Ferroptosis Participates in Coenzyme Q10-treated Silicosis Fibrosis in Mice

Ferroptosis Participates in Coenzyme Q10-treated Silicosis Fibrosis in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ferroptosis Participates in Coenzyme Q10-treated Silicosis Fibrosis in Mice Yue Sun, Mengxue Yu, Huning Zhang, Wenyue Zhang, Shengpeng Wen, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4415956/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Silicosis is the most common, fastest-progressing, and most severe type of occupational pneumoconiosis, which result in diffuse pulmonary fibrosis. However, there are no specific treatments for silicosis. Coenzyme Q10, as a component of the mitochondrial electron transport chain, can enhance mitochondrial quality and cellular energy supply, inhibit the production of ROS to reduce oxidative damage for reducing the risk of fibrosis. Ferroptosis is triggered by reactive oxygen species and lipid peroxidation induced by the overload of Fe 2+ and has tight correlation with pulmonary fibrosis. However, whether ferroptosis is involved in coenzyme Q10-treated silicosis fibrosis in mice remains unclear. Methods After intratracheal instillation of silica in C57BL/6J mice for 48 hours, CoQ10 was administered orally at a dose of 100 mg/kg•d. The mice were randomly divided into control group, saline group and CoQ10 treatment group, and there are 6 mice in each group. Lung injury and fibrosis in mice were observed by HE, Masson, and Sirius Red assays. Iron content was measured by colorimetry in lung tissue. The content of malondialdehyde (MDA) in lung tissue was detected by immunofluorescence staining. Protein and mRNA expression of aSMA, Collagen I, GPX 4 and p53 were determined by qRT-PCR and Western blotting. Multiple data comparisons were conducted using one-way ANOVA, meanwhile multiple comparisons were conducted using Tukey test. Result Histopathological staining assays showed that the normal lung tissues in control group exhibited a basically intact alveolar structure, thin alveolar walls, no obvious inflammatory cells aggregation, and no significant collagen fiber deposition in pulmonary mesenchyme. But after CoQ10 treatment, the alveolar structure was still acceptable and no silicosis nodules and reduced collagen deposition. qPCR and WB experiments showed that CoQ10 significantly reduced the expression levels of α-SMA and collagen I in silicosis lung tissues. It is worth noting that CoQ10 significantly inhibited the accumulation of lipid peroxidation and Fe 2+ and increased the expression of ferroptosis regulatory core enzyme GPX4 and reduced its upstream regulator p53 in silicosis lung tissues. Conclusion Ferroptosis is involved in coenzyme Q10-treated silicosis fibrosis and this finding is a new perspective for exploring the pathogenesis and treatment for silicosis. Silicosis Fibrosis Coenzyme Q10 Ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Silicosis, as the most common, fastest-progressing, and most severe type of occupational pneumoconiosis, is mainly caused by the invasion of ~ 0.5 -5 µm free silica into the lungs, resulting in diffuse pulmonary fibrosis [ 1 ] . The main clinical symptoms of silicosis are cough, phlegm, chest tightness, and secondary respiratory and circulatory disorders, and then progresses to various systemic diseases. Many researchers suggested that the mechanism of silicosis fibrosis is the continuous interaction between silica dust and pneumonocyte, rebuilding cytokine networks, which stimulates the proliferation of fibroblasts, increases the production and secretion of collagen, and forms fibrosis [ 2 , 3 ] .However, there are no specific treatments for silicosis. The current clinical treatment strategy mainly focuses on alleviating lung tissue inflammation, fibrosis, and other symptom, but the curative effects are not ideal [ 4 – 6 ] . Therefore, it is necessary to explore new anti-silicosis therapeutic drugs and elucidate corresponding molecular mechanisms. CoQ10, a lipid-soluble antioxidant, is synthesized by endogenous biosynthesis in the body, which can improve immunity, enhance antioxidant capacity, delay aging. It is widely used in the adjuvant treatment of cardiovascular system diseases in medicine [ 7 ] . Ferroptosis is an iron-dependent programmed cell death pattern and distinct from apoptosis and autophagy. Ferroptosis mechanism involves the accumulation of lipid peroxidation and Fe 2+ , and the inactivation of molecules such as glutathione peroxidase 4 (GPX4), which are involved in the pathophysiological processes of various organ injury [ 8 ] . Currently, there are no reports on whether ferroptosis is involved in coenzyme Q10-treated silicosis fibrosis in mice. Therefore, our study adopts a model of silicosis fibrosis by tracheal instillation of silica suspension to investigate whether ferroptosis participate in the treatment of silicosis fibrosis with CoQ10, providing a theoretical basis for the treatment of silicosis fibrosis (Schematic: Technical strategy of the study). Materials and methods 1.1 Materials 1.1.1 Experimental animals Twenty-four male C57BL/6 mice, aged 6–8 weeks and weighing 20 ± 2g, were purchased from the Beijing Wei Shang Li De Biotechnology Co., LTD. (production license number: SCXK (Beijing) 2016-0002). After one week of adaptive feeding under SPF conditions in Laboratory Animal Center of Ningxia Medical University, and they were randomly divided into three groups, with 6 mice in each group: (1) control group; (2) Saline group; (3) CoQ10 treatment group. The feeding conditions were 12h light, 12h darkness, temperature (23 ± 1) ℃, relative humidity 40%-50%, independent drinking and eating, feeding and model making followed the relevant regulations of the management and use of experimental animals in the Experimental Animal Center of Ningxia Medical University, and the test procedures were reviewed by the Experimental Animal Welfare Committee of the Experimental Animal Center of Ningxia Medical University. License number SYXK (NING) 2015-0001. 1.1.2 Experimental instrument ELIASA epoch, (Bio-Tek, USA); Electrophoretic, electrokinetic and gel imaging systems (Rio⁃Rad, USA); Ultraviolet-visible spectrophotometer DS-11 (DeNovix Inc., USA ); Laser confocal microscope LSM800 (Zeiss, Germany); Fluorescent quantitative PCR instrument QuantStudioTM5 (ThermoFisher, USA); Inverted microscope (Leica, Germany). 1.1.3 Experimental reagents SiO 2 (Sigma, USA), Coenzyme Q10 (Beijing Solaibao Biotech Co., LTD., China), Corn oil (Aladdin, China), Sirius Red kit, HE staining kit (Beijing Leegan Biotech Co., LTD., China), Hydroxyproline kit (alkaline hydrolysis method) (Nanjing Jianxian Institute of Biological Products, China), α-SMA rabbit primary antibody (Chengdu Zhengneng Co., LTD., China), Mouse goat serum, DAB color detection kit (Zhongshan Jinqiao Biotechnology Co., LTD., China), Reverse transcription kit, qPCR kit (TaRaKa, Japan), Whole protein extraction kit (KGI Biotech Co., LTD., China), Separation gel, concentration gel (Herix, China) China), Acrylamide (Beijing Bio Top Technology Co., LTD., China), ammonium persulfate, TEMED (Sigma, USA), 5×Loading buffer(Kangwei Century, China), High-sensitivity ECL chemiluminescence kit (New Saemi Biotechnology Co., LTD., China). Tissue iron and malondialdehyde (MDA) detection kits were purchased from Jiancheng Biological Company, Nanjing, China; Antibodies for aSMA, collengen I, p53, GPX4, MDA-FITC and Alexa Fluor® 488, purchased from abcam (UK); Total RNA extraction kit, purchased from Tiangen (China), Reverse transcription and fluorescence quantitative PCR kit, purchased from TaKaRa (Japan). 1.2 Methods 1.2.1 Preparation silicosis model in vivo After isoflurane anesthesia, the trachea was exposed, and 0.1mL normal saline was instilled into the control group, and 0.1mL SiO 2 suspension (50mg/mL) was instilled into the model control group and CoQ10 treatment group, respectively. The skin was sutured, and the survival status of the mice was observed. The mice in the treatment group were given CoQ10 (100mg/kg) by gavage 48 hours after operation, and the other mice were fed autonomously. On day 60 after operation, the mice were sacrificed by intraperitoneal injection of 0.3mL urethane, the right middle lobe of the lung was removed, rinsed with normal saline, fixed with 4% paraformaldehyde, and the remaining lung tissues were frozen at -80℃ for subsequent experiments. 1.2.2 Histopathological staining HE staining Paraffin sections of lung tissue were dewaxed and fixed, followed by gradient alcohol dehydration, hematoxylin staining for 6min, ethanol differentiation for 5s, running underwater washing for 3min, eosin staining for 5min, running underwater washing for 5s. After gradient alcohol treatment, xylene was used twice for transparency, three minutes at a time, and the tablets were sealed. Masson staining Paraffin sections of the kidney were deparaffinized and fixed, stained with Weigert iron hematoxylin staining solution, differentiated with acid ethanol differentiation solution, returned to blue with Masson blue solution, washed with distilled water, stained with Ponceaux-fuchsine-red staining solution, washed with phosphamoridic acid solution, stained with aniline blue staining solution, dehydrated, transparent, and sealed. Sirius red staining the mice were dewaxed, stained with Sirius Red droplets for 40min, washed for 2min, the surface staining solution was removed, dehydrated, transparent, and sealed with neutral gum. The changes of lung fibrosis in mice were observed. 1.2.3 Detection of collagen I and α-SMA in lung tissue The content of collagen I and a-SMA in lung tissue was detected by immunofluorescence assay. Portions of lung tissue were made into frozen sections, removed from − 80℃ and placed in a cassette for 25–30 min until room temperature was restored. Fixation with glacial acetone for 25 ~ 30min; Use immunohistochemical strokes to draw circles; Triton − 0.3% X100 incubation 10 ~ 20 min, PBS washed five times, each time 3 min. 5% serum closed for 30min; collagen I and a-SMA antibodies (1:100) were incubated overnight at 4℃. The fluorescent secondary antibody was incubated at 37℃ after rewarming. 4', 6-diaminyl-2-phenylindole (DAPI) was nucleated for 5 min and washed with PBS 5 times for 3 min each time. Fluorescence images were taken by laser confocal microscopy. 1.2.4 Determination of iron ion concentration in lung tissue of silicosis mice Part of the lung tissue of mice was weighed and recorded, and normal saline (volume ratio 1:9) was added to mix. Mechanical homogenization was performed on ice, centrifuged at 1200 g for 10 min, and the supernatant was removed. The blank tube, standard tube and sample tube to be tested were added according to the instructions. After mixing, the tubes were bathed at 99 ℃ for 5 min, cooled and centrifuged at 1 800 g for 10 min. The absorbance value of the supernatant was measured at 520 nm. The results were calculated as tissue iron content = (A determination -A blank )/ (A standard -A blank ) ×C standard ÷Cpr, where Cpr was the protein concentration in tissue homogenates. 1.2.5 Detection of MDA level in lung tissue The level of malondialdehyde (MDA) in lung tissue was detected by immunofluorescence assay. Part of the lung tissue was cut into 8µm frozen sections, removed from − 80 ℃, and placed in a cassette for 25–30 min to recover to room temperature. Fixed in ice-cold acetone for 25–30 min; Immunohistochemical staining circles; The cells were incubated with 0.3% Triton-X100 for 10–20 min and washed 5 times with PBS for 3 min each time. The cells were blocked with 5% serum for 30 min. Goat polyclonal anti-MDA-FITC antibody (1:100) was incubated overnight at 4 ℃. After rewarming, the cells were washed 5 times with PBS for 3 min each time. Nuclei were stained with 1 mg/L 4', 6-diamidino-2-phenylindole (DAPI) for 5 min and washed with PBS for 5 times, 3 min each time. Fluorescence images were taken by laser confocal microscopy. 1.2.6 Protein expression in lung tissue Western blotting was used to detect protein expression in lung tissue. Fifty mg of lung tissue was weighed, 500 µL of protein lysis buffer was added, homogenized with a tissue homogenizer, and protein extraction was performed according to the steps in the instructions of the whole protein extraction kit. The protein concentration was measured by ultramicro UV-visible spectrophotometer. The 5× loading buffer was mixed with the extracted tissue protein 1:4, and the protein was denatured by boiling at 99.5 ℃ for 10 min. The protein was electrophoresed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), and the pressure was constant. The protein in the gel was wet transferred to 0.22 µm polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk powder for 2 to 3 hours, incubated with primary antibody at 4℃ overnight, washed with secondary antibody at room temperature for 2 hours, washed with PBS 3 times, 10 min each time, and detected by chemiluminescence method. The relative content of the target protein was calculated as the ratio of gray value between the target protein and the internal reference protein (β-actin). Repeat 3 times. 1.2.7 Detection of mRNA levels in lung tissue Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression in lung tissue. Primer sequences (Table 1 ) : Table 1 Primer sequence used for qRT-PCR gene forward primer Reverse primer GPX4 5'-TGTGCATCCCGCGATGATT-3' 5'-CCCTGTACTTATCCAGGCAGA-3' p53 5'-CCCCTGTCATCTTTTGTCCCT-3' 5'-AGCTGGCAGAATAGCTTATTGAG-3' aSMA 5’-CCTTCGTGACTACTGCCGAG-3’ 5’-GTCAGCAATGCCTGGGTACAT-3’ Collagen I 5'-GCTCCTCTTAGGGGCCACT-3’ 5’-ATTGGGGACCCTTAGGCCAT-3’ GAPDH 5'AGGTCGGTGTGAACGGATTTG-3' 5'-GGGGTCGTTGATGGCAACA-3' The qRT-PCR reaction system included denaturation (95 ℃ for 30 s), annealing (95 ℃ for 5 s), and extension (60 ℃ for 34 s). A total of 40 cycles were set up, ΔCt = Ct(objective) -Ct (internal control). 1.2.8 Immunohistochemistry Paraffin-embedded sections of mouse lung tissue were dewaxed with xylene, dehydrated with gradient alcohol, antigen heat repaired with sodium citrate (pH = 6) for 15min, blocked with goat serum for 10 min, incubated with α-SMA/collagen I primary antibody (diluted 1:100) overnight, HRP-labeled goat anti-rabbit/anti-mouse secondary antibody for 30 min, and DAB for 6 min. The slices were dehydrated, transparent, and sealed. Images were taken under a 20x objective lens under an ordinary light microscope to observe the expression of α-SMA and collagen I protein in each group. The positive areas were counted by Image-Pro Plus 6.0 software. The results were quantified as the ratio of the IOD value of the positive area to the area of the picture. 1.3 Statistical Methods Statistical analysis was conducted using GraphPad 8.0 software, with data presented as mean ± standard deviation. One-way analysis of variance was employed for comparing multiple groups, followed by Tukey's post hoc test for pairwise comparisons. Results 2.1 CoQ10 Alleviates Silicosis Lung Injury and Collagen Deposition Firstly, we used HE, Masson, and Sirius red assays to assess silicosis lung injury and collagen deposition after CoQ10 treatments (Fig. 1 A-D). Histopathological staining assay showed that the normal lung tissues in control group exhibited a basically intact alveolar structure, thin alveolar walls, no obvious inflammatory cells aggregation, and no significant collagen fiber deposition in pulmonary mesenchyme. In the saline group, there were significantly dense and damaged the lung structure, a large number of inflammatory cells gathered in both the pulmonary mesenchyme and alveolar cavities, and uneven size silicosis nodules, and a large amount of collagen fiber deposition. In contrast, after CoQ10 treatment, the alveolar structure was still acceptable compared to the saline group, with no silicosis nodules observed, and reduced collagen deposition, but there were inflammatory cells aggregation. All above results indicated that CoQ10 could alleviate lung injury in silicosis mice. 2.2 CoQ10 Reduced the Expression of a-SMA and Collagen I in Silicosis Mice a-SMA and Collagen I plays an important role in the development of pulmonary fibrosis [ 9 ] . In general, a-SMA mainly plays a role in supporting and stabilizing cellular structures. However, once pulmonary fibrosis occurs, myofibroblasts, producing more a-SMA, and become over-activated and synthesize too much collagen and other extracellular matrix components to accelerate the development of pulmonary tissue fibrosis. Collagen I, as a marker molecule for fibrosis, it is positively correlated with the degree of fibrosis. Furtherly, we detected the expression level of α-SMA and Collagen I in silicosis mice to evaluate the ability of CoQ10 to alleviate fibrosis. qRT-PCR assay showed that compared to the saline group (0.263 ± 0.029), CoQ10 treatment significantly decreased the α-SMA mRNA expression level (0.118 ± 0.051) and higher than the control group (0.048 ± 0.009) ( F = 61.230 , P < 0.001 ). Meanwhile, CoQ10 also decreased the mRNA expression level of Collagen-1 (1.245 ± 0.874) relative to the saline group (3.400 ± 1.062) and higher than the control group (0.849 ± 0.486) ( F = 15.950, P < 0.001 ) (Fig. 2 A). Western blot assay showed that compared to the saline group (1.178 ± 0.253), CoQ10 treatment significantly decreased the α-SMA protein expression level (0.408 ± 0.184) and higher than the control group (0.502 ± 0.265) ( F = 18.86, P < 0.001 ). Meanwhile, CoQ10 also decreased the protein expression level of Collagen-1 (0.065 ± 0.031) relative to the saline group (0.135 ± 0.023) and higher than the control group (0.058 ± 0.027) ( F = 14.780, P < 0.001 ) (Fig. 2 B, C). The data of immunohistochemistry and fluorescence staining assays also showed that CoQ10 reduced the activation of fibroblasts in the silicosis lung and weaken the development of silicosis fibrosis (Fig. 2 D, E). 2.3 Effect of CoQ10 on the Content Levels of Fe 2+ and Malondialdehyde (MDA) in Silicosis Lung Ferroptosis is a non-apoptotic cell death form that relies on the large amount accumulation of Fe 2+ and the corresponding abnormal increased lipid peroxides in cells [ 10 ] . Some studies have reported that the presence of a large amount of Fe 2+ in cells might trigger the Fenton reaction, which in turn initiates the occurrence of lipid peroxidation [ 11 , 12 ] . In this study, compared with the content of Fe 2+ in the normal control group (0.068 ± 0.009 nmol/mg Prot), Fe 2+ content in the saline group increased significantly (0.205 ± 0.008 nmol/mg Prot), but after CoQ10 treatment, the content of Fe 2+ decreased significantly (0.098 ± 0.026 nmol/mg Prot) (Fig. 3 A). MDA, as one of the classic indicators for detecting lipid peroxidation in damaged tissues and cells, is widely used in the field of iron-dependent cell death research. Immunofluorescence assay also showed that compared to the saline group, CoQ10 decreased MDA level in lung tissue (Fig. 3 B). In brief, CoQ10 was involved in the occurrence of iron-dependent cell death via reducing the levels of lipid peroxidation and Fe 2+ in silicosis lung tissues. 2.4 CoQ10 Reduced the expression level of ferroptosis -related proteins GPX4 and p53 in Silicosis Mice To clarify whether ferroptosis is involved in the CoQ10-treatment silicosis fibrosis in mice, we further examined the expression level of ferroptosis core enzymes GPX4 and its upstream regulator p53. qRT-PCR assay showed that compared to the saline group (0.233 ± 0.116), CoQ10 treatment significantly increased the GPX4 mRNA expression level (0.777 ± 0.216) and lower than the control group (0.826 ± 0.127) ( F = 25.540, P < 0.0001 ). Meanwhile, CoQ10 also decreased the mRNA expression level of p53 (0.041 ± 0.029) relative to the saline group (0.086 ± 0.031) and higher than the control group (0.038 ± 0.0194) ( F = 6.996, P < 0.01 ) (Fig. 4 A,B). Western blotting assay showed that compared to the saline group (0.252 ± 0.116), CoQ10 treatment significantly increased the GPX4 protein expression level (1.215 ± 0.331) and lower than the control group (0.656 ± 0.129) ( F = 30.20, P < 0.0001 ). Meanwhile, CoQ10 also decreased the protein expression level of p53 (0.775 ± 0.506) relative to the saline group (1.730 ± 0.735) and higher than the control group (0.495 ± 0.287) ( F = 10.010, P < 0.01 ) (Fig. 4 C-E). These results suggested that ferroptosis may be involved in the process of CoQ10 alleviating pulmonary fibrosis in silicosis mice. Discussion With the rise of new industries, workers, such as miners, stone processing workers, construction workers, are still exposed to large amounts of silica for a long time. It has been reported that there are millions of dust-exposed workers in the United States, among whom one in ten suffers from silicosis. In addition, the situation of silicosis prevention and control in developing countries is more critical, such as India where the number of silicosis patients is as high as tens of millions, and China where the number of pneumoconiosis patients exceeds 850000, of which silicosis accounts for more than 50% [ 13 – 15 ] . So far, due to the limited effective treatment methods for silicosis, lung damage and fibrosis cannot be reversed. As a major public health problem, the current treatment strategies for silicosis are still mainly focused on anti-inflammatory, anti-fibrotic and other symptomatic treatments combined with lung lavage or combined with oxygen therapy and psychological counseling, but the effects are not ideal. In this study, after intratracheal instillation of silica for 48 hours, CoQ10 was administered orally at a dose of 100 mg/kg•d to treat silicosis mice. After 60 days, CoQ10 significantly improved the pathological characteristics of silicosis lung tissues and prevented the development of pulmonary fibrosis. Compared to the saline group, the alveolar wall structure in the CoQ10 treatment group was acceptable, no silicosis nodules were observed, and collagen deposition was reduced. qPCR and WB experiments showed that CoQ10 significantly reduced the expression levels of α-SMA and collagen I in silicosis lung tissues. It is worth noting that CoQ10 significantly inhibited the accumulation of lipid peroxidation and Fe 2+ and increased the expression of ferroptosis regulatory core enzyme GPX4 and downregulated its upstream regulator p53 in silicosis lung tissues. In summary, the above studies indicated that ferroptosis was involved in the process of silicosis fibrosis after treatment with CoQ10. CoQ10 is a lipid-soluble quinone compound that is present in the phospholipid bilayer of cell membranes and accumulates significantly in the inner mitochondrial membrane [ 16 ] . The pharmacological effects of CoQ10 mainly involve antioxidation, anti-fibrosis, scavenging free radicals, dilating blood vessels, reducing pro-inflammatory cytokines, etc [ 17 ] . Currently, it has been widely used to reduce blood viscosity, improve ischemia and reperfusion injury after coronary artery revascularization in the prevention and treatment of atherosclerosis [ 18 , 19 ] . Coenzyme Q10, as a component of the mitochondrial electron transport chain, can enhance mitochondrial quality, improve mitochondrial function, increase mitochondrial activity, enhance cellular energy supply, inhibit the production of ROS, help reduce oxidative damage to cells, thereby reducing the risk of fibrosis [ 20 ] . Coenzyme Q10 can also reduce different inflammatory mediators (such as TNFα, TGF-β, and MCP1) in liver and lung tissues, alleviate inflammation and fibrosis In addition, CoQ10 has also been shown to alleviate arginine-induced pancreatic fibrosis by reducing collagen deposition in pancreatic tissues [ 21 ] . In this study, CoQ10 significantly reduced the expression levels of pulmonary α-SMA and collagen I in the lung tissues of silicosis mice and could inhibit the level of lipid peroxidation in the lung of silicosis mice. Ferroptosis is involved in the regulation of liver and lung fibrosis processes. As a novel cell death mode which distinct from apoptosis, autophagy, and pyroptosis, ferroptosis is triggered by reactive oxygen species and lipid peroxidation induced by the overload of Fe 2+ . Meanwhile, the depletion of antioxidant glutathione or glutathione peroxidase 4 is also considered an essential condition for the initiation and progression of ferroptosis [ 22 ] . Numerous studies have shown that ferroptosis is an important mechanism in the progression of pulmonary diseases, participating in the regulation of various pulmonary diseases, including lung cancer [ 23 ] , lung infection [ 24 ] , chronic obstructive pulmonary disease [ 25 ] , acute lung injury [ 26 ] , etc. Moreover, ferroptosis plays an important role in pulmonary fibrosis. Gong [ 27 ] et al. treated human embryo lung fibroblasts (HFL1) with TGF-β to study the relationship between ferroptosis and pulmonary fibrosis. The inducer of ferroptosis, Erastin, can induce pulmonary fibrosis by stimulating fibroblasts to transform into myofibroblasts [ 28 ] . In addition, the inhibitor of ferroptosis, ferrostatin-1 (Fer-1), can block the accumulation of lipid peroxidation and increase GPX4 activity, alleviating the pulmonary fibrosis induced by ferroptosis [ 29 ] . Li's research results also suggested that ferroptosis exacerbates radiation-induced pulmonary fibrosis, while the inhibitor of ferroptosis, Liproxstatin-1, can significantly reduce RILF [ 30 ] . GPX4 is one of the most important antioxidant enzymes in mammals, and it is considered a core factor in ferroptosis. It converts lipid peroxides into lipid alcohols, preventing tissue and cell damage from lipid peroxidation, thereby inhibiting ferroptosis. The ferroptosis inducers Erastin and RSL3 can inactivate GPX4, leading to the induction of ferroptosis. In addition, p53 is an important tumor suppressor gene that can downregulate the expression of SLC7A11 and negatively regulate the activity of GPX4, resulting in a decrease in cellular antioxidant capacity, accumulation of ROS, and ultimately ferroptosis [ 31 ] . Our research found that CoQ10 significantly inhibits the lipid peroxidation and accumulation of Fe 2+ in silicosis lung tissues and increases the expression of the core ferroptosis regulatory enzyme GPX4 and downregulated its upstream regulator p53, indicating that ferroptosis is involved in the process of CoQ10-treated silicosis fibrosis. Recent research has shown that coenzyme Q10 not only plays a key role in maintaining mitochondrial function and the cell respiratory chain but also regulates the process of ferroptosis. CoQ10 enhances mitochondrial function and antioxidant properties, regulates the balance of ferrous iron in cells, thereby reducing the production of free radicals and the risk of mitochondrial damage [ 32 ] . In addition, CoQ10 can further regulate the levels and activity of intracellular ferrous iron by regulating the expression of genes related to intracellular iron metabolism, affecting the absorption, transport, and storage of iron [ 33 ] . In this way, CoQ10 can regulate the mechanism of ferroptosis, reduce the toxicity of ferrous iron, and protect cells from oxidative stress. Conclusions CoQ10 has the ability to regulate ferroptosis during the process of silicosis fibrosis via enhancing anti-oxidation capability, and regulating the expression of iron metabolism-related genes to achieve protect cells from free radical damage. This finding is a new perspective for exploring the pathogenesis and treatment for silicosis. Declarations Ethics approval and consent to participate Permission must be obtained from the Animal Ethics Committee of Ningxia Medical University before starting any animal experiments. All experiments were conducted in accordance with the guidelines of Ningxia Medical University Animal Ethics Committee. Consent for publication Written informed consent was obtained from all participants. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fundings The work was supported by the National Natural Science Foundation of China (82060264, 82271626, 82160088, 82260142, 82360639), China Postdoctoral Science Foundation (2023MD734192), National Natural Science Foundation Regional Innovation and Development Joint Fund (U21A20343), Ningxia Natural Science Foundation (2022AAC03128, 2022AAC05025), Ningxia Key Research and Development Projects (2020BEG03008, 2020BFH02003, 2021BEG02030), Open competition mechanism to select the best candidates for key research projects of Ningxia Medical University (XJKF230106, XJKF230125), Basic scientific research operating expenses from public welfare research institutes at the central level of the Chinese Academy of Medical Sciences (2019PT330002). Conflict of Interest The authors declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Contributions Yue Sun conceived and designed the study. Mengxue Yu, Shengpeng Wen, Huning Zhang, Wenyue Zhang, Sirong Chang, Fei Yang, Guangjun Qi and Xin Ma performed the experiments. Anning Yang and Zhihong Liu analyzed the data. Bin Liu and Yideng Jiang wrote the manuscript. All authors reviewed and approved the final version of the manuscript. Data availability Data will be made available on request. References Cheng D, Xu Q, Wang Y, Li G, Sun W, Ma D, Zhou S, Liu Y, Han L, Ni C (2021) Metformin attenuates silica-induced pulmonary fibrosis via AMPK signaling. J Transl Med 19:349 Hoy RF, Chambers DC (2020) Silica-related diseases in the modern world. Allergy 75:2805–2817 Yang G, Tian Y, Li C, Xia J, Qi Y, Yao W, Hao C (2022) LncRNA UCA1 regulates silicosis-related lung epithelial cell-to-mesenchymal transition through competitive adsorption of miR-204-5p. Toxicol Appl Pharmacol 441:115977 Huihui T, Hui Z, Aowei M, Luocheng S, Deyong G, Jiale L, Wenjian H, Ke X, Qianqian M, Wenfeng W et al (2023) VX-765 attenuates silica-induced lung inflammatory injury and fibrosis by modulating alveolar macrophages pyroptosis in mice. Ecotoxicology and Environmental Safety Yuan L, Sun Y, Zhou N, Wu W, Zheng W, Wang Y (2022) Dihydroquercetin Attenuates Silica-Induced Pulmonary Fibrosis by Inhibiting Ferroptosis Signaling Pathway. Front Pharmacol 13:845600 Zhou Y, Zhang Y, Zhao R, Cheng Z, Tang M, Qiu A, Dong Y, Lu Y, Lian Y, Zhuang X et al (2021) Integrating RNA-Seq With GWAS Reveals a Novel SNP in Immune-Related HLA-DQB1 Gene Associated With Occupational Pulmonary Fibrosis Risk: A Multi-Stage Study. Front Immunol 12:796932 Minjun L, Xueke H, Yangyang Z, Wenwen P, Xiaomei Z, Jibin M (2024) Coenzyme Q10 in atherosclerosis. Eur J Pharmacol Liu T, Bao R, Wang Q, Hao W, Liu Y, Chang S, Wang M, Li Y, Liu Z, Sun Y (2022) SiO 2 -induced ferroptosis in macrophages promotes the development of pulmonary fibrosis in silicosis models. Toxicol Res (Camb) 11:42–51 Q RB, Y WMY (2023) Z, S W, T L, M W, Y L, S C, H C, et al: AAV9-HGF cooperating with TGF-beta/Smad inhibitor attenuates silicosis fibrosis via inhibiting ferroptosis, vol 161. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, p 114537 Dempsey LA (2019) Death by iron. Nat Immunol 20:665 El-Horany HE, Atef MM, Abdel Ghafar MT, Fouda MH, Nasef NA, Hegab II, Helal DS, Elseady W, Hafez YM, Hagag RY et al (2023) Empagliflozin Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats by Modulating Sesn2/AMPK/Nrf2 Signaling and Targeting Ferroptosis and Autophagy. Int J Mol Sci 24 Pei Z, Qin Y, Fu X, Yang F, Huo F, Liang X, Wang S, Cui H, Lin P, Zhou G et al (2022) Inhibition of ferroptosis and iron accumulation alleviates pulmonary fibrosis in a bleomycin model. Redox Biol 57:102509 Abd-El-Fattah AA, El-Sawalhi MM, Rashed ER, El-Ghazaly MA (2010) Possible role of vitamin E, coenzyme Q10 and rutin in protection against cerebral ischemia/reperfusion injury in irradiated rats. Int J Radiat Biol 86:1070–1078 Liao M, He X, Zhou Y, Peng W, Zhao XM, Jiang M (2024) Coenzyme Q10 in atherosclerosis. Eur J Pharmacol 970:176481 Peng Z, Ding YN, Yang ZM, Li XJ, Zhuang Z, Lu Y, Tang QS, Hang CH, Li W (2024) Neuron-targeted liposomal coenzyme Q10 attenuates neuronal ferroptosis after subarachnoid hemorrhage by activating the ferroptosis suppressor protein 1/coenzyme Q10 system. Acta Biomater 179:325–339 Allen RM, Vickers KC (2014) Coenzyme Q10 increases cholesterol efflux and inhibits atherosclerosis through microRNAs. Arterioscler Thromb Vasc Biol 34:1795–1797 Ran X, Jianxin W, Liquan Y, Xinjuan L, Yan G, Yanbo P, Yanbin W, Jianyu H (2019) Coenzyme Q10 Ameliorates Pancreatic Fibrosis via the ROS-Triggered mTOR Signaling Pathway. Oxidative Medicine and Cellular Longevity Liu H, Liu S, Jiang J, Zhang Y, Luo Y, Zhao J, Xu J, Xie Y, Liao W, Wang W et al (2022) CoQ10 enhances the efficacy of airway basal stem cell transplantation on bleomycin-induced idiopathic pulmonary fibrosis in mice. Respir Res 23:39 Mohamed DI, Khairy E, Tawfek SS, Habib EK, Fetouh MA (2019) Coenzyme Q10 attenuates lung and liver fibrosis via modulation of autophagy in methotrexate treated rat. Biomed Pharmacother 109:892–901 Takahashi M, Mizumura K, Gon Y, Shimizu T, Kozu Y, Shikano S, Iida Y, Hikichi M, Okamoto S, Tsuya K et al (2021) Iron-Dependent Mitochondrial Dysfunction Contributes to the Pathogenesis of Pulmonary Fibrosis. Front Pharmacol 12:643980 Yu F, Hao S, Zhao Y, Ren Y, Yang J, Sun X, Chen J (2014) Mild maternal iron deficiency anemia induces DPOAE suppression and cochlear hair cell apoptosis by caspase activation in young guinea pigs. Environ Toxicol Pharmacol 37:291–299 Altamura S, Vegi NM, Hoppe PS, Schroeder T, Aichler M, Walch A, Okreglicka K, Hültner L, Schneider M, Ladinig C et al (2020) Glutathione peroxidase 4 and vitamin E control reticulocyte maturation, stress erythropoiesis and iron homeostasis. Haematologica 105:937–950 Kathula SK, Thomas DE, Anstadt MP, Khan AU (2011) Paraneoplastic cutaneous leukocytoclastic vasculitis and iron deficiency anemia as the presenting features of squamous cell lung carcinoma. J Clin Oncol 29:e83–85 Weiss G, Ganz T, Goodnough LT (2019) Anemia of inflammation. Blood 133:40–50 Inoue S (2023) Anemia and iron deficiency in chronic obstructive pulmonary disease. Respir Investig 61:485–486 Saccone N, Bass J, Ramirez ML (2022) Bleomycin-Induced Lung Injury After Intravenous Iron Administration. Cureus 14:e27531 Olga K, Alexey MN, Korzhenevskiy DA, Valeriy KS, Vasilisa MT, Vsevolod VB, Arina GS (2023) Proteomic Shift in Mouse Embryonic Fibroblasts Pfa1 during Erastin, ML210 and BSO-Induced Ferroptosis. null Gong Y, Wang N, Liu N, Dong H (2019) Lipid Peroxidation and GPX4 Inhibition Are Common Causes for Myofibroblast Differentiation and Ferroptosis. DNA Cell Biol 38:725–733 Song J, Chen Y, Chen Y, Wang S, Dong Z, Liu X, Li X, Zhang Z, Sun L, Zhong J (2024) Ferrostatin-1 Blunts Right Ventricular Hypertrophy and Dysfunction in Pulmonary Arterial Hypertension by Suppressing the HMOX1/GSH Signaling. J Cardiovasc Transl Res 17:183–196 Li X, Duan L, Yuan S, Zhuang X, Qiao T, He J (2019) Ferroptosis inhibitor alleviates Radiation-induced lung fibrosis (RILF) via down-regulation of TGF-β1. J Inflamm (Lond) 16:11 Bersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, Roberts MA, Tong B, Maimone TJ, Zoncu R et al (2019) The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 575:688–692 Xu R, Wang W, Zhang W (2023) Ferroptosis and the bidirectional regulatory factor p53. Cell Death Discov 9:197 Doll S, Freitas FP, Shah R, Aldrovandi M, da Silva MC, Ingold I, Goya Grocin A, da Xavier TN, Panzilius E, Scheel CH et al (2019) FSP1 is a glutathione-independent ferroptosis suppressor. Nature 575:693–698 Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Schematic: Technical strategy of the study supplementdata.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4415956","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":305786860,"identity":"690a9f25-37fa-4c92-a4cf-8a664d79dd1e","order_by":0,"name":"Yue Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYPACGyjNRryWNNK1HCZBi/yM3GPSPBXn8wyunTFg+FB2mIF/dgN+LQY38tKkec7cLpacnWPAOOPcYQaJOwcIaJHIMZPmbbud2C+dY8DM23YYKJJAyGEgLf/OJbaBtPwlRgvDDZCWhgMQWxiJ0WJw5o2x5ZxjyYkzZ6cVHOw5l84jcYOQw9pzDG+8qbFL3HA7eeODH2XWcvwzCDmMgYFFAsY6AMQ8BNUDAfMHYlSNglEwCkbBCAYAowE+/5vuLMIAAAAASUVORK5CYII=","orcid":"","institution":"Hunan University","correspondingAuthor":true,"prefix":"","firstName":"Yue","middleName":"","lastName":"Sun","suffix":""},{"id":305786867,"identity":"0bca1094-fe07-4580-bf70-5cc37ff63bf6","order_by":1,"name":"Mengxue Yu","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengxue","middleName":"","lastName":"Yu","suffix":""},{"id":305786869,"identity":"32c6769f-1ede-4c25-91b9-b57dd02c3ac6","order_by":2,"name":"Huning Zhang","email":"","orcid":"","institution":"Ningxia Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Ma","suffix":""},{"id":305786882,"identity":"8ea50ea5-474b-44bb-bc3b-a364850f7b6a","order_by":9,"name":"Zhihong Liu","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Liu","suffix":""},{"id":305786883,"identity":"81765004-bd60-4c2b-a876-06a1954fa3e0","order_by":10,"name":"Anning Yang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Anning","middleName":"","lastName":"Yang","suffix":""},{"id":305786886,"identity":"be5ad2dc-99e5-44a8-896c-90ca51bc78dc","order_by":11,"name":"Yideng Jiang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yideng","middleName":"","lastName":"Jiang","suffix":""},{"id":305786887,"identity":"06903e78-11ce-48bd-886d-80f1831e5a0e","order_by":12,"name":"Bin Liu","email":"","orcid":"","institution":"Hunan University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-05-14 02:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4415956/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4415956/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57083368,"identity":"257dbb8b-0300-4814-822a-c899e771fbff","added_by":"auto","created_at":"2024-05-24 11:10:23","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":430600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoQ10 alleviates silicosis lung injury and collagen deposition in mice. \u003c/strong\u003eA. HE staining B. Masson staining C. Sirius red staining. Control refers to the normal group, Saline refers to the model group, CoQ10 refers to the treatment group. scale bar: 100μm.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4415956/v1/100f99ee14429d543a367842.jpeg"},{"id":57083371,"identity":"f63bf00a-95d5-4001-8cb5-d3e7be2d0459","added_by":"auto","created_at":"2024-05-24 11:10:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1131692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoQ10 decreased the expression of aSMA and Collagen I in silicosis mice. \u003c/strong\u003eA. mRNA expression levels of aSMA and Collagen I; B and C. protein expression levels of aSMA and Collagen I; D: Immuno-histochemical staining assay to detect the content of aSMA; scale bar: 100μm.E. Immuno-fluorescent staining assay to detect the content of aSMA protein expression in silicosis lung. scale bar: 20μm. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4415956/v1/5ea116073941eb72e245b80f.png"},{"id":57083374,"identity":"b603f57e-51e4-47f2-b558-c07b3cd1a462","added_by":"auto","created_at":"2024-05-24 11:10:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":188992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipid peroxidation and Fe\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e content in silicosis mice.\u003c/strong\u003e A. Fe\u003csup\u003e2+\u003c/sup\u003e content in the silicosis lung mice by colorimetry; B. MDA content by immunofluorescence assay. scale bar: 20μm. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4415956/v1/146beb1a9e721c01e1037fe4.png"},{"id":57083372,"identity":"ae055ca6-71aa-46ee-8bc7-2950b93c6a82","added_by":"auto","created_at":"2024-05-24 11:10:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoQ10 reduced the expression level of ferroptosis -related proteins GPX4 and p53 in silicosis mice. \u003c/strong\u003eA-B, mRNA expression levels of GPX4 and p53; C-E western blot assay and quantitative analysis of p53 and GPX4, respectively. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4415956/v1/62ac8c5c497326f3ab1da3e3.png"},{"id":58373057,"identity":"327d20fc-3f26-484b-97e7-9bdbbe2ecd80","added_by":"auto","created_at":"2024-06-14 14:29:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2484562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4415956/v1/e3a7ba92-34ab-4dea-98f9-c17c9f235016.pdf"},{"id":57083367,"identity":"779b31f4-203a-4312-b2c3-1220ec8d5d84","added_by":"auto","created_at":"2024-05-24 11:10:23","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":340276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic: Technical strategy of the study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4415956/v1/9f6a95b66c1b572645367c47.png"},{"id":57083369,"identity":"04469425-6e3d-4682-a8c4-92a287ccdf94","added_by":"auto","created_at":"2024-05-24 11:10:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":946496,"visible":true,"origin":"","legend":"","description":"","filename":"supplementdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-4415956/v1/e4895107381330033551c13e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ferroptosis Participates in Coenzyme Q10-treated Silicosis Fibrosis in Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSilicosis, as the most common, fastest-progressing, and most severe type of occupational pneumoconiosis, is mainly caused by the invasion of ~\u0026thinsp;0.5 -5 \u0026micro;m free silica into the lungs, resulting in diffuse pulmonary fibrosis\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The main clinical symptoms of silicosis are cough, phlegm, chest tightness, and secondary respiratory and circulatory disorders, and then progresses to various systemic diseases. Many researchers suggested that the mechanism of silicosis fibrosis is the continuous interaction between silica dust and pneumonocyte, rebuilding cytokine networks, which stimulates the proliferation of fibroblasts, increases the production and secretion of collagen, and forms fibrosis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.However, there are no specific treatments for silicosis. The current clinical treatment strategy mainly focuses on alleviating lung tissue inflammation, fibrosis, and other symptom, but the curative effects are not ideal\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is necessary to explore new anti-silicosis therapeutic drugs and elucidate corresponding molecular mechanisms.\u003c/p\u003e \u003cp\u003eCoQ10, a lipid-soluble antioxidant, is synthesized by endogenous biosynthesis in the body, which can improve immunity, enhance antioxidant capacity, delay aging. It is widely used in the adjuvant treatment of cardiovascular system diseases in medicine\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Ferroptosis is an iron-dependent programmed cell death pattern and distinct from apoptosis and autophagy. Ferroptosis mechanism involves the accumulation of lipid peroxidation and Fe\u003csup\u003e2+\u003c/sup\u003e, and the inactivation of molecules such as glutathione peroxidase 4 (GPX4), which are involved in the pathophysiological processes of various organ injury\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Currently, there are no reports on whether ferroptosis is involved in coenzyme Q10-treated silicosis fibrosis in mice. Therefore, our study adopts a model of silicosis fibrosis by tracheal instillation of silica suspension to investigate whether ferroptosis participate in the treatment of silicosis fibrosis with CoQ10, providing a theoretical basis for the treatment of silicosis fibrosis (Schematic: Technical strategy of the study).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Materials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e1.1.1 Experimental animals\u003c/h2\u003e \u003cp\u003eTwenty-four male C57BL/6 mice, aged 6\u0026ndash;8 weeks and weighing 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2g, were purchased from the Beijing Wei Shang Li De Biotechnology Co., LTD. (production license number: SCXK (Beijing) 2016-0002). After one week of adaptive feeding under SPF conditions in Laboratory Animal Center of Ningxia Medical University, and they were randomly divided into three groups, with 6 mice in each group: (1) control group; (2) Saline group; (3) CoQ10 treatment group. The feeding conditions were 12h light, 12h darkness, temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1) ℃, relative humidity 40%-50%, independent drinking and eating, feeding and model making followed the relevant regulations of the management and use of experimental animals in the Experimental Animal Center of Ningxia Medical University, and the test procedures were reviewed by the Experimental Animal Welfare Committee of the Experimental Animal Center of Ningxia Medical University. License number SYXK (NING) 2015-0001.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.1.2 Experimental instrument\u003c/h2\u003e \u003cp\u003eELIASA epoch, (Bio-Tek, USA); Electrophoretic, electrokinetic and gel imaging systems (Rio⁃Rad, USA); Ultraviolet-visible spectrophotometer DS-11 (DeNovix Inc., USA ); Laser confocal microscope LSM800 (Zeiss, Germany); Fluorescent quantitative PCR instrument QuantStudioTM5 (ThermoFisher, USA); Inverted microscope (Leica, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e1.1.3 Experimental reagents\u003c/h2\u003e \u003cp\u003eSiO\u003csub\u003e2\u003c/sub\u003e (Sigma, USA), Coenzyme Q10 (Beijing Solaibao Biotech Co., LTD., China), Corn oil (Aladdin, China), Sirius Red kit, HE staining kit (Beijing Leegan Biotech Co., LTD., China), Hydroxyproline kit (alkaline hydrolysis method) (Nanjing Jianxian Institute of Biological Products, China), α-SMA rabbit primary antibody (Chengdu Zhengneng Co., LTD., China), Mouse goat serum, DAB color detection kit (Zhongshan Jinqiao Biotechnology Co., LTD., China), Reverse transcription kit, qPCR kit (TaRaKa, Japan), Whole protein extraction kit (KGI Biotech Co., LTD., China), Separation gel, concentration gel (Herix, China) China), Acrylamide (Beijing Bio Top Technology Co., LTD., China), ammonium persulfate, TEMED (Sigma, USA), 5\u0026times;Loading buffer(Kangwei Century, China), High-sensitivity ECL chemiluminescence kit (New Saemi Biotechnology Co., LTD., China). Tissue iron and malondialdehyde (MDA) detection kits were purchased from Jiancheng Biological Company, Nanjing, China; Antibodies for aSMA, collengen I, p53, GPX4, MDA-FITC and Alexa Fluor\u0026reg; 488, purchased from abcam (UK); Total RNA extraction kit, purchased from Tiangen (China), Reverse transcription and fluorescence quantitative PCR kit, purchased from TaKaRa (Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1.2 Methods\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e1.2.1 Preparation silicosis model in vivo\u003c/h2\u003e \u003cp\u003eAfter isoflurane anesthesia, the trachea was exposed, and 0.1mL normal saline was instilled into the control group, and 0.1mL SiO\u003csub\u003e2\u003c/sub\u003e suspension (50mg/mL) was instilled into the model control group and CoQ10 treatment group, respectively. The skin was sutured, and the survival status of the mice was observed. The mice in the treatment group were given CoQ10 (100mg/kg) by gavage 48 hours after operation, and the other mice were fed autonomously. On day 60 after operation, the mice were sacrificed by intraperitoneal injection of 0.3mL urethane, the right middle lobe of the lung was removed, rinsed with normal saline, fixed with 4% paraformaldehyde, and the remaining lung tissues were frozen at -80℃ for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e1.2.2 Histopathological staining\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eHE staining\u003c/strong\u003e \u003cp\u003eParaffin sections of lung tissue were dewaxed and fixed, followed by gradient alcohol dehydration, hematoxylin staining for 6min, ethanol differentiation for 5s, running underwater washing for 3min, eosin staining for 5min, running underwater washing for 5s. After gradient alcohol treatment, xylene was used twice for transparency, three minutes at a time, and the tablets were sealed.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMasson staining\u003c/strong\u003e \u003cp\u003eParaffin sections of the kidney were deparaffinized and fixed, stained with Weigert iron hematoxylin staining solution, differentiated with acid ethanol differentiation solution, returned to blue with Masson blue solution, washed with distilled water, stained with Ponceaux-fuchsine-red staining solution, washed with phosphamoridic acid solution, stained with aniline blue staining solution, dehydrated, transparent, and sealed.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSirius red staining\u003c/strong\u003e \u003cp\u003ethe mice were dewaxed, stained with Sirius Red droplets for 40min, washed for 2min, the surface staining solution was removed, dehydrated, transparent, and sealed with neutral gum. The changes of lung fibrosis in mice were observed.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e1.2.3 Detection of collagen I and α-SMA in lung tissue\u003c/h2\u003e \u003cp\u003eThe content of collagen I and a-SMA in lung tissue was detected by immunofluorescence assay. Portions of lung tissue were made into frozen sections, removed from \u0026minus;\u0026thinsp;80℃ and placed in a cassette for 25\u0026ndash;30 min until room temperature was restored. Fixation with glacial acetone for 25\u0026thinsp;~\u0026thinsp;30min; Use immunohistochemical strokes to draw circles; Triton \u0026minus;\u0026thinsp;0.3% X100 incubation 10\u0026thinsp;~\u0026thinsp;20 min, PBS washed five times, each time 3 min. 5% serum closed for 30min; collagen I and a-SMA antibodies (1:100) were incubated overnight at 4℃. The fluorescent secondary antibody was incubated at 37℃ after rewarming. 4', 6-diaminyl-2-phenylindole (DAPI) was nucleated for 5 min and washed with PBS 5 times for 3 min each time. Fluorescence images were taken by laser confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e1.2.4 Determination of iron ion concentration in lung tissue of silicosis mice\u003c/h2\u003e \u003cp\u003ePart of the lung tissue of mice was weighed and recorded, and normal saline (volume ratio 1:9) was added to mix. Mechanical homogenization was performed on ice, centrifuged at 1200 g for 10 min, and the supernatant was removed. The blank tube, standard tube and sample tube to be tested were added according to the instructions. After mixing, the tubes were bathed at 99 ℃ for 5 min, cooled and centrifuged at 1 800 g for 10 min. The absorbance value of the supernatant was measured at 520 nm. The results were calculated as tissue iron content = (A \u003csub\u003edetermination\u003c/sub\u003e-A \u003csub\u003eblank\u003c/sub\u003e)/ (A \u003csub\u003estandard\u003c/sub\u003e-A \u003csub\u003eblank\u003c/sub\u003e) \u0026times;C \u003csub\u003estandard\u003c/sub\u003e \u0026divide;Cpr, where Cpr was the protein concentration in tissue homogenates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e1.2.5 Detection of MDA level in lung tissue\u003c/h2\u003e \u003cp\u003eThe level of malondialdehyde (MDA) in lung tissue was detected by immunofluorescence assay. Part of the lung tissue was cut into 8\u0026micro;m frozen sections, removed from \u0026minus;\u0026thinsp;80 ℃, and placed in a cassette for 25\u0026ndash;30 min to recover to room temperature. Fixed in ice-cold acetone for 25\u0026ndash;30 min; Immunohistochemical staining circles; The cells were incubated with 0.3% Triton-X100 for 10\u0026ndash;20 min and washed 5 times with PBS for 3 min each time. The cells were blocked with 5% serum for 30 min. Goat polyclonal anti-MDA-FITC antibody (1:100) was incubated overnight at 4 ℃. After rewarming, the cells were washed 5 times with PBS for 3 min each time. Nuclei were stained with 1 mg/L 4', 6-diamidino-2-phenylindole (DAPI) for 5 min and washed with PBS for 5 times, 3 min each time. Fluorescence images were taken by laser confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e1.2.6 Protein expression in lung tissue\u003c/h2\u003e \u003cp\u003eWestern blotting was used to detect protein expression in lung tissue. Fifty mg of lung tissue was weighed, 500 \u0026micro;L of protein lysis buffer was added, homogenized with a tissue homogenizer, and protein extraction was performed according to the steps in the instructions of the whole protein extraction kit. The protein concentration was measured by ultramicro UV-visible spectrophotometer. The 5\u0026times; loading buffer was mixed with the extracted tissue protein 1:4, and the protein was denatured by boiling at 99.5 ℃ for 10 min. The protein was electrophoresed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), and the pressure was constant. The protein in the gel was wet transferred to 0.22 \u0026micro;m polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk powder for 2 to 3 hours, incubated with primary antibody at 4℃ overnight, washed with secondary antibody at room temperature for 2 hours, washed with PBS 3 times, 10 min each time, and detected by chemiluminescence method. The relative content of the target protein was calculated as the ratio of gray value between the target protein and the internal reference protein (β-actin). Repeat 3 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e1.2.7 Detection of mRNA levels in lung tissue\u003c/h2\u003e \u003cp\u003eQuantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression in lung tissue. Primer sequences (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) :\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequence used for qRT-PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003egene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward primer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse primer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGPX4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TGTGCATCCCGCGATGATT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-CCCTGTACTTATCCAGGCAGA-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CCCCTGTCATCTTTTGTCCCT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-AGCTGGCAGAATAGCTTATTGAG-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eaSMA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-CCTTCGTGACTACTGCCGAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GTCAGCAATGCCTGGGTACAT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollagen I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GCTCCTCTTAGGGGCCACT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ATTGGGGACCCTTAGGCCAT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'AGGTCGGTGTGAACGGATTTG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GGGGTCGTTGATGGCAACA-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe qRT-PCR reaction system included denaturation (95 ℃ for 30 s), annealing (95 ℃ for 5 s), and extension (60 ℃ for 34 s). A total of 40 cycles were set up, ΔCt\u0026thinsp;=\u0026thinsp;Ct(objective) -Ct (internal control).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e1.2.8 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin-embedded sections of mouse lung tissue were dewaxed with xylene, dehydrated with gradient alcohol, antigen heat repaired with sodium citrate (pH\u0026thinsp;=\u0026thinsp;6) for 15min, blocked with goat serum for 10 min, incubated with α-SMA/collagen I primary antibody (diluted 1:100) overnight, HRP-labeled goat anti-rabbit/anti-mouse secondary antibody for 30 min, and DAB for 6 min. The slices were dehydrated, transparent, and sealed. Images were taken under a 20x objective lens under an ordinary light microscope to observe the expression of α-SMA and collagen I protein in each group. The positive areas were counted by Image-Pro Plus 6.0 software. The results were quantified as the ratio of the IOD value of the positive area to the area of the picture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Statistical Methods\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using GraphPad 8.0 software, with data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way analysis of variance was employed for comparing multiple groups, followed by Tukey's post hoc test for pairwise comparisons.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.1 CoQ10 Alleviates Silicosis Lung Injury and Collagen Deposition\u003c/h2\u003e \u003cp\u003eFirstly, we used HE, Masson, and Sirius red assays to assess silicosis lung injury and collagen deposition after CoQ10 treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). Histopathological staining assay showed that the normal lung tissues in control group exhibited a basically intact alveolar structure, thin alveolar walls, no obvious inflammatory cells aggregation, and no significant collagen fiber deposition in pulmonary mesenchyme. In the saline group, there were significantly dense and damaged the lung structure, a large number of inflammatory cells gathered in both the pulmonary mesenchyme and alveolar cavities, and uneven size silicosis nodules, and a large amount of collagen fiber deposition. In contrast, after CoQ10 treatment, the alveolar structure was still acceptable compared to the saline group, with no silicosis nodules observed, and reduced collagen deposition, but there were inflammatory cells aggregation. All above results indicated that CoQ10 could alleviate lung injury in silicosis mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2\u003c/b\u003e CoQ10 Reduced the Expression of a-SMA and Collagen I in Silicosis Mice\u003c/p\u003e \u003cp\u003ea-SMA and Collagen I plays an important role in the development of pulmonary fibrosis\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In general, a-SMA mainly plays a role in supporting and stabilizing cellular structures. However, once pulmonary fibrosis occurs, myofibroblasts, producing more a-SMA, and become over-activated and synthesize too much collagen and other extracellular matrix components to accelerate the development of pulmonary tissue fibrosis. Collagen I, as a marker molecule for fibrosis, it is positively correlated with the degree of fibrosis. Furtherly, we detected the expression level of α-SMA and Collagen I in silicosis mice to evaluate the ability of CoQ10 to alleviate fibrosis. qRT-PCR assay showed that compared to the saline group (0.263\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029), CoQ10 treatment significantly decreased the α-SMA mRNA expression level (0.118\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051) and higher than the control group (0.048\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009) (\u003cem\u003eF\u0026thinsp;=\u0026thinsp;61.230\u003c/em\u003e, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Meanwhile, CoQ10 also decreased the mRNA expression level of Collagen-1 (1.245\u0026thinsp;\u0026plusmn;\u0026thinsp;0.874) relative to the saline group (3.400\u0026thinsp;\u0026plusmn;\u0026thinsp;1.062) and higher than the control group (0.849\u0026thinsp;\u0026plusmn;\u0026thinsp;0.486) (\u003cem\u003eF\u0026thinsp;=\u0026thinsp;15.950, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blot assay showed that compared to the saline group (1.178\u0026thinsp;\u0026plusmn;\u0026thinsp;0.253), CoQ10 treatment significantly decreased the α-SMA protein expression level (0.408\u0026thinsp;\u0026plusmn;\u0026thinsp;0.184) and higher than the control group (0.502\u0026thinsp;\u0026plusmn;\u0026thinsp;0.265) (\u003cem\u003eF\u0026thinsp;=\u0026thinsp;18.86, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Meanwhile, CoQ10 also decreased the protein expression level of Collagen-1 (0.065\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031) relative to the saline group (0.135\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023) and higher than the control group (0.058\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027) (\u003cem\u003eF\u0026thinsp;=\u0026thinsp;14.780, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). The data of immunohistochemistry and fluorescence staining assays also showed that CoQ10 reduced the activation of fibroblasts in the silicosis lung and weaken the development of silicosis fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3\u003c/b\u003e Effect of CoQ10 on the Content Levels of Fe\u003csup\u003e2+\u003c/sup\u003e and Malondialdehyde (MDA) in Silicosis Lung\u003c/p\u003e \u003cp\u003eFerroptosis is a non-apoptotic cell death form that relies on the large amount accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e and the corresponding abnormal increased lipid peroxides in cells\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Some studies have reported that the presence of a large amount of Fe\u003csup\u003e2+\u003c/sup\u003e in cells might trigger the Fenton reaction, which in turn initiates the occurrence of lipid peroxidation\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In this study, compared with the content of Fe\u003csup\u003e2+\u003c/sup\u003e in the normal control group (0.068\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009 nmol/mg Prot), Fe\u003csup\u003e2+\u003c/sup\u003e content in the saline group increased significantly (0.205\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 nmol/mg Prot), but after CoQ10 treatment, the content of Fe\u003csup\u003e2+\u003c/sup\u003e decreased significantly (0.098\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026 nmol/mg Prot) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). MDA, as one of the classic indicators for detecting lipid peroxidation in damaged tissues and cells, is widely used in the field of iron-dependent cell death research. Immunofluorescence assay also showed that compared to the saline group, CoQ10 decreased MDA level in lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In brief, CoQ10 was involved in the occurrence of iron-dependent cell death via reducing the levels of lipid peroxidation and Fe\u003csup\u003e2+\u003c/sup\u003e in silicosis lung tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4 CoQ10 Reduced the expression level of ferroptosis -related proteins GPX4 and p53 in Silicosis Mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo clarify whether ferroptosis is involved in the CoQ10-treatment silicosis fibrosis in mice, we further examined the expression level of ferroptosis core enzymes GPX4 and its upstream regulator p53. qRT-PCR assay showed that compared to the saline group (0.233\u0026thinsp;\u0026plusmn;\u0026thinsp;0.116), CoQ10 treatment significantly increased the GPX4 mRNA expression level (0.777\u0026thinsp;\u0026plusmn;\u0026thinsp;0.216) and lower than the control group (0.826\u0026thinsp;\u0026plusmn;\u0026thinsp;0.127) (\u003cem\u003eF\u0026thinsp;=\u0026thinsp;25.540, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e). Meanwhile, CoQ10 also decreased the mRNA expression level of p53 (0.041\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029) relative to the saline group (0.086\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031) and higher than the control group (0.038\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0194) (\u003cem\u003eF\u0026thinsp;=\u0026thinsp;6.996, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B). Western blotting assay showed that compared to the saline group (0.252\u0026thinsp;\u0026plusmn;\u0026thinsp;0.116), CoQ10 treatment significantly increased the GPX4 protein expression level (1.215\u0026thinsp;\u0026plusmn;\u0026thinsp;0.331) and lower than the control group (0.656\u0026thinsp;\u0026plusmn;\u0026thinsp;0.129) (\u003cem\u003eF\u0026thinsp;=\u0026thinsp;30.20, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e). Meanwhile, CoQ10 also decreased the protein expression level of p53 (0.775\u0026thinsp;\u0026plusmn;\u0026thinsp;0.506) relative to the saline group (1.730\u0026thinsp;\u0026plusmn;\u0026thinsp;0.735) and higher than the control group (0.495\u0026thinsp;\u0026plusmn;\u0026thinsp;0.287) (\u003cem\u003eF\u0026thinsp;=\u0026thinsp;10.010, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E). These results suggested that ferroptosis may be involved in the process of CoQ10 alleviating pulmonary fibrosis in silicosis mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Discussion","content":"\u003cp\u003eWith the rise of new industries, workers, such as miners, stone processing workers, construction workers, are still exposed to large amounts of silica for a long time. It has been reported that there are millions of dust-exposed workers in the United States, among whom one in ten suffers from silicosis. In addition, the situation of silicosis prevention and control in developing countries is more critical, such as India where the number of silicosis patients is as high as tens of millions, and China where the number of pneumoconiosis patients exceeds 850000, of which silicosis accounts for more than 50%\u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. So far, due to the limited effective treatment methods for silicosis, lung damage and fibrosis cannot be reversed. As a major public health problem, the current treatment strategies for silicosis are still mainly focused on anti-inflammatory, anti-fibrotic and other symptomatic treatments combined with lung lavage or combined with oxygen therapy and psychological counseling, but the effects are not ideal. In this study, after intratracheal instillation of silica for 48 hours, CoQ10 was administered orally at a dose of 100 mg/kg\u0026bull;d to treat silicosis mice. After 60 days, CoQ10 significantly improved the pathological characteristics of silicosis lung tissues and prevented the development of pulmonary fibrosis. Compared to the saline group, the alveolar wall structure in the CoQ10 treatment group was acceptable, no silicosis nodules were observed, and collagen deposition was reduced. qPCR and WB experiments showed that CoQ10 significantly reduced the expression levels of α-SMA and collagen I in silicosis lung tissues. It is worth noting that CoQ10 significantly inhibited the accumulation of lipid peroxidation and Fe\u003csup\u003e2+\u003c/sup\u003e and increased the expression of ferroptosis regulatory core enzyme GPX4 and downregulated its upstream regulator p53 in silicosis lung tissues. In summary, the above studies indicated that ferroptosis was involved in the process of silicosis fibrosis after treatment with CoQ10.\u003c/p\u003e \u003cp\u003eCoQ10 is a lipid-soluble quinone compound that is present in the phospholipid bilayer of cell membranes and accumulates significantly in the inner mitochondrial membrane\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The pharmacological effects of CoQ10 mainly involve antioxidation, anti-fibrosis, scavenging free radicals, dilating blood vessels, reducing pro-inflammatory cytokines, etc\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Currently, it has been widely used to reduce blood viscosity, improve ischemia and reperfusion injury after coronary artery revascularization in the prevention and treatment of atherosclerosis\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Coenzyme Q10, as a component of the mitochondrial electron transport chain, can enhance mitochondrial quality, improve mitochondrial function, increase mitochondrial activity, enhance cellular energy supply, inhibit the production of ROS, help reduce oxidative damage to cells, thereby reducing the risk of fibrosis\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Coenzyme Q10 can also reduce different inflammatory mediators (such as TNFα, TGF-β, and MCP1) in liver and lung tissues, alleviate inflammation and fibrosis In addition, CoQ10 has also been shown to alleviate arginine-induced pancreatic fibrosis by reducing collagen deposition in pancreatic tissues\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In this study, CoQ10 significantly reduced the expression levels of pulmonary α-SMA and collagen I in the lung tissues of silicosis mice and could inhibit the level of lipid peroxidation in the lung of silicosis mice.\u003c/p\u003e \u003cp\u003eFerroptosis is involved in the regulation of liver and lung fibrosis processes. As a novel cell death mode which distinct from apoptosis, autophagy, and pyroptosis, ferroptosis is triggered by reactive oxygen species and lipid peroxidation induced by the overload of Fe\u003csup\u003e2+\u003c/sup\u003e. Meanwhile, the depletion of antioxidant glutathione or glutathione peroxidase 4 is also considered an essential condition for the initiation and progression of ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Numerous studies have shown that ferroptosis is an important mechanism in the progression of pulmonary diseases, participating in the regulation of various pulmonary diseases, including lung cancer\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, lung infection\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, chronic obstructive pulmonary disease\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, acute lung injury\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, etc. Moreover, ferroptosis plays an important role in pulmonary fibrosis. Gong\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003eet al. treated human embryo lung fibroblasts (HFL1) with TGF-β to study the relationship between ferroptosis and pulmonary fibrosis. The inducer of ferroptosis, Erastin, can induce pulmonary fibrosis by stimulating fibroblasts to transform into myofibroblasts\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In addition, the inhibitor of ferroptosis, ferrostatin-1 (Fer-1), can block the accumulation of lipid peroxidation and increase GPX4 activity, alleviating the pulmonary fibrosis induced by ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Li's research results also suggested that ferroptosis exacerbates radiation-induced pulmonary fibrosis, while the inhibitor of ferroptosis, Liproxstatin-1, can significantly reduce RILF\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGPX4 is one of the most important antioxidant enzymes in mammals, and it is considered a core factor in ferroptosis. It converts lipid peroxides into lipid alcohols, preventing tissue and cell damage from lipid peroxidation, thereby inhibiting ferroptosis. The ferroptosis inducers Erastin and RSL3 can inactivate GPX4, leading to the induction of ferroptosis. In addition, p53 is an important tumor suppressor gene that can downregulate the expression of SLC7A11 and negatively regulate the activity of GPX4, resulting in a decrease in cellular antioxidant capacity, accumulation of ROS, and ultimately ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Our research found that CoQ10 significantly inhibits the lipid peroxidation and accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e in silicosis lung tissues and increases the expression of the core ferroptosis regulatory enzyme GPX4 and downregulated its upstream regulator p53, indicating that ferroptosis is involved in the process of CoQ10-treated silicosis fibrosis. Recent research has shown that coenzyme Q10 not only plays a key role in maintaining mitochondrial function and the cell respiratory chain but also regulates the process of ferroptosis. CoQ10 enhances mitochondrial function and antioxidant properties, regulates the balance of ferrous iron in cells, thereby reducing the production of free radicals and the risk of mitochondrial damage\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In addition, CoQ10 can further regulate the levels and activity of intracellular ferrous iron by regulating the expression of genes related to intracellular iron metabolism, affecting the absorption, transport, and storage of iron \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. In this way, CoQ10 can regulate the mechanism of ferroptosis, reduce the toxicity of ferrous iron, and protect cells from oxidative stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCoQ10 has the ability to regulate ferroptosis during the process of silicosis fibrosis via enhancing anti-oxidation capability, and regulating the expression of iron metabolism-related genes to achieve protect cells from free radical damage. This finding is a new perspective for exploring the pathogenesis and treatment for silicosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePermission must be obtained from the Animal Ethics Committee of\u0026nbsp;Ningxia Medical University\u0026nbsp;before starting any animal experiments. All experiments were conducted in accordance with the guidelines of\u0026nbsp;Ningxia Medical University\u0026nbsp;Animal Ethics Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the National Natural Science Foundation of China (82060264, 82271626, 82160088, 82260142, 82360639), China Postdoctoral Science Foundation (2023MD734192), National Natural Science Foundation Regional Innovation and Development Joint Fund (U21A20343), Ningxia Natural Science Foundation (2022AAC03128, 2022AAC05025), Ningxia Key Research and Development Projects (2020BEG03008, 2020BFH02003, 2021BEG02030), Open competition mechanism to select the best candidates for key research projects of Ningxia Medical University (XJKF230106, XJKF230125), Basic scientific research operating expenses from public welfare research institutes at the central level of the Chinese Academy of Medical Sciences (2019PT330002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYue Sun conceived and designed the study. Mengxue Yu, Shengpeng Wen, Huning Zhang, Wenyue Zhang, Sirong Chang,\u0026nbsp;Fei Yang, Guangjun Qi and Xin Ma\u0026nbsp;performed the experiments. Anning Yang and Zhihong Liu analyzed the data. Bin Liu and Yideng Jiang wrote the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCheng D, Xu Q, Wang Y, Li G, Sun W, Ma D, Zhou S, Liu Y, Han L, Ni C (2021) Metformin attenuates silica-induced pulmonary fibrosis via AMPK signaling. J Transl Med 19:349\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoy RF, Chambers DC (2020) Silica-related diseases in the modern world. Allergy 75:2805\u0026ndash;2817\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang G, Tian Y, Li C, Xia J, Qi Y, Yao W, Hao C (2022) LncRNA UCA1 regulates silicosis-related lung epithelial cell-to-mesenchymal transition through competitive adsorption of miR-204-5p. Toxicol Appl Pharmacol 441:115977\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuihui T, Hui Z, Aowei M, Luocheng S, Deyong G, Jiale L, Wenjian H, Ke X, Qianqian M, Wenfeng W et al (2023) VX-765 attenuates silica-induced lung inflammatory injury and fibrosis by modulating alveolar macrophages pyroptosis in mice. Ecotoxicology and Environmental Safety\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan L, Sun Y, Zhou N, Wu W, Zheng W, Wang Y (2022) Dihydroquercetin Attenuates Silica-Induced Pulmonary Fibrosis by Inhibiting Ferroptosis Signaling Pathway. Front Pharmacol 13:845600\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Zhang Y, Zhao R, Cheng Z, Tang M, Qiu A, Dong Y, Lu Y, Lian Y, Zhuang X et al (2021) Integrating RNA-Seq With GWAS Reveals a Novel SNP in Immune-Related HLA-DQB1 Gene Associated With Occupational Pulmonary Fibrosis Risk: A Multi-Stage Study. Front Immunol 12:796932\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinjun L, Xueke H, Yangyang Z, Wenwen P, Xiaomei Z, Jibin M (2024) Coenzyme Q10 in atherosclerosis. Eur J Pharmacol\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, Bao R, Wang Q, Hao W, Liu Y, Chang S, Wang M, Li Y, Liu Z, Sun Y (2022) SiO\u003csub\u003e2\u003c/sub\u003e -induced ferroptosis in macrophages promotes the development of pulmonary fibrosis in silicosis models. Toxicol Res (Camb) 11:42\u0026ndash;51\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQ RB, Y WMY (2023) Z, S W, T L, M W, Y L, S C, H C, et al: AAV9-HGF cooperating with TGF-beta/Smad inhibitor attenuates silicosis fibrosis via inhibiting ferroptosis, vol 161. Biomedicine \u0026amp; pharmacotherapy\u0026thinsp;=\u0026thinsp;Biomedecine \u0026amp; pharmacotherapie, p 114537\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDempsey LA (2019) Death by iron. Nat Immunol 20:665\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Horany HE, Atef MM, Abdel Ghafar MT, Fouda MH, Nasef NA, Hegab II, Helal DS, Elseady W, Hafez YM, Hagag RY et al (2023) Empagliflozin Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats by Modulating Sesn2/AMPK/Nrf2 Signaling and Targeting Ferroptosis and Autophagy. Int J Mol Sci 24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePei Z, Qin Y, Fu X, Yang F, Huo F, Liang X, Wang S, Cui H, Lin P, Zhou G et al (2022) Inhibition of ferroptosis and iron accumulation alleviates pulmonary fibrosis in a bleomycin model. Redox Biol 57:102509\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbd-El-Fattah AA, El-Sawalhi MM, Rashed ER, El-Ghazaly MA (2010) Possible role of vitamin E, coenzyme Q10 and rutin in protection against cerebral ischemia/reperfusion injury in irradiated rats. Int J Radiat Biol 86:1070\u0026ndash;1078\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao M, He X, Zhou Y, Peng W, Zhao XM, Jiang M (2024) Coenzyme Q10 in atherosclerosis. Eur J Pharmacol 970:176481\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng Z, Ding YN, Yang ZM, Li XJ, Zhuang Z, Lu Y, Tang QS, Hang CH, Li W (2024) Neuron-targeted liposomal coenzyme Q10 attenuates neuronal ferroptosis after subarachnoid hemorrhage by activating the ferroptosis suppressor protein 1/coenzyme Q10 system. Acta Biomater 179:325\u0026ndash;339\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllen RM, Vickers KC (2014) Coenzyme Q10 increases cholesterol efflux and inhibits atherosclerosis through microRNAs. Arterioscler Thromb Vasc Biol 34:1795\u0026ndash;1797\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRan X, Jianxin W, Liquan Y, Xinjuan L, Yan G, Yanbo P, Yanbin W, Jianyu H (2019) Coenzyme Q10 Ameliorates Pancreatic Fibrosis via the ROS-Triggered mTOR Signaling Pathway. Oxidative Medicine and Cellular Longevity\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu H, Liu S, Jiang J, Zhang Y, Luo Y, Zhao J, Xu J, Xie Y, Liao W, Wang W et al (2022) CoQ10 enhances the efficacy of airway basal stem cell transplantation on bleomycin-induced idiopathic pulmonary fibrosis in mice. Respir Res 23:39\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohamed DI, Khairy E, Tawfek SS, Habib EK, Fetouh MA (2019) Coenzyme Q10 attenuates lung and liver fibrosis via modulation of autophagy in methotrexate treated rat. Biomed Pharmacother 109:892\u0026ndash;901\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi M, Mizumura K, Gon Y, Shimizu T, Kozu Y, Shikano S, Iida Y, Hikichi M, Okamoto S, Tsuya K et al (2021) Iron-Dependent Mitochondrial Dysfunction Contributes to the Pathogenesis of Pulmonary Fibrosis. Front Pharmacol 12:643980\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu F, Hao S, Zhao Y, Ren Y, Yang J, Sun X, Chen J (2014) Mild maternal iron deficiency anemia induces DPOAE suppression and cochlear hair cell apoptosis by caspase activation in young guinea pigs. Environ Toxicol Pharmacol 37:291\u0026ndash;299\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltamura S, Vegi NM, Hoppe PS, Schroeder T, Aichler M, Walch A, Okreglicka K, H\u0026uuml;ltner L, Schneider M, Ladinig C et al (2020) Glutathione peroxidase 4 and vitamin E control reticulocyte maturation, stress erythropoiesis and iron homeostasis. Haematologica 105:937\u0026ndash;950\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKathula SK, Thomas DE, Anstadt MP, Khan AU (2011) Paraneoplastic cutaneous leukocytoclastic vasculitis and iron deficiency anemia as the presenting features of squamous cell lung carcinoma. J Clin Oncol 29:e83\u0026ndash;85\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiss G, Ganz T, Goodnough LT (2019) Anemia of inflammation. Blood 133:40\u0026ndash;50\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoue S (2023) Anemia and iron deficiency in chronic obstructive pulmonary disease. Respir Investig 61:485\u0026ndash;486\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaccone N, Bass J, Ramirez ML (2022) Bleomycin-Induced Lung Injury After Intravenous Iron Administration. Cureus 14:e27531\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlga K, Alexey MN, Korzhenevskiy DA, Valeriy KS, Vasilisa MT, Vsevolod VB, Arina GS (2023) Proteomic Shift in Mouse Embryonic Fibroblasts Pfa1 during Erastin, ML210 and BSO-Induced Ferroptosis. null\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong Y, Wang N, Liu N, Dong H (2019) Lipid Peroxidation and GPX4 Inhibition Are Common Causes for Myofibroblast Differentiation and Ferroptosis. DNA Cell Biol 38:725\u0026ndash;733\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong J, Chen Y, Chen Y, Wang S, Dong Z, Liu X, Li X, Zhang Z, Sun L, Zhong J (2024) Ferrostatin-1 Blunts Right Ventricular Hypertrophy and Dysfunction in Pulmonary Arterial Hypertension by Suppressing the HMOX1/GSH Signaling. J Cardiovasc Transl Res 17:183\u0026ndash;196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Duan L, Yuan S, Zhuang X, Qiao T, He J (2019) Ferroptosis inhibitor alleviates Radiation-induced lung fibrosis (RILF) via down-regulation of TGF-β1. J Inflamm (Lond) 16:11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, Roberts MA, Tong B, Maimone TJ, Zoncu R et al (2019) The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 575:688\u0026ndash;692\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu R, Wang W, Zhang W (2023) Ferroptosis and the bidirectional regulatory factor p53. Cell Death Discov 9:197\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoll S, Freitas FP, Shah R, Aldrovandi M, da Silva MC, Ingold I, Goya Grocin A, da Xavier TN, Panzilius E, Scheel CH et al (2019) FSP1 is a glutathione-independent ferroptosis suppressor. Nature 575:693\u0026ndash;698\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Silicosis Fibrosis, Coenzyme Q10, Ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-4415956/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4415956/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSilicosis is the most common, fastest-progressing, and most severe type of occupational pneumoconiosis, which result in diffuse pulmonary fibrosis. However, there are no specific treatments for silicosis. Coenzyme Q10, as a component of the mitochondrial electron transport chain, can enhance mitochondrial quality and cellular energy supply, inhibit the production of ROS to reduce oxidative damage for reducing the risk of fibrosis. Ferroptosis is triggered by reactive oxygen species and lipid peroxidation induced by the overload of Fe\u003csup\u003e2+\u003c/sup\u003e and has tight correlation with pulmonary fibrosis. However, whether ferroptosis is involved in coenzyme Q10-treated silicosis fibrosis in mice remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAfter intratracheal instillation of silica in C57BL/6J mice for 48 hours, CoQ10 was administered orally at a dose of 100 mg/kg\u0026bull;d. The mice were randomly divided into control group, saline group and CoQ10 treatment group, and there are 6 mice in each group. Lung injury and fibrosis in mice were observed by HE, Masson, and Sirius Red assays. Iron content was measured by colorimetry in lung tissue. The content of malondialdehyde (MDA) in lung tissue was detected by immunofluorescence staining. Protein and mRNA expression of aSMA, Collagen I, GPX\u003csub\u003e4\u003c/sub\u003e and p53 were determined by qRT-PCR and Western blotting. Multiple data comparisons were conducted using one-way ANOVA, meanwhile multiple comparisons were conducted using Tukey test.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eHistopathological staining assays showed that the normal lung tissues in control group exhibited a basically intact alveolar structure, thin alveolar walls, no obvious inflammatory cells aggregation, and no significant collagen fiber deposition in pulmonary mesenchyme. But after CoQ10 treatment, the alveolar structure was still acceptable and no silicosis nodules and reduced collagen deposition. qPCR and WB experiments showed that CoQ10 significantly reduced the expression levels of α-SMA and collagen I in silicosis lung tissues. It is worth noting that CoQ10 significantly inhibited the accumulation of lipid peroxidation and Fe\u003csup\u003e2+\u003c/sup\u003e and increased the expression of ferroptosis regulatory core enzyme GPX4 and reduced its upstream regulator p53 in silicosis lung tissues.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFerroptosis is involved in coenzyme Q10-treated silicosis fibrosis and this finding is a new perspective for exploring the pathogenesis and treatment for silicosis.\u003c/p\u003e","manuscriptTitle":"Ferroptosis Participates in Coenzyme Q10-treated Silicosis Fibrosis in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-24 11:10:19","doi":"10.21203/rs.3.rs-4415956/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a85f88f3-24c1-41f5-9a19-f6232f233f72","owner":[],"postedDate":"May 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-14T14:21:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-24 11:10:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4415956","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4415956","identity":"rs-4415956","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}