【作者】 凌琳；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2011， 硕士

【摘要】 目的:根据佐剂性关节炎（adjuvant arthritis,AA）大鼠的足爪肿胀、评分、关节滑膜及病理形态学等改变,观察芍药苷（paeoniflorin,Pae）对AA大鼠巨噬细胞分泌Il-1水平、T、B细胞增殖水平的影响;在整体、器官、细胞水平上明确Pae对AA大鼠的治疗作用;以AA发病的靶细胞—成纤维滑膜细胞（fibroblast-like synoviocytes,FLS）为突破口,观察Pae对AA大鼠FLS的增殖、cAMP水平、含有SH2结构域的酪氨酸蛋白磷酸酶（ Src homology 2 domain-containing protein tyrosine phosphatase-2,SHP-2）、G蛋白偶联受体激酶2（G-protein-coupled receptor kinases 2,GRK₂）表达的影响;分析Pae是否通过影响SHP-2在AA大鼠FLS中的表达情况而发挥效应。明确SHP-2在FLS中调节GRK₂的作用及Pae治疗大鼠AA的可能的分子机制。方法:大鼠左后足跖皮内注射弗氏完全佐剂（Freund’s complete adjuvant,FCA）制备大鼠AA模型;以Pae(25、50、100 mg·kg^-1)灌胃给药（intragastric administration, ig）（d14-d24）。计数法测定多发性关节炎指数,称重法测定脾脏指数, ELISA法检测巨噬细胞分泌IL-1水平,氚标记的胸腺嘧啶核苷掺入法测定T、B淋巴细胞增殖反应,HE染色法光镜观察关节病理形态学变化等。组织块法培养大鼠FLS,MTT法检测FLS的增殖反应;放射性免疫法测定FLS cAMP水平;Western blot技术检测FLS中GRK₂、SHP-2的表达变化。结果:1. Pae对AA大鼠的治疗作用Pae(25、50、100 mg·kg^-1)于造模后d14- d24灌胃给药均不同程度明显减轻AA大鼠多发性关节炎指数、继发侧肿胀度、升高的脾脏指数、升高的巨噬细胞分泌IL-1水平、增强的T、B淋巴细胞增殖反应及关节病理形态改变等方面都有不同程度的改善作用。光镜下观察关节滑膜组织中轻度的滑膜增生、淋巴细胞浸润减少;滑膜下有少量纤维增生、血管翳减轻;软骨受损程度减轻,仅见少量炎细胞;骨髓腔中脂肪细胞明显减少、骨骼细胞密集。充分显示了Pae对AA大鼠具有治疗作用。2. SHP-2抑制剂NSC-87877对AA大鼠FLS增殖、胞内cAMP水平、GRK₂及SHP-2表达的影响加入SHP-2抑制剂（NSC-87877）(10^-5 mol·L^-1)后,可显著抑制AA大鼠FLS的过度增殖,升高细胞内cAMP水平,降低AA大鼠FLS中GRK₂、SHP-2的高表达水平;分离FLS胞浆、胞膜蛋白,应用Western-blot方法,检测显示加入NSC-87877(10-5 mol·L^-1)能显著降低AA大鼠FLS细胞膜中GRK₂蛋白的高表达水平,显著升高AA大鼠FLS细胞浆中GRK₂蛋白的低表达水平。3. Pae对AA大鼠FLS增殖、胞内cAMP水平、GRK₂及SHP-2表达的影响Pae(10^-5、10^-6、10^-7、10^-8 mol·L^-1)体外给药后,可显著抑制EP2受体激动剂（Butaprost）刺激的AA大鼠FLS的过度增殖,升高细胞内cAMP水平,降低AAWestern-blot方法,检测显示Pae(10^-5、10^-6、10^-7、10^-8 mol·L^-1)体外给药能显著降低AA大鼠FLS细胞膜中GRK₂蛋白的高表达水平,显著升高AA大鼠FLS细胞浆中GRK₂蛋白的低表达水平。结论:1、Pae对AA大鼠有一定的治疗的作用,对其多发性关节炎指数、升高的脾脏指数、继发侧肿胀度、升高的巨噬细胞分泌IL-1水平、增强的T、B淋巴细胞增殖反应及关节病理形态改变具有明显的改善作用。2、SHP-2抑制剂可下调AA大鼠滑膜细胞GRK₂和SHP-2的高表达;降低细胞增殖水平,升高胞内cAMP水平;同时使胞膜GRK₂水平降低,而升高胞浆GRK₂水平。提示SHP-2可能促进了GRK₂的转膜,下调胞内cAMP水平,进而影响细胞增殖。3、Pae(10^-5、10^-6、10（-7）、10^-8 mol·L^-1)体外给药,可不同程度地降低Butaprost刺激的AA大鼠FLS的异常增殖,下调SHP-2和GRK₂的高表达,提示下调SHP-2和GRK₂可能是Pae抑制FLS异常增殖的机制之一。更多还原

【Abstract】 OBJECTIVEAccording to the changes that secondary paw swelling, polyarthritis index,and histological morphological of adjuvant arthritis（AA）rats,this study tends to clarify the therapeutic effects of paeoniflorin（Pae） on AA rats.Meanwhile,our study aimed to confirm the role of GRK₂ and SHP-2 playing in fibroblase-like synoviocytes（FLS） of AA rats and the effects of Pae.And further to analyze mechanism of Pae on AA rats,we investigate the role of SHP-2 on GRK₂ and its signalling pathways.METHODSFreund’s complete adjuvant （FCA）was used to induce AA in rats.Therapeutic treatment of interagastric administration Pae(12.5、25、50、100 mg·kg ^-1·d^-1, d14-d24 )were given after immunization. Hind paw volumes of rats were measured by volume meter.Arthritis index,spleen index were recorded. IL-1 level produced by thymus T cells was measured by ELISA and the proliferation of T and B lymphocytes were measured by [3H]-TdR incorporation. Pathological changes in synovium of joint were observed by light microscope. The proliferation of fibroblase-likesynoviocytes was assayed using the MTT assay.cAMP were measured by radioimmunoassay.The expression of GRK₂、SHP-2 were detected by westernblot analysis.RESULTS1. therapeutic effects of Pae on AA ratsPae(12.5、25、50、100 mg·kg ^-1·d^-1, d14-d24) significantly suppressed significantly the paw swelling as well as down-regulated the index of polyarthritis、spleen index、IL-1 produced by thymus T cells and proliferation of T and B lymphocytes in AA rats. The articular histopathological changes were also improved.This suggested that Pae had therapeutic effects on AA rats.2. Effects of SHP-2 inhibitor NSC-87877 on expression of GRK₂ and SHP-2、the level of cAMP、proliferation in synoviocytes from AA rats.SHP-2 inhibitor NSC-87877 significantly inhibited proliferation of FLS、increased the level of cAMP in synoviocytes and decreased the expression of GRK₂、SHP-2 in FLS.The result of western blot analysis demonstrated that SHP-2 inhibitor NSC-87877(10^-5mol·L^-1) in vitro significantly decreased the expression of GRK₂ in membrane and increased GRK₂ in cytoplasm.3. Effects of Pae on expression of GRK₂ and SHP-2、the level of cAMP、proliferation of FLS from AA rats.Pae significantly inhibited proliferation of FLS、increased the level of cAMP in FLS and decreased the expression of GRK₂、SHP-2.The result of western blot analysis demonstrated that SHP-2 inhibitor NSC-87877(10^-5mol·L^-1) in vitro significantly decreased the expression of GRK₂ in membrane and increased GRK₂ in cytoplasm. CONCLUSIONS1. Pae could alleviate the inflammation of joint at different degree and decrease the spleen index ,the secretion of IL-1,proliferation of T,B lymphocyte and articular histopathological changes in rats with AA.2. SHP-2 inhibitor could decrease the expression of GRK₂ and SHP-2 in FLS from AA rats; SHP-2 inhibitor inhibited proliferation of FLS by incpreasing the level of cAMP and improving the expresstion of GRK₂ in cytoplasm meanwhile decreased the expression of GRK₂ in membrane.Which indicated that SHP-2 inhibited proliferation of FLS by inhibiting transmembrane of GRK₂ and then increasing the level of cAMP in FLS.3.Pae could decreased the expression of SHP-2 in FLS,which may be one of the important mechanisms for the Pae on AA rats.更多还原