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, 44 (4), 1309-1324

Hydrogen Alleviates Mitochondrial Dysfunction and Organ Damage via Autophagy‑mediated NLRP3 Inflammasome Inactivation in Sepsis

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Hydrogen Alleviates Mitochondrial Dysfunction and Organ Damage via Autophagy‑mediated NLRP3 Inflammasome Inactivation in Sepsis

Hongguang Chen et al. Int J Mol Med.

Abstract

Sepsis is a highly heterogeneous syndrome that is caused by a dysregulated host response to infection. The disproportionate inflammatory response to invasive infection is a triggering event inducing sepsis. The activation of inflammasomes in sepsis can amplify inflammatory responses. It has been reported that damaged mitochondria contribute to NACHT, LRR and PYD domains‑containing protein 3 (NLRP3) inflammasome‑related sepsis. Our previous study revealed that hydrogen (H2) exerts anti‑inflammatory effects in sepsis but the detailed mechanism remains to be elucidated. In the present study, septic mice induced by cecal ligation and puncture (CLP) and macrophages induced by lipopolysaccharide (LPS) were used as models of sepsis in vivo and in vitro, respectively. An inducer and inhibitor of autophagy and the NLRP3 inflammasome were administered to investigate the detailed mechanism of action of H2 treatment in sepsis. The results demonstrated that LPS and ATP led to NLRP3 inflammasome pathway activation, excessive cytokine release, mitochondrial dysfunction and the activation of autophagy. CLP induced organ injury and NLRP3 pathway activation. H2 treatment ameliorated vital organ damage, the inflammatory response, mitochondrial dysfunction and NLRP3 pathway activation, and promoted autophagy in macrophages induced by LPS and in CLP mice. However, the inhibitor of autophagy and the inducer of NLRP3 reversed the protective effect of H2 against organ damage, the inflammatory response and mitochondrial dysfunction in vivo and in vitro. Collectively, the results demonstrated that H2 alleviated mitochondrial dysfunction and cytokine release via autophagy‑mediated NLRP3 inflammasome inactivation.

Figures

Figure 1
Figure 1
Cytokine and inflammasome protein expression in macrophages treated with LPS, ATP and H2. Macrophages were exposed to by LPS, ATP and H2. The culture supernatants were collected to detect cytokines (A) IL-1β, (B) IL-18, (C) TNF-α and (D) IL-6 by ELISA, and cells were harvested to measure the expression of (E) caspase-1, (F) NLRP3 and (G) ASC by western blotting. (H) Blots of caspase-1, NLRP3 and ASC in cells. Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. Con group, **P<0.05 vs. LPS group, ##P<0.05 vs. LPS+ATP group. LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; ASC, apoptosis-associated speck-like protein containing a CARD; NLRP3, NACHT, LRR and PYD domains-containing protein 3; H2, hydrogen; Con, control.
Figure 2
Figure 2
Mitochondria and mtDNA in macrophages treated with LPS, ATP and H2. Macrophages were treated by LPS, ATP and H2. Cells were harvested to measure (A) MMP, (B) RCR, (C) ATP, (D) mtDNA and (E) ROS release. Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. Con group, **P<0.05 vs. LPS group, ##P<0.05 vs. LPS+ATP group. mtDNA, mitochondrial DNA; LPS, lipopolysaccharide; MMP, mitochondrial membrane potential; RCR, respiratory control ratio; ROS, reactive oxygen species; H2, hydrogen; Con, control.
Figure 3
Figure 3
Autophagy-related protein expression in macrophages treated with LPS, ATP and H2. Macrophages were treated by LPS, ATP and H2. Cells were harvested to measure the (A) number of autophagosomes (indicated by yellow arrows; magnification, ×10,000), and the expression of (B) LC3, (C) Beclin 1, (D) PINK1, (E) Parkin and (F) VDAC by western blotting. (G) Representative blots of LC3, Beclin 1, Parkin and VDAC in cells. Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. Con group, **P<0.05 vs. LPS group. LPS, lipopolysaccharide; LC3, microtubule-associated protein 1 light chain 3; PINK1, PTEN-induced putative kinase 1; VDAC, voltage-dependent anion channel; H2, hydrogen; Con, control.
Figure 4
Figure 4
Effect of autophagy on inflammasome activation in macrophages induced by LPS and treated with H2. Macrophages were treated by LPS, ATP and H2, autophagy inducer Rap, and autophagy inhibitor 3-MA. The culture supernatants were collected to detect cytokines (A) IL-1β, (B) IL-18, (C) TNF-α and (D) IL-6 by ELISA, and cells were harvested to measure the expression of (E) caspase-1, (F) NLRP3 and (G) ASC by western blotting. (H) Representative blots of caspase-1, NLRP3 and ASC in cells. Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. LPS group, **P<0.05 vs. LPS+H2 group, ##P<0.05 vs. LPS+H2+NLRP3 inhibitor group. LPS, lipopolysaccharide; Rap, rapamycin; 3-MA, 3-methyladenine; IL, interleukin; TNF, tumor necrosis factor; NLRP3, NACHT, LRR and PYD domains-containing protein 3; ASC, apoptosis-associated speck-like protein containing a CARD; H2, hydrogen.
Figure 5
Figure 5
H2 alleviates mitochondrial dysfunction in macrophages induced by LPS via autophagy-mediated NLRP3 inactivation. Macrophages were treated with LPS, ATP and H2, autophagy inducer Rap and autophagy inhibitor 3-MA. Cells were harvested to measure (A) MMP, (B) RCR, (C) ATP, (D) mtDNA and (E) ROS release. Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. LPS group, **P<0.05 vs. LPS+H2 group, ##P<0.05 vs. LPS+H2+NLRP3 inhibitor group. LPS, lipopolysaccharide; Rap, rapamycin; 3-MA, 3-methyladenine; MMP, mitochondrial membrane potential; RCR, respiratory control ratio; mtDNA, mitochondrial DNA; ROS, reactive oxygen species; H2, hydrogen.
Figure 6
Figure 6
Effect of autophagy-mediated NACHT, LRR and PYD domains-containing protein 3 inactivation on lung injury, biochemical parameters of liver and kidney and survival rate in LPS-induced macrophages treated with H2. Sepsis was produced by CLP. Septic mice were treated with H2, autophagy inducer Rap and autophagy inhibitor 3-MA. After 24 h, lung tissues were collected to detect (A) pathological tissue changes by hematoxylin and eosin staining, (B) MPO activity and (C) W/D weight ratio; (D) bronchoalveolar lavage fluid was collected to analyze total proteins. (E) Liver and (F) kidney tissues were collected to investigate pathological scores. Blood was obtained to measure the biochemical parameters (G) ALT, (H) AST, (I) Cr and (J) BUN. (K) Survival rate was analyzed at 1, 2, 3, 5 and 7 days post-CLP (n=20). Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. Con group, **P<0.05 vs. CLP group, #P<0.05 vs. CLP+H2 group. LPS, lipopolysaccharide; CLP, cecal ligation and puncture; Rap, rapamycin; 3-MA, 3-methyladenine; MPO, myeloperoxidase, W/D, wet/dry; ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen; Cr, creatinine; H2, hydrogen; Con, control.
Figure 6
Figure 6
Effect of autophagy-mediated NACHT, LRR and PYD domains-containing protein 3 inactivation on lung injury, biochemical parameters of liver and kidney and survival rate in LPS-induced macrophages treated with H2. Sepsis was produced by CLP. Septic mice were treated with H2, autophagy inducer Rap and autophagy inhibitor 3-MA. After 24 h, lung tissues were collected to detect (A) pathological tissue changes by hematoxylin and eosin staining, (B) MPO activity and (C) W/D weight ratio; (D) bronchoalveolar lavage fluid was collected to analyze total proteins. (E) Liver and (F) kidney tissues were collected to investigate pathological scores. Blood was obtained to measure the biochemical parameters (G) ALT, (H) AST, (I) Cr and (J) BUN. (K) Survival rate was analyzed at 1, 2, 3, 5 and 7 days post-CLP (n=20). Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. Con group, **P<0.05 vs. CLP group, #P<0.05 vs. CLP+H2 group. LPS, lipopolysaccharide; CLP, cecal ligation and puncture; Rap, rapamycin; 3-MA, 3-methyladenine; MPO, myeloperoxidase, W/D, wet/dry; ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen; Cr, creatinine; H2, hydrogen; Con, control.
Figure 7
Figure 7
H2 alleviates cytokine release and NLRP3 inflammasome activation in the lungs of septic mice via increasing autophagy. Septic mice were treated with H2, autophagy inducer Rap and autophagy inhibitor 3-MA. After 24 h, lung tissues were collected to detect cytokines (A) IL-1β, (B) IL-18 and (C) TNF-α by ELISA and the expression of (D) caspase-1, (E) NLRP3 and (F) ASC by western blotting. Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. Con group, **P<0.05 vs. the CLP group, #P<0.05 vs. CLP+H2 group. Rap, rapamycin; 3-MA, 3-methyladenine; IL, interleukin; TNF, tumor necrosis factor; NLRP3, NACHT, LRR and PYD domains-containing protein 3; ASC, apoptosis-associated speck-like protein containing a CARD; CLP, cecal ligation and puncture; H2, hydrogen; Con, control.
Figure 8
Figure 8
H2 alleviates cytokine release and NLRP3 inflammasome activation in the livers of septic mice via increasing autophagy. Septic mice were treated with H2, autophagy inducer Rap, and autophagy inhibitor 3-MA. After 24 h, liver tissues were collected to detect cytokines (A) IL-1β, (B) IL-18 and (C) TNF-α by ELISA and the expression of (D) caspase-1, (E) NLRP3 and (F) ASC by western blotting. Data are expressed as mean ± standard deviation (n=6). *P<0.05 vs. Con group, **P<0.05 vs. CLP group, #P<0.05 vs. CLP+H2 group. Rap, rapamycin; 3-MA, 3-methyladenine; IL, interleukin; TNF, tumor necrosis factor; NLRP3, NACHT, LRR and PYD domains-containing protein 3; ASC, apoptosis-associated speck-like protein containing a CARD; CLP, cecal ligation and puncture; H2, hydrogen; Con, control.
Figure 9
Figure 9
H2 alleviates cytokine release and NLRP3 inflammasome activation in the kidneys of septic mice via increasing autophagy. Septic mice were treated with H2, autophagy inducer Rap and autophagy inhibitor 3-MA. After 24 h, kidney tissues were collected to detect cytokines (A) IL-1β, (B) IL-18 and (C) TNF-α by ELISA and the expression of (D) caspase-1, (E) NLRP3 and (F) ASC by western blotting. Data are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. Con group, **P<0.05 vs. CLP group, #P<0.05 vs. CLP+H2 group. Rap, rapamycin; 3-MA, 3-methyladenine; IL, interleukin; TNF, tumor necrosis factor; NLRP3, NACHT, LRR and PYD domains-containing protein 3; ASC, apoptosis-associated speck-like protein containing a CARD; CLP, cecal ligation and puncture; H2, hydrogen; Con, control.

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