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, 1 000, and 10 000 nM) or with neurokinin-1 receptor (NK-1R) 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 of α-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 of α-SMA 72 hours after 100 nM SP treatment. The NK-1R inhibitor (L732138) could inhibit promoting effect of SP on MRC-5 cell proliferation, collagenic fiber content and α-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.
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