Coenzyme Q10 Alleviates Silicosis Fibrosis via Inhibiting Ferroptosis in Mice

Abstract

Background/Aim

Silicosis, the most severe type of occupational pneumoconiosis, leads to diffuse pulmonary fibrosis without specific therapy. Ferroptosis is triggered by reactive oxygen species (ROS) and Fe²⁺ overload-induced lipid peroxidation, which is involved in the progression of pulmonary fibrosis. As an important coenzyme in the process of aerobic respiration, Coenzyme Q10 (CoQ10) can enhance mitochondrial function and energy supply and reduce malondialdehyde (MDA) to limit the risk of fibrosis. We aimed to clarify whether ferroptosis is involved in the process of coenzyme CoQ10-treated silicosis fibrosis.

Materials and Methods

C57BL/6J mice were divided in 3 groups (n=6 in each group). In the normal group, mice underwent sham operation; in the silicosis group, mice were tracheally instilled with SiO₂ suspension; in CoQ10 group, mice with silicosis were treated with CoQ10 solution. Histological analyses were performed to assess the lung injury level. Iron content was measured by colorimetry in lung tissue. The levels of MDA in lung tissue were characterized by immunofluorescence staining. The level of alpha smooth muscle actin (α-SMA), Collagen I, GPX4, p53 expression was analyzed by qRT-PCR and western blotting.

Results

CoQ10 significantly reduced the mRNA and protein 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 Fe²⁺ level by increasing the expression of ferroptosis regulatory core enzyme GPX4 and reducing its upstream regulator p53 in silicosis lung tissues.

Conclusion

CoQ10 alleviated silicosis fibrosis via inhibiting ferroptosis in mice. This finding is a new perspective for exploring the pathogenesis and treatment for silicosis.

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 is a lipid-soluble antioxidant that is synthesized by endogenous biosynthesis in the body. It has been shown to improve immunity, enhance antioxidant capacity, and delay aging. It is widely used in the adjuvant treatment of cardiovascular diseases in medicine (7). Ferroptosis is an iron-dependent programmed cell death pattern, distinct from apoptosis and autophagy. Ferroptosis mechanism involves the accumulation of lipid peroxidation and Fe²⁺, 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 CoQ10-treated silicosis fibrosis in mice. Therefore, our study adopts a model of silicosis fibrosis by tracheal instillation of silica suspension to investigate whether ferroptosis participates 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

Experimental animals. Twenty-four male C57BL/6 mice, aged 6-8 weeks and weighing 20±2 g, 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 specific-pathogen-free conditions in Laboratory Animal Center of Ningxia Medical University, they were randomly divided into three groups, with 6 mice in each group: (i) Normal group; (ii) Silicosis group; (iii) CoQ10 treatment group (Figure 1). The light conditions were 12 h light and 12 h darkness, temperature was at 23±1˚C, relative humidity was set at 40%-50%, and independent drinking and eating. All the experimental procedures were performed according to the guidelines for the management and use of experimental animals proposed by 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.

Figure 1.

Technical strategy of the study. The mice were randomly divided into three groups, with 6 mice in each group: (i) Normal group; (ii) Silicosis group; (iii) CoQ10 treatment group. In the normal group, mice underwent sham operation; in the silicosis group, mice were tracheally instilled with SiO₂ suspension; in CoQ10 treatment group, mice with silicosis were treated with CoQ10 solution. Lung tissue samples were histopathologically examined by hematoxylin-eosin (HE), Masson, and Sirus red. The expression levels of key fibrosis- and ferroptosis- related factors, including α-Smooth muscle actin (α-SMA), collagen I, glutathione peroxidase 4 (GPX4), and p53, were analyzed by quantitative real-time PCR (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF), and spectrophotometry.

Silica-induced silicosis mouse model. After isoflurane anesthesia in mice, tracheotomy was carried out and 0.1 ml SiO₂ (Sigma, St Louis, MO, USA) suspension (50 mg/ml) was instilled to cause silicosis fibrosis. After 48 h operation, the treatment mice were administrated CoQ10 (100 mg/kg; Solaibao Biotech Co., LTD., Beijing, PR China) by gavage for 8 weeks (twice a week). The skin was sutured, and the survival status of the mice was recorded. After 60 days, mice were sacrificed by intraperitoneal injection of 0.3 ml urethane, the right middle lobe of the lung was removed, rinsed with saline, fixed with 4% paraformaldehyde, and the remaining lung tissues were frozen at −80˚C for subsequent experiments.

Histopathological staining. Hematoxylin-eosin (HE) staining: lung sections fixed in 4% paraformaldehyde, were dehydrated by gradient alcohol, and followed by hematoxylin staining for 6 min, ethanol differentiation for 5 s, running underwater washing for 3 min, eosin staining for 5 min, running underwater washing for 5 s. After gradient alcohol treatment, xylene was used twice (3 min each time) for transparency and covered a coverslip to seal.

Masson staining: Paraffin sections of the lung were deparaffinized and fixed, stained with Weigert iron hematoxylin staining solution, differentiated with acid ethanol differentiation solution, returned to blue with Masson blue solution and washed with distilled water. Then the samples were stained with Ponceaux-fuchsine-red staining solution, washed with phosphamoridic acid solution, and stained with aniline blue staining solution. After staining, the samples were dehydrated through a graded ethanol series, cleared by xylene to make them transparent for microscopic examination, and then mounted on slides.

Sirius red staining: the lung tissue samples were dewaxed, stained with Sirius Red droplets for 40 min, washed for 2 min, then surface staining solution was removed, dehydrated, transparent, and sealed with neutral gum. The degree of pulmonary fibrosis in mice was captured under microscope (Zeiss LSM 800 Confocal Laser Scanning Microscopy, LSM 800, Carl Zeiss AG).

Detection of collagen I and alpha smooth muscle actin (α-SMA) in lung tissue samples. Collagen I and a-SMA expression in lung tissue were detected by immunofluorescence assay. Frozen lung tissue samples were placed in a cassette for 25-30 min until room temperature was restored. Samples were fixed with glacial acetone for 25-30 min and circled the area of interest with a histology pen. Then the samples were incubated with 0.3% Triton X-100 for 10-20 min, washed with PBS for 5 times (3 min each wash), closed with 5% serum for 30 min, and incubated with anti-α-SMA antibody (1:1,000, #AF1032, Affinity Biosciences, Jiangsu, PR China), anti-Collagen-І antibody (1:1,000, #ab260043, Abcam, Cambridge, UK) overnight at 4˚C. The fluorescent secondary antibody was incubated at 37˚C after rewarming. 4’, 6-diaminyl-2-phenylindole (DAPI) was nucleated for 5 min and washed with PBS about 5 times for 3 min each time. Fluorescence images were taken by laser confocal microscopy (Leica Microsystems, Wetzlar, Germany).

Determination of iron ion concentration in silicosis lung tissue samples. 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 1,200×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.

Detection of MDA level in lung tissue. The level of MDA in lung tissue was detected by immunofluorescence assay. Part of the lung tissue was cut into 8-μm frozen sections, removed from −80˚C, and placed in a cassette for 25-30 min to recover at room temperature. The samples were fixed in ice-cold acetone for 25-30 min, then were incubated with 0.3% Triton-X100 for 10-20 min and washed 5 times with PBS for 3 min each time. After blocking with 5% serum for 30 min, the samples were incubated with goat polyclonal anti-MDA-FITC antibody (1:100), overnight at 4˚C. After 5 washes with PBS (3 min each wash), nuclei were stained with 1 mg/l 4’, 6-diamidino-2-phenylindole (DAPI) for 5 min and washed with PBS (5×3 min). Fluorescence images were taken by laser confocal microscopy.

Protein expression in lung tissue. Western blotting was used to detect protein expression in lung tissue. A total of 50 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 ultra-micro 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˚C for 10 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the pressure was constant. Proteins were wet transferred to 0.22 μm polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk powder for 2 to 3 h, incubated with primary antibody, including anti-p53 (1:1,000, #ab26, Abcam), anti-GPX4 (1:1,000, #ab125066, Abcam), anti-α-SMA antibody and anti-Collagen-І antibody at 4˚C overnight. Finally, HRP-conjugated goat anti-rabbit secondary antibody (1:5,000, ZSGBBIO, Beijing, PR China) was added for 2 h at room temperature. The membranes were incubated with enhanced chemiluminescence solution to visualize the bands (Bio-Rad Laboratories, Inc. Hercules, CA, USA). Each experiment was repeated three times.

Detection of mRNA levels in lung tissue sample. Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression in lung tissue. Primer sequences are shown in Table I. The qRT-PCR reaction protocol included denaturation (95˚C for 30 s), annealing (95˚C for 5 s), and extension (60˚C for 34 s). A total of 40 cycles were set up, ΔCt=Ct(objective)-Ct (internal control).

Table I. Primer sequence used for qRT-PCR.

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 15 min, 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 20× objective lens under a light microscope (Leica Microsystems) 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 integrated optical density (IOD) value in the positive area of the picture.

Statistical analysis. Data are 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. Statistical analysis was conducted using GraphPad 8.0 software.

Ethics approval and consent to participate. All experiments were conducted in accordance with the guidelines of Ningxia Medical University Animal Ethics Committee. Prior to the experiments, permission was obtained from the Animal Ethics Committee of Ningxia Medical University.

Results

CoQ10 alleviated 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 treatment (Figure 2A-D). Histopathological staining assay showed that the normal lung tissue without silica stimulation exhibited a basically intact alveolar structure, thin alveolar wall, no obvious inflammatory cells aggregation, and no significant collagen fiber deposition in pulmonary mesenchyme. In the silicosis group, there were significantly dense and damaged lung structure, many 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 showed no significant silicosis and reduced collagen deposition, except for inflammatory cells aggregation, compared to the silicosis group. All the above results indicated that CoQ10 could alleviate lung injury in silicosis mice.

Figure 2.

Coenzyme Q10 (CoQ10) alleviated silicosis lung injury and collagen deposition in mice. Lung morphology (A). Lung tissues were examined with hematoxylin-eosin (HE) staining (B), Masson staining (C), and Sirius red staining (D). The mice were randomly divided into three groups: Normal: saline; Silicosis: silicosis non-treated group, CoQ10: CoQ10-treated group. Scale bar: 100μm.

Then, we investigated the effect to CoQ10 on the silicosis mice fibrosis by detecting α-SMA and Collagen I. qRT-PCR assay showed that CoQ10 significantly decreased the levels of α-SMA mRNA, compared to the silicosis group (0.118±0.051 vs. 0.263±0.029, p<0.001). Moreover, CoQ10 also decreased the mRNA expression level of Collagen-I compared to the silicosis group (1.245±0.874 vs. 3.400±1.062, p<0.001) (Figure 3A). In line with the gene expression results, the α-SMA protein expression levels were decreased in the CoQ10-treated group, compared to the silicosis group (0.408±0.184 vs. 1.178±0.253, p<0.001). Meanwhile, CoQ10 also decreased the protein expression level of Collagen-I, compared to the silicosis group (0.065±0.031 vs. 0.135±0.023, p<0.001) (Figure 3B, C). The data of immunohistochemistry and fluorescence staining assays also showed that CoQ10 reduced the activation of fibroblasts and weakened the development of silicosis fibrosis, which was reflected by the decreased level of α-SMA expression (Figure 3D, E).

Figure 3.

Coenzyme Q10 (CoQ10) decreased the expression of alpha smooth muscle actin (α-SMA) and collagen I (COL-1) in silicosis mice. The mRNA expression levels (A) and protein levels (B, C) of α-SMA and collagen I were examined. (B) Representative immunoblot images from three repeated experiments (B) and quantified results (C) are shown (B). Immunohistochemical staining assay to detect the content of α-SMA (D). Scale bar: 100 μm. Immuno-fluorescent staining assay to detect the levels of α-SMA protein in silicosis lung (E). Scale bar: 20 μm. Normal: saline; Silicosis: silicosis non-treated group, CoQ10: CoQ10-treated group. *p<0.05, **p<0.001.

CoQ10 reduced the level of Fe²⁺ and MDA in silicosis lung. Compared to the normal group (0.068±0.009 nmol/mg), Fe²⁺ content in the silicosis group was significantly increased (0.205±0.008 nmol/mg, p<0.01). In contrast, CoQ10 significantly decreased the content of Fe²⁺ compared to the silicosis group (0.098±0.026 vs. 0.205±0.008 nmol/mg, p<0.01) (Figure 4A). Immunofluorescence assay revealed that compared to the silicosis group, CoQ10 significantly decreased MDA level in lung tissue (33.31±4.554 vs. 12.27±2.350, p<0.01) (Figure 4B). In brief, CoQ10 was involved in the occurrence of ferroptosis via reducing the levels of lipid peroxidation and Fe²⁺ in silicosis lung tissues.

Figure 4.

Coenzyme Q10 (CoQ10) decreased the level of Fe²⁺ and malondialdehyde (MDA) in silicosis mice. Fe²⁺ content was measured in the lung tissue of the mice by colorimetry (A). MDA content was examined by immunofluorescence assay (B). Scale bar: 20μm. Normal: saline; Silicosis: silicosis non-treated group, CoQ10: CoQ10-treated group. *p<0.05, **p<0.001.

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 levels of ferroptosis core enzymes GPX4 and its upstream regulator p53. qRT-PCR assay showed that compared to the silicosis group, CoQ10 treatment significantly increased the GPX4 mRNA levels (0.233±0.116 vs. 0.777±0.216, p<0.0001). In addition, CoQ10 also decreased the mRNA levels of p53 (0.041±0.029), compared to the silicosis group (0.041±0.029 vs. 0.086±0.031, p<0.01) (Figure 5A, B). In agreement with these results, CoQ10 treatment significantly increased the GPX4 protein expression level, compared to the silicosis group (1.215±0.331 vs. 0.252±0.116, p<0.0001) and decreased the protein expression level of p53 (0.775±0.506 vs. 1.730±0.735, p<0.01) (Figure 5C-E). These results suggested that ferroptosis may be involved in the process of CoQ10 alleviating pulmonary fibrosis in silicosis mice.

Figure 5.

Coenzyme Q10 (CoQ10) reduced the expression level of ferroptosis -related proteins GPX4 and p53 in silicosis mice. mRNA expression levels of GPX4 (A) and p53 (B) were examined. Protein levels of GPX4 and p53 were investigated by Western blot assay and the results were quantified (C-E). Normal: saline; Silicosis: silicosis non-treated group, CoQ10: CoQ10-treated group. *p<0.05, **p<0.001.

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 (2). It has been reported that there are millions of dust-exposed workers in the United States, among whom one in ten suffers from silicosis (2). In India, the number of silicosis patients is as high as tens of millions, while in China pneumoconiosis patients exceed 850,000, of which silicosis accounts for more than 50% (7,9,10). So far, due to the limited effective treatment methods for silicosis, lung damage and fibrosis cannot be reversed (11). As a major public health problem, the current treatment strategies for silicosis are still mainly focused on anti-inflammatory, anti-fibrosis and other symptomatic treatments combined with lung lavage or oxygen therapy and psychological counseling, but the effects are not ideal (12). In this study, we found that CoQ10 mitigated silicosis pathology and fibrosis, including improved alveolar structure, reduced collagen deposition, and decreased α-SMA and Collagen I expression. In addition, CoQ10 inhibited lipid peroxidation and Fe²⁺ accumulation, enhanced GPX4 expression, and downregulated p53 in lung tissues. These findings suggested that ferroptosis participated in the process of CoQ10-mediated silicosis fibrosis.

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 (13). The pharmacological effects of CoQ10 mainly involve antioxidation, anti-fibrosis, scavenging free radicals, dilating blood vessels and reducing pro-inflammatory cytokines (14). 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 (15,16). CoQ10, 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 (17). CoQ10 can also reduce different inflammatory mediators (such as TNF-α, 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 (18). In this study, CoQ10 significantly reduced the expression levels of pulmonary α-SMA and Collagen I in the lung tissues of silicosis mice and inhibit the level of lipid peroxidation in the lung of silicosis mice.

Ferroptosis is involved in the regulation of liver and lung fibrosis processes (19,20). As a novel cell death mode, distincting from apoptosis, autophagy, and pyroptosis, ferroptosis is triggered by reactive oxygen species and lipid peroxidation induced by the overload of Fe²⁺. Meanwhile, the depletion of antioxidant glutathione or GPX4 is also considered an essential condition for the initiation and progression of ferroptosis (21). 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 (22), lung infection (23), chronic obstructive pulmonary disease (24) and acute lung injury (25). Moreover, ferroptosis plays an important role in pulmonary fibrosis. Gong et al. (26) 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 (27). 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 (28). Li’s et al. research results also suggested that ferroptosis exacerbates radiation-induced pulmonary fibrosis. The inhibitor of ferroptosis, Liproxstatin-1, can significantly reduce radiation-induced lung fibrosis (29).

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 (30). Our research found that CoQ10 significantly inhibited the lipid peroxidation and accumulation of Fe²⁺ in silicosis lung tissues and increased 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 CoQ10 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 (31). 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 (32). In this way, CoQ10 can regulate the mechanism of ferroptosis, reduce the toxicity of ferrous iron, and protect cells from oxidative stress.

Conclusion

CoQ10 can regulate ferroptosis during the process of silicosis fibrosis via enhancing anti-oxidation capability and regulating the expression of iron metabolism-related genes to protect cells from silicon dioxide damage. This finding is a new perspective for exploring the pathogenesis and treatment for silicosis.

Funding

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).

Conflicts of Interest

The Authors declare no conflicts of interest.

Authors’ 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.