目的:基于活化T细胞核因子2(NFAT2)/环氧化酶-2(COX-2)通路探讨雷公藤多苷片(TWPT)防治糖尿病肾病(DN)肾脏损伤的可能作用机制。方法:选取雄性清洁级SD大鼠42只,适应性喂养1周后随机分为正常组8只,造模组34只。正常组予以正常饲养,造模...目的:基于活化T细胞核因子2(NFAT2)/环氧化酶-2(COX-2)通路探讨雷公藤多苷片(TWPT)防治糖尿病肾病(DN)肾脏损伤的可能作用机制。方法:选取雄性清洁级SD大鼠42只,适应性喂养1周后随机分为正常组8只,造模组34只。正常组予以正常饲养,造模组采用高脂高糖饮食喂养1周后予腹腔注射链脲佐菌素(STZ)法建立DN大鼠模型,除去造模过程中死亡及失败,选取造模成功的24只随机分为模型组、缬沙坦(8.33 mg·kg^(-1)·d^(-1))组、TWPT(5 mg·kg^(-1)·d^(-1))组。正常组和模型组均予等体积生理盐水灌胃,6周后测量体质量,收集大鼠尿液,腹主动脉取血后处死取材,生化检测血清中的尿素氮(BUN)、肌酐(SCr)、丙氨酸氨基转移酶(ALT)、血脂血糖及尿液中的24 h尿蛋白总量(24 h UTP),苏木素-伊红(HE)及马松(Masson)染色观察肾脏病理,酶联免疫吸附测定法(ELISA)检测血清中的NFAT2、COX-2表达水平,蛋白免疫印迹法(Western blot)检测肾组织中NFAT2、COX-2蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测肾组织中NFAT2、COX-2 mRNA表达。结果:与正常组比较,模型组大鼠24 h UTP、BUN、SCr、CHO、TG、FBG及血清NFAT2、COX-2表达显著升高(P<0.01),肾组织中的NFAT2、COX-2蛋白及mRNA表达显著升高(P<0.01),肾脏病理示肾小球体积增大,系膜细胞轻度增生,系膜基质增宽;与模型组比较,TWPT组大鼠24 h UTP、BUN、SCr、CHO、TG、FBG均明显降低(P<0.05,P<0.01);肾脏病理示肾小球形态基本正常,血清中NFAT2、COX-2表达显著降低(P<0.01),肾组织中的NFAT2、COX-2 mRNA和蛋白表达显著下降(P<0.01)。结论:TWPT可减轻DN大鼠24 h UTP、保护肾功能、改善肾脏病理,其作用机制可能与下调血清及肾组织NFAT2/COX-2表达相关。展开更多
OBJECTIVE: To investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-...OBJECTIVE: To investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1). METHODS: Mice with closed impact injury with fracture in both hind limbs were adopted as the trauma model. Spleen lymphocytes were isolated from traumatized mice and stimulated with Con-A. Culture supernatants were assayed for IL-2 activity, and total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. DNA binding activity of NFAT and AP-1 were measured by electrophoretic mobility shift assay (EMSA). The expression of c-Fos, c-Jun and JunB proteins was determined by the Western blot analysis. RESULTS: DNA binding activity of NFAT and AP-1 gradually decreased to a minimum of 41% and 49%, respectively, of the control on the 4th day after injury, which was closely followed by the decline in IL-2 activity and IL-2 mRNA. A decrease in the expression of c-Fos on the 1st and 4th day after trauma had no significant effect on c-Jun expression; the increase in expression of JunB was only on the 1st day after injury. CONCLUSION: Decreased IL-2 expression is, at least in part, due to a decline in the activation of NFAT and AP-1 in traumatized mice. The decline in DNA binding activity of NFAT and AP-1 is partly due to a trauma-induced block in the expression of c-Fos.展开更多
文摘目的探讨不同浓度巴戟天含药血清对体外培养成骨-破骨细胞共育体系中碳酸酐酶Ⅱ(CAⅡ)、活化T细胞核因子(NFAT2)mRNA表达的影响。方法取24 h内新生SD乳鼠头盖骨分离培养成骨细胞,取5周龄SD大鼠四肢长骨骨髓基质细胞,加入集落细胞刺激因子(M-CSF)和细胞核因子κB受体活化因子配体(RANKL)诱导培养破骨细胞。采用ALP染色鉴定成骨细胞,TRAP染色、骨吸收陷窝甲苯胺蓝染色、电镜等扫描鉴定破骨细胞,体外建立成骨-破骨细胞共育体系,设置高、中、低3种浓度巴戟天含药血清组和对照组,干预3d后提取各组总RNA,应用Real Time PCR(RT-PCR)方法测定各组CAⅡ、NFAT2mRNA表达并进行统计学分析。结果不同浓度巴戟天含药大鼠血清对成骨-破骨细胞共育体系CAⅡ、NFAT2 mRNA的表达均有抑制作用,且其抑制作用表现出一定的浓度依赖性;各组间差异有统计学意义(P<0.05)。结论巴戟天含药血清可抑制成骨-破骨细胞共育体系CAⅡ、NFAT2mRNA表达,从而达到降低破骨细胞分化成熟及骨吸收活性。
文摘目的:基于活化T细胞核因子2(NFAT2)/环氧化酶-2(COX-2)通路探讨雷公藤多苷片(TWPT)防治糖尿病肾病(DN)肾脏损伤的可能作用机制。方法:选取雄性清洁级SD大鼠42只,适应性喂养1周后随机分为正常组8只,造模组34只。正常组予以正常饲养,造模组采用高脂高糖饮食喂养1周后予腹腔注射链脲佐菌素(STZ)法建立DN大鼠模型,除去造模过程中死亡及失败,选取造模成功的24只随机分为模型组、缬沙坦(8.33 mg·kg^(-1)·d^(-1))组、TWPT(5 mg·kg^(-1)·d^(-1))组。正常组和模型组均予等体积生理盐水灌胃,6周后测量体质量,收集大鼠尿液,腹主动脉取血后处死取材,生化检测血清中的尿素氮(BUN)、肌酐(SCr)、丙氨酸氨基转移酶(ALT)、血脂血糖及尿液中的24 h尿蛋白总量(24 h UTP),苏木素-伊红(HE)及马松(Masson)染色观察肾脏病理,酶联免疫吸附测定法(ELISA)检测血清中的NFAT2、COX-2表达水平,蛋白免疫印迹法(Western blot)检测肾组织中NFAT2、COX-2蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测肾组织中NFAT2、COX-2 mRNA表达。结果:与正常组比较,模型组大鼠24 h UTP、BUN、SCr、CHO、TG、FBG及血清NFAT2、COX-2表达显著升高(P<0.01),肾组织中的NFAT2、COX-2蛋白及mRNA表达显著升高(P<0.01),肾脏病理示肾小球体积增大,系膜细胞轻度增生,系膜基质增宽;与模型组比较,TWPT组大鼠24 h UTP、BUN、SCr、CHO、TG、FBG均明显降低(P<0.05,P<0.01);肾脏病理示肾小球形态基本正常,血清中NFAT2、COX-2表达显著降低(P<0.01),肾组织中的NFAT2、COX-2 mRNA和蛋白表达显著下降(P<0.01)。结论:TWPT可减轻DN大鼠24 h UTP、保护肾功能、改善肾脏病理,其作用机制可能与下调血清及肾组织NFAT2/COX-2表达相关。
文摘OBJECTIVE: To investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1). METHODS: Mice with closed impact injury with fracture in both hind limbs were adopted as the trauma model. Spleen lymphocytes were isolated from traumatized mice and stimulated with Con-A. Culture supernatants were assayed for IL-2 activity, and total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. DNA binding activity of NFAT and AP-1 were measured by electrophoretic mobility shift assay (EMSA). The expression of c-Fos, c-Jun and JunB proteins was determined by the Western blot analysis. RESULTS: DNA binding activity of NFAT and AP-1 gradually decreased to a minimum of 41% and 49%, respectively, of the control on the 4th day after injury, which was closely followed by the decline in IL-2 activity and IL-2 mRNA. A decrease in the expression of c-Fos on the 1st and 4th day after trauma had no significant effect on c-Jun expression; the increase in expression of JunB was only on the 1st day after injury. CONCLUSION: Decreased IL-2 expression is, at least in part, due to a decline in the activation of NFAT and AP-1 in traumatized mice. The decline in DNA binding activity of NFAT and AP-1 is partly due to a trauma-induced block in the expression of c-Fos.
