A polypeptide and its application in preparing LRP1 activator or medicine for treating intervertebral disc degeneration

Abstract

The present invention relates to the field of biotechnology, and specifically discloses a polypeptide and its application in preparing an LRP1 activator or a drug for treating intervertebral disc degeneration. The polypeptide has an amino acid sequence of: Ac-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu-Gln-Asn-Thr-Lys-NH2. In vitro experiments show that the polypeptide can activate LRP1; in vivo experimental studies show that the polypeptide has a therapeutic effect on intervertebral disc degeneration; therefore, the polypeptide of the present invention can be used to prepare an LRP1 activator or a drug for treating intervertebral disc degeneration.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide and application thereof in preparing an LRP1 activator or a medicine for treating intervertebral disc degeneration. Background Art

[0002] Low back pain (LBP) is one of the main causes of disability. Intervertebral disc degeneration (IDD) is closely related to low back pain, accounting for about 40% of all low back pain cases. At the same time, IDD is also the main cause of spinal degenerative diseases, such as lumbar disc herniation and lumbar spinal stenosis, which lead to severe LBP and even paralysis. The intervertebral disc is located between the cartilages of the adjacent vertebral endplates. It is composed of the surrounding annulus fibrosus and the central gelatinous nucleus pulposus tissue. It plays a role in load support and shock absorption in the spine and is an important guarantee for the normal activity of the spine. IDD changes the height of the intervertebral disc, destroys the biomechanical balance of the spine, and has an adverse effect on structures such as bones, muscles and ligaments. Nucleus pulposus cells play a key role in maintaining the normal function of the intervertebral disc, and their dysfunction is the main trigger for IDD. Although the pathogenesis of IDD is not fully understood, it has been confirmed that increased mechanical load, degeneration of the extracellular matrix, increased secretion of inflammatory factors, excessive aging and apoptosis of nucleus pulposus cells are the main causes of IDD. Studies have shown that inflammation is a key factor in nucleus pulposus cell apoptosis and imbalance between anabolism and catabolism, leading to the destruction of the extracellular matrix (ECM) and ultimately causing IDD.

[0003] At present, there is no specific medicine for disc degeneration, and the main treatment methods are surgical treatment and conservative treatment of anti-inflammatory and analgesic. Conservative treatment generally involves oral nonsteroidal anti-inflammatory drugs (NSAIDs) and physical therapy such as infrared ultrasound to relieve patients' pain, but it cannot stop the progression of disc degeneration, and surgical treatment is inevitable. Although oral NSAIDs can achieve a certain degree of pain relief, they have certain side effects, such as gastrointestinal side effects. Long-term use can cause gastric ulcers; there are also literature reports that there is a risk of causing cardiovascular disease. The main methods of surgical treatment are lumbar fusion and disc replacement. Lumbar fusion requires the removal of the disc, internal fixation and intervertebral fusion, which has large trauma, related intraoperative and postoperative complications, postoperative loss of activity joints and the risk of spondylosis; in terms of economic issues, the high cost of implants and instrument consumables undoubtedly brings a burden to society and families. Therefore, it is a common goal pursued by researchers and medical personnel to explore the molecular mechanisms behind intervertebral disc degeneration, explore possible new therapeutic targets, block its occurrence and development, repair and reconstruct the intervertebral disc, and avoid surgical treatment. Summary of the invention

[0004] In order to solve at least one of the technical problems pointed out in the prior art, the present invention provides a new polypeptide; the polypeptide can activate LRP1 and has a therapeutic effect on intervertebral disc degeneration.

[0005] The above technical problems to be solved by the present invention are achieved through the following technical solutions:

[0006] The present invention first provides a polypeptide, the amino acid sequence of which is:

[0007] Ac-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu-Gln-Asn-Thr-Lys-NH2

[0008] The present invention also provides the use of the polypeptide in preparing an LRP1 activator.

[0009] The polypeptide is hereinafter abbreviated as SP16.

[0010] Low Density Lipoprotein Receptor-Related Protein-1 (LRP1) plays an important regulatory role in various cellular processes such as cell proliferation, survival, motility, differentiation, and transdifferentiation, thereby participating in the pathogenesis of organ dysfunction and injury. Studies have shown that LRP1 controls inflammation, tissue remodeling, and clearance of extracellular molecules. LPR1-knocked-out chondrocytes have downregulated the expression of Aggrecan and Col-2, and increased the expression of MMP-9 and Col-1, leading to chondrocyte degeneration. In addition, studies have shown that LRP1 can inhibit TNF-α-induced chondrocyte apoptosis and inflammatory response. In summary, LRP1 may be a key molecule related to intervertebral disc degeneration. However, in the field of intervertebral disc degeneration research, there has been no research on the relevant intervention and exploration of LRP1.

[0011] Our study on LRP1 showed that after knocking out LRP1, apoptosis of nucleus pulposus cells increased, expression of matrix metalloproteinases was upregulated, and expression of collagen and proteoglycans was downregulated, confirming the importance of LRP1 in the normal maintenance of nucleus pulposus cells. Based on the beneficial effects of LRP1, activation of LRP1 may have a significant therapeutic effect on IDD.

[0012] The polypeptide of the present invention is an artificially synthesized polypeptide, which can activate LRP1 and is a promising polypeptide for treating intervertebral disc degeneration.

[0013] The present invention also provides the use of the polypeptide in preparing a medicine for treating intervertebral disc degeneration.

[0014] The inventors further demonstrated through in vivo experimental studies that the polypeptide of the present invention has a therapeutic effect on intervertebral disc degeneration.

[0015] Beneficial effects: The present invention provides a polypeptide with a novel amino acid sequence; studies have shown that the polypeptide can activate LRP1 and also has a therapeutic effect on intervertebral disc degeneration; therefore, the polypeptide of the present invention can be used to prepare an LRP1 activator or a drug for treating intervertebral disc degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure shows the changes in matrix metabolic phenotype and apoptotic phenotype of nucleus pulposus cells after LRP1 knockdown.

[0017] Figure 2 This figure shows the effect of SP16 on the matrix metabolism and apoptotic phenotype of nucleus pulposus cells under IL-1β stimulation.

[0018] Figure 3 This is the MRI manifestation of the intervertebral disc after 4 weeks of SP16 treatment after the degeneration model was established.

[0019] Figure 4 HE and safranin O staining images of the intervertebral disc after 4 weeks of SP16 treatment after the degeneration model was established. DETAILED DESCRIPTION

[0020] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

[0021] Example 1 Preparation of the polypeptide of the present invention (SP16)

[0022] The polypeptide of the present invention is synthesized by the Fmoc solid phase polypeptide synthesis method: the sequence of the polypeptide of the present invention is:

[0023] Ac-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu-Gln-Asn-Thr-Lys-NH2.

[0024] (1) Take 0.3 g of Fmoc-Linker-MBHA Resin (degree of substitution: about 0.3 mmol / g, total of about 0.09 mmol reaction sites) and swell it with DCM for 20 minutes;

[0025] (2) Add 20% Pip / DMF solution 3 times the volume of the resin, blow nitrogen for 30 minutes, and drain to obtain H2N-Linker-MBHA Resin. (Remove the Fmoc group);

[0026] (3) Wash the resin from the previous step five times with DMF twice the volume of the resin; (wash the resin to remove the residual solvent and prepare for the next reaction);

[0027] (4) Take 0.27mmol Fmoc-Lys(Boc)-OH amino acid, 0.54mmol DIPEA, 0.2565mmol HBTU; an appropriate amount of solvent DMF and react for 30 minutes to obtain Fmoc-Lys(Boc)-Linker-MBHA Resin; amino acid: DIPEA: resin = 3:6:2.85 (molar ratio)

[0028] (5) Wash the resin from the previous step three times with DMF twice the volume of the resin; (wash the resin to remove the residual solvent and prepare for the next reaction);

[0029] (6) Add 20% Pip / DMF solution 3 times the volume of the resin, blow nitrogen for 30 minutes, and drain to obtain H2N-Lys(Boc)-Linker-MBHAResin. (Removal of Fmoc group)

[0030] (7) Wash the resin from the previous step five times with DMF twice the volume of the resin. (Wash the resin to remove the residual solvent and prepare for the next reaction);

[0031] (8) Repeat steps 4, 5, 6, and 7, and add Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Nle-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Val ... oc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val- OH gets H2N-Val-Lys(Boc)-Phe-Asn(Trt)-Lys(Boc)-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu(OtBu)-Gl n(Trt)-Asn(Trt)-Thr(tBu)-Lys(Boc)-Linker-MBHA Resin.

[0032] (9) using 10% acetic anhydride / DMF solution twice the volume of the resin, the resin was capped for 30 minutes, and after washing, Ac-Val-Lys(Boc)-Phe-Asn(Trt)-Lys(Boc)-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu(OtBu)-Gln(Trt)-Asn(Trt)-Thr(tBu)-Lys(Boc)-Linker-MBHA Resin was obtained;

[0033] (10) Wash the resin with methanol three times and drain the resin (to prepare for cutting);

[0034] (11) Cutting: add 6 times the volume of resin with cutting solution (volume ratio; trifluoroacetic acid: thioanisole: 1,2-ethanedithiol: phenol: water = 87.5%: 5%: 2.5%: 2.5%: 2.5%), shake on a shaker for 2 hours, filter out the resin, precipitate the filtrate with ice-cold anhydrous ether, and wash the precipitate with ice-cold anhydrous ether 3 times. Finally, place the precipitate in a vacuum drying kettle and dry it at room temperature for 24 hours to obtain the crude product Ac-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu-Gln-Asn-Thr-Lys-NH2;

[0035] (12) The obtained crude product was purified by HPLC to obtain Ac-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu-Gln-Asn-Thr-Lys-NH2 with a purity of more than 95%;

[0036] Among them, the conditions of HPLC are as follows:

[0037] The chromatographic column is: ZORBAX SB-C18 4.6*250MM, 5um; the mobile phase A is acetonitrile containing 0.1 volume % trifluoroacetic acid, and the mobile phase B is water containing 0.1 volume % trifluoroacetic acid; the flow rate is 1.0 ml / min; the detection wavelength is 220 nm;

[0038] The gradient elution conditions are: at 0.0 min, the volume fraction of mobile phase A is 30%; at 20.0 min, the volume fraction of mobile phase A is 55%; at 20.1 min, the volume fraction of mobile phase A is 100%; at 30 min, it ends; the chromatographic peak retention time of the polypeptide is: 9.492 min.

[0039] Experimental Example 1 In vitro experiment

[0040] Effect of LRP1 on the functional phenotype of rat nucleus pulposus cells

[0041] The rat nucleus pulposus cell line used in the in vitro experiments was from Dr. Chen Di, Department of Orthopedics at Rush University Medical Center.

[0042] ① Knockdown of LRP1

[0043] Nucleus pulposus cells were seeded in 6-well plates one day before transfection, and the cell density on the day of transfection was about 50%. Lipo3000 and siRNA were mixed in Opti-Men medium, incubated at room temperature for 15 minutes, and then added to the well plate. After 4-6 hours, the medium was replaced with 10% FBS + 1% double-antibody DEME complete medium. After 48 hours, protein was extracted to detect changes in related functional phenotypes after LRP1 knockout. Wash with PBS three times, lyse on ice for 30 minutes with RIPA lysis buffer containing 1% protease inhibitors and 1% phosphatase inhibitors, collect the lysate, and centrifuge at 14000rpm for 20 minutes. Collect the supernatant and detect the protein concentration with a BCA kit. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h, the membrane was incubated with the following primary antibodies: Anti-LRP1 (1:10000, Abcam), Anti-COL2a (1:1000, Abcam), Anti-MMP13 (1:1000, Saiweier), Anti-bcl2 (1:1000, Immunoway), Anti-Actived Caspase3 (1:1000, Immunoway), Anti-GAPDH (1:5000, Immunoway) overnight at 4°C. After washing and incubation with horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h, the bands were detected on an automatic chemiluminescence imaging system (GelView 6000pro, Bo Lu Teng).

[0044] ② Activation of LRP1

[0045] Nucleus pulposus cells were seeded in 6-well plates one day before transfection, with a cell density of about 50%. The 10% FBS + 1% double-anti-DEME complete medium was replaced with 2% FBS + 1% double-anti-DEME medium, and the polypeptides prepared according to the method of Example 1 were added for pretreatment for 2 hours at 10 μM and 20 μM, and then IL-1β40 ng / ml was added for treatment for 48 hours to detect the expression of related proteins. Wash with PBS three times, lyse on ice for 30 minutes with RIPA lysis buffer containing 1% protease inhibitors and 1% phosphatase inhibitors, collect the lysate, and centrifuge at 14000 rpm for 20 minutes. Collect the supernatant and detect the protein concentration with a BCA kit. The protein sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk for 1 h, the membrane was incubated with the following primary antibodies: Anti-COL2a (1:1000, Abcam), Anti-Aggrecan (1:1000, Abcam), Anti-MMP13 (1:1000, Servicebio), Anti-bcl2 (1:1000, Immunoway), Anti-Actived Caspase3 (1:1000, Immunoway), Anti-GAPDH (1:5000, Immunoway) overnight at 4°C. The membrane was washed and incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h, and the bands were detected on an automatic chemiluminescence imaging system (GelView 6000pro, Bo Lu Teng).

[0046] The results of in vitro experiments showed that (see Appendix Figure 1 and 2 ):

[0047] ① Knockdown of LRP1

[0048] 1. After knocking down LRP1, the protein level of COL2A1 was downregulated, indicating that the anabolic function of nucleus pulposus cells was affected and the synthesis of extracellular matrix with COL2A1 as the main component was reduced.

[0049] 2. After knocking down LRP1, the protein level of MMP13 was upregulated, indicating that the catabolic function of nucleus pulposus cells was enhanced. MMP13 is an extracellular matrix degrading enzyme that degrades COL2A1.

[0050] 3. After knocking down LRP1, the protein level of BCL2 was downregulated. BCL2 is a protein that inhibits apoptosis, indicating that the nucleus pulposus cells are in a state of apoptosis.

[0051] 4. After knocking out LRP1, the expression of Cleaved-caspase3 was upregulated. This protein is the final executor of apoptosis, indicating that the cells underwent apoptosis.

[0052] In summary, after the expression of LRP1 was downregulated, the nucleus pulposus cells underwent apoptosis, which induced an imbalance in the synthesis and catabolism functions and ultimately led to the degeneration of the intervertebral disc. This indicates that LRP1 is a key molecule related to intervertebral disc degeneration.

[0053] ② Activation of LRP1

[0054] Inflammation is one of the main causes of intervertebral disc degeneration, and IL-1β is an important inflammatory factor and a classic inducer of nucleus pulposus cell degeneration model.

[0055] 1. After the application of SP16, the protein level of COL2A1 was upregulated, indicating that the activation of LRP1 under the stimulation of IL-1β inhibited the downregulation of COL2A1 and promoted its synthesis.

[0056] 2. After using SP16, the protein level of Aggrecan was upregulated. Aggrecan is another important protein in anabolism. This result shows that after activating LRP1, the effect of IL-1β on anabolism function is inhibited.

[0057] 3. After the application of SP16, BCL2 expression was upregulated, indicating that activation of LRP1 can inhibit IL-1β-induced apoptosis.

[0058] 4. After the application of SP16, the expression of Cleaved-caspase3 was downregulated, which once again verified that the activation of LRP1 inhibited the apoptosis of nucleus pulposus cells.

[0059] In summary: The polypeptide (SP16) of the present invention can activate LRP1, inhibit the apoptosis of nucleus pulposus cells in an inflammatory environment, and protect the functional phenotype of nucleus pulposus cells. From the positive and negative aspects of knockdown and activation, it is shown that LRP1 is a key regulatory molecule for intervertebral disc degeneration. Activating LRP1 may be an effective method for treating intervertebral disc degeneration.

[0060] Experimental Example 2 In vivo experiment

[0061] The experimental animals were SPF grade SD male rats.

[0062] ①Build an IDD model

[0063] Twenty-one rats were divided into three groups: normal control group, IDD group, and IDD+SP16 group. The IDD model was established by annulus puncture at 12 weeks of age. The specific steps of the establishment are as follows: After fasting for 12 hours and depriving water for 4 hours, the animals were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital. After disinfecting the skin with 75% ethanol, a 20g puncture needle was used to enter the center of the intervertebral disc vertically through the skin at the junction of the tail fur, one horizontal finger below. The puncture depth was about 4mm, and then the needle was held for 30 seconds after rotating 360 degrees clockwise. Finally, the puncture needle was pulled out to complete the modeling.

[0064] ② Intra-discal injection using the polypeptide of the present invention

[0065] The IDD group and IDD+SP16 group were both modeled using the above method. The control group was operated similarly to the other two groups, but the intervertebral disc was not punctured. One week after the model was established, 4 μL of SP16 (10 μM) was injected into the intervertebral disc of rats in the IDD+SP16 group, and 4 μl of PBS was injected into the optic disc of rats in the IDD group. In order to minimize secondary damage caused by acupuncture, a 33 g Hamilton syringe (Hamilton, USA) was used for injection.

[0066] ③MRI imaging and HE and Safranin O staining

[0067] After 4 weeks of treatment, rats were injected with an overdose of sodium pentobarbital (100 mg / kg), then killed, and the tails were cut off at the upper two segments of the puncture site, and magnetic resonance imaging was performed immediately to record the data. After the MRI was completed, the tail fur was removed, and the tissue was fixed for 48 hours using 4% paraformaldehyde. After 4 weeks of decalcification with EDTA decalcification solution, the tissue was embedded in paraffin and sliced. HE staining and safranin O staining were completed by Wuhan Sewell Company.

[0068] The results of in vivo experiments showed that (see Appendix Figure 3 and 4 ):

[0069] ①MRI results: The water content in the normal nucleus pulposus is high. The intervertebral discs in the normal control group showed high signals; the intervertebral discs in the IDD group showed black low signals, indicating that the water in the intervertebral discs was lost and the intervertebral discs were seriously degenerated. The intervertebral discs in the IDD+SP16 group had some high signals, which were between the normal group and the IDD group, indicating that the polypeptide described in the present invention had a certain alleviating effect on intervertebral disc degeneration.

[0070] ② Specimen staining results: From the staining results, it can be observed that the normal control group has a high content of nucleus pulposus and is full; the IDD group has a low content of nucleus pulposus and the nucleus pulposus is not replaced by the annulus fibrosus tissue; the IDD+SP16 group has an incomplete pulp morphology and a loss of nucleus pulposus tissue, but still maintains a good morphology and a high content of nucleus pulposus, which is significantly improved compared with the IDD group. This shows that the polypeptide of the present invention has a certain therapeutic effect on intervertebral disc degeneration.

Claims

1. Use of a polypeptide in the preparation of a drug for treating intervertebral disc degeneration, characterized in that: The polypeptide has an amino acid sequence of: Ac-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu-Gln-Asn-Thr-Lys-NH2.

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