Application of isotoosendanin in preparation of medicine for relieving neuropathic pain and application of TRPV1 and Nav1.7 as medicine targets
By using isonasalin to inhibit the expression of TRPV1 and Nav1.7 channel proteins, a drug for relieving neuropathic pain was prepared, which solved the problem of poor drug efficacy in the prior art and achieved effective relief of neuropathic pain and improved safety.
Patent Information
- Application Number
- CN202511496259.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-08-14
- Filing Date
- 2025-10-20
- Publication Date
- 2025-11-21
AI Technical Summary
Current technologies lack effective drugs with minimal side effects for relieving neuropathic pain, and the lack of clear drug targets leads to poor treatment outcomes.
Using isonasalin as the active ingredient, a drug to relieve neuropathic pain was prepared by inhibiting the expression of TRPV1 and Nav1.7 channel proteins. TRPV1 and Nav1.7 were used as drug targets for targeted drug design.
Isochondrial can significantly inhibit the reduction of mechanical and cold pain thresholds, reduce neuropathic pain, and has no toxicity to the liver and kidneys. It provides more precise pain signal intervention, improving treatment efficacy and safety.
Smart Images
Figure CN120983447A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, in particular to the use of isoxanthohumol in the preparation of a drug for relieving neuropathic pain and the use of TRPV1 and Nav1.7 as drug targets. BACKGROUND
[0002] Xanthium sibiricum is one of the main components of the Jinlingzi San prescription, which has a certain analgesic effect on abdominal pain and dysmenorrhea. In addition, xanthium sibiricum combined with orange peel decocted can be used to treat intercostal neuralgia. Xanthohumol is the main active ingredient of xanthium sibiricum, which has the effects of anti-inflammatory and anti-tumor. However, its severe hepatotoxicity is currently mainly studied in the field of anti-tumor. Recent studies have shown that the oral drug safety of isoxanthohumol, a chemical isomer of xanthohumol, is significantly higher than that of xanthohumol, and its hepatotoxicity within a certain dose is lower than that of xanthohumol.
[0003] Among them, the ion channel target is TRPV1 and SCN9A (gene encoding Nav1.7), and isoxanthohumol has a binding effect with TRPV1 and SCN9A. The expression change, excessive activation or dysfunction of cation channels on neurons and peripheral tissues is the basis for the excessive sensitivity of neurons, abnormal neurotransmitters and changes in nerve electrophysiology to become the process of pain transmission, perception and amplification. Therefore, by inhibiting the excessive expression of ion channels, regulating the electrophysiological activity of neurons and reducing the sensitivity of neurons to stimulation, it plays an important role in relieving neuropathic pain. Therefore, TRPV1 and Nav1.7 are used as drug targets for the preparation of a drug for relieving neuropathic pain with isoxanthohumol as the effective component. It is necessary to study the use of isoxanthohumol in the preparation of a drug for relieving neuropathic pain and the use of TRPV1 and Nav1.7 as drug targets. SUMMARY
[0004] Therefore, the present application aims at the defects in the prior art, and provides the use of isoxanthohumol in the preparation of a drug for relieving neuropathic pain and the use of TRPV1 and Nav1.7 as drug targets. Isoxanthohumol can relieve neuropathic pain, and can inhibit the expression of TRPV1 and Nav1.7. The effective component is found from traditional Chinese herbal medicine with analgesic effect, and the relationship between the expression of TRPV1 and Nav1.7 and neuropathic pain is verified. Isoxanthohumol can treat neuropathic pain by inhibiting the expression of TRPV1 and Nav1.7 channel proteins, thereby providing a new idea for the preparation of a drug for neuropathic pain.
[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The use of isoxanthohumol in the preparation of a drug for relieving neuropathic pain.
[0006] As a preferred solution: the isoyanhydrolide is used for preparing a medicine for relieving neuropathic pain.
[0007] As a preferred solution: the medicine prepared from the isoyanhydrolide can inhibit the expression of TRPV1 and Nav1.7 proteins of dorsal root ganglion (DRG) neurons.
[0008] As a preferred solution: the medicine prepared from the isoyanhydrolide can inhibit the decrease of mechanical pain threshold (PWMT) and cold pain threshold (PWCD).
[0009] As a preferred solution: the medicine prepared from the isoyanhydrolide has no toxic effect on liver and kidney.
[0010] As a preferred solution: the neuropathic pain includes pain after selective injury of sciatic nerve branches.
[0011] As a preferred solution: the dosage form of the medicine prepared from the isoyanhydrolide includes tablets, granules, pills, powders, capsules and suspensions.
[0012] As a preferred solution: the administration mode of the medicine prepared from the isoyanhydrolide includes oral administration and injection.
[0013] TRPV1 and Nav1.7 as a drug target are applied to the preparation of the medicine for relieving neuropathic pain.
[0014] As a preferred solution: TRPV1 and Nav1.7 as a drug target are applied to the preparation of the medicine for relieving neuropathic pain with isoyanhydrolide as an effective component.
[0015] Compared with the prior art, the present application has obvious advantages and beneficial effects. Specifically, from the above technical solution, it can be known that isorubins plays a role in relieving neuropathic pain, and isorubins can inhibit the expression of TRPV1 and Nav1.7; the relationship between the expression of TRPV1 and Nav1.7 and neuropathic pain is verified by searching for effective components from traditional Chinese herbal medicines with analgesic effect; isorubins can treat neuropathic pain by inhibiting the expression of TRPV1 and Nav1.7 channel proteins, thereby providing a new idea for preparing neuropathic pain drugs; as a natural component, isorubins has lower side effects and less burden on the patient's body, thereby reducing the risk of other health problems caused by drug treatment; as drug targets, TRPV1 and Nav1.7 provide a clear direction for drug research and development, targeted drug design is carried out, and the development process of new anti-neuropathic pain drugs is accelerated; TRPV1 and Nav1.7 play a key role in the occurrence and development of neuropathic pain, isorubins takes these two targets as the action sites, can more accurately intervene in the conduction and processing of pain signals, and compared with some non-specific analgesic drugs, it can more effectively block the transmission of pain signals, thereby more significantly relieving neuropathic pain and improving the specificity and effectiveness of drug treatment.
[0016] To make the structural features and effects of the present application clearer, the following will describe them in detail in combination with the drawings and specific examples. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 It is an experimental schematic diagram and a schematic diagram of pain threshold changes of rats in each group in the embodiment of the present application; Figure 2 It is a schematic diagram of pain threshold changes of SNI model rats in the embodiment of the present application; Figure 3 It is a schematic diagram of changes of TRPV1 and Nav1.7 proteins of DRG neurons of SNI rats in the embodiment of the present application; Figure 4 It is a schematic diagram of changes of TRPV1 and Nav1.7 mRNA of DRG neurons of SNI rats in the embodiment of the present application; Figure 5 It is an experimental schematic diagram and a schematic diagram of pain threshold changes of rats in each group in the embodiment of the present application; Figure 6 It is a schematic diagram of effects of isorubins on TRPV1 and Nav1.7 proteins of DRG neurons of SNI rats in the embodiment of the present application; Figure 7 It is a schematic diagram of effects of isorubins on the content of TRPV1 and Nav1.7 mRNA of DRG neurons of SNI rats in the embodiment of the present application; Figure 8 Figure 1 shows the changes of liver and kidney function indicators in the embodiment of the present application. DETAILED DESCRIPTION
[0018] The present application is as follows Figure 1 As shown in Figure 8: the use of isochewanlactone in the preparation of a drug for relieving neuropathic pain and its TRPV1 and Nav1.7 as drug targets.
[0019] The use of isochewanlactone in the preparation of a drug for relieving neuropathic pain.
[0020] The isochewanlactone is used for preparing a drug for relieving neuropathic pain.
[0021] The drug prepared from the isochewanlactone can inhibit the expression of TRPV1 and Nav1.7 proteins of dorsal root ganglion (DRG) neurons.
[0022] The drug prepared from the isochewanlactone can inhibit the decrease of mechanical pain threshold (PWMT) and cold pain threshold (PWCD).
[0023] The drug prepared from the isochewanlactone does not produce toxic effects on the liver and kidney.
[0024] The neuropathic pain includes pain after selective injury of sciatic nerve branches.
[0025] The dosage form of the drug prepared from the isochewanlactone includes tablets, granules, pills, powders, capsules and suspensions.
[0026] The administration mode of the drug prepared from the isochewanlactone includes oral administration and injection.
[0027] TRPV1 and Nav1.7 as drug targets are applied in the preparation of a drug for relieving neuropathic pain.
[0028] TRPV1 and Nav1.7 as drug targets are applied in the preparation of a drug for relieving neuropathic pain.
[0029] Embodiment: the use of isochewanlactone in the preparation of a drug for relieving neuropathic pain and its TRPV1 and Nav1.7 as drug targets Adult male Sprague-Dawley rats, 8 weeks old, weighing 180-220 g, were used. The animals required for the experiment were raised in a clean level experimental environment. The laboratory and animal living place maintained a temperature of 18-22℃, a relative humidity of 40%-85%, 12 hours light / 12 hours dark, good ventilation conditions, and free access to food and water, with bedding changed once a week. Before the experiment, the rats were ensured to adapt to the surrounding environment to avoid their being affected by external environmental factors; all animal experiments were performed in accordance with the International Association for the Study of Pain Research Ethical Guidelines.
[0030] Preparation of experimental model (1) Establishment of spared nerve injury (SNI) animal model: One day before the experiment, the body weight of rats in each group was measured and recorded. On the day of modeling, the laboratory temperature was controlled at 22±2℃, and the rats were anesthetized by inhaling isoflurane (4% concentration). After anesthesia, the rats in the experimental group were placed on the mouse plate, and the limbs were fixed with silk thread. Isoflurane (2.5% concentration) was continuously inhaled for anesthesia. The skin was exposed by shaving the surgical site and disinfecting with iodophor. The skin was incised parallel to the thigh at the posterolateral side of the left hind leg near the posterior superior iliac spine. Then the muscle was bluntly dissociated, and the sciatic nerve was fully exposed and separated into three branches: tibial nerve, common peroneal nerve, and sural nerve. The tibial nerve and common peroneal nerve were ligated with 4-0 medical silk knitting thread, tied tightly, and cut at the distal end of the ligation, leaving a distal stump of about 2-4 mm. The sural nerve was left intact to avoid any damage. Then the muscle and skin were sutured layer by layer. Before suturing, the wound was flushed with normal saline. The animals were raised separately in a single cage for the experiment and set as the Control group.
[0031] (2) Sham operation animal model: One day before the experiment, the body weight of rats in each group was measured and recorded. On the day of modeling, the laboratory temperature was controlled at 22±2℃, and the rats were anesthetized by inhaling isoflurane (4% concentration). After anesthesia, the rats in the experimental group were placed on the mouse plate, and the limbs were fixed with silk thread. Isoflurane (2.5% concentration) was continuously inhaled for anesthesia. The skin was exposed by shaving the surgical site and disinfecting with iodophor. The skin was incised parallel to the thigh at the posterolateral side of the left hind leg near the posterior superior iliac spine. Then the muscle was bluntly dissociated, and the sciatic nerve was fully exposed and separated into three branches: tibial nerve, common peroneal nerve, and sural nerve. The sural nerve was left intact to avoid any damage. Then the muscle and skin were sutured layer by layer. Before suturing, the wound was flushed with normal saline. The animals were raised separately in a single cage for the experiment and set as the Sham group.
[0032] (3) SNI+DMSO group: On the basis of the SNI model, 10% DMSO+90% Corn oil of the same volume as the drug was administered daily by gavage from the 8th day of SNI modeling.
[0033] (4) SNI + different concentrations of I TSN group: On the basis of the SNI model, the different groups were given intraperitoneal injection of I TSN (ITSN was dissolved in DMSO, and then the DMSO solution was dissolved in Corn oil, and the final amount of DMSO was 10%) (1 time / day) from the 8th day after the SNI modeling.
[0034] Gavage The rats in the SNI + I TSN 5 mg / kg group, the SNI + I TSN 10 mg / kg group, and the SNI + I TSN 20 mg / kg group were orally gavaged with I TSN solution, 1 ml per rat, once a day from the 8th to the 21st day after the operation. Before gavage, I TSN was dissolved in DMSO, and then the DMSO solution was dissolved in Corn oil (the final amount of DMSO was 10%) to prepare the I TSN solution. At the same time, the SNI + DMSO group was orally gavaged with the same proportion and volume of DMSO solution.
[0035] Mechanical pain threshold (PWMT) determination Each group was determined by mechanical stimulation with Von Frey fibers on the 1st day before the operation and the 1st, 7th, 14th, and 21st days after the operation. Each test was performed between 10:00 and 12:00, and the environmental temperature was controlled at 22-25°C. At the beginning of the behavioral test, the room was kept as quiet as possible, and the SD rats were placed in a transparent plastic box on a high metal grid (the adjacent two boxes were opaque plastic plates), and the cover was covered after the rats were calm and had no exploration and grooming behavior. A series of standardized Von Frey fibers (2.0, 4.0, 6.0, 8.0, 10.0, and 15.0 g) were used to stimulate the left hind foot of the rat through the slit of the bottom metal grid, and the fiber tip was placed in the center of the hind foot. The fiber was kept bent at a low force for 6-8 s. The initial stimulation intensity was 2.0 g. Five consecutive stimulations were performed with at least 30 s between each stimulation. If a rapid withdrawal reflex or licking reaction occurred three times in the five stimulations, it was recorded as a positive "X", and the stimulation intensity was reduced by one level for the next test. Conversely, a reaction was recorded as "O", i.e., the stimulation intensity was increased by one level. If the stimulation intensity was >15.0 g or <2.0 g, it was recorded as 15.0 g or 2.0 g. The final stimulation intensity was recorded. When the response to the stimulation was different from the previous one, four more measurements were made according to the above sequence, for a total of six measurements, and the final stimulation intensity was recorded. A computer program was used to calculate the mechanical pain threshold of each group of rats.
[0036] Thermal pain threshold (PTWL) determination The rats were placed in a quiet experimental environment for 30 minutes on the first day before surgery and on the first, seventh, fourteenth, and twenty-first days after surgery. The rats were not allowed to perform other behaviors such as exploration before the experiment began. The cold-heat plate pain tester was set to 55 ± 0.1°C and was warmed. After the temperature was constant, the rats were placed in the transparent glass box of the cold-heat plate pain tester. When the hind foot contacted the metal plate of the tester, the timer started. When the rats exhibited the following behaviors (lifting the foot, licking the foot, or running away), the timer stopped. The time at which the foot was lifted was recorded as the thermal pain threshold of the rat. To prevent the rat's foot from being burned and affecting the experimental results, the upper limit time should be set to 30 seconds. Each rat was measured three times, with an interval of 15 to 20 minutes between each measurement, allowing for a sufficient buffer rest before the next experimental measurement. The average value was taken as the final thermal pain threshold.
[0037] Cold pain threshold (PWCD) determination The rats were placed in a quiet experimental environment for 30 minutes on the first day before surgery and on the first, seventh, fourteenth, and twenty-first days after surgery. The cold-heat plate pain tester was set to 55 ± 0.1°C and was warmed. After the temperature was constant, the rats were placed in the transparent glass box of the cold-heat plate pain tester. When the hind foot contacted the metal plate of the tester, the timer started. When the rats exhibited the following behaviors (lifting the foot, licking the foot, or running away), the timer stopped. The time at which the foot was lifted was recorded as the thermal pain threshold of the rat. To prevent the rat's foot from being burned and affecting the experimental results, the upper limit time should be set to 30 seconds. Each rat was measured three times, with an interval of 15 to 20 minutes between each measurement, allowing for a sufficient buffer rest before the next experimental measurement. The average value was taken as the final thermal pain threshold.
[0038] DRG processing in Western blot and RT-qPCR experiments Isoflurane was used to induce deep anesthesia in rats, and immediately after the rats were decapitated, the chest was opened to expose the heart, which was perfused with 0.9% saline until the perfused liquid was colorless. The skin and deep fascia were cut, and about 4 cm of muscle was separated longitudinally on the left side of the spine (the modeling side) centered on the posterior superior iliac spine. The sciatic nerve in the muscle layer was exposed, and its L4, L5, and L6 branches were traced back to the lateral side of the intervertebral foramen. The surrounding residual muscle and tissue were carefully removed with forceps, and the lamina was carefully cut to expose the DRG tissue. The DRG was carefully clamped and the nerve root was cut with ophthalmic scissors. The dorsal root ganglion was removed and placed in a clean pre-cooled saline tray. The excess nerve fibers and tissue attached to the dorsal root ganglion were carefully trimmed with ophthalmic forceps and ophthalmic scissors, and finally placed in an EP tube. After being stored in liquid nitrogen overnight, it was stored in a -80°C refrigerator.
[0039] Western blot assay of TRPV1 and Nav1.7 protein expression in DRG DRG protein extraction: (1) Liquid preparation: add 10 μL PMSF (100 mM) and 10 μL phosphatase inhibitor to 1 ml lysis solution, shake well and place on ice.
[0040] (2) DRG lysis: Take DRG from -80°C refrigerator, weigh and place in an enzyme-free EP tube. Place the EP tube in an ice box. Add 8 μL lysis solution per 1 mg of tissue, carefully cut into homogenate with ophthalmic scissors, and place the EP tube on ice for 30 min for complete lysis.
[0041] Centrifugation: After lysis for 30 min, use a pipette to transfer the lysis solution to a 1.5 ml centrifuge tube, then centrifuge at 12000 rpm for 5 min at 4°C. Take the supernatant and divide it into 0.5 ml centrifuge tubes and store at -20°C.
[0042] Operation procedure: (1) Electrophoresis and electrotransfer: Fix the precast gel to the electrophoresis tank, and pour the electrophoresis solution into the reservoir. Use a micropipette to add the prepared protein sample and marker to the loading well, with a total protein amount of 30 μg. Electrophorese at a voltage of 80 V for 45 min; when the protein mixed with the loading buffer or bromophenol blue reaches the boundary between the concentrated gel and the separation gel, change to a voltage of 120 V for electrophoresis until the end of electrophoresis. Remove the glass plate on the electrophoresis frame, cut the gel, and only keep the gel in the marker and protein channels. Soak the PVDF membrane in methanol for 5 min, and let the membrane naturally bend to complete activation. Cover the PVDF membrane onto the gel, gently squeeze out the air bubbles with the gel cutting plate, and keep the membrane moist with the electrotransfer solution. According to the order of black clamp + sponge + filter paper + gel + PVDF membrane + filter paper + sponge, clamp them together, place them in the electrotransfer tank, and fill the tank with the prepared electrotransfer solution. Connect the wires, and electrotransfer at a constant current of 200 mA for 90 min. Place the electrotransfer tank in an ice-water mixture.
[0043] (2) Blocking: Prepare the blocking solution in advance, and wash the electrotransferred PVDF membrane with TBST for 5 min, a total of 3 times. Place the PVDF membrane in the blocking solution on a shaker, and slowly shake for 1 h. Block for 1 h.
[0044] (3) Primary antibodies: Prepare the primary antibodies in advance: rabbit anti-TRPV1 (1:1000, PAB42552; Bioswamp), rabbit anti-Nav1.7 (1:1000; PAB58552; Bioswamp), and mouse anti-β-actin (1:20000; 66009-1-lg; proteintech). After blocking, pick up the PVDF membrane with tweezers, put the primary antibody into it, and discard the blocking solution. Gently shake on a shaker at 4℃ for 14 hours.
[0045] (4) Secondary antibodies: Secondary antibodies were prepared in advance: goat anti-rabbit (1:20000; BL003A; Biosharp) and goat anti-mouse (1:20000; SAB43714; Bioswamp). After the primary antibody incubation, the PVDF membrane was washed with TBST for 10 min, a total of 3 times. The membranes were then placed in the secondary antibody solution and incubated on a shaker at room temperature for 2 h with gentle shaking.
[0046] (5) Exposure: Prepare the luminescent reagent in advance, and prepare it according to the ratio of solution A to solution B = 1:1 according to the required amount of PVDF film to be exposed. After incubation, wash the PVDF film with TBST for 10 minutes, and wash 3 times. Use a pipette to evenly drop the prepared luminescent reagent onto the PVDF film, and expose it automatically using a chemiluminescence analyzer.
[0047] Real-time quantitative PCR Rats were treated as described above. Left L4-L6 DRG tissue was removed and placed into EP tubes according to different components. DRG tissue was lysed, total RNA was extracted, and after detecting RNA concentration, the total RNA was reverse transcribed into cDNA using a reverse transcription kit. Amplification was then performed using a SYBR Green I real-time PCR kit. -ΔΔCt The method was used to assess the relative mRNA expression levels of TRPV1 and Nav1.7.
[0048] Liver and kidney function tests Blood was collected from the tail vein of rats on day 21 post-surgery. After collection, the blood was incubated at 4°C for 3 hours and then centrifuged at 12,000 rpm for 10 minutes. The supernatant serum was collected, and liver and kidney function indicators were measured using ALT, AST, BUN, and CRE kits. The specific procedures were performed according to the kit instructions.
[0049] Results Analysis DMSO had no significant effect on pain behavior in SNI model rats. Experimental diagram as follows Figure 1 As shown in Figure a, where Figure 1 In the figure, 'a' represents the time points for modeling, treatment, and behavioral measurement in the Control group, Sham group, SNI group, and SNI+DMSO group, as well as the time points for molecular experiments in the SNI group.Figure 1 Figure 16. PMWT, PWCD and PTWL thresholds of rats in Control, Sham, SNI and SNI+DMSO groups on different days. b-d, PMWT, PWCD and PTWL thresholds of rats in Control, Sham, SNI and SNI+DMSO groups on different days (n=6). *** P <0.001, SNI vs. Control; ### P <0.001, SNI+DMSO vs. Control; ns P >0.05. Figure 1 Figure 16. PMWT, PWCD and PTWL thresholds of rats in Control, Sham, SNI and SNI+DMSO groups on different days. e-g, PMWT, PWCD and PTWL thresholds of rats in Control, Sham, SNI and SNI+DMSO groups on day 21 (n=6, *** P <0.001, ns P >0.05. It was calculated that there was no statistical difference in PMWT, PWCD and PTWL of SD rats on day 1 before operation (n=6, P >0.05). There was no statistical difference in PMWT, PWCD and PTWL of rats in Control and Sham groups (n=6, P >0.05). Compared with Control group, PMWT and PWCD of rats in SNI group were significantly decreased on day 1, 7, 14 and 21 after operation (n=6, P <0.001); PMWT and PWCD of rats in SNI+DMSO group were also significantly decreased on day 1, 7, 14 and 21 after operation (n=6, P <0.001) as shown in b-g of Figure 16. Figure 1 <0.001, SNI vs. Control; P >0.05).
[0050] PMWT and PWCD of SNI rats were decreased, and PTWL had no obvious change It was calculated that there was no statistical difference in PMWT, PWCD and PTWL of SD rats on day 1 before operation (n=6, P <0.001) compared with day 1 before operation; PWCD was also continuously decreased (n=6, P <0.001) compared with day 1 before operation; PWCD was also continuously decreased (n=6, P >0.05) compared with day 1 before operation; PWCD was also continuously decreased (n=6, P >0.05) compared with day 1 before operation; PWCD was also continuously decreased (n=6,P > 0.05). There was no statistical difference in PTWL in SNI rats within 21 d after operation (n = 6, P > 0.05), as shown in Figure 2 , where Figure 2 a-c are the threshold values of PMWT, PWCD and PTWL in SNI rats on different days. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns P > 0.05.
[0051] Increased expression of TRPV1 and Nav1.7 proteins in DRG neurons of SNI rats As shown by Western blot results: compared with the 1st day before operation, the expression of TRPV1 protein in DRG gradually increased on the 1st and 7th days after SNI operation (n = 3, all P < 0.0001), and the expression of Nav1.7 protein also gradually increased (n = 3, all P < 0.0001). The expression of TRPV1 and Nav1.7 tended to be stable from the 7th day, as shown in Figure 3 , where Figure 3 a is the expression of TRPV1 and Nav1.7 proteins on different days, and β-actin was used as an internal control. Figure 3 b-c are the quantitative analysis of TRPV1 and Nav1.7 expression levels. n = 3, **** P < 0.0001, ns P > 0.05.
[0052] Increased expression of TRPV1 and Nav1.7 mRNA in DRG neurons of SNI rats As shown by qPCR results: compared with the 1st day before operation, the expression of TRPV1 mRNA in DRG gradually increased on the 1st and 7th days after SNI operation (n = 3, all P < 0.0001), and the expression of Nav1.7 mRNA also gradually increased (n = 3, all P < 0.0001). The expression of TRPV1 and Nav1.7 tended to be stable from the 7th day, as shown in Figure 4 , where Figure 4 a is the quantitative analysis of TRPV1 mRNA expression level. Figure 4 b is the quantitative analysis of Nav1.7 mRNA expression level. n = 3, ****P <0.0001, ns P >0.05. Based on the above results, the changes on the 21st day after surgery were most significant, so the rats on the 21st day after surgery were selected as the research object in the follow-up experiment.
[0053] Inhibition of ITSN on the decrease of PMWT and PWCD in SNI rats The experimental scheme is shown in Figure 5 There was no statistical difference in PTWL among the rats in each group (n=6, P >0.05) as shown in Figure 5 There was no statistical difference in PMWT and PWCD among the rats in each group on the 1st day and the 7th day before surgery (n=6, P >0.05). On the 21st day after surgery, there was no significant difference in PMWT and PWCD between the SNI group and the SNI+DMSO group (n=6, P >0.05). Compared with the SNI group, the PMWT and PWCD of the SNI+ITSN 5mg / kg group, the SNI+ITSN 10mg / kg group and the SNI+ITSN 20mg / kg group were significantly increased (n=6, all P <0.001). Compared with the SNI+ITSN 5mg / kg group, the PMWT and PWCD of the SNI+ITSN 10mg / kg group and the SNI+ITSN 20mg / kg group were significantly increased (n=6, all P <0.001). There was no statistical difference in PMWT and PWCD between the SNI+ITSN 10mg / kg group and the SNI+ITSN 20mg / kg group (n=6, P >0.05) as shown in Figure 5 e-f. Figure 5 a is the time node of modeling, treatment, behavioral determination and molecular experiment of rats in each group. Figure 5 b-d are the PMWT, PWCD and PTWL thresholds of rats in each group on different days (n=6). Figure 5 e-g are the PMWT, PWCD and PTWL thresholds of rats in each group on the 21st day (n=6). *** P <0.001, ns P >0.05.
[0054] Inhibition of ITSN on the expression of TRPV1 and Nav1.7 in DRG neurons of SNI rats Western blot results showed that there was no significant difference in the expression of TRPV1 and Nav1.7 proteins in DRG neurons between the Control group and the Sham group (n=3, P >0.05); the same was true for the SNI group and the SNI+DMSO group (n=3, P >0.05). The expression of TRPV1 protein was reduced in the SNI+ITSN 5mg / kg group, the SNI+ITSN 10mg / kg group, and the SNI+ITSN 20mg / kg group compared with the SNI group (n=3, P <0.05, P <0.0001, P <0.0001), and the expression of Nav1.7 protein was also reduced (n=3, P <0.001, P <0.0001, P <0.0001); the expression of TRPV1 protein was reduced in the SNI+ITSN 20mg / kg group, the SNI+ITSN 10mg / kg group, and the SNI+ITSN 5mg / kg group compared with the SNI+ITSN 20mg / kg group (n=3, P <0.05), and the expression of Nav1.7 protein was also reduced (n=3, P <0.05); there was no significant difference in the expression of TRPV1 and Nav1.7 proteins between the SNI+ITSN 10mg / kg group and the SNI+ITSN 20mg / kg group (n=3, P >0.05), as shown in Table 1, Figure 6 wherein Figure 6 a is the expression of TRPV1 and Nav1.7 proteins in each group, and β-actin is used as an internal control. Figure 6 b-c is the quantitative analysis of the expression levels of TRPV1 and Nav1.7. n=3, * P <0.05, *** P <0.001, **** P <0.0001 and ns P >0.05.
[0055] qPCR results showed that there was no significant difference in the content of TRPV1 and Nav1.7 mRNA between the Control group and the Sham group (n=3, P >0.05); the same was true for the SNI group and the SNI+DMSO group (n=3, P>0.05). Compared with the SNI group, the TRPV1 mRNA levels were decreased in the SNI+ITSN 5 mg / kg group, SNI+ITSN 10 mg / kg group, and SNI+ITSN 20 mg / kg group (n=3, all...). P <0.0001), and the Nav1.7 mRNA content was also decreased (n=3, all). P <0.0001); Compared with the SNI+ITSN 20 mg / kg group, the SNI+ITSN 10 mg / kg group and the SNI+ITSN 5 mg / kg group all showed decreased TRPV1 mRNA levels (n=3, all <0.0001). P <0.0001), and the Nav1.7 mRNA content was also decreased (n=3, all). P <0.0001); There was no statistically significant difference in TRPV1 mRNA levels between the SNI+ITSN 10 mg / kg group and the SNI+ITSN 20 mg / kg group (n=3, P >0.05), Nav1.7 mRNA levels decreased (n=3, P <0.01), such as Figure 7 As shown, Figure 7 In Figure 'a', the expression level of TRPV1 mRNA in each group is quantitatively analyzed. Figure 7 Figure b shows the quantitative analysis of Nav1.7 mRNA expression levels in each group. n=3. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 and ns P >0.05.
[0056] Drug liver and kidney toxicity testing like Figure 8 As shown, Figure 8 In the figure, 'a' represents the serum AST level of rats in each group. Figure 8 b represents the serum ALT level of rats in each group. Figure 8 c represents the serum BUN level of rats in each group. Figure 8 d represents the serum CRE level in each group of rats. n=6, ns P >0.05. There were no significant differences in ALT, AST, BUN, and CRE levels between the control and drug groups (n=6, all <0.05). P >0.05), indicating that ITSN does not have toxic effects on the liver and kidneys.
[0057] The design of the present application focuses on the fact that yuchuanleitin can relieve neuropathic pain, and can inhibit the expression of TRPV1 and Nav1.7; the effective components are found from traditional Chinese medicine prescriptions with analgesic effect, and the relationship between the expression of TRPV1 and Nav1.7 and neuropathic pain is verified; yuchuanleitin can treat neuropathic pain by inhibiting the expression of TRPV1 and Nav1.7 channel proteins, providing a new idea for the preparation of neuropathic pain drugs; yuchuanleitin, as a natural component, has lower side effects and less burden on the patient's body, reducing the risk of other health problems caused by drug treatment; TRPV1 and Nav1.7 as drug targets provide a clear direction for drug research and development, targeted drug design, and accelerate the development process of new anti-neuropathic pain drugs; TRPV1 and Nav1.7 play a key role in the occurrence and development of neuropathic pain, and yuchuanleitin takes these two targets as the action site, can more accurately intervene in the conduction and processing of pain signals, compared with some non-specific analgesic drugs, it can more effectively block the transmission of pain signals, thereby more significantly relieving neuropathic pain and improving the specificity and effectiveness of drug treatment.
[0058] The above is only a preferred embodiment of the present application, and does not limit the technical scope of the present application in any way, so any minor modification, equivalent change and modification made according to the technical essence of the present application to the above embodiment are still within the scope of the technical solution of the present application.
Claims
1. The use of isonasalin in the preparation of drugs for relieving neuropathic pain.
2. The use according to claim 1, characterized in that: The isonasalin is used to prepare a drug for relieving neuropathic pain.
3. The use according to claim 1, characterized in that: The drug prepared from isonasalin can inhibit the expression of TRPV1 and Nav1.7 proteins in dorsal root ganglion (DRG) neurons.
4. The use according to claim 1, characterized in that: The drug prepared from isonasalin can inhibit the decrease of mechanical pain threshold (PWMT) and cold pain threshold (PWCD).
5. The use according to claim 1, characterized in that: The drug prepared from isonasalin does not produce toxic effects on the liver and kidneys.
6. The use according to claim 1, characterized in that: The neuropathic pain includes pain following selective injury to branches of the sciatic nerve.
7. The use according to claim 1, characterized in that: The dosage forms of drugs prepared from isonasalin include tablets, granules, pills, powders, capsules, and suspensions.
8. The use according to claim 1, characterized in that: The drug prepared from isonasalin can be administered orally or by injection.
9. The use of TRPV1 and Nav1.7 as drug targets in the preparation of the drug for relieving neuropathic pain as described in any one of claims 1-8.
10. The application according to claim 9, characterized in that: TRPV1 and Nav1.7 are used as drug targets in the preparation of a drug for relieving neuropathic pain with isohyzapine as the active ingredient.