Effect Of Substance P On Human Embryo Lung Fibroblasts (MRC-5) And Its Mechanism

Objectives:To investigate the effect of substance P(SP)on the proliferation and activation of human embryonic lung fibroblasts(MRC-5)and its regulatory mechanism,in order to reveal the mechanism of action of substance P involved in pulmonary interstitial fibrosis,provide a novel method for the research and development of drugs for clinical treatment of pulmonary interstitial fibrosis.Methods:Human embryonic lung fibroblasts(MRC-5)were cultured in vitro and the cells in logarithmic growth phase were selected for the following studies:(1)MRC-5 cells were treated with a series of concentrations(1,10,100,1000,10000 n M)of substance P,CCK8 assay was used to detect the proliferation activity of MRC-5 treated with a series of concentrations of substance P at different time points(24,48,72h),so as to find out the optimal concentration and time of action of substance P on MRC-5.(2)The content of collagen fiber secreted by MRC-5 cells treated with serial concentrations of substance P was detected by Sirius scarlet staining at 72 h.(3)Western blot was performed to measure α-SMA protein expression of MRC-5 cells on at 72 h after treated with a series of concentrations of substance P。(4)To study the mechanism of substance P on MRC-5 cells,we divided the cells into three groups: control group,100 n M substance P group(referred to as substance P group),100 n M substance P + 10 μM L732138 group(NK-1R inhibitor)(Hereinafter referred to as substance P + L732138 group).After cells were cultured for 72 hours,the cell proliferation activity of each group was detected by the CCK8 assay.The content of collagen fibers secreted by the cells of each group was measured by Sirius scarlet staining.Meanwhile the expression ofα-SMA protein in each group was evaluated by Western blot,So as to elucidate the effect of substance P on the proliferation and activation of MRC-5 cells.(5)In order to study the role of SP/NK-1R in the TGF-β1/Smad3 signal pathway,The cells were divided into three groups: control group,100 n M substance P group(hereinafter referred to as substance P group)and 100 n M substance P +10μM SB431542 group(TGF-βR1 inhibitors)(hereinafter referred to as the substance P + SB431542 group),CCK8 assay was performed to measure cell proliferation activity,meanwhile Sirius scarlet staining was conducted to evaluate the content of collagen fibers in each group.Results:(1)After MRC-5 cells were treated with a series of concentrations of substance P(1,10,100,1000,10000 n M),there was no significant difference about the OD value after 24 h;While treated with substance P after 48 h and72h,compared with the control group,The OD value of 100 n M substance P group increased significantly,especially the OD value increased more significantly at 72h(P<0.01).(2)After MRC-5 cells were treated with serial concentrations of substance P for 72 h,compared with the control group,the content of collagen fibers of 100 n M substance P group increased obviously(P<0.001).(3)While cells were treatment with a series of concentrations of substance P for 72 hours,compared with the control group,both of the relative expression of α-SMA protein in the cells of the 10 n M substance P group and the 100 n M substance P group increased,especially the increasement in 100 n M substance P group was more remarkable(P<0.001).(4)As the cells were treated with drugs for 72 hours,compared with the substance P group,the content of collagen fibers of the substance P+L732138 group reduced outstandingly(P<0.001),the OD value of cells and the relative expression of α-SMA protein reduced obviously(P<0.01).(5)After the cells were treated with drugs for 72 hours,compared with the substance P group,the relative expression of TGFβ1 and p-Smad3 protein in the substance P+L732138 group reduced significantly(P<0.01).(6)While treated with drugs for 72 hours,compared with the substance P group,the OD value of cells and the content of collagen fibers of the substance P+SB431542 group reduced prominently(P<0.001).The differences of all the above were statistically significant.Conclusions:Substances P can promote the proliferation and activation of human embryonic lung fibroblasts(MRC-5),and the mechanism may be related to the TGF-β1/ Smad3 signaling pathway.