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Melatonin alleviates lipopolysaccharide (LPS) / adenosine triphosphate (ATP)-induced pyroptosis in rat alveolar Type II cells (RLE-6TN) through nuclear factor erythroid 2-related factor 2 (Nrf2)-driven reactive oxygen species (ROS) downregulation

ABSTRACT Pyroptosis has pivotal parts within disease development, rendering this attractive mechanism for novel therapeutics. This investigation aimed at analyzing melatonin roles within pyroptosis together with related mechanistics. RLE-6TN cultures were exposed to varying LPS doses for 4.5 h followed by concomitant culturing in the presence of ATP (5 mM) for 0.5 h to induce injury, and the roles of melatonin, N-Acety-L-cysteine (NAC – a ROS scavenger), ML385 (specific Nrf2 inhibitor) were examined. Apoptosis analysis was performed through lactate dehydrogenase (LDH) activity assays, together with propidium iodide (PI) stain-assay. Intracellular ROS were quantified through 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA). Pyrolysis-associated proteins, such as nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific protease-1 P20 (Caspase-1 P20), gasdermin D-N (GSDMD-N), and mature interleukin-1β (IL-1β), were identified through Western blotting. Dataset outcomes demonstrated LPS/ATP induce RLE-6TN cell pyroptosis, while melatonin alleviated this phenomenon, visualized through increased cell survival rate, reduction of LDH discharge and PI+ cellular count. Moreover, melatonin effectively reduced NLRP3 inflammasome triggering in RLE-6TN cells. Meanwhile, this study demonstrated melatonin thwarting over NLRP3 inflammasome triggering was depending on ROS. In addition, this study found that melatonin activated Nrf2/Heme Oxygenase-1 (HO-1) pathway, with pyroptotic-inhibiting function of melatonin was reverted through a bespoke Nrf2-inhibitor and siNrf2. In summary, this study concluded that melatonin prevents RLE-6TN cellular pyroptosis through Nrf2-triggered ROS downregulation.


Introduction
Pyroptosis consists of a recently identified proinflammation-based type of apoptosis [1], characterized by the loss of membrane integrity [2], and dependent on the inflammasome [3]. Pyroptosis participates in heart and vascular conditions, tumors, together with rheumatism-based conditions [4] and acute lung injury (ALI) [5], rendering this an attractive drug target.
Melatonin (MT) is well known for its role in regulating the circadian rhythm. Furthermore, melatonin also carries anti-oxidant, anti-apoptotic, neuroprotecting, and immune-modulation roles [6]. Melatonin also exhibited effective roles against inflammation within atherosclerosis [7], Parkinson's Disease [8], and acute intraocular hypertension [9], since it downregulates NLRP3-inflammasome constituent/pro-inflammation cytokine expression simultaneously. One study has shown that melatonin relieves severe pulmonary injuries by thwarting NLRP3 inflammasome [10]. However, precise mechanisms for such melatonin roles have not been identified as yet.
ROS can be deemed as a major triggering factor for NLRP3 inflammasome [15]. Overwhelming ROS generation and/or lack of anti-oxidant defensive activities lead to oxidative stress [16]. The Nrf2 transcription factor represents a master controller for preventing ROS-driven oxidative stress [17]. Following triggering, Nrf2 translocated within the nucleus and enhanced HO-1 proteomic expression [18,19]. Nrf2 triggering was revealed to provide protection for macrophages exposed to LPS-driven inflammatory action, through antioxidant defensive measures [20]. Furthermore, within foam-cells, Nrf2/HO-1 nexus influences ROS generation, downregulating pro-inflammation mediator expression [21]. It has been reported that Nrf2 triggers, including melatonin and other naturally occurring molecular players can thwart NLRP3 inflammasome triggering [22,23]. Presently, functions for Nrf2 transcription factor pertaining to regulatory influences over melatonin, consequently affecting NLRP3 inflammasome triggering in ALI, are still undiscovered.
This investigation hypothesized that LPS/ATP co-treatment induces pyroptosis in RLE-6TN cells and that melatonin exerts its anti-pyroptotic effect by activating Nrf2 to reduce ROS production and inhibit NLRP3 inflammasome. The study goals were to analyze melatonin roles within pyroptosis, together within related molecular mechanisms.

Animals
All in vivo studies were conducted in line with the National Institutes of Health Guidelines for the Use of Laboratory Animals. Male C57BL/6 murines (Seven-weeks-old) were procured through Chongqing Medical University Animal Center. Animals had ad libitum water/chow-food and were kept in a regulated environment (20 ± 22°C; 12-hlight/dark cycle), and they were randomized within three separate groups (n = 6/group): control-group; LPS-group; LPS + melatonin-group. Control-murines were treated with sterilized saline. ALI was induced as previously described [24]. Briefly, murines were given intra-tracheal administration of 1 mg/Kg LPS in 50 μL of sterilized saline. Regarding LPS + melatonin group, murines were anesthetized and treated with melatonin (30 mg/Kg) from the trachea at 60 minutes post-ALI triggering, together with a second melatonin dose administered after a further 24-hour period [10].

Histology
Lung tissues were fixated with 4% paraformaldehyde, followed by dehydration, paraffin-embedding, and slicing. Slices were stained with hematoxylin and eosin (H&E) and evaluated under a light microscope.

Cell culture treatment
LPS (Sigma TM , USA)/melatonin (Sigma TM , USA) were placed into solution containing sterilized/deionized water followed by serial dilution preparations as necessary. ATP, NAC, and ML385 were procured through Sigma-Aldrich TM (Shanghai, China). The classic two-step approach of NLRP3 inflammasome activation in alveolar macrophages (AMs) [5] with minute modification, was used to activate the NLRP3 inflammasome within RLE-6TN cultures. Cells were stimulated with differing LPS doses for 270 minutes, with subsequent cotreatment with ATP for 30 minutes to fullyactivate NLRP3 inflammasome. Following pretreatment with melatonin and other chemicals, LPS and ATP were added. All experimental runs were conducted post-LPS/ATP administration.

Apoptotic rate analysis
Apoptosis was assessed through LDH discharge/PI stain-testing. LDH present within cellular culture supernatants was evaluated through LDH assay kit® (Nanjing Jiancheng Biology Engineering Institute, Jiangsu, China) in line with kit protocols. Regarding PI staining, the methods were followed according to protocols by Su et al. [25]. In brief, poststimulation, all cells were harvested/triple-washed using 1× PBS. PI (25 µg/mL, Beyotime TM , China) was added each well at 37°C in dark conditions, for 15 minutes. Eventually, DAPI (Beyotime TM , China) was employed for staining cells (10 minutes in dark conditions). Post-staining, images of the cells were immediately acquired by fluorescence microscopy. The fluorescence intensity was assessed using ImageJ software.

Cell viability assay
The half-maximal inhibitory concentration (IC50) for LPS/ATP and optimal study-doses for melatonin were ascertained through CCK-8 assay as previously described [26]. CCK-8 assay kit (Jian Cheng Biology Engineering Institute, Nanjing, China) was employed in line with kit protocol.

Detection of oxidative stress
ROS was detected as previously described [27]. Photos were taken through fluorescence microscopy (Olympus Corporation TM , Tokyo, Japan). Mean fluorescence intensity was evaluated through ImageJ software.

Nrf2 siRNA transfection
Both siNrf2 and negative control (NC) siRNA were purchased from RiboBio TM (Guangzhou, China). RLE-6TN cells were transfected with siNrf2 and negative control (NC) siRNA using the Lipofectamine 3000 reagent (Invitrogen TM , USA), according to the manufacturer ' s protocols. Western blotting was applied to assess transfection efficiency.

Extraction of cytoplasmic and nuclear proteins
Cytoplasmic and nuclear proteins were extracted as previously described [28]. Nuclear and Cytoplasmic Protein Extraction Kit® (NCPE), obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China), was employed for extracting cytoplasmic and nuclear proteins. Assays were carried out in line with manufacturer protocols.

Immunofluorescence staining
Immunofluorescence staining was performed according to the previously described protocols [30]. RLE-6TN cells were seeded on glass coverslips in a 6-well plate and cultured for 24 hours, followed by treatment with melatonin, LPS and ATP as described above. Post-treatment, cells were thrice-washed with 1× PBS for five minutes, fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeated with 0.1% Triton X-100® (Solarbio Science & Technology TM ) for 20 minutes, blocked with 10% goat serum for 60 minutes, and consequently incubated with Nrf2 primary antibodies (1:200) overnight, in a humid chamber at 4°C. Consequently, cells were incubated with Cy3-conjugated secondary antibody (1:40) for 60 minutes, and subsequently stained using DAPI (Beyotime TM , China) in dark conditions, for 5 minutes. Images were taken by fluorescence microscopy (Olympus Corporation TM , Tokyo, Japan). ImageJ software was used to evaluate fluorescence intensity.

Statistical analyses
Datasets were presented as mean ± standarddeviation (SD) of three separate replicates. Group variations were assessed through one-way ANOVA/ Bonferroni's multiple comparisons test (GraphPad Prism TM version 8.0). The significance of the difference between negative control siRNA and siNrf2 was determined using Student's t-tests. P values <0.05 were deemed to confer statistical significance.

Results
This study revealed that LPS/ATP co-culture induced RLE-6TN pyroptosis. This phenomenon was inhibited by melatonin. Melatonin reduces ROS production, following LPS/ATP treatment, by activating Nrf2, with resultant inhibition of NLRP3 inflammasome triggering and mitigating pyroptosis. Nrf2 inhibition and knockdown significantly promoted ROS production, NLRP3 inflammasome activity and pyroptosis. In essence, melatonin reduces LPS/ATP-driven ROS production through targeting Nrf2 activation, resulting in thwarting NLRP3 inflammasome activation and pyroptosis, at in vitro and in vivo levels.

LPS/ATP reduced cell viability and triggered NLRP3-dependent pyroptosis in RLE-6TN cells
The IC50 of LPS/ATP was identified through CCK-8 assay. As shown in Figure 1a, RLE-6TN cellular viability was negatively correlated with the LPS dose. Consequent to 100 μg/mL LPS, cellular viability was severely reduced, though was still above 50%. In order to demonstrate whether pyroptosis could be induced in LPS/ATP-treated RLE-6TN cells, pyroptosis-related proteomic quantifications were performed. As shown in Figure 1b-C, LPS/ATP costimulation enhanced proteomic content of NLRP3, ASC, Caspase-1 P20, GSDMD-N, and mature IL-1β. Upregulated proteomic expression profiles were also positively linked to LPS dose. Moreover, LPS/ATP co-treatment increased LDH release (Figure 1d). All of the data above indicated that LPS/ATP activated the NLRP3 inflammasome and induced pyroptosis within RLE-6TN cultures through an LPS dosedependent route, with strongest influence occurring at 100 μg/mL. Therefore, 100 μg/mL was the standard dose within following experiments.

Melatonin promoted cell viability and prevented RLE-6TN cells pyroptosis
As shown in Figure 2a, the maximum non-cytotoxic melatonin dose reached 200 μmol/L through CCK-8 methodology, with this dose selected for subsequent investigations within this study. In comparison to LPS/ATP group, melatonin enhanced RLE-6TN cell viability by approximately 21% (Figure 2b). Moreover, melatonin reduced LPS/ATP-directed LDH discharge (Figure 2c). PI stain-testing was conducted for evaluating plasma membrane integrity. Melatonin reduced PI-positive cellular population level (Figure 2d-e). These results indicated that melatonin (200 µmol/L) effectively prevented RLE-6TN cells pyroptosis.

Nrf2 activation is triggered through melatonin
Nrf2 is a master controller averting oxidative stress repercussions, whereby this study postulated that Nrf2 orchestrates NLRP3 inflammasome triggering inhibition through melatonin. Figure 4a/b depict melatonin upregulating nuclear-located Nrf2 and downregulating cytoplasmic Nrf2 levels, consequently upregulating the expression of HO-1 and NQO1. Meanwhile, immunofluorescence also confirmed that melatonin promoted Nrf2 nuclear transcription (Figure 4c). As a means of crystallizing Nrf2 triggering functions within RLE-6TN pyroptosis, ML385 (a Nrf2-inhibitor), was employed for pre-treating cultures. Figure 4d depicts Western blot analysis demonstrating melatonin downregulation influence on GSDMD-N and Caspase-1 P20, to be rescued through ML385. Consequently, melatonin suppressive effect  (Figure 4h) assay showed that the prevention of melatonin on RLE-6TN cells pyroptosis was reversed by ML385. Additionally, Nrf2 siRNA was also employed for further investigation on the possibility of Nrf2 affecting protective effects of melatonin on cell pyroptosis. Nrf2 siRNA downregulated relative expression of Nrf2 by almost 51%, when compared with transfection using siRNA negative control (NC) (Figure 4i). Meanwhile, Nrf2 siRNA dampened the inhibitory effect of melatonin on NLRP3 inflammasome (Figure 4j). Furthermore, Nrf2 siRNA reversed the protective effect of melatonin on pyroptosis ( Figure 4k) and ROS generation (Figure 4l). Such dataset outcomes suggested that prophylaxis effects by melatonin over RLE-6TN pyroptosis depended upon Nrf2 triggering.

Melatonin inhibits inflammation and oxidative stress and upregulated Nrf2 in vivo
In order to assess whether melatonin has a protective effect on ALI, the HE staining showed leukocyte penetration and alveolar-wall swellings post-ALI. However, melatonin doses reduced ALI (Figure 5a). Simultaneously, pulmonary IL-1β ( Figure 5b) and IL-18 ( Figure 5c) levels were also significantly increased in ALI murines, whereas melatonin treatment distinctly downregulated IL-1β/IL-18. This study also found that melatonin increased SOD level (Figure 5d  lung tissues, with results showing that melatonin reduced oxidative stress in murines with ALI. This study further detected the protein level of NLRP3, Nrf2, ASC, GSDMD-N, and Caspase-1 P20 in lung tissues, discovering that Nrf2 was markedly decreased and the NLRP3, ASC, GSDMD-N, Caspase-1 P20 levels were highly upregulated within LPS group. Consequently, melatonin promoted Nrf2 activation and downregulated NLRP3, ASC, GSDMD-N, Caspase-1 P20 (figure 5f). All such data validated this postulation whereby melatonin attenuates pyroptosis through inhibiting ROS via Nrf2 activation.

Discussion
ALI/ARDS (Acute Respiratory Distress Syndrome) represents a highly acute inflammation status together with diffused alveolar/epithelial injuries [31]. Apoptosis of RLE-6TN cells was deemed to be a possible mechanism for ALI [32]. However, apoptosis is typically not present with inflammation responses and thus apoptosis cannot justify such intense inflammation manifestations during ALI/ ARDS. A study has confirmed that RLE-6TN can release pro-inflammatory orchestrators once triggered [33]. Moreover, researchers confirmed that NLRP3 can be expressed in RLE-6TN cells [34]. Therefore, it was reasonable to speculate that RLE-6TN cells can participate in the acute inflammatory process of ALI through NLRP3 inflammasome. In this study, classic two-step method, that is widely used in macrophages, was employed to active NLRP3 inflammasome in RLE-6TN cells. This study found that LPS/ATP induced RLE-6TN cells pyroptosis in a LPS concentration-dependent manner and the required LPS dose for RLE-6TN was 100 μg/mL, being well elevated in comparison to doses for alveolar macrophages (500 ng/mL [5]). Consequently, the above results show that although RLE-6TN can produce inflammatory mediators post-stimulation, the stimulation dose required is extremely high, in line with findings from past studies [35]. It might remind us that in the early stage of ALI, alveolar macrophages play an important role.  Following the release of inflammatory factors, inflammatory burst aggravates the condition, and whereby the role of alveolar epithelial cells cannot be ignored. These results can provide a new underlying mechanism and prospect for treating ALI. Meanwhile, this study also paves the way for future research on associations for macrophages/alveolar epithelial cells within ALI.
Immune-system dysregulated function was always deemed to be a major influencer for inflammation-related diseases. Inflammasomes can be intimately linked to inflammatory diseases. The term 'inflammasome' was originally coined by Martinon et al. in 2002 [36]. Presently, there are five types of inflammasomes that have been confirmed: NLRP1, NLRP2, NLRP3, AIM2, and PAF/NLRC4. NLRP3 inflammasome emerges as the most scrutinized one [37]. It has been postulated that two steps are required to completely activate the NLRP3 inflammasome [38,39]. The first step is also called the priming step: PAMPs, such as LPS, or DAMPs, that induce transcriptional upregulation and posttranslational modifications of NLRP3/pro-IL-1β/ pro-IL-18 by binding to Toll-like receptors (TLRs) [40]. In the second (activation) step, components of the inflammasome build up into a complete inflammasome, promoting the autocatalysis of pro-Caspase -1, which triggers the cleavage of pro-IL-1β/IL-18 Caspase-1 P20, protein quantitative histogram shown on the right. Datasets represented as mean ± SD (n = 6) for each group. ** P < 0.01 in comparison to control group. # # P < 0.01, compared to LPS group. and releasing matured IL-1β/IL-18, resulting in inflammation and cleaving of gasdermin-D (GSDM-D) into GSDMD-N concomitantly, ultimately inducing inflammatory apoptosis, called pyroptosis [37]. Limiting inflammatory reactions and protecting cells are vital strategies to control inflammatory dysregulations [41].
ROS are established to have pivotal parts for activating the NLRP3 inflammasome [42] together with driving NLRP3/caspase-1 complexation, which results in pyroptosis [43]. These were in line with our findings. In our study, we found that following treatment of RLE-6TN cells with LPS/ATP, ROS were continuously generated, leading to NLRP3 inflammasome triggering and pyroptosis. This study confirmed that halting melatonin activity on NLRP3-inflammasome triggering was ROS-dependent, validated through NAC. Following pre-treatment with NAC, ROS generation was reduced, together with NLRP3 inflammasome triggering and pyroptosis. This was in line with a recent review article, where the authors reviewed different signaling pathways that have been proposed to participate in triggering NLRP3 inflammasome, and developed a model in which a major factor for NLRP3 triggering was ROS upregulation [44]. However, accumulating evidence demonstrated ROS have counterintuitive parts concerning NLRP3 inflammasome triggering [45]. Multiple investigations highlighted NLRP3 inflammasome triggering is not affected (within rats/humans) though NADPH oxidase-inhibiting activities [46][47][48]. Notably, mitochondria represent another source of ROS. However, Munoz-Planillo and colleagues demonstrated that mitochondrial malfunctions/mtROS levels expendable regarding NLRP3 inflammasome triggering [43]. Therefore, all research results indicate that ROS have differing effects on the NLRP3 inflammasome, according to differing diseases and cells. Therefore, it is extremely important to continue to study the mechanism of the interaction between ROS and NLRP3 inflammasome.
Nrf2/HO-1 pathway serves as a central regulator of antioxidant activity [49]. Previous studies have mainly focused on its antioxidant effects, though with detailed research efforts, the cytoprotective and anti-inflammatory effects of Nrf2 have been continuously explored [50]. Among its anti-inflammatory effects, Nrf2 pathway has also been increasingly shown to interact with inflammasome pathways in multiple manners. A study revealed that in cerebral ischemia reperfusion injury, Nrf2 thwarted NLRP3 inflammasome triggering through regulating Trx1/TXNIP complex [51]. In addition, research has shown that suppressing Nrf2/HO-1 signaling in osteoarthritis enhances NLRP3 inflammasome signaling [52]. Therefore, activating Nrf2 for inhibiting NLRP3 inflammasome triggering could be a target for pro-inflammatory conditions.
Melatonin has been used for treating multiple inflammatory conditions due to its powerful antiinflammatory and antioxidant effects. This compound is able to scavenge free radicals directly or indirectly by stimulating antioxidant enzyme activity, as well as through inhibition of pro-oxidative enzyme activity [53]. Another indirect antioxidant melatonin influence is orchestrated through Nrf2 transcription-factor triggering, as validated through multiple studies [54], including the suggestion that Nrf2 activators (including melatonin) thwart NLRP3 inflammasome through downregulating ROS [22,55]. However, direct intermolecular associations for Nrf2-induction/NLRP3suppressor functions for melatonin over LPS/ATPexposed RLE-6TN pyroptosis, were not reported until now. This investigation highlighted that melatonin translocated Nrf2 into the nucleus and upregulated HO-1 within RLE-6TN cell lines. In order to confirm whether NLRP3 inflammasome thwarting functions by melatonin were through the triggering of Nrf2, ML385 and siNrf2 were employed, leading to partial rescuing effects. This suggested that Nrf2 has pivotal parts within melatonin inhibitory effects.
However, some studies have found that melatonin even promotes the production of ROS in several tumor and non-tumor cell lines [56]. These contradictory melatonin effects can be caused by different cell types/intracellular molecular network compositions. Therefore, the resulting effect of melatonin on ROS requires further research. In addition, one study found a controversial effect of Nrf2 on NLRP3 inflammasome: both Nrf2 ablation and Nrf2 activators inhibit activation of NLRP3 inflammasome [23]. This study pointed out that Nrf2 ablation only contused activation of NLRP3 inflammasome, and Nrf2 activators (depending on dose) thoroughly inhibit Caspase-1 activation, which suggested that two differing molecular mechanisms are involved in NLRP3 inflammasome inhibition -through Nrf2 ablation and by Nrf2-activating compounds. Therefore, it is paramount to explore how Nrf2 activators inhibit inflammasome activity in the near future.

Limitations of the study
We only discussed the role of a single cell. Alveolar macrophages were confirmed to have a pivotal part within ALI. Therefore, it is necessary to further investigate the associations for alveolar macrophages/epithelial cells and corresponding mechanisms.

Conclusions
In essence, this investigation revealed that melatonin lowers the generation of ROS, driven by LPS/ ATP, through targeting Nrf2 activity, thereby thwarting NLRP3 inflammasome to ease pyroptosis -in vitro and vivo. Moreover, these findings provided novel views into possible cellular and molecular mechanisms implicated within ALI/ ARDS. At the same time, it also paves the way for future research on the interaction between alveolar macrophages and epithelial cells.