ABSTRACT
AIM: To screen the lentiviral vector carrying siRNA with higher efficiency of suppressing the sphingosine-1-phosphate receptor 2 (S1P2) gene expression in the primarily cultured corpus cavernosum smooth muscle cells of spontaneously hypertensive rats (SHR).METHODS: SHR and SD rats (n=5 each) were used for primarily culturing corpus cavernosum smooth muscle cells.The cells were randomly divided into 6 groups: SHR siRNA-1, SHR siRNA-2, SHR siRNA-3, SHR GFP, SHR control (SHR non-transfection group), and SD control (SD rat control group).Each group had 5 samples with 1.0×105 cells of each sample.At 72 h after transfection (MOI=60) with lentiviral vectors carrying S1P2 siRNA into the SHR corpus cavernosum smooth muscle cells, the expression of GFP was observed under fluorescence microscope.The protein expression of S1P2, ROCK1, ROCK2 and eNOS in the corpus cavernosum smooth muscle cells, and the mRNA expression of S1P2, ROCK1 and ROCK2 were determined by by Western blot and RT-PCR.RESULTS: The transfection efficiency of the corpus cavernosum smooth muscle cells in SHR siRNA-1, SHR siRNA-2, SHR siRNA-3 and SHR GFP groups were>80%.Compared with SHR control group, the mRNA levels and the protein expression of S1P2, ROCK1 and ROCK2 in SHR GFP group showed no remarkable changes, while those in SHR siRNA-1, SHR siRNA-2, SHR siRNA-3 and SD control groups were significantly lower than those in SHR control group (P<0.05).The protein expression of eNOS in SHR siRNA-1, SHR siRNA-2, SHR siRNA-3 and SHR GFP groups were not significantly changed as compared with SHR control group, but that in SD control group was significantly higher than that in SHR control group.CONCLUSION: Three groups of siRNA lentiviral vectors targeting S1P2 inhibit the expression of S1P2 in the corpus cavernosum smooth muscle cells of SHR, and by silencing the S1P2 expression, the expression of ROCK1 and ROCK2 is inhibited.Among them, siRNA-1 has the highest inhibitory efficiency.
ABSTRACT
AIM:To investigate the effects and mechanisms of sphingosine-1-phosphate receptor-2 (S1P2R) on lipopolysaccharide (LPS)-induced acute lung injury (ALI).METHODS:ALI model was induced by intratracheal ad-ministration of LPS in both wild-type mice and S1P2R-deficient mice.The pathological changes in the lung tissues were ob-served, and the protein concentration , total cell number, neutrophil ratio, TNF-αlevel and IL-6 level were determined in the bronchoalveolar lavage fluid (BALF) 24 h after LPS injection.In order to investigate the mechanisms of S1P2R in LPS-induced ALI, 10 min before LPS injection, both wild-type mice and S1P2R-deficient mice were injected with nitric oxide synthase inhibitor by tail vein injection , the pathological changes of the lung tissues were observed , and the protein concen-tration and total cell number in BALF were determined 12 h after LPS injection .RESULTS: Compared with wild-type mice, S1P2R-deficient mice showed more severe LPS-induced ALI, and the protein concentration , neutrophils and inflam-matory cytokines in BALF were significantly increased in S1P2R-deficient mice.Administration of nitric oxide synthase in-hibitor Nω-L-nitro-arginine methyl ester protected S1P2R-deficient mice from aggravation of ALI .CONCLUSION:S1P2R mediates the protection from LPS-induced ALI possibly through inhibiting nitric oxide synthase .
ABSTRACT
Objective To explore the role of sphingosine-1-phosphate receptor (S1PR2) in human coronary artery endothelial cell proliferation in vitro. Methods MTT assay was used to detect cell proliferation in human coronary artery endothelial after treatment of S1P and S1PR2 antagonist JTE-013. Phosphor-ERK and total- ERK level were measured by western blot in endothelial after treatment of S1P and JTE-013. Results 1 μmol/L S1P significantly increased endothelial cells proliferation. S1PR2 antagonist JTE-013 inhibited S1P-induced endothelial cell proliferation in dose-dependent manner. S1PR2 antagonist JTE-013 significantly inhibited S1P-induced phosphor-ERK level in endothelial cells. Conclusion S1PR2 may involve in S1P-induced endothelial cell proliferation through activation of ERK pathway.