目的 探究NOD样受体蛋白3（NLRP3）炎症小体介导的细胞焦亡在痛风性关节炎（GA）中的调控机制。方法 通过Gene Cards数据库筛选GA相关靶点，GSEA数据库检索细胞焦亡相关靶点，借助STRING平台构建交集基因的蛋白质-蛋白质相互作用（PPI）网络，预测GA中细胞焦亡相关作用机制；将96只SPF级雄性大鼠适应性喂养1周后，分2批（每批48只），每批随机分为对照组、模型组、MCC950(NLRP3抑制剂，2、4、8、10 mg·kg^-1)组或LDC7559[(消皮素D（GSDMD）抑制剂，2、4、8、10 mg·kg^-1]组，每组8只。除对照组外，其余各组大鼠均通过右后肢踝关节注射尿酸钠（MSU）构建GA大鼠模型，以大鼠血清中白细胞介素-1β（IL-1β）含量及关节组织中IL-1β mRNA及蛋白表达水平为指标，筛选2种抑制剂的最佳作用剂量；另取32只SPF级雄性大鼠随机分为对照组、模型组、MCC950(8 mg·kg^-1)组、LDC7559(4 mg·kg^-1)组，除对照组外，其余各组均采用上述相同方法构建GA模型。计算各组大鼠关节肿胀度；采用苏木素-伊红（HE）染色法观察关节组织病理变化；全自动生化分析仪测定大鼠血清尿酸（SUA）、肌酐（SCr）、尿素氮（BUN）含量；酶联免疫吸附法（ELISA）检测大鼠血清IL-1β、IL-8、TNF-α水平；实时荧光定量PCR（q RT-PCR）法检测大鼠关节组织中凋亡相关斑点样蛋白（ASC）、含半胱氨酸的天冬氨酸蛋白水解酶-1（Caspase-1）、NLRP3、GSDMD、IL-1β的mRNA表达；Western blotting检测大鼠关节组织中NLRP3、GSDMD、消皮素D-N端（GSDMD-N）、IL-1β、白细胞介素1β前体（pro-IL-1β）、Caspase-1、半胱氨酸天冬氨酸蛋白水解酶-1前体（pro-Caspase-1）、ASC蛋白表达水平；免疫共沉淀（COIP）法验证NLRP3与GSDMD的结合情况。明确最佳抑制剂剂量干预下NLRP3/GSDMD介导的细胞焦亡在GA中的调控机制。结果 网络药理学分析显示，共筛选得到352个GA作用靶点、60个细胞焦亡作用靶点及14个交集基因，核心靶点与NOD样受体通路密切相关；MCC950、LDC7559的最佳剂量分别为8、4 mg·kg^-1。与对照组相比，MSU诱导的GA模型大鼠右后肢踝关节及足底均发生不同程度的红肿、发热，关节滑膜重度增厚，结缔组织增生，伴新生血管形成及淋巴细胞、中性粒细胞浸润，关节腔内可见坏死细胞碎片；血清SUA、SCr、BUN及IL-1β、IL-8、TNF-α水平均显著升高（P<0.01），关节组织中NLRP3、GSDMD、ASC、Caspase-1、IL-1β等焦亡关键基因及蛋白表达均显著上调（P<0.05、0.01、0.001），提示GA急性发作时NOD样受体通路激活并诱发焦亡；经MCC950或LDC7559干预后，NLRP3炎症小体的活化被抑制，GSDMD、ASC、Caspase-1、IL-1β蛋白表达降低；且抑制GSDMD激活后NLRP3蛋白表达也显著下调；COIP结果证实，NLRP3与GSDMD具有紧密的互作关系。结论 NLRP3/GSDMD通路可通过调控细胞焦亡参与GA疾病进程，为GA的治疗提供潜在靶点。

Objective To explore the regulatory mechanism of NOD-like receptor protein 3（NLRP3） inflammasome-mediated pyroptosis in gouty arthritis（GA）. Methods GA-related targets were screened through the GeneCards database, and pyroptosisrelated targets were retrieved from the GSEA database. The protein-protein interaction（PPI） network of the intersection genes was constructed with the STRING platform to predict the mechanism of pyroptosis in GA. Ninety-six SPF male rats were adaptively fed for one week and then divided into two batches（48 rats each）. Each batch was randomly divided into a control group, a model group, MCC950(NLRP3 inhibitor, 2, 4, 8, 10 mg·kg^-1) groups or LDC7559 [(Gasdermin D（GSDMD） inhibitor, 2, 4, 8, 10 mg·kg^-1) groups, with 8 rats in each group. Except for the control group, the other groups were injected with sodium monosulfate（MSU） into the right hind ankle joint to establish the GA rat model. The content of interleukin-1β（IL-1β） in rat serum and the expression levels of IL-1β mRNA and protein in joint tissues were used as indicators to screen the optimal doses of the two inhibitors. Another 32 SPF male rats were randomly divided into a control group, a model group, an MCC950(8 mg·kg^-1) group, and an LDC7559(4 mg·kg^-1) group. Except for the control group, the other groups were also established with the same method as above. The degree of joint swelling in each group was calculated. The pathological changes of joint tissues were observed by hematoxylin-eosin（HE） staining. The contents of serum uric acid（SUA）, creatinine（SCr）, and blood urea nitrogen（BUN） in rats were determined by an automatic biochemical analyzer. The levels of IL-1β, IL-8, and TNF-α in rat serum were detected by enzyme-linked immunosorbent assay（ELISA）. The mRNA expressions of apoptosis-associated speck-like protein（ASC）, Caspase-1, NLRP3, GSDMD, and IL-1β in rat joint tissues were detected by real-time fluorescence quantitative PCR（qRT-PCR）. The protein expressions of NLRP3, GSDMD, Gasdermin D-Nterminal（GSDMD-N）, IL-1β, pro-IL-1β, Caspase-1, pro-Caspase-1, and ASC in rat joint tissues were detected by Western blotting. The binding of NLRP3 and GSDMD was verified by co-immunoprecipitation（COIP）. The regulatory mechanism of NLRP3/GSDMDmediated pyroptosis in GA under the optimal inhibitor dose was clarified. Results Network pharmacological analysis showed that a total of 352 GA-related targets, 60 pyroptosis-related targets, and 14 intersection genes were screened, and the core targets were closely related to the NOD-like receptor pathway. The optimal doses of MCC950 and LDC7559 were 8 and 4 mg·kg^-1, respectively. Compared with the control group, the right hind ankle joint and plantar of the MSU-induced GA model rats showed varying degrees of redness, swelling, and fever, with severe thickening of the synovium, connective tissue hyperplasia, accompanied by neovascularization and infiltration of lymphocytes and neutrophils, and necrotic cell debris were visible in the joint cavity. The levels of serum SUA, SCr, BUN, IL-1β, IL-8 and TNF-α were all significantly increased（P ＜ 0.01）, and the expressions of key pyroptosis-related genes and proteins such as NLRP3, GSDMD, ASC, Caspase-1 and IL-1β in joint tissues were significantly upregulated（P ＜ 0.05, 0.01, 0.001）, suggesting that the NOD-like receptor pathway was activated and pyroptosis was induced during the acute attack of GA. After intervention with MCC950 or LDC7559, the activation of NLRP3 inflammasome was inhibited, and the expressions of GSDMD, ASC, Caspase-1 and IL-1β proteins were decreased. Moreover, the expression of NLRP3 protein was also significantly downregulated after inhibiting GSDMD activation. COIP results confirmed that NLRP3 and GSDMD have a close interaction. Conclusion The NLRP3/GSDMD pathway can participate in the disease process of GA by regulating GA cell pyroptosis, providing a potential target for the treatment of GA.

R684.3;R329.25

国家自然科学基金资助项目(82074149); 黑龙江省自然科学基金资助项目(PL2024H242)