Preparation method and application of conjugate targeting pelvic autonomic nerves

Abstract

The application discloses a preparation method and application of a conjugate targeting pelvic autonomic nerves, wherein the conjugate is NE-RH formed by coupling of rhein and norepinephrine, the conjugate can realize precise targeting of norepinephrine neurons, improve the accumulation effect of RH in the norepinephrine neurons, and further enhance the nerve protection effect of RH. The conjugate has a molecular weight of less than 1000 Da, can help to penetrate the blood-brain barrier, and can avoid the defects of being easily removed by a mononuclear phagocyte system and having a short half-life. The production process of the conjugate is simple, the chemical coupling reaction is clear, the yield can reach 38%, and the purity of the product is greater than 99%, so that the conjugate is convenient to popularize and use. The conjugate and the preparation method have important significance for the damage repair of pelvic autonomic nerves.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a method for preparing and applying a coupling agent targeting the pelvic autonomic nervous system. Background Technology

[0002] Damage to the pelvic autonomic nerves during gynecological surgeries, such as those for cervical cancer and deep invasive endometriosis, frequently leads to serious nerve-related complications. Classic radical hysterectomy and pelvic lymph node dissection are standard surgical procedures for treating early-stage cervical cancer, achieving good oncological outcomes. However, damage to the pelvic autonomic nerves running within the ligaments surrounding the cervix is ​​unavoidable during these procedures, resulting in severe bladder, rectal, and sexual dysfunction. This complication has a high incidence and is difficult to cure, with approximately 50% of patients experiencing it for life. Promoting the repair of pelvic nerve damage is key to improving postoperative functional impairment; however, current research focuses primarily on minimizing intraoperative damage while neglecting postoperative nerve repair.

[0003] Currently, there is a lack of effective drug treatments for pelvic autonomic nerve injury. M-receptor agonists and cholinesterase inhibitors can improve detrusor muscle contractility and enhance bladder emptying, but their effectiveness in promoting the repair of pelvic autonomic nerve damage is not widely recognized. Furthermore, cholinesterase inhibitors may cause adverse reactions such as dry mouth, blurred near vision, constipation, and tachycardia. These drugs can also cross the blood-brain barrier, acting on central muscarinic receptors and leading to alterations in cognition and consciousness. Studies have shown that some neurotrophic factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNE), neurotrophic factor 3 (NT-3), glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF), can increase the growth of major pelvic ganglion processes, promote neuronal differentiation, improve the expression of various neurotrophic factors after nerve injury, and promote nerve repair. However, these compounds are either not approved for clinical use, have intolerable complications, are difficult to preserve, and are expensive, thus limiting their clinical translation. Therefore, there is an urgent need to find a safe and effective drug to promote the repair of pelvic autonomic nerve damage and improve neurogenic bladder dysfunction.

[0004] Rhein (RH) is an anthraquinone compound isolated from traditional Chinese medicines such as aloe vera, rhubarb, cassia seed, and polygonum multiflorum. In recent years, researchers have discovered that RH possesses anti-fibrotic, antioxidant, and anti-tumor effects. Although high-dose RH shows significant efficacy in animal models, its pharmacokinetic defects—including a strong first-pass effect and low blood-brain barrier penetration (bioavailability <5%)—make it difficult for the drug concentration in nerve tissue to reach the therapeutic threshold at conventional doses. This may be because RH is easily metabolized by metabolic enzymes in the liver and intestine, undergoing metabolic reactions such as glucuronidation and sulfonation. The metabolites are transported by efflux transport proteins such as multidrug resistance-associated proteins in the liver and small intestine, and excreted into the bile or intestinal lumen. Domestic and international scholars have used mesoporous silica nanoparticles, liposome nanoparticles, polymer micelles, and hydrogels to improve the dosage form of RH and enhance its bioavailability. However, after these dosage form improvements, the drug particle size remains between 100-300 nm, making it difficult to cross the blood-nerve barrier and thus unsuitable for the treatment of pelvic autonomic nerve injury.

[0005] To address the aforementioned issues, this invention provides a method for preparing and applying a coupling agent targeting the pelvic autonomic nervous system, achieving precise targeting of noradrenergic neurons, improving the accumulation effect of RH within noradrenergic neurons, and further enhancing the neuroprotective effect of RH. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a method for preparing and applying a coupling device targeting the pelvic autonomic nervous system.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a coupling element, said coupling element being represented by the following formula I:

[0009] Formula I: ,

[0010] Where A is selected from the structure shown in the following formulas A-1, A-2 or A-3:

[0011] A-1: A-2: ;

[0012] A-3: .

[0013] In some embodiments of the present invention, when A is A-1, the chemical structural formula of the coupling compound is as follows:

[0014] .

[0015] In some embodiments of the present invention, when A is A-2, the chemical structural formula of the coupling compound is as follows:

[0016] .

[0017] In some embodiments of the present invention, when A is A-3, the chemical structural formula of the coupling compound is as follows:

[0018] .

[0019] In some embodiments of the present invention, the structure shown in A-1 is selected from norepinephrine.

[0020] In some embodiments of the present invention, the structure shown in A-2 is selected from dopamine.

[0021] In some embodiments of the present invention, the structure shown in A-3 is selected from 5-hydroxytryptamine.

[0022] A second aspect of the present invention provides a method for preparing the above-mentioned coupling compound, the method comprising the following steps:

[0023] (1) Rhein and neurotransmitters are dissolved in an organic solvent to carry out an amidation reaction;

[0024] (2) Add an activator to the product obtained in (1) to further activate the unreacted carboxyl group, so that it couples with the primary amino group to form an amide bond, thereby obtaining the coupling product.

[0025] In some embodiments of the present invention, the mass ratio of rhein to neurotransmitter is 1 to 5:1.

[0026] In some embodiments of the present invention, the mass ratio of rhein to neurotransmitter is 1 to 3:1.

[0027] In some embodiments of the present invention, the mass ratio of rhein to neurotransmitter is 1 to 2:1.

[0028] In some embodiments of the present invention, the organic solvent includes at least one of dichloromethane, trichloromethane, tetrahydrofuran, ethyl acetate, or dimethyl sulfoxide.

[0029] In some embodiments of the present invention, the activator includes at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxythiosuccinimide, 1-hydroxybenzotriazole or 4-dimethylaminopyridine.

[0030] In some embodiments of the present invention, the amount of organic solvent used is 5 to 15 mL.

[0031] In some embodiments of the present invention, the reaction temperature in step (1) is 20°C to 30°C.

[0032] In some embodiments of the present invention, the reaction temperature in step (1) is room temperature.

[0033] In some embodiments of the present invention, the room temperature is 20°C to 25°C.

[0034] In some embodiments of the present invention, the reaction time in step (1) is 20 to 40 minutes.

[0035] In some embodiments of the present invention, the reaction temperature in step (2) is 20°C to 30°C.

[0036] In some embodiments of the present invention, the reaction temperature in step (2) is room temperature.

[0037] In some embodiments of the present invention, the reaction time in step (2) is 10-14 hours.

[0038] A third aspect of the invention provides a pharmaceutical composition comprising the above-described conjugate, said pharmaceutical composition further comprising pharmaceutically acceptable excipients.

[0039] In some embodiments of the present invention, the excipients include one or more of diluents, stabilizers, osmotic pressure regulators, pH regulators, preservatives, and antioxidants.

[0040] In some embodiments of the present invention, the antioxidant includes at least one of ascorbic acid, sodium bisulfite, or tert-butylhydroxyanisole.

[0041] In some embodiments of the present invention, the dosage form of the drug includes tablets, injections, powders, oral liquids, or injections.

[0042] A fourth aspect of the invention provides the use of the above-described conjugate or pharmaceutical composition in the preparation of products for the prevention and / or treatment of pelvic nerve injury or related conditions.

[0043] In some embodiments of the present invention, the pelvic nerve injury-related conditions include neurogenic bladder dysfunction.

[0044] The beneficial effects of this invention are:

[0045] This invention provides a drug conjugate, which is formed by the conjugation of rhein and norepinephrine (NE) to form NE-RH. This drug conjugate utilizes "biomimetic camouflage" to mimic the structure of endogenous neurotransmitters, achieving precise targeting of noradrenergic neurons, improving the accumulation effect of RH within noradrenergic neurons, and further enhancing the neuroprotective effect of RH. Compared to traditional nanoparticle drug delivery systems, this drug conjugate has a molecular weight <1000 Da, which facilitates drug penetration of the blood-brain barrier; the drug conjugate utilizes neurotransmitter transporters to achieve targeting of specific types of neurons and enrichment in the lesion area; the drug conjugate avoids the drawbacks of nanomedicines, such as easy clearance by mononuclear phagocytic systems and short half-life.

[0046] The production process of the drug conjugate described in this invention is simple, the chemical coupling reaction is well defined, the yield can reach 38%, the product purity is >99%, and it is easy to promote and use.

[0047] The drug conjugates and their preparation methods described in this invention are of great significance for the repair of damage to the pelvic autonomic nerves. Attached Figure Description

[0048] Figure 1 The reaction formula for NE-RH provided in the embodiments of the present invention.

[0049] Figure 2 The NE-RH hydrogen NMR spectrum provided for embodiments of the present invention.

[0050] Figure 3 The NE-RH Fourier transform infrared spectrum provided for embodiments of the present invention.

[0051] Figure 4 The following are the immunofluorescence results of neurons in the pelvic ganglia of SD rats after NE-RH treatment, as provided in this embodiment of the invention: A is the NeuN staining image of the pelvic ganglia of normal SD rats; B is the DAPI staining image of the pelvic ganglia of normal SD rats; C is the fusion image of DAPI and NeuN staining of the pelvic ganglia of normal SD rats; D is the NeuN staining image of the pelvic ganglia of SD rats one week after the establishment of the pelvic nerve injury model; E is the DAPI staining image of the pelvic ganglia of SD rats one week after the establishment of the pelvic nerve injury model; F is the fusion image of DAPI and NeuN staining of the pelvic ganglia of SD rats one week after the establishment of the pelvic nerve injury model; G is the NeuN staining image of the ganglia of the SD rat pelvic nerve injury model after NE-RH treatment; H is the DAPI staining image of the ganglia of the SD rat pelvic nerve injury model after NE-RH treatment; I is the fusion image of DAPI and NeuN staining of the ganglia of the SD rat pelvic nerve injury model after NE-RH treatment.

[0052] Figure 5The image shows the Masson staining results of the bladder of SD rats after NE-RH treatment, as provided in an embodiment of the present invention.

[0053] Figure 6 The following are bar charts showing the detrusor muscle thickness and bladder volume of SD rats after NE-RH treatment, provided in this embodiment of the invention: A is a graph showing the results of detrusor muscle thickness in SD rats after NE-RH treatment; B is a bar chart showing the bladder volume of SD rats after NE-RH treatment.

[0054] Figure 7 Figure A shows the expression of NLRP3-mediated pyroptosis-related proteins and the detection results of macrophage phenotypes in the pelvic ganglia of SD rats after NE-RH treatment, as provided in Example 4 of this invention. Figure B shows the expression results of NLRP3-mediated pyroptosis-related proteins; Figure B shows the detection results of macrophage phenotypes in the pelvic ganglia of the control group; Figure C shows the detection results of macrophage phenotypes in the pelvic ganglia after NE-RH treatment.

[0055] Figure 8 The figure shows the results of neurite length in PC12 cells after treatment with different concentrations of NE-RH.

[0056] Figure 9 The figure shows the results of detecting the effect of 0.02 μM NE-RH on the phenotype of RAW264.7 macrophages in Example 6. Detailed Implementation

[0057] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0058] Example 1: Synthesis and purification of NE-RH

[0059] This embodiment provides a method for the synthesis and purification of norepinephrine-coupled rhein (NE-RH), as well as the detection and analysis of the prepared NE-RH. The specific experimental steps are shown below.

[0060] (1) In a 50 mL flask, add 17 mg norepinephrine, 30 mg rhein, and 10 mL dichloromethane, and react at room temperature (20-25 °C) for 0.5 hours;

[0061] (2) Add 15 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 11 mg of N-hydroxysuccinimide (NHS) to the product obtained from the reaction in (1), and stir at room temperature for 12 hours.

[0062] (3) After the reaction in (2) is completed, the reaction solution is washed with water, the organic phase is separated, the organic solvent is evaporated, and each fraction is separated by column chromatography (adsorbent: silica gel; eluent: dichloromethane). A small amount of each fraction and the reactants (norepinephrine and rhein) in the reaction system are spotted with a capillary tube and spotted on a silica gel plate. The plate is placed in a developing tank and developed with the same eluent system as column chromatography. The silica gel plate is removed, the solvent is evaporated, and the spots are observed by ultraviolet light (UV 254 / 365 nm). The fractions with the same spots as the reactants are removed, and the fractions with the same spots are combined. The solvent is removed by rotary evaporator. Then, each fraction is detected by nuclear magnetic resonance hydrogen spectrum and infrared spectrum to identify the target compound (NE-RH) and byproducts.

[0063] The obtained NE-RH was dissolved in deuterated DMSO solution for determination. 1 The obtained NE-RH was ground and compressed with potassium bromide powder, and analyzed by FTIR at 400-4000 cm⁻¹. -1 The infrared spectrum of NE-RH was obtained at a certain frequency.

[0064] The yield of norepinephrine-coupled rhein obtained in the experiment was 18 mg (38%). The reaction formula for norepinephrine-coupled rhein is as follows: Figure 1 As shown.

[0065] 1H NMR (1H NMR) 1 H NMR analysis such as Figure 2 As shown, the proton NMR spectrum reveals that the compound contains six active hydrogen atoms at δ 11.89 (2H, s, 1-OH, 9-OH) and 8.94 (1H, t, δ). J =5.6 Hz, NH), 8.83 (1H, s, 3'-OH), 8.71 (1H, s, 4'-OH), 5.32 (1H, s, 7'-OH), 8 aromatic hydrogens at δ 8.14 (1H, d, J =1.4 Hz, H-4), 7.84 (1H, t, J =7.9 Hz, H-7), 7.76(2H, m, H-2, 6), 7.42(1H, d, J =8.3 Hz, H-8), 6.79 (1H, d, J =1.9Hz, H-2'), 6.67(1H, d, J =8.0 Hz, H-5'), 6.60 (1H, dd, J=8.1, 1.9 Hz, H-6'), based on peak shape and coupling constant, the compound contains one ortho-trisubstituted benzene ring, one 1,2,3,5-tetrasubstituted benzene ring, and one 1,2,4-trisubstituted benzene ring. One hydroxymethyl hydrogen is at δ 4.62 (1H, m, H-7'), and two azo-methylene hydrogens are at δ 3.41 (1H, m, H-8') and 3.28 (1H, dd, ). J =8.1, 5.3 Hz, H-8'); In summary, the spectral data of the compound are consistent with the structural formula.

[0066] The NMR data are distributed as follows: 1 H NMR(600 MHz, DMSO)δ 11.89(2H, s, 1-OH, 9-OH),8.94(1H, t, J = 5.6 Hz, NH), 8.83(1H, s, 3'-OH), 8.71(1H, s, 4'-OH), 8.14(1H,d, J = 1.4 Hz, H-4), 7.84(1H, t, J = 7.9 Hz, H-7), 7.76(2H, m, H-2, 6), 7.42(1H,d, J = 8.3 Hz, H-8), 6.79 (1H, d, J = 1.9 Hz, H-2'), 6.67(1H, d, J = 8.0 Hz, H-5'), 6.60(1H, dd, J = 8.1, 1.9 Hz, H-6'), 5.32(1H, s, 7'-OH), 4.62(1H, m, H-7'), 3.41(1H, m, H-8'), 3.28(1H, dd, J = 8.1, 5.3 Hz, H-8').

[0067] Fourier transform infrared spectrum as Figure 3 As shown, 3386 cm -1 The location represents the OH stretching vibration / NH stretching vibration: 1627 cm. -1 The band at 1550 cm⁻¹ may be due to the C=O stretching vibration of amides (amide I band). -1 The location may be due to the CNH bending vibration of secondary amides (amide II band) / aromatic ring skeletal vibration; 1450 cm⁻¹ -1 The location may be due to aromatic ring skeletal vibration; 1375 cm -1 The location may be due to aromatic ring skeletal vibration; 1273 cm-1 The band at 1198 cm⁻¹ may represent the CN stretching vibration of amide (amide III band) or the C-OH stretching vibration of phenol. -1 The position may be due to the C-OH stretching vibration of phenol; 1076 cm⁻¹ -1 The location may be due to in-plane bending vibration of the aromatic ring (CH) / stretching vibration of C-OH; 1003 cm⁻¹ -1 The location may be due to in-plane bending vibration of the aromatic ring (CH) / stretching vibration of C-OH; 898 cm -1 The location may be due to the out-of-plane bending vibration of the aromatic ring (CH); 812 cm -1 The location may be due to the out-of-plane bending vibration of the aromatic ring (CH); 746 cm -1 The position may be due to the out-of-plane bending vibration of the CH group in the aromatic ring. Since the reactants contain a carboxyl group (-COOH), the carbonyl C=O stretching vibration of the carboxyl group is generally located at 1700 cm⁻¹. -1 The above wavenumbers were observed, but only 1627 cm⁻¹ was found in the infrared spectrum. -1 The characteristic peak at this point is because the carbonyl C=O stretching vibration shifts to a lower wavenumber due to the conjugation effect of N, indicating that the sample does not contain a carboxyl group, suggesting that the reaction synthesized the target substance.

[0068] Example 2: The protective effect of NE-RH on pelvic ganglion neurons

[0069] This embodiment provides the detection of the protective effect of NE-RH prepared in Example 1 on pelvic ganglion neurons. The specific experimental steps are as follows.

[0070] Establishment of a pelvic nerve injury model in SD rats: After surgically dissecting and separating the pelvic ganglia of rats, the pelvic ganglia were clamped with vascular clips for 2 minutes to establish a pelvic ganglion clamping injury model.

[0071] The experimental groups were as follows: Ten SD rats (2 months and older, female, 250-280g) were randomly divided into two groups: the experimental group (n=5) was administered NE-RH by gavage for 4 weeks at a dose of 0.5 mmol / kg based on the ratio of NE-RH usage to rat body weight; the control group (n=5) was administered the same amount of sterile water for injection by gavage for 4 weeks.

[0072] Four weeks after surgery, rats were sacrificed, and pelvic ganglia were isolated. The expression of NeuN was detected by immunofluorescence, and the changes in the number of neurons in the pelvic ganglia were analyzed.

[0073] The experimental steps for detecting NeuN expression by immunofluorescence are as follows:

[0074] First, the obtained pelvic ganglion tissue was rapidly fixed in 4% paraformaldehyde (PFA). After fixation, the tissue was dehydrated using a sucrose gradient and cryoprotected, then embedded in OCT embedding medium and rapidly frozen at low temperature. The tissue was cut into 10-micrometer sections using a cryostat and attached to anti-detachment slides. Before staining, the sections were rehydrated at room temperature and washed with PBS. To enhance antibody penetration, permeabilization was performed using PBS containing Triton X-100, followed by blocking of non-specific binding sites with blocking buffer containing normal serum. Diluted anti-NeuN primary antibody (abcam, ab177487, 1:200) was added, and the mixture was incubated overnight in a humidified chamber at 4°C. The next day, unbound primary antibody was thoroughly washed with PBS to remove it, and fluorescently labeled secondary antibody (Invitrogen, A-11011, 1:200) was added, and the mixture was incubated at room temperature in the dark for 1-2 hours. After thorough washing with PBS, the cell nuclei were counterstained with DAPI. Finally, the slide was sealed with an anti-quenching mounting medium, stored in the dark, and observed under a fluorescence microscope or confocal microscope after curing for the expression and localization of NeuN (positive signal in neuronal nuclei) and DAPI (all cell nuclei).

[0075] Experimental results are as follows Figure 4 As shown in the AI, compared with normal SD rats ( Figure 4 AC), control group ( Figure 4 DF) and NE-RH treatment group ( Figure 4 The number of neurons in the pelvic ganglion (GI) was reduced in both groups, indicating that constructing a pelvic ganglion injury model in SD rats using ganglion pinching is feasible. The number of neurons in the pelvic ganglion of the control group was significantly lower than that in the NE-RH treatment group, with a statistically significant difference. This suggests that NE-RH can promote neuronal survival after pelvic ganglion injury and exert a neuroprotective effect in vivo.

[0076] Example 3: NE-RH promotes bladder function recovery after pelvic nerve injury in SD rats.

[0077] This embodiment demonstrates the effect of NE-RH prepared in Example 1 on the recovery of bladder function in SD rats after pelvic nerve injury. The specific experimental steps are as follows. The SD rat pelvic nerve injury model was constructed in the same manner as in Example 2.

[0078] The experimental groups were as follows: 20 SD rats (2 months and older, female, 250-280g) were randomly divided into 4 groups: sham group, solvent group (physiological saline), rhein (RH) group (SD rats were gavaged daily at a dose of 0.5 mmol / kg based on the ratio of RH usage to mouse body weight for 4 weeks), and NE-RH group (SD rats were gavaged daily at a dose of 0.5 mmol / kg based on the ratio of NE-RH usage to mouse body weight for 4 weeks).

[0079] The bladder volume of SD rats in each group was measured 4 weeks after surgery. The measurement steps are as follows.

[0080] SD rats were anesthetized with isoflurane inhalation and fixed in a supine position. The urethral orifice was disinfected. A 19G cystometry catheter (connected to a three-way stopcock and pressure sensor) was gently inserted into the bladder through the urethra (approximately 3 cm deep), and the catheter tip was fixed after confirmation of its position. 37°C physiological saline was continuously infused through the catheter at a rate of 6 mL / h (approximately 100 μL / min) using a constant flow pump, while simultaneously recording changes in intravesical pressure in real time. Infusion was stopped immediately when leakage of urine was observed at the urethral orifice (i.e., fluid overflow due to detrusor muscle contraction). The total infused volume at this point is the maximum bladder capacity.

[0081] Rats were sacrificed 4 weeks after surgery, and bladder tissue was collected for Masson staining. The specific steps are as follows:

[0082] Bladder tissue from SD rats was fixed in 4% paraformaldehyde for 24 hours, then dehydrated using a series of ethanol solutions, embedded in paraffin, and sectioned to a thickness of 5 μm. After dewaxing to water, the following steps were performed: Weigert iron hematoxylin staining of nuclei for 5-10 minutes (nuclei appear dark blue), followed by washing and differentiation; Ponceau S-acid fuchsin staining for 5 minutes (muscle fibers / cytoplasm appear red), followed by rinsing with distilled water; differentiation with 1% phosphomolybdic acid solution for 1-2 minutes (to remove non-specific staining); counterstaining with 2% aniline blue solution for 3 minutes (collagen fibers appear blue), followed by a short rinse with 1% glacial acetic acid. The sections were then dehydrated using a series of ethanol solutions, cleared with xylene, and mounted with neutral resin. Microscopic examination revealed red smooth muscle fibers, blue collagen fibers, and dark blue-black nuclei, which were used for quantitative analysis of bladder wall fibrosis and muscle layer thickness.

[0083] Experimental results are as follows Figure 5 and Figure 6 As shown, Figure 5 Masson staining of bladder tissue showed that the thickness of the detrusor muscle in the sham-operated group and the NE-RH group was better than that in the solvent group and the RH group, while the degree of bladder fibrosis was less than that in the solvent group and the RH group. These experimental results further validated histologically that NE-RH can promote the recovery of bladder function. Figure 6 The thickness of the bladder detrusor muscle was shown in the sham surgery group and the NE-RH group. Figure 6 (A) and bladder capacity ( Figure 6 The results showed that, at conventional doses, RH could not promote the recovery of bladder function after pelvic nerve injury in SD rats, while the same molar dose of NE-RH could promote the recovery of bladder function.

[0084] Example 4: Effects of NE-RH on NLRP3-driven macrophage pyroptosis and macrophage M1 polarization following pelvic ganglion injury in SD rats.

[0085] This embodiment demonstrates the effects of NE-RH prepared in Example 1 on NLRP3-driven macrophage pyroptosis and macrophage M1 polarization following pelvic ganglion injury in SD rats. The SD rat pelvic nerve injury model was constructed as in Example 2.

[0086] The experimental groups were as follows: NE-RH group (SD rats were administered NE-RH by gavage at a dose of 0.5 mmol / kg per day for 4 weeks); control group (SD rats were administered physiological saline by gavage for 4 weeks).

[0087] Four weeks post-surgery, rats were sacrificed, and pelvic ganglia were harvested. Western blot analysis was used to detect the expression of NLRP3-driven pyroptosis-related proteins in the pelvic ganglia, including NLRP3, ASC, Caspase-1, Cleaved Caspase-1, and GSDMD. The specific experimental procedures were as follows: SD rats were euthanized by cervical dislocation, and pelvic ganglia tissue was rapidly dissected and obtained. The tissue was handled on ice, rinsed with physiological saline, blotted dry with filter paper, and immediately flash-frozen in liquid nitrogen and stored at -80°C. For protein extraction, an appropriate amount of tissue was weighed into pre-chilled lysis buffer (RIPA lysis buffer containing protease and phosphatase inhibitors), ground on ice, incubated at 4°C for 30-60 minutes, centrifuged at 12000-16000 g for 15 minutes at 4°C, and the supernatant was collected. The total protein concentration was determined using the BCA method. SDS-PAGE separating gels (8-15%) and 5% stacking gels were prepared according to the target protein molecular weight. Mix an equal volume of protein (50 μg) with loading buffer, boil to denature for 5-10 minutes, and then load the sample. Simultaneously add a pre-stained protein molecular weight marker and a housekeeping protein internal control (β-actin). Perform constant voltage electrophoresis (80-100V for stacking gel, 120-150V for separating gel), stopping when the target band migrates to the appropriate position. Transfer the protein to a PVDF membrane using a wet transfer method, performing the transfer in a 4°C ice bath or cold room. After transfer, rinse the membrane with TBST and block with 5% skim milk powder at room temperature for 1 hour. Dilute the specific primary antibodies (anti-NLRP3 antibody (Abcam, ab263899, 1:500), anti-ASC antibody (Abcam, ab283684, 1:500), anti-Caspase-1 antibody (Abcam, ab207802, 1:1000), anti-Cleaved caspase-1 antibody (Cell Signailng, 89332S, 1:500), anti-GSDMD antibody (Abcam, ab219800, 1:500), and anti-GAPDH antibody (Abcam)) with blocking buffer and incubate overnight at 4°C. After thorough washing with TBST, incubate with the corresponding species' HRP-labeled secondary antibody (Sangon Biotech (Shanghai) Co., Ltd., D110058) at room temperature for 1-2 hours, followed by another TBST wash. Cover the membrane with ECL chemiluminescent substrate working solution, incubate at room temperature for 1-5 minutes, capture the signal using a chemiluminescence imaging system, and save the image. ImageJ and other software were used to analyze the gray values ​​of the target band and the intrinsic reference band, calculate the relative expression level, and perform statistical analysis.

[0088] Macrophage polarization detection: Immunofluorescence detection of M1 macrophages (ED1) in pelvic ganglia + iNOS + M2 macrophages (ED1) + CD163+ The specific experimental steps are as follows: (The ratio of )

[0089] Pelvic ganglia from SD rats were fixed with 4% paraformaldehyde for 6 hours, dehydrated with 30% sucrose, and then embedded in OCT to prepare 10μm frozen sections. After antigen retrieval (0.01M citrate buffer, 95℃ for 15 minutes), the sections were blocked with PBS containing 10% serum + 0.3% Triton X-100 for 1 hour. Add a mixture of rabbit anti-ED1 (pan-macrophage marker, abcam, ab283654, 1:200) and mouse anti-iNOS (M1 marker, abcam, ab31630, 1:200) primary antibodies, and a mixture of rabbit anti-ED1 (pan-macrophage marker) and mouse anti-CD163 (M2 marker, abcam, ab289979, 1:200) primary antibodies, respectively, and incubate overnight at 4°C. After washing with PBS, incubate for 2 hours in the dark with Alexa Fluor 568-labeled goat anti-rabbit secondary antibody (Invitrogen, A-11011, 1:200) and Alexa Fluor 488-labeled goat anti-mouse secondary antibody (Invitrogen, A-11029, 1:200), respectively. Rinse thoroughly with PBS, stain with DAPI, and mount. Determine under a confocal microscope: M1 type (ED1) + iNOS + M2 type (ED1) + CD163 + ).

[0090] Experimental results are as follows Figure 7 As shown, Figure 7 Western blot analysis of the NE-RH group showed that, compared with the control group, the NE-RH group had low expression of NLRP3-driven pyroptosis-related proteins such as NLRP3, ASC, Caspase-1, Cleaved caspase-1, and GSDMD in the pelvic ganglia. Figure 7 Immunofluorescence assays in the pelvic ganglia of the NE-RH group showed that M2 macrophages (ED1) were the dominant macrophages. + CD163 + ) as the main ( Figure 7 In the middle C), while in the control group, the macrophages in the pelvic ganglia were mainly M1 type macrophages (ED1). + iNOS + ) as the main ( Figure 7 (Middle B). This result suggests that NE-RH may exert its neuroprotective effect by influencing NLRP3-driven pyroptosis and macrophage polarization.

[0091] Example 5: Effects of NE-RH on in vitro neuronal cell neurite growth

[0092] This embodiment demonstrates the effect of NE-RH on the growth of neuronal neurites in vitro. A PC12 cell line derived from rat adrenal pheochromocytoma was used to induce differentiation into PC12 cells to detect the effect of NE-RH on the growth of neuronal neurites in vitro. The specific experimental steps are as follows.

[0093] PC12 cells were spaced at 1 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates and pre-coated for 24 hours. The medium was then replaced with DMEM / F12 induction medium containing 1% FBS, 50 ng / mL NGF (nerve growth factor), 20 ng / mL BDNF (brain-derived neurotrophic factor), 15 μM forskolin, and 1% (v / v) Pen / Strep. The cells were cultured for 5 days (half the medium was changed every 48 hours) until >50% of the cells differentiated into neurites (length ≥ twice the cell body diameter).

[0094] The above-mentioned neurally differentiated PC12 cells in the 96-well plates were incubated for 24 hours with 200 μL of the above-mentioned induction culture medium containing different concentrations of NE-RH (0, 0.02, 0.04 and 0.08 μM). The control group was the induction culture medium.

[0095] Detection of neuronal dysplasia and regeneration capacity: The immunofluorescence experiment is the same as in Example 2, except that this example uses β-tubulin III antibody (abcam, ab18207, 1:200). After immunofluorescence staining, the length of neuronal cell dysplasia is observed under a microscope.

[0096] Experimental results are as follows Figure 8 As shown, the length of neuronal neurites was significantly concentration-dependent with NE-RH, indicating that NE-RH also has a significant effect on promoting neurite growth in vitro.

[0097] Example 6: Effect of NE-RH on inhibiting M1 polarization of macrophages in vitro

[0098] This embodiment demonstrates the effect of NE-RH on inhibiting M1 polarization of macrophages in vitro. RAW264.7 cells were treated with lipopolysaccharide and then co-incubated with a mechanically damaged PC12 neuron model incubated with NE-RH (NE-RH group), while the control group consisted of macrophages treated with PBS and co-incubated with a mechanically damaged PC12 neuron model (PBS group). The specific experimental steps are shown below.

[0099] (1) RAW264.7 cells cultured under normal conditions (passage ≤ 25, density 80% confluence) were gently washed twice with sterile PBS and replaced with DMEM complete medium containing 10% FBS + 1% penicillin / streptomycin. Lipopolysaccharide (LPS) was added to a final concentration of 100 ng / mL and the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The control group was washed with PBS and cultured under the same conditions.

[0100] (2) The mechanically damaged PC12 neuron model was incubated for 24 hours with 200 μL of induction culture medium containing different concentrations of NE-RH (0, 0.02, 0.04 and 0.08 μM) (DMEM / F12 medium containing 1% FBS, 50 ng / mL NFG, 20 ng / mL BDNF, 15 μM forskolin and 1% (v / v) Pen / Srep). The control group was incubated with induction culture medium without NE-RH. The mechanically damaged PC12 neuron model was constructed based on the neuron-induced differentiation into PC12 cell model in Example 5. A cross-shaped mechanical injury (1 mm wide) was vertically scratched with a 200 μL sterile pipette tip. After removing the debris with PBS, the medium was replaced with DMEM / F12 medium containing 1% FBS and stabilized for 2 hours.

[0101] (3) The RAW264.7 macrophages treated above and the PC12 neuron model after injury were co-incubated in a Transwell co-culture system (Corning, 0.4 μm pore size) for 12 hours at a cell count ratio of 1:1, with RAW264.7 in the upper chamber and PC12 in the lower chamber (both at a density of 2×10⁻⁶ cells / mL). 5 (cells / mL). After co-culture, RAW264.7 cells from the upper chamber were collected, and the proportion of M1 and M2 macrophages was detected by flow cytometry.

[0102] Experimental results are as follows Figure 9 As shown, compared with the control group (PBS group), the NE-RH group, after being treated with 0.02 μM NE-RH, showed a tendency of M2 polarization in macrophages after co-incubation with mechanically damaged PC12 neurons. This in vitro experimental result suggests that NE-RH may exert its therapeutic effect by affecting macrophage polarization.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of the coupling agent in the preparation of products for treating pelvic nerve injury, characterized in that, The coupling is shown in the following formula: 。

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