Fig. 4.
SP induces pro-fibrotic changes in corneal fibroblasts through activation of its receptor NK-1R. a Bovine collagen I liquid gel was mixed together with 0.25 × 106 corneal fibroblasts, FBS (10%), 0.5 mM VitC, and 0.25 ng/ml recombinant human TGF-β1. Experimental groups contained 10−5M SP or 10−6M of the NK-1R blocker L-733,060 together with 10−5M SP. Gels containing fibroblasts, 10% FBS, VitC, and TGF-β1 served as positive controls for the fibrosis model (ctrl) and treated gels were compared with them. Gel contraction assay showed that when NK-1R receptor was blocked with L-733,060, SP was unable to induce contraction of the gels. Gel contraction of L-733,060 treated gels remained at the level of control. b Supernatants collected 2 days and 4 days after induction of fibrosis and treatment with 10−5M SP or 10−6M L-733,060 together with 10−5M SP were subjected to pro-collagen I ELISA. Secretion of pro-collagen I was increased in supernatants collected from SP-treated cells both 2 days and 4 days after treatment. Treatment of cells with L-733,060 and SP together resulted in decreased secretion of pro-collagen I as compared with SP alone. c Supernatants collected 2 days and 4 days after induction of fibrosis and treatment with 10−5M SP or 10−6M L-733,060 together with 10−5M SP were subjected to fibronectin ELISA. Secretion of fibronectin was increased in supernatants collected from SP-treated cells both 2 days and 4 days after treatment. Treatment of cells with L-733,060 and SP together resulted in decreased secretion of fibronectin as compared with SP alone. d α-SMA protein expression was assessed by western blot 4 days after treatment with 10−5M SP or 10−6M L-733,060 together with 10−5M SP. Densitometry analysis was performed in order to quantify the western blot results. Intensity of α-SMA band was normalized to the intensity of β-actin band. SP treatment resulted in increased α-SMA (42 kDa) protein expression 4 days after treatment. Treatment of cells with L-733,060 and SP together resulted in decreased expression of α-SMA as compared with SP alone. β-actin (45 kDa) served as loading control. Values are means ± SD. n.s. (not significant); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001