CN117205187A - Application of 2,2’-methylenebis in the preparation of analgesic drugs - Google Patents

Abstract

The invention discloses application of 2,2' -methylenebis in preparing analgesic drugs. The invention applies the 2,2' -methylene bis to relieve neuropathic pain and provides a solution for the problem of lack of analgesic drug selection in clinical treatment. The market prospect of developing novel analgesic drugs is wide, and the novel analgesic drugs have medical application value and economic value.

Description

Application of 2,2’-methylenebis in the preparation of analgesic drugs

Technical field

The present invention relates to an analgesic drug, and specifically relates to the application of 2,2'-methylenebis in the preparation of analgesic drugs. Belongs to the field of medical technology.

Background technique

Pain is a complex sensory and emotional experience. Depending on the background, meaning and psychological state of the pain, there may be great differences between individuals or between different parts of the individual. Pain is a physiological response when the body tissue is damaged. The protection and defense of the body play a very important role. The International Pain Society calls pain “an unpleasant subjective and emotional experience, often accompanied by existing or latent tissue damage.” Pain is divided into acute pain and chronic pain according to the length of time. Chronic pain lasts for a long time, causing continuous damage to damaged tissues, continuous activation of pain signals, and superimposed pain, making the pain more difficult to control.

Chronic pain is defined by the biopsychosocial model as a heterogeneous disorder that lasts longer than three months and is characterized by multiple pathological mechanisms, including physical dysfunction, beliefs, coping strategies, distress, illness, behavior, and social interactions. Combination, difficult to treat, long-term and complex. Its core symptoms are long-lasting and persistent nerve pain and inflammation in the body, which is not conducive to the patient's physical and mental health. Neuropathic pain (NP) is the most typical chronic pain with the highest incidence rate. The disease is mainly characterized by spontaneous pain, hypoesthesia, allodynia and hyperalgesia. According to statistics, more than a quarter of the world's population suffers from NP, which has caused huge economic losses and waste of resources to society, and seriously affected social stability and development. The causes of NP are complex and involve many physiological processes. It has always been a difficult problem in clinical pain treatment. Current treatments for NP mainly include opioids, non-steroidal anti-inflammatory drugs, antidepressants, etc. However, certain drug dependence and adverse drug reactions, such as constipation, nausea, vomiting, etc., often occur during long-term medication treatment. It has serious side effects and is highly addictive. The disease is easy to relapse after stopping the drug. The overall safety and effectiveness are still controversial. There is an urgent need to develop new reliable and effective analgesic drugs. The market prospect of analgesic drug development is broad, and the development of new analgesic drugs or preparations has broad medical application value and economic value.

2,2′-methylenebis, English name 2,2′-methylenebis, is a synthetic antioxidant that exhibits good free radical scavenging ability in vitro and in animal models (zebrafish, mice and rats) Among them, 2,2′-methylenebis also exhibits low biological toxicity and good antioxidant properties. At present, there are few research reports on 2,2′ methylene bis. It is mainly used for anti-tumor research and enhancing the anti-tumor efficacy of anti-tumor drugs. It works by activating autophagy and apoptosis and has certain anti-tumor efficacy and Antioxidant capacity, there are no research reports on the application of 2,2′-methylene bis to neuropathic pain.

Oxidative stress plays an important role in the development of pain, with excess free radicals promoting sensitization through direct activation of neuronal ion channels, as well as through specific mechanisms of neuromodulation and dysfunctional synaptic plasticity. Addressing oxidative stress may be an effective strategy for alleviating neuropathic pain. 2,2′-Methylenebis may be one of the candidates for analgesic drugs because of its antioxidant properties.

Contents of the invention

The object of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide the application of 2,2'-methylenebis in the preparation of analgesic drugs.

In order to achieve the above objects, the present invention adopts the following technical solutions:

1. Application of 2,2’-methylenebis in the preparation of drugs for relieving neuropathic pain.

2. An analgesic drug whose active ingredient is 2,2’-methylene bis.

Beneficial effects of the present invention: The present invention applies 2,2'-methylene bis to relieve neuropathic pain, providing a solution to the problem of lack of analgesic drug selection in clinical treatment. The development of new analgesic drugs has broad market prospects and has medical application value and economic value.

Description of the drawings

Figure 1 is the structural formula of 2,2-' methylene bidrug.

Figure 2 shows the effects of intraperitoneal injection of 20 mg/kg 2,2-’methylene bis (Met) or normal saline on formalin-induced pain behavior in wild-type (WT) mice. A: 30 minutes after intraperitoneal injection of 20 mg/kg Met or normal saline (NC), 20 μl of 2% (v/v) formalin was injected into the plantar surface of the right hind paw of WT mice, and then measured every 5 minutes for 60 minutes. Formalin-induced pain behaviors include lifting, paw withdrawal, and paw licking. Data are expressed as mean±SEM (n=6). B: In two groups of mice, the total pain score was divided into the first stage (0-15min) and the second stage (15-60min). Data are expressed as mean±SEM, *P<0.05 (n=6).

Figure 3 shows the changes and development of pain behavior after chronic constriction injury (CCI) of the sciatic nerve in rats. After CCI, compared with the sham operation group, the model group showed mechanical withdrawal threshold (PWT, A in Figure 3), pressure (B in Figure 3) and thermal withdrawal latency (TWL, C in Figure 3) on the ipsilateral paw. )Obvious reduction. Measurements were performed before surgery (D0) and on days 3, 5, 7, and 14 after CCI or sham surgery. Data are expressed as mean ± SEM,

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

Figure 4 shows the effect of intraperitoneal injection of 20 mg/kg 2,2′-methylenebis on pain behavior in mice with chronic sciatic nerve injury. On the 14th day after surgery, the changes in pain behavior of mice were measured within 2 hours after intraperitoneal injection of 20 mg/kg 2,2′-methylenebis. Compared with the solvent group, the administration group showed a mechanical withdrawal threshold of the ipsilateral paw (PWT, Figure 4 A) and thermal withdrawal latency (PWL, B in Figure 4) were significantly reduced. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6).

Figure 5 shows the effect of intraperitoneal injection of 20 mg/kg 2,2′-methylenebis on pain behavior in rats with chronic sciatic nerve injury. On the 14th day after surgery, the changes in pain behavior of rats within 2 hours were measured after intraperitoneal injection of 20 mg/kg 2,2′-methylenebis. Compared with the solvent group, the administration group showed a mechanical withdrawal threshold of the ipsilateral paw (PWT, Figure 5 A), pressure (B in Figure 5) and thermal withdrawal latency (PWL, C in Figure 5) were significantly reduced, followed by continuous administration for 7 days, and the changes in rat pain behavior were measured before each administration. Compared with the solvent group In comparison, the drug administration group showed a significant decrease in the mechanical withdrawal threshold (PWT, D in Figure 5 ) pressure (E in Figure 5 ) and thermal withdrawal latency (PWL, F in Figure 5 ) of the ipsilateral paw. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6).

Figure 6 shows the effect 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 were increased in the spinal cord of rats with chronic sciatic nerve injury. Figure 6 A: p-ERK/ERK; Figure 6 B: p-P38/P38 after drug treatment. , Met can significantly inhibit 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).

Figure 7 shows the effect of intraperitoneal injection of 20 mg/kg 2,2′-methylenebis on the expression levels of inflammatory factor mRNA in the spinal cord of rats with chronic sciatic nerve injury. A in Figure 7: IL-6; B in Figure 7: IL-1β; C in Figure 7: TNF-α; D in Figure 7: CCL-2. Compared with the control group, a series of inflammations are upregulated after chronic injury of the sciatic nerve. After drug treatment, Met can significantly inhibit the up-regulation of inflammatory factor expression levels. Data are expressed as mean ± SEM,

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

Figure 8 shows the 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 increased significantly, and intraperitoneal injection of 20 mg/kg 2,2'-methylenebis could inhibit the up-regulation of MDA expression level. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=3).

Figure 9 shows the effect of Met on the survival rate of mouse microglioma cells (BV-2) in inflammation and oxidative stress models. Met was prepared with DMSO into a stock solution with a concentration of 10mM. After 24 hours of drug treatment, the cell survival rate was measured using CCK-8. Five secondary wells (n=5) were set for each treatment. A in Figure 9: The effect of Met on cell survival rate; B in Figure 9: The effect of Met on cell survival rate after applying 1 μg/ml LPS; C in Figure 9: The effect of Met on cells after applying 250 μM H ₂ O ₂ Impact on survival rates. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=5).

Detailed ways

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

Experimental materials and methods:

SPF grade C57BL/6 male mice (20), 6-8 weeks old, provided by Hunan Slack Jingda Experimental Animal Co., Ltd., purchased from the Experimental Animal Center of Central South University, fostered and produced in the barrier on the fourth floor of the Experimental Animal Center The license number is SCXK (Hunan) 2021-0002. Mice were kept in an independent environment day/night for 12 hours, kept at room temperature (24±1°C), allowed to drink and eat freely, and adapted to the environment 1 week in advance. All experimental procedures complied with the regulations of the Ethics Committee of the International Association for the Study of Pain.

SPF Sprague-Dawley (SD) male rats (60 rats), weighing 250-300 grams, provided by Hunan Slack Jingda Experimental Animal Co., Ltd., purchased from the Experimental Animal Center of Central South University, on the fourth floor of the Experimental Animal Center Fostering within the barrier, the production license number is SCXK (Hunan) 2021-0002. Rats were kept in an independent environment day/night for 12 hours, kept at room temperature (24 ± 1°C), drank and eaten freely, and adapted to the environment 1 week in advance. All experimental procedures complied with the regulations of the Ethics Committee of the International Association for the Study of Pain.

Mouse microglioma cell line (BV-2 cells) was purchased from WheLab Shanghai Yingwan Biotechnology Company.

1. HUF test

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

Twenty microliters of 2% (v/v) formalin (Sigma-Aldrich, St. Louis, MO, USA) was injected under the plantar surface of the mouse's right hind paw using a 26.5-gauge needle. Formalin-induced painful behaviors such as licking, paw raising, and flinching were observed at 5-minute intervals for 60 minutes in individual cages. For analysis, pain scores were summarized and divided into two phases: Phase 1 (0-15 minutes) and Phase 2 (15-60 minutes). All tests were conducted in a quiet room between 11:00 and 15:00.

2. CCI model construction

Reference Bennett (Bennett GJ, 6) method, follow the following steps:

Anesthetize the rat (0.1mL/100g) with 3% sodium pentobarbital solution (m/v), fix it on the operating table, prepare and disinfect the skin of the right thigh, and cut the middle and back of the skin of the right thigh. Bluntly separate the muscles, expose the upper part of the sciatic nerve, liberate the nerve from the bifurcation of the peroneal nerve, surround the nerve with 4-0 suture, and make 4 ligation rings with a spacing of 1 mm. The knot strength is determined by the knot. Close to the nerve, it is appropriate to twitch or kick the thigh muscles, which will not affect the blood supply of the tendon sheath. After local spraying of penicillin powder for disinfection, the skin was sutured with 3-0 silk suture. The rats in the Sham group did not have the sciatic nerve ligated, and the remaining steps were the same as those in the model group. Compared with the control group, the animals developed a series of self-protective behaviors such as obvious spontaneous pain (spontaneously lifting the injured limb, sometimes biting or kicking the leg), and the mechanical withdrawal threshold (MWT), paw withdrawal threshold (Pressure) and The thermal withdrawal latency (TWL) was significantly reduced compared with the Sham group, indicating the success of the model.

3. Animal grouping and treatment

After the CCI model and sham operation group were established, all experimental rats were divided into 5 groups, with 10 rats in each group (50 rats out of 60 rats were selected for follow-up experiments, excluding deaths caused by surgery or other reasons).

1) Normal control group (control group) without any treatment;

2) Sham operation group (sham operation group), the sciatic nerve is not ligated, and other steps are the same as the CCI model;

3) Sham operation + 0.9% normal saline (m/v);

4) CCI model group (CCI model), sciatic nerve ligation;

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

The mechanical withdrawal threshold (MWT), paw withdrawal threshold (Pressure) and thermal withdrawal latency (TWL) were tested before surgery and on days 3, 5, 7 and 14 after surgery to confirm the success of the model. Medication was started and continued until 21 days after surgery. Inject 20 mg/kg Met intraperitoneally, and measure the changes in mechanical withdrawal threshold (MWT), paw withdrawal threshold (Pressure) and thermal withdrawal latency (TWL) within 2 hours (0, 0.5, 1, 1.5, 2); followed by seven days of For continuous administration, changes in mechanical withdrawal threshold (MWT), paw withdrawal threshold (Pressure) and thermal withdrawal latency (TWL) were detected before administration. Real-time fluorescence quantitative PCR technology was used to detect changes in the transcription levels of inflammatory factors in rat spinal cord lumbar segments. Western Blot was used to detect the effects of ERK1/2 and p38 activation levels in the L3-L5 spinal cord of rats 21 days after surgery. The MDA kit was used to detect the changes in MDA levels in the L3-L5 lumbar segments of the rat spinal cord.

4. Real-time fluorescence quantitative PCR (QPCR)

RNA was extracted using RNAeasy ^TM animal RNA extraction kit to extract animal tissue RNA. Place the collected rat spinal cord L3-5 segments into 1.5 mL centrifuge tubes, add 1 mL Trizol to each tube, homogenize by ultrasonic at room temperature for about 1 minute, and incubate at room temperature for 5 minutes. Place on ice, add 0.2 mL of chloroform, vortex to mix the sample, about 15 seconds, let stand for 2 minutes, then repeat vortex and let stand 2-3 times. Centrifuge at 12,000 g for 15 minutes at 4°C and transfer the upper layer to a new 1.5 mL centrifuge tube. Add 0.5 mL isopropyl alcohol to the tube, mix thoroughly, and leave for 10 minutes to precipitate RNA. Centrifuge at 13,000 g for 10 minutes at 4°C, discard the supernatant, and collect the RNA pellet. After washing the precipitate with pre-cooled 75% ethanol (v/v), it was dried in an electric heating oven at 65°C to remove the remaining liquid. Add DEPC water to form an RNA solution and vortex to mix. Use a spectrophotometer to measure RNA concentration and purity, and store at -80°C.

cDNA synthesis: Use HiFiScript rapid de-genomic cDNA first-strand synthesis kit to reverse-transcribe and synthesize cDNA. Thaw the extracted RNA on ice, prepare a 20μL system of 1-5μg RNA, 4μL 5×PrimeSrip RTmix and RNase-free water, place it in a labeled centrifuge tube, centrifuge briefly and perform reverse transcription to synthesize cDNA in a PCR machine. The conditions were incubation at 37°C for 15 min and 85°C for 5 s, and the obtained cDNA was stored at -80°C.

Real-time fluorescence quantitative PCR: Thaw cDNA on ice, mix 2 μL of cDNA, 1 μL of forward and reverse primers, 10 μL of 2×iTaqSYBR Greensupermix, and 6 μL of DEPC water to make a 20 μL system. Set 3 secondary wells for each primer of each group of samples. Put the sample on a 96-well plate dedicated for QPCR, seal it with a special sealing template for QPCR, and perform the reaction on a QuantStudio 5 PCR machine. The conditions are pre-denaturation 95°C for 30s, denaturation 95°C 10s—annealing 58°C 30s—extension 72°C 32s, a total of 40 cycle. Subtract the Ct value of the internal reference 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 .

IL-6：Forward:5′-CAGACCCACATGCTCCGAGA-3′

Reverse:5′-CAAGGCTTGGCAACCCAAGTA-3′

CCL-2：Forward:5’-TAGCATCCACGTGCTGTCTC-3’

Reverse:5’-CAGCCGACTCATTGGGATCA-3’

IL-1β: Forward:5’-AGAGGTGTGGATCCCAAACAA-3’

Reverse:5’-AGTCAACTATGTCCCGACCA-3’

TNF-α: Forward:5’-TGATCGGTCCCAACAAGGA-3’

Reverse:5’-TGCTTGGTGGTTTGCTACGA-3’

5. Western Blot detects changes in protein expression levels.

Tissue collection: Rats were anesthetized with sodium pentobarbital and sacrificed. The spinal cord was quickly removed and immediately frozen on ice. The spinal cord from the lumbar segment L3-L5 was cut out, quickly frozen in liquid nitrogen, and stored at -80°C for later use.

Protein extraction and concentration determination: Take an appropriate amount of tissue and place it in a 1.5 mL centrifuge tube. Add PMSF-containing lysis solution that is about 10 times the volume of the tissue. Pre-cool the homogenizer on ice. Use an electric homogenizer to homogenize 5 s × 3 times with an interval of 30 s. , until the tissue is completely broken, place the evenly polished tissue on ice for 30 minutes, centrifuge at 12,000g for 15 minutes at 4°C and take the supernatant.

Concentration determination: Collect the supernatant and detect the protein content using the Beyotime BCA protein concentration quantification method. The specific method is as follows. Take a 96-well enzyme plate and prepare the bovine serum albumin (1 mg/mL BSA) standard into a concentration gradient of 0, 0.025, 0.125, 0.25, 0.5, 0.75, 1, 1.5, and 2 mg/mL. Add 4 μl of the prepared concentration gradient standard and the sample for measuring total protein to the 96-well plate in sequence, and make up to 20 μl with the standard diluent for marking. Prepare an appropriate amount of BCA working solution according to the volume ratio of BCA reagent A:B 50:1, then add 100μl BCA working solution into the enzyme label well where the standard and sample have been added, shake for 5 minutes and mix thoroughly, then place it in a 37°C incubator Incubate for 30 minutes, and measure the absorbance at a wavelength of 562 nm using a microplate reader. Using the abscissa as the protein concentration of the standard and the ordinate as the absorbance, draw a standard curve. Calculate the protein concentration of the sample based on the standard curve and the sample volume used. Use lysis buffer to prepare the sample into equal volumes and equal concentrations.

Sample processing: Add 1/3 volume of 4×Loading Buffer and 1/40 of β-mercaptoethanol to the immunoblot protein sample, mix well, and heat at 100°C for 10 minutes to completely denature the protein. After processing, the samples were aliquoted and stored at -80°C.

Western blot analysis: After equal amounts of protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to polyvinylidene fluoride (PVDF) membrane by wet method, 250mA 90min, 5% skimmed milk powder (m/v) Room temperature shaker was blocked for 1 hour, and anti-ERK1/2 (1:1000, CST), anti-p-ERK1/2 (1:1000, CST), anti-GAPDH (1:10000) were used respectively. Anti-p38 (1:1000), anti-p-p38 (1:1000), anti-β-actin (1:10000) (v/v) were incubated overnight at 4°C, washed 3 times with 1×TBST, 6 min each time , and 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, 6 minutes each time, and added ECL luminescent solution dropwise to the strip to detect the protein band signal with a Western-blot instrument. The resulting bands were analyzed using Image-J for grayscale density.

6. MDA kit detection

Beyotime's Lipid Peroxidation MDA Assay Kit uses a color reaction based on the reaction of MDA and thiobarbituric acid (TBA) to produce a red product. The absorption of this product The peak value is 532nm. Colorimetric methods are then used for the quantitative detection of MDA in plasma, serum, urine, animal and plant tissues or cell lysates. MDA is calculated using a standard curve according to the manufacturer's data sheet and is widely used in lipid oxidation (lipid). peroxidation) level detection kit.

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

Product packaging list: TBA 25mg, TBA diluent 15mL, TBA preparation solution 6.76mL, antioxidant 300μl, standard (1mM malondialdehyde) 200μl.

Sample preparation: Take an appropriate amount of tissue and use lysis solution for homogenization or lysis. The proportion of tissue weight in the homogenate or lysis solution is 10%. After homogenization or lysis, centrifuge at 10,000g–12,000g for 10 minutes and take the supernatant for subsequent use. Sample preparation steps such as assay, homogenization or lysis are performed in an ice bath or at 4°C.

Preparation of TBA storage solution: Weigh an appropriate amount of TBA and use TBA preparation solution to prepare a TBA storage solution (m/v) with a concentration of 0.37%. The TBA preparation solution must be completely dissolved before use. It can be heated to 70°C to promote dissolution.

Dilution of the standard: Take an appropriate amount of the standard and dilute it with distilled water to 1, 2, 5, 10, 20, 50, 100, 150, 200 μM for subsequent preparation of the standard curve.

Sample determination:

a. The reaction system is shown in Table 1.

Table 1. Reaction system

b. After mixing, heat at 100℃ or boiling water bath for 15 minutes. When heating, be sure to avoid boiling and splashing of liquid.

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

d. Calculation of MDA content: Calculate the MDA content in the sample solution based on the standard curve.

7. Cell activity detection and model construction

Cell culture: Place BV-2 cells in a constant temperature incubator containing 5% CO ₂ (v/v) and 37°C, using RPMI-1640 medium, supplemented with 10% PAN fetal bovine serum (v/v) Culture with 1% penicillin-streptomycin antibiotic (v/v). When cells are passaged, discard the culture medium first, rinse with PBS, then add trypsin for digestion for 20-25 seconds, and then add 2 mL of prepared culture medium to terminate digestion. , pipette the cells at the bottom of the culture dish until all the cells fall off. After pipetting evenly, divide the cells into the prepared culture dish at a ratio of 1:2 or 1:3. Add culture medium to make the liquid volume 5mL. Use a pipette to mix the cells. Can be put back into the incubator to continue culturing.

Cell viability test: Cells were subcultured in a 96-well plate at a density of 2.0×10 ⁴ per well. After the cells adhered, drugs prepared with DMSO were added. The concentration of the mother solution was 10mM, and diluted with culture medium to 0, 2.5, 5, and 10 , 15, 20 μM, set 5 replicates for each concentration. After incubation for 24 hours, add serum-free medium (v/v) containing 10% CCK8. After incubation for 1-2 hours, detect the OD value at a wavelength of 450 nm. Based on the OD value Calculate cell viability.

Oxidative stress model construction: Cells were subcultured in a 96-well plate at a density of 2.0×10 ⁴ cells per well, and were tested after being co-treated with 250 μM and 300 μM hydrogen peroxide and 0, 0.15, 0.3, 0.625, 1.25, 2.5, and 5 μM drugs for 24 hours. Cell viability to evaluate the antioxidant effect of the drug Met.

Experimental results:

1. Effect of intraperitoneal injection of Met on formalin-induced pain behavior in mice

Figure 2 shows the effect of intraperitoneal injection of 20 mg/kg Met or saline on formalin-induced pain behavior in wild-type mice. A: After intraperitoneal injection of 20 mg/kg Met or normal saline (NC) for 30 min, 20 μl of 2% formalin (v/v) was injected into the plantar surface of the right hind paw of WT mice, and then measured every 5 min for 60 min. Formalin-induced pain behaviors include lifting, paw withdrawal, and paw licking. Data are expressed as mean±SEM (n=6). B: In two groups of mice, the total pain score was divided into the first stage (0-15min) and the second stage (15-60min). Data are expressed as mean±SEM, *P<0.05, (n=6). Experimental results show that intraperitoneal injection of 2,2’-methylenebis can significantly alleviate formalin-induced pain behavior in mice. The pain behavior time caused by inflammation in mice decreased significantly in phase II, indicating that 2,2’-methylene bis has the ability to relieve inflammatory pain.

2. Pain hypersensitivity after chronic contraction of sciatic nerve in rats

Figure 3 shows the changes and development of pain behavior after chronic injury of the sciatic nerve in rats. After CCI, compared with the sham operation group, the model group showed significantly lower mechanical withdrawal threshold (PWT) (A), pressure (B), and thermal withdrawal latency (TWL) (C) on the ipsilateral paw. Measurements were performed before surgery (D0) and on days 3, 5, 7, and 14 after CCI or sham surgery. Data shows average

±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). The experimental results showed that after CCI, the PWT and PWL thresholds of rats were significantly reduced and stabilized after the 7th day, which was consistent with literature reports, indicating that the CCI model was successfully constructed.

3. Effects of intraperitoneal injection of Met on the pain behavior of mice with chronic sciatic nerve injury

Figure 4 shows the effect of intraperitoneal injection of 20 mg/kg 2,2′-methylenebis on pain behavior in mice with chronic sciatic nerve injury. On the 14th day after surgery, the changes in pain behavior of mice within 2 hours were measured after intraperitoneal injection of 20 mg/kg 2,2′-methylenebis. Compared with the solvent group, the administration group showed the mechanical withdrawal threshold (PWT) of the ipsilateral paw (A ) and thermal withdrawal latency (PWL) (B) were significantly increased. Data are expressed as average

±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). Experimental results show that a single intraperitoneal injection of 2,2'-methylene bis can significantly alleviate the pain behavior of mice induced by CCI surgery within 2 hours, indicating that 2,2'-methylene bis has the ability to alleviate neuropathy caused by mechanical injury. The ability to rationalize pain.

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

Figure 5 shows the effect of intraperitoneal injection of 20 mg/kg 2,2′-methylenebis on pain behavior in rats with chronic sciatic nerve injury. On the 14th day after surgery, the changes in pain behavior of rats within 2 hours were measured after intraperitoneal injection of 20 mg/kg 2,2′-methylenebis. Compared with the solvent group, the administration group showed the mechanical withdrawal threshold (PWT) of the ipsilateral paw (A ) Pressure (B) and thermal withdrawal latency (PWL) (C) were significantly increased, followed by continuous administration for 7 days. The changes in the pain behavior of rats were measured before each administration. Compared with the solvent group, the performance of the administration group The mechanical withdrawal threshold (PWT) (D) pressure (E) and thermal withdrawal latency (PWL) (F) of the ipsilateral paw were significantly increased. Data are expressed as average

±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). Experimental results show that a single intraperitoneal injection of 2,2'-methylene bis can significantly alleviate the pain behavior of rats induced by CCI surgery within 2 hours; during long-term continuous administration, 2,2'-methylene bis Both can also effectively relieve pain behavior in rats. This shows that 2,2’-methylene bis has the ability to relieve neuropathic pain caused by mechanical injury in both short and long periods of time.

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

Figure 6 shows the effect 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 were increased in the spinal cord of rats with chronic sciatic nerve injury. (A): p-ERK/ERK; (B): p-P38/P38. After drug treatment, Met Can significantly inhibit the activation of ERK1/2 and P38. Data are expressed as average

±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). The MAPK signaling pathway is one of the important pathways for intracellular and extracellular signal transduction in the body. It can be stimulated by a variety of extracellular signals such as cytokines, neurotransmitters, hormones, and cellular stress, and participates in the regulation of gene expression and cytoplasmic functional activities. Experimental results show that 2,2'-methylene bis can inhibit the phosphorylation of ERK and P38 proteins in the MAPK signaling pathway caused by CCI, thereby affecting the expression of downstream inflammation and antioxidant-related genes, and alleviating the neuroinflammatory response and inflammation caused by CCI. Oxidative stress on nerves, thereby relieving pain.

6. Effect of Met on the expression levels of inflammatory factors in the spinal cord of rats with chronic sciatic nerve injury

Figure 7 shows the effect of intraperitoneal injection of 20 mg/kg 2,2′-methylenebis on the expression levels of inflammatory factor mRNA 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, the mRNA expression levels of a series of inflammatory factors are upregulated after chronic injury of the sciatic nerve. After drug treatment, Met can significantly inhibit the up-regulation of the expression of inflammatory factors. Data are expressed as average

±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=6). Experimental results show that 2,2’-methylene bis can inhibit the neuroinflammatory response caused by CCI and inhibit the expression of inflammatory factors in neural tissue.

7. Effect of Met on the expression level of MDA in the spinal cord of rats with chronic sciatic nerve injury

Figure 8 shows the 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 increased significantly, and intraperitoneal injection of 20 mg/kg 2,2'-methylenebis could inhibit the up-regulation of MDA expression level. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=3). Experimental results show that MDA levels in the spinal cord are significantly increased after CCI, the degree of neurolipid peroxidation increases, and oxidative stress occurs. 2,2’-Methylenebis can significantly reduce MDA levels in the spinal cord, indicating that 2,2’-methylenebis can significantly inhibit neurolipid peroxidation and relieve oxidative stress in peripheral nerves.

8. The effect of Met on the survival rate of mouse microglioma cells (BV-2) in inflammation and oxidative stress models

Figure 9 shows the effect of Met on the survival rate of mouse microglioma cells (BV-2) in inflammation and oxidative stress models. Met was prepared with DMSO into a stock solution with a concentration of 10mM. After drug treatment for 24 hours, CCK-8 was used to determine the cell survival rate. Five secondary wells (n=5) were set for each treatment. (A): The effect of Met on cell survival rate; (B): The effect of Met on cell survival rate after applying 1 μg/mL LPS;

(C): Effect of Met on cell viability after applying 250 μM H ₂ O ₂ . Data are expressed as average

±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=5). LPS and hydrogen peroxide were used to induce BV-2 cells to construct an in vitro model of inflammation and oxidative stress. Experimental results show that 2,2'-methylene bis has no effect on cell survival in the concentration range of 0-5 μM and is not cytotoxic; in the concentration range of 0-5 μM, 2,2'-methylene bis cannot Inhibits cell proliferation caused by LPS-induced microglial activation, but cannot inhibit LPS-induced inflammatory response; 2,2'-methylene bis can significantly alleviate cellular oxidative stress caused by hydrogen peroxide in the concentration range of 0-5μM The resulting cell death can inhibit the oxidative stress of microglia in vitro and possess antioxidant capabilities. In vitro cell experiments have shown that 2,2’-methylene bis can relieve neuropathic pain by inhibiting oxidative stress in microglia.

Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, they do not limit the scope of the present invention. On the basis of the technical solutions of the present invention, those skilled in the art can make various modifications without any creative work. Modifications or variations are still within the scope of the invention.

Claims (2)

1. Application of 1.2,2' -methylenebis in preparing analgesic.
2. 2. An analgesic drug is characterized in that the effective component is 2,2' -methylenebis.