CN113181155A - Application of endogenous hydrogen sulfide donor in preparation of medicine for treating rheumatoid arthritis - Google Patents
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- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
本发明属于药物新用途领域，涉及内源性硫化氢供体在制备治疗类风湿性关节炎药物中的用途，其中NAV2作为靶点。本发明采用的试验包括SPRC抑制AIA大鼠炎症及NAV2表达试验、检测NAV2在类风湿性关节炎病人外周血中异常表达试验、NAV2通过激活Wnt/β－catenin信号通路促进MH7A细胞炎症反应试验等，结果表明：SPRC通过抑制NAV2表达缓解类风湿性关节炎，直接激活NAV2可以促进类风湿性关节炎发展，抑制NAV2表达可以对类风湿性关节炎起到显著的治疗效果。
The invention belongs to the field of new uses of medicines, and relates to the use of endogenous hydrogen sulfide donors in the preparation of medicines for treating rheumatoid arthritis, wherein NAV2 is used as a target. The tests used in the present invention include SPRC inhibiting AIA rat inflammation and NAV2 expression test, detecting NAV2 abnormal expression test in peripheral blood of rheumatoid arthritis patients, NAV2 promoting MH7A cell inflammatory response test by activating Wnt/β-catenin signaling pathway, etc. , the results showed that SPRC alleviates rheumatoid arthritis by inhibiting the expression of NAV2, directly activating NAV2 can promote the development of rheumatoid arthritis, and inhibiting the expression of NAV2 can have a significant therapeutic effect on rheumatoid arthritis.
Description
Technical Field
The invention relates to the field of new application of medicines, in particular to application of a novel endogenous hydrogen sulfide donor in preparing a medicine for treating rheumatoid arthritis with NAV2 as a target point.
Background
Neuron navigator 2(NAV2) was first identified as an all-trans retinoic acid (atRA) responsive gene in human neuroblastoma cells (retinoic acid-induced neuroblastoma 1, RAINB1) that extends neurites after exposure to atRA. It is structurally related to the caenorhabditis elegans unc-53 gene and is essential for cell migration and axon growth. The NAV2 protein is localized on the cell bodies and growing neurites of SH-SY 5Y cells in a pattern very similar to that of neurofilaments and tubulin. The vector deleted by NAV2 was transfected in Cos-1 cells, and a region of NAV2 protein (aa 837-1065) was found to be localized to the microtubule cytoskeleton. Hydrogen sulfide is a metabolite of cysteine and the like in the human body. Although the physiological concentration of the in vivo hydrogen sulfide is very low, researches show that the hydrogen sulfide is widely involved in the occurrence and development of various diseases, and particularly has obvious regulation effect on body inflammation.
Rheumatoid Arthritis (RA) is a common systemic autoimmune inflammatory disease characterized by painful, swollen joints that can severely impair bodily function and quality of life. Musculoskeletal pain, swelling and stiffness symptoms common in clinical practice, and thus familiarity with the diagnosis and management of RA is crucial. Patients with rheumatoid arthritis have a higher risk of developing severe infections, respiratory diseases, osteoporosis, cardiovascular diseases, cancer and death than the general population. RA seriously reduces the quality of life of patients and significantly increases the mortality of patients, with great threat to public health. In recent years, anti-rheumatoid arthritis drugs that are diagnosed early, actively treated, and expanded in treatment options have significantly improved the treatment and long-term prognosis of RA.
To date, treatment of RA patients has focused primarily on the basis of treatment of inflammation and indirect treatment of pain. At present, basic drugs such as antirheumatic drugs (DMARDs) and the like are mostly adopted clinically for treatment. These drugs typically take about 1 to 3 months to be effective. Some patients with poor curative effect need to take the medicine for a long time, and the life quality and the working capacity of the patients are seriously influenced. The burden on the family and the socioeconomic impact caused by this are not negligible. Therefore, rheumatism specialists at home and abroad try to develop a novel treatment scheme from various aspects. Patients expect that more safe and effective RA treatment drugs will be available early.
The existing research shows that hydrogen sulfide has a remarkable anti-inflammatory effect, but the curative effect of hydrogen sulfide on RA inflammation and pain is not clear.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the application of an endogenous hydrogen sulfide donor in treating the rheumatoid arthritis with NAV2 as a target point, and is beneficial to preparing a safer and more effective RA treatment medicament.
In order to achieve the purpose of the invention, the invention provides the application of an endogenous hydrogen sulfide donor in preparing a medicament for treating rheumatoid arthritis, wherein NAV2 is an action target point.
The further technical scheme is that the endogenous hydrogen sulfide donor can obviously reduce NAV2 protein expression, thereby relieving inflammation symptoms.
The further technical scheme is that the endogenous hydrogen sulfide donor is SPRC, namely propargyl cysteine.
In a further technical scheme, the NAV2 and the endogenous hydrogen sulfide donor are used for treating rheumatoid arthritis, including improving joint swelling and relieving pain reaction caused by inflammation.
The further technical proposal is that NAV2 and endogenous hydrogen sulfide donor can obviously reduce the expression of inflammatory protein and relieve inflammatory symptoms.
The further technical scheme is that the silent NAV2 expression can inhibit the proliferation and invasion of synovial fibroblasts and relieve inflammatory symptoms, and the endogenous hydrogen sulfide donor can reduce the NAV2 protein expression and also can inhibit the proliferation, migration and invasion of synovial fibroblasts so as to relieve inflammatory symptoms.
The further technical scheme is that the endogenous hydrogen sulfide donor reduces NAV2 expression, and can treat rheumatoid arthritis by reducing the activation of Wnt/beta-catenin pathway under inflammatory conditions.
Compared with the prior art, the invention can obtain the following beneficial effects:
the invention provides new application of NAV2 and hydrogen sulfide in inflammation, and particularly relates to application of NAV2 and hydrogen sulfide in preparation of a medicament for treating RA. According to the invention, the hydrogen sulfide endogenous donor SPRC can be used for treating RA and inhibiting inflammation, and is verified through rat arthritis model tests induced by AIA adjuvant, human rheumatoid arthritis MH7A cell RA model tests and the like, and test results show that the hydrogen sulfide endogenous donor SPRC can remarkably relieve inflammation symptoms of AIA rats and improve joint swelling; the high expression of NAV2 in RA can promote the development of inflammation, NAV2 can promote the proliferation, invasion and migration of MH7A cells by activating a Wnt/beta-catenin signal channel, which indicates that NAV2 is a novel therapeutic target of RA, and NAV2 can be used for preparing a medicament for treating RA; the endogenous hydrogen sulfide donor SPRC can be used for preparing a medicament for treating RA by inhibiting the expression of rat synovial tissue NAV2 and reducing the activation of a downstream Wnt/beta-catenin pathway.
Drawings
FIG. 1 shows SPRC dose-dependent relief of joint swelling in AIA rats; wherein FIG. 1(a) shows the results of the sham group, FIG. 1(b) shows the AIA group (model group), FIG. 1(c) shows AIA +25mg/kg SPRC, FIG. 1(d) shows AIA +50mg/kg SPRC, and FIG. 1(e) shows AIA +100mg/kg SPRC.
FIG. 2 shows the expression of the relevant inflammatory proteins in AIA rats after SPRC treatment; wherein FIG. 2(a) is a Western Blot chart, FIG. 2(b) is a relative expression level of MMP9, FIG. 2(c) is a relative expression level of ICAM-1, FIG. 2(d) is a relative expression level of IL-6, FIG. 2(e) is a relative expression level of Nrf2, and FIG. 2(f) is a relative expression level of CSE; in the figure, GAPDH is used as reference, and p < 0.05, p < 0.01 and p < 0.001, respectively, are indicated in comparison with the AIA rates group.
FIG. 3 shows that SPRC treatment can significantly reduce the expression of NAV2 protein in synovial tissue of AIA rats; protein expression levels were measured by Western Blot, and GAPDH was used as a reference.
FIG. 4 shows that NAV2 is abnormally highly expressed in the peripheral blood of rheumatoid arthritis patients; wherein FIG. 4(a) is a Western Blot chart, and FIG. 4(b) is the relative expression level of NAV 2; in the figure, p is < 0.001, compared with the health group or OA group.
FIG. 5 shows that NAV2 is abnormally highly expressed in synovial fibroblasts of patients with rheumatoid arthritis; wherein FIG. 5(a) is a Western Blot image, and FIG. 5(b) is a picture of the level of protein expression detected by immunofluorescence.
Fig. 6 shows that NAV2 may promote the development of MH7A cell inflammation; NAV2 and related inflammatory protein expression levels were measured as Western Blot, GAPDH as a reference.
FIG. 7 shows that NAV2 can activate Wnt/β -catenin signaling pathway; protein expression levels were measured by Western Blot.
Fig. 8 shows that NAV2 can promote MH7A cell proliferation, invasion and migration; detecting by a scratch experiment, a Transwell chamber experiment and a CCK-8 proliferation experiment; FIG. 8(a) is a graph showing the results of the experiment, FIG. 8(b) is cell proliferation, FIG. 8(c) is the rate of healing, and FIG. 8(d) is cell count; in the figure, p is < 0.001 compared to the TNF-alpha group.
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Detailed Description
Example 1
180g to 200g of SD rats were divided into 6 groups: (1) sham, (2) AIA, (3) AIA +1mg/kg MTX, (4) AIA +100mg/kg SPRC, (5) AIA +50mg/kg SPRC, (6) AIA +25mg/kg SPRC. Each group consisting of 15. Wherein the sham in the group (1) is a sham operation group; the AIA of group (2), model group, was established as an AIA adjuvant-induced arthritis model in rats by injecting 4mg/ml complete Freund's adjuvant subcutaneously in the tail root, and the pathological manifestations of AIA were similar to human rheumatoid arthritis; the group (3) is that methotrexate MTX with corresponding dose is injected into the abdominal cavity after the model is made; groups (4) to (6) were performed by oral gavage of the corresponding dose of SPRC after molding. After 28 days of continuous administration, relevant indexes were examined.
The results show that SPRC dose-dependently reduced the severity of joints in AIA rats, improved joint swelling, and was able to alleviate pain from inflammation, as shown in FIG. 1. As shown in FIG. 2, Western Blot showed that SPRC treatment dose-dependently decreased MMP-9, ICAM-1, and IL-6 expression in inflammatory joints, and in addition, SPRC treatment significantly increased CSE and Nrf2 expression in inflammatory joints. As shown in fig. 3, SPRC significantly reduced NAV2 protein expression.
This example demonstrates that SPRC is able to inhibit joint inflammation in AIA rats.
Example 2
Blood was collected from RA patients, Osteoarthritis (OA) patients and Healthy (health) volunteers in the Rheumatology department of Longhua hospital, Shanghai university of medicine, with the informed consent signed. All epidemiological investigations and classifications from volunteers, in which N is the number of people and NA indicates no assessment, were performed according to the criteria of the american society for rheumatic diseases, as shown in table 1 below.
TABLE 1 RA, OA and healthy volunteer data sheet
| Parameter(s) | RA(N＝20) | 0A(N＝9) | Healthy(N＝6) |
| Age (mean (range)) | 61(37～79) | 56(58～75) | 46(18～65) |
| Gender (number of people (woman/man)) | 18/2 | 7/2 | 3/3 |
| Rheumatoid factor > 20IU/mL | 16/20 | NA | NA |
The separation and extraction of leukocytes is performed first on clinical blood samples, followed by subsequent experiments. The Western Blot results show that NAV2 expression was significantly increased in the blood of RA patients compared to OA patients or healthy samples, as shown in figure 4.
This example shows that NAV2 is abnormally highly expressed in the peripheral blood of rheumatoid arthritis patients.
Example 3
Synovial tissue from 6 patients with Rheumatoid Arthritis (RA) following knee replacement was collected, and synovial tissue from 6 patients with Osteoarthritis (OA) synovial lesions following a synovium resection procedure was collected. Extraction and culture of primary fibroblast-like synoviocytes (FLS) were performed and subsequent experiments were performed. As shown in fig. 5, Western Blot and cellular immunofluorescence results showed a significant increase in expression of NAV2 in RA synovial fibroblasts relative to OA synovial fibroblasts.
This example demonstrates abnormally high expression of NAV2 in synovial fibroblasts of rheumatoid arthritis patients.
Example 4
Activation of the Wnt/β -catenin pathway in MH7A cells under inflammatory conditions leads to inflammation and enhanced proliferation, invasion and migration. MH7A cell RA model assay: (1) TNF- α + si NAV2 group: MH7A cells are transfected with si NAV2 small interfering RNA to silence the expression of NAV2, after transfection for 48 hours, an inflammation model is established by 20mg/kg TNF-alpha stimulation, and detection is carried out after 12 hours; (2) TNF-. alpha. + SiScr group: MH7A cells were transfected with siScr small interfering RNA to silence the expression of Scr, the remaining steps were identical to the TNF-. alpha. + si NAV2 group; (3) TNF- α group: an inflammation model is directly established by TNF-alpha stimulation; (4) control group, i.e. blank group. The Western Blot results show that NAV2 promotes inflammatory response (as shown in fig. 6), activates Wnt/β -catenin signaling pathway (as shown in fig. 7), and promotes proliferation, invasion and migration of MH7A cells (as shown in fig. 8).
This example demonstrates that NAV2 promotes MH7A cellular inflammatory responses by activating the Wnt/β -catenin signaling pathway.
The test results of the above examples are combined, and the hydrogen sulfide endogenous donor SPRC can remarkably relieve the inflammation symptoms of AIA rats and improve the joint swelling, and can be used for preparing the medicine for treating RA. Further research shows that the high expression of NAV2 in RA can promote the generation and development of inflammation, NAV2 can promote the proliferation, invasion and migration of MH7A cells by activating Wnt/beta-catenin signal channel, which indicates that NAV2 is a novel therapeutic target of RA, and NAV2 can be used for preparing a medicament for treating RA. The therapeutic effect of SPRC is achieved by inhibiting the expression of NAV2, which is capable of inhibiting the activation of the downstream Wnt/β -catenin pathway.
Finally, it should be emphasized that the above-described embodiments are merely preferred examples of the invention, which is not intended to limit the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
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| CN114246845A (en) * | 2021-11-01 | 2022-03-29 | 澳门科技大学 | Endogenous hydrogen sulfide sustained release preparation, and preparation method and application thereof |
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| CN110496105A (en) * | 2019-08-07 | 2019-11-26 | 澳门科技大学 | A microsphere preparation loaded with S-propargyl cysteine and its preparation method |
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| CN110478320A (en) * | 2019-08-07 | 2019-11-22 | 澳门科技大学 | Carry the meso-porous titanium dioxide silicon-agent and preparation method thereof of S- S-propargyl-cysteine |
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| CN114246845A (en) * | 2021-11-01 | 2022-03-29 | 澳门科技大学 | Endogenous hydrogen sulfide sustained release preparation, and preparation method and application thereof |
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