CN113577051A

CN113577051A - Application of verapamil in preparation of product for protecting chondrocytes and relieving osteoarthritis

Info

Publication number: CN113577051A
Application number: CN202111049774.XA
Authority: CN
Inventors: 赵杰; 杨二柱; 曹贤坤; 秦安
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2021-11-02

Abstract

The invention relates to an application of verapamil in preparing a product for protecting chondrocytes and relieving osteoarthritis, wherein the product is a composition or a preparation containing the verapamil, or an optical isomer thereof, or a pharmaceutically acceptable salt thereof. Compared with the prior art, the invention discovers that verapamil can promote the expression of the cartilage extracellular matrix by efficiently reducing the level of TXINIP protein in cartilage cells, thereby having the functions of protecting the cartilage cells and relieving osteoarthritis. Therefore, verapamil has a safe and effective active ingredient for protecting chondrocytes to alleviate osteoarthritis.

Description

Application of verapamil in preparation of product for protecting chondrocytes and relieving osteoarthritis

Technical Field

The invention relates to the field of medicines, in particular to application of verapamil in protecting chondrocytes and relieving osteoarthritis.

Background

Osteoarthritis (OA) is an irreversible degenerative disease, accompanied by progressive destruction of the articular cartilage. Currently, about 7% of the world's population is affected by OA, while women are more severely affected. Recent research data show that the number of people affected by OA worldwide has increased by 48% in the last 30 years, while OA is the 15 th most global disability factor in the statistics of 2019, and the number of disabilities accounts for 2% of the total number of disabilities worldwide. This problem may be exacerbated as the population ages and the growing population of obese people. Therefore, the emphasis and enhancement of research and exploration for OA have been reluctant. At present, clinically, aiming at patients with early mild degenerative OA, health promotion, rehabilitation exercise, weight control, some non-steroidal anti-inflammatory drugs, intra-articular drug injection and other symptomatic treatments are recommended; the operation treatment is recommended for patients with advanced degeneration, severe pain and obvious dysfunction. The development of OA is a complex process involving a variety of factors including genetic traits, aging, chronic inflammation and oxidative stress. Since the studies on the intrinsic mechanism of OA are not clear, specific drugs from which OA progression can be blocked are still lacking at the present stage.

The change of cartilage tissues in the pathophysiological process of OA can be found to be particularly obvious in clinical and basic researches, so that the metabolism, differentiation and maturation of chondrocytes and the secretion of cartilage matrixes are key points for researching the intrinsic mechanism of OA. Therefore, the research of clinically applied medicines has very important clinical diagnosis and treatment and basic scientific research values for the protection of the chondrocytes.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a safe and effective application of verapamil in preparing a product for protecting chondrocytes and relieving osteoarthritis.

The purpose of the invention can be realized by the following technical scheme:

in a first aspect of the invention, there is provided the use of verapamil, or an optical isomer thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a composition or formulation for protecting chondrocytes from osteoarthritis.

In another preferred embodiment, the composition or formulation is for one or more applications selected from the group consisting of:

(i) promoting articular chondrocyte matrix expression;

(ii) reducing the intracellular expression level of TXNIP;

(iii) reducing damage to chondrocytes from oxidative stress, or improving the physiological state of chondrocytes;

(iv) reducing damage to chondrocytes from inflammatory stimuli, or improving the physiological state of chondrocytes;

(v) increase the thickness of articular cartilage and slow down the development of osteoarthritis;

(vi) improving the stress and repair capability of the articular cartilage.

In another preferred embodiment, the composition is a pharmaceutical composition, nutraceutical composition, dietary supplement or food composition.

In another preferred embodiment, the formulation comprises a dietary supplement, a food additive, or a test agent.

In another preferred embodiment, the pharmaceutical composition comprises (a) verapamil or a pharmaceutically acceptable salt thereof as an active ingredient; and (b) a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the pharmaceutical composition further comprises an additional active ingredient for protecting chondrocytes and relieving osteoarthritis.

In another preferred embodiment, the verapamil is present in the pharmaceutical composition in an amount of 0.001 to 99 wt%, preferably 0.01 to 90 wt%, based on the total weight of the composition.

In another preferred embodiment, the pharmaceutical composition is an injectable or oral drug.

In another preferred embodiment, the pharmaceutical composition is in a form selected from the group consisting of: tablets, capsules, granules, suspensions, pills, solutions, syrups, or injections.

In another preferred embodiment, the composition or formulation is administered to a mammal.

In another preferred embodiment, the mammal comprises: a primate; more preferably the mammal is a human.

In another preferred embodiment, the cell is a somatic cell.

In another preferred embodiment, the cell is selected from the group consisting of: bone marrow mesenchymal stem cells, osteoprogenitor cells, osteogenic precursor cells, osteoblastic cells, chondroprogenitor cells, chondrocyte cells, or a combination thereof.

In another preferred embodiment, said protecting chondrocytes to relieve osteoarthritis comprises protecting chondrocytes from skin to relieve osteoarthritis.

In a second aspect of the present invention, there is provided a nutraceutical composition comprising verapamil as an effective ingredient for protecting chondrocytes and alleviating osteoarthritis.

In another preferred embodiment, the verapamil is present in the nutraceutical composition in an amount of 0.001 to 99 wt%, preferably 0.01 to 90 wt%.

In a third aspect of the present invention, there is provided a cosmetic composition comprising verapamil as an effective ingredient for protecting chondrocytes and relieving osteoarthritis.

In another preferred embodiment, the content of verapamil in the cosmetic composition is 0.001 to 99% by weight, preferably 0.01 to 90% by weight.

In a fourth aspect of the present invention, there is provided a use of verapamil for non-therapeutically reducing the expression level of TXNIP protein in vitro, the method comprising the steps of:

adding verapamil or a salt thereof to a cell culture system, thereby reducing the expression level of the TXNIP protein in the cells;

or culturing the cells in the presence of verapamil or a salt thereof, thereby reducing the expression level of the TXNIP protein in the cells.

In another preferred embodiment, verapamil is present in the culture system at a concentration of 0.01-100. mu.M, preferably 0.05-50. mu.M, more preferably 0.1-20. mu.M, most preferably 0.5-10. mu.M.

In another preferred embodiment, the cell is a eukaryotic cell.

In another preferred embodiment, the cells are derived from a mammal, preferably human cells.

In another preferred embodiment, the cell is a somatic cell.

In a fifth aspect of the present invention, there is provided a use of verapamil in protecting chondrocytes and alleviating osteoarthritis, comprising the steps of: verapamil, or an optical isomer thereof, or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof as an active ingredient for protecting chondrocytes from alleviating osteoarthritis.

In another preferred embodiment, the subject in need thereof is a mammal, said mammal comprising: a primate; more preferably the mammal is a human.

In a sixth aspect of the present invention, there is provided a pharmaceutical composition for protecting chondrocytes from osteoarthritis, the pharmaceutical composition comprising:

(a1) verapamil or a pharmaceutically acceptable salt thereof as an active ingredient;

(a2) an additional chondrocyte protective osteoarthritis relief active ingredient, said additional chondrocyte protective osteoarthritis relief active ingredient being selected from the group consisting of: melatonin (which can reduce TXNIP levels);

(b) a pharmaceutically acceptable carrier or excipient.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention proves that verapamil can effectively reduce the TXINIP level in chondrocytes and promote the expression of extracellular matrix for the first time. The verapamil can maintain the stability of the cartilage matrix and delay the development of joints by promoting the expression of cartilage extracellular matrix.

2. Through the mode of local administration in joints, the verapamil has the effects of protecting chondrocytes and relieving osteoarthritis, and avoids systemic influence caused by systemic administration.

3. The invention firstly discovers that the verapamil (or a composition and a product containing the verapamil) as an active ingredient can be applied to various aspects such as cartilage cell protection, osteoarthritis disease incidence delay and the like.

4. The verapamil or the optical isomer or the pharmaceutically acceptable salt thereof selected by the invention can be prepared into various pharmaceutical compositions, health care product compositions, dietary supplements, food additives, test reagents or cosmetic compositions, and can be applied to a needed object through various modes, so that the use is convenient. Joint replacement surgery, operative osteotomy to protect joints or mixed injection of multiple medicines in joints are generally selected for treating terminal osteoarthritis in clinical work.

5. The verapamil content in the composition or preparation is controlled to be 0.001-99 wt% according to the severity of arthritis, and only 0.001% of verapamil is added for the preventive protection of osteoarthritis and the effective differentiation of osteoprogenitor cells in subchondral bone into chondrocytes, and 99% of verapamil is added for the improvement of joint cartilage proliferation metabolism and extracellular matrix expression for osteoarthritis with clinical symptoms, and local intra-articular single drug injection is generally performed without combination of other drugs.

Drawings

FIG. 1 is a graph showing the results of in vitro chondrocyte line experiments

Wherein: TXNIP agonists inhibit extracellular cartilage matrix expression, TXNIP inhibitors have a protective effect; two inflammatory factors, namely IL-1 beta and TNF-alpha, stimulate an ATDC5 cell line, the TXNIP expression is increased, and the secretion of cartilage matrix in high-density culture in vitro is reduced; C. the fluorescent double-label indicates that TXINIP expression is increased, and the expression of chondrocyte Collagen II is reduced. Significant differences relative to control: denotes P < 0.05.

FIG. 2 is the mouse DMM joint model research picture

Wherein: A. the X-ray examination of the knee joint of the mouse shows that Verapamul has the protective effect on the articular cartilage of the mouse; B. mouse knee tissue sections were H & E stained. C. Safranin fast green staining, Verapamil inhibited TXNIP levels, promoted cartilage extracellular matrix expression, maintained cartilage tissue thickness. Denotes P < 0.05. Denotes P < 0.01.

FIG. 3 is the mouse DMM joint model research picture

Wherein: A. the mouse knee joint X-ray examination shows that joint degeneration is aggravated by the DZNep effect, and the gap collapse is obvious; B. mouse knee tissue sections were H & E stained. C. Safranin fast green staining, TXNIP agonists aggravate joint degeneration with a marked decrease in cartilage tissue thickness. Denotes P < 0.05. Denotes P < 0.01.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Through relevant and intensive research, the inventor unexpectedly finds that verapamil can promote the expression of the cartilage extracellular matrix by efficiently reducing the level of TXINIP protein in cartilage cells, thereby having the functions of protecting the cartilage cells and relieving osteoarthritis. Therefore, verapamil has a safe and effective active ingredient for protecting chondrocytes to alleviate osteoarthritis. On the basis of this, the present invention has been completed.

Specifically, the mouse is used as a model animal, and a classical arthritis model (DMM model) experiment of the mouse is established to prove that the verapamil can reduce the level of TXINIP protein in chondrocytes, promote the expression of chondrocyte matrixes, protect the chondrocytes and relieve osteoarthritis.

Term(s) for

As used herein, the terms "active ingredient", "chondrocyte-protecting ingredient for alleviating osteoarthritis", "composition", "chondrocyte-protecting composition or formulation" refer to verapamil, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or an optical isomer thereof, or a racemate thereof, or a crystal thereof, or a hydrate thereof, or a deuterated compound thereof, or a combination thereof.

As used herein, the term "TXNIP" refers to a Thioredoxin interacting protein (TXNIP).

Osteoarthritis and protection of chondrocytes for relief of osteoarthritis

As used herein, Osteoarthritis (OA) is an irreversible degenerative disease, accompanied by progressive destruction of the articular cartilage.

As used herein, "protecting chondrocytes from osteoarthritis" refers to delaying, arresting, reducing, halting and/or reversing the progression of degeneration.

The change of cartilage tissue is particularly obvious in the pathophysiological process of OA, so that the metabolism, differentiation and maturation of chondrocytes and the secretion of cartilage matrixes are key points for researching the internal mechanism of OA. The chondrocytes can normally maintain metabolic balance in a physiological environment and well control the oxidation-reduction reaction in the differentiation and maturation process.

TXNIP is an important physiological inhibitor of the intracellular Thioredoxin (TXN) redox system, is expressed in various organs and tissues, and plays a role in regulating intracellular redox reactions. TXNIP plays an important role in redox regulation and is also involved in many important physiological processes such as inflammatory reactions, carbohydrate metabolism, and the like. Mouse chondrocyte line (ATDC5)

ATDC5 cell is mouse chondrogenesis cell line from teratoma cell, under the stimulation of insulin, it differentiates into cartilage cell to form cartilage nodule, and expresses II type collagen and X type collagen to mineralize finally to reflect chondrogenesis process.

Mouse arthritis model (DMM)

Surgical construction of a mouse model of medial meniscal instability (DMM) has been widely used in the mechanistic study of osteoarthritis.

Active ingredient

The cartilage cell protecting and osteoarthritis relieving active ingredient is verapamil or a pharmaceutically acceptable salt thereof.

Verapamil (verapamil) is a class of phenylalkylamine type calcium channel blockers used clinically to treat hypertension, arrhythmia and angina pectoris. The chemical formula is as follows: C27H38N2O4, molecular weight: 454.6110. the structural formula is shown as the formula:

in the present invention, a preferred active ingredient is a pharmaceutically acceptable salt of verapamil, and a typical pharmaceutically acceptable salt is the hydrochloride salt, i.e., verapamil hydrochloride (B1867). The commonly used verapamil hydrochloride is the hydrochloride salt of 1 equivalent of verapamil with 1 equivalent of hydrochloric acid.

Verapamil is a calcium channel blocker (calcium antagonist). The slow release impulse from the sinoatrial node and slow conduction are due to the inhibition of calcium influx which reduces the rate of automatic depolarization during diastole. Forward conduction can be slowed down and atrioventricular node reentry can be eliminated. It has a dilating effect on peripheral blood vessels, causing a drop in blood pressure, but is weaker and generally causes a slower heart rate, but also a faster reflex heart rate due to a drop in blood pressure. Has dilating effect on coronary artery, increasing coronary blood flow, and improving myocardial oxygen supply.

Pharmaceutical compositions and methods of administration

The invention also provides a composition or a preparation or a product containing the active ingredients of the invention, and the composition or the preparation or the product can be used for protecting the chondrocytes and relieving osteoarthritis. Representative compositions or preparations or products comprise medicines and health products for protecting chondrocytes and relieving osteoarthritis.

One preferred composition is a pharmaceutical composition comprising an effective amount of verapamil or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in humans and/or animals (i.e., protects chondrocytes to relieve osteoarthritis) and is acceptable to humans and/or animals.

As used herein, an ingredient of the term "pharmaceutically acceptable" is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, gel, and combinations thereof. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the active ingredient of the invention is administered at a daily dose of about 0.01mg to 50mg per kg of animal body weight, preferably 0.1mg to 10mg per kg of animal body weight. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

Typically, when verapamil is administered by local intra-articular injection, the daily average dose in a subject (human) of 60kg body weight is usually 0.6-3000mg, preferably 6-600mg, more preferably 45-90 mg.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

Method for protecting chondrocytes and relieving osteoarthritis

The present invention provides a method of protecting chondrocytes from osteoarthritis (including therapeutic methods, or in vitro non-therapeutic methods), as well as a method of inhibiting TXNIP protein expression and/or activity.

Typically, the method comprises: adding the pharmaceutical composition of the present invention or the active ingredient of the present invention to the cultured cell system, thereby promoting the progress of the expression of the cartilage extracellular matrix; or reducing the expression amount of TXNNIP protein.

In another preferred embodiment, the cells are ATDC5 cells, especially normal somatic cells.

The invention also provides a method for protecting chondrocytes and relieving osteoarthritis, which comprises the following steps: verapamil, or an optical isomer thereof, or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof as an active ingredient for protecting chondrocytes from alleviating osteoarthritis.

Preferably, the method for protecting chondrocytes to relieve osteoarthritis is to administer (or apply) a health product containing verapamil or a salt thereof to the periphery of a joint of the subject.

In the present invention, representative cosmetic formulations may be selected from the following group (but are not limited to): solid dosage forms, semi-solid dosage forms, liquid dosage forms, or combinations thereof, such as solutions, gels, creams, lotions, essences, transdermal patches, masks, and the like.

The main advantages of the invention include:

(a) the verapamil is proved to be capable of effectively reducing the TXINIP level in chondrocytes and promoting the expression of extracellular matrix for the first time.

(b) The verapamil can maintain the stability of the cartilage matrix and delay the development of joints by promoting the expression of cartilage extracellular matrix.

(c) Through the mode of local administration in joints, the verapamil has the effects of protecting chondrocytes and relieving osteoarthritis, and avoids systemic influence caused by systemic administration.

(d) Verapamil has a high safety in use as an old drug which has been on the market.

Therefore, the verapamil (or the composition and the product containing the verapamil) which is the active ingredient can be applied to various aspects such as the protection of chondrocytes, the delay of the incidence rate of osteoarthritis and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1.

In vitro cell experiment of verapamil

1.1 methods

Verapamil hydrochloride (B1867) available from APExBIO corporation; the ATDC5 chondrocyte line was provided by the orthopedics laboratory of the ninth national hospital affiliated to shanghai university of transportation.

IC50 values for Verapamil (TXNIP inhibitor)/dzneep (TXNIP agonist) were determined by CCK 8. Verapamil (TXNIP inhibitor)/DZNep (TXNIP agonist) stimulates the ATDC5 chondrocyte line in vitro under the effective drug concentration, and the proliferation, differentiation and apoptosis conditions of the cells are observed. The TXNIP protein was detected as a change in level after drug stimulation using WB. The effect of Verapamil (TXNIP inhibitor)/dzneep (TXNIP agonist) on cartilage differentiation maturation and cartilage extracellular matrix secretion was confirmed by high density in vitro, daily dosing with ATDC5, stimulation for 7 days, Alician blue staining, and observation of extracellular matrix. The influence of Verapamil (TXNIP inhibitor)/DZNep (TXNIP agonist) on cartilage differentiation maturation and cartilage extracellular matrix secretion was verified by in vitro balling culture with ATDC5, stimulation by daily administration, and observation of extracellular matrix by section staining after 4 weeks of culture. Frozen sections were performed in combination with immunofluorescence to verify the effect of Verapamul (TXNIP inhibitor) on chondrocyte lineage differentiation maturation and cartilage matrix secretion.

1.2 results

The experimental data are shown in figure 1.

FIG. 1 is a graph showing the results of an in vitro chondrocyte line experiment.

Wherein: A. the upper left is a Western immunoblot (Western Blot, WB) diagram after stimulating the ATDC5 cell line with the in vitro TXNIP agonist and inhibitor, and the results show that the TXNIP expression is promoted after stimulation with dzzep and is inhibited by Verapamil, wherein beta-actin is the reference protein of the control; the upper right is a WB band gray value quantitative analysis, and the result shows that TXNIP expression is increased after DZNep stimulation, and TXNIP expression is reduced after Verapami stimulation. The low part is an in vitro high-density culture picture of ATDC 5. The results show that after DZNep stimulation, the extracellular cartilage matrix expression is reduced, the staining area is reduced, and the color depth is reduced; verapamul stimulates the increase of posterior volume and deepens the color. The TXNIP agonist DZNep promotes the expression of TXNIP by a chondrocyte line and inhibits the expression of cartilage extracellular matrix; verapamil reduces TXNIP expression, promotes cartilage extracellular matrix expression, and has protective effect.

B. The ATDC5 cell line was stimulated by two inflammatory factors, IL-1 β and TNF- α. The upper panel shows WB results under stimulation with two inflammatory factors showing increased TXNIP expression compared to control, where β -actin is the control reference protein. The lower part is an in vitro high-density culture picture of ATDC5, the expression of TXNIP is increased under the stimulation of two inflammatory factors, the secretion of cartilage matrix in vitro high-density culture is reduced, the picture display area is reduced, and the color is lightened.

C. Immunofluorescent double-label staining was performed on in vitro ATDC5 chondrocyte culture sections. The result shows that the TXNIP inhibitor Verapamil inhibits the TXNIP expression quantity and promotes the cartilage matrix expression (Coll 2); in contrast, the TXNIP agonist dzneep promotes increased TXNIP expression, inhibiting chondrocyte Collagen II expression. Significant differences relative to control: denotes P < 0.05.

Research on DMM joint model of mice

2.1 method

Selecting adult (12-week-old) wild-type mice as a blank control group, a surgery control group, establishing a DMM arthritis model for 15 mice respectively with low concentration (Verapamul-0.7 mg/KG) and high concentration (Verapamul-1.4 mg/KG), administrating drug in joints 2 times per week after modeling, administrating drug for 8 weeks totally, taking 3 random materials from each group at 4, 8 and 12 weeks after surgery respectively, performing knee joint histological staining, and observing the physiological phenotype of cartilage through H & E staining, safranin solid-green staining, sirius red staining and immunofluorescence staining. Performing knee joint histological staining, histological H & E staining, safranin fast green staining, sirius red staining and the like to observe differences of chondrocytes and extracellular matrix, performing quantitative analysis, and performing immunohistochemical and immunofluorescence to observe the expression condition of cartilage related protein.

The same approach verifies the effect of DZNEP on osteoarthritis after TXNIP agonism. Adult (12-week-old) wild-type mice are selected to be divided into a blank control group, a surgery control group, 15 DMM arthritis models are respectively established for low concentration (DZNep-0.1mg/KG) and high concentration (DZNep-1mg/KG), the DMM arthritis models are administrated in joints 2 times per week after modeling, the administration lasts for 8 weeks, 3 random materials are respectively taken from each group at 4, 8 and 12 weeks after surgery, the knee joint histological staining is carried out, and the physiological phenotype of cartilage is observed through H & E staining, safranin fast green staining, stellar red staining and immunofluorescence staining. Performing knee joint histological staining, histological H & E staining, safranin fast green staining, sirius red staining and the like to observe differences of chondrocytes and extracellular matrix, performing quantitative analysis, and performing immunohistochemical and immunofluorescence to observe the expression condition of cartilage related protein.

2.2 results

The results of the experiment are shown in fig. 2 and 3.

FIG. 2 is a graph showing the results of the mouse DMM joint model Verapamul intra-articular administration study (2 times per week, intra-knee injection administration, 8 weeks after total administration).

Wherein: A. performing X-ray examination on the knee joints of the mice, namely, respectively performing blank control (injecting PBS with the same volume in the non-operated joints), operation control (DMM operation + PBS), low concentration (DMM + Verapamul-0.7 mg/KG) and high concentration (DMM + Verapamul-1.4 mg/KG), wherein the result shows that the gap height of the knee joints of the mice in the blank control is well maintained, and no obvious bone is formed around the joints; narrowing the joint space and peripheral hyperosteogeny in the operation control group; the Verapamil treatment group (low and high concentration) had improved joint space height and reduced periarticular hyperplasia. The results prove that Verapamul has a protective effect on mouse articular cartilage.

B. H & E staining of mouse knee joint tissue sections shows that the blank control group mouse knee joint femur distal end and tibia proximal end tissue structure is complete, the shape is regular, and the articular cartilage surface is smooth; the joint clearance of the operation control group becomes narrow, the structure is disordered, and the surface of the articular cartilage is rough and degenerated; the structure forms of the distal femur and the proximal tibia in the joint space of the Verapamil treatment group (low concentration and high concentration) are improved, and the cartilage surface tends to be smooth and complete.

C. The safranin is subjected to fast green staining, and the result shows that (on the left side of the figure 2. C) the articular cartilage on the surfaces of the distal femur and the proximal tibia of the knee joint of the blank control group mice is regular in shape and good in thickness maintenance; the joint clearance of the operation control group becomes narrow, and the surface of the articular cartilage becomes rough and thin; the cartilage structure morphology of the surfaces of the distal femur and the proximal tibia in the joint space of the Verapamil treatment group (low concentration and high concentration) is improved, and the cartilage surface tends to be smooth and the whole thickness is increased. Statistical analysis of cartilage thickness on knee surface (right side of fig. 2. C) Verapamil treated DMM mouse arthritis model showed a significant improvement in cartilage thickness compared to the control group following surgery alone. The above results demonstrate that Verapamil inhibits TXNIP levels, promotes chondrocyte extracellular matrix expression, and maintains cartilage tissue thickness. Denotes P < 0.05. Denotes P < 0.01.

FIG. 3 is a graph showing the results of the intra-articular administration study of DZNep in a mouse DMM joint model (2 times a week, with intra-articular injection administration for 8 weeks).

Wherein: A. the X-ray examination of the knee joints of the mice is carried out by a blank control group (the non-operation intra-articular injection of PBS with the same volume), an operation control group (DMM operation + PBS), a low-concentration (DMM + DZNep-0.1mg/KG) and a high-concentration (DMM + DZNep-1mg/KG) drug group. The result shows that the knee joint gap height of the blank control group mouse is well maintained, and no obvious bone patch is formed around the joint; narrowing the joint space and peripheral hyperosteogeny in the operation control group; joint space collapse is aggravated and hyperplasia around joints is obvious in the DZNep administration group (low concentration and high concentration). The above results demonstrate that dzneep exacerbates the progression of osteoarthritis in mice by agonizing TXNIP.

B. Mouse knee tissue sections were H & E stained. The results show that the tissue structures of the knee joint distal femur and the tibia proximal end of the blank control group mouse are complete, the shapes are regular, and the articular cartilage surface is smooth; the joint clearance of the operation control group becomes narrow, the structure is disordered, and the surface of the articular cartilage is rough and degenerated; the structural morphology of the distal femur and the proximal tibia in the joint space of the DZNep administration group (low concentration and high concentration) is further disordered, and the cartilage surface is rough and collapsed.

C. The safranin is subjected to fast green staining, and the result shows that (the left side of the figure 3. C) the articular cartilage on the surfaces of the distal femur and the proximal tibia of the knee joint of the blank control group mice is regular in shape and good in thickness maintenance; the joint clearance of the operation control group becomes narrow, and the surface of the articular cartilage becomes rough and thin; the structural morphology of cartilage on the surfaces of the far end of femur and the near end of tibia in the joint space of the DZNep administration group (low concentration and high concentration) is further disordered, the cartilage surface is rough and obvious, and the thickness is thinned. Statistical analysis of cartilage thickness on knee surface (right side of fig. 3. C) the thickness of cartilage after DMM mouse arthritis model in dzneep-dosed group was thinner than that in the control group of simple surgery, and there was a statistical difference between the two groups. TXNIP agonists exacerbate joint degeneration with a marked decrease in cartilage tissue thickness. Denotes P < 0.05. Denotes P < 0.01.

Claims

1. The application of verapamil in preparing a product for protecting chondrocytes and relieving osteoarthritis is a composition or a preparation containing the verapamil or an optical isomer or a pharmaceutically acceptable salt thereof.

2. The use of verapamil as claimed in claim 1 for protecting chondrocytes from osteoarthritis, wherein the composition or formulation comprises any one of the following:

(i) promoting articular chondrocyte matrix expression;

(ii) reducing the intracellular expression level of TXNIP;

(vi) improving the stress and repair capability of the articular cartilage.

3. The use of verapamil as claimed in claim 1, wherein the composition is a pharmaceutical, cosmetic, dietary supplement, food composition or nutraceutical composition.

4. The use of verapamil as claimed in claim 3, wherein the pharmaceutical composition comprises (a) verapamil or a pharmaceutically acceptable salt thereof as an active ingredient; and (b) a pharmaceutically acceptable carrier or excipient.

5. The use of verapamil for protecting chondrocytes from osteoarthritis according to claim 4, wherein the pharmaceutical composition further comprises an additional active ingredient for protecting chondrocytes from osteoarthritis.

6. The use of verapamil as claimed in claim 5, wherein the additional chondrocyte-protective osteoarthritis-alleviating active ingredients are: melatonin can be reduced in TXNIP levels.

7. The use of verapamil as claimed in claim 3, wherein the pharmaceutical composition is in the form of a composition comprising: tablets, capsules, granules, suspensions, pills, solutions, syrups, or injections.

8. The use of verapamil as claimed in claim 3, wherein the nutraceutical composition comprises verapamil as an effective ingredient for protecting articular chondrocytes and alleviating osteoarthritis.

9. Use of verapamil for protecting chondrocytes to alleviate osteoarthritis according to claim 1, wherein the verapamil content of the composition or formulation is 0.001-99 wt%.

10. Use of verapamil for the non-therapeutic in vitro reduction of the expression level of a TXNIP protein by adding verapamil or a salt thereof to a cell culture system, thereby reducing the expression level of the TXNIP protein in said cells;