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Preconditioning of carbon monoxide releasing molecule-derived CO attenuates LPS-induced activation of HUVEC.

Sun B, Zou X, Chen Y, Zhang P, Shi G - Int. J. Biol. Sci. (2008)

Bottom Line: To investigate the effects and potential mechanisms of preconditioning of tricarbonyldichlororuthenium (III) dimer (CORM-2)-liberated CO on LPS-induced activation of endothelial cells (HUVEC).Preconditioning of CO liberated by CORM-2 elicited its anti-inflammatory effects by interfering with the induction of intracellular oxidative stress.In addition, it also supports the notion that CO is a potent inhibitor of iNOS and NF-kappaB.

View Article: PubMed Central - PubMed

Affiliation: Department of Burn and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang 212001, Jiangsu Province, PR China. sunbinwe@vip.sina.com

ABSTRACT

Objective: To investigate the effects and potential mechanisms of preconditioning of tricarbonyldichlororuthenium (III) dimer (CORM-2)-liberated CO on LPS-induced activation of endothelial cells (HUVEC).

Methods: HUVEC were pretreated with CORM-2 at the concentration of 50 or 100 microM for 2 hrs, washed and stimulated with LPS (10 microg/ml) for additional 4 hrs. Activation (oxidative stress) of HUVEC was assessed by measuring intracellular oxidation of DHR 123 or nitration of DAF-FM, specific H(2)O(2) and NO fluorochromes, respectively. The expression of HO-1, iNOS (Western blot) and ICAM-1 (cell ELISA) proteins and activation of inflammation-relevant transcription factor, NF-kappaB (EMSA) were assessed. In addition, PMN adhesion to HUVEC was also assessed.

Results: The obtained data indicate that pretreatment of HUVEC with CORM-2 results in: 1) decrease of LPS-induced production of ROS and NO; 2) up-regulation of HO-1 but decrease in iNOS at the protein levels; 3) inhibition of LPS-induced activation of NF-kappaB; and 4) downregulation of expression of ICAM-1, and this was accompanied by a decrease of PMN adhesion to LPS-stimulated HUVEC.

Conclusions: Preconditioning of CO liberated by CORM-2 elicited its anti-inflammatory effects by interfering with the induction of intracellular oxidative stress. In addition, it also supports the notion that CO is a potent inhibitor of iNOS and NF-kappaB.

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Effects of CORM-2 pretreatment on iNOS and HO-1 expression in LPS-stimulated HUVEC. The experimental conditions were the same as described in Fig.1. iNOS and HO-1 expression were determined by Western blot. Note that pretreatment of HUVEC with CORM-2 resulted in more effective inhibition of iNOS protein expression in LPS stimulated HUVEC compare to coincubation group,#p <0.05 as compared to LPS; *p <0.05 as compared to co-incubation with 50 μM CORM-2; * *p <0.05 as compared to co-incubation with 100 μM CORM-2 (A). Both pretreatment and coincubation of CORM-2 significantly upregulated expression of HO-1 in LPS-stimulated HUVECs. Shown is a representative image from three experiments. *p <0.05 as compared to control, # p <0.05 as compared to LPS (B).
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Figure 2: Effects of CORM-2 pretreatment on iNOS and HO-1 expression in LPS-stimulated HUVEC. The experimental conditions were the same as described in Fig.1. iNOS and HO-1 expression were determined by Western blot. Note that pretreatment of HUVEC with CORM-2 resulted in more effective inhibition of iNOS protein expression in LPS stimulated HUVEC compare to coincubation group,#p <0.05 as compared to LPS; *p <0.05 as compared to co-incubation with 50 μM CORM-2; * *p <0.05 as compared to co-incubation with 100 μM CORM-2 (A). Both pretreatment and coincubation of CORM-2 significantly upregulated expression of HO-1 in LPS-stimulated HUVECs. Shown is a representative image from three experiments. *p <0.05 as compared to control, # p <0.05 as compared to LPS (B).

Mentions: At 4h after LPS stimulation, the expression of iNOS (Fig.2 A) and HO-1 (Fig.2 B) in HUVEC significantly increased compared to the control. Both pretreatment and coincubation of CORM-2 in LPS-stimulated HUVECs significantly downregulated the expression of iNOS. However, CO preconditioning was more effective to downregulate the expression of iNOS compare to coincubation group. Interestingly, not only pretreatment, but also coincubation of CORM-2 in LPS-stimulated HUVECs significantly upregulated the expression of HO-1.


Preconditioning of carbon monoxide releasing molecule-derived CO attenuates LPS-induced activation of HUVEC.

Sun B, Zou X, Chen Y, Zhang P, Shi G - Int. J. Biol. Sci. (2008)

Effects of CORM-2 pretreatment on iNOS and HO-1 expression in LPS-stimulated HUVEC. The experimental conditions were the same as described in Fig.1. iNOS and HO-1 expression were determined by Western blot. Note that pretreatment of HUVEC with CORM-2 resulted in more effective inhibition of iNOS protein expression in LPS stimulated HUVEC compare to coincubation group,#p <0.05 as compared to LPS; *p <0.05 as compared to co-incubation with 50 μM CORM-2; * *p <0.05 as compared to co-incubation with 100 μM CORM-2 (A). Both pretreatment and coincubation of CORM-2 significantly upregulated expression of HO-1 in LPS-stimulated HUVECs. Shown is a representative image from three experiments. *p <0.05 as compared to control, # p <0.05 as compared to LPS (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2519837&req=5

Figure 2: Effects of CORM-2 pretreatment on iNOS and HO-1 expression in LPS-stimulated HUVEC. The experimental conditions were the same as described in Fig.1. iNOS and HO-1 expression were determined by Western blot. Note that pretreatment of HUVEC with CORM-2 resulted in more effective inhibition of iNOS protein expression in LPS stimulated HUVEC compare to coincubation group,#p <0.05 as compared to LPS; *p <0.05 as compared to co-incubation with 50 μM CORM-2; * *p <0.05 as compared to co-incubation with 100 μM CORM-2 (A). Both pretreatment and coincubation of CORM-2 significantly upregulated expression of HO-1 in LPS-stimulated HUVECs. Shown is a representative image from three experiments. *p <0.05 as compared to control, # p <0.05 as compared to LPS (B).
Mentions: At 4h after LPS stimulation, the expression of iNOS (Fig.2 A) and HO-1 (Fig.2 B) in HUVEC significantly increased compared to the control. Both pretreatment and coincubation of CORM-2 in LPS-stimulated HUVECs significantly downregulated the expression of iNOS. However, CO preconditioning was more effective to downregulate the expression of iNOS compare to coincubation group. Interestingly, not only pretreatment, but also coincubation of CORM-2 in LPS-stimulated HUVECs significantly upregulated the expression of HO-1.

Bottom Line: To investigate the effects and potential mechanisms of preconditioning of tricarbonyldichlororuthenium (III) dimer (CORM-2)-liberated CO on LPS-induced activation of endothelial cells (HUVEC).Preconditioning of CO liberated by CORM-2 elicited its anti-inflammatory effects by interfering with the induction of intracellular oxidative stress.In addition, it also supports the notion that CO is a potent inhibitor of iNOS and NF-kappaB.

View Article: PubMed Central - PubMed

Affiliation: Department of Burn and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang 212001, Jiangsu Province, PR China. sunbinwe@vip.sina.com

ABSTRACT

Objective: To investigate the effects and potential mechanisms of preconditioning of tricarbonyldichlororuthenium (III) dimer (CORM-2)-liberated CO on LPS-induced activation of endothelial cells (HUVEC).

Methods: HUVEC were pretreated with CORM-2 at the concentration of 50 or 100 microM for 2 hrs, washed and stimulated with LPS (10 microg/ml) for additional 4 hrs. Activation (oxidative stress) of HUVEC was assessed by measuring intracellular oxidation of DHR 123 or nitration of DAF-FM, specific H(2)O(2) and NO fluorochromes, respectively. The expression of HO-1, iNOS (Western blot) and ICAM-1 (cell ELISA) proteins and activation of inflammation-relevant transcription factor, NF-kappaB (EMSA) were assessed. In addition, PMN adhesion to HUVEC was also assessed.

Results: The obtained data indicate that pretreatment of HUVEC with CORM-2 results in: 1) decrease of LPS-induced production of ROS and NO; 2) up-regulation of HO-1 but decrease in iNOS at the protein levels; 3) inhibition of LPS-induced activation of NF-kappaB; and 4) downregulation of expression of ICAM-1, and this was accompanied by a decrease of PMN adhesion to LPS-stimulated HUVEC.

Conclusions: Preconditioning of CO liberated by CORM-2 elicited its anti-inflammatory effects by interfering with the induction of intracellular oxidative stress. In addition, it also supports the notion that CO is a potent inhibitor of iNOS and NF-kappaB.

Show MeSH
Related in: MedlinePlus