Application of pyrroloquinoline quinone in preparation of composition for treating and / or relieving spinal degenerative diseases

Abstract

The invention discloses an application of pyrroloquinoline quinone (PQQ) in preparation of a composition for treating and / or relieving spinal degenerative diseases. The PQQ is applied to a LSI (Lumbar Spine Instability) model of a mouse, and the research finds that the PQQ can be used for remarkably improving the spine anaphylaxis and anxiety behaviors caused by the LSI operation. PQQ treatment can relieve mechanical and heat pain sensitivity, improve the activity level of mice and reduce mechanical allergy symptoms. In addition, by reducing senescent osteoclasts (SnOCs) and regulating inflammatory response, the PQQ can be used for remarkably relieving the degenerative change of the lumbar vertebra end plate and protecting the spine microstructure. The PQQ also effectively reduces the porosity of the end plate and the separation of bone trabecula, and significantly reduces the number of TRAP + osteoclasts in the end plate, which further shows that the PQQ has a protective effect on degenerative diseases. As a potential disease correction treatment medicine, PQQ not only can relieve low back pain, but also can improve other symptoms related to spinal degenerative diseases, and a new strategy is provided for treatment of related diseases.

Description

Technical Field

[0001] The present invention relates to the fields of medicinal chemistry and pharmacotherapy, in particular to the field of treatment of spinal degenerative diseases, and more particularly to the use of pyrroloquinoline quinone in preparing a composition for treating and / or alleviating spinal degenerative diseases. Background Art

[0002] With aging or spinal injury, spinal degenerative lesions (such as lumbar disc degeneration, endplate injury, etc.) gradually affect the body's functions, causing patients to experience low back pain (LBP) and related anxiety symptoms.

[0003] For the treatment of spinal degenerative diseases, most oral traditional Chinese medicines contain highly toxic ingredients, and long-term use can cause gastrointestinal and other side effects. Conventional Chinese medicine patches can avoid systemic side effects, but they suffer from low percutaneous absorption, slow onset of action, poor efficacy, and a long treatment course. Unless the patient's condition is extremely severe and invasive surgery is necessary, doctors generally recommend conservative treatments. Currently, clinical treatment options include traction therapy, infrared thermotherapy, and transcutaneous electrical stimulation. However, these methods are limited to large tertiary hospitals or specialized rehabilitation centers due to the large size of the equipment used, high per-session costs, and some side effects. Furthermore, these methods do not allow for remote rehabilitation training guidance from doctors. Therefore, for most patients, the high cost, timeliness, and effectiveness of treatment may lead to reduced or even no treatment. Both oral traditional Chinese medicine and conservative treatments focus on symptom relief, but have limited effectiveness in structurally repairing spinal degenerative diseases and are associated with significant side effects. Recent research has found that senescent osteoclasts (SnOCs) in spinal degeneration play a key role in lumbar endplate damage and pain. Existing technologies have not yet proposed effective treatments targeting this mechanism. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a use of pyrroloquinoline quinone in the preparation of a composition for treating and / or alleviating spinal degenerative diseases, thereby alleviating low back pain caused by spinal degenerative diseases by clearing senescent osteoclasts in the lumbar endplate, and providing a new research direction for spinal degenerative diseases.

[0005] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0006] Use of a pyrroloquinoline quinone compound, or a pharmaceutically acceptable salt thereof, in preparing a composition for treating and / or alleviating spinal degenerative diseases.

[0007] As an improvement, the dosage form of the composition includes tablets, powders, injections, granules, ointments, suppositories, suspensions, syrups or capsules.

[0008] As an improvement, the content of the pyrroloquinoline quinone compound or its pharmaceutically acceptable salt in the pharmaceutical composition is 4 mg / kg.

[0009] A pharmaceutical composition comprises an active ingredient and a pharmaceutically acceptable excipient and / or carrier, wherein the active ingredient is the above-mentioned pyrroloquinoline quinone compound or a pharmaceutically acceptable salt thereof.

[0010] Beneficial effects:

[0011] Compared to existing technologies, the present invention's use of pyrroloquinoline quinone in the preparation of a composition for treating and / or alleviating spinal degenerative diseases has demonstrated, through animal experiments, that PQQ alleviates spinal pain through multiple mechanisms, including reducing the number of senescent osteoclasts, modulating inflammatory pathways, and influencing neurotrophic and angiogenic processes. By targeting these factors, PQQ significantly alleviates low back pain and related anxiety symptoms caused by spinal degenerative lesions. It can effectively reduce inflammatory factors associated with degenerative spinal lesions, modulate the immune response, and slow disease progression. It also eliminates senescent osteoclasts, improves spinal structure and function, and reduces lamina porosity and trabecular separation. This has potential disease-modifying effects and provides a potential strategy for addressing the structural degeneration and functional impairment associated with spinal instability. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of different treatments on LSI mice and the results of their behavioral effects;

[0013] Figure 2 This is the result of the protective effect of PQQ treatment on the lumbar endplate structure in LSI mice;

[0014] Figure 3 Schematic diagram of the effects of PQQ on aging osteoclasts and the regulation of inflammatory factors;

[0015] Figure 4 The result is that PQQ treatment reduces the growth of neural endothelium;

[0016] Figure 5 The results show the effects of PQQ treatment on angiogenesis and osteogenesis in LSI mice;

[0017] Figure 6 The mechanism by which PQQ treatment alleviates spinal pain in the LSI mouse model. DETAILED DESCRIPTION

[0018] The following examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0019] 1. Materials and Methods

[0020] 1. Materials

[0021] 1.1 Reagents and experimental equipment

[0022] 1.1.1 Main drugs and reagents

[0023] Pyrroloquinoline quinone (PQQ) was purchased from Sigma, USA; methylprednisolone was purchased from Pfizer, Belgium; mouse type I procollagen amino-terminal propeptide (PINP) and type I collagen cross-linked C-terminal peptide (CTX) enzyme-linked immunosorbent assay kits were purchased from Elabscience, China; 10% ethylenediaminetetraacetic acid (10% EDTA) decalcification solution was purchased from Nanjing Jiancheng, China; OCT embedding solution (OCT Compound) was purchased from Sakura Tissue Tek, USA; hematoxylin and eosin were purchased from Yuanye, China; and 10% paraformaldehyde, sucrose, anhydrous ethanol, distilled water, paraffin, phosphate-buffered saline (PBS), 10% chloral hydrate, neutral resin, and anti-fluorescence fading mounting medium.

[0024] 1.1.2 Main instruments

[0025] Micro-CT (SkyScan 1176, Belgium), paraffin slicer (Leica 2135, Germany), freezing slicer (Leica CM1520, Germany), slide baker (Leica 1120, Germany), paraffin embedding machine (BMJ-Ⅱ, Changzhou, China), Axiovert40C optical microscope (Zeiss, Germany), microplate reader (Biotec, USA), and a set of surgical instruments.

[0026] 1.2 Experimental animals

[0027] Thirty healthy male C57BL / 6J mice, weighing 25 ± 5 g, and aged 8 weeks, were provided by the Animal Experimental Center of Xuzhou Medical University. They were maintained under the following conditions: room temperature of 18–20°C, humidity of 50–60%, good ventilation, and free access to food and water. All procedures were performed in accordance with the Animal Care Committee and the Guide for the Care and Use of Animals of the U.S. National Institutes of Health.

[0028] 2. Experimental methods

[0029] 2.1 Experimental Animal Grouping

[0030] Thirty C57BL / 6J mice were randomly divided into the following three groups:

[0031] (1) Sham group (control group):

[0032] 10 rats, only the skin surface of the lumbar spinous processes 3-5 was cut. They were fed with normal feed every day until they were killed;

[0033] (2) LSI group (model group):

[0034] Ten rats were surgically removed from the lumbar spinous processes, supraspinous ligaments, and interspinous ligaments at L3-5 to establish a lumbar vertebral instability model. The rats were fed a normal diet daily until sacrifice.

[0035] (3) PQQ group (treatment group):

[0036] Ten rats underwent surgical resection of the lumbar spinous processes, supraspinous ligaments, and interspinous ligaments at L3-5 to establish a lumbar spinal instability model. Eight weeks later, PQQ (4 mg / kg) was added to the normal diet until sacrifice.

[0037] 2.2 Preparation of the mouse lumbar spine instability (LSI) model

[0038] The present invention uses surgery to destroy the posterior column structure of the lumbar spine (such as the supraspinous ligament, interspinous ligament, and articular process), simulates spinal biomechanical imbalance, and induces intervertebral disc degeneration and pain behavior. This model is consistent with the progressive pathological process of human intervertebral disc degeneration and is commonly used to study the degeneration mechanism and pain signal conduction. This application selects 8-week-old male mice, cuts the skin along the midline of the lumbar spine, and exposes the L3-L5 spinous process and lamina. The supraspinous ligament and interspinous ligament of the L3-L4 and L4-L5 segments are removed; the bilateral inferior articular processes of L4 are bitten off (destroying the stability of the articular process joints); the muscles and skin are sutured layer by layer, and antibiotics (cefotaxime, 50 mg / kg) are injected for 3 consecutive days after surgery. For the control group mice, only the skin of the waist is cut without destroying other structures.

[0039] 2.3 Specimen collection

[0040] Eight weeks after LSI surgery, no special treatment was given to simulate the course of chronic low back pain. After eight weeks, the mice were supplemented with a normal diet or PQQ (4 mg / kg) for another eight weeks. After the experiment, pain behavior monitoring was performed. Mechanical pain threshold was measured using von Frey fibers, and thermal pain latency was measured using the Hargreaves method to assess hyperalgesia. After behavioral testing, the mice were sacrificed and serum was collected: cardiac blood was collected using capillary blood collection tubes and placed in a 4°C refrigerator for 2 hours. After the blood coagulated and the clot shrank, it was centrifuged at 3000 rpm for 20 minutes. The upper layer of serum was collected for bone metabolism marker detection. Lumbar vertebrae 3, 4, and 5 were then collected and fixed in 4% neutral paraformaldehyde for 48 hours. Bone parameters were collected using Micro-CT scanning. The lumbar vertebrae were then decalcified with 10% EDTA for 4 weeks and embedded in paraffin for histological staining analysis.

[0041] Micro-CT scanning was used to observe endplate sclerosis, osteophyte formation, and changes in intervertebral disc height. HE staining was used to assess the structural integrity of the annulus fibrosus. Safranin fast green staining was used to detect proteoglycan content and verify the degree of nucleus pulposus degeneration. Immunohistochemistry was used to detect TRAP+ osteoclasts and CGRP+ nerve fiber infiltration in the endplates, indicating pain-related neural remodeling. RT-qPCR and west blot analysis revealed upregulated expression of degeneration markers (such as COX-2, IL-1β, and MMP-3).

[0042] 2.4 Pain behavioral testing

[0043] 2.4.1. Mechanical hyperalgesia test (von Frey fiber method)

[0044] (1) Place the mice on a metal mesh floor in a transparent plexiglass box (20 × 20 × 15 cm) and allow them to adapt for 20 minutes.

[0045] (2) Use von Frey fibers to vertically stimulate the central area of ​​the mouse's hind foot, starting from a low intensity (0.4 g) and gradually increasing the pressure, with each fiber staying for 5 seconds.

[0046] (3) Record the minimum threshold force (g) required for the mouse to withdraw, lick, or flick its paw, and repeat this three times to obtain the average value.

[0047] 2.4.2. Thermal hyperalgesia test (Hargreaves method)

[0048] (1) Place the mice on the glass bottom of a transparent plexiglass box and allow them to adapt for 20 minutes.

[0049] (2) Use Hargreaves instrument to focus the thermal light source (intensity 50-70mW / cm 2) irradiate the hind paw and record the latency (seconds) of the mouse's paw withdrawal response.

[0050] (3) Each foot was tested three times with an interval of 5 minutes and the average value was taken.

[0051] 2.4.3. Cold pain test (acetone evaporation method)

[0052] (1) Place the mice in a test box with a metal mesh bottom and allow them to adapt for 20 minutes.

[0053] (2) Use a micropipette to drop 50 μl of acetone on the sole of the hind paw, and record the number of times the mouse licks, lifts, or shakes its paw within 5 minutes.

[0054] Motor function assessment

[0055] (1) Balance beam test: The stability and time of mice walking on a 1 cm diameter circular balance beam were recorded to eliminate the influence of movement disorders on pain testing.

[0056] (2) Rotarod test: The time mice spend on a rotating rod (4-40 rpm) is measured to assess motor coordination.

[0057] 2.5 Anxiety Behavioral Testing Steps

[0058] 2.5.1. Elevated plus maze (EPM)

[0059] (1) Utilize the mouse's natural fear of open space to assess anxiety (the shorter the time spent in the open arm, the higher the anxiety level). Gently place the mouse in the center of the maze (facing the open arm) and start recording.

[0060] (2) Observe for 5 minutes and record the following indicators: open arm stay time (seconds); number of open arm entries (one entry is counted as one full entry of all four limbs); total movement distance (cm, excluding motor ability interference).

[0061] 2.5.2. Open field test (OFT)

[0062] (1) The exploratory behavior of mice in an open environment reflects their anxiety level (the shorter the time spent in the center area, the more obvious the anxiety). Gently place the mouse in the corner of the open field box (facing the wall) and start recording.

[0063] (2) Observe for 10 minutes and record the following indicators: time spent in the center area (seconds); number of entries into the center area; total movement distance (cm); number of upright positions (forelimbs ≥ 1 cm off the ground, reflecting exploratory motivation).

[0064] 2.5.3. Light-Dark Box Test (LDB)

[0065] (1) Mice prefer dark environments. The time and frequency of entering the light area reflect their anxiolytic ability. Place the mouse in the dark area, close the door, and allow it to adapt for 1 minute.

[0066] (2) Open the access door and start recording for 5 minutes, recording the following indicators: time spent in the bright area (seconds); number of times the animal shuttles between the bright and dark areas; and latency to first entry into the bright area (seconds).

[0067] 2.6 Micro-CT detection

[0068] After 48 hours of fixation, micro-CT scanning was performed with the following parameters: resolution 18 μm, voltage 50 kV, current 500 μA, exposure time 100 ms, and 0.9° / 8 images. The lumbar spine of the mouse was selected as the region of interest (ROI). Micro-CT image analysis software was used to perform three-dimensional reconstruction and analysis of the images, and bone mineral density (BMD, g / cm2) of the lumbar spine in the ROI region was recorded. 3 ), trabecular bone volume to total bone volume ratio (BV / TV), trabecular separation / spacing (Tb.Sp, μm -1 ), trabecular thickness (Trabecular thickness, Tb.Th, mm).

[0069] After anesthesia, mice were killed by cervical dislocation, and the lumbar vertebrae (L3-L3) were completely removed, preserving the paravertebral soft tissue (to avoid traction damage to the vertebral body). They were immediately placed in 4% paraformaldehyde (PFA) for fixation for 24-48 hours to maintain the stability of the vertebral anatomical structure. A high-resolution Micro-CT system (Bruker SkyScan 1176) was used for scanning. The scanning parameters were: voltage (kV) 70kV, current 100μA, exposure time 300ms, 0.3° step, 360° full-angle scanning, and resolution 9μm. Key indicators were measured using the equipment's supporting software (such as NRecon, Bruker CTvox): bone density (BMD, mg HA / cm 3 ) and bone volume fraction (BV / TV,%); osteophytes at the vertebral margin were marked in the 3D model and the osteophyte volume (mm 3 ) and number (≥3 consecutive levels were defined as 1 osteophyte); the mid-disc height of the L4-L5 intervertebral space was measured in sagittal images, and the differences were compared between the normal group and the model group.

[0070] 2.7 Histological staining

[0071] 2.7.1 H&E staining

[0072] The collected lumbar vertebrae were fixed with 10% paraformaldehyde for 48 hours and decalcified with 10% EDTA for 4 weeks. Then, a gradient ethanol dehydration process was initiated: immersion in 60%, 70%, 80%, 90%, and 100% ethanol for 1 hour each. The dehydrated vertebrae were then placed in xylene for 3 hours and then in 70°C paraffin for 8 hours. Finally, the vertebrae were placed in a mold, filled with paraffin, and cooled on a -20°C cooling block for 1 hour. The solidified tissue wax blocks were removed and stored at room temperature or sectioned and stained at a thickness of 6 μm.

[0073] H&E staining steps:

[0074] (1) Dewaxing and rehydration: After dewaxing with xylene (10 min × 3 times), the sections were rehydrated in 100%, 100%, 95%, 85%, and 70% ethanol to deionized water, respectively, for 10 min each time.

[0075] (2) Rinse with distilled water for 3 minutes, stain with hematoxylin solution for 5 minutes, and rinse with tap water for 2 minutes;

[0076] (3) Differentiation with 1% hydrochloric acid alcohol solution for 30 seconds, followed by rinsing with tap water for 1 minute;

[0077] (4) Bluing in 10% ammonia solution for 30 seconds, then rinse with tap water for 1 minute;

[0078] (5) Counterstain with 1% eosin solution for 5 minutes and rinse with tap water for 1 minute;

[0079] (6) Routine dehydration, transparency, and sealing.

[0080] Evaluation method: Select five consecutive sections and observe the porosity of the end plate under a 20× optical microscope.

[0081] 2.7.2 Safranin Fast Green Staining

[0082] After dewaxing the lumbar vertebrae with xylene, the blocks were hydrated with graded ethanol, rinsed with distilled water for 5 minutes, and immersed in 0.02% Fast Green staining solution (dissolved in 0.2% glacial acetic acid) for 5 minutes. The blocks were then differentiated with 1% glacial acetic acid for 10 seconds and rinsed with distilled water. The blocks were then immersed in 0.1% Safranin O staining solution (dissolved in 1% ethanol) for 5-10 minutes. The blocks were quickly rinsed with distilled water to remove the floating color, then dehydrated and mounted. The junction area between the endplate and the intervertebral disc was identified in the sagittal section (the endplate was red and the calcified area was green). The percentage of the red non-calcified area (proteoglycan-rich area) in the endplate to the total endplate area was calculated (threshold segmentation using ImageJ software). The thickness ratio of the calcified endplate (green) to the non-calcified cartilage (red) was measured.

[0083] 2.8 Immunofluorescence staining

[0084] After dewaxing the lumbar vertebrae paraffin blocks with xylene, they were hydrated with gradient ethanol and rinsed with distilled water for 5 minutes. Antigen retrieval was first performed using citrate buffer (pH 6.0) and high-pressure heat repair (121°C, 20 minutes), followed by natural cooling to room temperature. 5% BSA + 0.3% Triton X-100 in PBS blocking solution was added dropwise and incubated at room temperature for 1 hour. Primary antibody was added and incubated overnight at room temperature. The corresponding secondary antibody (1:500) was added dropwise in the dark and incubated at room temperature for 1 hour. DAPI (1:1000) was used to counterstain the cell nuclei for 5 minutes and the cells were rinsed with PBS. Anti-fluorescence quencher (ProLong Gold) was added dropwise, the sections were sealed with coverslips, and dried in the dark for 24 hours.

[0085] Primary antibodies included p16 (1:200, ab211542, Abcam), TRAP (1:200, ab191406, Abcam), CGRP (1:500, ab81887, Abcam), PGP9.5 (1:200, ab108986, Abcam), CD31 (1:1000, ab222783, Abcam), Endomucin (1:200, ab106100, Abcam), osterix (1:1000, ab22552, Abcam), and osteocalcin (1:500, M188, Takara). Secondary antibodies included Alexa Fluor 488 (green), 555 (red), and 647 (far red).

[0086] 2.9 Westblot experiment

[0087] (1) Mouse lumbar endplates were quickly frozen in liquid nitrogen and ground into powder. RIPA lysis buffer (containing 1% protease inhibitors and 1% phosphatase inhibitors) was added. The concentration was determined using the BCA assay and adjusted to a uniform concentration (4 μg / μL).

[0088] Add 4×LDS loading buffer (containing 10% β-mercaptoethanol) and denature at 95°C for 5 minutes.

[0089] (2) Prepare different concentrations of separating gel (7.5%-15%) and 5% stacking gel. Load 20-40 μg of total protein per well. Then perform constant voltage electrophoresis: 80V for stacking gel and 120V for separating gel (Mini-PROTEAN system).

[0090] (3) PVDF membrane (0.45 μm) was activated with methanol for 1 minute.

[0091] (4) Block with 5% skim milk (prepared with TBST) and shake at room temperature for 1 hour.

[0092] (5) Add primary antibody and incubate overnight (4°C). The antibodies used included: p16 (1:1000, ab211542, Abcam), p21 (1:200, ab109199, Abcam), p53 (1:500, ab26, Abcam), Sirt1 (1:1000, ab110304, Abcam), BMI-1 (1:1000, ab269678, Abcam), γH2A.X (1:1000, ab81299, Abcam), Endomucin (1:1000, ab106100, Abcam), CD31 (1:1000, sc-376764, Santa Cruz Biotechnology, Inc.), Osterix (1:1000, ab209484, Abcam), Osteocalcin (1:1000, ab309521, Abcam), and GAPDH (1:1000, ab8245, Abcam) was then incubated with HRP-conjugated secondary antibody (anti-rabbit / mouse / rat) at a dilution of 1:5000 for 1 hour at room temperature.

[0093] (6) Mix ECL A / B solution in a ratio of 1:1, cover the membrane surface, and react in the dark for 1 minute. Use the ChemiDoc imaging system in automatic exposure mode for image acquisition.

[0094] 2.10 Lumbar endplate ELISA test

[0095] (1) Place the lumbar endplates in 10% EDTA solution (pH 7.4) at 4°C for 48 hours, replacing the EDTA solution every 12 hours. Grind the decalcified tissue in liquid nitrogen and add 500 μL of RIPA lysis buffer (containing 1× protease inhibitor, Thermo#87786) for 30 minutes on ice. Ultrasonicate using a Qsonica sonicator (20% amplitude, 5-second pulses × 3 times). Centrifuge at 14,000 × g, 4°C, for 20 minutes, and collect the supernatant. Determine the total protein concentration by the BCA method and adjust to 1 mg / mL.

[0096] (2) Set up standard wells, blank wells, and sample wells separately: add 100 μL of serially diluted standard to the standard wells, 100 μL of sample diluent to the blank wells, and 100 μL of the sample to be tested to the remaining wells; coat the ELISA plate with film and incubate at 37°C for 90 minutes;

[0097] (3) Shake off the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, seal the membrane, and incubate at 37°C for 1 h.

[0098] (4) Shake off all liquid in the wells, add 350 μL of washing solution to each well and soak for 1 min, shake off the liquid, and repeat this washing step 3 times;

[0099] (5) Add 100 μL of enzyme conjugate working solution to each well, seal the film, and incubate at 37°C for 1 h;

[0100] (6) Shake off the liquid in the wells and wash the plate 5 times using the same method as step 4;

[0101] (7) Add 90 μL of substrate solution (TMB) to each well, cover the plate with film, seal the film, and incubate at 37°C for 15 min;

[0102] (8) Add 50 μL of stop solution to each well to terminate the reaction, and measure the optical density (OD value) of each well at 450 nm using a microplate reader.

[0103] (9) Kits used: IL-6 (BMS603-2, ThermoFisher), TNF-α (BMS607-3, ThermoFisher), Netrin-1 (EM56RB, ThermoFisher), prostaglandin E2 (PGE2) (SKGE004B, R&D Systems).

[0104] 2.11 Statistical Analysis

[0105] Data were analyzed using Graphpad Prism 9.0 statistical software. Data are expressed as mean ± standard deviation. Multiple group comparisons were performed using one-way ANOVA. Pairwise comparisons were performed using LSD and Dunnett's t-tests, provided that the overall variances were equal. A p < 0.05 was considered statistically significant.

[0106] 2. Results

[0107] 1. PQQ reduces hyperalgesia and anxiety-induced behavior caused by LSI surgery

[0108] Figure 1 (A) Schematic diagram of the surgical procedure and PQQ supplementation protocol for LSI mice.

[0109] Figure 1 (B)-(D) Compared with the sham-operated group, the activity levels of mice in the LSI group were significantly reduced, including active time, walking distance, and maximum speed, indicating that spinal cord injury caused a decrease in motor ability.

[0110] Figure 1 (E) The thermal response time of mice in the LSI group was significantly delayed in the hot plate test, indicating enhanced thermal sensitivity.

[0111] Figure 1(F)-(G) Mice in the LSI group exhibited significantly increased paw withdrawal frequency (PWF) in response to mechanical stimulation, indicating increased mechanical sensitivity. PQQ supplementation significantly improved these behavioral indices, including enhanced mobility, faster thermal response time, and reduced mechanical hypersensitivity, suggesting its potential in alleviating pain-related hypersensitivity.

[0112] Figure 1 (H)-(K) Eight weeks after LSI, anxiety-related behaviors were significantly increased in mice in the LSI group, as assessed by open field and elevated plus maze tests. PQQ supplementation significantly reduced these anxiety-related behaviors, as demonstrated by increased time and distance spent in the center area and prolonged time spent in the open arms.

[0113] 2. PQQ reduces endplate porosity and trabecular separation in the LSI model

[0114] Figure 2 (A) Three-dimensional CT images show that the endplate porosity of mice in the LSI group increased significantly, while the PQQ supplementation group maintained the bone volume fraction (BV / TV) and trabecular thickness (Tb.Th) and reduced trabecular separation (Tb.Sp).

[0115] Figure 2 (B)-(C) Safranin O / Fast Green and H&E staining, as well as histological analysis, confirmed degenerative changes in the endplates of mice in the LSI group, while the sham-operated group showed intact, homogeneous cartilage. PQQ supplementation significantly reduced the endplate score, indicating less structural degeneration.

[0116] Figure 2 (D)-(E) Immunofluorescence staining showed that the distribution of matrix metalloproteinase 13 (MMP13) and type X collagen (Col X) increased in the LSI group and was significantly reduced after PQQ supplementation.

[0117] Figure 2 (F) TRAP staining showed that TRAP+ osteoclasts increased in the endplates of LSI mice, while PQQ supplementation significantly reduced these osteoclasts.

[0118] 3. PQQ treatment significantly reduced the number of senescent osteoclasts in the endplate of LSI mice

[0119] like Figure 3 As shown in (A)-(B), PQQ treatment significantly reduced senescent osteoclasts in the endplates of LSI mice. Double immunofluorescence analysis of senescence markers (p16 and HMGB1) and an osteoclast marker (TRAP) revealed a significant increase in the number of p16+TRAP+ and HMGB1+TRAP+ double-positive cells in the lumbar endplates of LSI mice. However, PQQ supplementation significantly reduced this number.

[0120] SA-βGal staining showed ( Figure 3 (C) ), The number of SA-βGal+ cells in the spinal endplates of PQQ-supplemented LSI mice was significantly decreased.

[0121] Western blot analysis showed that ( Figure 3 (D) In ​​the LSI group, senescence-related proteins (p16, p21, p53, and γH2A.X) were significantly increased, while anti-senescence proteins (Sirt1 and BMI-1) were significantly decreased in the endplates. PQQ supplementation significantly reversed these changes in endplate proteins.

[0122] RT-PCR analysis showed that ( Figure 3 (E) ), PQQ treatment significantly inhibited the LSI-induced elevation of senescence-associated secretory phenotype (SASP) factors, including IL-1α, IL-1β, IL-6, IL-8, MCP-1, MMP-13, TNF-α, TGF-β, NF-κB, CEBPβ, GATA4, and PAI-1.

[0123] ELISA results showed that ( Figure 3 (F) Inflammatory markers, including IL-6 and TNF-α, were significantly elevated in the spinal endplates of mice in the LSI group. PQQ supplementation significantly reduced IL-6 and TNF-α levels, indicating a regulated inflammatory microenvironment.

[0124] 4. PQQ reduces sensory innervation in the LSI model.

[0125] Immunofluorescence staining of CGRP (a marker of pain-conducting C fibers) and PGP9.5 (a general nerve fiber marker) showed (eg Figure 4 (A) Sensory innervation increased in the pore endplates of LSI mice. PQQ supplementation significantly reduced the number of CGRP+PGP9.5+ nerve fibers, indicating that PQQ reduces sensory nerve infiltration into degenerating spinal tissue.

[0126] ELISA analysis showed (eg Figure 4 (B) Netrin-1 levels were elevated in LSI endplates, whereas PQQ supplementation significantly reduced this level.

[0127] Immunostaining showed (eg Figure 4 (C) ) The expression of the Netrin-1 receptor DCC was increased in the LSI group, while the expression of DCC was decreased in the PQQ-supplemented group, suggesting that PQQ may interfere with the Netrin-1-mediated neurotrophic signaling pathway and inhibit neural sprouting.

[0128] like Figure 4(D)-(F) The elevated expression of COX-2 and PGES mRNA and the increased PGE2 concentration in LSI endplates were significantly reduced by PQQ supplementation.

[0129] 5. PQQ reduces angiogenesis and osteogenesis in the LSI model.

[0130] like Figure 5 (A) Immunofluorescence staining of CD31+Emcn+ vessels showed that angiogenesis was significantly increased in the sclerotic endplates of LSI mice, whereas PQQ supplementation significantly reduced angiogenesis.

[0131] like Figure 5 (B) In the endplates of LSI mice, the number of osteoblast precursor cells (Osx+) and mature osteoblasts (OCN+) increased. PQQ supplementation significantly reduced the number of osteoblast precursor cells (Osx+) and mature osteoblasts (OCN+).

[0132] like Figure 5 (C) Western blotting and qRT-PCR analysis confirmed that LSI leads to upregulation of endomucin, CD31, Osx, and OCN expression in the lumbar endplate, suggesting that LSI leads to increased angiogenesis and osteogenesis. PQQ supplementation significantly reduces the abnormal increase in angiogenesis and osteogenesis.

[0133] In summary, animal studies have demonstrated that PQQ alleviates spinal pain through multiple mechanisms. PQQ exerts its effects through various mechanisms, including reducing the number of senescent osteoclasts, modulating inflammatory pathways, and influencing neurotrophic and angiogenic processes. By targeting these factors, PQQ offers a potential strategy for addressing the structural degeneration and functional impairment associated with spinal instability.

Claims

1. Use of a pyrroloquinoline quinone compound, or a pharmaceutically acceptable salt thereof, in the preparation of a composition for treating and / or alleviating spinal degenerative diseases.

2. The use according to claim 1, characterized in that The dosage forms of the composition include tablets, powders, injections, granules, ointments, suppositories, suspensions, syrups or capsules.

3. The use according to claim 1, characterized in that The content of the pyrroloquinoline quinone compound or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 4 mg / kg.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition or its pharmaceutically acceptable carrier comprises an active ingredient and a pharmaceutically acceptable excipient and / or carrier, wherein the active ingredient is the pyrroloquinoline quinone compound according to claim 1, or a pharmaceutically acceptable salt thereof.

Images