阅读说明：本技术 瑞香素在制备防治椎间盘退变药物中的应用 (Application of daphnetin in preparation of medicine for preventing and treating intervertebral disc degeneration ) 是由 王建儒 陈顺伦 陈凡 于 2021-09-27 设计创作，主要内容包括：本发明属于生物医药技术领域,具体涉及瑞香素在制备防治椎间盘退变药物中的应用,本发明提供了瑞香素的新用途,即可通过抑制NF-κB通路发挥抗氧化应激、抗炎和凋亡作用,从而延缓椎间盘退变,可用于防治椎间盘退变,特别是可制备成防治椎间盘退变药物的形式用于防治椎间盘退变。本发明不仅提供了瑞香素的新应用方向,也为椎间盘退变的治疗提供了一种新的治疗药物和治疗途径。(The invention belongs to the technical field of biological medicines, and particularly relates to application of daphnetin in preparation of a medicament for preventing and treating intervertebral disc degeneration. The invention not only provides a new application direction of daphnetin, but also provides a new therapeutic drug and a new therapeutic approach for the treatment of intervertebral disc degeneration.)

1. Application of daphnetin in preparing medicine for preventing and treating intervertebral disc degeneration is provided.

2. Use according to claim 1, characterized in that the concentration of daphnetin is 2-5 μ M.

3. The use as claimed in claim 1, wherein the prevention and treatment of disc degeneration is through inhibition of NF- κ B pathway exerting antioxidant stress, anti-inflammatory and apoptotic effects, thereby delaying disc degeneration.

4. A medicine for preventing and treating intervertebral disc degeneration is characterized in that daphnetin is taken as a main active ingredient.

5. The drug for preventing and treating intervertebral disc degeneration according to claim 4, further comprising other active ingredients which are compatible with daphnetin to play a synergistic effect.

6. The medicament for preventing and treating intervertebral disc degeneration according to claim 4, further comprising a pharmaceutically acceptable carrier and/or excipient.

7. The medicine for preventing and treating intervertebral disc degeneration according to claim 4, wherein the dosage form of the medicine includes but is not limited to injection, tablet, granule, capsule, dripping pill, sustained release agent, oral liquid preparation.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of daphnetin in preparation of a medicine for preventing and treating intervertebral disc degeneration.

Background

Intervertebral disc degeneration (IVDD) is one of the main causes of Low Back Pain (LBP), and the pathological manifestations are imbalance of synthesis and catabolism of extracellular matrix, apoptosis of nucleus pulposus cells, local inflammatory reaction, and enhancement of oxidative stress. The physical, psychological and economic burden is brought to IVDD, the specific mechanism of IVDD generation is still not clear at present, the research is mainly focused on the directions of biomechanics and inflammation pathology, and the like, and researchers begin to pay attention to the relation between the energy metabolism of nucleus pulposus and the generation of degeneration recently. At present, the curative effect of non-operative treatment is not clear clinically, most patients still need a nuclectomy operation in the later stage, and the operation inevitably has certain traumatism, so how to effectively prevent and treat IVDD becomes an important subject in the field of spinal surgery.

Daphnetin (Daphnetin), also known as Daphnetin A, is a natural extract of Daphne odora (Daphne Korean Nakai) belonging to the family daphneceae and having the chemical name of 7, 8-dihydroxycoumarin (7, 8-dithioxycoumarin), and belongs to coumarin compounds. Daphnetin has therapeutic effect on pain, malaria, thromboangiitis obliterans, angina pectoris, and rheumatoid arthritis. However, no report about the therapeutic effect of daphnetin on lumbago or intervertebral disc degeneration exists at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a new application of daphnetin, namely daphnetin can play the roles of resisting oxidative stress, resisting inflammation and apoptosis by inhibiting an NF-kB channel, thereby delaying the degeneration of intervertebral discs and providing a new treatment way for the degeneration of intervertebral discs.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides an application of daphnetin in preparing a medicament for preventing and treating intervertebral disc degeneration.

Preferably, the prevention and treatment of disc degeneration is achieved by inhibiting NF-kB pathway to exert antioxidant stress, anti-inflammatory and apoptosis effects, thereby delaying disc degeneration.

The invention researches the action and mechanism of daphnetin on intervertebral discs from two aspects of cells and animal models in vitro and in vivo, and the research result shows that (as shown in figure 1), daphnetin can play the roles of resisting oxidative stress, resisting inflammation and apoptosis by inhibiting NF-kB channels, thereby delaying the degeneration of intervertebral discs. The daphnetin is suggested to be used for preventing and treating the degeneration of the intervertebral disc, and particularly used for preventing and treating the degeneration of the intervertebral disc in the form of a medicament for preventing and treating the degeneration of the intervertebral disc.

Preferably, the concentration of daphnetin is 2-5 μ M. At the concentration, daphnetin has low toxicity to cells, and is safe and harmless.

The invention also provides a medicament for preventing and treating intervertebral disc degeneration, which takes daphnetin as a main active ingredient.

Preferably, the composition also comprises other effective components which are compatible with daphnetin to play a synergistic effect.

Preferably, a pharmaceutically acceptable carrier and/or excipient is also included. Namely, the medicine can be mixed with a pharmaceutically acceptable carrier or excipient to prepare a composition.

Further, the excipient refers to diluents, binders, lubricants, disintegrants, cosolvents, stabilizers and the like which can be used in the pharmaceutical field, and some pharmaceutical bases. The carrier is functional pharmaceutical adjuvant available in the pharmaceutical field, and comprises surfactant, suspending agent, emulsifier and some novel medicinal polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid polylactic acid copolymer (PLGA), hyaluronic acid, etc.

Preferably, the dosage form of the medicament comprises but is not limited to injection, tablets, granules, capsules, dripping pills, sustained release preparations and oral liquid preparations. The preparation is a clinically common preparation. Pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) and, if certain drugs are unstable under gastric conditions, may be formulated as enteric coated tablets.

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

the invention provides a new application of daphnetin, namely, the daphnetin can play the roles of resisting oxidative stress, resisting inflammation and apoptosis by inhibiting NF-kB channels so as to delay the degeneration of intervertebral discs, can be used for preventing and treating the degeneration of the intervertebral discs, and particularly can be prepared into a medicament for preventing and treating the degeneration of the intervertebral discs. The invention not only provides a new application direction of daphnetin, but also provides a new therapeutic drug and a new therapeutic approach for the treatment of intervertebral disc degeneration.

Drawings

FIG. 1 is a schematic diagram of daphnetin for retarding degeneration of intervertebral discs;

FIG. 2 is a graph of the toxic effect of daphnetin on nucleus pulposus cells;

FIG. 3 shows the reversal of TNF- α induced oxidative stress by daphnetin (β -actin is an internal reference protein);

FIG. 4 is a graph showing the inhibitory effect of daphnetin on H2O 2-induced oxidative stress;

FIG. 5 shows the results of RT-qPCR detection of the expression levels of inflammatory factor (IL-6 and IL-1. beta.) mRNA;

FIG. 6 shows the results of ELISA detection of the secretion level of IL-6 protein, an inflammatory factor;

FIG. 7 shows WB assay results of the expression level of IL-6 protein, an inflammatory factor (Tublin is an internal reference protein);

FIG. 8 is a graph of the anti-apoptotic effect of daphnetin;

FIG. 9 shows the NF-. kappa.B pathway inhibition by daphnetin (β -actin is an internal reference protein);

FIG. 10 shows the degree of degeneration of intervertebral discs of mice of the punctured IVDD model after daphnetin treatment (CTR is a control group, not punctured; punture is a punctured IVDD model group; punture + DAP is a modeled intraperitoneal DAP group).

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 Effect of daphnetin (abcam, ab143113) on nucleus pulposus cells

(1) Extraction and culture of nucleus pulposus cells

Nucleus pulposus cells are derived from nucleus pulposus tissue isolated under sterile conditions from an intervertebral disc specimen taken after clinical surgery (patient informed consent was signed before specimen acquisition and approved by the ethical committee of the first hospital affiliated to the university of zhongshan) or rat intervertebral disc isolated under sterile conditions (approved by the ethical committee of the center of experimental animals).

The method for separating nucleus pulposus cells from nucleus pulposus tissue comprises the following steps: the isolated nucleus pulposus tissue (one segment) was first placed at 37 ℃ in 5% CO₂In the incubator, 20mL DMEM (containing 0.2% protease, 10% FBS) was added to digest the nucleus pulposus tissue for 1 h. Followed by another 20mL DMEM (containing 0.025% collagenase, 10% FBS) for 15 h. Finally, the precipitate is collected into a centrifuge tube for centrifugation (900r/min), and the precipitate is added into DMEM medium containing 10% FBS after being resuspended and placed at 37 ℃ and 5% CO₂Subculturing in an incubator. Spreading 80-90% of culture dish for passage, freezing part of cells after the third generation, storing in liquid nitrogen, and recovering directly for subsequent use.

(2) Cytotoxicity of daphnetin

Human or rat nucleus pulposus cells are planted in a DMEM culture dish, replaced by new DMEM whole culture medium after 24h, then Daphnetin (DAP) with different concentrations (2 mu M,5 mu M,10 mu M,20 mu M,50 mu M) is added to treat the nucleus pulposus cells for 24h, and the toxicity of daphnetin on the nucleus pulposus cells is detected by using a cytotoxicity kit (CCK-8) (Dojindo Lab, Japan).

(3) Antioxidant stress, antiinflammatory and anti-apoptosis effects of daphnetin

Human or rat nucleus pulposus cells are planted in a DMEM culture dish, and are replaced by new DMEM full culture medium after 24 hours, and then the following treatment is carried out: the TNF-alpha (20 mu M) treatment group is an induced inflammation and apoptosis cell model group and is treated for 24 hours; h₂O₂The induction group (200 mu M) is an oxidative stress model group and is treated for 4 hours; the daphnetin treatment group comprises dissolving daphnetin in DMSO with mother liquor concentration of 10mM, respectively setting the final concentration of daphnetin as four gradients of 1 μ M,2 μ M,5 μ M and 10 μ M, treating with daphnetin for 30min, and adding TNF-alpha (24H) or H₂O (2 h). After the treatment is finished, extracting total protein or total RNA directly according to the experiment requirement, and finally detecting the expression condition of the target protein (superoxide dismutase SOD1) by Western Blot (WB); real-time fluorescent quantitative PCR (RT-qPCR) is used for detecting the expression of target mRNA (IL-1 beta, IL-6)(ii) a Detecting the level of intracellular oxidative stress with an intracellular oxidative stress probe DCFH-DA (Sigma, D6883); the secretion of inflammatory factors (IL-1. beta., IL-6) was measured using ELIA kit (MultiSciences, EK 301B/3-96; EK 306/3-96).

(4) Results of the experiment

1) Toxicity of daphnetin to nucleus pulposus cells

As shown in fig. 2, after treating nucleus pulposus cells with Daphnetin (DAP) at different concentrations for 24h, daphnetin in the range of 2-5 μ M was found to have low cytotoxicity, and no significant difference compared with the control group (CTR).

2) Daphnetin for promoting expression of superoxide dismutase SOD1

SOD1 is expressed more in vivo under oxidative stress, and thus exerts antioxidant stress. In a diagram of fig. 3, CTR is a control group, and is not treated, and the rest is total protein extracted after DAP (0.5-10uM) with different concentrations is added for 24 hours, and Western blot experiment detection is carried out, so that DAP can promote the expression level of SOD1 protein, and the effect of 0.5-5uM is obvious, thereby showing that DAP can play a role in resisting oxidative stress; the CTR in the b picture is a control group and is not treated, DAP is not added in the second group from left to right, only TNF-alpha is added, DAP (1-5uM) is added in the rest groups before the TNF-alpha is added for pretreatment for 2h, then total protein is extracted after TNF-alpha is added for treatment for 24h, Western blot experiment detection is carried out, the expression of SOD1 can be inhibited by TNF-alpha, DAP can weaken the inhibition effect of the TNF-alpha on SOD1, and the 2uM or 5uM effect is optimal, so that DAP can reverse a TNF-alpha induced model, and the anti-oxidative stress effect is exerted. It can be seen that the WB results in fig. 3 indicate that daphnetin itself can promote the expression of SOD1, while reversing the effects of TNF- α in the TNF- α treatment induction model.

3) Daphnetin has antioxidant stress effect

In FIG. 4, DCFH-DA is intracellular oxidative stress assay kit, H₂O₂Inducing an intracellular oxidative stress model, and indicating that Daphnetin (DAP) can inhibit intracellular oxidative stress and H₂O₂Induced oxidative stress.

4) Daphnetin has anti-inflammatory effect

As can be seen from the expression level of the inflammatory factor mRNA of fig. 5, the secretion level of the inflammatory factor protein of fig. 6, and the expression level of the inflammatory factor protein of fig. 7 (Tublin is an internal reference protein), daphnetin can inhibit the expression of inflammatory factors, and has an anti-inflammatory effect.

5) Daphnetin has anti-apoptosis effect

Cells were divided into control (CTR, no treatment), DAP (2 uM DAP added), TNF-. alpha. + DAP (TNF-. alpha.20 ng/mL after 2h of DAP addition at various concentrations (1-5 uM)), according to the treatment protocol, according to the apoptosis kit (MultiSciences, AP107) and detected by flow cytometry (Beckman Coulter, Fullerton). The data in FIG. 8 represent normal cell ratios, indicating that TNF- α can induce apoptosis, while Daphnetin (DAP) can exert an anti-apoptotic effect.

6) Daphnetin plays roles of resisting oxidative stress, resisting inflammation and resisting apoptosis by inhibiting NF-kB pathway

The results in fig. 9 show that TNF- α can promote phosphorylation of P65, increase expression of PP65 (PP 65 becomes PP65 after phosphorylation of P65, PP65 can enter cell nucleus to activate inflammation-related genes, thereby initiating biological processes such as inflammation, oxidative stress and apoptosis), and Daphnetin (DAP) can inhibit the action, so daphnetin can inhibit NF- κ B pathway, thereby exerting the effects of antioxidant stress, anti-inflammation and anti-apoptosis.

The analysis shows that daphnetin has the functions of resisting oxidative stress, resisting inflammation and resisting apoptosis.

Example 2 therapeutic Effect of daphnetin on IVDD model mice

(1) Establishment of animal model

The rat acupuncture degeneration model is widely applied and simulates human body IVDD to have higher similarity, and is the most accepted classic animal model for studying IVDD. In this study, a mouse needle degeneration model (IVDD mouse model) was established using C57BL/6N mice (Gemphamatech, China). The specific operation is as follows: the mice were anesthetized by intraperitoneal injection of pentobarbital (50mg/kg), prepared, fixed in supine position on an operating table, sterilized and draped conventionally. The skin, subcutaneous tissue, was incised to expose the L4/5 disc. The operation is carried out under an operation microscope, the depth is limited by using small vascular forceps, a 31G syringe needle is used for puncturing the intervertebral disc annulus fibrosis by about 3mm in parallel to the end plate, the intervertebral disc annulus fibrosis is slowly pulled out after staying for about 30 seconds, and the muscle is sutured by using a 3-0 silk thread and the skin is sutured by using a 4-0 silk thread. Raising the pigs in cages after the recovery from anesthesia. The control group exposed only the L4/5 disc as above, did not undergo a puncture procedure, and was then sutured.

(2) Intraperitoneal injection of daphnetin and Magnetic Resonance Imaging (MRI) examination of animals

The next day after the IVDD mouse model is established, the daphnetin is diluted by PBS, the model mouse is injected into the abdominal cavity according to the amount of 3mg/kg, the injection is continuously carried out for 2 weeks, then the feeding is continuously carried out, and the MRI detection is carried out 4 weeks and 8 weeks after the model is established, so as to determine the degeneration condition of the intervertebral disc.

(3) Results of the experiment

The results in fig. 10 show that white arrows indicate punctured discs, disc degeneration was evident in the pure group, and disc degeneration was alleviated in mice at 4 and 8 weeks after intraperitoneal injection of daphnetin.

In conclusion, the results of the examples 1 and 2 show that daphnetin can play the roles of resisting oxidative stress, resisting inflammation and apoptosis by inhibiting NF-kB channels, thereby delaying the degeneration of intervertebral discs.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.