Regulatory Effect Of CCK-8 On LPS-induced NOS In Vascular Endothelial Cells

Objective: To investigate the regulatory effect of CCK-8 on LPS-induced inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in vascular endothelial cells.Methods: Human veinous endothelium line ECV-304 was cultured and incubated with LPS, CCK-8, LPS+CCK-8, or vehicle for different times. The supernatant of culture medium and cells were collected to measure NO content and NOS activity spectrophotometrically and to detect NOS expression by immunocytochemistry and Western blot.Results: (1)The effect of LPS on NO content, NOS activity and expression in ECV-304: Incubation of ECV-304 with 1ug/ml LPS resulted in a gradual increase of NO content in a time-dependent manner, which was markedly higher than that of vehicle group. The NOS activity also obviously enhanced in ECV-304 exposed to LPS compared with vehicle group. Incubation of ECV-304 with 0.01ug/ml, 0.1ug/ml and 1ug/ml LPS for 2h and 16h led to an obvious increase of NO content and NOS activity in a dose-dependent manner compared with vehicle group. The result of immunocytochemistry showed that eNOS expressed in the cytoplasm and membrane of ECV-304 in the vehicle group, and the eNOS expression enhanced in the LPS-stimulated ECV-304 for 2h without localization changes. The quantity of iNOS expression in the ECV-304 of the vehicle group is very little, which was obviously increased in LPS-stimulated cells for 16h. The Western blot assay showed the same results as above. (2) The effect of CCK-8 on NO content, NOS activity and expression in ECV-304: Incubation of ECV-304 with 10-6mol/L,10-7mol/L and 10-8mol/L CCK-8 for 2h and 16h led to no difference compared with vehicle group on NO content and NOS. The result of immunocytochemistry showed that the eNOS expression has no alteration in the CCK-8-stimulated ECV-304 for 2h without localization changes compared with the vehicle group. The quantity of iNOS expression in the ECV-304 of vehicle group is very little, which was not different from CCK-8-stimulated cells for 16h. The Western blot assay showed the same results as above. (3) The effect of CCK-8 on NO content, NOS activity and expression in ECV-304 exposed to 0.1ug/ml LPS: Incubation of ECV-304 exposed to 0.1ug/ml LPS with 10-6mol/L,10-7mol/L and 10-8mol/L CCK-8 led to an obvious decrease of NO content compared with LPS-stimulated group. And it was not different from vehicle group. The NOS activity in CCK-8 and LPS group for 2h was not different from LPS-stimulated group and higher than that of vehicle group. But the NOS activity in CCK-8 and LPS group for 16h was lower than that of LPS-stimulated group and higher than that of vehicle group. The result of immunocytochemistry showed that the expression of eNOS in CCK and LPS group for 2h was not different from that of vehicle group. The quantity of iNOS expression was obviously increased in CCK-8 and LPS group compared with the vehicle group. The Western blot assay showed the same result as above.Conclusion: LPS could induce the upregulation of NOS and increased NOS activity and NO contents. CCK-8 had no effect on iNOS/eNOS expression, NOS activity and NO contents in ECV-304, but it could inhibit LPS-induced the upregulation of iNOS expression, increased NOS activity and NO contents.