p38MAPK信号转导通路在七氟烷后处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用

目的 评价p38丝裂原活化蛋白激酶(p38MAPK)信号转导通路在七氟烷后处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用.方法 健康新生SD大鼠,日龄1～3 d,处死后取心室肌组织,培养心肌细胞,随机分为7组:对照组(C组)于CO2培养箱中持续培养3 h;缺氧复氧组(AR组)细胞缺氧2 h,复氧1 h;七氟烷后处理组(SP组)细胞缺氧2 h,复氧开始即刻更换为3%七氟烷饱和的DMEM培养液,孵育20 min,再更换为无血清DMEM培养液,继续复氧40 min;七氟烷后处理+SB203580组(SP+SB组)于七氟烷后处理同时加入SB203580(p38MAPK特异性抑制剂)至5 μmol/L,孵育20 min;七氟烷后处理+二甲亚砜组(SP+DMSO组)于七氟烷后处理同时加入0.1%DMSO,孵育20 min;SB203580组(SB组)于复氧开始时加入SB203580至5 μmol/L,孵育20 min;二甲亚砜组(DMSO组)于复氧开始即刻加入0.1%DMSO,孵育20 min.各组细胞分别接种于24孔培养板(1 ml/孔)、35 mm培养皿(5 mlnd/皿)和50 ml培养瓶(8 ml/瓶)中,每组12孔、6皿和6瓶.于复氧结束后,采用比色法测定细胞培养液乳酸脱氢酶(LDH)活性;采用台盼蓝排斥实验测定细胞存活率;采用流式细胞仪测定细胞凋亡率;采用Western blot法测定磷酸化p38MAPK(p-p38MAPK)表达水平.结果 与C组比较,其余各组LDH活性升高,细胞存活率降低,细胞凋亡率升高,AR组、SP组、SP+SB组、SP+DMSO组和DMSO组p-p38MAPK表达上调(P<0.05);与AR组比较,SP组、SP+SB组和SP+DMSO组LDH活性降低,细胞存活率升高,细胞凋亡率降低,p-p38MAPK表达上调(P<0.05);与SP组比较,SP+SB组、SB组和DMSO组LDH活性升高,细胞存活率降低,细胞凋亡率升高,p-p38MAPK表达下调(P<0.05).结论 七氟烷后处理可通过激活p38MAPK信号转导通路减轻乳鼠心肌细胞缺氧复氧损伤.     

p38MAPK信号转导通路在七氟烷后处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用

Objective To evalnate the role of p38 mitogen-activated protein kinase (p38MAPK) signal pathway in the protective effect of sevoflurane postconditioning (S-Postcon) on cultured neonatal rat cardiomyocytes against anoxia/reoxygenation (A/R) injury. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into 7 groups: group Ⅰ normal control (C); group Ⅱ A/R; group Ⅲ S-Poatcan + A/R; group ⅣS-Postcon + SB203580 + A/R; group Ⅴ S-Postcon + DMSO + A/R; group Ⅵ SB203580 + A/R and group Ⅶ DMSO + A/R. GroupⅡ-Ⅶ were exposed to 2 h anoxia (95% N2-5% CO2) followed by 1 h reoxygenation. In group Ⅲ, Ⅳ and Ⅴ the cultured myocytes were postconditioned with 20 min 3% sevoflurane in 97% O2 alone (in group Ⅲ) or in conjunction with 5 μmol/L 5B203580 (a specific p38 MAPK inhibitor) (in group Ⅳ) or 0.1% DMSO (in group Ⅴ) followed by 40 min reoxygenation. The cardiomyocytes were reoxygenated in the presence of 5 μmol/L SB203580 in group Ⅵ or 0.1% DMSO in group Ⅶ. The LDH activity, cell survival rate and apoptotic rate were measured at the end of the experiment. The levels of phosphor-p38MAPK (p-p38MAPK) was detected by Western blotting. Results S-Postcon reduced LDH activity and apoptotic rate and increased cell survival rate and the level of p-p38MAPK as compared with group A/R (group Ⅱ). The myocardial protecfive effect of S-Posteon was eliminated by p38MAPK inhibltor-SB203580 and p-p38MAPK level was also decreased at the same time.Conclusion Sevoflurane postconditioning can attenuate anoxia/reoxygenation induced cardiomyocyte injury through activation of p38MAPK signal pathway.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

目的 探讨罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶（MAPK）信号通路的作用。 方法 分别用罗格列酮（RGZ,10 μmol/L）、过氧化物酶体增殖物活化受体γ（PPARγ）抑制剂GW9662（10 μmol/L）、RGZ（10 μmol/L）+GW9662（10 μmol/L）、p38MAPK特异性抑制剂SB203580（10 μmol/L）、SB203580（10 μmol/L）+RGZ（10 μmol/L）处理体外培养的多囊肾囊肿衬里上皮细胞（PKD细胞）2 h后,再用表皮生长因子（EGF）刺激不同时间,另设置空白对照组和单独EGF刺激组。采用Western印迹方法检测p38MAPK、磷酸化p38MAPK（p-p38）、增殖细胞核抗原（PCNA）表达;RT-PCR检测p38 mRNA表达;免疫细胞化学检测c-fos和c-jun的表达。 结果 (1) 与空白对照组相比,EGF显著上调p-p38、PCNA、 c-fos和c-jun的表达（P ＜ 0.01）。(2) 与EGF单独刺激相比,RGZ显著降低p38活化和基因表达（均P ＜ 0.01）。RGZ组、SB203580+RGZ组p-p38、PCNA、c-fos、c-jun表达明显下调（P ＜ 0.01）,两组间差异无统计学意义。(3) 与RGZ组相比,RGZ+GW9662组部分阻断RGZ的下调作用（P ＜ 0.05）。 结论 罗格列酮抑制多囊肾囊肿衬里上皮细胞增殖的作用机制可能与其降低p38MAPK活性,继而抑制PCNA、c-fos及c-jun表达有关。这种抑制作用是部分非PPARγ依赖的。     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.