Application of ligusticum chuanxiong hort extract in preparation of medicine for treating intervertebral disc degeneration

Abstract

The invention belongs to the field of medicine preparation, and particularly relates to application of a ligusticum chuanxiong hort extract to preparation of a medicine for treating intervertebral disc degeneration lesion, the ligusticum chuanxiong hort extract is ligusticum chuanxiong hort lactone, the ligusticum chuanxiong hort lactone can be combined with Atg5 protein and regulate and control interaction of the Atg5 protein and NLRP3 inflammasome, the NLRP3 inflammasome mediated pyroptosis is inhibited, and the NLRP3 inflammasome can be used for treating intervertebral disc degeneration lesion. ECM metabolism in IVDD is balanced again, and related inflammatory reactions are inhibited. Based on the discovery, the ligustralactone can be used as a potential therapeutic drug for the IVDD, and a new drug choice is provided for the treatment of the IVDD.

Description

Technical Field

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

[0002] Intervertebral disc degeneration (IVDD) is one of the most common degenerative diseases and is considered an important cause of chronic low back pain (LBP), which can seriously affect the quality of life and overall health of patients. Epidemiological data show that approximately 80% of the population experiences low back pain at least once in their lifetime, and 540 million patients worldwide are suffering from it for a long time. In China, the number of patients with lumbar diseases has exceeded 200 million, and with the progress of society and the popularization of computer work, the incidence rate is increasing year by year, thus causing a heavy social and economic burden.

[0003] As the core structure of the spine for mechanical load-bearing and buffering, the degenerative changes of the intervertebral disc are the main pathological basis of LBP. The pathogenesis of IVDD is complex and involves key links such as extracellular matrix (ECM) degradation, oxidative stress, imbalance of the inflammatory microenvironment, and programmed cell death (such as apoptosis and pyroptosis). Recent studies have revealed that pyroptosis can play a core role in the process of IVDD through the inflammatory cascade mediated by the NLRP3 inflammasome: pyroptosis triggers the maturation and release of pro-inflammatory factors such as IL-1β and IL-18, exacerbates ECM catabolism (such as high expression of MMP13), and at the same time inhibits anabolism (such as reduction of Collagen II), ultimately leading to the loss of function of nucleus pulposus cells (NPCs) and the collapse of the intervertebral disc structure, and finally causing further spinal diseases (such as lumbar disc herniation, lumbar spinal stenosis, and lumbar instability, etc.), thus resulting in persistent low back pain.

[0004] Due to the complex pathophysiological mechanism of IVDD, there is currently a lack of effective means for clinical treatment of IVDD targeting the etiology. The commonly used treatment strategies still mainly rely on symptomatic treatment (such as non-steroidal anti-inflammatory drugs) and surgical intervention (such as discectomy and fusion), but the former can only relieve symptoms and has significant side effects when used for a long time, and the latter is traumatic, has many complications, and cannot reverse the degeneration process. Therefore, developing new drugs targeting the etiology of IVDD is an urgent need to break through the current treatment dilemma.

[0005] In recent years, with the continuous in-depth research on traditional Chinese medicine, natural Chinese herbal medicine monomers have been considered as important sources of drugs and lead compounds due to their advantages such as safety, effectiveness, and immediate availability. However, due to the complex components of natural Chinese herbal medicines and the unclear molecular targets of Chinese medicine monomers, drug research based on traditional Chinese herbs in China still faces huge obstacles, restricting their clinical translation. The traditional Chinese medicine Chuanxiong Rhizoma has the effects of promoting blood circulation to remove blood stasis, anti-inflammatory and analgesic, and is widely used in the treatment of diseases such as low back pain. However, its active ingredients and mechanisms of action are not yet clear. Based on network pharmacology analysis and in vitro and in vivo experiments, our team first discovered that the key active ingredient of Chuanxiong Rhizoma, (Z)-Ligustilide (abbreviated as LIG), can inhibit pyroptosis of nucleus pulposus cells by targeting the regulation of the Atg5 / NLRP3 axis, block the inflammatory cascade reaction, reshape the ECM metabolic balance, and ultimately delay the progression of IVDD, providing a new strategy for the etiological treatment of IVDD and promising to fill the gap in drug development in this field. Summary of the Invention

[0006] The present invention provides an application of a Chinese herbal medicine monomer named ligustilide (LIG) in the preparation of a drug for inhibiting pyroptosis and improving intervertebral disc degeneration. The present invention focuses on the impact of pyroptosis on IVDD and NPCs, and studies the role played by ligustilide in regulating pyroptosis and IVDD.

[0007] The technical solution provided by the present invention is as follows:

[0008] The application of Chuanxiong Rhizoma extract in the preparation of a drug for treating intervertebral disc degenerative diseases, wherein the Chuanxiong Rhizoma extract is ligustilide, and the molecular formula of the ligustilide is C 12 H 14 O 2 , and the structural formula is:

[0009] .

[0010] Furthermore, the Chuanxiong Rhizoma extract is used to improve the extracellular matrix metabolism in intervertebral disc degeneration.

[0011] Furthermore, the Chuanxiong Rhizoma extract is used to inhibit the activation of NLRP3 inflammasome.

[0012] Furthermore, the Chuanxiong Rhizoma extract is used to inhibit pyroptosis of nucleus pulposus cells.

[0013] Furthermore, the Chuanxiong Rhizoma extract is used to block the assembly of NLRP3 inflammasome and inhibit pyroptosis signal transduction by regulating the interaction between Atg5 / NLRP.

[0014] Furthermore, the Chuanxiong Rhizoma extract is used to delay intervertebral disc degenerative diseases in mammals.

[0015] Furthermore, the Chuanxiong extract is used to inhibit intervertebral disc collapse and dehydration degeneration.

[0016] Furthermore, the concentration of the Chuanxiong extract is 10 - 50 μM.

[0017] Furthermore, the pharmaceutical dosage form is tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained release tablets, capsules, soft capsules, dripping pills, granules, injections, powder injections or aerosols.

[0018] Furthermore, the drug also contains a drug carrier, and the drug carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0019] According to the specific embodiments of the present invention, the drug is used to treat and / or relieve pyroptosis and the progression of IVDD, and the drug is a preparation prepared from ligustilide in an effective amount as the main or sole active ingredient and pharmaceutically acceptable excipients.

[0020] Beneficial effects

[0021] The present invention has confirmed through experiments that ligustilide can bind to the Atg5 protein and regulate its interaction with the NLRP3 inflammasome. By inhibiting pyroptosis mediated by the NLRP3 inflammasome, it rebalances the ECM metabolism in IVDD and inhibits related inflammatory reactions. Based on the above findings, ligustilide can be used as a potential therapeutic drug for IVDD, providing a new drug option for the treatment of IVDD. Description of the drawings

[0022] Figure 1 Ligustilide reshaped the ECM metabolic balance of nucleus pulposus cells; among which A is the result of protein gel electrophoresis detection; B is the result of immunofluorescence staining;

[0023] Figure 2 Ligustilide inhibited pyroptosis of nucleus pulposus cells mediated by the NLRP3 inflammasome; among which A is the comparison of apoptosis rate results; B is the result of protein gel electrophoresis detection; C is the result of immunofluorescence staining;

[0024] Figure 3 Ligustilide regulated the interaction between Atg5 / NLRP3 and inhibited pyroptosis; among which A is the binding situation of ligustilide and Atg5; B is the result of immunofluorescence staining; C is the result of protein gel electrophoresis detection;

[0025] Figure 4Ligustilide delays IVDD; where A is the experimental process; B is the results of X-ray imaging and MRI images; C is the comparison result of disc height; D is the comparison result of disc signal intensity; E is the result of histological staining; F is the result of histological grading. Detailed implementation mode

[0026] The technical solutions of the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the described embodiments.

[0027] The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product instructions. The reagents and raw materials used in the present invention are all commercially available.

[0028] Example 1

[0029] Ligustilide remodels the ECM metabolic balance in nucleus pulposus cells

[0030] Primary rat nucleus pulposus cells were isolated and cultured, and DMEM / F12 medium was used for cell culture. Passage was carried out when the density reached 80% - 90%. The P3 chondrocytes were seeded in 6-well / 24-well cell culture plates at 3000 cells / cm² and cultured with DMEM / F12 medium. RT-PCR, WB, and immunofluorescence methods were used to detect the effects of ligustilide on the anabolic metabolism of ECM in nucleus pulposus cells. The specific groups were: Control group, LIG group, LPS + ATP group, LPS + ATP + LIG group. After 3 days of culture, immunofluorescence staining, RT-PCR detection, and protein gel electrophoresis detection were carried out respectively. The main observation indexes were: the expression levels of Aggrecan, CollagenII, MMP9, and MMP13, and the fluorescence intensities of Collagen II and MMP13. The results are as Figure 1 shown. In vitro, ligustilide intervention can remodel the metabolic balance of extracellular ECM in nucleus pulposus cells.

[0031] In this example, ligustilide was purchased from MedChemExpress, product model HY-N0401A.

[0032] Example 2

[0033] Ligustilide inhibits NLRP3 inflammasome-mediated pyroptosis of nucleus pulposus cells

[0034] The P3 generation of chondrocytes was seeded at 3000 cells / cm² in 6-well / 24-well cell culture plates and cultured using DMEM / F12 medium. TUNEL staining, RT-PCR, WB, and immunofluorescence were used to detect the effect of ligustilide on pyroptosis of nucleus pulposus cells induced by LPS + ATP. The specific groups were: Control group, LIG group, LPS + ATP group, and LPS + ATP + LIG group. After 3 days of culture, immunofluorescence staining, RT-PCR detection, and protein gel electrophoresis detection were performed respectively. The main observation indicators were: the proportion of apoptotic cells, the expression levels of NLRP3, GSDMD-N, cle-IL-1β, and caspsae-1, and the fluorescence intensities of NLRP3 and GSDMD. The results are as Figure 2 shown, in vitro ligustilide intervention can inhibit pyroptosis of nucleus pulposus cells.

[0035] Example 3

[0036] Ligustilide regulates the interaction between Atg5 / NLRP3 and inhibits pyroptosis

[0037] Computer simulation was carried out using the structure of Atg5 (PDBId: 4tq1) in the database, and the ligustilide molecule was artificially simulated and sent into the Atg5 pocket to preliminarily study the binding of ligustilide and Atg5. The analysis showed that ligustilide can be embedded in the protein structure of Atg5 and form pi-alkyl (hydrophobic bond) interactions with the residues of TRP-166, ARG-170, and MET-173. The P3 generation of chondrocytes was seeded at 3000 cells / cm² in 6-well / 24-well cell culture plates and cultured using DMEM / F12 medium. Immunofluorescence and immunoprecipitation (Co-ip) were used to detect the effect of ligustilide on the interaction between Atg5 and NLRP3. The main observation indicators were: the expression levels and fluorescence intensities of Atg5 and NLRP3. The results are as Figure 3 shown, in vitro ligustilide can bind to Atg5 to promote its interaction with NLRP3 and delay pyroptosis.

[0038] Example 4

[0039] In vivo application of ligustilide delays the progression of IVDD

[0040] (1) Eighty-week-old SD rats were selected as experimental animals and grouped into: Control group, IVDD group, IVDD + LIG (Low) group, and IVDD + LIG (High) group. The rat IVDD model was established by damaging the lumbar intervertebral disc with the classic acupuncture method to cause its degeneration. Intraperitoneal injection treatment was started on the 3rd day after surgery, once a day. Knee joint specimens were collected at the 4th week and the 8th week after surgery for subsequent detection.

[0041] (2) Radiological evaluation of the effect of ligustilide on disc height and signal intensity. The specific implementation steps were as follows: at 4 weeks after surgery, the caudal vertebrae of rats were evaluated by X-ray and MRI. By analyzing the obtained X-ray images and MRI images of the T2-weighted (T2WI) sequence, the disc height index and optical density were calculated. The results were as Figure 4 shown in B-D, ligustilide inhibited disc collapse and dehydration degeneration in vivo.

[0042] (3) Histological evaluation of the effect of ligustilide on IVDD. The specific implementation steps were as follows: the caudal vertebrae of rats obtained at 4 weeks and 8 weeks after surgery were immersed in 10% formalin for 48 hours, and then decalcified with 10% EDTA. Finally, paraffin-embedded sections were made, and multiple histological stains (hematoxylin-eosin, safranin-fast green) were performed on sections with a thickness of 6 µm / slice. After examining the samples under a microscope, the histological grade was evaluated. The results were as Figure 4 shown in E-F, ligustilide inhibited disc collapse and dehydration degeneration in vivo.

Claims

1. The use of Chuanxiong extract in the preparation of a drug for treating intervertebral disc degeneration, characterized in that: The Chuanxiong extract is Chuanxiong lactone, and the structural formula of Chuanxiong lactone is: 。 2. The use according to claim 1, characterized in that: The Chuanxiong extract is used for improving the metabolism of extracellular matrix in degenerative intervertebral disc lesions.

3. The use according to claim 1, characterized in that: The Chuanxiong extract is used to inhibit NLRP3 inflammasome activation.

4. The use according to claim 1, characterized in that: The Chuanxiong extract is used for inhibiting pyroptosis of nucleus pulposus cells.

5. The use according to claim 1, characterized in that: The Chuanxiong extract is used to block NLRP3 inflammasome assembly and inhibit pyroptosis signal transduction by regulating Atg5 / NLRP interaction.

6. The use according to claim 1, characterized in that: The Chuanxiong extract is used for delaying degenerative lesions of intervertebral discs in mammals.

7. The use according to claim 1, characterized in that: The Chuanxiong extract is used for inhibiting intervertebral disc collapse and dehydration degeneration.

8. The use according to claim 1, characterized in that: The concentration of the Chuanxiong extract is 10-50 μM.

9. The use according to claim 1, characterized in that: The drug dosage form is tablet, dispersible tablet, lozenge, orodisintegrating tablet, sustained-release tablet, capsule, soft capsule, dripping pill, granule, injection, powder injection or aerosol.

10. The use according to claim 1, characterized in that: The medicine further comprises a drug carrier, which includes one or more of fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents commonly used in medicine.

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