Application of Phillyrin in the Preparation of Drugs for Treating Intervertebral Disc Degeneration

Abstract

The present invention discloses the application of forsythin in the preparation of a medicament for treating intervertebral disc degeneration, belonging to the field of medical technology. In the study of intervertebral disc degeneration, it is found that forsythin can significantly inhibit the increase in catabolism and the decrease in anabolism of nucleus pulposus cells during the progression of intervertebral disc degeneration, and also has a significant inhibitory effect on the activation of the NF-κB pathway and the increase in ROS, thereby regulating the synthesis and catabolism of the extracellular matrix and cell apoptosis. Moreover, through verification, it is found that it has a therapeutic effect in a rat model of intervertebral disc degeneration, confirming that it also has a rescue effect on intervertebral disc degeneration in vivo.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of phillyrin in the preparation of drugs for treating intervertebral disc degeneration. Background Art

[0002] Low back pain is the most common disabling factor in the world, bringing a lot of health and economic burdens to the world. Statistical data shows that 80% of adults have suffered from low back pain during their lifetime. Clinically, it is recognized that 40% of low back pain is caused by intervertebral disc degeneration, which is a spinal-related degenerative musculoskeletal disease that may be secondary to a series of diseases such as spinal stenosis, lumbar disc herniation, and lumbar spondylolisthesis. At present, the treatment methods for intervertebral disc degeneration are limited, mainly including physical therapy, non-steroidal anti-inflammatory drug treatment, and surgical treatment. The treatment methods are highly invasive and have limited efficacy. Currently, there is no effective drug to relieve the progression of intervertebral disc degeneration, and drugs for the study of intervertebral disc degeneration have great social and clinical significance. Summary of the Invention

[0003] To solve the above problems in the prior art, the present invention provides the application of phillyrin in the preparation of drugs for treating intervertebral disc degeneration, and uses the anti-inflammatory and antioxidant effects of phillyrin to inhibit the occurrence and development of intervertebral disc degeneration.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides the application of phillyrin in the preparation of drugs for treating intervertebral disc degeneration.

[0006] The present invention also provides a drug for treating intervertebral disc degeneration, and the drug contains phillyrin.

[0007] As a preferred embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier or excipient and is formulated into a pharmaceutically acceptable dosage form.

[0008] As a preferred embodiment of the present invention, the pharmaceutically acceptable carrier or excipient includes one or more solid, semi-solid or liquid adjuvants.

[0009] As a preferred embodiment of the present invention, the pharmaceutically acceptable dosage form includes tablets, capsules, granules, injections, pills, syrups, powders, ointments or liquid preparations.

[0010] As a further preferred embodiment of the present invention, the pharmaceutically acceptable dosage form is an injection.

[0011] As a further preferred embodiment of the present invention, the injection is an injection prepared from phillyrin and dimethyl sulfoxide, and the concentration of phillyrin is 5-10 μM, preferably 10 μM.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] Through research, it is found that inflammatory factors are correlated with intervertebral disc degeneration. After stimulation by inflammatory factors, the concentration of reactive oxygen species in nucleus pulposus cells increases, and at the same time, the activation of downstream inflammatory pathways is induced. Subsequently, changes in the expression of extracellular matrix-related proteins, an increase in apoptosis, and a decrease in proliferation of nucleus pulposus cells occur, leading to the progression of intervertebral disc degeneration. Therefore, inhibiting the increase in the level of reactive oxygen species (ROS) and the activation of inflammatory pathways caused by inflammatory factors can effectively slow down the progression of intervertebral disc degeneration. Forsythoside, as a traditional Chinese medicine, has obvious anti-inflammatory and antioxidant effects. Its extract, forsythin, is a small molecule compound that shows obvious effects in anti-inflammatory, antioxidant, and anti-obesity aspects. In the study of intervertebral disc degeneration, it is found that forsythin can significantly inhibit the increase in catabolism and the decrease in anabolism of nucleus pulposus cells during the progression of intervertebral disc degeneration, and has obvious inhibitory effects on the activation of the NF-κB pathway and the increase in ROS, thereby regulating the synthesis and catabolism of the extracellular matrix and apoptosis of cells. Through verification, it is found that it has a therapeutic effect in a rat model of intervertebral disc degeneration, confirming its rescue effect on intervertebral disc degeneration in vivo.

[0014] The forsythin provided by the present invention is a drug that can effectively alleviate the progression of intervertebral disc degeneration, which has great social and clinical significance for the research of drugs for intervertebral disc degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0016] Figure 1 It is a diagram showing the effects of forsythin on the protein-level expression of Aggrecan, MMP3, MMP9, MMP13, and ADAMTS5 in nucleus pulposus cells under the action of IL-1β in Example 1;

[0017] Figure 2 It is a diagram showing the effects of forsythin on the activation of the inflammatory pathway in nucleus pulposus cells stimulated by inflammation in Example 1;

[0018] Figure 3 It is a diagram showing the effects of forsythin on the ROS level caused by inflammatory stimulation in Example 1;

[0019] Figure 4 It is a flowchart of the experiment in Example 2;

[0020] Figure 5 It is the MRI scan image of the rat caudal vertebra in Example 2;

[0021] Figure 6 It is the safranin O-fast green and HE staining images of the paraffin sections of the rat intervertebral disc tissue in Example 2. Detailed implementation manners

[0022] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention. It should be understood that the terms described in the present invention are only used to describe specific implementation manners and are not used to limit the present invention.

[0023] In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0025] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0026] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, that is, they are meant to include but not be limited to.

[0027] The present invention provides the application of phillyrin in the preparation of drugs for treating intervertebral disc degeneration.

[0028] The present invention also provides a drug for treating intervertebral disc degeneration, and the drug contains phillyrin.

[0029] As a preferred scheme of the present invention, the drug also contains a pharmaceutically acceptable carrier or excipient and is made into a pharmaceutically acceptable dosage form.

[0030] As a preferred embodiment of the present invention, the pharmaceutically acceptable carrier or excipient includes one or more solid, semi-solid or liquid adjuvants.

[0031] As a preferred embodiment of the present invention, the pharmaceutically acceptable dosage forms include tablets, capsules, granules, injections, pills, syrups, powders, ointments or liquid preparations.

[0032] In some preferred embodiments, the pharmaceutically acceptable dosage form is an injection.

[0033] In some preferred embodiments, the injection is an injection prepared with forsythin and dimethyl sulfoxide, and the concentration of forsythin is 10 μM.

[0034] In the following examples and comparative examples, the forsythin used was purchased from SELLECK with a purity of 99.34%. This will not be repeated below.

[0035] Example 1

[0036] Through in vitro experiments, the effect of forsythin on nucleus pulposus cells was studied

[0037] Experimental method:

[0038] (1) Extraction and culture of primary cells

[0039] After separating the nucleus pulposus tissue from the lumbar intervertebral discs of 8-week-old male SD rats (Animal Laboratory of Zhongshan School of Medicine, Sun Yat-sen University), the tissue was digested with 0.2% protease at 37 °C for 1 hour, and then digested with 2.5% collagenase II at 37 °C for 15 minutes. The nucleus pulposus cells were collected and cultured in DMEM medium supplemented with 10% serum and 1% antibiotic at 37 °C, 5% CO 2 and 20% O 2 The medium was changed every two days until a cell density of 80 - 90% was reached.

[0040] (2) Cell treatment

[0041] The nucleus pulposus cells were seeded into six-well plates. After adherence, the cells were pretreated with forsythin and DMSO for 2 hours according to the grouping, then stimulated with 40 ng / mL IL-1β, and western blot and flow cytometry were performed after a certain time.

[0042] (3) Western blot analysis

[0043] The nucleus pulposus cells were lysed in RIPA lysis buffer containing 1% phosphatase inhibitor and 1% protease inhibitor. After lysis, the proteins were centrifuged and combined with loading buffer followed by heating. Finally, 10 μL of the sample was subjected to protein electrophoresis on an SDS-PAGE gel for 90 minutes, transferred to a PVDF membrane, incubated with the primary antibody overnight, washed three times with TBST, then incubated with the secondary antibody and washed three times with TBST again. Finally, the exposure solution was added and the signal was detected and analyzed using an ECL imager.

[0044] (4) Flow cytometry

[0045] The nucleus pulposus cells were digested with trypsin, resuspended and washed twice with PBS, and incubated with the ROS probe for 20 minutes, then resuspended and washed twice before being loaded onto the machine for detection.

[0046] 1. Observe the effect of forsythin on the extracellular matrix of nucleus pulposus cells stimulated by inflammation

[0047] Forsythin improves the synthesis and catabolism of the extracellular matrix of nucleus pulposus cells under the action of IL-1β. The expression levels of AGGRECAN, MMP3, MMP9, MMP13, and ADAMTS5 at the protein level were evaluated in nucleus pulposus cells. The results are as Figure 1 shown. It can be Figure 1 seen that pretreatment with forsythin can prevent the degradation of the extracellular matrix of nucleus pulposus cells induced by IL-1β. Through Western blot experiments, it was found that forsythin can reduce the degradation of the extracellular matrix caused by IL-1β.

[0048] 2. Observe the effect of forsythin on the activation of the inflammatory pathway in nucleus pulposus cells stimulated by inflammation

[0049] Forsythin reduces the activation of the inflammatory pathway caused by IL-1β in nucleus pulposus cells. The activation of the NF-κB pathway was evaluated by the phosphorylation of P65. The results are as Figure 2 shown. It can be Figure 2 seen that nucleus pulposus cells pretreated with forsythin showed lower phosphorylation of P65 after stimulation with IL-1β, indicating that forsythin can reduce the activation of the NF-κB pathway caused by IL-1β.

[0050] 3. Observe the effect of forsythin on the ROS level caused by inflammatory stimulation

[0051] The results are as Figure 3 shown. It can be Figure 3 seen that nucleus pulposus cells pretreated with forsythin showed a reduced increase in ROS level after stimulation with IL-1β, indicating that forsythin can reduce the oxidative stress caused by IL-1β.

[0052] In summary, forsythin can alleviate IL-1β-induced intervertebral disc degeneration and has potential effects on the treatment of intervertebral disc degeneration.

[0053] Example 2

[0054] In this example, a method of constructing a rat model of intervertebral disc degeneration - applying forsythin or control for local injection in vivo - detecting the conditions of each group of rat models was used to verify the effect of forsythin on treating the rat model of intervertebral disc degeneration. The experimental flow chart is as Figure 4 shown, and the specific steps are as follows:

[0055] 1) Construct a rat model of intervertebral disc degeneration

[0056] Eighteen 8-week-old male SD rats (from the Animal Laboratory of Zhongshan School of Medicine, Sun Yat-sen University) were kept at 26-28 °C and 50-65% humidity for 12 hours a day and night. The animals were fed a standard rodent diet and had free access to fresh water. Before the operation, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (5 mg / 100 g body weight). The tail was disinfected, and the acupuncture segment (Co8 / 9) was determined by fingertip palpation. A (21G) puncture needle was inserted into the center of the intervertebral disc and passed through the entire annulus fibrosus (AF) layer. The needle insertion depth was about 4 mm. After the needle was rotated 720 degrees, it was left in the intervertebral disc for 1 minute, and then the puncture needle was withdrawn. The puncture site was disinfected, and the intervertebral disc degeneration model was established. The 18 male SD rats were divided into groups: Group 1 (control group, hereinafter referred to as CTR), Group 2 (simple intervertebral disc degeneration group, hereinafter referred to as IDD), and Group 3 (intervertebral disc degeneration + forsythin treatment group, hereinafter referred to as IDD + forsythin).

[0057] 2) Apply forsythin and control for injection in vivo

[0058] After the intervertebral disc model was established, 5 μL of forsythin (prepared with dimethyl sulfoxide at a concentration of 10 μM) and DMSO (control) were injected into the intervertebral disc respectively.

[0059] 3) Detect the intervertebral disc degeneration of each group of rats

[0060] After 4 weeks, the caudal vertebrae of the rats were scanned by MRI, and the results are as Figure 5 shown. Then all the rats were sacrificed, the tails were taken, and then the soft tissues were removed. After the spine was fixed with 4% PFA for 48 h, it was soaked in the decalcifying solution for 30 days. The intervertebral disc tissue was sectioned into 5-μm-thick paraffin sections for histological evaluation. The paraffin sections were stained with safranin O-fast green and HE according to standard laboratory procedures. The results are as Figure 6 shown.

[0061] From Figure 5 and Figure 6It can be seen that the construction of the intervertebral disc degeneration model by acupuncture modeling was successful. After acupuncture, the intervertebral disc space decreased significantly, the MRI signal weakened significantly, and a significant decrease in the extracellular matrix was visible in the staining. After local injection of forsythin in the treatment group, both the disc height and signal were increased compared with the acupuncture modeling group, and the decrease in the content of the extracellular matrix was also significantly rescued. It was confirmed that forsythin has a therapeutic effect on intervertebral disc degeneration in vivo.

[0062] As described above, only the preferred specific embodiments of the present invention are provided, and the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes should be covered within the protection scope of the present invention.

Claims

1. Use of forsythin as the only active ingredient in the preparation of a medicament for treating intervertebral disc degeneration.

2. The use according to claim 1, wherein, the medicament further contains a pharmaceutically acceptable carrier or excipient and is made into a pharmaceutically acceptable dosage form.

3. The use according to claim 2, wherein, the pharmaceutically acceptable carrier or excipient includes one or more solid, semi-solid or liquid adjuvants.

4. The use according to claim 2, wherein, the pharmaceutically acceptable dosage form includes tablets, capsules, granules, injections, pills, syrups, powders, ointments or liquid preparations.

5. The use according to claim 2, wherein, the pharmaceutically acceptable dosage form is an injection.

6. The use according to claim 5, wherein, the concentration of forsythin is 5 - 10 μM.

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