Korean J Crit Care Med.  2015 Aug;30(3):151-157. 10.4266/kjccm.2015.30.3.151.

The Anti-Inflammatory Effect of Arginine-Vasopressin on Lipopolysaccharide-Induced IkappaBalpha/Nuclear Factor-kappaB Cascade

• Park J
• Eo EY
• Lee KH
• Park JS
• Lee JH
• Yoo CG
• Lee CT
• Cho YJ

• KMID: 2227639
• DOI: http://doi.org/10.4266/kjccm.2015.30.3.151

Abstract

BACKGROUND
Arginine vasopressin (AVP) is widely used as a vasopressor agent. Some recent studies have suggested that AVP may exert an immunomodulatory effect. However, the mechanism about the anti-inflammatory effect of AVP is not well known. We investigated the effect of AVP on the ihibitor of kappa B (IkappaBalpha)/nuclear factor-kappa B (NF-kappaB) pathway in RAW 264.7 cells.
METHODS
Cultured RAW 264.7 cells were pretreated with AVP and stimulated with lipopolysaccharide (LPS). To evaluate the effect of AVP on inflammatory cytokines, the concentration of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were assessed by an enzyme-linked immunosorbent assay technique. The expression of IkappaBalpha and nuclear translocation of NF-kappaB p65 were measured by Western blotting, and IkappaB kinase (IKK) activity was analyzed by an in vitro immune complex kinase assay. To confirm the AVP effect on IkappaBalpha/NF-kappaB cascade and via V2 receptor, we added tolvaptan (V2 receptor antagonist) after AVP pretreatment.
RESULTS
The increase of IL-6 and TNF-alpha in LPS-stimulated RAW 264.7 cells was suppressed by a treatment with AVP. Pretreatment of AVP inhibited increasing of IKK activity and IkappaBalpha degradation induced by LPS in RAW 264.7 cells. Furthermore, LPS induced and NF-kappaB transcription was inhibited by AVP pretreatment. The observed changes in IKK activity, IkappaBalpha degradation and NF-kappaB transcription by AVP was abolished by tolvaptan treatment.
CONCLUSIONS
Our results suggest that AVP showed anti-inflammatory effect on LPS-induced IkappaBalpha/NF-kappaB cascade in mouse macrophages via V2 receptors.

Keyword

anti-inflammatory effect; arginine Vasopressin; IkappaBalpha; NF-kappaB

MeSH Terms

Animals
Antigen-Antibody Complex
Arginine Vasopressin
Blotting, Western
Cytokines
Enzyme-Linked Immunosorbent Assay
I-kappa B Kinase
Interleukin-6
Macrophages
Mice
NF-kappa B
Phosphotransferases
Receptors, Vasopressin
Tumor Necrosis Factor-alpha
Antigen-Antibody Complex
Arginine Vasopressin
Cytokines
I-kappa B Kinase
Interleukin-6
NF-kappa B
Phosphotransferases
Receptors, Vasopressin
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1. IL-6 and TNF-α expression after vasopressin and/or LPS by ELISA. After stimulation with LPS 1 μg for 6 hours, inflammatory cytokines (IL-6, TNF-α) were increased. However, AVP (10 nM for 4 hours) inhibits inflammatory cytokines production in LPS-induced RAW 264.7 cells compared with the level of LPS alone. The IL-6 level was increased after only AVP applying, but it was not significant (p = 0.199). (A) IL-6, (B) TNF-α. *p < 0.05, compared to the value of LPS alone. IL, interleukin; TNF, tumor necrosis factor, c, control; LPS, lipopolysaccharide; AVP, arginine vasopressin.

• 

  Fig. 2. IκB kinase (IKK) activity and IκBα expression after AVP and/or LPS by Western blotting. (A) IKK activity was enhanced by LPS (1 μg/mL for 5 minutes) but pretreated AVP (10 nM for 4 hours) down-regulated IKK activity. (B) IκBα was decreased by treatment with LPS for 10 minutes and pretreatment with AVP blocked IκBα degradation.

• 

  Fig. 3. NF-κB p65 transactivity after AVP and/or LPS by Western blotting. LPS stimulation (1 μg/mL for 30 minutes) alone decreased cytosolic p65 and increased nuclear p65. However, pretreatment with AVP attenuated the NF-κB p65 level changes when LPS was applied in the cytosol and nucleus. A: arginine vasopressin; L: lipopolysaccharide.

• 

  Fig. 4. Expression of COX-2 after AVP and/or LPS by Western blotting. AVP has no effect on the expression of COX-2. COX: cyclooxygenases.

• 

  Fig. 5. IκBα expression and NF-κB p65 transactivity after AVP and vasopressin receptor antagonist (Tolvaptan) treatment and/or LPS by Western blotting. AVP, tolvaptan, LPS treated cells showed no difference with LPS only stimulated cells on the value of IκBα activation and NF-κB p65 transactivity. (A) IκBα expression. (B) NF-κB p65 transactivity. A, arginine vasopressin; L, lipopolysaccharide; T, Tolvaptan.

Reference

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