建肾虚证CIA模型,共分为4组,每组6只,分别为空白对照组(control组)、肾虚证CIA组(model组)、肾虚证CIA+WBT组(M+WBT组)、肾虚证CIA+甲氨蝶呤(methotrexate,MTX)组(M+MTX组)。评估各组肾虚证和关节炎评分;酶联免疫吸附测定(ELISA)法检测血清白细胞介素-18(IL-18)、白细胞介素-1β(IL-1β)水平;苏木精-伊红(hematoxylin-eosin,HE)染色、番红-固绿染色观察踝关节、滑膜及软骨的病理变化情况;免疫组化(immunohistochemistry,IHC)检测踝关节骨组织内DNA聚合酶β(polymerase beta,Polβ)、环磷酸鸟苷-腺苷合成酶(cGAS)的表达水平;蛋白免疫印迹(Western blot)法检测Polβ、cGAS、干扰素刺激因子(STING)、磷酸化STING(p-STING)、核因子κB(nuclear factor-kappa B,NF-κB)、磷酸化NF-κB(phospho-NF-κB,p-NF-κB)、干扰素调节因子3(interferon regulatory factor 3,IRF3)、消皮素D(gasdermin D,GSDMD)、GSDMD蛋白N端(GSDMD-NT)、半胱氨酸蛋白酶-1(caspase-1)的表达水平;TUNEL染色检测踝关节骨组织细胞凋亡率。结果显示,与control组相比,model组肾虚和关节炎症状和体征变化最严重,经WBT干预后关节变形、精神状态、毛色有一定程度改善;与model组相比,WBT能够显著改善肾虚和关节炎症状。与control组相比,model组血清IL-18、IL-1β水平升高;与model组相比,M+WBT组IL-18水平显著降低,IL-1β有降低趋势。与control组相比,model组踝关节骨组织内可见大量炎细胞浸润伴结构破坏,软骨基质已破坏,WBT能够减轻model组大鼠踝关节骨组织的炎症、骨与软骨破坏。与control组相比,model组cGAS、IRF3、p-NF-κB/NF-κB、caspase-1、cleaved-caspase-1水平显著升高,p-STING/STING、GSDMD、GSDMD-NT升高;与model组相比,WBT干预后的model组大鼠踝关节骨组织中Polβ表达显著增强,cGAS、IRF3、GSDMD、GSDMD-NT、caspase-1表达水平显著降低,p-STING/STING、p-NF-κB/NF-κB、cleaved-caspase-1有降低趋势。与control组比,model组骨组织内、滑膜、新生血管内皮处细胞凋亡显著降低,WBT的干预能够增加滑膜等处的细胞凋亡。综上得出,WBT对肾虚证CIA大鼠有治疗作用,其机制体现在显著提高踝关节骨组织的Polβ的表达水平,抑制cGAS-STING信号通路,降低焦亡标志蛋白的表达,减少细胞因子的释放,从而减轻炎症反应,延缓骨破坏的进展,发挥防治RA的作用。

This study aims to investigate the therapeutic effect of Wangbi Tablets(WBT)on the inflammation in the rat model of collagen-induced arthritis(CIA)with the syndrome of kidney deficiency based on the cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)-stimulator of interferon genes(STING)signaling pathway.Eighteen rats were randomly chosen from 24 SPF-grade rats for the modeling of CIA with the syndrome of kidney deficiency.The 24 SPF-grade rats were randomized into 4 groups:control(normal rats),model(CIA with syndrome of kidney deficiency),model+WBT(M+WBT),and model+methotrexate(M+MTX).The syndrome score of kidney deficiency and arthritis index were recorded.The serum levels of interleukin-18(IL-18)and interleukin-1β(IL-1β)were measured by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin staining and safranin O-fast green staining were employed to observe the pathological status of ankle joints,synovium,and cartilage.Immunohistochemistry(IHC)was used to detect the expression of polymerase beta(Polβ)and cGAS in rats.The protein levels of polymerase beta(Polβ),cGAS,STING,phospho-STING(p-STING),nuclear factor-kappa B(NF-κB),phospho-NF-κB(p-NF-κB),interferon regulatory factor 3(IRF3),gasdermin-D(GSDMD),GSDMD N-terminus(GSDMD-NT),and cysteinyl aspartate specific proteinase-1(caspase-1)were determined by Western blot.Terminal-deoxynucleoitidyl transferase-mediated dUTP nick end labeling(Tunel)was employed to examine the apoptosis in the articular joints of rats.The results showed that compared with the control group,the model group showed obvious symptoms and signs of kidney deficiency and arthritis.The ankle joint deformity,mental condition,and coat color were improved by WBT.Compared with the model group,WBT alleviated the symptoms and signs of kidney deficiency and arthritis.Compared with the control group,the modeling elevated the serum levels of IL-18 and IL-1β,which were reduced by WBT,especially the level of IL-18.Compared with the control group,the model group showed a large number of inflammatory cells and damage of the cartilage layer in ankle joint,while WBT alleviated the pathological damage.Compared with the control group,the modeling significantly up-regulated the protein levels of cGAS,IRF3,p-NF-κB/NF-κB,caspase-1,and cleaved-caspase-1 and slightly up-regulated the protein levels of p-STING/STING,GSDMD,and GSDMD-NT.Compared with the model group,WBT significantly up-regulated the protein level of Polβ,significantly down-regulated the protein levels of cGAS,IRF3,GSDMD,and GSDMD-NT,and caspase-1,and slightly down-regulated the protein levels of p-STING/STING,p-NF-κB/NF-κB,and cleaved-caspase-1.Compared with the control group,the model group demonstrated decreased apoptosis in the ankle joint,synovium,and neovascularized endothelium,while WBT mitigated these situations.In conclusion,WBT has a therapeutic effect on CIA rats with the syndrome of kidney deficiency.Specifically,WBT may up-regulate the expression of Polβin the ankle joint and inhibit the cGAS-STING signaling pathway to down-regulate the expression of executive protein of pyroptosis and reduce the release of cytokines,thus inhibiting inflammation and slowing down the progression of bone destruction.