Melatonin Alleviates Lps-induced Ali By Inhibiting Pyroptosis Via Activating Nrf2/HO-1 Pathway

Background It is well-known that acute lung injury（ALI）/ acute respiratory distress syndrome（ARDS）is a fatal clinical syndrome characterized by progressive hypoxemia,dyspnea,and increased work of breathing,which caused by infection,trauma,blood transfusion,drug poisoning and other pathogenic factors.Epidemiological investigations indicated that ALI/ARDS represented over 10% of total intensive care unit（ICU）admissions with a mortality approximately 40%.In recent years,various therapeutic strategies including protective ventilation measures have greatly improved the survival rates of patients in hospital,there is still no specific therapeutic measures,and the underlying pathophysiological mechanism remains unclear.Therein,severe inflammatory responses and oxidative stress are the primary pathophysiology of ALI/ARDS progression.Melatonin（Mel,N-acetyl-5-methoxytryptamine）,a neurosecretory hormone,is mainly secreted from pineal gland with protective biological functions of anti-inflammation and antioxidants.The study aims to explore the protective effects of melatonin in LPSinduced acute lung injury,and its potential mechanism.ObjectiveTo investigate whether melatonin was capable of alleviating LPS-induced ALI by inhibiting pyroptosis via activating the Nrf2/HO-1 pathway in vivo and in vitro.Materials and Methods1.In vivo,8-week-old male C57BL/6 mice were randomly divided into four groups,including the PBS group,the melatonin+PBS group,the LPS stimulation group and the melatonin+LPS group（8 mice each group）.Mice were injected with melatonin（30 mg/kg）intraperitoneally for consecutive five days at 6 pm before LPS instillation intratracheally（5mg/kg,50μl）.On the fifth day,mice were given one dose of melatonin,1 h later,mice were instilled intratracheally with PBS or LPS（5 mg/kg）dissolved in 50 μL of PBS,and the mice were sacrificed 24 h later.The bronchoalveolar lavage fluid（BALF）was collected,inflammatory cells in BALF were counted and smeared.The levels of IL-1βand TNF-α in BALF were detected by ELISA.MDA,GSH and GSSG levels in serums were detected.Lung injury scoring was calculated by HE staining of lung sections.And we also proceeded the elevation of the wet and dry lung weight ratio（W/D）and the survival analysis.Western blot and immunohistochemistry were used to detect the activation of Nrf2/HO-1 and ROS-NLRP3/GSDMD pathway in mice lung tissues.The mRNA expressions of inflammatory cytokines（IL-1β and TNF-α）and antioxidant genes（HO-1,NQO1,SOD and Catalase）in mouse lung tissues were detected by RT-PCR.2.In vitro,human alveolar epithelial cell（AECⅡ）A549 cell lines and mouse macrophage Raw264.7 cell lines were pretreated with melatonin（400 μM）before LPS（10 μg/ml）stimulation.ROS and MDA contents were detected.The expression of Nrf2 and HO-1were detected by western blot and immunofluorescence,and the expression of NLRP3,Caspase-1 and GSDMD-N were detected by western blot.RT-PCR was used to determine mRNA expressions of IL-1β and TNF-α as well as antioxidant genes（HO-1,NQO1,SOD and Catalase）.TUNEL staining and LDH releasing detection were used to calculate the degrees of of cell pyroptosis.Results1.In vivo,LPS stimulation induced obvious pulmonary neutrophils infiltration,alveolar damage with formation of hyaline membranes and edema.Compared to LPS-stimulated mice,melatonin pretreatment significantly down-regulated lung injury scoring and the lung W/D ratio,alleviated lung edema;reduced the counting of neutrophils and macrophages in BALF;inhibited the levels of IL-1β and TNF-α in BALF;inhibited the contents of MDA and GSSG in serum.Mechanistically,melatonin pretreatment activated Nrf2/HO-1 signaling axis,promoted mRNA expression of HO-1,NQO1,Mn-SOD and Catalase;inhibited the protein expression of iNOS and NLRP3,Caspase-1 and GSDMDN as well as the mRNA expression TNF-α,IL-1β and IL-18 in mice lung tissues.2.In vitro,western blot and immunofluorescence showed that melatonin pretreatment induced the significant up-regulation of Nrf2 and HO-1 expression,and drove antioxidant genes expression including HO-1,NQO1,Mn-SOD and Catalase in LPS-stimulated A549 cells and Raw264.7 cells.And,melatonin inhibited the ROS levels and MDA contents in the medium.Additionally,melatonin also inhibited LPS-induced pyroptosis by reversing the overexpression of NLRP3,Caspase-1 and GSDMD-N,as well as IL-1β,IL-18,LDH release and TUNEL-positive cells in A549 cells and Raw264.7 cells.ConclusionsMelatonin alleviates LPS-induced pyroptosis in acute lung injury by inhibiting NLRP3/GSDMD pathway via activating the Nrf2/HO-1 pathway.