Application of 3, 6-dihydroxyflavone in preparation of medicine for treating neuropathic pain

Abstract

The invention provides an application of 3, 6-dihydroxyflavone in preparation of a medicine for treating neuropathic pain. Experimental results show that the 3, 6-dihydroxyflavone can remarkably relieve hyperalgesia and hypersensitivity symptoms of mice; pathological injury changes of dorsal root ganglions and spinal dorsal horns are improved; the recovery of the sciatic nerve function has an analgesic effect on mice suffering from neuropathic pain induced by chronic compression injury; the 2, 3, 6-dihydroxyflavone can regulate the release of spinal cord IL-1beta through a microRNA-21 / A20 / NLRP3 signal channel, so as to achieve an analgesic effect on a mouse with neuropathic pain.

Description

Technical Field

[0001] This application belongs to the field of medicine. Specifically, this application provides the use of 3,6-dihydroxyflavone in the preparation of drugs for treating nervous system diseases. Background Art

[0002] Neuropathic pain (NP) is caused by damage or diseases of the somatic sensory nervous system and is one of the common chronic pains clinically, mainly including spontaneous pain, hyperalgesia, allodynia, and paresthesia, which cause sleep disorders, anxiety, and depression in patients and seriously affect the quality of life of patients. According to epidemiological statistics, the prevalence of NP in the global general population is as high as 10%, and there are approximately 90 million NP patients in China. With the aging of the population, the improvement of cancer survival rate will continue to increase the prevalence of neuropathic pain. Drugs are currently the main means of clinical treatment of NP. In China, for the treatment of NP, mainly based on the NP treatment guidelines issued by the International Association for the Study of Pain (IASP), first-line drugs represented by pregabalin, nortriptyline, and venlafaxine and second- and third-line drugs represented by capsaicin and opioid drugs are used to treat NP patients. However, currently, the first-line treatment drugs can only relieve the pain symptoms of 40% of the patients, and the second- and third-line drugs often have serious adverse reactions, which limit the clinical application of these drugs. This makes it an urgent problem in the medical field to find safe and effective new drugs for anti-NP.

[0003] Neuroinflammation is considered to be a key factor in the development of neuropathic pain. During the process of neuropathic pain, IL-1β acting on the spinal dorsal horn (SDH) can cause neuroinflammation, further increasing the synaptic connection and the strength of excitatory synaptic transmission.

[0004] miRNA participates in the occurrence of neuroinflammation by regulating the expression of inflammatory factors. miRNA mainly acts by binding to its target gene, resulting in mRNA degradation or translation inhibition. Silencing or inhibition of miR-21 in the dorsal root ganglion / sciatic nerve can relieve pain hypersensitivity. Nuclear factor kB regulatory protein 3 (TNFAIP3 / A20) participates in regulating inflammatory signal transduction. Dual-luciferase assay shows that A20 is a direct target of miR-21. Research shows that intrathecal injection of A20 can relieve mechanical hypersensitivity and thermal hyperalgesia. miR-21 can target A20 and inhibit its expression, thereby promoting the activation of the NF-kB and NLRP3 signaling pathways. NLRP3 is a multimeric protein composed of the receptor protein NLRP3, the adaptor protein ASC, and the effector protein caspase-1. Activation of the NLRP3 inflammasome mediates the release of IL-1β.

[0005] The expression changes of miR-21 in the spinal cord of neuropathic pain mice have been studied. However, the reasons for the changes in miRNA-21 expression have rarely been explored. Pre-miRNA is transported from the nucleus to the cytoplasm under the action of exportin 5. Pre-miRNA located in the cytoplasm is cleaved by Dicer enzyme to produce mature miRNA. Most miRNAs are dicer1-dependent. Studies have shown that Dicer1 can promote liver regeneration by increasing miR-21. However, the reasons for the elevation of miR-21 in the spinal cord and how miR-21 in the spinal cord participates in the occurrence and development of neuropathic pain are still unclear.

[0006] Phytochemicals extracted from plants may regulate and reverse gene transcription and abnormal epigenetic changes, including alterations in miRNAs. 3,6-Dihydroxyflavone (3,6-DHF) is a natural flavonol compound that can regulate miRNA expression, such as upregulating miR-34a and downregulating miR-21. Based on the above evidence, we speculate that 3,6-DHF has an analgesic effect on CCI-induced neuropathic pain mice, which may be related to neuroinflammation mediated by the miR-21 / A20 / NLRP3 signaling pathway. Summary of the Invention

[0007] Through the pharmacological action research on 3,6-dihydroxyflavone, the present application discovers that 3,6-dihydroxyflavone can relieve mechanical / cold allodynia, heat hyperalgesia, improve the pathological damage changes of dorsal root ganglion and spinal dorsal horn tissues, and restore the function of the sciatic nerve, thereby providing the use of 3,6-dihydroxyflavone in the preparation of drugs for treating neuropathic pain.

[0008] On the one hand, the present invention provides the use of 3,6-dihydroxyflavone in the preparation of drugs for treating neuropathic pain, and the structural formula of the 3,6-dihydroxyflavone is shown as formula (1):

[0009]

[0010] Furthermore, the 3,6-dihydroxyflavone in the drug is the only active ingredient.

[0011] Furthermore, the neuropathic pain is allodynia and hyperalgesia.

[0012] Furthermore, the allodynia and hyperalgesia are manifested as a decrease in the mechanical paw withdrawal threshold, a shortening of the thermal paw withdrawal latency, an increase in the number of cold paw lifts, as well as sciatic nerve function injury and pathological damage of the dorsal root ganglion and / or spinal cord tissue.

[0013] Further, the drug is in the form of an injection or oral dosage form; the preferred dosage forms are tablets, capsules, oral liquids, injections, and powder injections; the particularly preferred dosage form is the oral dosage form.

[0014] Further, the drug includes various pharmaceutically acceptable excipients; the preferred excipients are selected from one or more of solvents, solubilizers, binders, stabilizers, antioxidants, pH regulators, and flavoring agents; particularly preferred are the excipients for oral dosage forms.

[0015] On the other hand, the present invention provides a non-therapeutic method for improving allodynia and hyperalgesia, including administering 3,6-dihydroxyflavone.

[0016] Further, the single-dose application of 3,6-dihydroxyflavone is 6.67 - 20 mg / kg.

[0017] Further, the single-dose application of 3,6-dihydroxyflavone is 20 - 60 mg / kg.

[0018] Further, the single-dose application of 3,6-dihydroxyflavone is 60 mg / kg.

[0019] The non-therapeutic methods in this application include but are not limited to health care methods and scientific research methods, and the subjects of implementation can be humans or other mammals such as mice, rabbits, dogs, etc.

[0020] The symptoms or indications of neuropathic pain in this application include but are not limited to mechanical / cold allodynia, heat hyperalgesia, pathological damage to the dorsal root ganglion and spinal dorsal horn tissues, and abnormal sciatic nerve function.

[0021] The available drug dosage forms in this application include various injection and oral dosage forms, including but not limited to tablets, capsules, oral liquids, injections, powder injections, transdermal drug delivery preparations, and the particularly preferred dosage form is the oral dosage form.

[0022] In addition to 3,6-dihydroxyflavone, the drugs described in this application may also include various pharmaceutically acceptable excipients, including but not limited to coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH regulators, flavoring agents, etc., and particularly preferred are the various excipients for oral dosage forms.

[0023] The drugs of the present invention may contain other Chinese and Western drugs or health products for treating allodynia and hyperalgesia, or the drugs of the present invention may be used in combination with these Chinese and Western drugs or health products or treatment means such as surgery. The Chinese and Western drugs or health products include but are not limited to anti-infective drugs, gonadotropin drugs, antioxidant drugs, energy metabolism and blood circulation improvers.

[0024] In another aspect, the present application provides a pharmaceutical composition for treating neuropathic pain, and the pharmaceutical composition contains 3,6-dihydroxyflavone.

[0025] Furthermore, the 3,6-dihydroxyflavone in the pharmaceutical composition is the only active ingredient.

[0026] Furthermore, the pharmaceutical composition further contains other drugs for treating neuropathic pain. Preferably, the other drugs and / or health products are selected from one or more of anti-infective drugs, gonadotropin drugs, antioxidant drugs, and agents for improving energy metabolism and blood circulation.

[0027] The use of 3,6-dihydroxyflavone provided by the present invention in the preparation of a drug for treating neuropathic pain has the following beneficial effects:

[0028] (1) 3,6-Dihydroxyflavone can significantly relieve mechanical / cold hyperalgesia and heat hyperalgesia;

[0029] (2) The analgesic effect of 3,6-dihydroxyflavone will not cause inhibitory effects on the central nervous system;

[0030] (3) 3,6-Dihydroxyflavone significantly improves the pathological damage of dorsal root ganglion and spinal dorsal horn tissues;

[0031] (4) 3,6-Dihydroxyflavone significantly restores the function of the sciatic nerve;

[0032] (5) 3,6-Dihydroxyflavone can inhibit the expression of miR-21 and the protein expression of IL-1β;

[0033] The present invention first confirms that 3,6-dihydroxyflavone has the effect of treating neuropathic pain in mice and can be used for the preparation of a therapeutic drug for neuropathic pain, providing a new option for such drugs. Description of the Drawings

[0034] Figure 1 Effects of 3,6-dihydroxyflavone on hyperalgesia and allodynia in mice with CCI-induced neuropathic pain; part a: mechanical withdrawal reflex threshold; part b: thermal withdrawal reflex latency; part c: cold foot lifting times; part d: spontaneous activity times; the results are expressed as mean±SEM (n = 10), # P<0.05, ## P<0.01, ### P<0.001 indicates comparison with the control group, * P<0.05, ** P<0.01, *** P<0.001 indicates comparison with the model group.

[0035] Figure 2 Effects of 3,6-dihydroxyflavone on SNCV and CAMP in mice with CCI-induced neuropathic pain; Part A: Representative diagrams of sciatic-sensory nerve action potentials in mice of each group (a: control group, b: control + solvent group, c: model + solvent group, d: model + pregabalin group, and e-g: model + 3,6-dihydroxyflavone groups (6.67 mg / kg, 20 mg / kg, 60 mg / kg)). The maximum mV, minimum mV, average mV, and peak-to-peak mV of each group were a: 36.03, -26.91, -0.20, 64.94; b: 32.07, -26.48, -0.26, 58.55; c: 7.40, -4.60, -0.26, 12.00; d: 24.89, -17.36, -0.10, 42.25; e: 9.96, -5.37, -0.01, 15.33; f: 13.72, -10.26, -0.04, 23.98; g: 25.72, -15.92, -0.10, 41.56); Part B: Statistical chart of SNCV in mice of each group; Part C: Statistical chart of CAMP in mice of each group; Results are expressed as mean ± SEM (n = 6), ### P < 0.001 indicates comparison with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001 indicates comparison with the model group.

[0036] Figure 3 Showing the effects of 3,6-dihydroxyflavone on histopathological damage of dorsal root ganglion in mice with CCI-induced neuropathic pain; Part a: control group, Part b: control + solvent group, Part c: model + solvent group, Part d: model + pregabalin group, and Parts e-g: model + 3,6-dihydroxyflavone groups (6.67 mg / kg, 20 mg / kg, 60 mg / kg))

[0037] Figure 4 Showing the effects of 3,6-dihydroxyflavone on histopathological damage of spinal dorsal horn in mice with CCI-induced neuropathic pain; Part a: control group, Part b: control + solvent group, Part c: model + solvent group, Part d: model + pregabalin group, and Parts e-g: model + 3,6-dihydroxyflavone groups (6.67 mg / kg, 20 mg / kg, 60 mg / kg))

[0038] Figure 5Effect of 3,6-dihydroxyflavone on the expression of pre-miRNA-21, miRNA-21, dicer1 mRNA and dicer1 protein in the spinal cord of CCI-induced neuropathic pain mice; Part a: Statistical chart of miR-21 expression in the spinal cord of mice in each group; Part b: Statistical chart of pre-miR-21 expression in the spinal cord of mice in each group; Part c: Representative immunoblotting of dicer1 protein; Part d: Dicer1 protein band; Part e: Statistical chart of dicer1 mRNA expression in mice in each group; The results are expressed as mean±SEM (n = 6), # P<0.05, ## P<0.01 indicates comparison with the control group, * P<0.05, ** P<0.01, *** P<0.001 indicates comparison with the model group.

[0039] Figure 6 Show the effect of 3,6-dihydroxyflavone on the expression of A20 mRNA and A20 protein in the spinal cord of CCI-induced neuropathic pain mice; Part a: Statistical chart of A20 mRNA expression in mice in each group; Part b: Representative immunoblotting of A20 protein; Part c: Statistical chart of A20 protein band; The results are expressed as mean±SEM (n = 3), # P<0.05 indicates comparison with the control group, * P<0.05 indicates comparison with the model group.

[0040] Figure 7 Show the effect of 3,6-dihydroxyflavone on the expression of NLRP3, ASC, caspase1, cleaved-caspase1, IL-1β, IL-18 proteins in the spinal cord of CCI-induced neuropathic pain mice; Part a: Representative immunoblotting of each protein; Part b: Statistical chart of NLRP3 protein band; Part c: Statistical chart of ASC protein band; Part d: Statistical chart of caspase1 protein band; Part e: Statistical chart of cleaved-caspase1 protein band; Part f: Statistical chart of IL-1β protein band; The results are expressed as mean±SEM (n = 3), # p<0.05 indicates comparison with the control group.

[0041] Figure 8 Show the effect of 3,6-dihydroxyflavone on the interaction between NLRP3 and A20 proteins in the spinal cord of CCI-induced neuropathic pain mice.

[0042] Figure 9To show the effects of 3,6-dihydroxyflavone on the co-expression of NLRP3 with neuronal marker (NeuN), astrocyte marker (GFAP), and microglia marker (IBA-1) in the spinal dorsal horn of CCI-induced neuropathic pain mice; Part a: Representative immunofluorescence image of the co-expression of NLRP with astrocyte marker (GFAP); Part b: Representative immunofluorescence image of the co-expression of NLRP with microglia marker (IBA-1); Part c: Representative immunofluorescence image of the co-expression of NLRP with neuronal marker (NeuN); Part d: Statistical chart of the positive cell rate of NLRP3 and GFAP in the spinal dorsal horn of mice in each group; Part e: Statistical chart of the positive cell rate of NLRP3 and IBA-1 in the spinal dorsal horn of mice in each group; Part f: Statistical chart of the positive cell rate of NLRP3 and NeuN in the spinal dorsal horn of mice in each group; The results are expressed as mean ± SEM (n = 3). Detailed implementation manners

[0043] The present invention will be further described in detail below in conjunction with embodiments. The 3,6-dihydroxyflavone used in the following embodiments is the compound shown in the foregoing formula (1) and can be obtained through commercial purchase.

[0044] Embodiment 1 Experimental materials

[0045] Animal treatment

[0046] SPF-grade adult male C57BL / 6 mice weighing between 20 - 22 g, bred in the Experimental Animal Center of Ningxia Medical University (qualified animal incubation batch number: SCXK(Ning)2020 - 0001). The animals were raised according to the experimental animal breeding standard. The lighting in the breeding room was artificially controlled with a 12-hour day-night cycle, and the mice had free access to water.

[0047] Experimental drugs and instruments

[0048] The main drugs and reagents involved in this experiment include: 3,6-dihydroxyflavone (purchased from Shanghai Yuanye Bio-Technology Co., Ltd., purity ≥ 98%); pregabalin (purchased from Pfizer); DMSO (purchased from Solarbio); Tween 80 (purchased from Solarbio); PEG300 (purchased from MACKLIN); FreeZol Reagent (purchased from Novoprotein), EasyScript One-Step gDNA Removal, cDNA Synthesis SuperMix EasyScript / All-in-One First-Strand cDNA Synthesis SuperMix, Green qPCR SuperMix (purchased from TRANS); antibodies Dicer1, A20, IL-18, ASC, β-tubulin, fluorescent secondary antibody Fluorescein (FITC)-conjugated Affinipure Goat Anti-Rabbit IgG(H+L), HRP-conjugated secondary antibodies, ultra-wide molecular weight marker (10 - 310KDa) (purchased from proteintech); antibodies NLRP3, antibody cleaved-caspase1, antibody caspase1, antibody IL-1β (purchased from Affinity); rapid blocking solution, ECL luminescence solution, protein Marker (10 - 180KDa) (purchased from SEVEN); PBS, TBST (purchased from Servicebio Co., Ltd.); magnetic beads (purchased from Selleck, USA); DAPI (purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.); total protein extraction kit (purchased from Nanjing KeyGen Biotech Co., Ltd.); total protein quantification kit (purchased from Nanjing KeyGen Biotech Co., Ltd.); NC membrane (purchased from Pall Corporation); tissue fixative (purchased from Servicebio Co., Ltd.).

[0049] The main instruments involved in this experiment include: mechanical stinger (purchased from North Coast Medical, USA), radiant heat stinger (purchased from Chengdu Taimeng Software Co., Ltd.), cold and heat pain detector (purchased from Bioseb, France), microplate reader (purchased from Thermo scientific); electrothermal constant temperature water bath (purchased from Shanghai Precision Experimental Equipment Co., Ltd.); automatic sample cryogenic grinder (purchased from Shanghai Jingxin Technology Co., Ltd.); SDS-PAGE electrophoresis apparatus, electrotransfer apparatus (purchased from Bio-RAD, USA); electrothermal constant temperature forced air drying oven (purchased from Shanghai Langgan Experimental Equipment Co., Ltd.); fluorescence microscope (OLYMPUS); high-speed low-temperature centrifuge (Eppendorf, Germany).

[0050] Establish a chronic constriction injury model of the sciatic nerve

[0051] Construct a CCI model according to the method of Bennett and Xie. Anesthetize male C57BL / 6 mice with isoflurane, place them in the prone position on a clean operating table, prepare the skin at the root of the right thigh of the mice, disinfect with iodophor, and then incise the skin at the lower edge of the femur. Use a glass pick to bluntly separate the biceps femoris and gluteus muscles, expose the main trunk of the right sciatic nerve, and loosely ligate 3 loops at the main trunk of the sciatic nerve with 4-0 chromic catgut (sterilized in advance and softened with normal saline), with an interval of about 1 mm. Then, reset the sciatic nerve and suture the incision. Place the animals in a comfortable environment and transfer them to a normal breeding environment after they wake up. In the control group, only expose the sciatic nerve trunk without ligation, and perform CCI operations on the other groups.

[0052] Grouping and administration of experimental animals

[0053] After adaptively feeding male adult C57BL / 6 mice weighing 20 - 22 g in a clean breeding room for 7 days, randomly divide them into a control group, a control + solvent group, a model + solvent group, a model + pregabalin group, and a model + 3,6-dihydroxyflavone group (6.67 mg / kg, 20 mg / kg, 60 mg / kg) according to the body weight gradient sampling method. The positive drug group is intraperitoneally injected with pregabalin (20 mg / kg), and the model + 3,6-dihydroxyflavone group (6.67 mg / kg, 20 mg / kg, 60 mg / kg) is respectively given 3,6-dihydroxyflavone (6.67 mg / kg, 20 mg / kg, 60 mg / kg) by gavage, with 15 mice in each group. Administer the drugs continuously for 8 days. The mice in the control group and the model group are both given the same volume of solvent, and the other experimental conditions are the same.

[0054] Experimental process and result analysis of Example 2

[0055] Use Von Frey filaments to detect the mechanical withdrawal threshold of the paws in each group of mice

[0056] Experimental method:

[0057] Before the experiment, place the mice in a transparent plastic chamber on the metal mesh cage frame for the Von Frey filaments experiment to adapt for 30 min. Apply pressure at a 90° angle with the filament in contact with the plantar surface of the hind paw, gradually increase the pressure, and observe the discomfort reactions of the mice such as paw withdrawal or paw lifting. Conduct the experiment according to the up and down method, measure each paw 10 times, measure at intervals of 1 min, and take the average value as the final force (g).

[0058] Experimental results:

[0059] The results are asFigure 1 As shown, compared with the Sham+vehicle group, there was no significant change in PWT in the Sham+3,6-DHF (60 mg / kg) group; after modeling, PWT decreased significantly (p<0.001). Compared with the CCI+vehicle group, intragastric administration of 3,6-DHF (60 mg / kg) for 8 consecutive days significantly increased PWT (p<0.05), and intraperitoneal injection of pregabalin (20 mg / kg) for 8 consecutive days had a better effect than 3,6-DHF (60 mg / kg). It is suggested that 3,6-dihydroxyflavone has an improving effect on CCI mouse-induced mechanical hyperalgesia.

[0060] The thermal withdrawal reflex threshold of mice in each group was detected by the thermal radiation stimulation experiment

[0061] Experimental method

[0062] The mice were placed on a flat glass plate with a glass bell jar, and their activity range was restricted. After 30 min of adaptation, the PWL of the mice was measured according to the Hargreaves method. The thermal radiation instrument was pre-set (power: 100 W; stimulation intensity: 50%, the upper cut-off time was 14 sec), and the radiation beam was irradiated on the plantar surface of the surgical side of the mouse. When the foot on the surgical side of the mouse left the radiation beam, the irradiation automatically stopped, and it was judged whether it was a pain behavior. If so, the time displayed on the instrument was recorded, and the PWL value of the mouse was the recorded time displayed on the instrument. It was measured 3 times and the average value was taken. The measurement interval was 5 min, and the average value was the PWL of the mouse.

[0063] Experimental results

[0064] The results are as Figure 1 As shown, compared with the Sham+vehicle group, there was no significant change in PWL in the Sham+3,6-DHF (60 mg / kg) group; 7 days after modeling, the PWL of the mice decreased significantly (p<0.001). Compared with the CCI+vehicle group, intragastric administration of 3,6-DHF (60 mg / kg) for 8 consecutive days significantly increased PWL (p<0.05), and intraperitoneal injection of pregabalin (20 mg / kg) for 8 consecutive days had the same effect as 3,6-DHF (60 mg / kg) (p<0.05). It is suggested that 3,6-dihydroxyflavone has an improving effect on CCI mouse-induced thermal hyperalgesia.

[0065] The cold plate experiment was used to detect the number of cold foot lifts of mice in each group

[0066] Experimental method

[0067] The number of cold foot lifts in mice was measured according to the Jasmin method. The hot / cold plate instrument was turned on in advance to set the plate temperature at 4.0 ± 0.2 °C. The mice were placed on the hot / cold plate pain threshold meter and covered with a transparent plastic box to restrict their free movement. After each mouse to be tested adapted to the temperature of 4.0 ± 0.2 °C for 5 min, the number of foot lifts within 5 min was recorded.

[0068] Experimental results

[0069] The results were as Figure 1 shown. Compared with the Sham+vehicle group, there was no significant change in the number of cold foot lifts in the Sham+3,6-DHF (60 mg / kg) group. After modeling, the number of cold foot lifts in the CCI+vehicle group increased significantly (p < 0.05). Compared with the CCI+vehicle group, the number of cold foot lifts in mice significantly decreased after continuous intragastric administration of 3,6-DHF (60 mg / kg) for 8 days (p < 0.05 / p < 0.01), and continuous intraperitoneal injection of pregabalin (20 mg / kg) for 8 days had the same effect as 3,6-DHF (60 mg / kg) (p < 0.05 / p < 0.01). It is suggested that 3,6-dihydroxyflavone has an improving effect on cold allodynia induced by CCI in mice.

[0070] The sensory nerve conduction velocity and compound muscle action potential amplitude of mice in each group were measured by electrophysiological experiments

[0071] Experimental method:

[0072] Mice were anesthetized with isoflurane, placed in the prone position, their hair was shaved and their skin was prepared. After disinfection with iodophor, the skin of the hind limb on the CCI side was incised, and the sciatic nerve was separated with a glass pick needle, and the ligature was removed. The electrophysiological detection parameters of the software BL-420F were set. The stimulating electrode, recording electrode (between the ankle and toes 3 and 4 of the mouse), and reference electrode were inserted, and the reference electrode was gently placed on the gastrocnemius muscle. During the experiment, liquid paraffin at 37 °C was dropped on the sciatic nerve to keep it moist, and normal saline was dropped at the electrode to conduct electricity. There should be an interval between each electrical stimulation and the stimulation should be completed as soon as possible. The body temperature of the mouse needed to be maintained during the experiment. After the experiment, the data was saved.

[0073] Experimental results:

[0074] Compared with the Sham+vehicle group, there were no significant differences in CAMP and SNCV in the Sham+3,6-DHF (60 mg / kg) group of mice; after modeling, CMAP and SNCV in the CCI+vehicle group of mice were significantly decreased (P<0.001); compared with the CCI+vehicle group, CMAP and SNCV in the CCI+Pregabalin group of mice were significantly increased (P<0.01, P<0.001), CMAP in the CCI+3,6-DHF (6.67, 20, 60 mg / kg) group was significantly increased (P<0.05, P<0.01, P<0.001), and SNCV in the CCI+3,6-DHF (20, 60 mg / kg) group was significantly increased (P<0.05, P<0.001)( Figure 2 ).

[0075] HE staining was used to observe the pathological changes of the dorsal root ganglion and spinal dorsal horn tissues of mice in each group

[0076] Experimental method:

[0077] Preparation of paraffin sections

[0078] ① After the behavioral experiment on the 14th day, the spinal cord (L3-L5) and sciatic nerve (including the injured site) of mice in each group were taken out on ice and directly immersed in 4% paraformaldehyde for fixation (the fixation time was 24 h); ② After rinsing with running water for 15 min, dehydration was carried out with ethanol of different concentrations, 50%, 75%, 80%, 95%, absolute ethanol I, II, and the time for each gradient was 30 min; ③ Immerse in xylene I and II for 20 min each for tissue clearing; (The dehydration and clearing operations were all carried out in the fume hood) ④ Immerse the tissue in paraffin I and II for 2 h each; ⑤ Use an embedding cassette to embed on an automatic embedding machine and place it indoors until it solidifies into a wax block. ⑥ Set the section thickness to 4 μm on a paraffin slicer to cut the wax block

[0079] HE staining steps

[0080] ① Deparaffinization: First, immerse the sciatic nerve and spinal cord sections in xylene I and II for 10 min each, then in absolute ethanol I and II for 6 min each, then in 95%, 85%, 75% ethanol for 6 min each, and finally rinse with purified water for 2 min; ② Hematoxylin staining: Immerse the sections in hematoxylin solution for 4 min, rinse with running water for 5 min, and then rinse with purified water; ③ Differentiation with differentiation solution for 8 min, rinse with running water for 5 min, and rinse with purified water until blue returns; ④ Eosin staining: Immerse the sections in eosin staining solution for 12 min and rinse with purified water for 1 sec; ⑤ Hydration and clearing: Immerse the sections in 75%, 85%, 95% ethanol, absolute ethanol II for 1 sec - absolute ethanol I for 30 sec - xylene I and II for 1 min each, and seal the sections with neutral gum; ⑥ Observe under the microscope and collect images

[0081] Experimental results:

[0082] As Figure 3 , the neurons in the dorsal root ganglia of mice in the Sham+vehicle group and the Sham+3,6-DHF (60 mg / kg) group had regular shapes, were closely arranged, and the stained cell nuclei were clearly visible with the nucleoli located in the center of the neurons. After modeling, compared with the Sham+vehicle group, the cells in the dorsal root ganglia of mice in the CCI+vehicle group were loosely arranged, the cells were severely shrunk into clusters, and the neuronal nucleoli could not be clearly identified. Compared with the CCI+vehicle group, 3,6-DHF dose-dependently improved the pathological damage of the dorsal root ganglia. CCI+Pregabalin (20 mg / kg) improved the traumatic pathological changes of neurons in the dorsal root ganglia of mice, the number of normal neurons increased significantly, the cell arrangement was neat and the gaps became smaller, and the cell nuclei could be discerned by partial staining, and the phenomenon of nuclear condensation was also improved.

[0083] As Figure 4 , the neurons in the dorsal horn of the spinal cord of mice in the Sham+vehicle group and the Sham+3,6-DHF (60 mg / kg) group had regular shapes, and the stained cell nuclei were clearly visible with the nucleoli located in the center of the neurons. After modeling, compared with the Sham+vehicle group, the neuronal cells in the CCI+vehicle group of mice were severely shrunk into clusters, the staining was uneven, and the neuronal nucleoli could not be clearly identified. Compared with the CCI+vehicle group, there was no obvious pathological improvement in the dorsal horn of the spinal cord of mice in the CCI+3,6-DHF 6.67 mg / kg group, and there were still phenomena of neuronal cell shrinkage and disappearance of nucleoli. CCI+3,6-DHF (20, 60 mg / kg) and CCI+Pregabalin (20 mg / kg) both improved the traumatic pathological changes of neurons in the dorsal horn of the spinal cord of mice, the number of normal neurons increased significantly, the cell nuclei could be discerned by partial staining, and the phenomenon of nuclear condensation was also improved.

[0084] qPCR experiment was used to detect the expression of pre-miR-21, miR-21, A20 mRNA, and dicer1 mRNA

[0085] Experimental method:

[0086] Extraction of miRNA: Use a miRNA extraction kit and operate according to the instructions.

[0087] Total RNA extraction: Prepare reagents: chloroform substitute, isopropanol, 75% ethanol, RNase-free water (all reagents used need to be prepared with DEPC-treated water) 1. Homogenization treatment: Place the spinal cords of each group in an enzyme-free grinding tube, add grinding beads treated with RNase inhibitor, add 500 μL of Trizol to each tube, and perform homogenization treatment with a cryogenic grinder; let stand at room temperature for 10 min; add chloroform substitute, and vigorously shake the centrifuge tube by hand; let stand at room temperature for 10 min; centrifuge according to the centrifuge parameters set in the instruction manual. The RNA in the sample will be separated into the supernatant, while DNA and proteins will remain in the substrate. Add isopropanol in a volume ratio of 1:1, then gently shake the centrifuge tube to mix the RNA with isopropanol. Then place the mixture in a -20 °C refrigerator to fully precipitate the RNA. Add 75% ethanol in a volume ratio of 1:1, mix well, and centrifuge at 12000 rcf for 5 min to remove the residual Trizol reagent and isopropanol, and repeat twice. Open the lid of the centrifuge tube, place the centrifuge tube on the rack to promote ethanol evaporation; add 40 μL of enzyme-free water to dissolve the RNA. Detect the RNA concentration of each group.

[0088] Reverse transcription: Use the EasyScript One-Step gDNA Removal, cDNA Synthesis SuperMix EasyScript / All-in-One First-Strand cDNA Synthesis SuperMix kits to perform reverse transcription on mRNA and miRNA respectively. Set the reverse transcription conditions: 42 °C for 5 min, 85 °C for 2 min. After completion, perform fluorescence quantitative PCR or store at -80 °C for later use.

[0089] Fluorescence quantitative qPCR: Prepare a 20 μL reaction system (2 μL of sample, 4 μL of each forward and reverse primer (1 μM), 10 μL of Green qPCR SuperMix); Amplification conditions (qPCR three-step method): 94 °C for 30 sec; 94 °C for 5 sec; 58 °C for 15 sec; 72 °C for 10 sec; Repeat steps 2 - 4 for 45 cycles. Save the results.

[0090] Experimental results:

[0091] We used qPCR to detect the effects of 3,6-dihydroxyflavone on the expression of miR-21, pre-miR-21, dicer1, and A20 mRNA. The experimental results showed that after modeling, the expression of miR-21 and dicer1 mRNA increased, while the expression of pre-miR-21 and A20 mRNA decreased; after treatment with 60 mg / kg of 3,6-dihydroxyflavone, the expression of miR-21 and dicer1 mRNA decreased, and the expression of pre-miR-21 and A20 mRNA increased significantly ( Figure 5, 6).

[0092] Experimental method for detecting the protein expression of A20, dicer1, NLRP3, ASC, caspase1, and cleaved-caspase1 by Western blot:

[0093] Take the spinal cord tissues of each group and place them in a grinding tube. Add grinding beads, add lysis buffer according to the spinal cord weight (1 mL for every 100 mg), and put them into a tissue grinder for grinding (65 HZ, 45 s) twice. Let it stand on ice for 15 min, centrifuge (15 min, 12000 rcf, 4 °C), and take the supernatant and place it on ice for later use.

[0094] Determine the total protein concentration by BCA method: (1) Prepare the protein standard solution: Dilute the protein standard solution according to the gradient with reference to the kit instruction manual; (2) Prepare the sample to be tested: Take 2 μL of the protein stock solution of each group, and then add 78 μL of Lysis buffer and vortex to mix evenly to obtain the diluted stock solution for later use; Add 20 μL of the protein standard solution / sample diluent to each well of the microplate; (3) According to the kit instruction manual, mix solution A and solution B proportionally and evenly, add the BCA working solution to the microplate, incubate at 37 °C with low-speed intermittent oscillation for 30 min, and measure the OD value (562 nm); (4) Calculate the protein concentration of the sample: Use the OD values of the gradient-diluted protein standard solution to establish a standard curve, and substitute the OD value of the sample into the calculation of the protein concentration.

[0095] Determine the tissue protein loading concentration. According to the actual protein concentration, determine a unified concentration according to the lowest concentration among all groups. Add 6× loading buffer and Lysis Buffer solution to the supernatant to unify the protein samples of each group to the same concentration (tissue: 7 μg / μL), mix evenly and seal, place on a floating plate and boil in boiling water for 10 min, aliquot 25 μL per tube, and store at -80 °C for use as soon as possible in the short term.

[0096] SDS-PAGE gel electrophoresis: (1) Prepare 10% separating gel solution and 5% stacking gel solution with reference to the instruction manual: After mixing evenly, quickly place it in the glass plate on a non-sloping horizontal plane. Add the separating gel to at least 2 / 3 of the plate surface, and press the gel with methanol; After the separating gel is completely solidified, aspirate the methanol and rinse with double-distilled water; (2) Add the stacking gel to the gel plate in the same operation, place the comb obliquely, and insert the comb while removing air bubbles; (3) Loading: Add 2.5 μL of protein Marker to both sides of the well, and add 8 μL of the sample to the remaining wells in the group order; (4) Electrophoresis: Run at a low voltage of 15 V for 20 min, change to 65 V until the protein Marker is completely separated, then change to 90 V until the internal reference and the target band are completely separated, stop electrophoresis, and take out and cut the gel.

[0097] Protein band transfer to membrane: Moisten the sandwich clip with the transfer buffer placed in the refrigerator. Soak the NC membrane in the transfer buffer 20 minutes in advance. Prepare a compact transfer clip, transfer the gel to the transfer clip in sequence, add ice cubes to the tank, and add buffer to cover the transfer clip; Transfer at 230 mA. The transfer time varies slightly for each protein and is determined according to the protein molecular weight.

[0098] Blocking and incubating antibodies: Completely cover the NC membrane with the rapid blocking solution and shake slowly on a shaker for 20 minutes to ensure blocking of non-specific sites; Wash the residual rapid blocking solution 3 times with TBST, 5 minutes each time; Dilute the antibodies of each protein according to the instructions and incubate at 4 °C in a refrigerator with slow shaking for 16 hours to ensure sufficient binding of antibody and antigen; Wash the primary antibody, wash the residual primary antibody 3 times with TBST, 10 minutes each time; Incubate the secondary antibody: Incubate at room temperature for 2 hours; Wash the residual antibody with TBST 10 minutes each time and repeat 3 times.

[0099] Exposure and imaging of protein bands: Add 4 mL of hypersensitive ECL luminescent reaction solution to the antibody incubation box and soak the protein bands in it in the order of exposure. Start with automatic exposure. If the obtained bands are not appropriate, manually adjust the exposure time according to the bands, and finally save the pictures. Analyze the picture results. Use ImageJ software to analyze the protein bands. Normalize the ratio of gray values of the three samples.

[0100] Experimental results: We used Western blot to detect the effects of 3,6-dihydroxyflavone on the protein expressions of NLRP3, ASC, caspase-1, cleaved-caspase-1, and IL-1β. The experimental results showed that after modeling, there were no significant changes in NLRP3, ASC, and caspase-1, while cleaved-caspase-1 and IL-1β increased significantly; After treatment with 60 mg / kg 3,6-dihydroxyflavone, there were no significant changes in NLRP3, ASC, and caspase-1, while cleaved-caspase-1 and IL-1β decreased significantly ( Figure 7 ). Co-IP experiment was used to detect the interaction between NLRP3 and A20

[0101] Experimental method:

[0102] Take 20 μL of Protein A / G immunoprecipitation magnetic beads for each group. After washing with PBS buffer, add them to the above-mentioned tissue fluid, and then add Cyclin D1 antibody to prepare an antibody working solution. Place it on a rotary mixer at room temperature or gently invert the EP tube by hand for 30 min to allow the antigen and antibody to fully bind. If the binding force is weak, the reaction can be carried out at room temperature for 1 h or overnight at 4°C. Magnetically separate the above-mentioned magnetic bead-antibody-antigen complex that has completed antigen adsorption. After washing with the eluent, add loading buffer and boil the protein at high temperature for 10 min to denature it, and collect the supernatant. The subsequent result verification is carried out by Western blotting method.

[0103] Experimental results:

[0104] We used Co-IP to detect the protein interaction between NLRP3 and A20 and explored whether 3,6-dihydroxyflavone inhibits NLRP3 activation. The experimental results showed that in the input group, compared with the control group, the expression of NLRP3 in the model group increased, and the expression of A20 decreased. After treatment with 60 mg / kg 3,6-dihydroxyflavone, the expression of NLRP3 decreased and the expression of A20 increased. In the IP group, we found that the interaction between NLRP3 and A20 in the model group was enhanced, and after treatment with 3,6-dihydroxyflavone, the interaction between NLRP3 and A20 was weakened. The above experimental results indicate that 3,6-dihydroxyflavone can inhibit the binding of NLRP3 and A20 and inhibit NLRP3 activation ( Figure 8 ).

[0105] Experimental method for immunofluorescence detection of co-expression of NLRP3 with mouse spinal dorsal horn neurons, microglia, and astrocytes:

[0106] Place the paraffin sections in a 60°C constant temperature oven for 2 h for dewaxing. Place the sections of each group successively into xylene solution I / II for 20 min each; place the sections successively into absolute ethanol I / II, 100%, 95%, 85%, 75%, ddH 2Soak in O for 5 minutes respectively; cover the sample with peroxidase remover, seal it in the dark at room temperature for 10 minutes, and place it in PBS buffer for rinsing 5 minutes × 3 times. Place the section in the sodium citrate buffer that has been pre-boiled in a microwave oven and boil for 30 minutes; place it at room temperature and wait for it to cool naturally, then rinse it with PBS buffer 5 minutes × 3 times. Take out the section after it has cooled sufficiently, drain it, cover the sample with goat serum blocking solution, and block it at 37°C for 30 minutes; according to the recommended dilution of the primary antibodies NLRP3 (1:100), IBA-1 (1:500), GFAP (1:200), NeuN (1:100) in the antibody instruction manual, mix NLRP3 with the triple cell marker mixed antibodies respectively to cover the sample for primary antibody incubation, and incubate overnight in a 4°C refrigerator; the next day, warm the section at room temperature for 30 minutes, then rinse the section with PBS buffer, 10 minutes each time, for a total of 3 washes; incubate with biotin-labeled secondary antibody for 2 hours and incubate at room temperature in a humid box for 60 minutes. Rinse with PBS buffer for 5 minutes for a total of three times; seal with DAPI; take pictures using a fluorescence microscope.

[0107] Experimental results:

[0108] We found that NLRP3 was widely distributed in the dorsal horn of the spinal cord of male C57 mice and was distributed in neurons, astrocytes, and microglia ( Figure 9 ).

Claims

1. Use of 3,6-dihydroxyflavone in the preparation of drugs for treating neuropathic diseases, wherein the structural formula of the 3,6-dihydroxyflavone is as shown in Formula I:

2. The use according to claim 1, wherein 3,6-dihydroxyflavone is the only active ingredient in the drug.

3. The use according to claim 1 or 2, wherein the neuropathic pain is a chronic nervous system disease.

4. The use according to claim 1 or 2, wherein the neuropathic pain is mechanical / cold allodynia, thermal hyperalgesia; pathological damage to dorsal root ganglia and spinal cord dorsal horn tissue, and abnormal sciatic nerve function.

5. The use according to any one of claims 1 to 4, wherein the drug is in the form of an injection or oral dosage form; preferably, the dosage form is tablets, capsules, oral liquids, injections, powder injections; oral dosage forms are particularly preferred.

6. The use according to claim 5, wherein the drug comprises various pharmaceutically acceptable excipients / excipients; preferably, the excipients / excipients are selected from one or more of solvents, solubilizers, binders, stabilizers, antioxidants, pH regulators, and flavoring agents; particularly preferred are excipients for oral dosage forms.

7. The use according to any one of claims 1 to 6, wherein the drug has one or more of the following effects: (1) Relieve mechanical / cold allodynia and thermal hyperalgesia; (2) No inhibitory effect on the central nervous system (3) Improve pathological changes in dorsal root ganglia and spinal cord dorsal horn tissue; (4) restore sciatic nerve function; (5) Inhibit the expression of miR-21, reduce the protein expression of the inflammatory factor IL-1β through the miR-21 / A20 / NLRP3 signaling pathway, and alleviate neuroinflammation.

8. A non-therapeutic method for alleviating allodynia and hyperalgesia in a subject, comprising administering 3,6-dihydroxyflavone to the subject.

9. The method according to claim 8, wherein the single application dose of 3,6-dihydroxyflavone is 6.67-20 mg / kg.

10. The method according to claim 8, wherein the single application dose of 3,6-dihydroxyflavone is 20-60 mg / kg, preferably 60 mg / kg.

11. A pharmaceutical composition for treating neuropathic pain, characterized in that: The pharmaceutical composition comprises 3,6-dihydroxyflavone. 12 . The pharmaceutical composition according to claim 11 , wherein 3,6-dihydroxyflavone is the only active ingredient in the pharmaceutical composition.

13. The pharmaceutical composition according to claim 11 or 12, further comprising other drugs for treating neuropathic pain; preferably, the other drugs and / or health products are selected from one or more of anti-infective drugs, gonadotropin drugs, antioxidant drugs, energy metabolism and blood circulation improvers.

14. The pharmaceutical composition according to any one of claims 11 to 13, which is in the form of an injection or oral dosage form; Preferred dosage forms are tablets, capsules, oral liquids, injections, and powder injections; oral dosage forms are particularly preferred.

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