Heme oxygenase-1 protects against endotoxin-induced acute lung injury depends on NAD+-mediated mitonuclear communication through PGC1α/PPARγ signaling pathway

Abstract

Endotoxin-induced acute lung injury (ALI) is a challenging life-threatening disease for which no specific therapy exists. Mitochondrial dysfunction is corroborated as hallmarks in sepsis which commonly disrupt mitochondria-centered cellular communication networks, especially mitonuclear crosstalk, where the ubiquitous cofactor nicotinamide adenine dinucleotide (NAD⁺) is essential for mitonuclear communication. Heme oxygenase-1 (HO-1) is critical for maintaining mitochondrial dynamic equilibrium and regulating endoplasmic reticulum (ER) and Golgi stress to alleviating acute lung injury. However, it is unclear whether HO-1 regulates NAD⁺-mediated mitonuclear communication to exert the endogenous protection during endotoxin-induced ALI. In this study, we observed HO-1 attenuated endotoxin-induced ALI by regulated NAD⁺ levels and NAD⁺ affected the mitonuclear communication, including mitonuclear protein imbalance and UPR^mt to alleviate lung damage. We also found the protective effect of HO-1 depended on NAD⁺ and NAD⁺-mediated mitonuclear communication. Furtherly, the inhibition of the PGC1α/PPARγ signaling exacerbates the septic lung injury by reducing NAD⁺ levels and repressing the mitonuclear protein imbalance and UPR^mt. Altogether, our study certified that HO-1 ameliorated endotoxin-induced acute lung injury by regulating NAD⁺ and NAD⁺-mediated mitonuclear communications through PGC1α/PPARγ pathway. The present study provided complementary evidence for the cytoprotective effect of HO-1 as a potential target for preventing and attenuating of endotoxin-induced ALI.

Keywords: Acute lung injury; Heme oxygenase-1; Mitonuclear communication; NAD+; Sepsis.

Fig. 1

HO-1 attenuated the septic lung injury and increased NAD⁺ levels. A Expressions of the mRNA levels of HO-1. B Representative bands and quantification of HO-1. C Semiquantitative evaluation 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%). D The image of histopathologic changes of lung tissue with H&E staining (× 200). E Expressions of the mRNA levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. F The W/D ratio. G MPO activity. H The contents of NAD⁺ were determined with an NAD⁺/NADH Assay Kit. Values are expressed as mean ± SD and were analyzed by one-way ANOVA corrected with Bonferroni coefficient. ^*P < 0.05, ^**P < 0.01 versus WT group and ^#P < 0.05, ^##P < 0.01 versus WT + LPS group, respectively

Fig. 2

Effect of NAD⁺ levels on sepsis-related ALI. A Schematic of the NAD⁺ biosynthetic pathway. B NAD⁺ contents in lung tissue. C Relative mRNA levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 were determined. D Representative hematoxylin/eosin (H&E) staining of lung sections (× 200). E Semiquantification of pathological scores according to the pathological observation. Data are presented as mean ± SD and group comparisons were analyzed by one-way ANOVA followed by Bonferroni post hoc test. ^*P < 0.05, ^**P < 0.01 versus WT group, ^#P < 0.05, ^##P < 0.01 versus WT + LPS group, and ^{^}P < 0.05, ^{^^}P < 0.01 versus NMNAT1^−/− + LPS group, respectively

Fig. 3

NAD⁺ protected against mitochondrial dysfunction. A Mitochondrial ROS detected spectrofluorometrically using DCFH-DA as a fluorescent dye. B Mitochondrial DNA copy number (mtDNA-CN) in each group was measured using RT-PCR. C The columnar picture is the statistical analysis of the mitochondrial membrane potential. D The mitochondrial membrane potential (ΔΨm) changes in lung tissue were evaluated using JC-1 fluorescence dye by flow cytometry. Q1, red fluorescence + /green fluorescence, polarized ΔΨm; Q4, red fluorescence + /green fluorescence, depolarized ΔΨm. E The morphological alterations of mitochondria by transmission electron microscopy (Scale bar: 500 nm). Data are presented as mean ± SD and group comparisons were analyzed by one-way ANOVA followed by Bonferroni post hoc test. ^*P < 0.05, ^**P < 0.01 versus WT group, ^#P < 0.05, ^##P < 0.01 versus WT + LPS group, and ^{^}P < 0.05, ^{^^}P < 0.01 versus NMNAT1^−/− + LPS group, respectively

Fig. 4

NAD⁺ levels preserved the mitonuclear communication. A Representative Western blot of mitonuclear protein imbalance (MTCO1 and SDHA) and UPR^mt (HSP60, LONP1, HSP90) markers. B–E Quantification of the ratio of SDHA/MTCO1 and expressions of HSP60, LONP1, HSP90. Band intensity on the Western blotting images was expressed as their relative ratio compared with β-actin. Data are presented as mean ± SD and statistical analysis was performed by one-way analysis of variance followed by Bonferroni post hoc test. ^*P < 0.05, ^**P < 0.01 versus WT group, ^#P < 0.05, ^##P < 0.01 versus WT + LPS group, and ^{^}P < 0.05, ^{^^}P < 0.01 versus NMNAT1^−/− + LPS group, respectively

Fig. 5

The effect of HO-1 depended on NAD⁺ levels. A NAD⁺ contents in lung tissue. B Photomicrographs of histopathologic changes of lung sections stained with hematoxylin and eosin (× 200). C Semiquantitative analysis of lung tissues by lung injury scores. D The morphological alterations of mitochondria by transmission electron microscopy (Scale bar: 500 nm). E Mitochondrial ROS detected spectrofluorometrically using DCFH-DA as a fluorescent dye. F Mitochondrial DNA copy number (mtDNA-CN) in each group was measured using RT-PCR. G The mitochondrial membrane potential (ΔΨm) changes in lung tissue were evaluated using JC-1 fluorescence dye by flow cytometry. H The columnar picture is the statistical analysis of the mitochondrial membrane potential. Results are presented as mean ± SD and statistical analysis was performed by paired t-test. ^*P < 0.05, ^**P < 0.01

Fig. 6

The effect of HO-1 required NAD⁺-mediated mitonuclear protein imbalance and UPR^mt. A The protein expression levels of SDHA, MTCO1, HSP60, LONP1, HSP90 in lung extracts were detected by Western blotting. Band intensity on the western blotting images was expressed as their relative ratio compared with β-actin. B–E Quantification from Western blotting to assess the expressions of proteins. All data are presented as mean ± SD, and statistical analysis was performed by paired t-test. ^*P < 0.05, ^**P < 0.01

Fig. 7

PGC1α/PPARγ axis involved the regulation of HO-1/NAD⁺-mediated mitonuclear communication. A NAD⁺ levels. B Pathological changes of lung sections stained with H&E were observed by light microscopy (× 200). C Semiquantitative analysis of lung tissues by lung injury scores. D RT-PCR was used to measure the mitochondrial DNA (mtDNA) levels in the lung extracts. E Transmission electron microscopy (TEM) was utilized to investigate the ultrastructural changes of the mitochondria. (Scale bar: 500 nm). F, G The relative expression of PGC1α, PPARγ, HO-1, mito-encoded MTCO1 and nc-encoded SDHA, UPR^mt-related proteins HSP60, LONP1, and HSP90. Data are represented as mean ± SD and were analyzed by paired t-test. ^*P < 0.05, ^**P < 0.01