N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸对肺成纤维细胞P38分裂原活化蛋白酶蛋白核转位及其通路活化的调节

Regulation of AcSDKP on P38MAPK pathway activity through nuclear translocation in pulmoanry fibroblast

目的：观察P38分裂原活化蛋白酶和Ⅰ型、Ⅲ型胶原在肺成纤维细胞的分布与表达，探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸（AcSDKP）对P38MAPK信号转导通路活化的调节与其抗矽肺纤维化作用的关系。方法：培养新生大鼠肺成纤维细胞，分为对照组、转化生长因子-β1（TGF-131）刺激组、通路抑制剂组和AcSDKP干预组。采用激光扫描共聚焦显微镜检测磷酸化P38在肺成纤维细胞的分布与核转位的情况；免疫印迹检测P38MAPK蛋白以及I型、Ⅲ型胶原蛋白的表达；MTT法检测细胞增殖情况。结果：与对照组比较，TGF-131诱导刺激了肺成纤维细胞的磷酸化P38MAPK蛋白由胞质向胞核的转位，使胞核中磷酸化P38MAPK蛋白荧光强度增加，核浆比值升高，同时肺成纤维细胞增殖以及Ⅰ型、Ⅲ型胶原蛋白的表达上调。而AcsDKP减少了由TGF-β1诱导刺激的磷酸化P38MAPK蛋白由胞质向胞核的转位，使磷酸化P38MAPK蛋白在胞核的表达减少，核浆比值下降。同时也使细胞增殖下降以及I型、Ⅲ型胶原蛋白的表达下调。这些结果与P38MAPK通路抑制剂具有相同的作用。结论：AcSDKP通过抑制P38MAPK蛋白的核转位阻抑了P38信号转导通路的活化，进而抑制了肺成纤维细胞增殖与胶原蛋白的表达，这可能与AcSDKP抗矽肺纤维化的作用密切相关。

Abstract Objective： To observe distribution and expression of P38MAPK and collagen type I and type Ⅲ in cultured fibroblasts, and to investigate anti-silicotic fibrosis role of N-acetyl-seryl-aspartyl-lysyl-proline （AcSDKP） through inhibition of P38MAPK pathway activity mediated TGF-β1. Methods： Cultured neonatal rat pulmonary fibroblasts were divided into control group, TGF-β1 stimulated group, P38MAPK inhibition treated group and AcSDKP treated group. The distribution and nuclear translocation of phospho-p38 protein in the cultured fibroblasts were measured by laser scanner confocal microscopy. The protein expression of P38MAPK and collagen type I and type IlI were detected with Western blotting. The proliferation of fibroblast was detected with MTT assay. Results： Compared with the control group, TGF-131 was induced and phospho-p38 protein translocation was stimulated from cytoplasma to nucleus and increased fluorescence intensity in the nucleus and the ratio of fluorescence intensity in the nucleus and plasma was increased and,the expression of phospho-p38 protein in cultured fibroblast was up-regulated. TGF-131 also promoted the cell proliferation and expression of collagen type I and type Ⅲ in cultured fibroblast. However, AcSDKP obviously inhibited the nuclear translocation of phospho-p38 protein and decreased fluorescence indencity in nucleus and ratio of fluorescence intencity in nucleus and plasma and down-regulated expression of phospho-p38 protein in the cultured fibroblasts stimulated by TGF-β1. AcSDKP also reduced ceil proliferation and expression of collagen type Ⅰ and Ⅲ, which was as the same result in P38MAPK inhibition treatment. Conclusion： AcSDKP can block P38MAPK pathway activity through inhibition of phospho-p38 protein nuclear translocation and then reduced the proliferation and expressiong of collagen type Ⅰ and type Ⅲ of fibroblast, which is possiblely related witheffect of AcSDKP anti-silicotic fibrosis.