蛋白激酶A部分通过环磷腺苷效应元件结合蛋白信号防止足细胞凋亡
Prevention of apoptosis of podocytes by protein kinase A via cAMP response element binding protein signaling pathway

目的 探讨转录因子环磷腺苷效应元件结合蛋白（CREB）在蛋白激酶A（PKA）信号防止足细胞损伤中的作用。方法 使用条件永恒的小鼠足细胞株（5P12）14~25代分化的足细胞进行研究，细胞分为对照组、阿霉素（ADR）处理组（ADR组）和PKA特异性激动剂(pCPT-cAMP)预孵育+ADR刺激组（pCPT-cAMP+ADR组）。CCK-8法检测细胞毒性； Western blotting检测足细胞内被切割的半胱氨酸蛋白酶-3（cleaved caspase-3）表达；siRNA抑制细胞内CREB表达，免疫荧光共聚焦显微镜和Western blotting检测磷酸化CREB（p-CREB）的定位和表达。结果 ADR诱导足细胞内cleaved caspase-3表达升高，pCPT-cAMP预孵育可显著降低ADR诱导的cleaved caspase-3表达上升。ADR使足细胞减少为原来的（40.45±6.78）%，pCPT-cAMP预孵育使细胞数量恢复为原来的（69.8±5.48）%。Western blotting检测结果显示：pCPT-cAMP分别预孵育5、15 min可使足细胞总蛋白中p-CREB表达分别上升（105.64±38.21）%和(98.61±41.03)%，细胞核内p-CREB的表达上升（114.52±11.28）%和（118.84±14.44）%。免疫荧光共聚焦显微镜检测显示pCPT-cAMP主要引起细胞核内p-CREB表达上升。siRNA可使足细胞CREB蛋白表达下降为原来的（25.1±2.44）%，且pCPT-cAMP预孵育不能防止ADR诱导的cleaved caspase-3表达升高。结论 转录因子CREB介导了PKA信号对足细胞的保护作用。
： Objective To explore the effects of cAMP response element binding protein (CREB) signaling pathway on the protection of podocytes by the protein kinase A (PKA). Methods The 14-25 generations of mouse podocytes 5P12 differentiated under constant condition were selected and divided into the control group, adriamycin (ADR) treatment group (ADR group), and PKA specific agonist (pCPT-cAMP) pre-culture+ADR stimulation group (pCPT-cAMP+ADR group). Cell toxicity was detected by CCK-8 method. The expression of cleaved caspase-3 in podocytes was detected by Western blotting. The expression of CREB in podocytes was inhibited by siRNA and the localization and expression of phosphorylated CREB (p-CREB) were detected by immunofluorescence confocal microscopy and Western blotting. Results ADR induced up-regulation of the expression of cleaved caspase-3 in podocytes and pre-culture by pCPT-cAMP could significantly decrease the elevated expression of cleaved caspase-3 induced by ADR. ADR decreased the number of podocytes to (40.45±6.78)% of its original number and pre-culture by pCPT-cAMP increased the number of podocytes to (69.8±5.48)% of its original number. Results of Western blotting showed that pre-culture by pCPT-cAMP for 5 and 15 min could increase the expressions of p-CREB in total protein of podocytes by (105.64±38.21)% and (98.61±41.03)% and those in nuclei by (114.52±11.28)% and (118.84±14.44)%. Immunofluorescence confocal microscopy showed that pCPT-cAMP mainly increased the expression of p-CREB in nuclei. siRNA could decreased the protein expression of CREB in podocytes to (25.1±2.44)% of its original expression and pre-culture by pCPT-cAMP could not prevent the ADR-induced up-regulation of the expression of cleaved caspase-3. Conclusion Transcription factor CREB mediates the protective effect of PKA signaling