Application of phentolamine in preparation of medicine for treating intervertebral disc degeneration

Abstract

The invention discloses application of phentolamine in preparation of a medicine for treating intervertebral disc degeneration, and relates to the technical field of biomedicine.The phentolamine is applied to preparation of the medicine for treating intervertebral disc degeneration, inflammation inhibition, matrix expression promotion, matrix degradation inhibition and nucleus pulposus cell aging delaying are facilitated, and the effect of treating intervertebral disc degeneration is achieved. The height of the intervertebral disc can be increased, the integrity degree of a cell structure in the intervertebral disc can be improved, and the content of aggregation proteoglycan can be recovered.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of phentolamine in the preparation of a drug for treating intervertebral disc degeneration. BACKGROUND

[0002] Intervertebral disc degeneration is a kind of degenerative disease characterized by aging and biochemical imbalance of intervertebral disc tissue, mainly manifested as lower back pain and lower extremity nerve root pain, and its pathological mechanism involves mechanical compression theory and inflammatory reaction. Studies have found that the activity of inflammatory factors such as phospholipase A2 (PLA2), interleukin (IL-1, IL-6), and tumor necrosis factor alpha (TNFα) significantly increases during intervertebral disc degeneration, and these factors cause pain by inducing nerve root inflammation and sensitization. At present, the treatment methods for intervertebral disc degeneration mainly focus on physical therapy, painkillers or surgical intervention, such as the use of corticosteroids and other anti-inflammatory drugs to suppress inflammation, the use of intervertebral fusion to connect adjacent vertebral bodies, and there is still no effective drug intervention means.

[0003] Phentolamine is a classic non-selective alpha-adrenergic receptor blocker, commonly used for the treatment of hypertensive crisis, pheochromocytoma and other vascular-related diseases, and has been widely used for many years. Its safety and efficacy have been subjected to long-term clinical tests, but its research in the field of intervertebral disc degeneration is limited. SUMMARY

[0004] In view of the above-mentioned deficiencies of the related art, the present application provides the application of phentolamine in the preparation of a drug for treating intervertebral disc degeneration. The application of phentolamine in the preparation of a drug for treating intervertebral disc degeneration is beneficial to inhibit inflammation, promote matrix expression and inhibit matrix degradation, and delay the aging of nucleus pulposus cells, which is beneficial to improve the intervertebral disc height and improve the integrity of the cell structure in the intervertebral disc, and restore the content of aggregated proteoglycans.

[0005] In one aspect, the present application provides the application of phentolamine in the preparation of a drug for treating intervertebral disc degeneration.

[0006] Preferably, the administration mode of the phentolamine is intervertebral disc injection.

[0007] Preferably, the administration amount of the phentolamine is 0.2-2.5 mg / kg.

[0008] Preferably, the administration amount of the phentolamine is 0.5 mg / kg.

[0009] Preferably, the administration concentration of the phentolamine is 5-50 μg / μL.

[0010] Preferably, the administration concentration of the phentolamine is 15 μg / μL.

[0011] Preferably, the treating intervertebral disc degeneration comprises treating intervertebral disc degeneration by inhibiting expression of inflammatory factors.

[0012] Preferably, the treating intervertebral disc degeneration comprises treating intervertebral disc degeneration by promoting matrix synthesis.

[0013] Preferably, the treating intervertebral disc degeneration comprises treating intervertebral disc degeneration by inhibiting matrix degradation.

[0014] Preferably, the treating intervertebral disc degeneration comprises treating intervertebral disc degeneration by inhibiting expression of senescence phenotype.

[0015] Preferably, the intervertebral disc degeneration comprises one or more of intervertebral disc herniation, degenerative scoliosis, spinal stenosis and discogenic pain.

[0016] In another aspect, the present application provides a medicine for treating intervertebral disc degeneration, which comprises the following technical solution: A medicine for treating intervertebral disc degeneration, comprising phentolamine.

[0017] In summary, the present application comprises at least one of the following beneficial technical effects: 1. The present application uses phentolamine to prepare a medicine for treating intervertebral disc degeneration, which is beneficial to improve the intervertebral disc height and the integrity of the cell structure in the intervertebral disc, and restore the proteoglycan content.

[0018] 2. The present application uses phentolamine to prepare a medicine for treating intervertebral disc degeneration, which is beneficial to inhibit inflammation, promote matrix expression and inhibit matrix degradation, and delay the aging of the nucleus pulposus cells. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a comparison chart of cell activity of nucleus pulposus cells treated by phentolamine treatment solution at different treatment times with changes in phentolamine concentration; Figure 2 is a comparison chart of real-time fluorescence quantitative detection results of interleukin-6 (IL-6) in the control group, the model group and the treatment group in the cell experiment; Figure 3 is a comparison chart of real-time fluorescence quantitative detection results of tumor necrosis factor alpha (TNF-α) in the control group, the model group and the treatment group in the cell experiment; Figure 4 is a comparison chart of real-time fluorescence quantitative detection results of tumor necrosis factor alpha (TNF-α) in the control group, the model group and the treatment group in the cell experiment; Figure 5is a comparison chart of real-time fluorescence quantitative detection results of collagen Ⅱ of the control group, the model group and the treatment group in the cell experiment; Figure 6 is a comparison chart of real-time fluorescence quantitative detection results of aggrecan of the control group, the model group and the treatment group in the cell experiment; Figure 7 is a comparison chart of Western blot detection results of IL-6, TNF-α, Collagen Ⅱ, ACAN and MMP13 of the control group, the model group and the treatment group in the cell experiment; Figure 8 is a comparison chart of Western blot detection results of senescence phenotypes P16, P21 and P53 of the control group, the model group and the treatment group in the cell experiment; Figure 9 is a comparison chart of β-galactosidase activity staining results of the control group, the model group and the treatment group in the cell experiment; Figure 10 is a CT chart of the tail intervertebral disc of the control group, the model group and the treatment group in the animal experiment; Figure 11 is a comparison chart of intervertebral space height index of the tail intervertebral disc of the control group, the model group and the treatment group in the animal experiment; Figure 12 is a nuclear magnetic resonance chart of the tail intervertebral disc of the control group, the model group and the treatment group in the animal experiment; Figure 13 is a comparison chart of HE staining results of the tail intervertebral disc section of the control group, the model group and the treatment group in the animal experiment; Figure 14 is a comparison chart of ponceau solid green staining results of the tail intervertebral disc section of the control group, the model group and the treatment group in the animal experiment; Figure 15 is a comparison chart of immunohistochemical Collagen II staining results of the tail intervertebral disc section of the control group, the model group and the treatment group in the animal experiment; Figure 16 is a comparison chart of immunohistochemical ACAN staining results of the tail intervertebral disc section of the control group, the model group and the treatment group in the animal experiment. DETAILED DESCRIPTION

[0020] The application will be further described in conjunction with the following examples. The following examples are only used to illustrate the application, and should not be considered as limiting the scope of the application. The specific conditions are not specified in the following examples, which are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The methods used are conventional methods known in the art unless otherwise specified. The consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used herein have the same meaning as understood by those skilled in the art. In addition, any method or material similar or equivalent to that described can also be applied in the present application.

[0021] Phentolamine, also known as 3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]phenol, has a chemical formula of C 17 H 19 N3O, CAS No.: 50-60-2, and a chemical structure of: .

[0022] Example 1 Cell experiment Extraction and washing of nucleus pulposus tissue: 6-week-old SD rats (purchased from Hubei Center for Disease Control and Prevention) were sacrificed by spinal dislocation, and then immersed in alcohol for disinfection. The hair behind the spine was removed, the skin was cut, the muscle was bluntly separated, the spine was exposed, the D1-D7 segments were taken, the intervertebral disc was exposed, and the nucleus pulposus tissue in the center was semi-transparent and gelatinous. The nucleus pulposus tissue was gently separated from the intervertebral disc with a sterile spatula, and then washed with physiological saline until no blood was found. The nucleus pulposus tissue was placed in a centrifuge tube.

[0023] Digestion of nucleus pulposus tissue: In a cell clean bench, PBS solution was added to the centrifuge tube containing the nucleus pulposus tissue, and the tissue was cut into 1mm 3 size tissue pieces using sterile surgical instruments. A solution of type II collagenase (Sevier GC305014) with a concentration of 1-2 mg / ml was prepared and added to the centrifuge tube and mixed well. The digestion was carried out for 2-3 h, and the mixture was shaken every 15 min.

[0024] Extraction of nucleus pulposus cells: After the nucleus pulposus tissue was digested according to the above digestion scheme, a turbid cell suspension was formed. A 10 μm cell sieve was used to filter out the tissue pieces. The filtered cell suspension was centrifuged at 1200 rpm for 5 min to remove the supernatant. 2 mL of complete medium was added and mixed well. The cell culture bottle was divided into one cell culture bottle per mL of complete medium, and 4 mL of complete medium was added to the cell culture bottle.

[0025] Nucleus pulposus cell culture and passage: Samples were taken from the cell culture flasks for testing. When the density under a microscope reached 80%-90%, 2 mL of 0.25% trypsin-EDTA digestion solution (Seville G4001) was added to the cell culture flasks to ensure that the trypsin covered all cell surfaces. Digestion was carried out for 2 min until the nucleus pulposus cells shrank and became round. 2 mL of complete culture medium was added to the cell culture flasks to stop the digestion. The cell culture flasks were pipetted and the nucleus pulposus cells were collected into centrifuge tubes. The tubes were centrifuged at 1200 rpm for 5 min. The supernatant was removed, and the cells were resuspended in 6 mL of complete culture medium and plated into 6-well plates.

[0026] Nucleus pulposus cells were treated with phentolamine at different concentrations (0 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, and 10 μM) for 24 h and 48 h, and cell viability was assessed. The results are as follows: Figure 1 As shown. According to Figure 1 It is known that when the concentration of phentolamine treatment solution is 0.5-3 μM, the cell viability is still maintained at over 90%. Considering the dosage and cell viability, the preferred concentration of phentolamine treatment solution is 2 μM.

[0027] Drug treatment: When the cells grew to a density of 50%-60%, an IL-1β treatment solution with a concentration of 10 ng / mL was prepared using PBS buffer and IL-1β stock solution. A phentolamine treatment solution with a concentration of 1 μM was prepared by dissolving phentolamine in DMSO. The control group was not treated with drugs. The model group (labeled as IL-1β) was treated with IL-1β treatment solution to induce inflammatory damage. The treatment group (labeled as PM+IL-1β) was treated with IL-1β stock solution and phentolamine treatment solution.

[0028] Twenty-four hours after drug administration, real-time quantitative PCR of interleukin-6 (IL-6) was performed on the control group, model group, and treatment group, respectively. The results are as follows: Figure 2 As shown; real-time fluorescence quantitative PCR was performed on the control group, model group, and treatment group, respectively, and the results are as follows. Figure 3 As shown; real-time fluorescence quantitative PCR was performed on the control group, model group, and treatment group, respectively, and the results are as follows. Figure 4 As shown; real-time fluorescence quantitative PCR was performed on the control group, model group, and treatment group, respectively, and the results are as follows. Figure 5 As shown; real-time fluorescence quantitative PCR was performed on the matrix-degrading enzyme MMP13 in the control group, model group, and treatment group, and the results are as follows. Figure 6 As shown; Western blot analysis was performed on IL-6, TNF-α, Collagen II, ACAN, and MMP13 in the control group, model group, and treatment group, respectively. The results are as follows. Figure 7As shown; Western blot analysis was performed on the aging phenotypes P16, P21, and P53 in the control group, model group, and treatment group, respectively. The results are as follows. Figure 8 As shown; the control group, model group, and treatment group were stained using the β-galactosidase activity staining method, and the staining results are as follows. Figure 9 As shown.

[0029] Reference Figures 2-3 as well as Figure 7 It can be seen that, compared with the model group, the expression of inflammatory factors IL-6 and TNF-α in the treatment group was significantly reduced, approaching that of the control group. Figure 2 and Figure 3 The results and Figure 7 The results were consistent, indicating that phentolamine use is beneficial in inhibiting the expression of inflammatory factors in nucleus pulposus cells; (Refer to...) Figures 4-5 as well as Figure 7 It can be seen that, compared with the model group, the expression of matrix Collagen II and ACAN in the treatment group was upregulated, approaching that of the control group. Figure 4 and Figure 5 The results and Figure 7 The results were consistent, indicating that phentolamine use is beneficial in promoting matrix synthesis in nucleus pulposus cells; (Refer to...) Figure 6 and Figure 7 It can be seen that, compared with the model group, the expression of matrix-degrading enzyme MMP13 in the treatment group was reduced, approaching that of the control group. Figure 6 The results and Figure 7 The results were consistent, indicating that the use of phentolamine was beneficial in inhibiting the expression of matrix-degrading enzymes in nucleus pulposus cells, thereby inhibiting the reduction of matrix; (Refer to...) Figure 8 and Figure 9 It can be seen that, Figure 8 Compared to the model group, the expression of aging phenotypes P16, P21, and P53 was significantly reduced in the treatment group, approaching that of the control group. Figure 9 It can be clearly observed that the number of positive staining cells in the treatment group is significantly less than that in the model group. Figure 8 The results were consistent, indicating that the use of phentolamine is beneficial in inhibiting the aging of nucleus pulposus cells.

[0030] Example 2 Animal experiments Construction of the intervertebral disc degeneration model: Eight-week-old SD rats (280-320g) were selected, and the caudal vertebrae were labeled Co-3, Co-4, Co-5, and Co-6 from top to bottom. The Co4-5 and Co5-6 caudal intervertebral discs were randomly punctured with a 22G needle. The puncture needle was inserted parallel to the cartilaginous endplate and perpendicular to the skin. The needle was rotated 360° and held for 30 seconds before being withdrawn. On the first day after the operation, 5μL of IL-1β-infused PBS buffer solution (20ng / μL) was injected into the nucleus pulposus of the Co5-6 and Co6-7 caudal intervertebral discs to obtain the IL-1β-induced intervertebral disc degeneration model. The Co3-4 caudal intervertebral disc without treatment was used as the control group, and the Co4-5 caudal intervertebral disc was used as the model group.

[0031] Phentolamine preparation and injection: 5 mg phentolamine was dissolved in 30 μL LDMSO and 300 μL of normal saline to prepare a phentolamine injection solution with a concentration of approximately 15 μg / μL. On day 7 after establishing the intervertebral disc degeneration model, 10 μL of phentolamine injection solution was injected into the Co5-6 coccygeal intervertebral disc, which served as the treatment group.

[0032] Examination: Five weeks after establishing the intervertebral disc degeneration model, a coccyx was harvested for CT scan, and the results were as follows. Figure 10 As shown, the Co3-4 coccygeal intervertebral discs were labeled as the control group, the Co4-5 coccygeal intervertebral discs were labeled as IL-1β, and the Co5-6 coccygeal intervertebral discs were labeled as PM+IL-1β; for Figure 10 The analysis yielded the intervertebral disc height index, such as... Figure 11 As shown, the Co3-4 coccygeal intervertebral discs were labeled as the control group, the Co4-5 coccygeal intervertebral discs as IVDD, and the Co5-6 coccygeal intervertebral discs as IVDD+PM; MRI of the coccyx was performed, and the results are as follows. Figure 12 As shown, the Co3-4 coccygeal intervertebral discs were labeled as the control group, the Co4-5 coccygeal intervertebral discs were labeled as IL-1β, and the Co5-6 coccygeal intervertebral discs were labeled as PM+IL-1β. Sections of the coccygeal intervertebral discs were stained with hematoxylin and eosin (HE), and the results are as follows. Figure 13 As shown, the Co3-4 coccygeal intervertebral discs were labeled as the control group, the Co4-5 coccygeal intervertebral discs were labeled as IL-1β, and the Co5-6 coccygeal intervertebral discs were labeled as PM+IL-1β. Sections of the coccygeal intervertebral discs were stained with Safranin and Fast Green, and the results are as follows. Figure 14 As shown, the Co3-4 coccygeal intervertebral discs were labeled as the control group, the Co4-5 coccygeal intervertebral discs were labeled as IL-1β, and the Co5-6 coccygeal intervertebral discs were labeled as PM+IL-1β. Coccygeal intervertebral disc sections were stained with Collagen II and subjected to immunohistochemical staining. The results are as follows. Figure 15The results are shown in Figure 6, wherein the Co3-4 coccygeal disc is marked as the control group, the Co4-5 coccygeal disc is marked as IL-1β, and the Co5-6 coccygeal disc is marked as PM+IL-1β. The coccygeal disc sections were taken for immunohistochemical detection of ACAN staining, and the results are shown in Figure 7. Figure 16 The results are shown in Figure 6, wherein the Co3-4 coccygeal disc is marked as the control group, the Co4-5 coccygeal disc is marked as IL-1β, and the Co5-6 coccygeal disc is marked as PM+IL-1β.

[0033] Referring to Figures 10-12 It can be seen that Figure 11 The height score of the treatment group was higher than that of the model group, and the height score of the treatment group was close to that of the control group, indicating that the use of phentolamine was beneficial to restore the height of the intervertebral disc to achieve a therapeutic effect. It is analyzed that phentolamine restores the height of the intervertebral disc by inhibiting water loss from the nucleus pulposus, Figure 10 and Figure 12 The results are shown in Figure 6, wherein the Co3-4 coccygeal disc is marked as the control group, the Co4-5 coccygeal disc is marked as IL-1β, and the Co5-6 coccygeal disc is marked as PM+IL-1β. Figure 11 Referring to Figures 13-16 It can be seen that the nucleus pulposus part of the control group has a complete nucleus pulposus structure, the nucleus pulposus structure of the nucleus pulposus part of the model group completely disappears, and the nucleus pulposus structure of the nucleus pulposus part of the treatment group still exists, indicating that phentolamine is beneficial to inhibit the disappearance of the nucleus pulposus and has a therapeutic effect on intervertebral disc degeneration.

[0034] The above are optional embodiments of the present application, and do not limit the protection scope of the present application, therefore: any equivalent changes made in accordance with the structure, shape, principle of the present application should be covered within the protection scope of the present application.

Claims

1. Application of phentolamine in the preparation of drugs for treating intervertebral disc degeneration.

2. The application according to claim 1, characterized in that: The administration method of phentolamine is intervertebral disc injection.

3. The application according to claim 2, characterized in that: The dosage of phentolamine is 0.2-2.5 mg / kg.

4. The application according to claim 2, characterized in that: The dosage of phentolamine is 5-50 μg / μL.

5. The application according to claim 1, characterized in that: The treatment of intervertebral disc degeneration includes treating intervertebral disc degeneration by inhibiting the expression of inflammatory factors.

6. The application according to claim 1, characterized in that: The treatment of intervertebral disc degeneration includes treating intervertebral disc degeneration by promoting matrix synthesis.

7. The application according to claim 1, characterized in that: The treatment of intervertebral disc degeneration includes treating intervertebral disc degeneration by inhibiting matrix degradation.

8. The application according to claim 1, characterized in that: The treatment of intervertebral disc degeneration includes treating intervertebral disc degeneration by inhibiting the expression of senescence phenotypes.

9. The application according to claim 1, characterized in that: The degenerative disc disease includes one or more of the following: disc herniation, degenerative scoliosis, spinal stenosis, and discogenic pain.

10. A drug for treating intervertebral disc degeneration, characterized in that: Including phentolamine.

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