P物质对人胚肺成纤维细胞作用及机制

Effect of substance P on human embryo lung fibroblasts MRC-5 and its mechanism

目的探讨P物质(SP)对人胚肺成纤维细胞(MRC-5)增殖与活化的作用及其调控机制。方法体外培养MRC-5细胞,以P物质或与NK-1R抑制剂(L732138)及TGFβR1抑制剂(SB431542)联合培养细胞后,利用CCK8法检测细胞增殖活性,天狼猩红染色检测胶原纤维含量,Western blot检测α-SMA、TGFβ1、Smad3及p-Smad3蛋白的表达。结果MRC-5细胞经P物质(1、10、100、1000、10000 nmol/L)处理后,24 h细胞增殖率无明显变化;在处理48及72 h后,100 nmol/L P物质可促进细胞增殖。与对照组比较,100 nmol/L P物质处理72 h后的细胞胶原蛋白含量(54.04±1.25)、α-SMA蛋白表达水平(2.48±0.07)明显升高(P<0.001)。与100 nmol/L P物质+L732138组比较,100 nmol/L P物质组细胞胶原纤维含量、α-SMA、TGFβ1、p-Smad3蛋白相对表达量[分别为(56.14±1.25)、(0.89±0.04)、(0.87±0.05)、(1.06±0.03)]明显升高(P<0.001);与100 nmol/L P物质组比较,100 nmol/L P物质+SB431542组细胞A450值、胶原纤维含量明显降低(P<0.001)。结论P物质可以通过TGFβ1/Smad3信号通路促进人胚肺成纤维细胞(MRC-5)增殖及活化。

Objective To investigate the effect of substance P(SP)on the proliferation and activation of human embryonic lung fibroblasts(MRC-5)and its mechanism.Methods MRC-5 cells were cultured in vitro and treated with SP(at dosages of 1,10,100,1000,and 10000 nM)or with neurokinin-1 receptor(NK-1 R)inhibitors(L732138)and transforming growth factor-β1(TGFβ1)inhibitors(SB431542).The proliferation activity of the cells was measured with cell counting kit-8(CCK8)assay;the cells′collagenic fiber secretion was detected with Sirius red staining and expression ofα-smooth muscle actin(SMA),TGFβ1,solvated metal atom dispersion family number 3(Smad3)and phosphorylated(p)-Smad3 proteins was performed with Western blot.Results CCK8 assay demonstrated no significant changes in proliferation of MRC-5 cells 24 hours after the treatment of various doses SP;while,48 hours after the treatment of 100 nm SP,promoted proliferation of MRC-5 cells were detected and the increased proliferation was much more obvious 72 hours after the treatment.With Sirius red staining,obviously increased content of collagenic fibers was observed in MRC-5 cells 72 hours after 100 nM SP treatment.Western blot testing revealed remarkably increased expression ofα-SMA 72 hours after100 nM SP treatment.The NK-1 R inhibitor(L732138)could inhibit promoting effect of SP on MRC-5 cell proliferation,collagenic fiber content andα-SMA protein expression;L732138 could also significantly attenuate the expression of TGFβ1 and p-Smad3 proteins in MRC-5 cells treated with SP.In addition,SB431542(TGFβ1 inhibitor)could significantly inhibit the effect of SP on proliferation and collagen fiber content of MRC-5 cells.Conclusion Substance P can promote the proliferation and activation of human embryonic lung fibroblasts(MRC-5)through TGFβ1/Smad3 signaling pathway.