CN117298136A - Application of miR-143-3p in preparation of medicine for treating temporomandibular joint osteoarthritis - Google Patents

Abstract

The invention provides a novel application of a biological preparation miR-143-3p in preparation of a medicine for treating temporomandibular joint osteoarthritis, and belongs to the field of biological medicines. According to the research of the invention, miR-143-3p can effectively relieve the surface abrasion and congestion degree of the TMJOA joint disc; simultaneously miR-143-3p can well relieve degradation and damage of condylar cartilage tissue in temporomandibular joint osteoarthritis by inhibiting the iron death process of chondrocytes; and miR-143-3p can obviously inhibit the change of subchondral bone in temporomandibular joint osteoarthritis, reduce the osteoclast activity in subchondral bone, further relieve pain in joint areas, relieve osteoarthritis, and provide a new choice for the therapeutic drug for temporomandibular joint osteoarthritis. Therefore, miR-143-3p has high application value as a potential intervention means of temporomandibular joint osteoarthritis.

Description

Use of miR-143-3p in the preparation of drugs for the treatment of temporomandibular joint osteoarthritis

Technical field

The invention belongs to the field of biomedicine, and specifically relates to the new use of miR-143-3p in the preparation of drugs for the treatment of temporomandibular joint osteoarthritis.

Background technique

MicroRNA (miRNA) is a type of small non-coding RNA that does not directly encode proteins but regulates other RNAs. It silences target genes by binding to the 3' untranslated region (3'UTR) or open reading frame (ORF) region of mRNA. Expression, the role of miRNA in life activities has attracted more and more attention. Studies have confirmed that miRNA is involved in a variety of physiological and pathological activities, affecting the proliferation, differentiation, migration of different cells and the initiation and progression of various diseases. Disease treatment strategies based on miRNA have shown great potential in the treatment of various diseases.

Temporomandibular joint osteoarthritis (TMJOA) is a common degenerative joint disease. As the disease progresses, TMJOA patients' mandibular movements will be limited and accompanied by severe pain, which seriously affects the patient's physical and mental health. The main histopathological characteristics of TMJOA include progressive degeneration and destruction of condylar cartilage, abnormal subchondral bone remodeling, and obvious synovial inflammation. Articular cartilage is the main part of the joint that bears biological stress. Its damage or degradation will cause the joint to lose its normal structure and function, causing joint pain and movement disorders.

However, the avascular nature of cartilage tissue is not conducive to the conduction of signal molecules, cell migration, and transportation of nutrients or oxygen. As a result, the self-healing ability of cartilage is very limited, and it is difficult to effectively regenerate damaged cartilage tissue.

At present, the cause and mechanism of TMJOA is still unclear. Clinical treatments for TMJOA are mostly symptomatic, which cannot effectively reverse the structural and functional damage to joints caused by the disease. Therefore, finding a new therapeutic intervention for TMJOA is an important direction in the field of clinical research on osteoarthritis.

miR-143-3p, a type of miRNA, is currently reported to be relevant to the evaluation of efficacy in the treatment of schizophrenia. Liu Lang's dissertation from Nanchang University's Department of Surgery (Orthopedics) "Study on the mechanism of long non-coding RNA SNHG1 reducing IL-1β-induced chondrocyte inflammatory damage by targeting the miR-143-3p/KLF2 axis" (Medical and Health Science and Technology Issue, Master Electronic Journal Publishing (Issue 08, 2020)), disclosed that SNHG1 can reduce the inflammatory damage of chondrocytes induced by IL-1β, and miR-143-3p can inhibit the expression of SNHG1, leading to enhanced inflammation.

There are currently no reports directly studying the relationship between miR-143-3p and arthritis.

Contents of the invention

Based on the discovery that miR-143-3p can reduce temporomandibular joint osteoarthritis, the present invention provides a new drug for treating temporomandibular joint osteoarthritis.

The present invention provides the use of a substance that increases the expression level of miR-143-3p in preparing a drug for treating temporomandibular joint osteoarthritis.

The substances that increase the expression level are miR-143-3p, miR-143-3p mimics or miR-143-3pagomir.

The nucleotide sequence of the miR-143-3p is shown in SEQ ID NO.1; the nucleotide sequence of the miR-143-3p mimics is shown in SEQ ID NO.2; the miR-143-3p agomir includes the forward direction The sequence, reverse sequence and nucleotide sequence are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

The drug is a drug for treating temporomandibular joint osteoarthritis.

The drug is a drug that alleviates the surface wear and congestion of the articular disc; the drug is a drug that can significantly increase the type II collagen content in cartilage.

The drug is a drug that can alleviate the degeneration and degradation of cartilage tissue in temporomandibular joint osteoarthritis.

The drug is a drug that enhances the activity of chondrocytes; the drug is a drug that reduces the osteoclastic activity of subchondral bone in temporomandibular joint osteoarthritis.

The drug is a drug that increases the expression level of GPX4 in chondrocytes and/or increases the activity of GPX4 enzyme; the drug is a drug that reduces the level of malondialdehyde in chondrocytes; the drug is a drug that reduces the level of lipid reactive oxygen species in chondrocytes Drug; the drug is a drug that reduces the expression level of Mfsd8 in chondrocytes.

The drug is a drug that improves subchondral bone density in temporomandibular joint osteoarthritis.

The drug is a drug that relieves pain in the joint area of temporomandibular joint osteoarthritis.

Experimental results show that by administering AgomiR-143-3p or miR-143-3pmimics, the present invention increases the expression level of miR-143-3p, effectively alleviates the surface wear and congestion of TMJOA articular discs, and significantly increases type II collagen in cartilage. The content can alleviate the degeneration and degradation of cartilage tissue in temporomandibular joint osteoarthritis, reduce pain in the joint area, increase cartilage bone density, and treat temporomandibular joint osteoarthritis.

The present invention found that miR-143-3p can reduce temporomandibular joint osteoarthritis, which is contrary to the revelation of increased inflammation reported in the above-mentioned degree thesis of Liu Lang Department of Surgery (Orthopedics) of Nanchang University.

Obviously, according to the above content of the present invention, according to the common technical knowledge and common means in the field, without departing from the above basic technical idea of the present invention, various other forms of modifications, replacements or changes can also be made.

The above contents of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 shows the therapeutic effect of miR-143-3p on articular disc morphology and cartilage repair in temporomandibular joint osteoarthritis. (A) Gross image of the articular disc; (B) Safranin O fast green staining and Osteoarthritis Research Society International (OARSI) score and immunofluorescence staining of COL2A1, a molecule related to chondrocyte matrix synthesis.

Figure 2 shows the inhibitory effect of miR-143-3p on ferroptosis of chondrocytes in arthritis. (A) Immunofluorescence staining of ferroptosis-related protein GPX4; (B) Changes in chondrocyte activity; (C) Changes in chondrocyte ferroptosis-related indicator malondialdehyde (MDA) levels; (D) Changes in chondrocyte reactive oxygen species ROS .

Figure 3 shows that miR-143-3p acts on the downstream target gene Mfsd8 to inhibit ferroptosis of chondrocytes. (A) Immunofluorescence staining of Mfsd8 in chondrocytes; (B) Changes in the transcription level of Mfsd8 in chondrocytes; (C) Changes in the transcription level of Mfsd8 in chondrocytes; (D) Changes in Mfsd8 protein levels in chondrocytes; (E) Mfsd8 protein in chondrocytes Changes in levels; (F) Prediction of the action site of Mfsd8 by miR-143-3p; (G) (H) Dual-luciferase reporter gene verifies that Mfsd8 is a target gene of miR-143-3p; (I) Chondrocyte activity Changes; (J) Changes in chondrocyte ferroptosis-related index MDA levels; (K) Changes in chondrocyte reactive oxygen species ROS.

Figure 4 shows the effect of miR-143-3p on alleviating pain in the joint area of temporomandibular joint osteoarthritis and promoting subchondral bone density in arthritis. (A) Joint region pain pattern is modeled after Vonfrey pain threshold; (B) Micro-CT scan reconstruction; (C) Micro-CT scan bone analysis.

Figure 5 shows the inhibitory effect of a biological agent, miR-143-3p, on osteoclastic activity and CGRP expression of subchondral bone in arthritis. (A) TRAP staining; (B) CGRP immunofluorescence staining.

Detailed ways

The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

The Mfsd8 gene, major facilitator superfamily domain containing 8, can activate transmembrane transporter activity and is predicted to participate in transmembrane transport and play a role in the following life activities, including the TORC1 signaling pathway, autophagosome maturation, and the regulation of cellular catabolic processes. Located in lysosomes and membranes, the human homolog of this gene has been associated with neuronal-like lipofuscinosis.

Malondialdehyde (MDA) is a compound formed after the breakdown of lipid peroxides. Therefore, MDA is one of the most common indicators for detecting lipid peroxides in cells and tissues.

TRAP staining: also known as tartrate-resistant acid phosphatase staining, is used to detect the staining of osteoclasts in bones and osteocytes. It makes the osteoclasts red and the nuclei light blue to show the distribution and number changes of osteoclasts.

CGRP: Calcitonin gene-related peptide, a potent vasodilator-related molecule that can cause pain sensitization and serve as an indicator of nociceptive sensory innervation.

Sequence of miR-143-3p (SEQ ID NO.1): ugagaugaagcacuguagcuca

AgomiR is miR-143-3p agomir, sequence:

Sense(SEQ ID NO.3):5’-ugagaugaagcacuguagcuca-3’

Anti-sense(SEQ ID NO.4):5’-agcuacagugcuucaucucauu-3’

miR-143-3p mimics sequence (SEQ ID NO.2):

5’-ugagaugaagcacuguagcuca-3’

miR-143-3p inhibitors sequence: 5’-ugagcuacagugcuucaucuca-3’

MUT-Mfsd8 plasmid vector FR02, sequence: cactaacttatgtttctaatagcaagtggattcttaaaattactatttttcaagttctttaaactctagtgcaaactctgacctttgaatgccccttgtcagtaatgacagagaagaatagcagtttgcaacctacctgtagacccagtcacagcagcagagctgaagttggaccatgattgct ctcgggcagtctcatctcatcaggtgagacttgaagcagaatggccaaaggtgactactgatcaaactagtttctacctctctgtgtgaatggaacactggtatgtgtacgggaatctgtaaaacatgcagaagtgctttcctgtcttgttatcatggtttcaaatctgagtgtgaacataatattctccacctcatactctggtctgctgaag ctattagcaccacgtgatggcagcattttgcttcagtttgttttgtcatgccctggacgtcctcttgtcctcttacctgctgttc

WT-Mfsd8 plasmid vector FR02, sequence: cactaacttatgtttctaatagcaagtggattcttaaaattactatttttcaagttctttaaactctagtgcaaactctgacctttgaatgccccttgtcagtaatgacagagaagaatagcagtttgcaacctacctgtagacccagtcacagcagcagagctgaagttggaccatgattgct ctcggtcaggtgagacttgaagcagaatggccaaaggtgactactgatcaaactagtttctacctctgtgtgaatggaacactggtatgtgtacgggaatctgtaaaacatgcagaagtgctttcctgtcttgttatcatggtttcaaatctgagtgtgaacataatattctccacctcatactctggtctgctgaagctattagcaccacg tgatggcagcattttgcttcagtttgttttgtcatgccctggacgtcctcttgtcctcttacctgctgttc

Example 1: miR-143-3p can alleviate the degeneration and degradation of cartilage tissue in temporomandibular joint osteoarthritis and inhibit the ferroptosis process of chondrocytes.

1. Experimental methods of animal experiments

(1) Experimental animals

Eight-week-old female C57BL/6J mice were used in the experiment. All animals were raised at the West China Animal Experiment Center of Sichuan University and were raised under SPF standards.

(2) Experimental methods

1. Surgical modeling

When the mice were 9 weeks old, the unilateral anterior crossbite (UAC) model was constructed in the mice under general anesthesia, thereby inducing the formation of TMJOA and constructing the experimental TMJOA model mice.

2. Experimental grouping and intervention methods

The mice were randomly divided into three groups:

1) Negative control group (NC group): The same amount of AgomiR-NC was injected into the temporomandibular joint (TMJ) joint cavity, with a dosage of 20 μM;

2) TMJOA group: The same amount of AgomiR-NC was injected into the TMJ joint cavity of TMJOA model mice, with a dosage of 20 μM;

3) TMJOA+Agomir group: AgomiR-143-3p was injected into the TMJ joint cavity of TMJOA model mice for intervention, with a dosage of 20 μM.

(3) Results evaluation method

1. Histological evaluation of cartilage destruction in osteoarthritis

Tissue processing method 1: After 3 weeks of modeling induction, each experimental group started to receive different intervention intra-articular injections of TMJ every three days, and the mice were killed at 6 weeks, and the temporomandibular joint tissue was collected and treated with 4% paraformaldehyde. Fixed for 48 hours, decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for one month, dewaxed and embedded, sectioned with a thickness of 4um.

The sections were stained with Safranin O fast green, observed under a microscope, photographed and analyzed. The severity of osteoarthritis cartilage damage was evaluated according to the Osteoarthritis Research Association International (OARSI) scoring system.

The scoring details are:

0 points: normal cartilage;

0.5 points: Safranin O coloring is slightly reduced, and the cartilage tissue remains unchanged;

1 point: Small part of fibrosis on the cartilage surface but no cartilage defect;

2 points: Longitudinal cracks appear on the surface of the cartilage tissue;

3-6 points: Cartilage tissue cracks or defects reach directly into the calcified cartilage layer, and the defect area accounts for less than 25% of the cartilage surface area (3 points), 25-50% (4 points), 50-75% (5 points), >75 %(6 points).

2. Immunofluorescence staining to evaluate changes in cartilage tissue-related proteins in each group

Tissue processing was the same as the above tissue processing method 1. The sections of each experimental group were immunofluorescently stained for type II collagen α1 (COL2A1), glutathione peroxidase 4 (GPX4), and Mfsd8, and statistics were collected.

2. Experimental methods for cell experiments

(1) Cell grouping and processing

The present invention uses temporomandibular joint cartilage primary cells. The cartilage primary cells are obtained from the temporomandibular joint of 7-day-old newborn experimental animals, and are digested and cultured in a solution containing 10% fetal bovine serum and double antibodies (penicillin 50U/mg, streptomycin 50U/mg). ml) of low-sugar DMEM medium and cultured in a CO ₂ incubator (37°C, 5% CO ₂ , 21% O ₂ , 95% humidity).

1. In order to detect the regulatory effect of miR-143-3p on ferroptosis of chondrocytes in osteoarthritis, chondrocytes cultured in vitro were randomly divided into the following three groups for intervention:

1)miR-NC group: add mimics NC for intervention, mimics NC, dosage 20nM;

2)miR-NC+IL-1β group: Add IL-1β for intervention, IL-1β, dosage 10ng/ml; mimics NC, dosage 20nM;

3) IL-1β+miR-143-3p mimics group: IL-1β and miR-143-3p mimics were added for intervention at the same time. IL-1β, the dosage was 10ng/ml; miR-143-3p mimics, the dosage was 20nM.

2. In order to detect the effect of miR-143-3p on the mRNA and protein levels of the downstream target gene Mfsd8, chondrocytes cultured in vitro were randomly divided into the following interventions:

Intervention 1:

1)miR-NC group: add mimics NC for intervention, mimics NC, dosage 20nM;

2)miR-NC+IL-1β group: Add IL-1β for intervention, IL-1β dosage is 10ng/ml; mimics NC, dosage is 20nM;

3) IL-1β+miR-143-3p mimics group: IL-1β and miR-143-3p mimics were added for intervention at the same time. IL-1β, the dosage was 10ng/ml; miR-143-3p mimics, the dosage was 20nM.

Intervention 2:

1)miR-NC group: add inhibitors NC for intervention, inhibitors NC, dosage 20nM;

2)miR-NC+IL-1β group: Add IL-1β for intervention, IL-1β, dosage 10ng/ml; inhibitors NC, dosage 20nM;

3) IL-1β+miR-143-3p inhibitors group: IL-1β and miR-143-3pinhibitors were added for intervention at the same time. IL-1β, the dosage was 10ng/ml; miR-143-3p inhibitors, the dosage was 20nM.

3. In order to verify the binding site of miR-143-3p to the target gene Mfsd8, the HEK293 cell line was randomly divided into the following interventions:

Intervention 1:

1)miR-NC+WT-Mfsd8 group: add mimics NC and WT-Mfsd8 plasmid for intervention, mimics NC, dosage 20nM; WT-Mfsd8 plasmid, dosage 50nM;

2)miR-143-3p mimics+WT-Mfsd8 group: add miR-143-3p mimics and WT-Mfsd8 plasmid for intervention, miR-143-3p mimics, dosage 20nM; WT-Mfsd8 plasmid, dosage 50nM;

3)miR-NC+MUT-Mfsd8 group: add mimics NC and MUT-Mfsd8 plasmid for intervention, mimics NC, dosage 20nM; WT-Mfsd8 plasmid, dosage 50nM;

4)miR-143-3p mimics+MUT-Mfsd8 group: add miR-143-3p mimics and WT-Mfsd8 plasmid for intervention, miR-143-3p mimics, dosage 20nM; MUT-Mfsd8 plasmid, dosage 50nM;

Intervention 2:

1)miR-NC+WT-Mfsd8 group: add inhibitors NC and WT-Mfsd8 plasmid for intervention, inhibitorsNC, dosage 20nM; WT-Mfsd8 plasmid, dosage 50nM;

2)miR-143-3p inhibitors+WT-Mfsd8 group: add miR-143-3p inhibitors and WT-Mfsd8 plasmid for intervention, miR-143-3p inhibitors, dosage 20nM; WT-Mfsd8 plasmid, dosage 50nM;

3)miR-NC+MUT-Mfsd8 group: add inhibitors NC and MUT-Mfsd8 plasmid for intervention, inhibitors NC, dosage 20nM; WT-Mfsd8 plasmid, dosage 50nM;

4)miR-143-3p inhibitors+MUT-Mfsd8 group: add miR-143-3p inhibitors and WT-Mfsd8 plasmid for intervention, miR-143-3p inhibitors, dosage 20nM; MUT-Mfsd8 plasmid, dosage 50nM;

4. In order to detect the effect of miR-143-3p on chondrocyte ferroptosis in osteoarthritis by regulating the expression of Mfsd8, chondrocytes cultured in vitro were randomly divided into the following interventions:

1) NC group: Add inhibitors NC and Si-NC for intervention. Inhibitors NC, the dosage is 20nM; Si-NC, the dosage is 20nM;

2) NC+IL-1β group: Add IL-1β, inhibitors NC, and Si-NC for intervention. IL-1β, dosage 10ng/ml; inhibitors NC, dosage 20nM; Si-NC, dosage 20nM;

3) IL-1β+miR-143-3p inhibitors group: IL-1β, miR-143-3p inhibitors, and Si-NC were added for intervention at the same time. IL-1β, dosage 10ng/ml; miR-143-3p inhibitors, dosage 20nM; Si-NC, dosage 20nM.

4) IL-1β+miR-143-3p inhibitors+Si group: IL-1β, miR-143-3pinhibitors, and Si-Mfsd8 were added for intervention at the same time. IL-1β, dosage 10ng/ml; miR-143-3p inhibitors, The dosage is 20nM; Si-Mfsd8, the dosage is 20nM.

(2) Results evaluation method

1. In order to detect the changes in ferroptosis of chondrocytes during osteoarthritis and the regulatory effect of miR-143-3p on it, cell viability experiments (CCK8), lipid reactive oxygen species (lipid ROS) and lipid reactive oxygen species (lipid ROS) were performed after 48 hours of intervention on chondrocytes. ), MDA testing.

2. In order to detect the effect of miR-143-3p on the expression of Msfd8 in the process of osteoarthritis, chondrocytes were intervened for 48 hours and cell mRNA and protein were extracted. qPCR and western-blot were used to detect the expression changes of Msfd8 respectively.

3. In order to verify that Mfsd8 is the target gene of miR-143-3p in inhibiting ferroptosis of chondrocytes in osteoarthritis, the dual-luciferase reporter gene was used to confirm the effect of miR-143-3p on Mfsd8; intervention treatment was performed on chondrocytes 48 Hours later, CCK8, lipid ROS, and MDA were detected.

2. Experimental results

(1) Histological evaluation of cartilage destruction in osteoarthritis

1. As shown in Figure 1A, the results under the articular disc body microscope show that the shape of the articular disc in the NC group is complete and there is no congestion and perforation on the surface; the articular disc in the TMJOA group has obvious congestion and a rough surface; the surface wear and congestion of the articular disc in the TMJOA+Agomir group Compared with the TMJOA group, it was somewhat relieved.

2. As shown in Figure 1B, the results of Safranin O fast green staining showed that the cartilage surface in the NC group was smooth and complete, with no obvious wear and cracks; in the TMJOA group, the cartilage layer was significantly thinner, the cartilage was partially peeled off, and the cartilage appeared throughout the entire thickness. There are fewer cracks and detachments in the cartilage in the TMJOA+Agomir group than in the TMJOA group. The scope is smaller and the degree is lighter. OARSI score statistics show that the difference between the TMJOA group and the TMJOA+Agomir group is statistically significant (p<0.05). AgomiR-143-3p can effectively alleviate the surface wear and congestion of TMJOA articular disc.

(2) Immunofluorescence staining to evaluate changes in cartilage tissue-related proteins in each group

As shown in Figure 1B, COL2A immunofluorescence staining showed that the COL2A positive rate of chondrocytes in the NC group was higher, the COL2A positive rate of chondrocytes in the TMJOA group decreased significantly, and the COL2A positive rate of chondrocytes in the TMJOA+Agomir group was significantly higher than that in the TMJOA group. The differences are Statistical significance (p<0.05). The above results show that miR-143-3p can significantly increase the content of type II collagen in cartilage and alleviate the degradation of condylar cartilage in temporomandibular joint osteoarthritis.

(3) The regulatory effect of miR-143-3p on ferroptosis of condylar chondrocytes in osteoarthritis

1. As shown in Figure 2A, GPX4 immunofluorescence staining showed that the NC group had a higher GPX4 positive rate of chondrocytes, the TMJOA group had a significantly lower GPX4 positive rate of chondrocytes, and the TMJOA+Agomir group had a significantly higher GPX4 positive rate than the TMJOA group. The difference is statistically significant (p<0.05). This indicates that miR-143-3p regulates ferroptosis in condylar chondrocytes.

2. CCK8 detects the cell activity of each group after chondrocyte intervention. As shown in Figure 3B, IL-1β significantly reduced the cell activity of chondrocytes, while miR-143-3p could reverse the effect of IL-1β, and the difference was statistically significant (p<0.05).

3. Use MDA detection kit to detect changes in ferroptosis-related indicators in each group after chondrocyte intervention. As shown in Figure 3C, IL-1β induces ferroptosis and upregulates MDA concentration in chondrocytes, while miR-143-3p can reverse the effect of IL-1β and significantly reduce MDA concentration. The difference is statistically significant (p<0.05). .

4. Use the ROS detection kit to detect the changes in ROS in each group after chondrocyte intervention. As shown in Figure 3D, IL-1β induction significantly up-regulated ROS levels in chondrocytes, while miR-143-3p could reverse the effects of IL-1β and significantly reduce ROS levels, and the difference was statistically significant (p<0.05).

(4) The effect of miR-143-3p on ferroptosis of chondrocytes in osteoarthritis by regulating the target gene Mfsd8

1. As shown in Figure 3A, Mfsd8 immunofluorescence staining showed that the positive rate of Mfsd8 in chondrocytes in the NC group was lower, the positive rate of Mfsd8 in chondrocytes in the TMJOA group was significantly increased, and the positive rate of Mfsd8 in chondrocytes in the TMJOA+Agomir group was lower than that in the TMJOA group. The difference is statistically significant (p<0.05). The higher the positive rate of Mfsd8, the more serious TMJOA is.

2. Use qPCR to detect the changes in Mfsd8 transcription levels of cells in each group after chondrocyte intervention. As shown in Figure 3B and C, IL-1β can increase the expression of Mfsd8 transcript level, and the miR-143-3p mimic can reverse the effect of IL-1β and significantly reduce the expression of Mfsd8. The miR-143-3p inhibitor can further promote the increase in Mfsd8 expression induced by IL-1β, and the difference is statistically significant (p<0.05).

3. Use western-blot (immunoblotting test) to detect the changes in Mfsd8 protein levels of cells in each group after chondrocyte intervention. As shown in Figure 3D and E, IL-1β can increase the expression of Mfsd8 protein levels, and the miR-143-3p mimic can reverse the effects of IL-1β and significantly reduce the expression of Mfsd8. The miR-143-3p inhibitor can further promote the increase in Mfsd8 expression induced by IL-1β, and the difference is statistically significant (p<0.05).

4. As shown in Figure 3F, there are interaction sites between miR-143-3p and Mfsd8 that may bind to each other. The dual-luciferase reporter gene was used to verify the effect of miR-143-3p on Mfsd8. As shown in Figure 3G and H, the miR-143-3p mimic can significantly reduce the expression of Mfsd8, but this effect disappears when the binding site on Mfsd8 is mutated; the miR-143-3p inhibitor can significantly increase the expression of Mfsd8. , but this effect disappeared when the binding site on Mfsd8 was mutated, and the difference was statistically significant (p<0.05).

5. CCK8 detects the cell activity of each group after chondrocyte intervention. As shown in Figure 3I, both IL-1β and miR-143-3p inhibitors can significantly reduce the cell activity of chondrocytes; after knocking down Mfsd8, the effect of miR-143-3p inhibitor on reducing chondrocyte activity is reversed, and the difference is Statistical significance (p<0.05).

6. Use MDA detection kit to detect changes in ferroptosis-related indicators in each group after chondrocyte intervention. As shown in Figure 3J, IL-1β induces ferroptosis, and the miR-143-3p inhibitor can further promote the IL-1β-induced increase in MDA concentration in chondrocytes; after knocking down Mfsd8, the miR-143-3p inhibitor induces chondrocyte ferroptosis. The effect of cell ferroptosis was reversed, and the MDA concentration was significantly reduced, and the difference was statistically significant (p<0.05).

7. Use ROS detection kit to detect ROS changes in each group after chondrocyte intervention. As shown in Figure 3K, IL-1β induction significantly up-regulates ROS levels in chondrocytes, and the miR-143-3p inhibitor can further promote the up-regulation of ROS levels induced by IL-1β in chondrocytes; after knocking down Mfsd8, miR-143-3p inhibits The effect of drug-induced upregulation of ROS levels was reversed, ROS levels were significantly reduced, and the difference was statistically significant (p<0.05).

The above results show that in animal experiments on TMJOA model mice, increasing the expression level of miR-143-3p in the body by administering AgomiR-143-3p can effectively alleviate the surface wear and congestion of the TMJOA articular disc, and significantly improve cartilage The content of type II collagen can alleviate the degeneration and degradation of cartilage tissue in temporomandibular joint osteoarthritis and treat temporomandibular joint osteoarthritis; it has been further verified through cell experiments that miR-143-3p can effectively increase chondrocyte activity and reduce cartilage damage. Ferroptosis.

Example 2: Verification that miR-143-3p can inhibit the osteoclast activity of subchondral bone and reduce pain in the joint area of temporomandibular joint osteoarthritis

1. Experiments and methods

(1) Experimental animals

Eight-week-old female C57BL/6J mice were used in the experiment. All animals were raised at the West China Animal Experiment Center of Sichuan University and were raised under SPF standards.

(2) Experimental methods

1. Surgical modeling

When the mice were 9 weeks old, the unilateral anterior crossbite (UAC) model was constructed in the mice under general anesthesia, thereby inducing the formation of TMJOA and constructing the experimental TMJOA model mice.

2. Experimental grouping and intervention methods

The mice were randomly divided into three groups:

1) Negative control group (NC group): Inject the same amount of AgomiR-NC into the TMJ joint cavity for intervention, the dosage is 20 μM;

2) TMJOA group: The same amount of AgomiR-NC was injected into the TMJ joint cavity of TMJOA model mice for intervention, the dosage was 20 μM;

3) TMJOA+Agomir group: UAC was performed to construct the TMJOA model, and AgomiR-143-3p was injected into the TMJ joint cavity for intervention, with a dosage of 20 μM.

(3) Results evaluation method

1. Behavioral evaluation of the effect of miR-143-3p on temporomandibular joint osteoarthritis pain. After modeling, the pain threshold test (Vonfry test) in the temporomandibular joint area was performed every week.

2. Imaging evaluation of changes in bone density of subchondral bone tissue in each group

Three-dimensional X-ray microscopy (Micro-CT) was used to reconstruct and analyze the condylar morphology and surface bone changes in each group, and quantitative bone quality analysis of bone volume/total volume, trabecular thickness and trabecular separation was performed.

3. Histological evaluation of osteoclastic activity of subchondral bone in osteoarthritis

After 3 weeks of modeling induction, each experimental group started to receive different intervention intra-articular injections of TMJ every three days, and the mice were sacrificed at 6 weeks, and the temporomandibular joint tissue was collected and fixed with 4% paraformaldehyde for 48 hours and 10 Decalcify with % EDTA for one month, dewax, embed, and slice into 4um thick sections. TRAP staining was performed on the sections in each group and statistics were collected.

4. Histological evaluation of human calcitonin gene-related peptide (CGRP) expression in osteoarthritic cartilage

After 3 weeks of modeling induction, each experimental group started to receive different intervention intra-articular injections of TMJ every three days, and the mice were sacrificed at 6 weeks, and the temporomandibular joint tissue was collected and fixed with 4% paraformaldehyde for 48 hours and 10 Decalcify with % EDTA for two and a half months, dewax and embed, slice into 4um thick sections, and perform CGRP immunofluorescence staining on the sections of each group and statistics.

2. Experimental results

(1) Behavioral assessment of changes in pain thresholds in joint areas in each group

As shown in Figure 4A, the Vonfry pain threshold shows that the pain threshold of mice in the NC group increased with age in the first 14 days after the start of the study, and then remained in a relatively stable state; the pain thresholds of the TMJOA group and TMJOA+Agomir group at the start of the study were the same as those of the mice in the NC group. The NC group is comparable, but the pain threshold does not increase with age in the first 14 days; after modeling, it can be seen that the pain threshold of the TMJOA group fluctuates downward, and the decrease is larger; after modeling the pain threshold of the TMJOA+Agomir group, the pain threshold There was a slight decrease, but it was significantly higher than that in the TMJOA group.

(2) Imaging evaluation of changes in subchondral bone quality in each group

As shown in Figure 4B, Micro-CT three-dimensional reconstruction and analysis showed that the subchondral bone bone density of the TMJOA group decreased significantly, and the subchondral bone bone density of the TMJOA+Agomir group increased significantly compared with the TMJOA group, and the difference was statistically significant (p<0.05 ).

(3) Histological evaluation of subchondral bone osteoclast activity and CGRP expression changes in each group

1. As shown in Figure 5A, TRAP staining showed that the positive rate of TRAP in subchondral bone in the NC group was lower, the positive rate of TRAP in subchondral bone in the TMJOA group increased significantly, and the positive rate of TRAP in the subchondral bone in the TMJOA+Agomir group was significantly lower than that in the TMJOA group. The difference is statistically significant (p<0.05).

2. As shown in Figure 5B, CGRP immunofluorescence staining showed that the CGRP positive rate of subchondral bone in the NC group was lower, the CGRP positive rate of subchondral bone in the TMJOA group increased significantly, and the CGRP positive rate of subchondral bone in the TMJOA+Agomir group was significantly higher than that of the TMJOA group. decreased, the difference was statistically significant (p<0.05).

The above results show that miR-143-3p can reduce joint area pain, increase cartilage bone density, and relieve osteoarthritis by inhibiting the osteoclastic activity of subchondral bone.

In summary, the present invention increases the expression level of miR-143-3p by administering AgomiR-143-3p or miR-143-3p mimics, effectively alleviates the surface wear and congestion of TMJOA articular discs, and significantly increases type II collagen in cartilage. The content can alleviate the degeneration and degradation of cartilage tissue in temporomandibular joint osteoarthritis, reduce pain in the joint area, increase cartilage bone density, and treat temporomandibular joint osteoarthritis.

Claims (10)

1. Application of a substance for improving miR-143-3p expression level in preparation of medicines for treating temporomandibular joint osteoarthritis.

2. The use of claim 1, wherein the agent that increases expression levels is miR-143-3p, miR-143-3p micrometers, or miR-143-3p agomir.

3. The use of claim 1, wherein the nucleotide sequence of miR-143-3p is set forth in SEQ ID No. 1; the miR-143-3p micrometers nucleotide sequence is shown as SEQ ID NO. 2; miR-143-3p agomir comprises a forward sequence and a reverse sequence, and the nucleotide sequences are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

4. The use according to claim 1, wherein the medicament is a medicament for the treatment of temporomandibular joint osteoarthritis.

5. The use according to claim 1, wherein the medicament is a medicament for alleviating surface wear and hyperemia of a joint disc; the medicine can obviously improve the content of type II collagen in cartilage.

6. The use according to claim 1, wherein the medicament is a medicament capable of alleviating the degenerative degradation of cartilage tissue in temporomandibular joint osteoarthritis.

7. The use according to claim 1, wherein the medicament is a medicament that increases chondrocyte cell activity; the medicine is a medicine for reducing the activity of the subchondral bone of the temporomandibular joint osteoarthritis.

8. The use according to claim 1, wherein the medicament is a medicament that increases the level of GPX4 expression in chondrocytes and/or increases GPX4 enzymatic activity; the drug is a drug that reduces malondialdehyde levels in chondrocytes; the drug is a drug that reduces the level of lipid active oxygen in chondrocytes; the drug is a drug that reduces the expression level of Mfsd8 in chondrocytes.

9. The use according to claim 1, wherein the medicament is a medicament for increasing the density of subchondral bone of temporomandibular joint osteoarthritis.

10. The use according to claim 1, wherein the medicament is a medicament for alleviating pain in the arthritic joint region of the temporomandibular joint.