CN111000986A - Application of irisin in preparing medicine for preventing and treating osteoarthritis - Google Patents
Application of irisin in preparing medicine for preventing and treating osteoarthritis Download PDFInfo
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- CN111000986A CN111000986A CN201911259464.3A CN201911259464A CN111000986A CN 111000986 A CN111000986 A CN 111000986A CN 201911259464 A CN201911259464 A CN 201911259464A CN 111000986 A CN111000986 A CN 111000986A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
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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
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Abstract
The invention discloses an application of Irisin in preparing a medicament for preventing and treating osteoarthritis and a medicinal preparation for preventing and treating osteoarthritis. The experimental result shows that the Irisin can effectively inhibit the apoptosis of articular subchondral osteocyte, further improve the structures of cartilage and subchondral bone in arthritis, relieve the state of an illness and achieve the purpose of treatment. Therefore, Irisin provides a new approach for treating osteoarthritis.
Description
Technical Field
The invention belongs to the technical field of new application of medicaments, and particularly relates to application of irisin in preparation of medicaments for preventing and treating osteoarthritis.
Background
Irisin (Irisin) is a protein molecule which is mainly secreted by muscle tissues of human bodies and other mammals in motion and is sheared from fibronectin type III domain protein 5(FNDC5), previous researches find that Irisin plays an important role in the process of browning white fat, and brown fat can increase the fat metabolism of organisms and is considered as a potential treatment direction for diseases such as obesity and the like.
Osteoarthritis is an aging disease, the incidence of which in the population increases with the progress of aging of the population, placing a great burden on individuals and society. There is currently no effective medical/drug treatment to alleviate the condition of osteoarthritis.
Irisin is produced in large quantities during exercise, and moderate exercise is also an important intervention for preventing and treating osteoarthritis. The role of irisin in bone tissue has been studied in recent years, which can increase bone mass of skeletal cortical bone, and as an important molecule in skeletal muscle interaction process, which is expected to have therapeutic significance in bone tissue-related diseases.
The existing non-operative treatment of osteoarthritis mainly comprises the application of non-steroidal anti-inflammatory drugs (acetaminophen) and disease-improving drugs (glucosamine and sodium hyaluronate), but the treatment means cannot prevent the progress of the disease, and after the degeneration reaches a certain degree, the drug treatment cannot meet the treatment requirement, and still needs to be performed with surgical operation (artificial joint replacement), so that the later is not only high in economic cost, but also the physical condition of many old patients does not allow the operation.
Disclosure of Invention
In order to solve the existing problems, the invention aims to provide the application of irisin in preparing the medicines for preventing and treating osteoarthritis, thereby providing a new way for treating osteoarthritis.
In order to achieve the purpose, the invention provides the following technical scheme:
application of irisin in preparing medicine for preventing and treating osteoarthritis is provided.
A pharmaceutical preparation for preventing and treating osteoarthritis comprises effective amount of irisin or irisin and pharmaceutically acceptable adjuvants; the pharmaceutical preparation can be prepared by adopting the conventional preparation method in the field on the premise of not reacting with the active ingredient of the invention or influencing the curative effect of the medicament.
Further, the pharmaceutical preparation is suitable for pharmaceutical preparations for gastrointestinal or parenteral administration.
Furthermore, the pharmaceutical preparation is a freeze-dried preparation, an injection, a tablet, a granule or a capsule.
In the process of onset and progression of arthritis, in addition to cartilage degeneration, reconstruction change of subchondral bone is an important marker of the disease, and studies prove that the change is earlier than cartilage change and the subchondral bone can influence the severity of cartilage degeneration.
In vitro, the present invention uses the osteocyte cell line MLO-Y4 present in subchondral bone to simulate the excessive mechanical stimulation experienced in traumatic osteoarthritis by excessive tensile mechanical stimulation and also by H2O2Stimulation to mimic bone cell apoptosis caused by excessive stimulation of reactive oxygen species. The bone cell apoptosis after mechanical stimulation is verified by cck-8, flow cell number and PCR. Thereafter, we treated MLO-Y4 cells with Irisin at the indicated concentration (100ng/mL) and evaluated the effect of Irisin on bone cell apoptosis.
In vivo, the present invention uses a pre-crossed-ligament resection to induce a model of osteoarthritis in mice that causes knee instability and, in turn, arthritic cartilage and subchondral bone lesions. After the operation, Irisin is injected into the abdominal cavity, and the material is taken for 4 weeks for detection, so as to evaluate the effect of Irisin on the pathological changes of cartilage and subchondral bone.
Has the advantages that: the invention provides application of Irisin in preparing a medicament for preventing and treating osteoarthritis, and the Irisin can effectively relieve the phenotype of bone cell apoptosis by testing the effect of the Irisin on an MLO-Y4 cell apoptosis model through an in vitro experiment; meanwhile, the effect of Irisin on a mouse osteoarthritis model is tested through in vivo experiments, Irisin can effectively relieve cartilage degeneration, the expression of an apoptosis index Caspase-3 in subchondral bone is reduced after intervention by Irisin, and results of micro-CT and bone morphometry show that Irisin relieves bone loss of the subchondral bone of a mouse after operation and weakens bone absorption. According to the experimental results in vitro and in vivo, Irisin can be used as a medicine for inhibiting the apoptosis of articular subchondral bone osteocytes, further improving the structures of cartilage and subchondral bone in arthritis, relieving the state of illness and achieving the purpose of treatment. Therefore, Irisin provides a new approach for treating osteoarthritis.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of Irisin on the cell viability of the osteocyte in vitro cell line MLO-Y4.
FIG. 2 is a confocal microscope observation picture of Irisin after acting on an in vitro osteocyte cell line MLO-Y4.
FIG. 3 shows Irisin vs. H2O2Flow cytometric results after stimulation induced bone cell action.
FIG. 4 is a block diagram of the ShellPaPro apparatus.
FIG. 5 is a graph showing the flow cytometry results of Irisin on stretch-induced bone cells.
FIG. 6 is a graph showing the results of detection of an apoptosis-related indicator after the effect of Irisin on elongation-induced osteocytes.
Figure 7 is a graph of immunohistochemical results following Irisin's effect on a mouse osteoarthritis model.
Fig. 8 is a graph showing the results of analysis of subchondral bone of the tibia and cancellous bone below the growth plate after Irisin has acted on a mouse osteoarthritis model.
FIG. 9 is a graph showing the results of Trap staining of picric acid fuchsin and soft-cutting of hard tissue sections of mice after Irisin was applied to a mouse osteoarthritis model.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Example 1 in vitro experiment of the Effect of Irisin
First, the Effect of Irisin on the proliferation of the osteocyte in vitro cell line MLO-Y4
Recovering and culturing an in-vitro bone cell line MLO-Y4, inoculating the in-vitro bone cell line MLO-Y4 into a 96-well plate, after 12h, allowing cells to adhere to the wall, adding α -MEM culture medium (0-200ng/mL) containing Irisin with different concentrations during liquid change, after 24h, changing the liquid of the well plate into α -MEM culture medium containing 10% cck-8 reagent, placing the well plate in a 37-degree incubator for 2h, and detecting the absorbance at 450nm by using a microplate reader (650 nm is selected as a reference).
Cck-8 results of cell viability assay are shown in FIG. 1, and FIG. 1 shows that Irisin can promote the activity of MLO-Y4 cells within a certain concentration range (20, 50, 100ng/mL), so that the anti-apoptotic effect of Irisin can be tested by selecting a proper concentration according to the result.
An in vitro cultured MLO-Y4 cell line is used and inoculated into a collagen-I glue-spread confocal dish, after 12 hours, the cells are attached to the wall, the medium is changed into α -MEM medium containing 100ng/mL of Irisin, Edu staining is carried out for 24 hours, the cell nucleus is counterstained by using hochests, and observation is carried out by using a confocal microscope (Leica). the result of Edu staining is shown in figure 2, and the Irisin has obvious effect of promoting proliferation on MLO-Y4.
Second, effect of Irisin on apoptosis of bone cells
1. Irisin for H2O2Effect of stimulation of apoptosis of bone cells
Reference and self-validation, establishment H2O2Cell models that stimulate induction of bone cell apoptosis.
Using MLO-Y4 cell plate to collagen-I glue-spread 6-well plate, after 12h, cell adherence, changing the solution to Irisin containing 100ng/mL, after 24h culture, adding 300 mu M H2O2After 3h of stimulation, cells were collected, stained with FITC and PI, and observed on a flow machine. Flow cytometry observation showed that H is shown in FIG. 32O2After stimulation, the number of cells in early apoptosis and late apoptosis was significantly increased (3.6% vs 12.5%, 6.3% vs 12.9%), while in the Irisin intervention group (Irisin + H)2O2) Compared with H2O2The group then significantly reduced the apoptotic phenotype (12.9% vs 7.2%, 12.5% vs 7.2%).
2. Effect of Irisin on tension-induced apoptosis of osteocytes
A Minicon cell stretcher (ShellPaPro, Japan) shown in FIG. 4 is used for establishing stretching in vitro to induce MLO-Y4 cell apoptosis, MLO-Y4 cells are inoculated into a special stretching groove, after the cells are attached to the wall, 20% stretching amplitude, 1Hz stretching frequency and 24h stretching time are selected, and mechanical stimulation is carried out on the MLO-Y4 cells in vitro. And (3) changing the liquid in the tank after MLO-Y4 adheres to the wall, adding Irisin containing 100ng/mL, and then carrying out an in vitro stretching experiment according to the method. Collecting cells after stretching, and respectively carrying out (1) FITC-PI staining and flow-type on-machine detection; (2) PCR detects changes in apoptosis-related indicators.
The flow cytometry results are shown in fig. 5, from which it is concluded that MLO-Y4 cells undergo apoptosis after overstretch mechanical stimulation, whereas Irisin stem prognosis can significantly suppress the apoptotic phenotype.
The results of PCR detection of cells collected by tensile stimulation are shown in FIG. 6, and it is shown that tensile mechanical stimulation significantly increases the expression of the pro-apoptotic index Bax, while Irisin is used for dry prognosis, the change of Bax is not significantly changed compared with the control group, and the anti-apoptotic index Bcl-2 is significantly increased.
Example 2 in vivo experiment of Irisin Effect
In vivo animal experiments, we used pre-mouse cross-excision (ACLT) surgery to induce the osteoarthritic phenotype in mice.
The operation process is as follows: a12 w C57 male mouse was anesthetized by intraperitoneal injection with 7% chloral hydrate, then the mouse was disinfected, prepared for skin, the skin around the knee joint was exposed, a longitudinal incision of about 1cm was made in the inner side of the knee joint on the right side of the mouse using a scalpel, the patellar ligament was dissociated and dislocated, the anterior cruciate ligament was exposed, it was cut short using an ophthalmic scissors, the joint was repositioned, and the skin was sutured.
After surgery, mice were injected intraperitoneally with Irisin 4w continuously, once a week, 100. mu.g/time. And 4w later, taking materials from the right lower limb of the mouse, and detecting the change of the related indexes.
First, immunohistochemical staining analysis
The results of immunohistochemical staining of the joint of the mouse after decalcification are shown in fig. 7, and it is found from the figure that Irisin can significantly reduce the expression of MMP-13 in the cartilage tissue, thereby improving degeneration in the arthritic cartilage. In subchondral bone, a significant decrease in the expression of Caspase-3, an apoptosis indicator, was also observed, while this change was not observed in cartilage, considering that Irisin alleviates the symptoms of osteoarthritis by inhibiting apoptosis of subchondral bone.
Second, analysis of the tibial subchondral bone and cancellous bone below the growth plate
The analysis of the subchondral bone of the tibia and the cancellous bone below the growth plate of the mouse is performed, and the result is shown in fig. 8, and it is found from the figure that Irisin can improve the subchondral bone structure of the arthritic knee joint: the Irisin treated group had increased subchondral bone BV/TV, increased Tb.N, increased Conn-Dens, and decreased SMI. A similar phenomenon is also observed in cancellous bone.
Then, as a result of picric acid magenta staining and Trap staining for soft cutting on the hard tissue sections, as shown in fig. 9, it was observed that the bone resorption index (ES/BS) was significantly decreased after the action of Irisin, and it was also found that Irisin inhibited osteoclast activity during the progression of arthritis by Trap quantification.
In summary, the following conclusions are drawn from the above results:
(1) the following results were obtained by in vitro experiments: the model of simulating the apoptosis of osteocytes of an osteocyte cell line MLO-Y4 existing in subchondral bone through external stimulation, and after the MLO-Y4 cells are treated by Irisin, the Irisin can effectively relieve the apoptosis phenotype of the osteocytes.
(2) The following results were obtained by in vitro experiments: using a pre-crossed ligament resection to induce an osteoarthritis model in mice, surgery resulted in abrasion of mouse cartilage and in an increase in the ratio of calcified cartilage to hyaline cartilage, after treatment of MLO-Y4 cells with Irisin, Irisin alleviated cartilage degeneration in cartilage, in subchondral bone, the apoptosis indicator Caspase-3 was reduced in expression following intervention with Irisin, and the results of micro-CT and bone morphometry also indicate that Irisin alleviated bone loss and reduced bone resorption in subchondral bone of mice following surgery.
According to the experimental results in vitro and in vivo, Irisin can be used as a medicine for inhibiting the apoptosis of articular subchondral bone osteocytes, further improving the structures of cartilage and subchondral bone in arthritis, relieving the state of illness and achieving the purpose of treatment.
Claims (4)
1. Application of irisin in preparing medicine for preventing and treating osteoarthritis is provided.
2. A pharmaceutical preparation for preventing and treating osteoarthritis is characterized by comprising effective dose of irisin or irisin and pharmaceutically acceptable auxiliary materials.
3. The pharmaceutical formulation of claim 2, wherein the pharmaceutical formulation is suitable for parenteral administration.
4. The pharmaceutical preparation of claim 2, wherein the pharmaceutical preparation is a lyophilized preparation, an injection, a tablet, a granule or a capsule.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112773888A (en) * | 2021-02-04 | 2021-05-11 | 上海交通大学医学院附属第九人民医院 | Application of irisin in preparation of medicine for preventing and treating bone fracture |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170028018A1 (en) * | 2015-04-16 | 2017-02-02 | Universita' Degli Studi Di Bari | Irisin for care and prevention of osteoporosis |
| CN110101845A (en) * | 2019-05-24 | 2019-08-09 | 北京大学 | Application of the irisin in the drug that preparation prevention and treatment Postoperative Cognitive Dysfunction and blood-brain barrier are damaged mediated encephalopathy |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170028018A1 (en) * | 2015-04-16 | 2017-02-02 | Universita' Degli Studi Di Bari | Irisin for care and prevention of osteoporosis |
| CN110101845A (en) * | 2019-05-24 | 2019-08-09 | 北京大学 | Application of the irisin in the drug that preparation prevention and treatment Postoperative Cognitive Dysfunction and blood-brain barrier are damaged mediated encephalopathy |
Non-Patent Citations (1)
| Title |
|---|
| Z. HE等: "IRISIN attenuates osteoarthritis by inhibiting apoptosis of osteocytes through activating erk signaling pathway", 《OSTEOARTHRITIS AND CARTILAGE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112773888A (en) * | 2021-02-04 | 2021-05-11 | 上海交通大学医学院附属第九人民医院 | Application of irisin in preparation of medicine for preventing and treating bone fracture |
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