CN112791080A

CN112791080A - Application of resveratrol dimer TVN in preparation of medicine for treating osteoarthritis

Info

Publication number: CN112791080A
Application number: CN202110080192.1A
Authority: CN
Inventors: 孔令义; 杨蕾; 邹紫铃
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-01-21
Filing date: 2021-01-21
Publication date: 2021-05-14
Anticipated expiration: 2041-01-21
Also published as: CN112791080B

Abstract

The invention discloses an application of resveratrol dimer TVN in preparing a medicine for treating osteoarthritis. The TVN improves bone loss and protects articular cartilage by inhibiting bone resorption and bone remodeling; and delay the progression of osteoarthritis by improving cartilage degradation and cartilage damage; meanwhile, the degradation of the cartilage matrix is inhibited by maintaining the steady state of the cartilage matrix, and the method can be used for developing a medicament capable of delaying or reversing the progress of osteoarthritis.

Description

Application of resveratrol dimer TVN in preparation of medicine for treating osteoarthritis

Technical Field

The invention relates to a traditional Chinese medicine technology, in particular to an application of resveratrol dimer TVN in preparing a medicine for treating osteoarthritis.

Background

Osteoarthritis (OA) is the most common age-related degenerative disease, with the incidence of OA in older adults over 65 years of age reaching over 37%, with OA in older adults over 75 years of age reaching over 80%, with women having a 2-fold higher incidence of OA than men. It is characterized by synovial inflammation, cartilage degradation, cartilage matrix degradation, subchondral bone reconstruction and osteophyte formation, ultimately leading to joint pain and dysfunction. The symptoms of joint pain, swelling, stiffness, joint movement limitation and the like which are slowly progressed are frequently clinically manifested, and severe patients are manifested by joint deformity and joint function loss, which are main sources of social and economic costs, seriously affect the movement function of patients and reduce the life quality. OA is a multifactorial disease, the major causative factors including: age, sex, obesity, trauma, genetic and metabolic factors, the pathogenesis of which has not been established to date, and thus no established protocol exists for the prevention and treatment of OA. The current drugs for treating OA are mainly non-steroidal anti-inflammatory drugs, steroids, and the like. Joint replacement is an effective treatment for symptomatic end-stage disease, but the effect is not ideal and the life of the prosthesis is limited. Therefore, the emphasis on the study of osteoarthritis has shifted to disease prevention and treatment of early osteoarthritis.

The resveratrol dimer, (7R,8R) -Trans-delta-viniferin, TVN for short, has the following structural formula:

the compound is a natural product separated from grapes, and can also be used for carrying out biotransformation on resveratrol by balsam pear peroxidase to obtain TVN. The patent CN 201310238137.6 shows that the TVN can obviously reduce mouse hyperglycemia caused by cane sugar through pharmacodynamic experiments. On this basis, the applicants further investigated the biological activity of TVN against osteoarthritis.

Disclosure of Invention

The purpose of the invention is as follows: in view of the above prior art, the present application provides an application of resveratrol dimer TVN in the preparation of a medicament for treating osteoarthritis.

The technical scheme is as follows: the invention discloses an application of resveratrol dimer TVN in preparing a medicament for treating osteoarthritis.

The resveratrol dimer TVN can delay or reverse the progress of osteoarthritis.

Further, the resveratrol dimer TVN can inhibit bone remodeling by inhibiting bone resorption, and relieve the progress of osteoarthritis.

Further, the resveratrol dimer TVN exerts an anti-osteoarthritis effect by alleviating cartilage degradation.

Further, the resveratrol dimer TVN can protect articular cartilage by inhibiting the degradation of cartilage matrix and delay the progression of osteoarthritis.

Has the advantages that: experiments prove that the resveratrol dimer TVN can inhibit bone remodeling by inhibiting bone absorption and relieve the progress of osteoarthritis; can relieve cartilage degradation and play a role in resisting osteoarthritis; can inhibit the degradation of cartilage matrix to protect articular cartilage and delay the process of osteoarthritis, so that the resveratrol dimer TVN can be used for developing related anti-osteoarthritis medicines.

Drawings

FIG. 1 is a BV/TV quantitative analysis of the effect of TVN on bone density of mice modeled by ACLT;

FIG. 2 is a graph showing the effect of Tb.N on the number of trabeculae in ACLT-modeled rats;

FIG. 3 is a graph showing the effect of Tb.Th on the trabecular bone thickness of ACLT-modeled rats;

FIG. 4 is a graph showing the effect of Tb.Sp in quantitative analysis of TVN on the trabecular bone resolution of ACLT-modeled rats;

FIG. 5 is a graph showing the effect of safranin fast green on the structural changes of cartilage and subchondral bone in ACLT-modeled rats;

FIG. 6 is a graph showing toluidine blue to evaluate the protective effect of TVN on subchondral bone of ACLT-modeled rats;

FIG. 7 is a graph showing the effect of OARSI in the quantitative analysis of TVN on cartilage degradation in ACLT-modeled rats;

FIG. 8 is MMP13, and Collagen II immunohistochemistry was performed to evaluate the effect of TVN on cartilage matrix degradation in ACLT-modeled rats.

Detailed Description

The present application will be described in detail with reference to specific examples.

The TVN used in the examples was prepared by the method described in patent CN 201310238137.6.

Example 1 establishment of anterior cross ligament resection (ACLT) animal model

All animal experimental procedures were in compliance with the approval of the animal ethics committee of the university of chinese medical science. 50 SPF-grade SD male rats of 180-. Then, 40 rats were subjected to ACLT modeling simulation OA, the specific protocol: the rat is anesthetized and then fixed, hairs near the knee joint of the right leg of the rat are removed completely, the hairs are wiped by alcohol, the sterile environment is guaranteed as far as possible, a longitudinal incision with the length of about 1cm is made on the inner side skin beside the patella of the rat by using a surgical knife, then the knee joint is exposed, the patella and the patellar ligament are dislocated towards the outer side gently, the knee joint capsule is opened, the anterior cruciate ligament can be observed clearly by naked eyes, the fat pad is poked slightly by using micro forceps, and the anterior cruciate ligament is cut. Sterile normal saline is dripped on the surface of the articular cartilage to avoid the articular fluid attached to the surface of the articular cartilage from being completely dried in the operation process. Then the patella is reset, the muscle is sewed by 4-0 absorbable suture, the iodophor is sterilized, and then the patella is sewed by aseptic needle and thread. Penicillin is given 10 ten thousand U/time for three consecutive days after molding, once a day.

EXAMPLE 2TVN prophylaxis treatment of osteoarthritis

One week after molding, 50 rats were randomly grouped as: normal group, ACLT surgical model group, sodium hyaluronate positive control group (2.5 mL: 25mg), TVN (0.5mg/kg), TVN (1mg/kg), 10 pieces per group. TVN was dissolved with 40% PEG400, and administered by knee injection at doses of 0.5mg/kg and 1mg/kg, 50. mu.L each, for 5 weeks, 2 times per week. The sodium hyaluronate group was injected with 50. mu.L each time for 5 weeks, 1 time each time. The model group was injected with the same volume of 40% PEG 400. After 5 weeks of administration, the rats were euthanized and the right leg knee joint was fixed in 4% paraformaldehyde for later histological evaluation.

Example 3Micro-CT evaluation of the Effect of TVN on bone Density and subchondral bone microstructure in rats

The specimens were scanned using a Scanco viva CT 80 instrument. Setting parameters: resolution, 21 μm; voltage, 70 kV; current, 113 μ A. The parameters tested were: bone density (BV/TV), trabecular bone separation (Tb.Sp), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th).

BV/TV results are shown in FIG. 1, the model group (0.5072 + -0.03585) was significantly lower (P <0.001) than the normal group (0.6720 + -0.01821), significantly lower (P <0.001) than the sodium hyaluronate group (0.6264 + -0.04524), and significantly lower (P <0.001) than the TVN (0.5mg/kg) group (0.5767 + -0.03377) and TVN (1mg/kg) group (0.6229 + -0.03187). Tb.n and tb.th gave results consistent with BV/TV results (fig. 2, 3). Sp results as shown in fig. 4, the significance of the model group (0.3102 ± 0.02452) was increased (P <0.001) compared to the normal group (0.2838 ± 0.02157), and the significance of the sodium hyaluronate group (0.3836 ± 0.02649) and TVN (0.5mg/kg) group (0.3320 ± 0.03286) and (1mg/kg) group (0.3059 ± 0.02456) was decreased (P <0.001) compared to the model group. These results indicate that after ACLT molding, knee joint instability is caused, and the microstructure of the subchondral bone changes in the early stage, mainly cartilage destruction, which is manifested in bone remodeling of the subchondral bone. TVN (0.5,1mg/kg) can play a role in resisting bone resorption to inhibit bone remodeling of subchondral bone, relieve bone destruction and resorption, and protect bone structure of the subchondral bone, thereby playing a role in protecting articular cartilage.

Example 4 safranine fast green and toluidine blue staining to assess the protective effect of TVN on subchondral bone

The 4% paraformaldehyde fixed specimen was decalcified by changing to 10% EDTA for 4 weeks. After decalcification, the slices are dehydrated step by using ethanol with different concentrations, then paraffin embedding is carried out, and continuous longitudinal section sampling is carried out, wherein the section thickness is about 5 mu m. Each group randomly selected 6 specimens for sectioning, each specimen sectioned into 6 pieces. The resulting sections were dewaxed with xylene and then hydrated with a gradient of ethanol to remove residual xylene. Staining with safranine fast green and toluidine blue, then dehydrating the sections with gradient ethanol, then permeabilizing with xylene, and finally mounting with neutral gum. Panoramic scanning is performed using a slice scanner. The safranin can be combined with proteoglycan to show red color, and safranin fast green staining can visually reflect the changes of articular cartilage and subchondral bone structures, and the results are shown in figure 5, wherein the subchondral bone surface of a normal group is smooth, the boundary between cartilage and subchondral bone is clear, the tide line is complete, and the joint gap is large. And the color of the model group becomes light, the surface of the model group is festered and uneven, cracks appear, cell clusters appear irregularly, and the joint space is obviously narrowed. The TVN (0.5,1mg/kg) administration group was able to significantly improve these changes compared to the model group, while there was no significant difference between the sodium hyaluronate group and the TVN (0.5,1mg/kg) administration group. Toluidine blue staining reflects mainly glycosaminoglycan changes in the cartilage matrix, consistent with the results obtained with safranin fast green (fig. 6). Finally, scoring the image according to an OARSI (Osteoarthritis Research Society International) cartilage histopathology scoring system, and grading and evaluating the damage degree, wherein the scoring grade is as follows:

0 minute: normal cartilage

0.5 min: intact cartilage but loss of proteoglycans

1 minute: without cartilage loss but with fibrillation

And 2, dividing: vertical cracking and skin panel loss

And 3, dividing: the calcified layer lesion accounts for 1-25% of the whole articular surface

And 4, dividing: the calcified layer lesion accounts for 25-50% of the whole articular surface

And 5, dividing: the calcified layer lesion accounts for 50-75% of the whole articular surface

6 min: calcified layer lesion > 75%

The results are shown in FIG. 7: the OARSI score of the normal group is 0.5 +/-0.4472, the model group is 4.833 +/-0.7528, the score of the model group is obviously increased compared with the normal group (P <0.001), and the score of the model group is also obviously increased compared with the score of the sodium hyaluronate group (3.000 +/-0.8944). The TVN (0.5mg/kg) group scores 3.167 + -0.7528, and the TVN (1mg/kg) group scores 2.833 + -0.7528, and the TVN (1mg/kg) group scores significantly lower than the model group without significant difference with the sodium hyaluronate group. These results show that joint cavity injection of TVN can significantly improve cartilage degradation of ACLT model-making rats and reduce cartilage damage, thereby delaying the progression of osteoarthritis.

Example 5 immunohistochemical evaluation of the inhibitory Effect of TVN on cartilage matrix degradation

Immunohistochemical staining of the above sections was performed by incubating with 3% hydrogen peroxide for 5min, then soaking in PBS, further incubating with 10% goat serum at room temperature for 10min, adding MMP13(DF6494, 1:100), collagen II (ab34712, 1:100) primary antibody, and incubating overnight at 4 ℃. The next day, the cells were washed 3 times with PBS, and then biotin-labeled secondary antibody was added dropwise and incubated at room temperature for 30 min. Washing with PBS, labeling with horseradish peroxidase for 30min, washing with PBS, DAB developing, washing, counterstaining with hematoxylin, and sealing. The staining results of MMP13 are shown in fig. 8, and the protein expression of MMP13 in the model group was significantly increased as compared with the normal group, and also significantly increased as compared with the sodium hyaluronate group and TVN (0.5,1mg/kg) administration group. As a result of staining with Collagen II, as shown in FIG. 8, the protein expression in the model group with Collagen II was significantly reduced as compared with the normal group, and also significantly reduced as compared with the sodium hyaluronate group and TVN (0.5,1mg/kg) administration group. Collagen II is the major component of cartilage matrix (ECM) composition, and MMP13 is the most major metal matrix enzyme degrading Collagen II. These results show that intra-articular injection of TVN is effective in inhibiting degradation of ECM, and thus, in inhibiting osteoarthritis progression.

Claims

1. Application of resveratrol dimer TVN in preparing medicine for treating osteoarthritis is provided.

2. The use according to claim 1, wherein the resveratrol dimer TVN inhibits bone remodeling by inhibiting bone resorption, alleviating the progression of osteoarthritis.

3. The use according to claim 1, wherein the resveratrol dimer TVN exerts an anti-osteoarthritis effect by alleviating cartilage degradation.

4. The use according to claim 1, wherein the resveratrol dimer TVN is used for protecting articular cartilage and delaying the progress of osteoarthritis by inhibiting the degradation of cartilage matrix.