Application of anwuligan in preparation of medicine for treating or preventing neuroinflammation-related diseases and bone cancer pain

Abstract

The invention relates to the technical field of medicines, in particular to application of anwuligan in preparation of medicines for treating or preventing neuroinflammation related diseases and bone cancer pain. Animal experiments prove that the anwuligan can relieve nerve cell inflammatory response, inhibit tumor cell growth, prevent bone destruction caused by tumors and inhibit mechanical pain sensitivity, heat pain sensitivity and spontaneous pain of bone cancer pain rats. Results show that the anwuligan has potential anti-inflammatory, anti-cancer and analgesic effects and has an application prospect of being developed into a medicine for preventing and treating cancer pain.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of anwuzhisu in preparing medicines for treating or preventing diseases related to neuroinflammation and bone cancer pain. Background Art

[0002] Pain is a common symptom in patients with advanced cancer, which affects the effectiveness of treatment and reduces the patient's overall quality of life. Bone cancer pain is a special pain condition with overlapping effects of inflammatory pain and neuropathic pain but distinct characteristics. Initially, bone cancer pain originates from malignant tumors or metastatic bone cancer that develop in the bones. Cancer pain is characterized by fluctuations and rapid progression, making it more challenging to manage than pain caused by inflammation or trauma. Opioids remain the first-line treatment for cancer pain, but are accompanied by serious side effects. Therefore, new treatments are urgently needed to deal with cancer pain. Ideal bone cancer pain treatment should not only relieve cancer-related pain, but also reduce tumor burden and minimize bone damage.

[0003] In cancer treatment, natural compounds are gaining increasing attention due to their sustainability, minimal side effects, affordability, and ability to simultaneously target multiple pathways. Pentasin, a derivative extracted from the spice Myristica fragrans, is recognized for its ability to effectively combat inflammation, oxidative stress, and tumor cell proliferation. Studies have shown that pentasin inhibits the growth of non-small cell lung cancer cells, slows tumor progression, and suppresses the metastasis of colorectal cancer. Studies have shown that lipopolysaccharide (LPS)-induced effects on inducible nitric oxide synthase (iNOS) expression and nitrite formation in BV2 microglia. Excessive release of NO in the central nervous system is associated with the development of neuroinflammation, further contributing to various inflammatory diseases. However, the specific role of pentasin in neuroinflammation, tumor burden, and bone cancer pain, and its underlying mechanisms, are not fully understood. Summary of the Invention

[0004] In view of this, the present invention provides the use of anpentazhisu in the preparation of a drug for treating or preventing neuroinflammatory diseases and bone cancer pain. Through experiments, the present invention found that anpentazhisu can inhibit the inflammatory response of nerve cells, reduce tumor burden and tumor-induced bone destruction, and relieve bone cancer pain.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of anwuzhisu in preparing medicines for treating or preventing diseases related to neuroinflammation and bone cancer pain.

[0007] In some specific embodiments of the present invention, the treatment or prevention of a neuroinflammation-related disease comprises any of the following:

[0008] (I) inhibiting the expression of iNOS and / or COX-2 proteins; and / or

[0009] (II) Reduce the release of NO.

[0010] In some embodiments of the present invention, the neural cells include LPS-induced BV2 microglial cells.

[0011] In some specific embodiments of the present invention, the neuroinflammation-related disease is selected from Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke or traumatic brain injury.

[0012] In some specific embodiments of the present invention, the treatment or prevention of bone cancer pain includes any of the following:

[0013] (I) inhibiting tumor-induced bone tissue destruction; and / or

[0014] (II), reducing tumor burden in bone tissue; and / or

[0015] (III) Relieve pain caused by bone cancer.

[0016] In some embodiments of the invention, the tumor comprises primary bone cancer and / or metastatic bone cancer.

[0017] In some embodiments of the invention, the primary bone cancer comprises osteosarcoma cancer; and / or

[0018] The metastatic bone cancer includes one or more of breast cancer, prostate cancer, lung cancer, kidney cancer, liver cancer, renal cancer, bladder cancer, thyroid cancer, cervical cancer, colon cancer, esophageal cancer or nasopharyngeal cancer.

[0019] In some specific embodiments of the present invention, the dosage form of the drug includes an oral preparation or an injection.

[0020] In some specific embodiments of the present invention, the administration of the pentachloroquine includes subarachnoid administration.

[0021] In some specific embodiments of the present invention, the effective dose of the pentaerythrin is 75 mM to 200 mM / 10 μl / rat / day.

[0022] The present invention also provides a method for preventing and / or treating tumor-related bone diseases, comprising administering anpentazhisulphide to a subject.

[0023] The present invention includes but is not limited to providing the following beneficial effects:

[0024] Patients with advanced cancer often suffer from severe pain due to bone metastasis and destruction. Currently, there are few effective treatments for bone cancer pain. Pentachlorophenol, a natural lignan, is recognized for its potential antioxidant, anti-inflammatory and anti-cancer properties. However, the effect of Pentachlorophenol on bone cancer pain is not yet fully understood. The present invention has demonstrated through animal experiments that Pentachlorophenol can reduce mechanical allodynia, thermal allodynia and spontaneous pain in rats with bone cancer pain, prevent bone destruction caused by tumors, and inhibit local tumor growth in rats with bone cancer pain. The results show that Pentachlorophenol has potential analgesic and anti-cancer effects and has the application prospect of being developed into a drug for the prevention and treatment of cancer pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0026] Figure 1 Shown is a simplified diagram of the structure of Anpentaliptin;

[0027] Figure 2 Figure 4: Anpentaliprin inhibits the inflammatory response of neurons. A shows that Anpentaliprin effectively reduces the release of NO in BV2 cells induced by LPS. B shows that Anpentaliprin has no toxicity to BV2 cells. C shows that 10 μM Anpentaliprin effectively reduces the iNOS protein content in BV2 cells. D shows that 5 μM and 10 μM Anpentaliprin effectively reduce the COX-2 protein content in BV2 cells, ***p<0.001, vs. control group; #p<0.05, ##p<0.01, vs. LPS group, unpaired t-test, n=3.

[0028] Figure 3 The results show that Anwuzhisu reduces tumor burden; A shows optical microscopy images showing colony formation of MRMT-1 tumor cells after treatment with different concentrations of Anwuzhisu (0-80 μM); B shows that cell proliferation is significantly reduced after Anwuzhisu treatment; C shows that Anwuzhisu can inhibit the survival rate of MRMT-1 tumor cells; D shows that Anwuzhisu can increase the death rate of MRMT-1 tumor cells; **p<0.01, ***p<0.001, vs. control group, one-way analysis of variance, n=3-6; E shows in vivo imaging of tibial bone tumors in rats treated with Anwuzhisu on day 14 after bone cancer pain rat modeling; F shows quantitative analysis, Anwuzhisu can inhibit bone tumor burden in rats with bone cancer pain, ***P<0.001 vs. sham operation + solvent group, ##P<0.01 vs. bone cancer pain + solvent group, unpaired t-test, n=8;

[0029] Figure 4Figure 4: Anpentaliprine inhibits bone destruction induced by MRMT-1 tumor cells. A shows representative X-ray images of the tibiae of rats in the sham surgery + solvent, sham surgery + anpentaliprine, bone cancer pain + solvent, and bone cancer pain + anpentaliprine groups. Scale bar: 5 mm. Arrows indicate areas of bone damage. B shows quantitative analysis of X-rays. Anpentaliprine alleviates bone destruction in the tibiae of rats with bone cancer pain. ***P<0.001 vs. sham surgery + solvent group, #P<0.5 vs. bone cancer pain + solvent group, unpaired t-test, n=3-5. C shows a three-dimensional CT image taken 14 days after MRMT-1 tumor cell inoculation, showing that anpentaliprine alleviates the deterioration of cancellous and cortical bone in the tibiae of rats with bone cancer pain. Scale bar: 2 mm.

[0030] Figure 5 The results showed that Anpentaliptin can alleviate the pain behavior of rats with bone cancer pain; A showed that Anpentaliptin can alleviate the mechanical hyperalgesia of rats with bone cancer pain; B showed that Anpentaliptin can alleviate the thermal hyperalgesia of rats with bone cancer pain; CD showed that Anpentaliptin can alleviate the spontaneous pain behavior of rats with bone cancer pain, such as withdrawal and defense; *p<0.05, **p<0.01, ***p<0.001 vs. sham operation + solvent group, %p<0.05, %%p<0.01, %%%p<0.001, #p<0.05, ##p<0.01, ###p<0.001, &p<0.05, &&p<0.01, &&&p<0.001 vs. bone cancer pain + solvent group, two-way ANOVA, n=7. DETAILED DESCRIPTION

[0031] The present invention discloses the use of anpentaliptin in the preparation of drugs for treating or preventing neuroinflammatory related diseases and bone cancer pain. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] The technical problem to be solved by the present invention is to provide a medicine with definite efficacy for treating bone cancer pain.

[0033] 1. Evaluate the ability of Anwuzhisu to alleviate neuronal inflammation

[0034] The specific method is as follows:

[0035] (1) Establishment of a lipopolysaccharide (LPS)-induced BV2 cell inflammation model

[0036] BV2 cells were grown at 2×10 4 Cells were seeded into 96-well plates and incubated in a 37°C incubator with 5% CO₂. After 24 hours of incubation, the corresponding concentration (10 μM) of anpentasil was added to the drug-treated group and incubated for 2 hours. Subsequently, LPS was added to the drug-treated group and the LPS model group at a final concentration of 100 ng / mL. After 24 hours of incubation, the supernatant from each group was reacted with an equal volume of Griess buffer, and the OD value of NO release in each group was measured at a wavelength of 540 nm using a microplate reader (BioTek).

[0037] (2) Cell viability assay: BV2 cells were seeded in 96-well culture plates and cultured in a 37°C constant temperature incubator containing 5% CO2. After 24 hours of culture, 10 μM of anpentasilate was added to the drug-treated group and incubated for 2 hours. Subsequently, LPS was added to the drug-treated group and the LPS model group at a final concentration of 100 ng / mL. MTT was added to each well at a final concentration of 0.5 mg / mL. After incubation in the incubator, the culture medium was discarded, and 100 μL of DMSO was added to each well. The plates were shaken on a shaker to fully dissolve the DMSO. The OD value of each group was measured at a wavelength of 490 nm using a microplate reader (BioTek).

[0038] (3) Western Blot Detection of the Effect of Anwuzhisu on the Expression of Inflammatory Proteins

[0039] BV2 cells were treated with 10 μM and 20 μM anpentaliprin for 24 hours, after which proteins were extracted and subjected to SDS-PAGE electrophoresis. After electrophoresis, proteins were transferred to 0.2 μm nitrocellulose membranes by wet blotting. The membranes were blocked with either 5% skim milk or 5% BSA blocking buffer for 1 hour at room temperature. The membranes were then incubated overnight at 4°C with the corresponding primary antibodies: anti-iNOS antibody (1:500, BD Biosciences #610328) or anti-COX-2 antibody (1:1000, CST #12282). The membranes were washed three times (10 minutes each) in 1× TBST buffer on a shaker for 10 minutes. Following these washes, the membranes were incubated with the corresponding secondary antibody solution at room temperature for 2 hours, followed by three 10-minute washes in 1× TBST buffer. Protein bands were visualized using an automated chemiluminescence imaging system (Tanon), and grayscale analysis of each protein band was performed using ImageJ software.

[0040] 2. To investigate the effects of Anwuzhisu on local tumor burden and cancer-induced bone destruction

[0041] (1) Quantify the effect of local tumor burden on bone metabolism and analyze the intervention effect of anwuzhisu.

[0042] (2) To study the protective effects of anwuzhisu on bone tissue structure and function, and to evaluate its effect in inhibiting tumor-related bone destruction.

[0043] The specific method is as follows:

[0044] (1) Effect of Anwuzhisu on tumor burden after tibia injection of cancer cells in rats:

[0045] a. Cell Cloning: Rat mammary carcinoma MRMT-1 cells were dissociated using 0.25% trypsin during their exponential growth phase. Cell density was then carefully adjusted to meet experimental needs. Cells were counted using a hemocytometer, and 500 cells were seeded into each well of a 6-well culture plate. After cells attached, the culture medium was replaced with fresh medium containing various concentrations of pentapeptin ranging from 0 to 80 μM, and the cells were incubated in this medium for 48 hours. Subsequently, the treatment medium was replaced with standard growth medium, and the incubation period was extended for seven days to promote the generation of independent cell clones. After washing the cells three times with PBS to remove residual culture medium, the cells were fixed with 4% paraformaldehyde solution for 20 minutes at room temperature to preserve cell shape. To ensure thorough washing, the cells were rinsed three times with PBS for five minutes each. To more effectively visualize cell colonies, the cells were stained with 0.1% crystal violet solution for 15 minutes at room temperature. Subsequently, the cells were rinsed with PBS to remove residual dye, allowed to dry, and photographed and analyzed. The clone formation rate was calculated using the following formula: Clone formation rate = (number of clones formed / number of cells initially seeded) × 100%. The results were analyzed using ImageJ software.

[0046] b. CCK-8 experiment: Rat breast cancer MRMT-1 cells were cultured at 4×10 3 Cells were seeded into 96-well plates at 100 μL per well, with 100 μL of cell suspension added to each well for initial seeding. Subsequently, cells underwent a 24-hour preincubation period to ensure cell attachment and initial growth. Following this preincubation period, various concentrations of anpentaliprin were introduced into the wells to assess their effects on cell viability. To quantify cell viability, 10 μL of CCK-8 reagent was accurately added to each well, taking extreme care to avoid the formation of bubbles that could interfere with the assay. The plates were then returned to the incubator for 1–4 hours. Absorbance was measured at a precise wavelength of 450 nm using a microplate spectrophotometer. The percentage of viable cells was calculated using the following formula: [(As-Ab) / (Ac-Ab)] × 100%, where As represents the absorbance of the experimental sample, Ab represents the absorbance of the blank well, and Ac represents the absorbance of the untreated control well. The inhibition rate, used to quantify the extent of growth inhibition, was calculated using the following formula: [(Ac-As) / (Ac-Ab)] × 100%.

[0047] c. Establishment of a rat model of bone cancer pain induced by rat breast cancer MRMT-1 cells:

[0048] The cultured MRMT-1 cells were collected and the cell density was adjusted to 1x10 cells / mL using sterile phosphate buffered saline (PBS) solution. 7 A rat model of bone cancer pain was established by injecting 4 μL of MRMT-1 cells into the tibial bone marrow cavity of the left hind limb using a microinjector. A control group received an equal volume of PBS. Three, seven, 14, and 21 days after surgery, mechanical pain thresholds, thermal pain thresholds, and spontaneous pain were measured using von Frey filaments, a thermal analgesia meter, and an open field test to assess the success of the model.

[0049] d. In vivo animal imaging: 3, 7, 14, and 21 days after the rat bone cancer pain model was established, the rats were anesthetized with isoflurane (induction 5%, maintenance 3%, 2 L / min O2) and injected with rat Cy5.5 fluorescent probe reagent (0.2 mg / kg) via the tail vein. The in vivo imaging of the animal model was observed using the IVISSpectrum imaging system.

[0050] (2) Effect of Anwuzhisu on bone destruction in rats after injection of cancer cells into the tibia:

[0051] Radiographic Examination: 3, 7, 14, and 21 days after rat bone cancer pain modeling, rats were anesthetized with isoflurane (5% induction, 3% maintenance, 2 L / min O₂). After adequate anesthesia, the rats were carefully placed on an X-Viewer instrument, and radiographic examinations of the tibia were performed to assess the extent of bone degeneration. The degree of bone damage in tumor-affected tibia radiographs was scored from 0 to 5; 0 indicates normal baseline bone without tumor; 1 indicates one to three radiolucent lesions indicating initial bone degeneration; 2 indicates an increase in the number of lesions to three to six with associated medullary bone loss; 3 indicates the absence of medullary bone with initial cortical bone erosion; 4 indicates full-thickness single-layer cortical bone loss; and 5 indicates full-thickness double-layer cortical bone loss with fracture displacement. Quantitative radiographic assessments were performed by an investigator blinded to the experimental conditions.

[0052] b. Micro-computed tomography (Micro-CT) examination: At representative time points, 7 and 21 days after rat bone cancer pain modeling (early and mid-late stages of bone cancer pain), rats were anesthetized with isoflurane (5% induction, 3% maintenance, 2 L / min O₂). After adequate anesthesia, the rats were carefully placed on a Micro-CT instrument and their tibias were imaged. Images were analyzed using CTAn (Bruker micro-CT, Belgium, V1.16.9.0) software, and 3D models were analyzed using CTvox (Bruker micro-CT, Belgium, V3.3) software.

[0053] 3. To clarify the effects of anwuzhisu on pain symptoms and functional impairment of bone cancer pain in rats:

[0054] To evaluate the effects of anpentaliprine on neuropathic pain symptoms and their maintenance in a rat bone cancer pain model, including behavioral assessment of pain. To analyze the therapeutic efficacy of anpentaliprine in alleviating cancer pain and explore its preventive effects on pain induction and maintenance.

[0055] A rat model of bone cancer pain induced by rat breast cancer cells (MRMT-1) was established. The rats were randomly divided into sham operation + solvent group, sham operation + anpentaliptin group, bone cancer pain + solvent group, bone cancer pain + anpentaliptin low-dose group (25 mM / 10 μL), bone cancer pain + anpentaliptin medium-dose group (75 mM / 10 μL), bone cancer pain + anpentaliptin high-dose group (200 mM / 10 μL), and bone cancer pain + positive control group (gabapentin 100 µg / 10 μL). Subarachnoid administration of gabapentin was started when the rats with bone cancer pain showed obvious pain (10 days after surgery) and was given once a day for three consecutive days. After the third day of administration, the changes in mechanical pain threshold, thermal pain threshold, and spontaneous pain of the rats were behaviorally detected.

[0056] This invention is expected to reveal the potential biological mechanism of the novel compound Anwuzhisu in neuroinflammation and bone cancer pain, providing an important basis for the development of more effective targeted treatments for cancer pain.

[0057] Unless otherwise specified, the raw materials and reagents used in the application of the anwuzhisu provided by the present invention in the preparation of drugs for treating or preventing neuroinflammatory-related diseases and bone cancer pain can all be purchased from the market.

[0058] The present invention will be further described below in conjunction with the embodiments:

[0059] Example 1

[0060] 1. Materials and Methods

[0061] 1.1 Establishment of a lipopolysaccharide (LPS)-induced BV2 cell inflammation model:

[0062] BV2 cells were grown at 2×10 4 Cells were seeded into 96-well plates and incubated in a 37°C incubator with 5% CO₂. After 24 hours of incubation, the corresponding concentration (10 μM) of anpentasil was added to the drug-treated group and incubated for 2 hours. Subsequently, LPS was added to the drug-treated group and the LPS model group at a final concentration of 100 ng / mL. After 24 hours of incubation, the supernatant from each group was reacted with an equal volume of Griess buffer, and the OD value of NO release in each group was measured at a wavelength of 540 nm using a microplate reader (BioTek).

[0063] 1.2 Cell Viability Assay: BV2 cells were seeded in 96-well culture plates and cultured in a 37°C incubator containing 5% CO2. After 24 hours of culture, 10 μM anpentasilate was added to the drug-treated group and incubated for 2 hours. Subsequently, LPS was added to the drug-treated group and the LPS model group at a final concentration of 100 ng / mL. MTT was added to each well at a final concentration of 0.5 mg / mL. After incubation in the incubator, the culture medium was discarded, and 100 μL of DMSO was added to each well. The plates were shaken on a plate shaker to fully dissolve the DMSO. The OD value of each group was measured at a wavelength of 490 nm using a microplate reader (BioTek).

[0064] 1.3 Western Blot Detection of the Effect of Anwuzhisu on the Expression of Inflammatory Proteins

[0065] BV2 cells were treated with 10 μM and 20 μM anpentaliprin for 24 hours, after which proteins were extracted and subjected to SDS-PAGE electrophoresis. After electrophoresis, proteins were transferred to 0.2 μm nitrocellulose membranes by wet blotting. Membranes were blocked with either 5% skim milk or 5% BSA blocking buffer for 1 hour at room temperature. The membranes were then incubated overnight at 4°C with the corresponding primary antibodies: anti-iNOS antibody (1:500, BD Biosciences #610328) or anti-COX-2 antibody (1:1000, CST #12282). The membranes were washed three times (10 minutes each) with 1× TBST buffer on a shaker for 10 minutes. Following these washes, the membranes were incubated with the corresponding secondary antibody solution at room temperature for 2 hours, followed by three washes (10 minutes each) with 1× TBST buffer. Protein bands were visualized using an automated chemiluminescence imaging system (Tanon), and grayscale analysis of each protein band was performed using ImageJ software.

[0066] 1.4 Cell cloning

[0067] During the exponential growth phase, rat mammary carcinoma MRMT-1 cells were dissociated using 0.25% trypsin. Cell density was then carefully adjusted to meet experimental needs. Cells were counted using a hemocytometer, and 500 cells were seeded into each well of a 6-well culture plate. After cells attached, the original culture medium was replaced with DMEM (Catalog #11875500, Gibco) supplemented with 10% fetal bovine serum (FBS) and incubated for 48 hours. Subsequently, the treatment medium was replaced with the original culture medium, and the incubation period was extended for seven days to promote the generation of independent cell clones. After washing the cells three times with PBS to remove residual culture medium, the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature to preserve cell shape. To ensure thorough washing, the cells were rinsed three times with PBS for five minutes each. To more effectively visualize cell colonies, the cells were stained with 0.1% crystal violet solution for 15 minutes at room temperature. The cells were then rinsed with PBS to remove any residual dye, allowed to dry naturally, and photographed and analyzed. The colony formation rate was calculated using the following formula: colony formation rate = (number of colonies formed / number of cells initially seeded) × 100%. The results were analyzed using Image J software.

[0068] 1.5 CCK-8 assay

[0069] Rat breast cancer MRMT-1 cells were cultured at 4 × 10 3 Cells were seeded into wells of a 96-well plate, with 100 μL of cell suspension added to each well for initial seeding. Subsequently, cells underwent a 24-hour preincubation period to ensure cell attachment and initial growth. Following this preincubation period, various concentrations of anpentaliprin were introduced into the wells to assess their effects on cell viability. To quantify cell viability, 10 μL of CCK-8 reagent was accurately added to each well, taking extreme care to avoid the formation of bubbles that could interfere with the assay. The plates were then returned to the incubator for 1–4 hours. Absorbance was measured at a precise wavelength of 450 nm using a microplate spectrophotometer. The percentage of viable cells was calculated using the following formula: [(As-Ab) / (Ac-Ab)] × 100%, where As represents the absorbance of the experimental sample, Ab represents the absorbance of the blank well, and Ac represents the absorbance of the untreated control well. The inhibition rate, used to quantify the extent of growth inhibition, was calculated using the following formula: [(Ac-As) / (Ac-Ab)] × 100%.

[0070] 1.6 Animals

[0071] Female Sprague-Dawley (SD) rats, weighing 160–200 g, were purchased from the Experimental Animal Center of Zhengzhou University. Rats were housed in plastic boxes lined with corn cobs, with 2–4 rats per cage. Rats were provided with natural lighting and room temperature of 22.0 ± 1.0°C. Water and food were freely available, and the rats' feeding status was checked regularly daily. All animal experimental protocols were designed in accordance with the ethical guidelines of the International Association for the Study of Pain and were reviewed and approved by the Animal Ethics Committee of Zhengzhou University (Ethics Approval Number 2023-KY-1163-002).

[0072] 1.7 Establishment of a rat model of bone cancer pain induced by MRMT-1 breast cancer cells:

[0073] The cultured MRMT-1 cells were collected and the cell density was adjusted to 1x10 cells / mL using sterile phosphate buffered saline (PBS) solution. 7 A rat model of bone cancer pain was established by injecting 4 μL of MRMT-1 cells into the tibial bone marrow cavity of the left hind limb using a microinjector. A control group received an equal volume of PBS. Three, seven, 14, and 21 days after surgery, mechanical pain thresholds, thermal pain thresholds, and spontaneous pain were measured using von Frey filaments, a thermal analgesia meter, and an open field test to assess the success of the model.

[0074] 1.8 Drug preparation and subarachnoid administration

[0075] Anwuzhisu (Catalog #JOT-10501, Chengdu Pufeide), CAS No. 107534-93-0, chemical structure see Figure 2 A, dissolved in dimethyl sulfoxide (DMSO); the positive control drug, gabapentin, was dissolved in saline. The rats' basal pain thresholds were measured before establishing a bone cancer pain model and intrathecal administration. Ten days after surgery, the fused tip of the PE-10 tubing was removed, and low-dose (25 mM / 10 μl), medium-dose (75 mM / 10 μl), and high-dose (200 mM / 10 μl) anpentasilate, as well as gabapentin (100 μg / 10 μl), were injected into the subarachnoid space of the rats using a microsyringe inserted through the PE-10 tubing. 10 μl of air, previously withdrawn, was then injected to push the remaining drug into the subarachnoid space. Administration was slow and uniform over approximately 1 minute, and the tubing was resealed after administration. An equal volume of solvent was administered to the control group. Administration began on day 10 after bone cancer pain model establishment and continued once daily for three consecutive days.

[0076] 1.9 In vivo animal imaging

[0077] On days 3, 7, 14, and 21 after the rat bone cancer pain model was established, the rats were anesthetized with isoflurane (induction 5%, maintenance 3%, 2 L / min O2), and the rat Cy5.5 fluorescent probe reagent (0.2 mg / kg) was injected through the tail vein. The in vivo imaging of the animal model was observed using the IVIS Spectrum imaging system.

[0078] 1.10X-ray radiographic examination

[0079] On days 3, 7, 14, and 21 after induction of the rat bone cancer pain model, the rats were anesthetized with isoflurane (5% induction, 3% maintenance, 2 L / min O₂). After the rats were properly anesthetized, they were carefully placed on an X-Viewer instrument, and radiographic examinations of the tibia were performed to assess the extent of bone degeneration. The degree of bone damage in X-ray images of the tumor-affected tibia was scored from 0 to 5; 0 indicates normal baseline bone without tumor; 1 indicates one to three radiolucent lesions indicating initial bone degeneration; 2 indicates an increase in the number of lesions to three to six with associated medullary bone loss; 3 indicates no medullary bone with initial cortical bone erosion; 4 indicates full-thickness single-layer cortical bone loss; and 5 indicates full-thickness double-layer cortical bone loss with fracture displacement. Quantitative radiographic assessments were performed by an investigator blinded to the experimental conditions.

[0080] 1.11 Micro-computed tomography (Micro-CT) testing

[0081] Representative time points were selected: 7 and 21 days after rat bone cancer pain modeling (early and mid-late stages of bone cancer pain). Rats were anesthetized with isoflurane (5% induction, 3% maintenance, 2 L / min O₂). After adequate anesthesia, the rats were carefully placed on a micro-CT instrument and their tibias were imaged. Images were analyzed using CTAn (Bruker micro-CT, Belgium, V1.16.9.0) software, and 3D models were analyzed using CTvox (Bruker micro-CT, Belgium, V3.3) software.

[0082] 1.12 Mechanical pain threshold measurement

[0083] Three days prior to pain testing, animals were acclimated to the behavioral testing environment. Mechanical pain thresholds in rats were measured using the method described by Chaplan et al., with the results reflected by the 50% paw withdrawal threshold (PWT). Rats were placed on a metal grid and covered with a transparent plexiglass cover (18 cm × 8 cm × 8 cm). They were allowed to acclimate for approximately 20 minutes. After the grooming and exploratory activities of the rats had largely ceased, a series of standardized von Frey fibers (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10 g) were applied to the mid-plantar region of the hind paw until it bent into an S-shape. The stimulation lasted for 6–8 seconds, and the rats were observed for withdrawal. Positive pain responses included paw lifting, paw flicking, and paw licking.

[0084] The measurement begins with a moderate force (2.00 g) of von Frey fibers, using an "up and down" method. Specifically, if stimulation of a given von Frey fiber force fails to elicit a positive response, the next higher force is applied. If a positive response is observed, the next lower force is applied. This process continues until the first positive and negative (or negative and positive) response is achieved, at which point the measurement is repeated four times. Stimuli are typically separated by 3–5 minutes to prevent sensitization of the animal through repeated stimulation. If no positive response is observed at either the minimum (0.41 g) or maximum (15.10 g) von Frey fiber force, the rat's 50% withdrawal threshold is calculated as 0.25 g or 15.10 g. A positive response is denoted by an X, and a negative response by an O. The 50% withdrawal threshold is calculated using the following formula: 50% withdrawal threshold (g) = 10[Xf + kδ] / 10,000

[0085] Xf is the logarithm of the initial von Frey fiber force (2 g), δ is the average difference between the logarithm of adjacent von Frey fibers (i.e., 0.224), and k is the lookup table value for positive and negative response types (Chaplan et al., 1994). 15.10 g is the maximum 50% withdrawal threshold achievable with this method, and 0.25 g is the minimum 50% withdrawal threshold. A 50% withdrawal threshold less than 4 g is considered hyperalgesia.

[0086] References: Chaplan SR, Bach FW, Pogrel JW, Chung JM and Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J NeurosciMethods. 1994, 53(1): 55-63

[0087] 1.13 Determination of the Pain Latency of Foot Lifting Induced by Radiant Heat

[0088] In this experiment, the paw withdrawal latency (PWL) after thermal radiant stimulation was used to assess the degree of thermal allodynia in rats. The ambient temperature was maintained at 20 ± 0.5°C. Rats were placed in a plexiglass enclosure (18 cm × 8 cm × 8 cm) and allowed to acclimate for 30 minutes before testing. Testing began after the rats were quiet. Before testing, the intensity of the radiant heat lamp was adjusted to a level that would elicit a baseline paw withdrawal of 12–18 seconds in normal rats. This intensity was maintained throughout the experiment, and animals exceeding this range were eliminated. During testing, the lamp was placed directly beneath the rat's left paw pad. The lamp was turned on and the timer was started. A positive response was defined as a paw withdrawal. The lamp was immediately turned off after a positive response. Each rat was measured 3–5 times, with at least 5 minutes between tests. The average PWL was used as the average PWL.

[0089] 1.14 Spontaneous pain behavior detection

[0090] The spontaneous pain response of the rats after modeling was observed by video recording, including the frequency and duration of paw withdrawal and the protective action of the ipsilateral hind limb. The observation time was 10 minutes.

[0091] Effect Example 1: Anwuzhisu inhibits the inflammatory response of nerve cells ( Figure 2 )

[0092] Anwuzhisu can effectively reduce the release of NO in BV2 microglial cells induced by LPS, and has no toxicity to BV2 cell viability. Compared with the blank control, it can also promote BV2 cell growth.

[0093] Because impaired activity of inflammatory factors such as iNOS and COX-2 significantly impacts the development of inflammatory diseases, this study used Western Blot analysis to assess whether pentapeptide regulates iNOS and COX-2 protein levels in LPS-induced BV2 cells. In the group of cells pre-treated with pentapeptide, the expression of iNOS and COX-2 decreased in a dose-dependent manner compared to the group stimulated with LPS alone, indicating that pentapeptide can act as a COX-2 inhibitor to suppress inflammatory responses.

[0094] The results showed that anpentasiloxane could inhibit the inflammatory response of nerve cells.

[0095] Effect Example 2: Anwuzhisu reduces tumor burden ( Figure 3 )

[0096] Cells were treated with 0-80 μM concentrations of anpentaliprin for 24-48 hours. The results showed that anpentaliprin inhibited the colony formation and proliferation of MRMT-1 cells in a dose-dependent manner. Furthermore, in vivo imaging showed that when 0.1 mg / kg body weight of free Cy5.5 dye was injected intravenously into the tail vein of rats, the fluorescence intensity of the tumor at the injection site was significantly reduced in the anpentaliprin-treated group compared to the bone cancer pain + vehicle group.

[0097] The results showed that anwuzhisu could reduce tumor burden.

[0098] Effect Example 3: Anwuzhisu inhibits bone destruction caused by tumors ( Figure 4 )

[0099] X-ray imaging assessments revealed a significant increase in bone destruction in the proximal tibia of rats with bone cancer pain 14 days after modeling. The bone destruction score in rats treated with anpentaliprine was lower than that in the vehicle control group. To further investigate bone microarchitecture, micro-CT three-dimensional reconstructions of the proximal tibia of rats with bone cancer pain were performed. On day 14 after modeling, the anpentaliprine-treated group showed reduced trabecular bone loss and cortical bone lesions compared to the vehicle control group.

[0100] The results showed that anpentasiloxane could inhibit bone destruction caused by tumors.

[0101] Effect Example 4: Anwuzhisu alleviates pain behavior in rats with bone cancer pain ( Figure 5 )

[0102] On the 10th to 14th day after tumor cell implantation, rats with bone cancer pain were injected with pentosan (25mM, 75mM or 200mM) or 100 μg of gabapentin into the spinal cord daily. Compared with the control group, the mechanical allodynia, thermal allodynia and spontaneous allodynia of rats receiving different doses of pentosan or gabapentin were significantly reduced.

[0103] The results showed that anpentasiloxane could relieve bone cancer pain.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of anwuzhisu in the preparation of drugs for treating or preventing neuroinflammatory-related diseases and bone cancer pain.

2. The use according to claim 1, characterized in that The treatment or prevention of neuroinflammation-related diseases includes any of the following methods to achieve the treatment or prevention of the neuroinflammation-related diseases: (I) inhibiting the expression of iNOS and / or COX-2 proteins; and / or (II) Reduce the release of NO.

3. The use according to claim 1 or 2, characterized in that The neuroinflammation-related disease is selected from Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke or traumatic brain injury.

4. The use according to claim 1, wherein The treatment or prevention of bone cancer pain includes any of the following: (I) inhibiting tumor-induced bone tissue destruction; and / or (II), reducing tumor burden in bone tissue; and / or (III) Relieve pain caused by bone cancer.

5. The use according to claim 4, characterized in that The tumor includes primary bone cancer and / or metastatic bone cancer.

6. The use according to claim 5, characterized in that The primary bone cancer comprises osteosarcoma cancer; and / or The metastatic bone cancer includes one or more of breast cancer, prostate cancer, lung cancer, kidney cancer, liver cancer, renal cancer, bladder cancer, thyroid cancer, cervical cancer, colon cancer, esophageal cancer or nasopharyngeal cancer.

7. The use according to any one of claims 1 to 6, characterized in that The dosage form of the drug includes oral preparations or injections.

8. The use according to any one of claims 1 to 7, characterized in that The administration method of the anpentaliprin includes subarachnoid administration.

9. The use according to any one of claims 1 to 8, characterized in that The effective dose of the pentaerythrin is 75 mM to 200 mM / 10 μl / rat / day.

10. A method for preventing and / or treating tumor-related bone diseases, characterized in that: The method comprises administering anpentasone to a subject.

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