Relationship between autophagy and NLRP3 inflammasome during hydrogen-rich normal saline-induced reduction of lung injury in mice

Abstract

Objective To evaluate the relationship between autophagy and NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome during hydrogen-rich normal saline-induced reduction of lung injury in mice. Methods Thirty-six clean-grade healthy male C57BL mice, aged 6 weeks, weighing 20-25 g, were divided into 6 groups (n=6 each) by a random number table method: sham operation group (group Sham), sepsis group (group Sep), sepsis plus hydrogen-rich normal saline group (group Sep+ H2), sepsis plus hydrogen-rich normal saline plus rapamycin group (group Sep+ H2+ Rap), sepsis plus hydrogen-rich normal saline plus 3-methyladenine (3-MA) group (group Sep+ H2+ 3-MA) and sepsis+ hydrogen-rich normal saline plus MCC950 group (group Sep+ H2+ M). Sepsis was produced by cecal ligation and puncture (CLP) in mice anesthetized with chloral hydrate.Hydrogen-rich normal saline 5 ml/kg was intraperitoneally injected at 1 and 6 h after CLP in Sep+ H2, Sep+ H2+ Rap, Sep+ H2+ 3-MA and Sep+ H2+ M groups.At 1 h after CLP, autophagy agonist rapamycin 10 mg/kg was intraperitoneally injected in group Sep+ H2+ Rap, autophagy inhibitor 3-MA 15 mg/kg was intraperitoneally injected in group Sep+ H2+ 3-MA, and NLRP3 inflammasome inhibitor MCC950 50 mg/kg was intraperitoneally injected in group Sep+ H2+ M.At 24 h after establishing the model, bronchoalveolar lavage fluid (BALF) was collected to determine the protein concentration.The animals were then sacrificed, and the lung tissues were removed for microscopic examination of pathologic changes which were scored and for determination of wet/dry weight ratio (W/D ratio), activity of myeloperoxidase (MPO), contents of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay), and expression of pro-caspase-1 P10, NLRP3 and apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain (ASC) (using Western blot). Results Compared with group Sham, the protein concentration in BALF, pathological score, W/D ratio, activity of MPO, contents of IL-β, IL-18 and TNF-α and expression of pro-caspase-1 P10, NLRP3 and ASC were significantly increased in Sep and Sep+ H2 groups (P<0.05). Compared with group Sep, the protein concentration in BALF, pathological score, W/D ratio, activity of MPO, contents of IL-β, IL-18 and TNF-α and expression of pro-caspase-1 P10, NLRP3 and ASC were significantly decreased in Sep+ H2, Sep+ H2+ Rap and Sep+ H2+ 3-MA groups (P<0.05). Compared with group Sep+ H2, the protein concentration in BALF, pathological score, W/D ratio, activity of MPO, contents of IL-β, IL-18 and TNF-α and expression of pro-caspase-1 P10, NLRP3 and ASC were significantly decreased in Sep+ H2+ Rap and Sep+ H2+ M groups (P<0.05), and the protein concentration in BALF, pathological score, W/D ratio, activity of MPO, contents of IL-β, IL-18 and TNF-α and expression of pro-caspase-1 P10, NLRP3 and ASC were significantly increased in group Sep+ H2+ 3-MA (P<0.05). Conclusion The mechanism by which hydrogen-rich normal saline reduces lung injury is associated with inhibiting autophagy-mediated activation of NLRP3 inflammasome in septic mice. Key words: Hydrogen; Sepsis; Acute lung injury; Autophagy; NOD like receptor pynn domain containing 3