Nicotinamide mononucleotide alleviates endotoxin-induced acute lung injury by modulating macrophage polarization via the SIRT1/NF-κB pathway

Abstract

Context: Sepsis-induced acute lung injury (ALI) is a severe condition with limited effective therapeutics; nicotinamide mononucleotide (NMN) has been reported to exert anti-inflammatory activities.

Objective: This study explores the potential mechanisms by which NMN ameliorates sepsis-induced ALI in vivo and in vitro.

Materials and methods: Cultured MH-S cells and a murine model were used to evaluate the effect of NMN on sepsis-induced ALI. MH-S cells were stimulated with LPS (1 μg/mL) and NMN (500 μM) for 12 h grouping as control, LPS, and LPS + NMN. Cell viability, apoptotic status, and M1/2 macrophage-related markers were detected. The mice were pretreated intraperitoneally with NMN (500 mg/kg) and/or EX-527 (5 mg/kg) 1 h before LPS injection and randomized into 7 groups (n = 8): control, LPS, LPS + NMN, NMN, LPS + NMN + EX-527 (a SIRT1 inhibitor), LPS + EX-527, and EX-527. After 12 h, lung histopathology, W/D ratio, MPO activity, NAD⁺ and ATP levels, M1/2 macrophage-related markers, and expression of the SIRT1/NF-κB pathway were detected.

Results: In MH-S cells, NMN significantly decreased the apoptotic rate from 12.25% to 5.74%. In septic mice, NMN improved the typical pathologic findings in lungs and reduced W/D ratio and MPO activity, but increased NAD⁺ and ATP levels. Additionally, NMN suppressed M1 but promoted M2 polarization, and upregulated the expression of SIRT1, with inhibition of NF-κB-p65 acetylation and phosphorylation. Furthermore, inhibition of SIRT1 reversed the effects of NMN-induced M2 macrophage polarization.

Conclusions: NMN protects against sepsis-induced ALI by promoting M2 macrophage polarization via the SIRT1/NF-κB pathway, it might be an effective strategy for preventing or treating sepsis-induced ALI.

Keywords: NAD+; NMN; septic lung injury.

Figure 1.

Effect of NMN on cell proliferation and apoptosis of LPS-stimulated MH-S lines. (A) Cell viability of differentially treated MH-S cells was determined by CCK8 kit. (B–C) Apoptosis of MH-S cells induced by different groups as indicated was measured by Annexin V-FITC/PI. Data are portrayed as mean ± SD and statistical analysis was performed by one-way ANOVA followed by the Bonferroni post hoc test. *p < 0.05, **p < 0.01 vs. controls, ^#p < 0.05, ^##p < 0.01 vs. LPS group.

Figure 2.

NMN promotes M2-type macrophage polarization in LPS-stimulated MH-S cells. (A) Relative mRNA expressions of IL-1β, TNF-α, IL-6 and IL-10 were detected. (B) Representative mRNA levels of iNOS. (C) Representative mRNA levels of Arg1. (D–F) Representative blots and relative expressions of iNOS and Arg1 in MH-S cells. Data are presented as mean ± SD and analysed by one-way ANOVA corrected with Bonferroni test. *p < 0.05 and **p < 0.01 relative to controls, ^#p < 0.05 and ^##p < 0.01 relative to LPS group, respectively.

Figure 3.

NMN attenuates lung injury induced by LPS. (A) The image of histopathologic changes of lung sections with H&E staining (×400). (B) Semiquantitative assessment of ALI using lung injury scores. The grading scale of 0 = minimal damage, 1+ =mild damage (25%), 2+ =moderate damage (50%), 3+ =severe damage (75%), and 4+ =maximal damage (almost 100%). (C) The W/D ratio. (D) MPO activity. Data are presented as mean ± SD and analysed by one-way ANOVA followed with Bonferroni test. *p < 0.05, **p < 0.01 vs. control and ^#p < 0.05, ^##p < 0.01 vs. LPS group.

Figure 4.

The impact of NMN on NAD⁺ and ATP levels in lung tissues. (A) The levels of NAD⁺ were determined with an NAD/NADH Assay Kit. (B) NAD⁺/NADH ratio in lung tissues. (C) ATP content was also detected in this part of the study. Data are expressed as mean ± SD using one-way ANOVA followed with Bonferroni test. *p < 0.05, **p < 0.01 vs. control and ^#p < 0.05, ^##p < 0.01 vs. LPS group.

Figure 5.

Effect of NMN on macrophage differentiation during LPS-induced ALI. (A) Relative mRNA levels of inflammatory cytokines IL-1β, TNF-α, IL-6, and IL-10 were determined. (B–D) Western blots of iNOS and Arg1 and quantitation. (E–G) The levels of CD86 (for M1 macrophages) and CD206 (for M2 macrophages) were evaluated by flow cytometric analysis. Values are expressed as mean ± SD and analysed by one-way ANOVA corrected with Bonferroni coefficient. *p < 0.05, **p < 0.01 vs. control group and ^#p < 0.05, ^##p < 0.01 vs. LPS group, respectively.

Figure 6.

NMN activated the SIRT1/NF-κB pathway in sepsis-related ALI. (A–E) Representative bands and quantification of SIRT1, acetylated, phosphorylated NF-κB-p65, and NF-κB-p65. (F–G) Levels of acetylated NF-κB-p65 were shown by immunofluorescence (scale bar = 50 μm). Data are presented as means ± SD and analysed by one-way ANOVA followed with Bonferroni coefficient. **p < 0.01 compared with control; ^#p < 0.05, ^##p < 0.01 compared with LPS group.

Figure 7.

The SIRT1/NF-κB pathway was associated with the NMN-mediated M2 macrophage polarization. (A–G) Representative bands and quantification of SIRT1, acetylated, phosphorylated NF-κB-p65, NF-κB-p65, iNOS, and Arg1. (H–J) The levels of CD86 (for M1 macrophages) and CD206 (for M2 macrophages) were evaluated by flow cytometric analysis. Data are presented as means ± SD and analysed by one-way ANOVA followed with Bonferroni coefficient. *p < 0.05, **p < 0.01 compared with control; ^#p < 0.05, ^##p < 0.01 compared with LPS group.