{"paper_id":"3ebae0ea-5b80-426c-abe5-2925d1f89159","body_text":"Potential mechanisms and effects of melatonin-regulated Nrf2/HO-1 pathway on \nacute lung injury due to formaldehyde exposure\nBihong Wang1,#, Jianguo Lv2,3,#, Miao Xu1, Dewei Chang1, Zhe Wu4, Yanling Sun3,4,*\n1. School of Pharmacy, Xianning Medical College, Hubei University of Science and \nTechnology, Xianning, China\n2. School of Clinical Medical Sciences, Xianning Medical College, Hubei University \nof Science and Technology, Xianning, China\n3. School of Dentistry and optometry Xianning Medical College, Hubei University of \nScience and Technology, Xianning, China\n4.School of Basic Medicine Sciences, Xianning Medical College, Hubei University of \nScience and Technology, Xianning, China\n*Correspondence: Yanling Sun, sunstonesyl@163.com.\n#B. Wang and J. Lv contributed equally to this work.\nAbstract: Acute lung injury is a topic of great interest in critical care medicine due to \nits high mortality rates. The lungs are the immediate target organ for formaldehyde \ninhalation damage. Lung damage and fibrosis are the most important outcomes of \nsevere and acute lung disease and pose a serious threat to human health. Melatonin \n(MT), a natural bioactive compound with anti-inflammatory and antioxidant \nproperties, However, it is not clear whether MT can prevent FA-induced acute lung \ninjury (ALI). Therefore, in this study, we aimed to evaluate the protective effects of \nMT and the potential mechanisms against FA-induced ALI. An environmental \nexposure bin was used to inhale 3 mg∙m3 FA-induced ALI, which was given \nintraperitoneally with different doses of MT (5/10/20 mg/kg) after successful \nmodeling. In addition, rats were treated with Nrf2 inhibitor (ML385) to validate the \nsignaling pathway. Lung function was measured, histopathological/morphological \nchanges in lung tissue were assessed, and inflammatory expression and oxidation \nlevels in lung tissue were detected. We observed that MT greatly alleviated the lung \ndysfunction, pathological lung injury, pulmonary edema and inflammatory response \nafter successful modeling of FA. In additional, MT played a role in modulating the \nNrf2/HO-1 signaling pathway, which effectively inhibit oxidative stress caused by \nFA-induced lung tissue injure. Moreover, we found that activation of the NF-κB \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\npathway is associated with inflammation caused by this injury. Overall, our data \nsuggest that MT inhibits the expression of oxidative stress and inflammation in lung \ntissue through the institutional or Nrf2/HO-1 pathway, alleviating FA-induced ALI.\n1. Introduction\nAcute lung injury (ALI) is an early lesion of ARDS, ALI and more severe ARDS \nas common mortality and life-threatening lung diseases. Although some progress has \nbeen made in the diagnosis and treatment of ALI/ARDS, the pathogenesis is very \ncomplex due to its many causative factors.1 There are still no effective therapeutic \nmeasures, which allows the mortality rate of the disease to remain as high as 40% and \nseriously affects the prognosis of critically ill patients.2 The protein-rich edematous \nfluid in ALI/ARDS is associated with large numbers of neutrophils, pro-inflammatory \ncytokines and cytokines, proteases and oxidants.3 \nFormaldehyde (FA) is widely used in modern industry and is a widespread \nenvironmental and occupational pollutant, and among all known health effects of FA, \nlung injury is one of the most serious risks. Millions of people worldwide are exposed \nto FA every day.4 Studies have shown that FA leads to ALI through reduced \ntransalveolar Na+ transport, reduced human epithelial sodium channel activity and \nenhanced membrane depolarization, and increased ROS production.5-6 ROS \nupregulate inflammatory cytokines and perpetuate malignancy by recruiting more \ninflammatory cells to perpetuate the vicious cycle, ultimately leading to severe tissue \ndamage.7 Addressing inflammation and oxidative stress, which can lead to lung injury, \nis a desirable goal in the treatment of FA-induced ALI. Currently, there are no \neffective therapeutic agents and preventive strategies in clinical practice. And \nmultiple drug candidates with novel and unique mechanisms of action are needed.\nMelatonin (MT) has been reported to play a key role in various physiological \nactivities, including the regulation of circadian rhythms, immune responses, oxidative \nprocesses, apoptosis or mitochondrial homeostasis,8 and its most prominent \npharmacological effects are the scavenging of free radicals and the inhibition of \ninflammatory responses.9 Recent studies have shown that MT is an important \nantioxidant and anti-inflammatory carrier that plays a crucial role in alleviating \noxidative stress and overproduction of pro-inflammatory cytokines and chemokines in \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\nlung tissues,10 while the beneficial effect of MT is associated with nuclear factor \nerythroid 2-related factor 2 (Nrf2) activation.11\nNuclear factor E2-related factor 2 (Nrf2) is a major transcriptional regulator that \nensures the protection of a large number of tissues and cells from ROS-mediated \ninduction due to its various antioxidants and phase II detoxification enzymes,12 \n12activates the transcription of antioxidant genes and is also involved in the regulation \nof cell proliferation and inflammatory gene expression.13 Large amounts of ROS \nactivate tyrosine kinases to dissociate the Nrf2: Keap1 complex, nuclear import of \nNrf2 and coordinated activation of cytoprotective gene expression.14 At the same \ntime, oxidative stress activates cellular NF-κB inflammatory signaling and leads to \nchronic inflammation.15 Nrf2 promotes anti-inflammatory processes through \ncross-talk with the NF-κB pathway.16\nConsidering the ubiquity of FA in urban areas due to environmental pollution, it \nis important to explore effective strategies to stop the health hazards associated with \nFA. Based on this, we hypothesized that MT could exert a protective effect on \nFA-induced ALI through activation of Nrf2. Therefore, our study aimed to investigate \nthe protective role of MT in FA-induced ALI and to explore the underlying molecular \nmechanisms.\n2. Materials and Methods\n2.1 Animals and treatment \nA total of 60 famale Wistar rat weighing 130-150g (5-6 weeks old) were \npurchased from Hubei Province Experimental Animal Center (Wuhan, China). All \nanimals were housed in a 12 h light/dark circumstance with food and water ad libitum. \nAll experimental procedures were performed according to the local and international \nguidelines on the ethical use of animals, and all efforts were made to minimize the \nnumber of animals used and their sufferings. Ethics approval was obtained from the \nLaboratory Animal Ethics Committee of Hubei University of Science and Technology \n(2019-03-021). After a week of acclimatization feeding, we randomly divided 60 \nfemale Wistar rats into six groups: Control, FA, FA+MT5mg/kg, FA+MT10mg/kg, \nFA+MT20mg/kg, FA+MT10mg/kg+ML385 (APEXBIO; B8300; America), with 10 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\nrats in each group. All groups, except for the Control group, were exposed to FA \n(aladdin; F11941; China) 3mg/m317-18 through intranasal inhalation for 4 hours per \nday for 21 days (IES-NI; China). Following this, they were treated with \nintraperitoneal injections of MT (aladdin; M18674; China) at doses of 5/10/20mg/kg \n19-20 for 14 days, while continuing to be exposed to FA. The Control group, on the \nother hand, was injected intraperitoneally with an equal volume of 0.9% sodium \nchloride solution. Refer to (Fig.1) for further detail.\n2.2 Measurement of Airway hyperresponsiveness (AHR)\nAccording to the manufacturer’s instructions of the AniRes2005 lung function \nsystem (Bestlab, version 2.0, China), Rats were anesthetized by intraperitoneal \ninjection of 1% pentobarbital sodium (Urchem, China). The respiratory rate was \npre-set at 90/min, and the time ratio of expiration/inspiration was 20: 10. AHR was \nassessed by the indexes of Re, Ri, and the minimum value of Cldyn. Ri and Re \nR-areas, the graph area between the peak value and baseline, and the valley of Cldyn \nwere recorded for further analysis.\n2.3 Lung Histological Assay\nAfter ventilator testing, lung tissues were removed and fixed in 4% \nparaformaldehyde (PFA, 0.1 M phosphate buffer, pH 7.4) at 4°C for 12 h. The tissues \nwere then embedded in paraffin wax and cut into 4-μm sections with a microtome \n(RM 2165; Leica Microsystems GmbH). Sections were stained with haematoxylin \nand hemoglobin (H&E), periodic acid-Schiff (PAS), and Masson's trichrome to assess \nthe level of inflammation or fibers in the lungs (Solarbio; G1120; G1285; G1346; \nChina). Briefly, the sections were deparaffinized with xylene, 100% ethanol, 90% \nethanol, and 70% ethanol, and then treated with staining solutions, stained, and sealed \nwith neutral resin, and then visualized with a fluorescence microscope (Olympus \nIX73; Olympus). The degree of alveolar edema, intra-alveolar congestion, interstitial \nedema, and intra-alveolar congestion were assessed and scored separately in this study. \nScore of 0 indicates no change or very slight change, 1 indicates slight change, 2 \nindicates moderate change, 3 indicates severe change, and 4 indicates very severe \nchange. The scores of these four items were averaged to obtain the H&E staining lung \ninjury score.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n2.4 Lung wet/dry (W/D) weight measurement\nThe body mass was first weighed, and then the anterior lobe of the right lung was \nextracted to calculate the lung coefficient, which is the ratio of lung weight to body \nmass. To determine the surface water content of the lung tissue, filter paper was used \nto absorb the wet weight, which was then recorded. The tissue was then dried in a \nconstant temperature oven at 70℃ for 48 hours and weighed again to obtain the dry \nweight, which was used to calculate the W/D ratio (wet weight divided by dry weight). \nThe lung water content was calculated to reflect the degree of pulmonary edema.\n2.5 Molecular docking\nThe X-ray crystal structure of Nrf2 was obtained from the Protein Data Bank \n(PDB ID: 1X2R https: //www.rcsb.org/). The structure of MT was downloaded from \nthe PubChem database (https: //www.pubchem.ncbi.nlm.nih.gov/compound) and \noptimized using ChemBio3D Ultra 14.0 software (PerkinElmer Informatics). Auto \nDock Vina 1.1.2 software (Center for Computational Structural Biology) was used to \ndock conformation between Nrf2 and MT. PyMOL 2.2.3 was used to visualize the \nconformation.\n2.6 Immunohistochemistry (IHC) \nlung tissue sections were dewaxed, conducted to antigen retrieval (Beyotime \nBiotech; P0083; China), treated with 3% hydrogen peroxide for 10 min, closed with \n10% goat serum closure solution (Concentrated SABC-POD Rabbit IgG Kit; Boster \nBiolTech; SA2002; China) for 1 h, and then incubated with primary antibody \novernight at 4°C, After the primary antibody was applied, the sample was incubated \nwith secondary antibodies at room temperature for 1h. The peroxidase present in the \nsecondary antibodies was utilized to oxidize the DAB, resulting in the formation of a \nbrownish-yellow precipitate with the DAB chromogenic solution. Following this, the \nnuclei were stained blue with hematoxylin and observed under a fluorescence \nmicroscope (Olympus IX73; Olympus Corporation). The fluorescence intensities \nwere analyzed using ImageJ 1.51j8 (National Institutes of Health). The following \nprimary antibodies were used: anti-Nrf2 (1: 100; proteintech; 16396-1-AP; America), \nanti-HO-1 (1: 100; proteintech; 10701-1-AP; America) and anti-NF-κB (1: 100; \nproteintech; 10745-1-AP; America).\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n2.7 Western blotting\nThe study utilized the right posterior lobe of the animal's lung, homogenized in \nRIPA lysis buffer containing protease inhibitors (SEVEN Biotech; SW105; \nSW107-02; China), centrifugated at 12,000 g, 4°C for 20 min. Then the supernatant \nwas collected, separated on SDS-PAGE, and transferred to 0.22 μm PVDF \nmembranes. Protein concentration was quantified using a BCA analysis kit (Abbkine; \nKTD3001; America). Then the membranes were blocked with QuickBlockTM \nBlocking Buffer for Western Blot (Biosharp Life Sciences; BL502A, China), \nincubated with the appropriate primary antibodies overnight at 4°C. And \nHRP-conjugated secondary antibodies in TBST (1: 5,000) at room temperature for 1 h. \nProtein bands were visualized using ECL detection reagent (Abbkine; K22030; \nAmerica) and detected with an iBright 1500 instrument (Invitrogen; Thermo Fisher \nScientific, Inc). The grey values of bands were analyzed using ImageJ 1.51j8 software \n(National Institutes of Health). β-actin was used as a loading control. The following \nprimary antibodies were used: anti-HO-1 (1: 1000; proteintech; 10701-1-AP; \nAmerica), anti-Nrf2 (1: 1000; Abbkine; ABP0106; America), anti-NF-κB (1: 1000; \nproteintech; 10745-1-AP; America) and anti-p-NF-κB (1: 1000; Abbkine; ABP0043; \nAmerica).\n2.8 Fluorescence quantitative PCR\nThe study utilized the right posterior lobe of rat lung, from which RNA was \nextracted and purified using the Tissue Extraction RNA Kit (Dakewe Biotech; \n8034111; China). Reverse transcription was performed using the All-in-one First \nStrand cDNA Synthesis Kit (SEVEN Biotech; SM31-02; China) to obtain cDNA. The \ncDNA obtained from the reverse transcription was used as a template and detected \nusing the perfectStartTM Green qpCR SuperMix kit (TransGen Biotech; AQ601-02; \nChina). Table 1 displays all primers used in the study. The mRNA levels were \ncalculated with the 2-△△Ct method and normalized to β-actin. The primer sequences \nwere as follows: Nrf2 5'-TTCAAGCCGATTAGAGG-3', reverse \n5'-TTGCTCCTTGGACATCA-3'; HO-1: forward \n5'-GGTCCTGAAGAAGATTGCG-3', reverse 5'-GATGCTCGGGAAGGTGAA-3'; \nKeap1: forward 5'-CGCCCTGTGCCTCTATG-3', reverse \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n5'-AGGTGCCACTCGTCTCG-3';  and β-actin: forward \n5'-CGTTGACATCCGTAAAGACC-3', reverse  \n5’-GGAGCCAGGGCAGTAATCT-3'.\n2.9 Enzyme-linked immunosorbent assay (ELISA)\nAfter ventilator assay, the upper end of the tracheal cannula was then inserted \nusing a syringe, and 8 ml of saline was injected into the left lung in three separate \ndoses. The lavage fluid was flushed and recovered three times, and the supernatant \nwas centrifuged for 10 minutes at 12000 r-min-1 under 4 ℃. Finally, the levels of \nTNF-α, IL-6, and IL-1β were measured using the ELISA kit (Abbkine; America) \ninstructions.\n2.10 superoxide dismutase (SOD), glutathione (GSH), and 8-Hydroxydeoxyguanosine \n(8-OHdG) analyses in the lung tissues\n   To detect oxidative stress indicators, some biomarkers of lipid peroxidation \nincluding SOD (Shanghai Biyuntian Biotechnology Institute; S0101S; China), GSH \nand 8-OHdG (Nanjing Jiancheng Bioengineering Institute; A061-1/H165-1-1; China) \nwere detected using commercial assay kits according to the manufacturer’s \ninstructions.\n2.11 Statistical analysis\nData were expressed as mean ± standard deviation (SD) and analyzed using \nGraphpad Prism 9.0. Normal distribution was assessed with the Shapiro-Wilk test, \nand multiple comparisons were made using one-way ANOVA, followed by \nBonferroni test to compare data across multiple groups. Finally, we used a two-way \nANOVA with multiple comparison test to analyze the AHR results.\n3. Results\n3.1 MT treatment alleviates FA-induced lung function abnormalities\nTo assess the changes in AHR, we compared airway responses to MeCh in \ndifferent groups (Fig.2). In all experimental groups, both the expiratory and \ninspiratory resistance increased with an increase in the MeCh dose, while the trough \nvalue of Cldyn decreased. At each point, FA exposure had significant effects on Ri, \nRe, and Cldyn (p < 0.05 or p < 0.01) in each treatment group. Compared with the FA \ngroup, MT could significantly reduce the changes in lung function (p < 0.05 or p < \n0.01), mainly including reduced Ri and Re and increased dynamic lung compliance. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\nThis indicated that the MT could effectively reduce the changes in lung function \ninduced by FA.\n3.2 MT treatment attenuates FA-induced ALI\nIn H&E staining lung tissue sections, we observed thickening of alveolar walls, \ncollapse of alveoli, and massive infiltration of inflammatory cells in the FA group \n(Fig.3A). The W/D ratio and lung factor of lung tissue in FA group were increased, \nindicating the occurrence of pulmonary edema. MT treatment reduced lung injury \nscores and greatly attenuated the development of pulmonary edema (Fig.3B-D). \nMasson staining demonstrated collagen fiber deposition and structural damage, and \nPAS staining showed significant glycans, which indicated pathological changes such \nas chronic inflammation in the lung tissue (Fig. 3E). However, treatment with MT \nreduced these tissue structural abnormalities and inflammatory response induced by \nFA.\n3.3 MT antagonizes FA-induced ALI through the Nrf2/HO-1 pathway\nA molecular docking assay was performed on the X-ray crystal structures of \nNrf2 and the ligand MT (Fig.4A). Auto Dock data showed that MT formed two \nelectrovalent bonds with Nrf2 at residues CLY-367 and ARG-415.The electrovalent \nbond distances were measured to be 2.2 ng strom between Nrf2 CLY-367 and MT, \nand 2.3 ng strom between Nrf2 ARG-415 and MT. And the binding affinity was \n-7.6 kcal/mol. It indicates that Nrf2 has a higher affinity for MT. To investigate the \nprotective mechanism of MT against FA-induced ALI, the level and localization of \nNrf2/HO-1 were analyzed by immunohistochemical analysis. The expression level of \nNrf2/HO-1 in the lung tissue of rats in FA group was lower than that in the control \ngroup, but MT could promote the entry of Nrf2 into the nucleus and up-regulate the \nexpression of Nrf2, suggesting that the protective effect of MT on FA-induced ALI \nmight be related to the up-regulation of Nrf2 (Fig.4B-C). In addition, we \ndemonstrated the protective effect of Nrf2 against FA-induced ALI with Nrf2 \ninhibitor ML385, which significantly reversed FA-induced oxidative stress in lung \ntissue. These findings suggest that Nrf2 plays an important role in the development of \nFA-induced ALI, and that MT may ameliorate FA-induced ALI by activating Nrf2, \nthereby reducing oxidative stress. The changes of Nrf2/HO-1 expression levels in the \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\nresults of Western blot and qPCR were consistent with the results of \nimmunohistochemistry (Figure 4D-H), and the mRNA expression level of keap1, the \ndownstream molecule of Nrf2/HO-1, also showed significant changes. (Fig.4I).\n3.4 MT alleviates inflammation and oxidative stress through Nrf2\n Western blot analysis showed that the expression level of p-NF-κB in lung \ntissue of the FA group was higher than that of the control group, which was \nsignificantly reduced by MT treatment; however, Nrf2 inhibitor ML385 could reverse \nthe effect of MT (Fig.5A-B). In addition, we also found the changes of inflammatory \nfactors TNF-α, IL-6, IL-1β and oxidation indicators GSH, SOD, 8-OHDG in lung \ntissue. FA resulted in the decrease of TNF-α, IL-6, and IL-1β products, as well as the \ndecrease of GSH content and SOD activity, and the increase of 8-OHDG content, \nwhich is a marker of DNA oxidative damage. Levels of inflammation and oxidative \nstress were significantly reduced when MT was administered, and similarly the Nrf2 \ninhibitor ML385 reversed the effects of M (Fig.5C-H). These results suggest that MT \nalleviates FA-induced lung tissue damage by alleviating inflammatory response and \noxidative stress through Nrf2.\n3.5 Antagonistic effect of ML385 on the protective effect of MT against acute lung \ninjury\nAHR results showed that lung function was improved in the MT group, but \ndecreased after Nrf2 inhibitor administration, which was close to the level of the FA \ngroup, suggesting that inhibiting Nrf2 pathway may antagonize the improvement \neffect of MT on FA-induced lung function abnormalities (Fig.6A). HE staining of \nlung tissue showed that the degree of lung tissue damage in the Nrf2 inhibitor group \nwas similar to that in the FA group, indicating that Nrf2 inhibitor ML385 could \nantagonize the protective effect of MT. In addition, lung tissue score, W/D ratio, lung \ncoefficient, Masson and PAS staining further confirmed that MT alleviated \nFA-induced ALI by activating the Nrf2 pathway (Fig.6B-F).\n4. Discussion \nALI/ARDS is an important clinical syndrome associated with high morbidity and \nmortality, especially in critically ill patients.21 The pathological manifestations of ALI \nprimarily involve acute inflammatory responses and dysfunction of alveolar epithelial \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\nmembrane due to tissue damage.22 Moreover, cytokine mediated inflammatory \nprocesses aggravate the damage of epithelial and endothelial cells in the pathogenesis \nof ALI.23 FA is a prevalent environmental pollutant found in various sources such as \nwood products, plastics, synthetic fibers, insulation materials, upholstery, paints, \nvarnishes, household cleaning products, and cigarettes.24 Exposure to FA exacerbates \nthe inflammatory response, which is closely associated with its potential mechanism \nfor inducing lung injury. Furthermore, FA exposure can enhance the growth of indoor \nbacterial communities, and long-term exposure may lead to the development of \nbacterial communities that pose a high risk to human health.25 Although FA is known \nto cause lung damage, the specific molecular mechanisms underlying this effect \nremain largely unknown. Therefore, multifaceted validation of new drugs for \nantagonizing FA-induced ALI is essential. \nMT has attracted attention for its various biological activities, such as its \nanti-inflammatory and antioxidant properties.27 However, the potential of MT in the \ntreatment of FA-induced ALI has not been clearly defined. To date, studies have \nshown that MT has been investigated as a potential treatment for various cross-organ \nsystemic pathological conditions27 and that it is also effective in patients infected with \nneocoronary pneumonia by reducing vascular permeability, anxiety, sedative use and \nimproving sleep quality.28 And MT is also a very effective scavenger of superoxide \nand hydroxyl radicals.29 Our study found that this compound also blocks the \nproduction of pro-oxidant enzymes by indirectly inhibiting NF-κB. Another indirect \nantioxidant effect of MT is mediated by Nrf2 transcription factor activation. For \nexample, MT can attenuate diabetes-related restenosis in rats by activating Nrf2 \nsignaling.30 Indeed, MT also exhibits critical potential in various respiratory diseases \nbecause of the abundance of high-affinity MT receptors captured in lung tissue.31\nIn our study, we discovered that MT played a crucial role in mitigating \nFA-induced ALI. Through a series of experiments, we observed that MT significantly \nenhanced lung function. Specifically, it decreased inspiratory and expiratory \nresistance while increasing lung compliance, thereby reducing airway \nhyperresponsiveness. This suggests that MT has a positive impact on respiratory \nfunction. Additionally, we assessed the structure and function of lung tissue using HE \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\nstaining, Masson staining, PAS staining, lung injury score, W/D ratio, and lung \ncoefficient. These results indicated that MT reduced inflammatory cell infiltration and \ncollagen fiber proliferation in lung tissue, leading to improved alveolar structure and \nalleviation of lung injury. Notably, MT decreased the lung injury score and W/D ratio \nwhile improving the lung coefficient, indicating its ability to protect the structure and \nfunction of lung tissue. \nSeveral studies have shown that Nrf2 is a signaling coordinator that attenuates \nenvironmental particulate matter PM2.5-induced lung tissue damage by suppressing \ninflammation and oxidative stress.32 Under normal conditions, Nrf2 binds to \nKerch-like ECH-associated protein 1 (Keap1), and when the organism is under \noxidative stress, Nrf2 segregates from its negative regulator cytoskeleton-associated \nprotein Kelch-like ECH-associated protein 1 (Keap1) and translocates to the nucleus, \nwhere it further promotes transcription of downstream antioxidant genes such as \nHO-1 upon entry.33 To further investigate the role of Nrf2 in MT treatment of \nFA-induced ALI, we used Nrf2 inhibitor ML385 as an antagonist group. Through \nIHC, WB and qPCR analysis, we found that ML385 significantly inhibited the \nincrease of Nrf2/HO-1 expression in lung tissue after MT treatment. These results \nsuggest that Nrf2 plays a crucial role in alleviating FA-induced ALI after MT \ntreatment, and provide a valuable reference for further exploring the therapeutic \npotential of Nrf2 in respiratory diseases.\nNF-κB is a nuclear transcription factor that is key signaling molecule of the \nclassical inflammatory pathway that regulates the expression of several genes in the \ninflammatory response.34 Nrf2/Keap1/HO-1 signaling negatively regulates NF-κB \ntransmission in oxidative stress and inflammatory responses, initiating \nNF-κB-dependent transcriptional pathways that rapidly induce the secretion of \ninflammatory factors.35 Upon activation, NF-κB translocates to the nucleus and binds \nto specific DNA sequences, leading to the transcriptional expression of inflammatory \nfactors such as IL-1β, TNF-α, and IL-6. This activation also triggers a positive \nfeedback mechanism, further amplifying the inflammatory response. NF-κB plays a \ncrucial role in the body's immune response and disease progression. It is important to \nnote that the inflammatory response can induce oxidative stress, which further \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\nexacerbates inflammation, creating a vicious cycle.36 Our study showed that MT \nreduced expression of inflammatory and increases level of antioxidant in lung tissue, \nwhile Nrf2 inhibitor ML385 can reverse this effect, suggesting that MT regulated \ninflammation and oxidative stress by activating the Nrf2 pathway, thereby alleviating \nFA-induced ALI.\nStudies have shown that FA leads to inflammation closely associated with \noxidative stress and the development and progression of ALI. MT, a novel agonist of \nNrf2, can resist FA-induced inflammation and oxidative stress, thereby alleviating \nacute lung injury (Fig.7). This study discloses for the first time that MT \nsupplementation can prevent the deleterious effects of FA on lung tissue, and it is \nexpected that MT could be a possible candidate for the prevention of \npollution-induced lung injury.\nFigure Notes\nFig.1 Schematic diagram of the experimental procedures\nFig.2 MT treatment alleviates FA-induced lung function abnormalities\n (A) R-area of Ri, (B) R-area of Re, and (C) peak value of Cldyn at different doses of \nMeCh Animal groups (in all panels): n = 3 rat per group. (*: p < 0.05, **: p < 0.01, \ncompared with the FA group; ##: p < 0.01, compared with the control group).\nFig.3 MT treatment attenuates FA-induced ALI\n (A) Representative H&E staining images of each group of lung tissue sections. Scale \nbar = 50μm. (B) Quantitative analysis of inflammation score for the H&E staining in \neach group. (C) Lung wet/dry (W/D) weight ratio. (D) Lung coefficient measurement. \n(E) Representative Masoon/PAS-stained images of lung tissue sections from various \ngroups. Scale bar = 50μm.\nAnimal groups (in all panels): n = 3 rat per group. (*: p < 0.05, **: p < 0.01, \ncompared with the FA group; #: p < 0.05, ##: p < 0.01, compared with the control \ngroup).\nFig.4 MT antagonizes FA-induced ALI through the Nrf2/HO-1 pathway\n (A) The docking results of MT with Nrf2. The modelled 3D structure of Nrf2 \ndocked with MT. he enlarged view of binding site in box. Nrf2 protein was shown in \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\ncolor cyan. MT was colored green. The interaction residues showed as color red, \nbonds showed as yellow dotted lines, and bond lengths were presented as numbers. \n(B-C) Immunohistochemistry-based images of Nrf2/HO-1 in the lung tissues. Scale \nbar = 50μm. (D-F) Western blot analysis and quantification of relative grayscale \nvalues of Nrf2/HO-1 expression levels for each group. (G) Nrf2 qPCR results. (H) \nHO-1 qPCR results; (I) Keap1 qPCR results. Animal groups (in all panels): n = 3 rat \nper group. (*: p < 0.05, **: p < 0.01, compared with the FA group; #: p < 0.05, ##: p \n< 0.01, compared with the control group. &: p < 0.05).\nFig.5 MT alleviates inflammation and oxidative stress through Nrf2\n(A-B) Western blot analysis and quantification of relative grayscale values of \nNFĸB/p-NFĸB expression levels for each group. (C-E) TNF-α, IL-1β and IL-6 \nconcentrations in the BALF. (F-H) Analysis of SOD, GSH and 8-OHdG levels in rat \nlung tissue by kit. Animal groups (in all panels): n = 3 rat per group. (*: p < 0.05, **: \np < 0.01, compared with the FA group; #: p < 0.05, ##: p < 0.01, compared with the \ncontrol group. &: p < 0.05, &&: p < 0.01, compared with the FA+MT10mg/kg \ngroup).\nFig.6 Antagonistic effect of ML385 on the protective effect of MT against acute lung \ninjury\n(A) Representative H&E staining images of each group of lung tissue sections. Scale \nbar = 50μm. (B) Quantitative analysis of inflammation score for the H&E staining in \neach group. (C) Lung wet/dry (W/D) weight ratio. (D) Lung coefficient measurement. \n(E) Representative Masoon/PAS-stained images of lung tissue sections from various \ngroups. Scale bar = 50μm. (F) R-area of Ri, (G) R-area of Re and (H) peak value of \nCldyn (in all panels): n = 3 rat per group. (&&: p < 0.01, compared with the \nFA+MT0mg/kg group).\nFig.7 Schematic diagram of the potential mechanisms of MT treatment for acute lung \ninjury caused by FA.\nFunding：National Natural Science Foundation of China (81902937), Hubei College \nof Science and Technology, School of Ophthalmology and Stomatology (2020WG06), \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\nHubei College of Science and Technology, School of Ophthalmology and \nStomatology (2021WG10).\nReferences \n1. Liu R, Luo X, Li J, Lei Y, Zeng F, Huang X, Yang F. Melatonin: A window into \nthe organ-protective effects of sepsis. J Biomedicine & Pharmacotherapy, 2022, \n154, 113556. \n2. 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Activation of Nrf2/HO-1 \nsignaling: An important molecular mechanism of herbal medicine in the \ntreatment of atherosclerosis via the protection of vascular endothelial cells from \noxidative stress. J Journal of advanced research, 2021, 34, 43-63. \n36. Li J, Deng S H, Li J, Li L, Zhang F, Zou Y, Xu Y. Obacunone alleviates \nferroptosis during lipopolysaccharide-induced acute lung injury by upregulating \nNrf2 dependent antioxidant responses. J Cellular & Molecular Biology \nLetters, 2022, 27 (1), 1-20. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 18, 2024. ; https://doi.org/10.1101/2024.12.17.629008doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}