Javascript is currently disabled in your browser. When javascript is disabled, some functions of this website will not work.

Open access for scientific and medical research

From submission to the first editing decision.

From editor acceptance to publication.

The above percentage of manuscripts have been rejected in the past 12 months.

Open access to peer-reviewed scientific and medical journals.

Dove Medical Press is a member of OAI.

Batch reprints for the pharmaceutical industry.

We provide real benefits for authors, including fast processing of papers.

Register your specific details and specific drugs of interest, and we will match the information you provide with articles in our extensive database and send you a PDF copy via email in a timely manner.

Back to Journal »Journal of Inflammation Research» Volume 14

Platelet-derived growth factor induces autophagy to regulate the biological behavior of oral mucosal fibroblasts and its mechanism

Authors: Wang Jie, Yang Li, You Jie, Wen Ding, Yang Yi, Jiang Bing

Published on July 17, 2021, the 2021 volume: 14 pages 3405-3417

DOI https://doi.org/10.2147/JIR.S313910

Single anonymous peer review

Reviewing editor: Professor Quan Ning

Wang Jie,1,2 Yang Lina,2 Jia Ling,2 Wen Dada,2 Yang Bo,2 Jiang Canhua1 1Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Central South University, Changsha, 410078; 2Department of Immunology, Xiangya Medical College, Central South University, Changsha 410078, Republic of China Tel/Fax +86-731-89753046 Email [email protected] Objective: To investigate the effect of platelet-derived growth factor (PDGF) on autophagy of oral mucosal fibroblasts, and to further clarify that PDGF-BB regulates oral mucosal fibroblasts The molecular mechanism of autophagy. Oral mucosal fibroblasts induce the biological behavior of autophagy. Methods: The tissue block method and trypsin method were used to isolate and culture primary oral mucosal fibroblasts, and indirect immunofluorescence vimentin detection was used for identification. We detected the autophagy marker Beclin-1 and fibrosis marker Col-I of primary oral mucosal fibroblasts at different time points after different concentrations of PDGF-BB stimulated fibroblasts by Western blotting, and determined the best PDGF-BB Experimental concentration and stimulation time. Then, indirect immunofluorescence, Western blotting, and quantitative real-time polymerase chain reaction (PCR) were used to detect autophagy-related proteins and fibrotic proteins before and after 3-methyladenine (3-MA) ​​by PDGF-BB The influence of expression interferes. In addition, the effects of 3-MA on the proliferation and migration of primary oral mucosal fibroblasts stimulated by PDGF-BB were tested by MTT method and scratch experiment. The effect of PDGF-BB on the interaction between Beclin-1 and phosphatidylinositol 3 kinase 3 (PI3KC3) was detected by immunoprecipitation. Results: The results showed that PDGF-BB can induce autophagy in oral mucosal fibroblasts with a certain time and dose correlation. It induces autophagy through the interaction of Beclin-1 and PI3KC3, and promotes the proliferation, migration, transformation and collagen synthesis of fibroblasts. However, 3-MA inhibits the binding of Beclin-1 and PI3KC3 and weakens the proliferation, migration, transformation and collagen synthesis activities of fibroblasts. Conclusion: In general, PDGF-BB induces autophagy through Beclin-1 pathway, thereby regulating the biological behavior of oral mucosal fibroblasts. Keywords: PDGF-BB, primary oral fibroblasts, autophagy, 3-MA, biological behavior, signaling pathway

Oral submucosal fibrosis (OSF) is an insidious chronic oral mucosal disease, which has been recognized as a precancerous lesion of the oral cavity. Its incidence is increasing year by year, and its biological behavior is highly malignant, which brings a huge physical and psychological burden to patients. It is mainly prevalent in East Asia and Southeast Asia, with a high incidence in Taiwan and Hunan1,2. Among people who chew betel nuts, the risk of OSF increases by 109 to 287 times. 3 Studies have found that the arecoline in betel nut is the main factor that induces OSF, which is mainly due to continuous chemical and mechanical stimulation that causes chronic persistent inflammation and induces cells to release inflammatory mediators. At the same time, collagen synthesis increases and degradation decreases, leading to the deposition of collagen fibers in the oral tissues, which is an important cause of fibrosis. 4,5 OSF is a process involving multiple cells and multiple molecules, but the exact pathogenesis of OSF is not yet fully understood.

Various fibrotic diseases have one thing in common at the cellular level: under the action of certain stimulating factors, fibroblasts (FBs) in a static state undergo phenotypic conversion to myofibroblasts (MFBs). Compared with FBs, MFBs have increased smooth muscle cell contractility (α-smooth muscle actin (α-SMA) expression) and can synthesize a large amount of ECM; 6,7 at the same time, ECM degradation is reduced, leading to fibrosis. Studies have found that the process of OSF is related to a variety of inflammatory cytokines, such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β). It is up-regulated in tissues, mainly distributed in the membrane or cytoplasm of FBs and vascular endothelial cells. 8,9 PDGF is a strong stimulator of the proliferation and differentiation of FBs in oral mucosal tissues. It activates PDGF-receptor tyrosine phosphorylation by binding to the corresponding receptor, promotes cell mitosis, and increases ECM synthesis and secretion. 10,11 PDGF has five subtypes: PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and PDGF-AB. Among them, PDGF-BB plays an important role in fibrotic diseases. Studies have shown that in tissue damage and inflammation, PDGF-BB can stimulate the proliferation and migration of hepatic stellate cells (HSC) 12 or renal tubular mesenchymal cells 13 and induce their conversion into MFBs, thereby synthesizing large amounts of collagen, leading to organs Structural dysfunction promotes the occurrence of fibrotic diseases. According to related literature reports, PDGF-BB can induce autophagy in vascular smooth muscle cells14, and Li et al.15 found that TGF-β induces an increase in the autophagy level of oral mucosal FBs, and inhibiting autophagy can reduce type I levels. Collagen expression. Therefore, we speculate that PDGF-BB may participate in the occurrence and development of OSF by inducing autophagy in oral mucosal FBs.

In the cells of higher vertebrates, autophagy is ubiquitous, 16 mainly through the lysosomal degradation pathway to deal with damaged, degraded, senescent and out-of-function organelles, denatured proteins, nucleic acids and other biological macromolecules. 17,18 It includes micro-autophagy, macroautophagy, and chaperone-mediated autophagy. Among them, the study of macroautophagy is the most extensive, which is what we usually call autophagy19. The formation and regulation of autophagy is a complex process involving multiple molecules. Among the many autophagy pathways, mammalian target of rapamycin (mTOR), Beclin-1 and p53 are the three most common. Under physiological conditions, cells maintain a low level of background autophagy intensity. When the body is infected, mechanically damaged or nutritionally deficient, autophagy is activated to provide essential nutrients for cell reconstruction, regeneration and repair, thereby maintaining a stable cell environment. However, the abnormal activation of autophagy is widely involved in the pathophysiological process of infection, tumor, organ fibrosis and other diseases. Studies have found that the regulation of miR-200b by DNMT3A can control cardiac FB autophagy in the process of cardiac fibrosis, and slow down the occurrence and development of the disease. When HSC is damaged in a mouse liver fibrosis model, the lack of autophagy in mice reduces fibrogenesis and matrix accumulation. Therefore, selectively reducing the autophagy activity of fibrotic cells in the liver and other tissues can be used to treat fibrotic diseases. 21 The role of autophagy in renal fibrosis is two-way. The unilateral ureteral obstruction model exhibits time-dependent induction of autophagy and tubular atrophy, tubular cell death and interstitial fibrosis, and the autophagy inhibitor 3-methyladenine (3-MA) ​​can enhance obstructive renal tubules Apoptosis and interstitial fibrosis. However, on the contrary, some studies have shown that the activation of autophagy can lead to renal fibrosis. 22,23 According to reports, in pulmonary fibrosis, ATG4B protease and autophagy protect epithelial cells from bleomycin-induced stress and apoptosis, and also play a vital role. 24 Multiple studies have shown that autophagy is related to the fibrosis of the heart, liver, kidney, lung and other organs. More and more evidences show that the mechanism of autophagy is related to the occurrence, development and outcome of fibrotic diseases. 25

In essence, PDGF-BB works by binding to PDGF receptor-β (PDGFR-β). Han et al. 8 found that in the membranes of FB, epithelium and tissue vascular endothelial cells of OSF patients, the expression of PDGFR-β was up-regulated. The interaction of PDGF and PDGFR can induce phosphorylation of PDGFR and promote the activation of phosphatidylinositol-β. 3 Kinase (PI3K). 26 PI3K has three subtypes: PI3K type 1 (PI3KC1), PI3KC2 and PI3KC3 (the homolog of Vps34). After phosphorylation, it forms a complex with Beclin-1 to promote the occurrence of autophagy. 27,28 Therefore, this study aimed to explore the effect of PDGF-BB on the autophagy of FBs in the oral mucosa, and to further clarify the molecular mechanism of its action. The interaction of Beclin-1 and PI3KC3 induces autophagy to regulate the biological behavior of oral mucosal FBs. In summary, these findings indicate that the interaction between autophagy and PDGF-BB is a key mechanism for the development of OSF, and inhibition of the autophagy pathway may provide a new way to prevent fiber formation.

The specimen is the gum tissue obtained from a healthy 22-year-old Chinese woman with periodontal disease on October 23, 2019. The patient is healthy and free of systemic diseases and periodontal inflammation. This research was conducted in accordance with the Declaration of Helsinki. This study was approved by the ethics committee of Xiangya Hospital of Central South University. Written informed consent was obtained from the participant.

Pack normal human oral mucosal tissue blocks in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) containing 15% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin. The tissue pieces were washed 3 times with phosphate buffered saline (PBS) containing double antibodies, minced, inoculated into culture flasks, and then incubated in a 37°C incubator in a humid environment containing 5% CO2 for 15-20 sky. Then it was digested with 0.25% trypsin; the digestion was stopped with DMEM containing 15% fetal bovine serum (FBS). After centrifugation and resuspension, the cells were cultured in a 37°C incubator in a humid environment containing 5% CO2. The primary oral mucosal FBS is automatically purified after two passages to obtain a uniform oral mucosal FBS. The oral mucosa FBS after two passages was used in subsequent experiments.

Use the total RNA extractor lysate to extract the total messenger RNA (mRNA) of the cells, and reverse transcribed it into complementary deoxyribonucleic acid (cDNA) according to the instructions of the cDNA kit used. The mRNA level was quantified by Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) and detected by RT-qPCR (ABI7500, USA). The PCR reaction conditions were: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 10 s, 60°C annealing/extension for 30 s, and a total of 40 cycles of denaturation to extension. The relative expression levels of LC3 mRNA, Beclin-1 mRNA, COl-1 mRNA and α-SMA mRNA were calculated by the 2-ΔΔCt method relative to the expression of GAPDH mRNA. The list of primers is shown in Table 1. Table 1 List of primers

The radioimmunoprecipitation analysis (RIPA) lysate was used to separate the protein from the cell lysate, and the protein concentration was determined by the bicinchoninic acid (BCA) kit. Then, 30 μg of protein was separated by 12% SDS-PAGE and transferred to PVDF membrane. Incubate the membrane in blocking buffer containing 5% skimmed milk for 1 hour, then incubate with the primary antibody (including GAPDH, LC3, Beclin-1 and COl-I) at 4°C overnight, and then incubate the antibody with the secondary antibody. Place in the dark at room temperature for two hours. The membrane was washed 3 times with 0.1% Tween 20/TBS ​​solution. The blot was visualized by an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL). Each blot was repeated 3 times.

The cells were seeded at 1×104 in a preselected 24-well plate with spread glass slides and incubated with 10 mM 3-MA for 2 hours, and then the corresponding conditions were added to the culture for 24 hours. Then, the cell slides were washed with pre-cooled PBS and fixed with 4% paraformaldehyde at room temperature for 30 minutes, then 0.2% Triton-100 was used to penetrate the membrane for 10 minutes at 4°C and blocked with 1% BSA in 37 Leave it at °C for one hour. Incubate the cells with primary antibodies vimentin (1:100, CST), Beclin-1 (1:200, Bioworld) and LC3 (1:500, CST) overnight at 4°C, and then with fluorescently labeled secondary antibodies Incubation (Cwbiotech, China) and Hoechst (1:500) are performed simultaneously. Analyze the images under a fluorescence microscope (Ficol, LEICA DMi8).

The MTT method is used to evaluate cell proliferation. The cells were seeded in a 96-well plate at 200 μL/well (1 × 103 cells/well). When the conditioned medium is added for 0, 24, 48, 72, and 96 h, the cells are incubated with 20 μL of 5-mg/mL MTT solution for 4 h, and added to the test well on the day of measurement. After discarding the old medium, transfer 150 μL of DMSO solution to a new 96-well plate, shake at low speed for 10 minutes, and measure the absorbance at 490 nm with a microplate reader. The experiment was repeated three times.

The scratch test is used to evaluate cell migration. The cells were evenly seeded in a six-well culture plate, and conditioned medium was added to culture for 24 hours. When the cell density reaches more than 90%, use a P200 pipette tip to scrape the cells to create a scratch. The cells were washed 3 times with PBS to remove suspended cells. The adherent cells were incubated with DMEM 0.1% FBS medium and photographed under a 10x objective lens at 0, 12, 18, and 24 hours after the scratch.

The protein interaction was evaluated by immunoprecipitation. RIPA lyses cells to obtain total protein, and BCA method determines protein concentration. The concentration of each group is 1 μg/μL. Add agarose beads for adsorption and shake at 4°C for two hours. The total protein was centrifuged at 2,500 rpm × 30 s at 4°C, and then the supernatant was left and incubated with the primary antibody (Beclin-1 = 1:200, PI3KC3 = 1:50) at 4°C overnight. Then, add the beads again and shake at 4°C for two hours. Discard the supernatant by centrifugation at 4°C, add the remaining cells to the EP tube containing the loading buffer, boil the water bath, and denature for 5 minutes.

GraphPad Prism software was used for data plotting, and all statistical analysis was performed using SPSS 18.0 (SPSS, Chicago, IL, USA). The results are expressed as mean ± standard deviation (±S). The comparison between the two groups was evaluated by the Student's t-test. The inspection level is α = 0.05, and a P value of <0.05 indicates statistical significance.

In order to obtain primary oral mucosal FBs, the oral mucosal FBs were separated by tissue block and trypsin method, and observed under an inverted microscope 15 days later. Figure 1Aa and b show the morphology of cells swimming out of the tissue mass at different magnifications. After digestion, the separated cells are bright and spherical, with clear outlines, single or clustered into clusters. Figure 1Ac and d show the growth status of FB under different magnifications after purification. The cells are uniform, long and spindle-shaped, with clear boundaries between cells (Figure 1A). The expression of vimentin in primary cultured cells was further detected by immunofluorescence analysis (IFA). As shown in Figure 1B, vimentin is expressed in the cytoplasm of almost all cells (>99%). The cells have obvious synapses and typical FB features (Figure 1B). The results showed that the primary oral mucosa FBs were successfully isolated and cultured. Figure 1 Isolation and culture of high-purity primary oral mucosa FBs. (A) Morphological observation of primary cells under an inverted microscope. (a and b) The cells swim out of the tissue mass and expand to the surrounding area. (a) ×100; (b) ×200. (c and d) Morphological observation of cell subculture. (c) ×100; (d) ×200. (B) Vimentin expression in primary cells under a fluorescence microscope. When the cell density reaches 60-80%, remove the cells and use rabbit anti-human vimentin antibody as the primary antibody; goat anti-rabbit IgG-Cy2 as the secondary antibody to detect the expression of vimentin. Blue represents the nucleus and green represents vimentin. The results showed that vimentin was positively expressed in cells, with an expression rate of over 99%.

Figure 1 Isolation and culture of high-purity primary oral mucosa FBs. (A) Morphological observation of primary cells under an inverted microscope. (a and b) The cells swim out of the tissue mass and expand to the surrounding area. (a) ×100; (b) ×200. (c and d) Morphological observation of cell subculture. (c) ×100; (d) ×200. (B) Vimentin expression in primary cells under a fluorescence microscope. When the cell density reaches 60-80%, remove the cells and use rabbit anti-human vimentin antibody as the primary antibody; goat anti-rabbit IgG-Cy2 as the secondary antibody to detect the expression of vimentin. Blue represents the nucleus and green represents vimentin. The results showed that vimentin was positively expressed in cells, with an expression rate of over 99%.

In order to screen out the best experimental concentration of 3-MA, the concentration was divided into 7 groups: 0, 2.5, 5, 7.5, 10, 15, 20 and 25 mmol/L. After stimulating the oral mucosal FBs with the above 3-MA concentration for 24 h, the cell proliferation was detected by the MTT method, and the OD490nm value was detected by the microplate reader. The results showed that the half inhibitory concentration of 3-MA was about 10 mmol/L (Figure 2), which was used as the subsequent experimental concentration. Figure 2 The effect of different concentrations of 3-MA on the content of FBs in the oral mucosa.

Figure 2 The effect of different concentrations of 3-MA on the content of FBs in the oral mucosa.

Different concentrations of PDGF-BB (0, 10, 20, 30, 40, 60 ng/mL) stimulated oral mucosa FBs for 24 h. After extracting total cell protein, the expression of fibrosis-related protein Col-I and autophagy were analyzed by Western blot Detection-related protein Beclin-1. The results showed that the expression of COl-I increased with the concentration, first increased (40 ng/mL reached the peak) and then decreased. The expression of Beclin-1 also changed with concentration, and was also highest at 40 ng/mL (Figure 3). Figure 3 Western blotting to detect the protein expression of Col-I and Beclin-1 in oral mucosa FBs stimulated by different PDGF-BB concentrations. (A) Different concentrations of PDGF-BB (0, 10, 30, 40, 60 ng/mL) were used to stimulate FBs for 24 hours. (B) The effect of different PDGF-BB concentrations on the expression of Col-I and Beclin-1 proteins in FB. Comparing Col-I in the 40-ng/mL group with Col-I in the 0-, 10-, and 30-ng/mL group, ***P<0.001; when Beclin-1 and 0- in the 40-ng/mL group When comparing Beclin-1 in the, 10-, 30- and 60-ng/mL groups, #P <0.05.

Figure 3 Western blotting to detect the protein expression of Col-I and Beclin-1 in oral mucosa FBs stimulated by different PDGF-BB concentrations. (A) Different concentrations of PDGF-BB (0, 10, 30, 40, 60 ng/mL) were used to stimulate FBs for 24 hours. (B) The effect of different PDGF-BB concentrations on the expression of Col-I and Beclin-1 proteins in FB. Comparing Col-I in the 40-ng/mL group with Col-I in the 0-, 10-, and 30-ng/mL group, ***P<0.001; when Beclin-1 and 0- in the 40-ng/mL group When comparing Beclin-1 in the, 10-, 30- and 60-ng/mL groups, #P <0.05.

PDGF-BB (40 ng/mL) was used to stimulate the primary oral mucosa FB at different times (6, 12, 24, 48, 72 hours); a negative control group without stimulation was also created. Western blotting was used to detect the expression of Col-I and Beclin-1 in cells at different time points. Compared with the control group, the fibrosis-related protein Col-I increased with the increase of stimulation time. In addition, as the stimulation time increased, the autophagy-related protein Beclin-1 first increased and then decreased, reaching a peak at 24 hours (Figure 4). These findings indicate that the longer PDGF-BB stimulates primary oral mucosa FB, the higher the incidence of fibrosis. In addition, PDGF-BB can induce the occurrence of autophagy, which is time-dependent and increases first and then decreases. Figure 4 Western blotting detected the expression of Col-I and Beclin-1 protein in oral mucosa FBs after different PDGF-BB stimulation time. *** indicates the comparison of Col-I between the 24-h group and the 6-12-h group; *** P <0.001. * Indicates the comparison between Col-I in the 48-72-h group and Col-I in the 24-h group; * P <0.05. # Indicates the comparison between Beclin-1 in the 24 hour group and Beclin-1 in the rest time group; #P <0.05.

Figure 4 Western blotting detected the expression of Col-I and Beclin-1 protein in oral mucosa FBs after different PDGF-BB stimulation time. *** indicates the comparison of Col-I between the 24-h group and the 6-12-h group; *** P <0.001. * Indicates the comparison between Col-I in the 48-72-h group and Col-I in the 24-h group; * P <0.05. # Indicates the comparison between Beclin-1 in the 24 hour group and Beclin-1 in the rest time group; #P <0.05.

Regarding the effect of PDGF-BB on primary oral mucosal FBs, RT-qPCR showed that the expressions of Col-I, Beclin-1, and LC3 in PDGF-BB group were significantly higher than those in control group, and the difference was statistically significant (P <0.05) (Figure 5A). The expression levels of Col-I, Beclin-1 and LC3 were also detected by Western blot. Compared with the control group, the expression of these proteins in the PDGF-BB group increased significantly, and the difference was statistically significant (P <0.05) (Figure 5B). Indirect immunofluorescence results showed that the control group had no obvious red fluorescence; however, the PDGF-BB group showed red fluorescence. The red fluorescence intensity of the PDGF-BB group was significantly higher than that of the control group (Figure 5C). Overall, these findings indicate that PDGF-BB can promote the autophagy of primary oral mucosal FBs, and at the same time stimulate primary oral mucosal FB fibrosis. Figure 5 PDGF-BB promotes fibrosis and activates autophagy of FBs in the oral mucosa. (A) Detect the expression of Col-I, Beclin-1 and LC3 at the mRNA level by RT-qPCR. Compared with the control group, the data of the PDGF-BB group is expressed as the mean ± SD of at least three independent experiments. P <0.05; (*** P <0.01). (B) Western blot detection of autophagy and fibrosis in primary oral mucosa FB. After PDGF-BB stimulation for 24 h, the expressions of fibrosis-related protein Col-I and autophagy-related proteins Beclin-1 and LC3 in primary oral mucosal FBs were detected. PDGF-BB group vs. control group, P <0.05. (* P <0.05, *** P <0.01). (C) Indirect immunofluorescence detection of autophagy (×200). Indirect immunofluorescence was used to detect the expression and localization of autophagy-related proteins LC3 and Beclin-1 in primary oral mucosa FBs after PDGF-BB stimulation for 24 h. Hoechst 33342 is used to stain cell nuclei; rabbit anti-LC3 and rabbit anti-Beclin-1 are the primary antibodies, and Cy3-goat anti-rabbit IgG is the secondary antibody. LC3 and Beclin-1 show red fluorescence.

Figure 5 PDGF-BB promotes fibrosis and activates autophagy of FBs in the oral mucosa. (A) Detect the expression of Col-I, Beclin-1 and LC3 at the mRNA level by RT-qPCR. Compared with the control group, the data of the PDGF-BB group is expressed as the mean ± SD of at least three independent experiments. P <0.05; (*** P <0.01). (B) Western blot detection of autophagy and fibrosis in primary oral mucosa FB. After PDGF-BB stimulation for 24 h, the expressions of fibrosis-related protein Col-I and autophagy-related proteins Beclin-1 and LC3 in primary oral mucosal FBs were detected. PDGF-BB group vs. control group, P <0.05. (* P <0.05, *** P <0.01). (C) Indirect immunofluorescence detection of autophagy (×200). Indirect immunofluorescence was used to detect the expression and localization of autophagy-related proteins LC3 and Beclin-1 in primary oral mucosa FBs after PDGF-BB stimulation for 24 h. Hoechst 33342 is used to stain cell nuclei; rabbit anti-LC3 and rabbit anti-Beclin-1 are the primary antibodies, and Cy3-goat anti-rabbit IgG is the secondary antibody. LC3 and Beclin-1 show red fluorescence.

After autophagy inhibitor 3-MA and primary oral mucosal FBs were pre-cultured for 2 h, they were cultured in PDGF-BB (40 ng/mL) conditioned medium for 24 h. RT-qPCR detects the expression of Col-I, α-SMA, Beclin-1 and LC3 at the mRNA level. The results showed that the expression level of PDGF-BB group was significantly higher than that of control group, 3-MA group and 3-MA/PDGF-BB group; the difference was statistically significant (P <0.05) (Figure 6A). Figure 6 The effect of autophagy inhibitor 3-MA on PDGF-BB-induced oral mucosal FB fibrosis and autophagy. (A) Detect the expression of Col-I, α-SMA, Beclin-1 and LC3 at the mRNA level by RT-qPCR. The effects of PDGF-BB and 3-MA on autophagy and fibrosis of primary oral FBs. Data are expressed as the mean ± SD of at least three independent experiments. * Indicates compared with the control group, P <0.05; # indicates compared with the PDGF-BB group, P <0.05. (B) Western blot was used to detect autophagy and fibrosis in primary oral mucosa FB; the effects of PDGF-BB and 3-MA on autophagy and fibrosis of FBs. * Indicates compared with the control group, P <0.05; # indicates compared with the PDGF-BB group, P <0.05. (C) Indirect immunofluorescence was used to detect the effect of 3-MA on PDGF-BB-stimulated autophagy and autophagy-related proteins LC3 and Beclin-1 expressed by FB. Hoechst 33342 is used to stain cell nuclei; rabbit anti-LC3 and rabbit anti-Beclin-1 are the primary antibodies, and Cy3-goat anti-rabbit IgG is the secondary antibody. LC3 and Beclin-1 show red fluorescence.

Figure 6 The effect of autophagy inhibitor 3-MA on PDGF-BB-induced oral mucosal FB fibrosis and autophagy. (A) Detect the expression of Col-I, α-SMA, Beclin-1 and LC3 at the mRNA level by RT-qPCR. The effects of PDGF-BB and 3-MA on autophagy and fibrosis of primary oral FBs. Data are expressed as the mean ± SD of at least three independent experiments. * Indicates compared with the control group, P <0.05; # indicates compared with the PDGF-BB group, P <0.05. (B) Western blot was used to detect autophagy and fibrosis in primary oral mucosa FB; the effects of PDGF-BB and 3-MA on autophagy and fibrosis of FBs. * Indicates compared with the control group, P <0.05; # indicates compared with the PDGF-BB group, P <0.05. (C) Indirect immunofluorescence was used to detect the effect of 3-MA on PDGF-BB-stimulated autophagy and autophagy-related proteins LC3 and Beclin-1 expressed by FB. Hoechst 33342 is used to stain cell nuclei; rabbit anti-LC3 and rabbit anti-Beclin-1 are the primary antibodies, and Cy3-goat anti-rabbit IgG is the secondary antibody. LC3 and Beclin-1 show red fluorescence.

After extracting the total protein of each group for Western blot analysis, the expression of Col-I, Beclin-1, and LC3 in the PDGF-BB group were significantly higher than those in the control group, and the difference was statistically significant (P <0.05). In addition, the protein expressions of Col-I, Beclin-1, and LC3 in the 3-MA and 3-MA/PDGF-BB groups were lower than those in the PDGF-BB group, and the difference was statistically significant (P <0.05) (Figure 6B).

The IFA results showed that for LC3 and Beclin-1, the red fluorescence of the PDGF-BB group was the strongest, the 3-MA group had almost no fluorescence, and the fluorescence of the 3-MA/PDGF-BB group was stronger than that of the control and 3-MA groups (Figure 6C ). These results further indicate that PDGF-BB can induce autophagy in oral mucosa FB, and 3-MA can inhibit this autophagy. Therefore, PDGF-BB may regulate the biological behavior of oral mucosal FBs by inducing autophagy.

The MTT method is used to examine the effect of 3-MA on PDGF-BB's stimulation of primary oral mucosa FB proliferation. The results showed that the OD490nm value of the PDGF-BB group was higher than that of the control group and the 3-MA group at 48 h (P <0.05), and the difference between the groups increased with time. The difference is very significant (P <0.01). At 72 and 96 hours, the OD490nm value of the 3-MA/PDGF-BB group was significantly lower than that of the PDGF-BB group (Figure 7A). These data indicate that 3-MA can reduce the ability of PDGF-BB to promote the proliferation of FBs in the oral mucosa. Figure 7 3-MA attenuates the effect of PDGF-BB on the proliferation and migration of FBs in the oral mucosa. (A) MTT method detects the effect of PDGF-BB on the proliferation of FBs at different time points. * Indicates P <0.05 compared with the control group; # indicates P <0.05 compared with the PDGF-BB group. (B) Detecting the migration of FBs in the oral mucosa by cell scratch test. Real-time photographs were taken to detect the relative migration width of each group of cells (×50), and the average relative migration rate of cells at 6, 12, and 24 h was compared. Data are expressed as the mean ± SD of at least three independent experiments. * P <0.05 compared with the control group; #P <0.05 compared with the PDGF-BB group.

Figure 7 3-MA attenuates the effect of PDGF-BB on the proliferation and migration of FBs in the oral mucosa. (A) MTT method detects the effect of PDGF-BB on the proliferation of FBs at different time points. * Indicates P <0.05 compared with the control group; # indicates P <0.05 compared with the PDGF-BB group. (B) Detecting the migration of FBs in the oral mucosa by cell scratch test. Real-time photographs were taken to detect the relative migration width of each group of cells (×50), and the average relative migration rate of cells at 6, 12, and 24 h was compared. Data are expressed as the mean ± SD of at least three independent experiments. * P <0.05 compared with the control group; #P <0.05 compared with the PDGF-BB group.

The cell scratch test is a common method to detect the ability of cells to migrate. The stronger the cell migration ability, the faster the growth of peripheral cells to the central scratch area, and the smaller the average scratch width. In this study, as the incubation time increased, the scratch width of the PDGF-BB group gradually decreased. In addition, the scratch width of the 3-MA and 3-MA/PDGF-BB groups also gradually decreased with the increase of incubation time, but was significantly smaller than that of the PDGF-BB group (Figure 7B). The average relative migration rate of cells in the PDGF-BB group was significantly higher than that of the control group, 3-MA and 3-MA/PDGF-BB groups (P <0.05 for each group) (Figure 7B). These findings confirm that 3-MA can attenuate the ability of PDGF-BB to promote the migration of FBs in the oral mucosa.

The cell lysates of each group were extracted and analyzed by mutual immunoprecipitation with Beclin-1 and PI3KC3 antibodies. The results showed that there was no protein band in the negative control mixed with IgG, indicating that there was no non-specific protein interference. But Beclin-1 protein was detected in PI3KC3 precipitated protein, indicating that there is an interaction between the two. The interaction between Beclin-1 and PI3KC3 in the PDGF-BB group was significantly stronger than that in the control, 3-MA and 3-MA/PDGF-BB groups (Figure 8). The combination of the two decreases with the addition of 3-MA. These results indicate that PDGF-BB can induce the interaction between Beclin-1 and PI3KC3, promote oral mucosal FB autophagy, and 3-MA can reduce the intensity of autophagy by inhibiting protein binding. Figure 8 Co-immunoprecipitation to detect the expression level and interaction of Beclin-1 and PI3KC3 in different groups. The figure shows the result of detecting PI3KC3 with anti-PI3KC3 antibody as the primary antibody; inorganic phosphorus precipitates Beclin-1 protein. Here, 1, 2, 3, and 4 respectively represent the PDGF-BB group, the control group, the PDGF-BB+3-MA group, and the 3-MA group.

Figure 8 Co-immunoprecipitation to detect the expression level and interaction of Beclin-1 and PI3KC3 in different groups. The figure shows the result of detecting PI3KC3 with anti-PI3KC3 antibody as the primary antibody; inorganic phosphorus precipitates Beclin-1 protein. Here, 1, 2, 3, and 4 respectively represent the PDGF-BB group, the control group, the PDGF-BB+3-MA group, and the 3-MA group.

Platelet-derived growth factor is a cytokine that promotes cell activation, division and proliferation, while PDGF-BB is a common pro-fibrotic factor. Its expression increases in fibrotic diseases and is closely related to the formation of OSF. 8,9 Various physical, chemical, genetic, immune defense and nutritional factors affect the occurrence and development of OSF. However, the body is immune to fibrosis caused by PDGF-BB through a series of mechanisms, in which autophagy plays an important role.

In this study, we provided strong evidence that PDGF-BB can induce autophagy in primary oral mucosa FB. First, we separated and purified the primary oral mucosa FB by the tissue block method combined with trypsin digestion. To identify primary oral mucosa FB, we use vimentin antibodies. Vimentin is mainly expressed in mesenchymal cells. 29 Oral mucosal FB can express vimentin in the cytoplasm, but epithelial cells cannot. Therefore, the results proved that high-purity primary oral mucosa FB was obtained.

In order to study the effect of PDGF-BB on the autophagy and collagen synthesis and transformation of FBs in oral mucosa, we used RT-qPCR, Western blot and IFA experiments to verify that PDGF-BB stimulated cells to trigger a significant autophagy response. After PDGF-BB stimulation for 24 hours, the expression of Beclin-1 and LC3 was significantly increased by RT-qPCR and Western blotting, accompanied by the increase of Col-I and α-SMA expression. The expression of PDGF-BB histone protein was significantly stronger than that of the control group (P <0.05). In addition, IFA significantly enhanced the fluorescence of Beclin-1 and LC3 in the PDGF-BB group. Increased expression of autophagy-related markers also indicates increased levels of autophagy. Therefore, these results indicate that PDGF-BB promotes the occurrence of primary oral mucosal FB fibrosis and enhances the autophagy level of FBs.

In order to select the best experimental PDGF-BB concentration and the best detection time point, we reviewed the literature and found that PDGF-BB can induce autophagy in human umbilical vein endothelial cells, and its intensity is significantly higher than that of human umbilical vein endothelial cells stimulated by PDGF-BB. The middle stage of stimulation (24 h) and the early stage (6 h). 30 The purpose of this experiment is to study the difference in the expression of Beclin-1 and Col-I in primary oral mucosal FBs stimulated by different concentrations of PDGF-BB. Second, western blotting was performed. The expression of Col-I and Beclin-1 in the 40 ng/mL group increased significantly. The expression of Col-I increases over time, while Beclin-1 first increases and then decreases, and has a certain time-dose correlation. Therefore, we used 40 ng/mL PDGF-BB to stimulate oral mucosal FBs for 24 hours. The results show that in the early stage of PDGF-BB stimulation, FBs have increased nutrient requirements, so PDGF-BB induces autophagy to maintain cell survival. The level of autophagy reaches its peak at 24 h; therefore, related proteins are degraded and the intensity of autophagy gradually weakens. After 24 h, the cells gradually adapted to the environment, and autophagy was further weakened. It is reported in the literature that autophagy is related to the pathophysiological process of many diseases, such as cancer, metabolic and neurodegenerative diseases, as well as cardiovascular and pulmonary diseases. 31,32 Therefore, it is important to study the role of autophagy in OSF. Overexpression of the autophagy marker LC3 has been observed in tissue samples from OSF patients. Many studies have shown that pro-fibrotic cytokines (IL-1, IL-6, TGF-β, PDGF, bFGF and IGF) promote collagen synthesis. 33 According to relevant literature reports, TGF-β stimulation of primary oral mucosa FBs can induce the overexpression of Beclin-1 and LC3. 15 Our research shows that PDGF-BB can induce autophagy of FBs in the oral mucosa and promote the synthesis of Col-I. Therefore, the increase of Beclin-1 and LC3 mediated by PDGF-BB may be a potential molecular mechanism that promotes the development of fibrosis.

According to relevant literature reports, pathologically activated autophagy is related to the release of collagen in various fibrotic diseases 34-36 and OSF. 37 Therefore, we further studied the relationship between OSF and autophagy. After 3-MA intervention, the results of IFA showed that the level of autophagy decreased, while the results of RT-qPCR and Western blotting showed that the expression of Col-I and α-SMA decreased significantly. When α-SMA increases, it means that FBs have been converted into MFBs, which can greatly enhance collagen synthesis, participate in wound reconstruction, and aggravate organ fibrosis. These results indicate that 3-MA can inhibit PDGF-BB-induced autophagy of primary oral mucosal FBs, reduce the ability of PDGF-BB-stimulated oral mucosal FBs to synthesize collagen, and slow down the occurrence of fibrosis. We explored the effect of 3-MA on PDGF-BB-induced oral mucosal FB proliferation and migration, and found that inhibition of autophagy attenuated these processes. The results show that PDGF-BB promotes the proliferation and migration of primary oral mucosal FBs by inducing autophagy. The key role of autophagy in cell survival was confirmed in the study of Atg knockout mice. Mice lacking Atg3, Atg5, Atg7, Atg9, or Atg16L1 failed to induce autophagy due to starvation after the interrupted placental nutrient supply, and died on the day of birth. 38,39 In short, autophagy is a complex process, and its mechanism remains to be elucidated. Our current research shows that inhibiting autophagy can reduce the expression of Col-I and weaken the proliferation and migration of PDGF-BB on oral mucosal FBs. Therefore, autophagy may mediate a mechanism of fibrosis.

The activation of autophagy pathway and its role in promoting fibrosis indicate that autophagy may be a potential target of new anti-fibrosis methods. Beclin-1 plays an important role in the regulation of autophagy and is a BH3-only protein. In addition, PI3K subtype PI3KC3 can phosphorylate the third site protein of phosphatidylinositol in eukaryotes, thereby forming a complex with Beclin-1 (Beclin-1–PI3KC3) to promote the occurrence of autophagy. 27,28 Intracellular environmental disorders, neurodegenerative diseases, aging, and tumors are related to the loss and abnormality of the structure and function of the Beclin-1–PI3KC3 complex. 40 Our previous study found that PDGF-BB can activate the PI3K pathway and its phosphorylation, indicating that this PI3K subtype may be combined with Beclin-1 to induce autophagy. Therefore, this study tested the interaction between Beclin-1 and PI3KC3 in each group through co-immunoprecipitation (Co-ip) experiments, and found that PDGF-BB can induce autophagy through the interaction between them. Co-ip results showed that PDGF-BB activates PI3KC3, and PI3KC3 forms a complex with Beclin-1, which promotes autophagy to regulate the proliferation, migration, transformation and collagen synthesis of oral mucosal FBs. In summary, these findings indicate that the interaction between autophagy and PDGF-BB is a key mechanism for the occurrence and development of OSF, and inhibition of the autophagy pathway may provide a new method for preventing fibrosis.

In this study, we verified in vitro that PDGF-BB induces autophagy through the interaction of Beclin-1 and PI3KC3, thereby regulating the biological behavior of oral mucosal FBs. However, autophagy is a complex process, and the activation of different pathways will lead to different biological effects. This study lacks in vivo animal experiments to further clarify the pathogenesis of PDGF-BB/PiskC3/Beclin-1 axis in OSF. Therefore, our research results should be explained carefully. In order to clearly understand the mechanism of autophagy in fibrotic diseases, it is necessary to conduct a more comprehensive study of the signaling pathways of this process.

We would like to thank all the staff who implemented the intervention and evaluation part of this research for their hard work and dedication.

National Natural Science Foundation of China, No. 81271154. Student Innovation Project of Central South University, S2020105330686. Natural Science Foundation of Changsha City: kq2014140.

The authors declare that they have no competing interests.

1. Shih YH, Wang TH, Shieh TM, etc. Oral submucosal fibrosis: a review of pathogenesis. diagnosis. 2019;20(12):2940.

2. Tang Jianguo, Jian Xiaofeng, Gao Meilin, etc. Epidemiological survey of oral submucosal fibrosis in Xiangtan City, Hunan Province. Community Dent Oral Epidemiology. 1997;25(2):177-180. doi:10.1111/j.1600-0528.1997.tb00918.x

3. Jian Xia, Zheng Li. Research progress of oral submucosal fibrosis. Chinese Journal of Stomatology. 2011;4(2):65-68.

4. Anand R, Dhingra C, Prasad S, etc. Betel nut chewing and its harmful effects on the oral cavity. J Cancer Research Center. 2014;10(3):499–505. doi:10.4103/0973-1482.137958

5. Gao Yingjie, Peng Haihui, Yin Xiaomin, etc. Epidemiological survey of betel nut chewing among primary and middle school students in Loudi City, Hunan Province[J]. Chinese Journal of Stomatology. 2009;44(11):686–689. [Chinese].

6. Harris AK. Fibroblasts and myofibroblasts. Method enzyme. 1988; 163: 623-642.

7. Sun YB, Qu X, Caruana G, et al. The origin of renal fibroblasts/myofibroblasts and the signals that trigger fibrosis. Differentiation. 2016;92(3):102-107. doi:10.1016/j.diff.2016.05.008

8. Han Wennan, Peng Jingying, Liu Shunfeng. Expression and distribution of platelet-derived growth factor-BB in oral submucosal fibrosis. Chinese Journal of Pathophysiology. 2002;112(2-3):112-130.

9. Yin-Fang W, Jie-Ying P, Wei-Nong H. The relationship between the expression of oral mucosal platelet-derived growth factor BB receptor and oral submucosal fibrosis. J Clin Stomatol. 2004; 1:48-51.

10. Peppel K, Zhang LS, Orman ES, etc. TNF and PDGF activate vascular smooth muscle cells: overlapping and complementary signal transduction mechanisms. Cardiovascular Research 2005;65(3):674–682. doi:10.1016/j.cardiores.2004.10.031

11. Wu JH, Goswami R, Kim LK, etc. Platelet-derived growth factor receptor-β phosphorylates and activates g protein-coupled receptor kinase-2. J Biochemistry. 2005;280(35):31027–31035. doi:10.1074/jbc.M501473200

12. Si HF, Lv X, Guo A, et al. The inhibitory effect of leflunomide on the proliferation of rat hepatic stellate cells involves the activation of three mitogen-activated protein kinases triggered by PDGF-BB. Cytokines. 2008;42(1):24-31. doi:10.1016/j.cyto.2008.01.017

13. Roeyen CRCV, Ostendorf T, Floege J. The platelet-derived growth factor system in kidney disease: the emerging role of endogenous inhibitors. European Journal of Cell Biology. 2012;91(6–7):542–551. doi:10.1016/j.ejcb.2011.07.003

14. Salabei J, Cummins T, Singh M, etc. PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress. Biochem J. 2013;451(3):375-388. doi:10.1042/BJ20121344

15. Li Jie, Zhao Tao, Zhang Ping, etc. Autophagy mediates oral submucosal fibrosis. Exp Ther Med. 2016;11(5):1859–1864. doi:10.3892/etm.2016.3145

16. Kourtis N, Tavernarakis N. Autophagy and cell death in model organisms. Cell death is different. 2009;16(1):21-30. doi:10.1038/cdd.2008.120

17. Hamaï A, Botti J, Codogno P. Autophagy and inflammation. 2016.

18. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. nature. 2011;469(7330):323-335. doi:10.1038/nature09782

19. Levine B, Kroemer G. Autophagy in disease pathogenesis. cell. 2008;132(1):27–42. doi:10.1016/j.cell.2007.12.018

20. Zhao Xiaodong, Qin RH, Yang Jie, etc. DNMT3A controls miR-200b in cardiac fibroblast autophagy and cardiac fibrosis. Inflammation Research 2018; 67(8): 681-690. doi:10.1007/s00011-018-1159-2

21. Hernandez-Gea V, Ghiassi-ne Z, Rozenfeld R, etc. Autophagy releases lipids and promotes fibrosis by activating hepatic stellate cells in mouse and human tissues. Gastroenterology. 2012;142(4):938-946. doi:10.1053/j.gastro.2011.12.044

22. Kimura T, Isaka Y, Yoshimori T. Autophagy and kidney inflammation. Autophagy. 2017; 13(6): 997-1003. doi:10.1080/15548627.2017.1309485

23. Livngston MJ, Ding HF, Huang S, etc. During unilateral ureteral obstruction, the continuous activation of autophagy in renal tubular cells promotes renal interstitial fibrosis. Autophagy. 2016;12(6):976-998. doi:10.1080/15548627.2016.1166317

24. Cabrera S, Maciel M, Herrera I, etc. The important role of ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis. Autophagy. 2015; 11(4): 670–684. doi:10.1080/15548627.2015.1034409

25. Ghavami S, Yeganeh B, Zeki AA, etc. Autophagy and unfolded protein response promote the pro-fibrotic effect of TGF-β1 in human lung fibroblasts. Am J Physiol Molecular Physiology of Lung Cells. 2018; 314(3): L493–L504. doi:10.1152/ajplung.00372.2017

26. Heldin CH, Ostman A, Rönnstrand L. Signal transduction occurs through platelet-derived growth factor receptors. Journal of Biochim Biophys. 1998; 1378(1): F79–F113. doi:10.1016/s0304-419x(98)00015-8

27. Kong Qiang, Xu LH, Xu Wei, etc. HMGB1 translocation is involved in the transformation of autophagy complexes and promotes leukemia chemoresistance. International Journal of Oncology. 2015;47(1):161–170. doi:10.3892/ijo.2015.2985

28. Wang B, Yang Hong, Fan Y, et al. 3-Methyladenine improves liver fibrosis through autophagy regulated by NF-κB signaling pathway on hepatic stellate cells. Tumor target. 2017; 8(64): 107603–107611. doi:10.18632/oncotarget.22539

29. Chang YC, Tsai CH, Tai KW, et al. The increased vimentin expression of arecoline in oral fibroblasts in vitro may be the pathogenesis of oral submucosal fibrosis. Oral tumors. 2002;38(5):425–430. doi:10.1016/S1368-8375(01)00083-5

30. Dai Zhi, Zhu Bin, Yu Hua, et al. The role of arecoline-induced autophagy in the angiogenesis of oral submucosal fibrosis. Arch oral biologics. 2019; 102: 7-15. doi:10.1016/j.archoralbio.2019.03.021

31. Wu YC, Wang WT, Lee SS, etc. Glucagon-like peptide-1 receptor agonists attenuate autophagy through Drp1/NOX and Atg-5/Atg-7/Beclin-1/LC3β pathways to improve pulmonary hypertension. International J Molecular Science. 2019;20(14):3435–3450.

32. Giampieri F, Afrin S, Forbes-Hernandez TY, etc. Autophagy in human health and disease: new therapeutic opportunities. Anti-oxidant redox signal. 2019;30(4):577–634. doi:10.1089/ars.2017.7234

33. Pant I, Rao SG, Kondaiah P. The role of betel nut-induced JNK/ATF2/Jun axis in TGF-β pathway activation in precancerous oral submucosal fibrosis. Sci Rep. 2016;6(1):1-15. doi:10.1038/srep34314

34. He Ying, Jin Li, Wang Jie, etc. The mechanism of fibrosis in acute liver failure. Liver International 2015;35(7):1877–1885. doi:10.1111/liv.12731

35. De Stefano D, Villella VR, Esposito S, etc. Restore CFTR function in cystic fibrosis patients with F508del-CFTR mutation. Autophagy. 2014;10(11):2053-2074. doi:10.4161/15548627.2014.973737

36. He L, Livingston MJ, Dong Z. Autophagy in acute kidney injury and repair. Clinical practice of renal function. 2014;127(1–4):56–60. doi:10.1159/000363677

37. Junkins RD, McCormick C, Lin TJ. The emerging potential of autophagy-based therapies in the treatment of cystic fibrosis lung infections. Autophagy. 2014; 10(3): 538–547. doi:10.4161/auto.27750

38. Hara T, Nakamura K, Matsui M, etc. Inhibition of basal autophagy in nerve cells can lead to neurodegenerative diseases in mice. nature. 2006;441(7095):885-889. doi:10.1038/nature04724

39. Pua HH, Dzhagalov I, Chuck M, etc. The key role of autophagy gene Atg5 in T cell survival and proliferation. J Exp Med. 2007;204(1):25-31. doi:10.1084/jem.20061303

40. Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med. 2013; 368(7): 651–662. doi:10.1056/NEJMra1205406

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and include the Creative Commons Attribution-Non-commercial (unported, v3.0) license. By accessing the work, you hereby accept the terms. The use of the work for non-commercial purposes is permitted without any further permission from Dove Medical Press Limited, provided that the work has an appropriate attribution. For permission to use this work for commercial purposes, please refer to paragraphs 4.2 and 5 of our terms.

Contact Us• Privacy Policy• Associations and Partners• Testimonials• Terms and Conditions• Recommend this site• Top

Contact Us• Privacy Policy

© Copyright 2021 • Dove Press Ltd • Software development of maffey.com • Web design of Adhesion

The views expressed in all articles published here are those of specific authors and do not necessarily reflect the views of Dove Medical Press Ltd or any of its employees.

Dove Medical Press is part of Taylor & Francis Group, the academic publishing department of Informa PLC. Copyright 2017 Informa PLC. all rights reserved. This website is owned and operated by Informa PLC ("Informa"), and its registered office address is 5 Howick Place, London SW1P 1WG. Registered in England and Wales. Number 3099067. UK VAT group: GB 365 4626 36

In order to provide our website visitors and registered users with services that suit their personal preferences, we use cookies to analyze visitor traffic and personalize content. You can understand our use of cookies by reading our privacy policy. We also retain data about visitors and registered users for internal purposes and to share information with our business partners. By reading our privacy policy, you can understand which of your data we retain, how to process it, with whom to share it, and your right to delete data.

If you agree to our use of cookies and the content of our privacy policy, please click "Accept".