Melanocortins protect against multiple organ dysfunction syndrome in mice

Abstract

Background and purpose: Melanocortins reverse circulatory shock and improve survival by counteracting the systemic inflammatory response, and through the activation of the vagus nerve-mediated cholinergic anti-inflammatory pathway. To gain insight into the potential therapeutic value of melanocortins against multiple organ damage following systemic inflammatory response, here we investigated the effects of the melanocortin analogue [Nle⁴ D-Phe⁷]α-MSH (NDP-α-MSH) in a widely used murine model of multiple organ dysfunction syndrome (MODS).

Experimental approach: MODS was induced in mice by a single intraperitoneal injection of lipopolysaccharide followed, 6 days later (= day 0), by zymosan. After MODS or sham MODS induction, animals were randomized to receive intraperitoneally NDP-α-MSH (340 µg·kg⁻¹ day) or saline for up to 16 days. Additional groups of MODS mice were concomitantly treated with the melanocortin MC₄ receptor antagonist HS024, or the nicotinic acetylcholine receptor antagonist chlorisondamine, and NDP-α-MSH.

Key results: At day 7, in the liver and lung NDP-α-MSH, significantly reduced mRNA expression of tumour necrosis factor-α (TNF-α), increased mRNA expression of interleukin-10 and improved the histological picture, as well as reduced TNF-α plasma levels; furthermore, NDP-α-MSH dose-dependently increased survival rate, as assessed throughout the 16 day observation period. HS024 and chlorisondamine prevented all the beneficial effects of NDP-α-MSH in MODS mice.

Conclusions and implications: These data indicate that NDP-α-MSH protects against experimental MODS by counteracting the systemic inflammatory response, probably through brain MC₄ receptor-triggered activation of the cholinergic anti-inflammatory pathway. These findings reveal previously undescribed effects of melanocortins and could have clinical relevance in the MODS setting.

Figure 1

Experimental schedule and treatments.

Figure 2

NDP-α-MSH improved survival rate in MODS mice. HS024 and chlorisondamine counteracted the life-saving effect of NDP-α-MSH. Kaplan-Meier curves. At day 0, mice were 20 per group; each time point represents percentage of animals surviving. NDP-α-MSH: daily administration of 85, 170 or 340 µg·kg⁻¹ i.p. throughout the observation period (up to 16 days after zymosan, where indicated); saline: 1 mL·kg⁻¹ i.p.; HS024: 130 µg·kg⁻¹ i.p. 1 min before each NDP-α-MSH or saline administration; chlorisondamine: 0.25 mg·kg⁻¹ s.c. 1 min before each NDP-α-MSH or saline administration. *P < 0.01 vs. MODS + saline (Log Rank test).

Figure 3

Treatment with NDP-α-MSH reduced TNF-α mRNA expression in the liver (A) and lung (B) of MODS mice. Pretreatment with the melanocortin MC₄ receptor antagonist HS024 and the nicotinic acetylcholine receptor antagonist chlorisondamine prevented the effect of NDP-α-MSH. Values are means ± SEM for 6 mice per group at day 7 after zymosan. NDP-α-MSH: 340 µg·kg⁻¹ i.p. for 7 days after zymosan; saline: 1 mL·kg⁻¹ i.p. for 7 days after zymosan; HS024: 130 µg·kg⁻¹ i.p. 1 min before each NDP-α-MSH or saline administration; chlorisondamine: 0.25 mg·kg⁻¹ s.c. 1 min before each NDP-α-MSH or saline administration. *P < 0.01 vs. MODS + saline; #P < 0.01 vs. MODS + NDP-α-MSH (Student-Newman-Keuls test).

Figure 4

Treatment with NDP-α-MSH increased IL-10 mRNA expression in the liver (A) and lung (B) of MODS mice. Pretreatment with the melanocortin MC₄ receptor antagonist HS024 and the nicotinic acetylcholine receptor antagonist chlorisondamine prevented the effect of NDP-α-MSH. Values are means ± SEM for 6 mice per group at day 7 after zymosan. NDP-α-MSH: 340 µg·kg⁻¹ i.p. for 7 days after zymosan; saline: 1 mL·kg⁻¹ i.p. for 7 days after zymosan; HS024: 130 µg·kg⁻¹ i.p. 1 min before each NDP-α-MSH or saline administration; chlorisondamine: 0.25 mg·kg⁻¹ s.c. 1 min before each NDP-α-MSH or saline administration. *P < 0.01 vs. MODS + saline; #P < 0.01 vs. MODS + NDP-α-MSH (Student-Newman-Keuls test).

Figure 5

Treatment with NDP-α-MSH reduced TNF-α plasma levels. Pretreatment with the melanocortin MC₄ receptor antagonist HS024 and the nicotinic acetylcholine receptor antagonist chlorisondamine prevented the effect of NDP-α-MSH. Values are means ± SEM for 6 mice per group at day 7 after zymosan. NDP-α-MSH: 340 µg·kg⁻¹ i.p. for 7 days after zymosan; saline: 1 mL·kg⁻¹ i.p. for 7 days after zymosan; HS024: 130 µg·kg⁻¹ i.p. 1 min before each NDP-α-MSH or saline administration; chlorisondamine: 0.25 mg·kg⁻¹ s.c. 1 min before each NDP-α-MSH or saline administration. *P < 0.01 versus MODS + saline; #P < 0.01 vs. MODS + NDP-α-MSH (Student-Newman-Keuls test).

Figure 6

Treatment with NDP-α-MSH (340 µg·kg⁻¹ i.p. for 7 days after zymosan) reduced histological damage in the liver and lung of MODS mice. Representative light microscopy images of experiments of Table 1. (A) Normal liver histology at day 7 from sham MODS mice. (B) Representative liver from saline-treated MODS mice: presence of inflammatory cell infiltrates, steatosis, necrosis and ballooning degeneration. (C) Representative liver from NDP-α-MSH-treated MODS mice: few inflammatory cells, reduced steatosis, absence of necrosis and/or ballooning degeneration. (D) Representative liver from MODS mice treated with the nicotinic receptor antagonist chlorisondamine 1 min before each administration of NDP-α-MSH: massive necrosis, cellular degeneration, strong presence of inflammatory cells. (E) Normal lung histology at day 7 from sham MODS mice. (F) Representative lung from saline-treated MODS mice: presence of a sustained inflammatory cells infiltrate, vascular congestion and interstitial oedema. (G) Representative lung from NDP-α-MSH-treated MODS mice: reduction of inflammatory cells infiltrate, vascular congestion and interstitial oedema. (H) Representative lung from MODS mice treated with the nicotinic receptor antagonist chlorisondamine 1 min before each administration of NDP-α-MSH: strong inflammatory cells infiltrate, vascular congestion and interstitial oedema. Haematoxylin-Eosin staining; original magnification: liver X 40 and lung X 20.