加味四妙散对急性痛风性关节炎大鼠的疗效及对滑膜组织中嘌呤能离子通道型受体7 NOD样受体热蛋白结构域相关蛋白3表达的影响

Therapeutic effect of Jiawei Simiao Powder on acute gouty arthritis rats and its influence on the expression of P2X7R and NLRP3 protein in synovial tissue

目的通过研究加味四妙散干预急性痛风性关节炎大鼠滑膜组织中嘌呤能离子通道型受体7(P2X7R)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)表达的影响,探讨加味四妙散在防治急性痛风性关节炎的疗效及抗炎作用机制。方法选择雄性SD大鼠60只,按照随机数字表法分为6组,分别为空白对照组、模型组、秋水仙碱组、加味四妙散低剂量组、加味四妙散中剂量组、加味四妙散高剂量组,每组10只。空白对照组给予等体积0.9%氯化钠注射液在相应的部位,其余5组复制高尿酸血症并急性痛风性关节炎大鼠模型。加味四妙散组予加味四妙散低、中、高剂量水提液(4.24 g/kg、8.48 g/kg、16.96 g/kg)灌胃、秋水仙碱组予秋水仙碱混悬液(3 mg/kg)灌胃。模型组予等量0.9%氯化钠注射液灌胃。经过4 h、8 h、12 h、24 h和48 h的治疗,对右踝关节的肿胀情况及大鼠步态进行观察。末次给药2 h后,通过腹主动脉采血,采用生化法检测血清尿酸浓度、肝肾功能及血清三磷酸腺苷(ATP)水平,酶联免疫吸附试验(ELISA)测定血清中白细胞介素(IL)-1β、IL-8含量,取受试踝关节滑膜组织,苏木精-伊红染色(HE)观察滑膜组织病理情况,利用蛋白免疫印迹法检测滑膜组织中NLRP3、P2X7R蛋白的表达。结果除空白对照组外,各组大鼠的踝关节肿胀率从造模后4~8 h开始上升,24 h达高峰后下降。在造模48 h后,秋水仙碱组和加味四妙散的低、中、高剂量组的肿胀率减轻,步态逐渐改善(P<0.05),与空白组比较,模型组中滑膜组织表现出关节炎病理改变,显微镜下可见滑膜脱落、坏死,滑膜细胞增生,相较于模型组,加味四妙散的低、中、高剂量组以及秋水仙碱组在血清ATP、IL-1β、血尿酸、IL-8均降低,滑膜组织关节炎病理情况得到改善。滑膜组织P2X7R、NLRP3蛋白表达均降低(P<0.05),与秋水仙碱组相比,加味四妙散对大鼠肝肾功能的损害较小(P<0.05)。结论加味四妙散可以降低高尿酸血症和痛风模型大鼠的血尿酸水平,减少对肝肾功能的损害,抑制急性痛风性关节炎大鼠踝关节滑膜组织中NLRP3、P2X7R蛋白的表达,降低局部组织炎症反应,从而发挥治疗作用。

Objective To study the therapeutic effect of Jiawei Simiao Powder on acute gouty arthritis rats and its influence on the expression of P2X7R and NLRP3 protein in synovial tissue.Methods A total of 60 male SD rats were divided into 6 groups using the random number table method,with 10 rats in each group.The rat model with hyperuricemia and acute gouty arthritis was replicated.The normal control group was given an equal volume of physiological saline in the corresponding parts.While the Jiawei Simiao Powder group was given a low,medium or high dose of water extract(4.24 g/kg、8.48 g/kg、16.96 g/kg).The colchicine group was given colchicine suspension(3 mg/kg).The model group was adminstered an equal volume of 0.9%sodium chloride injection via gavage.After 4 h,8 h,12 h,24 h and 48 h treatment,the swelling of the right ankle joint and the gait of the rats were observed.Two hours after the last administration,blood samples were collected from the abdominal aorta,serum uric acid concentration,liver and kidney function and serum ATP level were detected by biochemical method,serum IL1βand IL8 levels were determined by enzymelinked immunosorbent assay(ELISA),and ankle synovium tissues were collected.Hematoxylineosinstaining(HE)was used to observe the pathology of synovial tissue,and the expression of NLRP3 and P2X7R proteins in synovial tissue was detected by Western Blot(WB).Results Except for the normal control group,the ankle joint swelling rate of rats in each group began to increase from 48 h after modeling and peaked at 24 h before decreasing.After 48 hours of modeling,the swelling rates of the colchicine group and the low,medium,and highdose groups of Jiawei Simiao powder were significantly reduced,and the gait was gradually improved(P<0.05).Compared with the blank group,the synovial tissue in the model group showed obvious pathological changes of arthritis,and the synovial tissue was exfoliated,necrosis,and synovial cell proliferation were observed under the microscope.The levels of serum ATP,IL1β,blood uric acid and IL8 in the low,medium and high dose groups of supplemented Simiao Powder and colchicine group were significantly reduced,and the pathological condition of synovial arthritis was improved.The protein expressions of P2X7R and NLRP3 in synovial tissue were significantly decreased(P<0.05).Compared with colchicine group,supplemented Simiao powder had less damage to liver and kidney function(P<0.05).Conclusion Jiawei Simiao Powder can reduce the blood uric acid level of hyperuricemia and gout model rats,reduce the damage to liver and kidney function,inhibit the expression of NLRP3 and P2X7R proteins in ankle synovium tissue of rats with acute gouty arthritis,and reduce local tissue inflammatory response,and thus exert therapeutic effects.