ABSTRACT
Objective To investigate the effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on LPS induced barrier injure of Caco-2 cells.Methods The model of Caco-2 monolayer cells damage induced by LPS was established by using 50 μg/ml LPS for 24 hours.After preconditioning with different concentrations (10 μmol/L,50 μmol/L,and 100 μ mol/L) of CORM-2 for 1 hour,the cultured well-grown Caco-2 monolayer cells were stimulated with 50 μ g/ml lipopolysaccharides (LPS) for 24 hour.The 100 μmol/L CORM-2 was put into 37℃ and 5％ CO2 incubator for 18 hours until the CO has been fully released,and it became an inactive CORM-2(iCORM-2).The cultured Caco-2 monolayer cells were divided into six groups:the control group,the LPS group,the LC1(10 μmol /L CORM-2 preconditioning) group,the LC2(50 μmol/L CORM-2 preconditioning) group,the LC3(100 μmol/L CORM-2 preconditioning) group,and the LC4 (iCORM-2 preconditioning) group.The apoptosis rates of different groups of Caco-2 monolayer cells were detected using flow cytometry.Cytokines (TNF-α,IL-1β and HMGB-1) levels of different groups were detected using ELISA kits.The levels of tight junction proteins (occludin ZO-1,claudin-1 and claudin-4) of every group were detected using Western blotting with specific antibodies.The structural changes of tight junction proteins were visualized by immunofluorescence technique.Results Compared with control group,cell apoptosis rate and release of inflammatory cytokines such as TNF-α,IL-1β and HMGB-1 in LPS group were significantly higher,and the levels of tight junction proteins were apparently decreased (P＜0.05) in LPS group.Compared with LPS group,cell apoptosis rate and release of inflammatory cytokines such as TNF-α,IL-1β and HMGB-1 decreased,and the levels of tight junction proteins were attenuated obviously,P＜0.05,in CORM-2 preconditioning groups.And the higher the concentration of CORM-2,the more obvious the protective effects.Conclusions This study demonstrates that CORM-2,as one of exogenous CO-releasing molecules,has the capacity to protect the barrier damage of LPS-stimulated Caco-2 monolayer cells in a concentration dependent manner.The higher the concentration of CORM-2 was,the stronger the protective effects were.The protective effects of CORM-2 include reducing Caco-2 monolayer cells apoptosis rate,inhibiting inflammatory cytokines production and release,and restoring distribution and levels of tight junction proteins.
ABSTRACT
Objective To investigate the suppressive effect of exogenous carbon monoxide (CO) on abnormal platelet exocytosis and its possible molecular mechanism. Methods Venous blood was collected from healthy volunteers. Platelet-rich plasma (PRP) was isolated from the blood by differential centrifugation. The PRP was randomly divided into five groups by random number table, namely normal control group, lipopolysaccharide (LPS) group (challenged with 10 mg/L LPS), inactively exogenous carbon monoxide releasing molecule 2 (iCORM-2) group (given 10 mg/L LPS + 50 μmol/L iCORM-2 for intervention), exogenous carbon monoxide releasing molecule 2 (CORM-2) 10 μmol/L and 50 μmol/L groups (given 10 mg/L LPS + CORM-2 10 μmol/L or 50 μmol/L for intervention). After 30 minutes, enzyme linked immunosorbent assay (ELISA) was used to determine the platelet-derived growth factor BB (PDGF-BB) and matrix metalloproteinase 2 (MMP-2). Chemical fluorescein method was used to determine the platelet adenosine triphosphate (ATP). Flow cytometer was used to determine the expression of P-selectin. The expressions of Toll-like receptor 4 (TLR4), phosphorylation of protein kinase Cθ (PKCθ) and syntaxin binding protein 1 (STXBP-1) were determined by Western Bolt. The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) complex formation [syntaxin 2-synaptosomal-associated protein 23-vesicle associated membrane protein 8 (STX2-SNAP23-VAMP8)] mediated by STXBP-1 was determined by immunoprecipitation. Results ① Compared with normal control group, the platelet release of PDGF-BB, MMP-2 and ATP was significantly increased after LPS challenge, and the P-selectin expression of platelet was also obviously up-regulated [PDGF-BB (μg/L): 127.53±1.78 vs. 94.35±5.84, MMP-2 (ng/L): 51.87±9.20 vs. 35.83±3.17, ATP (μmol/L): 1.288±0.056 vs. 0.975±0.010, P-selectin: (3.93±0.19)% vs. (0.44±0.10)%, all P < 0.05]. The increases in platelet release of PDGF-BB, MMP-2 and ATP were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration, as well as high-expression of P-selectin in a dose-dependent manner [PDGF-BB (μg/L): 114.68±1.35, 97.08±6.14 vs. 127.53±1.78, MMP-2 (ng/L): 32.67±8.00, 24.63±1.63 vs. 51.87±9.20, ATP (μmol/L): 0.999±0.015, 0.965±0.008 vs. 1.288±0.056, P-selectin: (1.95±0.27)%, (0.94±0.11)% vs. (3.93±0.19)%, all P < 0.05]. ② Compared with normal control group, LPS challenge resulted in a significant increase in the expression of TLR4 and the phosphorylation of PKCθ and STXBP-1 [TLR4 (gray value): 1.21±0.38 vs. 0.67±0.06, p-PKCθ (gray value): 1.36±0.20 vs. 0.44±0.03, p-STXBP-1 (gray value): 1.13±0.06 vs. 0.59±0.04, all P < 0.05]. The increases in above parameters were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [TLR4 (gray value): 0.76±0.05, 0.65±0.04 vs. 1.21±0.38; p-PKCθ (gray value): 0.71±0.07, 0.47±0.10 vs. 1.36±0.20; p-STXBP-1 (gray value): 0.56±0.02, 0.48±0.01 vs. 1.13±0.06, all P < 0.05]. ③ Compared with normal control group, the SNAREs proteins in platelet that combined with STXBP-1, including STX2, SNAP23 and VAMP8, were obviously increased after LPS challenge [STX2 (gray value): 1.35±0.06 vs. 0.57±0.04, SNAP23 (gray value): 0.97±0.04 vs. 0.30±0.12, VAMP8 (gray value): 1.37±0.12 vs. 0.77±0.10, all P < 0.05]. The increases in SNAREs complex formation were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [STX2 (gray value): 0.77±0.02, 0.39±0.03 vs. 1.35±0.06, SNAP23 (gray value): 0.41±0.03, 0.22±0.08 vs. 0.97±0.04, VAMP8 (gray value): 0.85±0.07, 0.66±0.07 vs. 1.37±0.12, all P < 0.05]. There was no significant difference in the above mentioned parameters between iCORM-2 group and LPS group. Conclusions LPS-induced abnormal secretion of platelet was suppressed by CORM-2 administration. The mechanism may involve the TLR4/PKCθ/STXBP-1 signaling pathway activation and the SNAREs complex formation.