Nicotinamide mononucleotide ameliorates acute lung injury by inducing mitonuclear protein imbalance and activating the UPRmt

Abstract

Mitochondria need to interact with the nucleus under homeostasis and stress to maintain cellular demands and nuclear transcriptional programs. Disrupted mitonuclear interaction is involved in many disease processes. However, the role of mitonuclear signaling regulators in endotoxin-induced acute lung injury (ALI) remains unknown. Nicotinamide adenine dinucleotide (NAD⁺) is closely related to mitonuclear interaction with its central role in mitochondrial metabolism. In the current study, C57BL/6J mice were administrated with lipopolysaccharide 15 mg/kg to induce endotoxin-induced ALI and investigated whether the NAD⁺ precursor nicotinamide mononucleotide (NMN) could preserve mitonuclear interaction and alleviate ALI. After pretreatment with NMN for 7 days, NAD⁺ levels in the mitochondrial, nucleus, and total intracellular were significantly increased in endotoxemia mice. Moreover, supplementation of NMN alleviated lung pathologic injury, reduced ROS levels, increased MnSOD activities, mitigated mitochondrial dysfunction, ameliorated the defects in the nucleus morphology, and these cytoprotective effects were accompanied by preserving mitonuclear interaction (including mitonuclear protein imbalance and the mitochondrial unfolded protein response, UPR^mt). Furthermore, NAD⁺-mediated mitonuclear protein imbalance and UPR^mt are probably regulated by deacetylase Sirtuin1 (SIRT1). Taken together, our results indicated that NMN pretreatment ameliorated ALI by inducing mitonuclear protein imbalance and activating the UPR^mt in an SIRT1-dependent manner.

Keywords: Nicotinamide mononucleotide; acute lung injury; lipopolysaccharide; mitochondria; mitochondrial unfolded protein response.

Figure 1.

Administration of NMN increased NAD⁺ levels in mice challenged by LPS. (a) NAD⁺-biosynthesis salvage pathways and NAD⁺ catabolism. (b) The animal experimental flowchart. C57BL/6 mice (20–25 g) were subjected to different doses of NMN (100, 300, and 500 mg/kg/day) or PBS intraperitoneally at 18:00 daily for 7 days. LPS (15 mg/kg) or 0.9% normal saline were injected via caudal vein, respectively, after 1 h. Then, mice were sacrificed 12 h after LPS injection and lung tissues were collected. (c) Pulmonary total NAD⁺ contents and ratios of NAD⁺/NADH in mice. (d) Mitochondrial NAD⁺ contents and ratios of NAD⁺/NADH in mice in each group. (e) Nuclear NAD⁺ contents and ratios of NAD⁺/NADH in mice in each group. (f) The ratios of mitochondrial NAD⁺ contents/ nuclear NAD⁺ contents in mice in each group. Statistical difference was calculated by one-way ANOVA (n = 5/group). (A color version of this figure is available in the online journal.)

Figure 2.

NMN prevented oxidative damage and alleviated lung injury in endotoxemia mice. (a) The Kaplan–Meier survival curve for NMN pretreated mice and PBS pretreated mice after LPS administration. n = 15 for the control group, n = 18 for the LPS group, n = 16 for the LPS + NMN group, ^#p < 0.05, *p < 0.01. (b) HE staining and corresponding lung histology scores. Scale bar, 100 μm. (c) ROS levels. (d) MnSOD activity. Statistical difference was calculated by one-way ANOVA (n = 5/group). (e) Representative TUNEL staining of formalin-fixed lung sections. Scale bar, 100 μm and (f) counted percentages of TUNEL-positive cells (n = 3/group, Power calculation for statistic differences between groups = 1). (A color version of this figure is available in the online journal.)

Figure 3.

Effects of NMN on mitochondrial function. (a) Mitochondrial membrane potential (ΔΨm) analyzed by flow cytometry. Q2-2, red fluorescence+/green fluorescence+, polarized ΔΨm; Q2-4, red fluorescence–/green fluorescence+, depolarized ΔΨm. (b) Quantitative histogram of ΔΨm. (c) Relative mtDNA content using real-time PCR. (d) Cytochrome c oxidase activity in mice. Statistical difference was calculated by one-way ANOVA (n = 5/group). (e) The morphological alterations of mitochondria and the nucleus were observed by transmission electron microscopy. Scale bars, 2 μm. Red arrows denoted abnormal mitochondria, which were manifested as isolated, swollen, and vacuolization mitochondria without clear cristae. White arrows denoted unusual nucleus size and shape, pyknotic nuclei, and irregular nuclear membrane. All the results were obtained from three independent experiments. (A color version of this figure is available in the online journal.)

Figure 4.

NMN mitigated LPS-induced oxidative injury by inducing mitonuclear protein imbalance and activating the UPR^mt in mice. (a) The protein levels of mtDNA-encoded and nuclear DNA-encoded mitochondrial proteins (MTCO1, SDHA) from the lung tissues of mice. (b) The protein ratios of MTCO1 to SDHA (n = 3/group, Power calculation for statistic differences between groups = 0.987). (c) qRT-PCR assay was applied to detect the mRNA expression levels of HSP60 (n = 5/group). Statistical difference was calculated by one-way ANOVA. HSP60, heat shock protein 6; MTCO1, cytochrome c oxidase subunit I; SDHA, succinate dehydrogenase A.

Figure 5.

NMN protected LPS-induced acute lung injury in an SIRT1-dependent manner. (a) Representative immunoblots of SIRT1 in lung tissues (n = 3/group, power calculation for statistic differences between groups = 0.999). (b) HE staining and corresponding lung histology scores. Scale bar: 100 μm. (c) MnSOD activity (n = 5/group). (d) The morphological alterations of mitochondria and the nucleus were observed by transmission electron microscopy. Scale bars, 2 μm. Red arrows denoted abnormal mitochondria, which were manifested as isolated, swollen, and vacuolization mitochondria without clear cristae. White arrows denoted unusual nucleus size and shape, pyknotic nuclei, and irregular nuclear membrane. All the results were obtained from three independent experiments. (e) Relative mtDNA content using real-time PCR (n = 5/group). (f) The protein levels of MTCO1 and SDHA from lung tissues of mice. (g) The protein ratios of MTCO1 to SDHA (n = 3/group, Power calculation for statistic differences between groups = 0.830). (h) qRT-PCR assay was applied to detect the mRNA expression levels of HSP60 (n = 5/group). Statistical difference was calculated by one-way ANOVA. (A color version of this figure is available in the online journal.)

Figure 6.

Protective effects of NMN on LPS-induced acute lung injury and the underlying mechanism. When LPS was administrated to C57BL/6J mice induced the production of a large amount of ROS. Meanwhile, pretreatment with NMN increased the levels of NAD⁺, up-regulated SIRT1 expression, improved mitochondrial morphology and dysfunction, accompanied by inducing mitonuclear protein imbalance and activating the mitochondrial unfolded protein response in endotoxemia mice. Thus, NMN administration might be an effective strategy for preventing endotoxemia-induced lung injury. (A color version of this figure is available in the online journal.)