KR20220165026A - Composition for Protecting or Regenerating Cartilage containing Ginsenoside-Rb1 and Hydrogel, and Pharmaceutical Composition for Preventing or Treating Degenerative Arthritis - Google Patents

Abstract

The present invention relates to a composition for protecting or regenerating cartilage containing ginsenoside-Rb1 and a hydrogel, and a pharmaceutical composition for preventing or treating degenerative arthritis, wherein ginsenoside-Rb1 is continuously released to have excellent effects of restoring joint function that is not functioning properly due to the occurrence of osteoarthritis. Therefore, the composition can be effectively used for the prevention or treatment of articular cartilage-related diseases such as degenerative osteoarthritis and joint cartilage damage caused by exercise.

Description

Composition for Protecting or Regenerating Cartilage containing Ginsenoside-Rb1 and Hydrogel, and Pharmaceutical Composition for Preventing or Treating Degenerative Arthritis }

The present invention relates to a composition for protecting or regenerating cartilage containing ginsenoside-Rb1 and a hydrogel, and a pharmaceutical composition for preventing or treating degenerative arthritis.

Degenerative arthritis is a chronic disease accompanied by inflammation and pain caused by damage to bones and ligaments due to gradual damage or degenerative changes in the cartilage that protects joints. It is a degenerative disease. Recently, patients with degenerative arthritis are rapidly increasing by more than 5% every year even under the age of 55 due to the increase in obesity patients due to westernized eating habits, surgical joint damage, excessive joint movement, and genetic problems. However, severe chronic joint pain has become a major social problem, such as restricting economic activities.

As a pathological cause of degenerative arthritis, it has been reported that the natural aging process and menopause are closely related, but it is difficult to develop clinical therapeutic agents for the treatment of degenerative arthritis because the clear pathological cause is unclear. Therefore, current clinical treatment for degenerative arthritis focuses on relieving chronic joint pain, and physical therapy for degenerative joints, joint cleaning using anti-pain agents, and artificial joint replacement are the main ones. However, since this procedure provides only a temporary joint pain relief effect, repeated treatment is required, and in the case of artificial joint replacement, there is a problem in that it is performed limitedly according to the age and medical history of the patient.

In order to overcome this, research on transplanting stem cells is being actively conducted, but the survival of the transplanted mesenchymal stem cells cannot be guaranteed, and the differentiation into chondrocytes and the efficiency of cartilage tissue formation in vivo after transplantation have not been proven. It is a difficult situation to expect from a human treatment method. In order to overcome the problems of in vivo distribution and differentiation of cell therapy products, technology to deliver cells using various types of biomaterials as scaffolds is being used, and furthermore, artificial cartilage tissue with a three-dimensional structure in vitro is used. Engineered cartilage) is being developed. However, existing hydrogel-type scaffolds do not have sufficient cell survival rate and cartilage differentiation rate, and membrane-type scaffolds cannot form 3-dimensional cartilage tissue, and 3-dimensional sponge or mesh scaffolds do not. ) type support, the bonding force between the artificial cartilage and the original host tissue is low, and the cartilage regeneration rate is not sufficient.

On the other hand, ginsenoside Rb1 (G-Rb1) has been reported to have cartilage regeneration function in vitro, but when ginsenoside Rb1 is applied to cartilage, it is difficult to show continuous pharmacological action, which is clinically sufficient for cartilage regeneration. And it is known that it does not exhibit an arthritis treatment effect.

Accordingly, the present inventors completed the present invention by conducting research to develop a therapeutic agent that can be effectively applied to articular cartilage-related diseases such as degenerative osteoarthritis and joint cartilage damage caused by exercise.

Korean Patent Publication No. 10-2004-0065979

One object of the present invention is to provide a composition for cartilage protection or regeneration comprising ginsenoside-Rb1 and a hydrogel.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative arthritis comprising ginsenoside-Rb1 and a hydrogel.

One aspect of the present invention provides a composition for protecting or regenerating cartilage comprising ginsenoside-Rb1 and a hydrogel.

The term "cartilage" used in the present invention refers to hyaline cartilage, fibrocartilage, and/or elastic cartilage, and includes articular cartilage, ear cartilage, nasal cartilage, and elbow cartilage. , meniscus, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage and spinal cartilage.

As used herein, the term "protection of cartilage" refers to thickening of cartilage, and includes delaying or maintaining the reduction of existing cartilage thickness or preventing or delaying the deterioration of pathological conditions of existing cartilage.

The term "cartilage regeneration" used in the present invention means that cartilage is regenerated when transplanted to a cartilage defect or damaged part, and includes showing an improvement or treatment effect for cartilage damage.

According to one embodiment of the present invention, the hydrogel may be made of one or more materials selected from the group consisting of agarose, alginate, chitosan, collagen, fibrin, gelatin and hyaluronic acid.

Gelation of the hydrogel may be achieved by chemically or physically crosslinking polymer chains to form a network structure by being induced by chemical agents (eg, crosslinking agents) or physical stimuli (eg, pH and/or temperature). In addition, during the manufacturing process of the hydrogel, 2D and 3D structures can be formed using hydrogel patterning technology, and one or more patterns can be integrated by changing the length, width, and height.

According to one embodiment of the present invention, the weight ratio of the ginsenoside-Rb1 and the hydrogel may be 3:100000000 to 10:100000000.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating degenerative arthritis comprising ginsenoside-Rb1 and a hydrogel.

Since degenerative arthritis is accompanied by inflammation and pain caused by damage to bones and ligaments due to gradual damage or degenerative change of cartilage that protects joints, it is possible to improve the symptoms of degenerative arthritis by inducing differentiation and/or regeneration of cartilage cells. can

A pharmaceutical composition according to one embodiment of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate , microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto not. The pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (22th ed., 2013).

The pharmaceutical composition according to one embodiment of the present invention may further contain a substance exhibiting at least one preventive or therapeutic activity of degenerative arthritis. In addition, the pharmaceutical composition according to one embodiment of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and/or biological response modifiers for the treatment of degenerative arthritis.

The pharmaceutical composition according to one embodiment of the present invention may include various bases and/or additives necessary and appropriate for formulation of the dosage form, and may include a nonionic surfactant, a silicone polymer, and a sieve within a range that does not impair its effectiveness. Pigments, fragrances, preservatives, bactericides, oxidative stabilizers, organic solvents, ionic or nonionic thickeners, softeners, antioxidants, free radical destroyers, opacifiers, stabilizers, emollients, silicones, α-hydroxy acids, antifoaming agents, humectants, vitamins, insect repellents, fragrances, preservatives, surfactants, anti-inflammatory agents, Substance P antagonists, fillers, polymers, propellants, basifying or acidifying agents, or colorants. .

The pharmaceutical composition according to one embodiment of the present invention can be administered parenterally, for example, it can be injected and applied to the affected area.

A suitable dosage of the pharmaceutical composition according to one embodiment of the present invention depends on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, route of administration, excretion rate and reaction sensitivity. can be prescribed in a variety of ways. A preferred dosage of the pharmaceutical composition according to one embodiment of the present invention is within the range of 0.001 to 1000 mg/kg based on adults.

According to one embodiment of the present invention, the hydrogel may be made of one or more materials selected from the group consisting of agarose, alginate, chitosan, collagen, fibrin, gelatin and hyaluronic acid.

According to one embodiment of the present invention, the weight ratio of the ginsenoside-Rb1 and the hydrogel may be 3:100000000 to 10:100000000.

According to one embodiment of the present invention, the composition may be a sustained-release formulation.

According to one embodiment of the present invention, the duration of ginsenoside-Rb1 release of the formulation may be 4 to 8 weeks.

Since ginsenoside-Rb1 is stably present in the hydrogel and can be released in a sustained release form for 4 to 8 weeks, the composition of the present invention continuously induces cartilage regeneration of ginsenoside-Rb1 at the site of damaged articular cartilage. It can maximize the treatment effect of degenerative arthritis.

According to the composition for cartilage protection or regeneration containing ginsenoside-Rb1 and a hydrogel, and the pharmaceutical composition for preventing or treating degenerative arthritis, ginsenoside-Rb1 is continuously released to prevent joints that do not function properly due to osteoarthritis. Since it is excellent in the effect of restoring function, it can be effectively used for the prevention or treatment of articular cartilage-related diseases such as degenerative osteoarthritis and joint cartilage damage caused by exercise.

1 is a photograph showing the preparation of a ginsenoside-Rb1/hydrogel complex.
Figure 2 is a photograph showing the osteoarthritis induction process in the right femoral condile using trephine.
3 is a photograph showing an experimental group and transplantation process for transplantation of the hydrogel/ginsenoside-Rb1 complex.
Figure 4 is a graph showing macroscopic evaluation (A) and quantitative indicators (B) for the efficacy of ginsenoside-Rb1/hydrogel complexes:
Values are mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.
5 is a photograph showing the results of Micro-CT analysis for evaluating the efficacy of ginsenoside-Rb1/hydrogel composite cartilage formation in a cartilage regeneration animal model.
Figure 6 is a graph and a photograph showing the measurement of reactive oxygen species (A) and quantitative indicators (B) for evaluating the effectiveness of chondrogenic Hydrogel -Rb1 in a cartilage regeneration animal model:
Values are means ± SD, (*p < 0.05, **p < 0.01 and ***p < 0.001).
Figure 7 is a graph showing the results of analyzing the changes in MMP-3 (A) and PGE2 (B) according to the administration of Hydrogel G-Rb1 in the cartilage regeneration animal model.
8 is a graph showing the effect of Hydrogel G-Rb1 on the expression of MMPs and TIMP-1 in a chondrocyte regeneration animal model.
9 is a graph showing the effect of Hydrogel G-Rb1 on the expression of Caspase-3, BAX and TNF-α in a chondrocyte regeneration animal model.
10 is a graph showing the effect of Hydrogel G-Rb1 on the expression of PI3K/Akt, NF-κB, and MAPK signaling in a chondrocyte regeneration animal model: Western blot analysis (A) and p-P38/P38 (B); Quantitative analysis of p-Akt/Akt (C) and NF-κB (D) expression.
11 is a photograph showing the results of H&E staining for analyzing the articular cartilage regeneration efficacy of the ginsenoside-Rb1/hydrogel complex.
12 is a photograph showing the results of Masson staining for collagin analysis in articular cartilage in a cartilage regeneration animal model.
13 is a photograph showing the results of Safranin O staining for analyzing the proteoglycan regeneration efficiency of articular cartilage.
Figure 14 is a photograph showing the results of immunostaining of MMP-1 for the analysis of cartilage regeneration efficacy in articular cartilage.

Hereinafter, the present invention will be described in more detail through one or more embodiments. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Preparation Example 1. Preparation of ginsenoside-Rb1/hydrogel composite

A slow-release ginsenoside-Rb1/hydrogel complex exhibiting a therapeutic effect on arthritis by intrachondral administration was prepared.

Specifically, a ginsenoside-Rb1/hydrogel composite was prepared as shown in FIG. 1 by mixing ginsenoside-Rb1 with a hygienic body-compatible hydrogel at a concentration of 30 or 100 μg/kg.

Specifically, the hydrogel/ginsenoside-Rb1 complex was prepared by mixing (weight/weight) ginsenoside-Rb1 in a uniform manner using gelatin (Gelatin, Sigma, USA) obtained from bovine skin. The manufacturing method is as follows. First, gelatin was swelled in distilled water for 20 minutes, and then gelatin was slowly dissolved at 60° C. for 1 hour using a magnetic stirrer. A gelatin-ginsenoside Rb1 mixture (hydrogel/ginsenoside Rb1 complex) was prepared by mixing 2% gelatin solution and ginsenoside Rb1 based on the gelatin solution, and then dried at 37°C. The concentration of ginsenoside Rb1 was prepared so that 30 μg/kg and 100 μg/kg could be administered per body weight (Kg) of the animal.

Example 1. Osteoarthritis animal model preparation

1-1. osteoarthritis induction

As shown in FIG. 2, osteoarthritis was induced in the rabbit's right femoral condile with a diameter of 5 mm and a depth of 2 mm using a trephine instrument, and the rabbit was anesthetized with isoflurane during surgery.

1-2. Experimental group design for osteoarthritis efficacy evaluation

In order to evaluate the efficacy of the ginsenoside-Rb1/hydrogel composite prepared in Preparation Example 1 on osteoarthritis, an experimental group was randomly designed for rabbits whose osteoarthritis was induced in Example 1-1 as follows.

No division note One Normal control (N/C) No damage to articular cartilage 2 Osteoarthritis (OA) control group Articular cartilage damage group (sham) 3 Hydrogel alone transplant group Free of ginsenoside Rb1 4 Hydrogel/G-Rb1 (low) composite graft group Intra-articular implantation of ginsenoside-Rb1 (30 μg/kg)/hydrogel complex 5 Hydrogel/G-Rb1 (high) composite graft group Intra-articular implantation of ginsenoside-Rb1 (100 μg/kg)/hydrogel complex 6 Diclofenac (positive control) Diclofenac (5mg/kg/day) intramuscular injection for 10 days

1-3 Implantation of ginsenoside-Rb1/hydrogel complex

Implantation of the ginsenoside-Rb1/hydrogel complex was performed in the following manner.

Specifically, the ginsenoside-Rb1/hydrogel complex prepared in Preparation Example 1 was transplanted as shown in FIG. 3 between the articular cartilages of the osteoarthritis animal model induced in Example 1-1.

New Zealand white rabbits (12 weeks, weight 3.5 ± 0.2 kg, male) were used to evaluate the cartilage regeneration efficacy of the hydrogel/ginsenoside Rb1 complex, which is the composition of the present invention. All animal testing procedures were approved by the Animal Ethics Committee of Chonbuk National University (IACUC number: CBNU 2018-111). For implantation of the hydrogel/ginsenoside Rb1 complex into the animal, the animal (rabbit) was first safely anesthetized with isoflurane. Thereafter, after washing with a 10% betadine solution, each rabbit's right knee joint was covered with aseptic vinyl in an aseptic manner and then accessed and opened. A degenerative arthritis model was induced by separating the side of the knee joint, pulling the muscle with a traction device, and carefully damaging the knee cartilage (diameter: 5 mm, depth: 2 mm) with an electric trephine in a vertical direction. After that, the cartilage and bone debris were removed, and the boundary of the drill hole was trimmed using a surgical knife and washed. Then, the hydrogel and the hydrogel-Rb1 sheet were implanted in the defect area of each experimental group. After transplantation, the surrounding muscle membrane and muscle tissue were sutured, and then the skin was sutured and corrected by banding. After this, the animals (rabbits) were allowed to freely move their knee joints in cages without any fixation device. Afterwards, two injections of antibiotics suppressed the infection.

Example 2. Confirmation of articular cartilage damage recovery effect according to visual evaluation

In order to confirm the effect of the ginsenoside-Rb1/hydrogel complex of Preparation Example 1 on the restoration of articular cartilage damage, visual evaluation was conducted.

Specifically, the depth of destruction of articular cartilage was assessed on a 4-point scale (0 = normal-stained surface, 1 = negligible fibrosis or slight yellow discoloration, 2 = damage visible only to the superficial or central layers, 3 = damage visible to the deep layers, 4 = extensively diffuse damage).

As a result, when the ginsenoside-Rb1/hydrogel complex was transplanted into the knee joint, it was confirmed that cartilage damage was visually recovered after 4 weeks and 8 weeks (FIG. 4).

Example 3. Confirmation of articular cartilage damage recovery effect according to Micro-CT analysis

Micro-CT analysis was performed to confirm the articular cartilage damage recovery effect of the ginsenoside-Rb1/hydrogel complex of Preparation Example 1.

Specifically, euthanasia was induced by anesthetizing the animals after completing all experimental procedures. Then, after separating the animal's knee, muscle and connective tissue of the knee joint were removed and fixed in 10% formaldehyde (Sigma Aldrich, USA) buffer. Subsequently, the degree of joint recovery in the knee region was evaluated using the Micro-CT analysis program of the Micro-CT (SKYSCAN, Belgium) equipment for the femoral condyles. As an analysis program for this, CTvox software (Sky Scan Company) was used.

As a result, the normal control group showed regular and smooth cartilage on the femur, whereas the arthritis-induced group in the articular cartilage damage model showed cartilage damage characterized by cartilage surface. On the other hand, when the ginsenoside-Rb1/hydrogel complex was implanted into the knee joint cavity, cartilage damage was confirmed to be restored on micro-CT after 4 weeks and 8 weeks (FIG. 5).

Example 4. Confirmation of the effect of reducing the level of reactive oxygen species in articular cartilage

In order to confirm the articular cartilage production effect of the ginsenoside-Rb1/hydrogel complex of Preparation Example 1, reactive oxygen species (ROS) analysis was performed.

Specifically, the ROS generation level of cartilage tissue was evaluated using a ROS detection kit equipped with Life Technologies' CellROX Green reagent (Invitrogen) according to the manufacturer's instructions. After 4 weeks and 8 weeks, the cartilage tissues were treated with 5 μM CellROX reagent in PBS at 37° C. for 60 minutes and used.

As a result, it was confirmed that the generation of reactive oxygen species harmful to chondrocytes was significantly suppressed after 4 weeks and 8 weeks when the ginsenoside-Rb1/hydrogel complex was implanted into the knee joint cavity (FIG. 6).

Example 5. Confirmation of cartilage destruction inhibitory effect according to MMP-3 and PGE2 expression reduction

In order to confirm the cartilage destruction inhibitory effect of the ginsenoside-Rb1/hydrogel complex of Preparation Example 1, the expression of MMP-3 and Prostaglandins E2 (PGE2) related to cartilage destruction were analyzed.

Specifically, blood samples were collected after euthanasia of animals in each group after all experiments, and serum was obtained from these samples by centrifugation at 3,000 rpm for 10 minutes and stored at -80 °C until analysis. Prostaglandin E2 (PGE2) (R&D Systems, Minneapolis, Minn., USA) and MMP-3 (CUSABIO, Wuhan, China) were measured by ELISA detection kits according to the manufacturer's recommendations. All evaluations were performed in triplicate.

As a result, compared to the concentrations of MMP and PGE2 in the normal control group, the concentrations of MMP and PGE2 were significantly increased in the arthritis induction group. On the other hand, when treating each concentration of ginsenoside-Rb1/hydrogel complex, it was confirmed that the concentrations of MMP and PGE2 after 4 weeks and 8 weeks were significantly reduced compared to the arthritis induction group (FIG. 7), and ginsenoside-Rb1/ The hydrogel composite was confirmed to have an effective effect on articular cartilage formation.

Example 6. Confirmation of articular cartilage production effect according to MMP expression reduction

Inflammatory mediators stimulate the production of matrix metalloproteinases (or matrix metalloproteases, MMPs), and MMP-1 and MMP-13 induce arthritis through collagen degradation. In particular, MMP-13 aggravates inflammation by destroying cartilage matrix by degrading proteoglycan in addition to degrading collagen. In addition, the expression of MMP-3 is increased in arthritis, and this enzyme damages articular cartilage by reducing the non-collagen matrix component of the joint (El-Sayed E. Mehana et. al., Life Sciences 234, 2019).

Therefore, in order to confirm the recovery effect of articular cartilage damage, MMP concentration was analyzed.

Specifically, in order to evaluate the degree of osteoarthritis recovery, the animals were euthanized, and then the knee cartilage was collected and the mRNA levels of MMP-1, MMP-3, and MMP-13, which are joint damage indicators, were analyzed. To this end, the mRNA isolated from cartilage tissue was subjected to reverse transcription polymerase chain reaction (RT-PCR) according to the manufacturer's analysis method (IntRON Biotechnology) to quantify the mRNA levels of MMP-1, MMP-3 and MMP-13. did The specific test method is as follows. After mixing mRNA (500 μg), 1 μL dNTPs (10 mM), and 1 μL primers, they were added to a 200 μL RNase-free centrifuge tube, 15 μL DEPC-treated water was added, and at 70 °C. Reacted for 5 minutes. The next step was to add 4 μL 5 Х 1st-stand buffer, 2 μL 0.1 MDTT, 25 units of RNase inhibitor and 200 units of superscript II reverse transcription enzyme (Invitrogen), finally RNA was reverse converted to cDNA. Expressions were evaluated in triplicate for each gene to reduce processing errors. The mRNA expression of MMP-1, MMP-3 and MMP-13 was evaluated quantitatively based on the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Band quantification and analysis were performed using Image J software (ver. 1.49). Accordingly, the mRNA expression levels of MMP-1, MMP-3, and MMP-13, which are indicators of cartilage damage, were evaluated.

As a result, it was confirmed that TIMP-1 expression decreased and MMP-13, MMP-3, and MMP-1 expression increased in osteoarthritis animal models. On the other hand, compared to the osteoarthritis induction group, TIMP-1 gene expression increased 4 weeks and 8 weeks after ginsenoside-Rb1/hydrogel complex transplantation, and MMP-13, MMP-3, and MMP-1 expressions were significantly (FIG. 8), it was confirmed that the ginsenoside-Rb1/hydrogel complex contributes to the production of articular cartilage by reducing MMP expression and increasing TIMP-1 gene expression.

Example 7. Caspase-3, BAX, and TNF-α Expression Reduction Confirmation of Articular Cartilage Tissue Cell Death Inhibitory Effect

Cell death (apoptosis) induces intracellular inflammation (inflammatory). Caspase-3 induces cell death, and BAX causes cell death through the mitochondrial pathway. In addition, TNF-α is an inflammatory cytokine and increases during inflammation.

Therefore, in order to confirm the effect of inhibiting articular cartilage tissue cell death, the concentrations of Caspase-3, BAX and TNF-α were analyzed.

To evaluate the degree of recovery from osteoarthritis, animals were euthanized and knee cartilage was collected and caspase-3, Bax, and TNF-α, which are indicators of articular cartilage death, were collected. mRNA levels were analyzed. To this end, mRNA isolated from cartilage tissue was subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to the manufacturer's analysis method (IntRON Biotechnology), and caspase-3, Bax, and TNF-α levels were quantified. The specific test method is as follows. After mixing mRNA (500 μg), 1 μL dNTPs (10 mM), and 1 μL primers, they were added to a 200 μL RNase-free centrifuge tube, 15 μL DEPC-treated water was added, and at 70 °C. Reacted for 5 minutes. The next step was to add 4 μL 5 Х 1st-stand buffer, 2 μL 0.1 MDTT, 25 units of RNase inhibitor and 200 units of superscript II reverse transcriptase (Invitrogen), finally RNA was reverse converted to cDNA. Expressions were evaluated in triplicate for each gene to reduce processing errors. The mRNA expression of caspase-3, Bax and TNF-α was evaluated quantitatively based on the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Band quantification and analysis were performed using Image J software (ver. 1.49). Accordingly, the mRNA expression levels of caspase-3, Bax, and TNF-α, which are indicators of cartilage damage, were evaluated.

As a result, it was found that the expression of TNF-α, caspase-3, and BAX significantly decreased 4 weeks and 8 weeks after transplantation of the ginsenoside-Rb1/hydrogel complex (FIG. 9), and ginsenoside-Rb1/hydrogel It was confirmed that cell death of cartilage tissue was inhibited by the administration of the complex.

Example 8. Confirmation of inhibitory effect on cartilage tissue inflammation according to inhibition of PI3K/Akt, NF-κB, and MAPK signaling pathways

Cell death is caused by various signaling pathways such as the PI3K/AKT signaling pathway, and AKT is known to be associated with apoptosis associated with proteins such as Bcl-2, Bax, and Caspase-3. NF-κB is also known to increase during inflammation. Mitogen-activated protein (MAP) kinases include ERKs, JNKs, and p38 proteins, and perform functions such as cell survival, differentiation, and death.

Therefore, in order to confirm the effect of inhibiting cartilage tissue inflammation, protein expression of PI3K/Akt, NF-κB and MAPK signaling pathways was analyzed.

Specifically, the Western blot method for quantifying protein concentration is as follows. First, to perform electrophoresis, after making a gel containing SDS in polyacrylamide gel, separating the cartilage tissue, extracting the protein with a buffer (Pro-Prep, Intron Biology, Korea), and quantifying the protein. Thereafter, proteins are separated through electrophoresis and subjected to a protein blotting process, which is a process of moving them to a filter membrane. Thereafter, 5% skim milk is used for 1 hour to block non-specific proteins. Then, it is reacted with a primary antibody that specifically binds to PI3K/Akt, NF-κB, and MAPK signaling proteins, so that the primary antibody binds to PI3K/Akt, NF-κB, and MAPK signaling proteins. That is, membranes containing proteins are Akt (1:500), p-Akt (1:1000), ABCam (UK) NF-κB (1:500), p-P65 (1:1000), P38 (1:1000) ), the primary antibody against p-P38 (1:800) of USA (MA) was reacted overnight at 4°C. Thereafter, excess primary antibodies that do not bind to PI3K/Akt, NF-κB, and MAPK signaling proteins are washed away, and then secondary antibodies that specifically bind to the primary antibodies are coupled to the primary antibodies. Membranes are incubated with horseradish peroxidase peroxidase (HRP)-conjugated secondary antibody for 2 hours at room temperature. Then, the target protein is detected through a secondary antibody detection reaction. The image of the membrane was quantitatively analyzed for protein concentration using an ImageQuant LAS-500 image analyzer (GE Healthcare, Sweden). Band quantification and analysis of membranes were performed using Image J software (ver. 1.49).

As a result, the expression of p-Akt/Akt, NF-κB, and p-P38/P38 was reduced when the ginsenoside-Rb1/hydrogel complex was transplanted (FIG. 10), and ginsenoside-Rb1/hydrogel The inhibitory effect of the complex on cartilage tissue inflammation was confirmed.

Example 9. Confirmation of chondroprotective effect according to histological evaluation in osteoarthritis animal model

Histological evaluation of the degree of cartilage damage was performed 4 weeks and 8 weeks after the implantation of the ginsenoside-Rb1/hydrogel composite of Preparation Example 1.

9-1. Chondrogenesis assay by H&E staining

The degree of cartilage formation was evaluated by hematoxylene-eosin (H&E) staining of joint tissue.

Specifically, after the end of the entire experiment, the animals were euthanized and the knee region was separated. In order to observe the shape of the cartilage tissue in each group, the knee tissue was fixed in 10% formalin, and Hematoxylin and Eosin (H&E) staining was performed. The staining method is as follows. After the knee region was separated, muscles and surrounding tissues were removed and fixed in a 10% buffered formalin solution (Sigma-Aldrich, USA). Thereafter, the tissue was maintained for 7 days by mixing 8% formic acid and 8% HCl to remove calcium. Afterwards, it was dehydrated using ethanol, and the tissue treatment process namely deparaffinization (xylene) - hydrolysis (alcohol) - water washing - Hematoxylin nuclear staining - water washing - fractionation - washing - neutralization - washing - Eosing (cytoplasm staining other than the nucleus) - dehydration (100 % alcohol) - Transparent (xylene) - Through the encapsulation process using encapsulant, a block was produced using paraffin wax. Then, the paraffin-containing part (thickness: 5 μm) was cut and deparaffinized using a micro-cutting machine. All subsequent samples of H&E staining were mounted on coverslips using permanent mounting medium (Vector Laboratories, USA). The intensity of H/E was observed with an inverted contrast microscope (Zeiss Corporation, Germany) at a fixed 200x magnification.

As a result, when the ginsenoside-Rb1/hydrogel complex was treated at different concentrations, it was confirmed that cartilage damage was visually significantly recovered after 4 weeks and 8 weeks. 11 points (4 weeks) and 7 points (8 weeks) when the concentration of ginsenoside Rb1 contained in the hydrogel is 30 μg/kg, 6 points (4 weeks) when the concentration of ginsenoside is 100 μg/kg, A score of 4 (8 weeks) was obtained. On the other hand, single administration of 100 μg/kg ginsenoside Rb1 alone without using a hydrogel showed results of 12 points (4 weeks) and 11 points (8 weeks), and ginsenoside-Rb1/hydrogel complex It was confirmed that the use of ginsenoside Rb1 increased the formation of cartilage tissue in a concentration-dependent manner compared to the case of single transplantation (FIG. 11).

9-2. Cartilage collagen analysis through Masson staining

Masson staining was performed on joint tissue to confirm changes in cartilage collagen.

Specifically, quantitative evaluation of collagen concentration is divided into normal tissue (12 points) and lesion tissue (1 point), divided into 1-12 points (mild collagen formation up to 1-4 points, moderate collagen formation up to 5-8 points) Formation and 9-12 points were highly advanced) and recovery of cartilage tissue was evaluated.

As a result, when the ginsenoside-Rb1/hydrogel complex was transplanted into the joint cavity, the formation of collagen was visually clearly recovered after 4 weeks and 8 weeks (FIG. 12), indicating that the ginsenoside-Rb1/hydrogel complex The concentration-dependent effect of increasing cartilage tissue formation was confirmed.

9-3. Cartilage proteoglycan analysis by Safranin O staining

Safranin O staining was performed on joint tissue to confirm changes in cartilage collagen.

Specifically, Safranin O staining was performed in each group 4 weeks and 8 weeks after transplantation for the analysis of proteoglycan, a component of cartilage. Cartilage tissue was fixed in 10% buffered formalin solution (Sigma-Aldrich, USA), digested with 8% formic acid and 8% HCl, and dehydrated in ethanol. Then, after sealing with paraffin wax, the paraffin-embedded part (5 μm in thickness) was thinly cut with a micro cutter. To detect cartilage matrix formation, sections were deparaffinized in distilled water, hydrated, stained with 0.1% safranin-O solution (Sigma-Aldrich, USA) for 5 min, dehydrated, 95% ethyl alcohol, absolute ethyl alcohol, and xylene. treated with. All samples were mounted on cover lips using inverted mounting medium (Vector Laboratories, USA). The intensity of Safranin-O staining was observed with an inverted contrast microscope (Zeiss Corporation, Germany) at a fixed 200x magnification.

As a result, when the concentration of ginsenoside Rb1 contained in the hydrogel was 30 μg/kg, 4 points (4 weeks) and 8 points (8 weeks), and when 100 μg/kg, 4 weeks (5 points) and 8 weeks ( 7 points) (FIG. 13), the concentration-dependent effect of increasing proteoglycan formation in cartilage tissue of the ginsenoside-Rb1/hydrogel complex was confirmed.

9-4. Evaluation of articular cartilage regeneration through immunostaining of MMP-1

MMP-1 has been implicated in the development of articular cartilage osteoarthritis.

Therefore, the joint tissue was subjected to MMP-1 immunostaining to evaluate the articular cartilage regeneration ability.

Specifically, tissue immunochemical staining for observing MMP-1 (Bioss Antivodies Inc., USA), an arthritis damage index, was performed according to a manual. Full-thickness samples (cartilage and bone) were collected from each group 4 weeks and 8 weeks after the hydrogel/ginsenoside-Rb1 complex was implanted. It was fixed in 10% buffered formalin solution (Sigma-Aldrich, USA), and bone calcium was extracted with 8% formic acid and 8% HCl. Thereafter, the tissue was dehydrated in ethanol, encapsulated with paraffin wax through a tissue processing process, and a block was fabricated, and the paraffin-embedded portion (thickness: 5 μm) was cut using a tissue cutter. After that, it was incubated at 4 °C using MMP-1 antibody for 24 hours, and the reactivity was confirmed using Vector ABC kit (Vector Lab). All samples are mounted on cover lips using permanent mounting medium according to the manufacturer's protocol (Vector Laboratories, USA). The expression level of MMP-1 was observed at a fixed magnification of about 200 times with a reverse contrast microscope (Zeiss Corporation, Zeiss Corporation, Germany).

As a result, almost no MMP-1 positive cells were observed in the cartilage layer of normal animals. On the other hand, in the articular cartilage damage group (OA control), MMP-1 positive cells were not observed, but cartilage damage was confirmed to be very severe. On the other hand, cartilage was regenerated when the ginsenoside-Rb1/hydrogel complex was transplanted, and MMP-1-positive cells were confirmed (FIG. 14), confirming the regenerative effect of the ginsenoside-Rb1/hydrogel complex on articular cartilage. It became.

So far, the present invention has been examined focusing on the embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

Claims (8)

A composition for protecting or regenerating cartilage comprising ginsenoside-Rb1 and a hydrogel.
The composition for protecting or regenerating cartilage according to claim 1, wherein the hydrogel is made of one or more materials selected from the group consisting of agarose, alginate, chitosan, collagen, fibrin, gelatin and hyaluronic acid.
The composition for protecting or regenerating cartilage according to claim 1, wherein the weight ratio of ginsenoside-Rb1 and the hydrogel is 3:100000000 to 10:100000000.
A pharmaceutical composition for preventing or treating degenerative arthritis comprising ginsenoside-Rb1 and a hydrogel.
The pharmaceutical composition for preventing or treating degenerative arthritis according to claim 4, wherein the hydrogel is made of one or more materials selected from the group consisting of agarose, alginate, chitosan, collagen, fibrin, gelatin and hyaluronic acid. .
The pharmaceutical composition for preventing or treating degenerative arthritis according to claim 4, wherein the weight ratio of ginsenoside-Rb1 and the hydrogel is 3:100000000 to 10:100000000.
The pharmaceutical composition for preventing or treating degenerative arthritis according to claim 4, wherein the composition is a sustained-release formulation.
[Claim 8] The pharmaceutical composition for preventing or treating degenerative arthritis according to claim 7, wherein the formulation has a release duration of ginsenoside-Rb1 of 4 to 8 weeks.

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