Application of N-acetylcysteine in preparation of medicine for treating temporomandibular joint disorder

Abstract

The invention discloses application of NAC in preparation of a medicine for treating temporomandibular joint disorder, and particularly discloses application of the medicine in effectively improving TMD joint tissue oxidation resistance, inhibiting inflammation level, promoting extracellular matrix secretion and inhibiting iron (ferrous) ions and lipid peroxides so as to improve cartilage injury, relieve pain in a condylar process region and improve osteoarthritis. As a clinical common medicine, NAC is low in cost, high in availability, excellent in cell and tissue safety and high in practicability. The medicine can be safely applied to TMD treatment by means of condyle local injection or condyle region skin application (microneedle) and the like, has important significance in clinical medication of TMD, and has good medication value and application prospect.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically... N- The use of acetylcysteine ​​in the preparation of drugs for the treatment of temporomandibular joint disorders. Background Technology

[0002] Temporomandibular joint disorder (TMD) is a broad category of complex diseases affecting the temporomandibular joint and masticatory muscles. Its clinical manifestations are diverse, including joint pain, clicking sounds, and limited mouth opening. Pathological changes in TMD include cartilage degeneration, bone remodeling, disc displacement, synovitis, and masticatory muscle dysfunction. Unlike the knee joint, which bears the main weight, the temporomandibular joint is the only bilaterally linked joint in the human body. Its function is complex, involving both gliding and rotational movements, and the joint disc tissue exists in a unique hypoxic microenvironment. This unique biomechanical environment and metabolic characteristics mean that the pathogenesis and treatment strategies for TMD cannot be simply modeled after other joint diseases. Currently, there are no drugs that can cure TMD clinically; treatment mainly involves symptomatic relief or surgery, but the effects are limited and certain side effects exist.

[0003] Mandibular joint chondrocytes (MCCs) are one of the main cell types in articular cartilage, and their functional state directly affects the integrity of the cartilage. During the pathogenesis of temporomandibular joint disorder (TMD), chondrocytes and articular disc cells are stimulated by various pathological factors, including abnormal mechanical stress, inflammatory cytokine stimulation, and oxidative stress, leading to cell dysfunction, increased apoptosis, and accelerated extracellular matrix degradation. In particular, recent studies have found that ferroptosis plays a crucial role in the pathogenesis of osteoarthritis. Ferroptosis is an iron-dependent lipid peroxidation-driven cell death mechanism closely related to chondrocyte death. Furthermore, the temporomandibular joint is physiologically in a hypoxic environment, and cellular energy metabolism is easily disrupted under pathological conditions. In the pathological state of TMD, abnormal loads and other factors can exacerbate cellular metabolic disorders, leading to insufficient energy supply and further accelerating cellular degeneration. Reactive oxygen species (ROS) play a key role in the pathogenesis of TMD. Mechanical stress, inflammatory cytokines, and other factors can lead to a large production of ROS within chondrocytes, exceeding the cell's antioxidant defense capacity and causing oxidative stress damage. Oxidative stress not only directly causes cell damage, but also activates inflammatory signaling pathways, promotes the expression of inflammatory factors, and creates a vicious cycle. In addition, ROS accumulation can induce ferroptosis, accelerating chondrocyte death. NN-Acetylcysteine ​​(NAC) is a thiol-containing compound that, as a precursor to glutathione, possesses potent antioxidant and anti-inflammatory effects. NAC can promote glutathione synthesis by replenishing cellular cysteine ​​levels, thereby scavenging reactive oxygen species and protecting cells from oxidative stress damage. Furthermore, the thiol group inherent in NAC also contributes to its excellent antioxidant activity. In the condition of transarterial malignancy (TMM), joint cells exhibit abnormal redox homeostasis and energy metabolism, leading to ferroptosis and impaired extracellular matrix synthesis.

[0004] Therefore, there is an urgent need in this field for a drug that can regulate the redox homeostasis and energy metabolism of joint cells under TMD conditions, alleviate ferroptosis, and prevent the synthesis of extracellular matrix. This is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides the following technical solutions: This invention provides N- The use of acetylcysteine ​​in the preparation of drugs for treating temporomandibular joint disorders; Furthermore, the intended use is to promote the proliferation of MCCs, inhibit cell death, or suppress inflammation; Furthermore, the intended use is to improve the antioxidant capacity of TMD joint tissues; Furthermore, the intended use is to promote the secretion of extracellular matrix in MCCs; Furthermore, the intended use is to promote redox homeostasis in MCCs cells; Furthermore, the intended use is to promote oxidative phosphorylation in MCCs cells; Furthermore, the intended use is to inhibit glycolysis in MCCs cells; Furthermore, the intended use is to inhibit ferroptosis in MCCs cells; Furthermore, the intended use is to promote mitochondrial function in MCCs; Furthermore, the inflammation-inhibiting effect is to improve cartilage damage and relieve condylar pain; further still, the use is to inhibit the secretion of iron and ferrous ions and the level of lipid peroxides; Furthermore, the intended use is to promote the function of nuclear factor Nrf2 and membrane protein SLC7A11; Furthermore, the drug is an injectable dosage form or a skin patch; The present invention has achieved the following beneficial effects: 1. This invention applies the clinical drug NAC to the treatment of TMD. Utilizing systematic cell biology experiments, omics sequencing, bioinformatics analysis, and animal experiments, by detecting cell proliferation levels, redox levels at the cellular and tissue levels, inflammation levels, oxidative phosphorylation levels, glycolysis levels, iron and ferrous ion levels, lipid peroxide levels, extracellular matrix secretion, and cartilage damage, it was found that NAC can effectively promote the proliferation activity of MCCs and inhibit cell death processes. In an inflammatory state, NAC can improve cellular energy metabolism processes and improve cellular and tissue antioxidant levels by promoting the functional expression of nuclear factor Nrf2 and membrane protein SLC7A11, inhibiting the levels of iron and ferrous ions and lipid peroxides in cells and tissues, and promoting extracellular matrix secretion, thereby promoting TMD tissue repair and relieving pain.

[0006] 2. The NAC described in this invention is a commonly used clinical drug, which is inexpensive, readily available, has excellent cell and tissue safety, and is highly practical.

[0007] 3. The drug of this invention can be safely applied to the treatment of TMD through local injection into the condyle or skin application (microneedle) to the condyle area. It has important significance in the clinical use of TMD and has good medicinal value and application prospects. Attached Figure Description

[0008] Figure 1 To illustrate the effect of different concentrations of NAC on the activity of MCCs in this invention, (A) is a schematic diagram of cell activity results under normal conditions, and (B) is a schematic diagram of cell activity results under inflammatory conditions. Figure 2 The effect of NAC on the death level of MCCs in this invention is illustrated by fluorescence staining of live and dead cells. Figure 3 To illustrate the effect of NAC on the redox homeostasis of MCCs in this invention, (A) schematic diagram of DCFH-DA probe fluorescence staining in cells, (B) quantitative analysis of DCFH-DA fluorescence intensity, (C) GSH content in cells, (D) GSSG content in cells, and (E) GPx enzyme activity in cells; Figure 4 The effects of NAC on cellular inflammation levels and extracellular matrix secretion function, as described in this invention, include (A) extracellular matrix secretion factor Col2a1 and inflammatory factor IL-1. 1β (A) Western blot image of protein bands; (B) Quantitative analysis of the grayscale value of the Western blot image of Col2a1, an extracellular matrix secretory factor; (C) inflammatory factor IL-1 1β Quantification of protein blot band gray values; (D) relative RNA expression level of extracellular matrix secretory factor Col2a1; (E) inflammatory factor IL- 1β The relative expression level of RNA; Figure 5 To illustrate the effects of NAC on cellular iron, ferrous ion, and lipid peroxide levels, the following diagrams are presented: (A) Schematic diagram of FerroOrange probe fluorescence staining in cells; (B) Total iron concentration in cells; (C) Iron concentration in cells; (D) Ferrous ion concentration in cells; (E) Schematic diagram of BDP 581 / 591 C11 probe fluorescence staining in cells; (F) Quantitative fluorescence intensity analysis, with green / red fluorescence reflecting lipid peroxide levels; (G) Malondialdehyde (MDA) level, a marker of lipid peroxide levels in cells; (H) 4-hydroxynonenoic acid (4-HNE) level, a marker of lipid peroxide levels in cells. Figure 6 The effects of NAC on cellular oxidative phosphorylation and glycolysis levels are as follows: (A) Total ATP and glycolysis-derived ATP content in cells; (B) Mitochondrial-derived ATP and glycolysis-derived ATP content in cells. Figure 7 To illustrate the effect of NAC on mitochondrial function in this invention, (A) is a schematic diagram of cell transmission electron microscopy, (B) is a schematic diagram of mitochondrial ROS level detection and MitoSox / MitoTracker probe fluorescence staining, (C) is a schematic diagram of mitochondrial membrane potential level detection and JC-1 probe fluorescence staining, and (D) is a quantitative analysis of JC-1 fluorescence intensity, with green / red fluorescence reflecting mitochondrial membrane potential level. Figure 8 To illustrate the effects of NAC on nuclear factor Nrf2 and membrane protein SLC7A11 in this invention, (A) Wayne diagram showing the number of GO entries and KEGG pathways co-enriched across groups, (B) major GO entries co-enriched, with red boxes indicating differentially expressed genes mainly enriched in GO entries related to extracellular matrix synthesis and cellular oxidative stress, (C) major KEGG signaling pathways co-enriched, with red boxes indicating differentially expressed genes mainly enriched in signaling pathways related to ferroptosis and maintaining redox homeostasis, (D) Western blot diagram showing that NAC significantly activated the expression levels of nuclear factor Nrf2 and membrane protein SLC7A11 in normal and inflammatory cells; Figure 9 This invention relates to the effect of NAC on the expression levels of key factor proteins in the Nrf2-SLC7A11-GPX4 signaling pathway. (A) Quantitative bar chart of Nrf2 protein band gray value shows that NAC significantly activates Nrf2 protein expression; (B) Quantitative bar chart of SLC7A11 protein band gray value shows that NAC significantly activates SLC7A11 protein expression; (C) Quantitative bar chart of GPX4 protein band gray value shows that NAC significantly activates GPX4 protein expression; (D) Quantitative bar chart of ACSL4 protein band gray value shows that NAC significantly inhibits ACSL4 protein expression. Figure 10 To investigate the effects of NAC on the expression levels of key genes in the Nrf2-SLC7A11-GPX4 signaling pathway, (A) the bar chart showing Nrf2 relative to RNA expression levels indicates that NAC significantly activates Nrf2 gene expression; (B) the bar chart showing SLC7A11 relative to RNA expression levels indicates that NAC significantly activates SLC7A11 gene expression; (C) the bar chart showing GPX4 relative to RNA expression levels indicates that NAC significantly activates GPX4 gene expression; and (D) the bar chart showing ACSL4 relative to RNA expression levels indicates that NAC significantly inhibits ACSL4 gene expression. Figure 11 To illustrate the efficacy of NAC in treating TMD, the following data were collected: (A) Micro-CT images of the condyle of rats in each group; (B) H&E staining and Safranin / Fixed Green staining of condyle tissue sections from rats in each group; (C) Immunohistochemical staining of condyle tissue sections from rats in each group; (D) Bone mineral density (BMD) of condyle tissue; (E) Trabecular bone thickness (Tb.Th) of condyle tissue; (F) Number of trabecular bones (Tb.N) of condyle tissue; (G) Trabecular bone separation (Tb.Sp) of condyle tissue; (H) Total iron concentration of condyle tissue; (I) Iron concentration of condyle tissue; (J) Ferrous ion concentration of condyle tissue; (K) 4-HNE content in condyle tissue; (L) MDA content in condyle tissue; (M) GSH content in condyle tissue; (N) GSSG content in condylar tissue, (O) inflammatory factor IL-1 in condylar tissue β Level, (P) condylar pain threshold. Detailed Implementation

[0009] Unless otherwise specified, the experimental methods used in the following implementation methods are all conventional methods; unless otherwise specified, the materials and reagents used in the following implementation methods are all commercially available. Example

[0010] (I) Experimental conditions and grouping Main experimental materials: NAC was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; MCCs were obtained from rat condylar tissue with the approval of the ethics committee of a certain university; SD rats were purchased from Beijing Spaford Biotechnology Co., Ltd.

[0011] Using PBS as solvent, add HEPES and adjust the final concentration to 20 mM. Weigh NAC in the dark and add it to the solution, adjusting the final NAC concentration to 500 mM. Adjust the pH of the solution to 7.0 with sodium bicarbonate. μ After filtration using an m filter, the container is sealed and ready for use.

[0012] Cell experiment grouping: Group 1, grouped according to the final NAC concentration added to the culture medium, without IL-1. β Induced cells: 0 μ Group M (blank group); 250 μ Group M; 500 μ Group M; 1mM group; 2mM group; 4mM group; Group II, grouped according to the final NAC concentration added to the culture medium and administered IL-1. β Induced cells: CON group (blank group); IL-1 β Group; 250 μ M+IL-1 β Group; 500 μ M+IL-1 β Group; 1mM+IL-1 β Group; 2mM+IL-1 β Group; 4mM+IL-1 β Group 3, grouped according to the components added to the culture medium: (1) CON group (blank group); (2) IL-1 β Group; (3) NAC group; (4) NAC+IL-1 β Group; rat MCCs were extracted and identified and then placed in an incubator at 37℃ and 5wt% CO2, and cultured in the above-mentioned culture medium with different concentrations of NAC added; Animal experimental grouping: Based on the intra-articular injection solution, rats were grouped as follows: CON group (blank group); PBS+TMD group; NAC+TMD group. All rats were housed under standard conditions with free access to food and water.

[0013] (II) Experimental Testing 1. Detection of cell viability and death levels The rat MCCs from groups one and two of the above cell experiments were seeded at 5000 cells / well in 96-well plates. After culturing for 1 day, the original culture medium was removed, and the cells were washed three times with PBS. Then, a culture medium containing 10% PBS was added. μ The culture medium containing LCCK-8 solution was incubated in the dark for 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Rat MCCs from group three of the above cell experiments were seeded into 6-well plates and cultured to 80% confluence. Cells were then stained using the Calcein / PI method provided by the Beyotime Calcein / PI Cell Viability and Cytotoxicity Assay Kit, and the fluorescence intensity was observed using a laser confocal microscope.

[0014] Experimental results: such as Figure 1 As shown in Figure A, this represents the results of CCK-8 assay in cells after 72 hours, compared to 0. μ Group M, after 500 μ The activity level of MCCs treated with M-2mM NAC increased, at 500 μ M NAC showed significant effects; such as Figure 1 As shown in B, 250 μM-4mMNAC can effectively restore the body after IL-1 β The activity levels of MCCs after treatment were measured, with 1 mM NAC showing the best effect. Figure 2 The image shows the results of cell liveness and death staining fluorescence. NAC can alleviate IL-1. β This leads to an increase in the proportion of cell death.

[0015] 2. Detection of cellular redox homeostasis Rat MCCs from cell experiment group three were seeded into 6-well plates and cultured until 60% confluence. The original culture medium was removed, and the cells were washed three times with PBS. Then, 1 mL of 10% PBS was added to each well. μ The DCFH-DA cell reactive oxygen species probe was incubated at 37°C in the dark for 30 minutes. After removing the probe working solution and washing three times, the ROS fluorescence intensity of the cells was observed using a laser confocal microscope. Rat MCCs from the above cell experiment group three were seeded into 6-well plates and cultured until 80% confluence. The original culture medium was removed, cells were collected by scraping, and the supernatant was removed by centrifugation. Following the instructions of the Beyotime GSH and GSSG detection kit, after protein removal and rapid freeze-thaw operations, the cells were centrifuged at 10000g for 10 minutes, and the supernatant was used to detect the content of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the cells, and the GSH / GSSG ratio was calculated. The rat MCCs from the above cell experiment group three were seeded into 6-well plates and cultured until 80% confluence. The original culture medium was removed, cells were collected by scraping, and the supernatant was removed by centrifugation. 150 mol / L of the solution was added per million cells. μ Cells were lysed using Beyotime Western blotting and IP cell lysis buffer, followed by centrifugation at 12000g for 10 minutes at 4°C. The supernatant was collected, and cell samples were processed stepwise according to the Beyotime Total Glutathione Peroxidase Detection Kit instructions. The absorbance at 340nm was measured using an ELISA reader, and the total glutathione peroxidase activity in each group of cell samples was calculated.

[0016] Experimental results: such as the cell fluorescence image of the reactive oxygen species probe ( Figure 3 A) and its fluorescence intensity quantitative histogram ( Figure 3 As shown in B), with IL-1 β Compared to the treated MCCs, NAC+IL-1 β The ROS fluorescence intensity in the NAC group was weakened, and the level was basically the same as that in the CON group. Furthermore, compared to the CON group, the ROS fluorescence intensity in the NAC group was also weakened, suggesting that NAC can effectively scavenge ROS in MCCs. (See the bar chart for total glutathione quantification in cells.) Figure 3 C) Quantitative histogram of oxidized glutathione ( Figure 3 D) and quantitative bar chart of glutathione peroxidase activity ( Figure 3 As shown in E), IL-1β Compared to the CON group, the antioxidant levels in the group were significantly lower, manifested as decreased GSH levels and total glutathione peroxidase activity, and increased GSSG levels; NAC+IL-1 β In the group, we found that the antioxidant level of cells was restored; in addition, compared with the CON group, the GSH level and total glutathione peroxidase activity of cells in the NAC group increased, while the GSSG level decreased, suggesting that NAC can not only improve the antioxidant level of cells under inflammatory conditions, but also enhance the antioxidant capacity of cells under normal conditions.

[0017] 3. Detection of cellular inflammation level and extracellular matrix secretion function The rat MCCs from group three of the above cell experiments were seeded into 6-well plates and cultured until 80% confluence. The original culture medium was removed, cells were collected by scraping, and the supernatant was removed by centrifugation. 150 mg / L of the culture medium was added per million cells. μ Lysis was performed using Beyotime Western blotting and IP cell lysis buffer (containing 10% PMSF), followed by centrifugation at 12000g for 10 minutes at 4°C. The supernatant was used for Western blotting and enzyme-linked immunosorbent assay (ELISA): 1. In the Western blotting experiment, the protein concentration of each group of samples was determined using the Solarbio BCA protein quantification kit. The protein samples were diluted with 5x loading buffer, incubated in a 100°C metal bath for 5 minutes, followed by 10wt% SDS-PAGE electrophoresis, transfer to a PVDF membrane at 300mA for 2 hours, and blocked with Beyotime rapid blocking buffer for 15 minutes before adding the primary antibody (inflammatory factor IL-1). β Incubate with extracellular matrix secretion-related factor Col2a1 at 4°C overnight. After washing the membrane three times, add secondary antibody and incubate at room temperature for 1 hour. Develop and expose using Beyotime ECL chromogenic kit. β -actin is an internal reference used to detect IL-1. β 1. And Col2a1 protein expression level; 2. In the enzyme-linked immunosorbent assay (ELISA), IL-1 in each sample was quantitatively detected according to the Solarbio rat interleukin 1β ELISA kit instructions. β The absorbance at 450 nm was measured using an ELISA reader, and the IL-1 concentration in the sample was calculated using a standard sample concentration curve. β Content. Rat MCCs from group three of the above cell experiments were seeded into 6-well plates and cultured until 80% confluence. The original culture medium was removed, and the cells were washed three times with PBS. Total RNA was collected using the Trizol method. IL-1 levels were measured using a gold-based reverse transcription kit and a quantitative real-time immunoassay kit. β and RT-qPCR expression quantification of Col2a1, in order to β -actin is an internal parameter.

[0018] Experimental results: as shown in the protein blot image ( Figure 4A) Quantitative bar chart of the gray value of Col2a1, a marker of extracellular matrix synthesis (A) Figure 4 B) and the inflammatory marker IL-1 β Quantitative histogram of grayscale values ​​of strips ( Figure 4 As shown in C), compared to IL-1 β Group, NAC+IL-1 β IL-1 in the group β Protein expression levels decreased, while Col2a1 protein expression levels increased. (See the quantitative bar chart of Col2a1 gene expression). Figure 4 D) and IL-1 β Quantitative bar chart of gene expression levels ( Figure 4 As shown in E), compared to IL-1 β Group, NAC+IL-1 β IL-1 in the group β Gene expression levels decreased, while Col2a1 gene expression levels were upregulated.

[0019] 4. Detection of cellular iron and ferrous ion and lipid peroxide levels The rat MCCs from experimental group 3 were seeded into 24-well plates and cultured until the cells reached 80% confluence. Fluorescent staining was performed according to the instructions of the Tongren Chemical Intracellular Ferrous Ion Fluorescent Probe (FerroOrange) Kit and the Lipid Peroxidation Detection Probe (BDP 581 / 591C11) Kit to detect the levels of ferrous ions and lipid peroxides in the cells.

[0020] Rats' MCCs from group three of the above-mentioned cell experiments were seeded into 6-well plates and cultured until 80% confluence. Cell clumps were collected by scraping, and the absorbance at 593 nm was measured using an ELISA reader according to the instructions of the Elabscience Total Iron Colorimetric Assay Kit and Ferrous Ion Colorimetric Assay Kit. The levels of total iron, ferric ions, and ferrous ions in the cells of each group were quantitatively detected based on the standard curve. Rats' MCCs from group three of the above-mentioned cell experiments were seeded into 6-well plates and cultured until 80% confluence. The lipid oxidation level of the cells in each group was quantitatively detected according to the instructions of the Beyotime Lipid Oxidation Malondialdehyde (MDA) Detection Kit. Cell protein supernatants from each group were prepared for enzyme-linked immunosorbent assay (ELISA), and the specific steps were the same as the aforementioned experimental methods. The protein content of the lipid peroxidation marker (4-hydroxynonenal, 4-HNE) was detected using the Beyotime ELISA kit.

[0021] Experimental results: such as the fluorescence image of FerroOrange cells using the ferrous ion probe ( Figure 5 A) Quantitative column chart of total iron ions ( Figure 5 B), Quantitative columnar iron ion analysis ( Figure 5 C) and quantitative histogram of ferrous ions ( Figure 5As shown in D), compared to IL-1 β Group, NAC+IL-1 β The levels of total iron ions, ferric ions, and ferrous ions in the group decreased significantly, as evidenced by a decrease in fluorescence intensity and a decrease in the concentrations of total iron ions, ferric ions, and ferrous ions. (See the cellular fluorescence graph of lipid peroxide markers and its quantitative fluorescence intensity bar graph.) Figure 5 E and Figure 5 F), quantitative histogram of lipid peroxide marker MDA ( Figure 5 G) and quantitative histogram of lipid peroxide marker 4-HNE (G) Figure 5 As shown in H), compared to IL-1 β Group, NAC+IL-1 β The levels of lipid peroxides (MDA and 4-HNE) in the group decreased significantly.

[0022] 5. Detection of cellular oxidative phosphorylation and glycolysis levels The rat MCCs from cell experiment group three were seeded into white 96-well plates and cultured overnight. The culture medium was then removed, and 100 mg / L of the medium was added to each plate. μ l Standard culture medium, containing 1.25 μ The medium containing mol / L Oligomycin (+) and 25 mmol / L 2-DG (+) was incubated at 37°C for 4 h, and 20 mol / L was recovered from each well. μ The culture supernatant was diluted 10-fold with ultrapure water to prepare the Lactate assay: Following the instructions of the Tongren Chemical Glycolysis / Oxidative Phosphorylation Detection Kit, the Lactate standard solution, the sample to be tested, and ultrapure water were mixed at a 1:1:1 ratio to prepare the assay solution. Using a 96-well plate, 20 μL of Lactate was added to each well. μ Incubate the test solution and 80 μl of Lactate working solution at 37°C for 30 min, and measure the absorbance at 450 nm using a microplate reader. The recovered white 96-well plate is then used for ATP assay: add 100 μl of Lactate working solution to each well. μ 1. Prepare ATP working solution, shake for 2 min, incubate at 25°C for 10 min, and use an ELISA reader to detect the chemiluminescence value (RLU).

[0023] Experimental results: such as bar charts of total ATP and ATP from glycolysis ( Figure 6 A) Quantitative bar chart of mitochondrial oxidative phosphorylation ATP and glycolytic ATP ( Figure 6 B) shows that, compared with normal MCCs, the main metabolic pathway of inflammatory MCCs shifts from oxidative phosphorylation to glycolysis, and the level of glycolysis in the cells is abnormally increased. Cells treated with NAC can effectively reverse this process: NAC restores the oxidative phosphorylation function in inflammatory cells and restores the level of glycolysis.

[0024] 6. Detection of cellular mitochondrial function Rat MCCs from group three of the above cell experiments were seeded into 6-well plates and cultured until 80% confluence. Cell clumps were collected, fixed with 2.5 wt% glutaraldehyde fixative, dehydrated, coated with clear resin, and ultrathin sections were prepared for transmission electron microscopy to observe mitochondrial structure. Rat MCCs from group three of the above cell experiments were seeded into 6-well plates and cultured until 60% confluence. The mitochondrial membrane potential and mitochondrial ROS levels of each group were detected using the Beyotime mitochondrial membrane potential (JC-1) assay kit and the mitochondrial superoxide dismutase (MitoSO Red) assay kit, respectively.

[0025] Experimental results: such as Figure 7 A transmission electron microscope image, IL-1 β Severe mitochondrial damage was observed in the induced cells, such as cristae disappearance and membrane rupture, and NAC+IL-1 levels decreased. β This phenomenon was somewhat alleviated in the group of cells. Figure 7 B MitoSox cell immunofluorescence image, Figure 7 C JC-1 cell immunofluorescence staining pattern and its quantitative fluorescence intensity bar chart ( Figure 7 D), IL-1 β The levels of mitochondrial ROS and mitochondrial membrane potential in the group cells were significantly increased, and these changes could be improved by NAC. The results suggest that NAC can effectively improve mitochondrial function in MCCs in an inflammatory state.

[0026] 7. Mechanisms for mining omics through sequencing and their validation Total RNA was extracted from cells in each group, following the same experimental procedures as described above. Transcriptome sequencing of the total RNA was performed on the Illumina NovaSeq6000 platform. Data were used to screen for differentially expressed genes in each component using DESeq2 software. GO enrichment analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes using TopGO and ClusterProfler packages. The IL-1 pathway was visualized using a Venn diagram. β "vs CON" and "NAC+IL-1" β Group vs IL-1 β "The intersection of GO entries and KEGG signaling pathways between groups was finally visualized using the ggplot2 package. Cell protein and total RNA samples were collected from each group for Western blot and RT-qPCR experiments, with the specific steps being the same as the aforementioned experimental methods, to detect the protein and gene expression levels of relevant factors (Nrf2, SLC7A11, GPX4, ACSL4).

[0027] Experimental results: such as Figure 8 A Wayne diagram, "IL-1" β"vs CON" and "NAC+IL-1" β Group vs IL-1 β "A total of 1279 GO entries and 118 KEGG pathways were enriched across the groups. For example..." Figure 8 The bubble diagram of B GO enrichment analysis shows that most of the enriched GO items involve processes such as extracellular matrix secretion and cellular oxidative stress response. Figure 8 C KEGG enrichment analysis bubble diagram, the KEGG pathway reveals the potential role of ferroptosis in regulatory mechanisms. (See Western blot image for details.) Figure 8 D) and the corresponding strip gray value quantitative analysis results show that, compared with IL-1 β Group, NAC can promote Nrf2 ( Figure 9 A), SLC7A11 ( Figure 9 B) and GPX4 Figure 9 C) Protein expression level, inhibition of ACSL4 ( Figure 9 D) Protein expression level; RT-qPCR results, compared with IL-1 β Group, NAC can promote Nrf2 ( Figure 10 A), SLC7A11 ( Figure 10 B) and GPX4 Figure 10 C) Gene expression levels, inhibition of ACSL4 ( Figure 10 D) Gene expression levels suggest that NAC can improve IL-1 by activating the Nrf2-SLC7A11-GPX4 signaling pathway. β The resulting MCCs iron death.

[0028] 8. Detection of joint tissue redox levels, inflammation levels, extracellular matrix secretion levels, iron and ferrous ion levels, lipid peroxide levels, cartilage repair levels, and condylar pain threshold. The animal experiments were reviewed and approved by the ethics committee of a university. Six-week-old SD rats were randomly assigned to three groups: the CON group (blank group); the PBS+TMD group; and the NAC+TMD group, with five rats in each group. The rats were injected intra-articularly with 50... μ A rat model of TMD was established using 10 mg / mL sodium iodoacetate solution. The PBS+TMD group received intra-articular injections of 50 mg / mL sodium iodoacetate solution. μ L PBS solution, NAC+TMD group received intra-articular injection of 50 μ L2mM NAC solution was injected weekly. After 4 weeks, condylar tissue was obtained from each group of rats. Micro-CT and tissue section staining were used to assess extracellular matrix secretion levels and cartilage damage levels. Joint tissue homogenates were prepared and analyzed using Beyotime GSH and GSSG detection kits, Beyotime total glutathione peroxidase detection kits, and Solarbiol rat interleukin-1 assays. βELISA kits, Elabscience total iron colorimetric assay kit and ferrous ion colorimetric assay kit, Beyotime malondialdehyde (MDA) lipid peroxidation assay kit, and Beline rat 4-HNE ELISA kit were used to detect tissue redox levels, inflammation levels, iron and ferrous ion levels, and lipid peroxide levels, respectively. The specific steps were the same as described in the previous experimental methods. The pain threshold in the bilateral temporomandibular joint region of rats was detected using a Von Frey fiber pain threshold test kit. Rats were placed in a cage with open sides, and the testing environment was kept quiet and free of irritants. After the rats had adapted to the environment for 30 minutes, Von Frey fibers were applied to the temporomandibular joint region through the holes in order of increasing force. Once the rat's head retraction response was observed, the force value at that moment was recorded as the pain threshold for that temporomandibular joint region. The threshold was recorded three times for each region, with a 30-second interval between each test.

[0029] Experimental results: like Figure 11 As shown in a Micro-CT image, NAC can increase condylar bone density in TMD rats, restore the smoothness of condylar cartilage surface, and alleviate subchondral bone resorption. Figure 11 H&E staining and Safranin-Fixed Green staining of tissue sections showed that NAC effectively reduced the level of tissue inflammation and alleviated the damage to the condylar structure of TMD, and promoted the synthesis of new collagen, suggesting that NAC can alleviate the level of articular cartilage damage by improving the secretory function of extracellular matrix. Figure 11 Immunohistochemical staining showed that NAC can effectively improve the inflammation level and extracellular matrix secretion level of TMD and promote the expression of Nrf2, SLC7A11 and GPX4. Figure 11 D, 11E, 11F, and 11G show the microstructural parameters of condylar bone detected by Micro-CT, indicating that NAC increases bone mineral density (BMD), trabecular thickness (Tb.Th), and trabecular number (Tb.N) in TMD tissue, while decreasing trabecular separation (Tb.Sp). Quantitative bar chart of total iron ions in tissues ( Figure 11 H), quantitative columnar section of iron ions ( Figure 11 I) and quantitative columnar section of ferrous ions ( Figure 11 J) indicates that NAC can effectively reduce the levels of iron and ferrous ions in TMD tissues; Figure 11 K, Figure 11 L indicates that NAC can effectively reduce the content of major lipid peroxides 4-HNE and MDA in TMD tissues; Figure 11 M, Figure 11N represents that NAC can increase the content of total glutathione (GSH) and decrease the content of oxidized glutathione (GSSG), respectively. Figure 11 O-NAC can effectively reduce the inflammatory factor IL-1 in TMD tissues. β level; Figure 11 P indicates that NAC can effectively reduce the pain threshold level in the condylar region of TMD (Von Frey method).

[0030] In summary, NAC can effectively improve the antioxidant capacity of TMD joint tissues, inhibit inflammation levels, promote extracellular matrix secretion, inhibit ferrous (ferrous) ion and lipid peroxide levels, improve cartilage damage, and relieve condylar pain. The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. N- The use of acetylcysteine ​​in the preparation of drugs for the treatment of temporomandibular joint disorders.

2. The use according to claim 1, characterized in that, The intended uses are to promote the proliferation of MCCs, inhibit cell death, or suppress inflammation.

3. The use according to claim 1, characterized in that, The stated purpose is to improve the antioxidant capacity of TMD joint tissues.

4. The use according to claim 1, characterized in that, The intended use is to promote the secretion of extracellular matrix in MCCs.

5. The use according to claim 1, characterized in that, The intended use is to inhibit glycolysis in MCCs (Multi-Cell Cells).

6. The use according to claim 1, characterized in that, The intended use is to inhibit ferroptosis in MCCs (medium-cell ferritin) cells.

7. The use according to claim 1, characterized in that, The anti-inflammatory effect is to improve cartilage damage and relieve pain in the condylar region.

8. The use according to claim 1, characterized in that, The stated purpose is to inhibit the secretion of iron and ferrous ions and the level of lipid peroxides.

9. The use according to claim 1, characterized in that, The intended use is to promote the function of nuclear factor Nrf2 and membrane protein SLC7A11.

10. The use according to claim 1, characterized in that, The drug is in injectable form or as a skin patch.

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