Forskolin激活cAMP信号通路调控小鼠心肌成纤维细胞结缔组织生长因子的表达

摘要

目的：研究胞内环磷酸腺苷（cAMP）水平提高对小鼠心肌成纤维细胞（MCFs）表达结缔组织生长因子（CT‐GF）的影响及其信号通路。方法采用出生1周内C57BL乳鼠心脏分离培养原代MCFs，第3代MCFs使用腺苷酸环化酶激活剂Forskolin干预培养后检测细胞CTGF、蛋白激酶A（PKA）、p44/42丝裂素活化蛋白激酶（MAPK）及磷酸化p44/42MAPK表达情况。分别给予磷酸化p44/42MAPK抑制剂PD98509及PKA抑制剂Rp‐cAMPS阻断相关信号节点，检测细胞内PKA、p44/42MAPK、磷酸化p44/42MAPK以及CTGF表达变化。结果Forskolin干预培养MCFs细胞后，胞内cAMP水平提高，细胞活力下降，PKA表达上升，p44/42MAPK总蛋白无明显改变，p44/42MAPK磷酸化水平下降，CTGFmRNA及蛋白表达下降；给予PD98509抑制p44/42MAPK磷酸化后，PKA表达上升，CTGFmRNA及蛋白表达下调；Rp‐cAMPS抑制PKA活化后，p44/42MAPK磷酸化水平升高，CTGFmRNA及蛋白表达上调。结论Forskolin可以通过提高MCFs胞内cAMP水平抑制CTGF的表达，其作用信号通路为高水平cAMP上调PKA表达，降低下游p44/42MAPK磷酸化水平，抑制CTGF的表达。%Objective To examine the effects of the increased levels of cyclic adenosine monophosphate (cAMP)on the ex‐pression of connective tissue growth factor(CTGF)in mouse cardiac fibroblasts(MCFs)and the related signal transduction path‐way.Methods MCFs from the hearts of 1‐week‐old C57BL mice were isolated and primarily cultured.The expression levels of CTGF ,protein kinase A(PKA) ,p44/42 mitogen activated protein kinase(MAPK)and phospho‐p44/42 MAPK were detected in 3‐generation MCFs treated with Forskolin(an activating agent of cAMP) ,PD98509(an inhibitor of phospho‐p44/42 MAPK)or Rp‐cAMPS(an inhibitor of PKA).Results The levels of cAMP were increased ,the cell viability decreased ,the expression levels of PKA and CTGF increased and the expression levels of phospho‐p44/42 MAPK declined in MCFs treated with Forskolin. The PKA expression levels were increased while the expression levels of CTGF decreased in MCFs treated with PD 98509.Rp‐cAMPS could inhibit the expression of PKA ,and upregulate the expressions of phospho‐p44/42 MAPK and CTGF.Conclusion Forskolin can inhibit the CTGF expression levels by increasing the intracellular level of cAMP.The involved mechanism is that high levels of cAMP upregulate the PKA expression level ,reduce the phosphorylation level of the downstream target p44/42 MAPK and inhibit the CTGF expression.