2,2'-methylenebis in the manufacture of analgesic medicaments

Abstract

The application discloses application of 2,2'-methylene bis in preparation of analgesic drugs. The application applies 2,2'-methylene bis to relieve neuropathic pain, and provides a solution to the problem of lack of analgesic drugs in clinical treatment. The new analgesic drug has a wide market prospect, and has medical application value and economic value.

Description

Technical Field

[0001] This invention relates to an analgesic drug, specifically to the use of 2,2'-methylenebis in the preparation of analgesic drugs. It belongs to the field of pharmaceutical technology. Background Technology

[0002] Pain is a complex sensory and emotional experience. Depending on the context, meaning, and psychological state of the pain, it can vary greatly between individuals or even between different parts of the body. Pain is a physiological response to tissue damage and plays a vital role in the body's protection and defense. The International Association for the Study of Pain defines pain as "an unpleasant subjective feeling and emotional experience, usually accompanied by existing or latent tissue damage." Pain is categorized into acute and chronic pain based on its duration. Chronic pain persists for a longer period, causing continuous damage to tissues and persistent activation of pain signals, resulting in cumulative pain that is more difficult to control.

[0003] Chronic pain, defined by the biopsychosocial model, is a heterogeneous disease lasting longer than three months, characterized by multiple pathological mechanisms, including a combination of physical dysfunction, beliefs, coping strategies, pain, illness, behavior, and social interactions. It is characterized by its difficulty in treatment, long duration, and complexity. Its core symptoms are persistent neuropathic pain and inflammation, which negatively impact the patient's physical and mental health. Neuropathic pain (NP) is the most typical and prevalent form of chronic pain, characterized primarily by spontaneous pain, sensory dullness, paresthesia, and hyperalgesia. Statistics show that over a quarter of the global population suffers from NP, causing significant economic losses and resource waste, and seriously affecting social stability and development. The pathogenesis of NP is complex, involving numerous physiological processes, and has long been a challenging problem in clinical pain management. Current treatments for analgesics (NP) primarily include opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), and antidepressants. However, long-term medication often leads to drug dependence and adverse reactions such as constipation, nausea, and vomiting. These medications are highly addictive, and relapse is common after discontinuation. The overall safety and efficacy remain controversial, highlighting the urgent need to develop new, reliable, and effective analgesics. The market prospects for analgesic drug development are vast, and the development of novel analgesics or formulations has significant medical and economic value.

[0004] 2,2′-methylenebis is a synthetic antioxidant that exhibits good free radical scavenging ability in vitro. In animal models (zebrafish, mice, and rats), 2,2′-methylenebis also shows low biotoxicity and good antioxidant properties. Currently, there are few research reports on 2,2′-methylenebis, mainly focusing on its use in anti-tumor research and enhancing the anti-tumor efficacy of anti-tumor drugs. It exerts its effects by activating autophagy and apoptosis, possessing certain anti-tumor efficacy and antioxidant capacity. There are no research reports on the application of 2,2′-methylenebis in neuropathic pain.

[0005] Oxidative stress plays a crucial role in pain development. Excessive free radicals promote sensitization by directly activating neuronal ion channels and by inducing neuromodulation and dysfunctional synaptic plasticity through specific mechanisms. Addressing oxidative stress may be an effective strategy for alleviating neuropathic pain. 2,2′-Methylenebis(2,2′) may be a candidate analgesic drug due to its antioxidant properties. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of 2,2'-methylenebis in the preparation of analgesic drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of 1,2,2'-methylenebis in the preparation of drugs for relieving neuropathic pain.

[0009] 2. An analgesic drug, the active ingredient of which is 2,2'-methylenebis.

[0010] The beneficial effects of this invention are as follows: This invention applies 2,2'-methylenebismuth to relieve neuropathic pain, providing a solution to the problem of limited analgesic drug options in clinical treatment. The development of novel analgesics has broad market prospects and possesses both medical and economic value. Attached Figure Description

[0011] Figure 1 It is a 2,2-'methylene bidrug structure.

[0012] Figure 2This study investigated the effect of intraperitoneal injection of 20 mg / kg 2,2'-methylenebis(Met) or saline on formalin-induced pain behavior in wild-type (WT) mice. A: Following intraperitoneal injection of 20 mg / kg Met or saline (NC) 30 min later, 20 μl of 2% (v / v) formalin was injected into the plantar surface of the right hind paw of WT mice. Formalin-induced pain behavior, including paw lifting, paw retraction, and paw licking, was measured every 5 min for 60 min. Data are expressed as mean ± SEM (n = 6). B: In both groups of mice, total pain scores were divided into two phases: Phase I (0–15 min) and Phase II (15–60 min). Data are expressed as mean ± SEM, *P < 0.05 (n = 6).

[0013] Figure 3 This study investigated the changes and development of pain behavior in rats following chronic sciatic nerve constriction injury (CCI). After CCI surgery, compared to the sham-operated group, the model group exhibited a significantly higher mechanical withdrawal threshold (PWT) on the ipsilateral paw. Figure 3 (A) Pressure ( Figure 3 (B) and thermal recession latency (TWL) Figure 3 C) was significantly reduced. Measurements were taken before surgery (D0) and on days 3, 5, 7, and 14 after CCI or sham surgery. Data are expressed as mean ± SEM.

[0014] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6).

[0015] Figure 4 This study investigated the effect of intraperitoneal injection of 20 mg / kg 2,2′-methylenebis on pain behavior in mice with chronic sciatic nerve injury. Fourteen days post-surgery, changes in pain behavior were measured within 2 hours following intraperitoneal injection of 20 mg / kg 2,2′-methylenebis. Compared to the solvent group, the treated group showed a significantly higher mechanical withdrawal threshold (PWT) in the ipsilateral paw. Figure 4 (A) and heat depression latency (PWL) Figure 4 (B) decreased significantly. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6).

[0016] Figure 5The effect of intraperitoneal injection of 20 mg / kg 2,2′-methylenebis on pain behavior in rats with chronic sciatic nerve injury was investigated. Fourteen days post-surgery, rats received an intraperitoneal injection of 20 mg / kg 2,2′-methylenebis. Changes in pain behavior were measured within 2 hours afterward. Compared to the solvent group, the treated group showed a significantly higher mechanical withdrawal threshold (PWT) in the ipsilateral paw. Figure 5 (A) Pressure ( Figure 5 (B) and heat depression latency (PWL) Figure 5 The rats showed a significant decrease in pain threshold (C) in the solvent group, followed by 7 consecutive days of administration. Changes in pain behavior were measured before each administration. Compared to the solvent group, the administration group exhibited a significantly lower mechanical withdrawal threshold (PWT) in the ipsilateral paw. Figure 5 (D) pressure ( Figure 5 (E) and heat depression latency (PWL) Figure 5 The mean (F) was significantly reduced. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6).

[0017] Figure 6 Effects of intraperitoneal injection of 20 mg / kg 2,2′-methylenebis on the activation of ERK1 / 2 and p38 in the spinal cord of rats with chronic sciatic nerve injury. Compared with the control group, the phosphorylation levels of ERK1 / 2 and p38 in the spinal cord of rats with chronic sciatic nerve injury were increased. Figure 6 A: p-ERK / ERK; Figure 6 In the B group: p-P38 / P38, after drug treatment, Met significantly inhibited the activation of ERK1 / 2 and p38. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6).

[0018] Figure 7 Effects of intraperitoneal injection of 20 mg / kg 2,2′-methylenebis on the expression levels of inflammatory cytokines mRNA in the spinal cord of rats with chronic sciatic nerve injury. Figure 7 Middle A: IL-6; Figure 7 IL-1β; Figure 7 C: TNF-α; Figure 7 In the middle jiao (CCL-2), compared with the control group, the expression levels of a series of inflammatory cytokines mRNA were upregulated after chronic sciatic nerve injury. After drug treatment, Met significantly inhibited the upregulation of inflammatory cytokine expression. Data are expressed as mean ± SEM.

[0019] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6).

[0020] Figure 8 Effect of Met on the expression level of MDA in the spinal cord of rats with chronic sciatic nerve injury. Compared with the control group, the expression level of MDA in the spinal cord of rats with chronic sciatic nerve injury model was significantly increased, and the up-regulation of MDA expression level could be inhibited after intraperitoneal injection of 20 mg / kg 2,2′-methylenebis. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n = 3).

[0021] Figure 9 Effect of Met on the survival rate of mouse microglial cells (BV-2) in inflammation and oxidative stress models. Met was prepared into a stock solution with a concentration of 10 mM using DMSO, and the cell survival rate was measured by CCK-8 after 24 h of drug treatment. Five replicates were set for each treatment (n = 5). Figure 9 A: Effect of Met on cell survival rate; Figure 9 B: Effect of Met on cell survival rate after applying 1 μg / ml LPS; Figure 9 C: Effect of Met on cell survival rate after applying 250 μM H2O2. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n = 5). Detailed implementation manners

[0022] The present invention will be further described below in conjunction with the drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0023] Experimental materials and methods:

[0024] SPF-grade C57BL / 6 male mice (20), 6 - 8 weeks old, provided by Hunan Slack Jingda Experimental Animal Co., Ltd., purchased on behalf by the Experimental Animal Center of Central South University, fostered in the barrier on the fourth floor of the Experimental Animal Center, and the production license number is SCXK(Xiang)2021 - 0002. The mice were housed in an independent environment for 12 hours during day / night, housed at room temperature (24 ± 1°C), with free access to drinking water and food, and acclimated to the environment 1 week in advance. All experimental operations followed the regulations of the Ethics Committee of the International Association for the Study of Pain.

[0025] SPF Sprague-Dawley (SD) male rats (60), weighing 250 - 300 g, were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., purchased on behalf by the Experimental Animal Center of Central South University, and fostered in the barrier on the fourth floor of the Experimental Animal Center. The production license number is SCXK(Xiang)2021 - 0002. The rats were housed in an independent environment with a 12-hour light / dark cycle, at room temperature (24 ± 1°C), with free access to drinking water and food, and were allowed to acclimatize to the environment for 1 week in advance. All experimental procedures followed the regulations of the Ethics Committee of the International Association for the Study of Pain.

[0026] The mouse microglial cell line (BV-2 cells) was purchased from WheLab Shanghai Yingwan Biotechnology Co., Ltd.

[0027] 1. Folin test

[0028] Performed according to the reference (Zhang E., Kim J.-J., Shin N., Yin Y., Nan Y., Xu Y., Hong J., Hsu T.M., Chung W., Ko Y., et al. High Omega-3 Polyunsaturated Fatty Acids in fat-1 Mice Reduce Inflammatory Pain. J. Med. Food. 2017; 20:535–541. doi:10.1089 / jmf.2016.3871.).

[0029] 20 μL of 2% (v / v) formalin (Sigma-Aldrich, St. Louis, Missouri, USA) was injected subcutaneously on the plantar surface of the right hind paw of the mice using a 26.5-gauge needle. In a single cage, pain behaviors induced by formalin, such as licking, lifting the paw, and withdrawal, were observed every 5 minutes for 60 minutes. For analysis, the pain scores were summarized and divided into two phases: the first phase (0 - 15 minutes) and the second phase (15 - 60 minutes). All tests were conducted between 11:00 and 15:00 in a quiet room.

[0030] 2. Construction of the CCI model

[0031] Following the method of Bennett (Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988; 33(1):87–107. doi:10.1016 / 0304-3959(88)90209-6), the following steps were followed:

[0032] Rats were anesthetized with 3% sodium pentobarbital solution (m / v) (0.1 mL / 100 g), fixed on the operating table, and the skin of the right thigh was prepared and disinfected. The middle and back of the right thigh skin were incised, and the muscles were bluntly dissected to expose the upper half of the sciatic nerve. The nerve was freed from the bifurcation of the peroneal nerve and surrounded with 4-0 sutures. Four ligation loops with a spacing of 1 mm were made. The knot strength was such that the knot was close to the nerve, causing the thigh muscles to twitch or kick, without affecting the blood supply to the tendon sheath. After local disinfection with penicillin powder, the skin was sutured with 3-0 silk sutures. In the Sham group, the sciatic nerve was not ligated, and the remaining steps were the same as in the model group. Compared with the control group, the animals exhibited a series of self-protective behaviors with obvious spontaneous pain (spontaneous lifting of the injured limb, sometimes biting the foot or kicking the leg), and the mechanical withdrawal threshold (MWT), pressure threshold, and thermal withdrawal latency (TWL) were significantly reduced compared with the Sham group, indicating that the model was successful.

[0033] 3. Animal grouping and treatment

[0034] After the CCI model and sham-operated group were established, all experimental rats were divided into 5 groups of 10 rats each (50 rats out of 60 were selected for follow-up experiments, excluding those that died due to surgery or other reasons).

[0035] 1) Normal control group (control group), no treatment required;

[0036] 2) Sham surgery group (sham surgery group): the sciatic nerve was not ligated, and other procedures were the same as in the CCI model;

[0037] 3) Sham surgery + 0.9% saline (m / v);

[0038] 4) CCI model group (CCI model), sciatic nerve ligation;

[0039] 5)CCI+20mg / kg 2,2′-methylenebis(CCI+Met).

[0040] Mechanical withdrawal threshold (MWT), claw withdrawal threshold (Pressure), and thermal withdrawal latency (TWL) were measured preoperatively and on postoperative days 3, 5, 7, and 14 to confirm model success. Drug treatment was initiated and continued until day 21 postoperatively. Changes in MWT, Pressure, and TWL were measured within 2 hours (0, 0.5, 1, 1.5, 2 hours) after intraperitoneal injection of 20 mg / kg Met. Subsequent seven-day continuous drug administration followed by pre-administration measurement of MWT, Pressure, and TWL. Real-time quantitative PCR was used to detect changes in the transcriptional levels of inflammatory factors in the lumbar spinal cord of rats. Western blotting was used on day 21 postoperatively to detect the effects of ERK1 / 2 and p38 activation levels in the L3-L5 lumbar spinal cord of rats. The changes in MDA levels in the lumbar segments L3-L5 of the rat spinal cord were detected using an MDA kit.

[0041] 4. Real-time quantitative PCR (QPCR)

[0042] RNA extraction using RNAeasy TM RNA was extracted from animal tissues using an animal RNA extraction kit. Rat spinal cord segments L3-5 were collected and placed in 1.5 mL centrifuge tubes. 1 mL of Trizol was added to each tube, and the mixture was sonicated for approximately 1 minute at room temperature, followed by incubation at room temperature for 5 minutes. The tubes were then placed on ice, and 0.2 mL of chloroform was added. The sample was vortexed for approximately 15 seconds, allowed to stand for 2 minutes, and this vortexing and standing process was repeated 2-3 times. The tubes were centrifuged at 12,000 g for 15 minutes at 4°C, and the supernatant was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added to the tube, and the mixture was thoroughly mixed and allowed to stand for 10 minutes to precipitate the RNA. The precipitate was then centrifuged at 13,000 g for 10 minutes at 4°C, the supernatant was discarded, and the RNA precipitate was collected. The precipitate was washed with pre-cooled 75% ethanol (v / v) and dried in a 65°C incubator to remove residual liquid. DEPC-treated water was added to form an RNA solution, which was then vortexed. RNA concentration and purity were determined using a spectrophotometer, and the solution was stored at -80°C.

[0043] cDNA synthesis: cDNA was synthesized by reverse transcription using the HiFiScript rapid degenomic cDNA first-strand synthesis kit. The extracted RNA was thawed on ice, and a 20 μL mixture of 1-5 μg RNA, 4 μL of 5×PrimeSrip RTmix, and RNase-free water was placed in a labeled centrifuge tube. After brief centrifugation, cDNA was synthesized by reverse transcription using a PCR instrument under the following conditions: 37°C for 15 min, 85°C for 5 s. The resulting cDNA was stored at -80°C.

[0044] Real-time quantitative PCR: Thaw cDNA on ice. Prepare a 20 μL system with 2 μL cDNA, 1 μL each of forward and reverse primers, 10 μL 2×iTaqSYBR Greensupermix, and 6 μL DEPC water. Load the sample into three wells per sample group using each primer, and then onto a 96-well qPCR plate. Seal the plate with a qPCR sealing film and run the reaction on a QuantStudio 5 PCR instrument. The conditions are: pre-denaturation 95℃ for 30 s, denaturation 95℃ for 10 s—annealing 58℃ for 30 s—extension 72℃ for 32 s, for a total of 40 cycles. Subtract the Ct value of the internal control RNA from the Ct value of the target RNA in each group to obtain the ΔCt value. Subtract the ΔCt value of the control group from the ΔCt value of the target RNA to obtain the ΔΔCt value. The relative expression level of the target RNA is 2. -ΔΔCt .

[0045] IL-6:Forward:5′-CAGACCCACATGCTCCGAGA-3′

[0046] Reverse:5′-CAAGGCTTGGCAACCCAAGTA-3′

[0047] CCL-2:Forward:5'-TAGCATCCACGTGCTGTCTC-3'

[0048] Reverse:5'-CAGCCGACTCATTGGGATCA-3'

[0049] IL-1β:Forward:5'-AGAGGTGTGGATCCCAAACAA-3'

[0050] Reverse:5'-AGTCAACTATGTCCCGACCA-3'

[0051] TNF-α: Forward:5'-TGATCGGTCCCAACAAGGA-3'

[0052] Reverse:5'-TGCTTGGTGGTTTGCTACGA-3'

[0053] 5. Western blot was used to detect changes in protein expression levels.

[0054] Tissue sampling: Rats were euthanized after being anesthetized with sodium pentobarbital. The spinal cord was quickly removed and immediately frozen on ice. The L3-L5 lumbar spinal cord was cut and quickly placed in liquid nitrogen for freezing and storage at -80°C for later use.

[0055] Protein extraction and concentration determination: Take an appropriate amount of tissue and place it in a 1.5 mL centrifuge tube. Add lysis buffer containing PMSF about 10 times the volume of the tissue. Pre-cool the homogenizer on ice and homogenize with an electric homogenizer for 5 seconds × 3 times, with an interval of 30 seconds, until the tissue is completely broken. Place the homogenized tissue on ice for lysis for 30 minutes. Centrifuge at 12,000 g for 15 minutes at 4°C and collect the supernatant.

[0056] Concentration determination: The collected supernatant was analyzed for protein content using the Beyotime BCA protein concentration quantification method. The specific method is as follows: A 96-well microplate was prepared with bovine serum albumin (1 mg / mL BSA) standards at concentrations of 0, 0.025, 0.125, 0.25, 0.5, 0.75, 1, 1.5, and 2 mg / mL. The prepared concentration gradient standards and 4 μl of the total protein sample were added sequentially to the wells of the 96-well plate, and the volume was brought up to 20 μl with standard dilution buffer. An appropriate amount of BCA working solution was prepared according to a BCA reagent A:B volume ratio of 50:1. Then, 100 μl of BCA working solution was added to the wells containing the standards and sample. The mixture was shaken for 5 min to mix thoroughly, and then incubated at 37°C for 30 min. The absorbance at 562 nm was measured using a microplate reader. A standard curve was plotted with the standard protein concentration on the x-axis and the absorbance on the y-axis. The protein concentration of the sample was calculated based on the standard curve and the sample volume used. The sample was then prepared into equal volumes and concentrations using lysis buffer.

[0057] Sample preparation: Add 1 / 3 volume of 4× loading buffer and 1 / 40 volume of β-mercaptoethanol to the immunoblot protein sample, mix well, and heat at 100℃ for 10 min to completely denature the protein. After sample preparation, aliquot and store at -80℃.

[0058] Immunoblot analysis: After separation by SDS-PAGE, equal volumes of protein samples were transferred to a polyvinylidene fluoride (PVDF) membrane using a wet method at 250 mA. Blocked with 5% skim milk powder (m / v) in a shaker at room temperature for 1 hour for 90 min, then incubated overnight at 4°C with anti-ERK1 / 2 (1:1000, CST), anti-p-ERK1 / 2 (1:1000, CST), anti-GAPDH (1:10000), anti-p38 (1:1000), anti-p-p38 (1:1000), and anti-β-actin (1:10000) (v / v). Washed three times with 1×TBST for 6 min each time, then incubated with HRP-labeled goat anti-rabbit and goat anti-mouse IgG at room temperature with shaking for 1 hour, washed three times with 1×TBST for 6 min each time. The protein band signals were detected by Western blot using ECL chemiluminescence solution. The obtained bands were analyzed for grayscale density using Image-J.

[0059] 6. MDA reagent kit detection

[0060] Beyotime's Lipid Peroxidation MDA Assay Kit employs a colorimetric reaction based on the reaction of MDA and thiobarbituric acid (TBA) to produce a red product, with an absorption peak at 532 nm. Subsequently, a colorimetric method is used to quantify MDA in plasma, serum, urine, animal and plant tissues, or cell lysates. MDA levels are calculated using a standard curve based on the manufacturer's datasheet. This kit is widely used for detecting lipid peroxidation levels.

[0061] Malondialdehyde can react with TBA at higher temperatures and in acidic environments to form a red MDA-TBA adduct.

[0062] Product Packaging List: TBA 25mg, TBA diluent 15mL, TBA preparation solution 6.76mL, antioxidant 300μl, standard (1mM malondialdehyde) 200μl.

[0063] Sample preparation: Take an appropriate amount of tissue and homogenize or lyse it using lysis buffer. The tissue weight should account for 10% of the homogenate or lysis buffer. After homogenization or lysis, centrifuge at 10,000g–12,000g for 10 minutes and collect the supernatant for subsequent determination. Sample preparation steps such as homogenization or lysis should be performed in an ice bath or at 4°C.

[0064] Preparation of TBA stock solution: Weigh an appropriate amount of TBA and prepare a 0.37% TBA stock solution (m / v) using TBA preparation solution. The TBA preparation solution must be completely dissolved before use; heating to 70°C can promote dissolution.

[0065] Dilution of standards: Take an appropriate amount of standard and dilute it with distilled water to 1, 2, 5, 10, 20, 50, 100, 150, or 200 μM for subsequent preparation of standard curves.

[0066] Sample determination:

[0067] a. The reaction system is shown in Table 1.

[0068] Table 1. Reaction System

[0069]

[0070]

[0071] b. After mixing, heat at 100°C or in a boiling water bath for 15 minutes. Take care to avoid the liquid boiling over and splashing during heating.

[0072] c. Cool to room temperature in a water bath, then centrifuge at 1000g for 10 min at room temperature. Add 200 μl of supernatant to a 96-well plate, and then measure the absorbance at 532 nm using a microplate reader.

[0073] d. Calculation of MDA content: The MDA content in the sample solution is calculated based on the standard curve.

[0074] 7. Cell viability detection and model construction

[0075] Cell culture: BV-2 cells were placed in a constant temperature incubator containing 5% CO2 (v / v) at 37°C and cultured in RPMI-1640 medium supplemented with 10% PAN fetal bovine serum (v / v) and 1% penicillin-streptomycin antibiotic (v / v). When passaged, the culture medium was discarded first, the cells were washed with PBS, and then trypsin was added for 20-25 seconds. Then, 2 mL of the prepared culture medium was added to stop the digestion. The cells at the bottom of the culture dish were pipetted until they were all detached. After being pipetted evenly, the cells were dispensed into the prepared culture dishes at a ratio of 1:2 or 1:3. Culture medium was added to make the liquid volume 5 mL. The cells were mixed by pipetting and then put back into the incubator for further culture.

[0076] Cell viability assay: Cells were passaged and seeded in 96-well plates at a density of 2.0 × 10⁶ cells / well. 4For each well, after the cells adhered, the drug prepared in DMSO was added. The stock solution concentration was 10 mM, which was diluted with culture medium to 0, 2.5, 5, 10, 15, and 20 μM. Five replicates were set up for each concentration. After incubation for 24 h, serum-free culture medium (v / v) containing 10% CCK8 was added. After incubation for 1-2 h, the OD value was measured at a wavelength of 450 nm, and the cell viability was calculated based on the OD value.

[0077] Construction of oxidative stress model: Cells were passaged and seeded in 96-well plates at a density of 2.0 × 10⁶ cells / well. 4 Cell viability was measured after each well was treated with 250 μM or 300 μM hydrogen peroxide and 0, 0.15, 0.3, 0.625, 1.25, 2.5, or 5 μM of the drug for 24 h to evaluate the antioxidant effect of the drug Met.

[0078] Experimental results:

[0079] 1. Effects of intraperitoneal injection of Met on formalin-induced pain behavior in mice

[0080] Figure 2 Effects of intraperitoneal injection of 20 mg / kg Met or saline on formalin-induced pain behavior in wild-type mice. A: 30 min after intraperitoneal injection of 20 mg / kg Met or saline (NC), 20 μl of 2% formalin (v / v) was injected into the plantar surface of the right hind paw of WT mice. Formalin-induced pain behavior, including paw lifting, paw retraction, and paw licking, was measured every 5 min for 60 min. Data are expressed as mean ± SEM (n=6). B: In both groups of mice, the total pain score was divided into two phases: Phase I (0-15 min) and Phase II (15-60 min). Data are expressed as mean ± SEM, *P<0.05 (n=6). The results showed that intraperitoneal injection of 2,2'-methylenebismuth significantly alleviated formalin-induced pain behavior in mice. The duration of pain behavior caused by inflammation in Phase II was significantly reduced, indicating that 2,2'-methylenebismuth has the ability to alleviate inflammatory pain.

[0081] 2. Pain hypersensitivity following chronic sciatic nerve contraction in rats

[0082] Figure 3 This study investigated the changes and development of pain behavior in rats following chronic sciatic nerve injury (CCI). Compared to the sham-operated group, the model group showed significantly lower mechanical withdrawal threshold (PWT) (A), pressure (B), and thermal withdrawal latency (TWL) (C) on the ipsilateral paw after CCI or sham-operation. Measurements were taken before surgery (D0) and on days 3, 5, 7, and 14 after CCI or sham-operation. Data showed that the mean...

[0083] ±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). Experimental results showed that after CCI surgery, the PWT and PWL thresholds of rats were significantly reduced and tended to stabilize after day 7, consistent with literature reports, indicating that the CCI model was successfully established.

[0084] 3. Effects of intraperitoneal injection of Met on pain behavior in mice with chronic sciatic nerve injury

[0085] Figure 4 The effect of intraperitoneal injection of 20 mg / kg 2,2′-methylenebis on pain behavior in mice with chronic sciatic nerve injury was investigated. Fourteen days post-surgery, mice received an intraperitoneal injection of 20 mg / kg 2,2′-methylenebis. Changes in pain behavior were measured within 2 hours afterward. Compared to the solvent group, the treated group showed significantly increased mechanical withdrawal threshold (PWT) (A) and thermal withdrawal latency (PWL) (B) of the ipsilateral paw. Data are expressed as mean.

[0086] ±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). The experimental results showed that a single intraperitoneal injection of 2,2'-methylenebismuth significantly alleviated CCI-induced pain behavior in mice within 2 hours, indicating that 2,2'-methylenebismuth has the ability to alleviate neuropathic pain caused by mechanical injury.

[0087] 4. Effects of intraperitoneal injection of Met on pain behavior in rats with chronic sciatic nerve injury

[0088] Figure 5 The effect of intraperitoneal injection of 20 mg / kg 2,2′-methylenebis on pain behavior in rats with chronic sciatic nerve injury was investigated. Fourteen days post-surgery, rats received an intraperitoneal injection of 20 mg / kg 2,2′-methylenebis. Changes in pain behavior were measured within 2 hours afterward. Compared to the solvent group, the treated group showed significantly higher mechanical withdrawal threshold (PWT) (A), pressure (B), and thermal withdrawal latency (PWL) (C) in the ipsilateral paw. Subsequent administration for 7 consecutive days, with changes in pain behavior measured before each administration, showed significantly higher mechanical withdrawal threshold (PWT) (D), pressure (E), and thermal withdrawal latency (PWL) (F) in the ipsilateral paw compared to the solvent group. Data are expressed as mean.

[0089] ±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). The results showed that a single intraperitoneal injection of 2,2'-methylenebismuth significantly alleviated CCI-induced pain behavior in rats within 2 hours; during prolonged continuous administration, 2,2'-methylenebismuth also effectively alleviated pain behavior in rats. This indicates that 2,2'-methylenebismuth possesses the ability to alleviate neuropathic pain caused by mechanical injury in both short and long-term durations.

[0090] 5. Effects of Met on ERK / 12 and p38 activation in the spinal cord of rats with chronic sciatic nerve injury

[0091] Figure 6 Effects of intraperitoneal injection of 20 mg / kg 2,2′-methylenebis on the activation of ERK1 / 2 and p38 in the spinal cord of rats with chronic sciatic nerve injury. Compared with the control group, the phosphorylation levels of ERK1 / 2 and p38 in the spinal cord of rats with chronic sciatic nerve injury were increased (A): p-ERK / ERK; (B): p-P38 / P38. After drug treatment, Met significantly inhibited the activation of ERK1 / 2 and p38. Data are expressed as mean.

[0092] ±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). The MAPK signaling pathway is one of the important intracellular and extracellular signal transduction pathways in the body. It can be stimulated by various extracellular signals such as cytokines, neurotransmitters, hormones, and cellular stress, and participates in gene expression regulation and cytoplasmic function activities. Experimental results showed that 2,2'-methylenebis can inhibit the phosphorylation of ERK and p38 proteins in the MAPK signaling pathway induced by CCI, thereby affecting the expression of downstream inflammation and antioxidant-related genes, alleviating the neuroinflammatory response and neurooxidative stress induced by CCI, and thus relieving pain.

[0093] 6. Effects of Met on the expression levels of inflammatory factors in the spinal cord of rats with chronic sciatic nerve injury

[0094] Figure 7 The effect of intraperitoneal injection of 20 mg / kg 2,2′-methylenebis on the mRNA expression levels of inflammatory factors in the spinal cord of rats with chronic sciatic nerve injury. (A): IL-6; (B): IL-1β; (C): TNF-α; (D): CCL-2. Compared with the control group, chronic sciatic nerve injury upregulated the mRNA expression levels of a series of inflammatory factors. After drug treatment, Met significantly inhibited the upregulation of inflammatory factor expression. Data are expressed as mean.

[0095] ±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). The experimental results show that 2,2'-methylenebis can inhibit CCI-induced neuroinflammatory responses and suppress the expression of inflammatory factors in nerve tissue.

[0096] 7. Effects of Met on MDA Expression Levels in the Spinal Cord of Rats with Chronic Sciatic Nerve Injury

[0097] Figure 8 This study investigated the effect of 2,2'-methylenebis on MDA expression levels in the spinal cord of rats with chronic sciatic nerve injury. Compared with the control group, MDA expression levels in the spinal cord of rats with chronic sciatic nerve injury were significantly increased. Intraperitoneal injection of 20 mg / kg 2,2'-methylenebis inhibited the upregulation of MDA expression levels. Data are presented as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=3). The results showed that MDA levels in the spinal cord were significantly upregulated after CCI, and the degree of neurolipid peroxidation increased, indicating oxidative stress. 2,2'-methylenebis significantly reduced MDA levels in the spinal cord, indicating that 2,2'-methylenebis can significantly inhibit neurolipid peroxidation and alleviate oxidative stress in the peripheral nerves.

[0098] 8. Effects of Met on the survival rate of mouse microglioma cells (BV-2) in inflammation and oxidative stress models.

[0099] Figure 9 To investigate the effect of methicillin (Met) on the survival rate of mouse microglioma cells (BV-2) in an inflammation and oxidative stress model. Met was prepared as a 10 mM stock solution using DMSO. After 24 h of treatment, cell viability was measured using a CCK-8 assay. Five accessory wells (n=5) were set up for each treatment. (A): Effect of Met on cell viability; (B): Effect of Met on cell viability after application of 1 μg / mL LPS;

[0100] (C): Effect of Met on cell viability after application of 250 μM H2O2. Data are expressed as mean.

[0101] ±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=5). An in vitro model of inflammation and oxidative stress was established in BV-2 cells induced by LPS and hydrogen peroxide. Experimental results showed that 2,2'-methylenebismuth substituent (MMDS) had no effect on cell survival and was not cytotoxic within the 0-5 μM concentration range. Within this range, MMDS could not inhibit LPS-induced microglia activation-induced cell proliferation or LPS-induced inflammatory responses. However, within the 0-5 μM concentration range, MMDS significantly alleviated cell death caused by hydrogen peroxide-induced oxidative stress and inhibited oxidative stress in microglia in vitro, demonstrating antioxidant capacity. In vitro cell experiments demonstrated that MMDS can alleviate neuropathic pain by inhibiting oxidative stress in microglia.

[0102] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1.2,2'-Methylenebis in the preparation of analgesic drugs.

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