Application of medicine composition in preparation of medicine for treating multiple sclerosis

Abstract

The invention relates to application of a pharmaceutical composition in preparation of a medicine for treating multiple sclerosis. The pharmaceutical composition comprises at least one of an SPAK kinase inhibitor, a down-regulating agent for reducing SPAK kinase expression, a down-regulating agent for reducing MMP2 expression and a down-regulating agent for reducing MMP9 expression. According to the SPAK inhibitor and the down-regulating agent for reducing SPAK expression, the expression of MMP2 and MMP9 is reduced by reducing phosphorylation of SPAK, so that the effect of treating multiple sclerosis is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of a pharmaceutical composition in preparing a drug for treating multiple sclerosis. Background Art

[0002] Multiple sclerosis (MS) is one of the common idiopathic inflammatory demyelinating diseases of the central nervous system (CNS). Its pathogenesis is mainly that autoreactive T lymphocytes in the human peripheral blood migrate to the CNS through the damaged blood-brain barrier, forming immune infiltration, attacking the myelin sheath and axons that protect the nerves, causing damage and shedding, and inhibiting nerve signal conduction. Typical clinical manifestations include but are not limited to visual impairment, limb weakness or sensory loss, cognitive dysfunction and potential disability.

[0003] Experimental autoimmune encephalomyelitis (EAE) is a disease characterized by specific sensitization of CD4 + EAE is an autoimmune disease of the central nervous system mediated by T lymphocytes, characterized by mononuclear cell infiltration around CNS blood vessels and white matter demyelination. Its pathological changes are similar to those of MS. Therefore, EAE animal models are widely used in the pathogenic mechanism and preclinical research of MS.

[0004] The choroid plexus (ChP) is an indispensable component for maintaining the immune status of the CNS. The tight junctions between the ChP epithelial cells form the blood-cerebrospinal fluid barrier to prevent biomacromolecules and immune cells from entering the cerebrospinal fluid. The "first wave" of T cell infiltration in the ChP indicates the decisive role of the ChP in EAE.

[0005] SPAK (Ste20-associated proline / alanine-rich kinase) can regulate the secretion of cerebrospinal fluid by regulating its downstream gene NKCC1 (sodium-potassium-chloride cotransporter 1). Studies have found that activation of the SPAK-NKCC1 cascade in ChP can promote immune inflammatory responses and aggravate stroke. Other studies have shown that activation of SPAK can lead to increased infiltration of immune cells in ChP, increased cerebrospinal fluid production in choroid plexus epithelial cells, and thus aggravated hydrocephalus. In addition, pharmacological inhibition of SPAK reduces cerebrospinal fluid secretion, alleviates stroke-related brain edema, and maintains the integrity of the ChP barrier.

[0006] Currently, the role of SPAK in ChP gateway activity remains largely unknown, and there have been no reports on the use of drugs to treat multiple sclerosis by regulating the barrier function of choroid plexus epithelial cells by modulating SPAK protein activity. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an application of a pharmaceutical composition in the preparation of a drug for treating multiple sclerosis in view of the current status of the prior art.

[0008] The technical solution adopted by the present invention to solve the above technical problems is: use of a pharmaceutical composition in the preparation of a drug for treating multiple sclerosis, the pharmaceutical composition comprising at least one of a SPAK kinase inhibitor, a down-regulator for reducing the expression of SPAK kinase, a down-regulator for reducing the expression of MMP2, and a down-regulator for reducing the expression of MMP9. The SPAK kinase inhibitor and the down-regulator for reducing the expression of SPAK kinase reduce the phosphorylation of SPAK, thereby reducing the expression of MMP2 and MMP9, regulating the barrier function of choroid plexus epithelial cells, thereby achieving the effect of treating multiple sclerosis.

[0009] Preferably, the inhibitor of SPAK kinase comprises ZT-1a.

[0010] Preferably, the administration of ZT-1a is intraperitoneal injection. ZT-1a can penetrate the blood-brain barrier, which is convenient for administration.

[0011] Preferably, the pharmaceutical composition comprises a down-regulator that reduces the expression of MMP2 and a down-regulator that reduces the expression of MMP9. Simultaneously reducing the expression of MMP2 and MMP9 can increase the effect of treating multiple sclerosis.

[0012] Preferably, the down-regulator for reducing the expression of SPAK kinase comprises at least one of active peptides, proteins, oligonucleotides and small molecule compounds that inhibit the expression of SPAK kinase.

[0013] Preferably, the down-regulator for reducing MMP2 expression comprises at least one of active peptides, proteins, oligonucleotides and small molecule compounds that inhibit MMP2 expression.

[0014] Preferably, the down-regulator for reducing the expression of MMP9 kinase includes at least one of active peptides, proteins, oligonucleotides and small molecule compounds that inhibit the expression of MMP9.

[0015] Compared with the prior art, the advantages of the present invention are: revealing that in the pathological process of EAE / MS, choroid plexus SPAK is highly expressed, promoting the expression of MMP2 / 9, degrading choroid plexus matrix proteins and tight junction proteins between epithelial cells, thereby causing damage to the choroid plexus barrier function and aggravating immune cell infiltration and damage; SPAK inhibitor ZT-1a penetrates the blood-brain barrier to target the choroid plexus for treatment, which can effectively alleviate the pathological process and reduce the inflammatory response; the present invention is the first to explore the mechanism of the involvement of choroid plexus SPAK protein in the pathological process of multiple sclerosis, providing a new idea for the treatment of multiple sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing changes in the expression of SPAK, p-SPAK, and p-NKCC1 proteins in the choroid plexus in Example 1 of the present invention from 0 to 20 days after EAE induction; wherein, Figure 1 A is the distribution and fluorescence intensity of SPAK protein in the choroid plexus at different phases of EAE induction; Figure 1 B is Figure 1 A is a quantitative statistical graph of the relative fluorescence intensity of SPAK in the choroid plexus; Figure 1 C is the distribution and fluorescence intensity of p-SPAK protein in the choroid plexus at different phases of EAE induction; Figure 1 D is Figure 1 C is a quantitative statistical graph of the relative fluorescence intensity of p-SPAK in the choroid plexus; Figure 1 E is the distribution and fluorescence intensity of p-NKCC1 protein in the choroid plexus at different phases of EAE induction; Figure 1 F is Figure 1 Quantitative statistical graph of the relative fluorescence intensity of p-NKCC1 in the choroid plexus in E; (Two-Way ANOVA Repeated Measures, *P<0.05, **P<0.01, ***P<0.001, n=5 / group);

[0017] Figure 2 This is a diagram of choroid plexus SPAK signals before and after choroid plexus specific knockdown in Example 2 of the present invention; Figure 2 In A, the SPAK knockdown virus was injected into the unilateral lateral ventricle of mice. Two weeks later, green fluorescent protein was specifically expressed in the choroid plexus tissue. SPAK immunofluorescence staining was performed on the choroid plexus tissue, and it was found that the red fluorescence intensity of the SPAK knockdown group was significantly reduced. Figure 2 B is Figure 2 Quantitative statistical graph of relative fluorescence intensity of SPAK in choroid plexus in A (Unpaired T test, ***P<0.001, n=5 / group); Figure 2 In C, mice were injected with SPAK knockdown virus into the unilateral lateral ventricle, and choroid plexus tissue was obtained by PBS perfusion 2 weeks later. Q-PCR results showed that the mRNA level of SPAK was reduced (Unpaired T test, **P<0.01, n=5 / group);

[0018] Figure 3 This is a graph showing the effect of targeted inhibition of choroid plexus SPAK signaling on EAE in Example 3 of the present invention; Figure 3 In A, targeted inhibition of SPAK signaling at the choroid plexus can effectively alleviate the pathological score of EAE (Two-Way ANOVA RepeatedMeasures, n=5 / group); Figure 3In B and 3C, HE staining showed that the infiltration of spinal cord immune cells decreased after inhibiting the SPAK signal at the choroid plexus (Unpaired T test, n = 5 / group); Figure 3 D, 3E, inhibition of SPAK signal in the choroid plexus improved the degree of spinal cord demyelination (Unpaired T test, n = 5 / group); Figure 3 F, 3G, inhibition of SPAK signaling in the choroid plexus reduces CD4 + T lymphocyte infiltration in the spinal cord (Unpaired T test, n = 5 / group); Figure 3 In H and 3I, inhibition of SPAK signaling in the choroid plexus reduced CD4 + T lymphocyte infiltration in the choroid plexus (Unpaired T test, n = 5 / group); Figure 3 In J and 3K, inhibition of SPAK signaling in the choroid plexus significantly inhibited the activation of spinal macrophages and microglia (Unpaired T test, n = 5 / group); Figure 3 In L and 3M, after inhibiting the SPAK signal at the choroid plexus, 3D reconstruction showed that the activation of spinal microglia was inhibited (Unpaired Ttest, n = 30 / group);

[0019] Figure 4 This is a result diagram of immune cells in the cerebrospinal fluid and spinal cord that target and inhibit SPAK signals in the choroid plexus in Example 3 of the present invention; wherein, Figure 4 In A, flow cytometry was used to detect CD4 + T cells, IFN-γ + T cells and IL-17 + T cell count; Figure 4 In B, 4C, and 4D, inhibition of SPAK expression in the choroid plexus reduced CD4 + T cells, IFN-γ + T cells and IL-17 + T cell count; Figure 4 In E, 4F, and 4G, inhibition of SPAK expression in the choroid plexus reduced CD4 + T cells, IFN-γ + T cells and IL-17 + T cell number; (Unpaired Ttest, *P<0.05, **P<0.01, n=5 / group);

[0020] Figure 5 This is a diagram showing the effect of targeted inhibition of choroid plexus SPAK signaling on tight junction protein and MMP protein expression in Example 4 of the present invention; Figure 5In A-5G, 2 weeks after intracerebroventricular injection of SPAK knockdown virus, choroid plexus tissues were obtained for Q-PCR analysis of OCLN, ZO-1, Claudin1, Claudin3, Claudin5, MMP2, and MMP9 protein expression levels (n=5 / group); Figure 5 In H , OCLN immunofluorescence images of ChP in the untreated group, after ICV injection of SPAK knockdown virus or control virus under physiological conditions ( n = 5 / group); Figure 5 In I, MMP2 immunofluorescence images of ChP in the untreated group, after ICV injection of SPAK knockdown virus or control virus under physiological conditions (n ​​= 5 / group); Figure 5 In J, MMP9 immunofluorescence images of ChP in the untreated group, after ICV injection of SPAK knockdown virus or control virus under physiological conditions; Figure 5 K, quantitative analysis as Figure 5 H shows the OCLN fluorescence intensity (n=5 / group); Figure 5 L, quantitative analysis as Figure 5 I shows the fluorescence intensity of MMP2 (n=5 / group); Figure 5 M, quantitative analysis as Figure 5 J shows the fluorescence intensity of MMP9 (n=5 / group); Figure 5 In N , OCLN immunofluorescence images of ChP in the untreated group, after ICV injection of SPAK knockdown virus or control virus under pathological conditions ( n = 5 / group); Figure 5 In O , MMP2 immunofluorescence images of ChP in the untreated group, after ICV injection of SPAK knockdown virus or control virus under pathological conditions ( n = 5 / group); Figure 5 In P, immunofluorescence images of MMP9 in ChP of untreated group, ICV injection of SPAK knockdown virus or control virus under pathological conditions; Figure 5 In Q, quantitative analysis is as follows Figure 5 N indicates OCLN fluorescence intensity (n=5 / group); Figure 5 In R, quantitative analysis is performed as Figure 5 O shows the fluorescence intensity of MMP2 (n=5 / group); Figure 5 S, quantitative analysis as Figure 5 MMP9 fluorescence intensity shown in P (n=5 / group); (Unpaired T test, *P<0.05, **P<0.01, ***P<0.001);

[0021] Figure 6 This is a result diagram of the effect of the SPAK-NKCC1 antagonist on EAE pathological damage in Example 5 of the present invention; wherein, Figure 6In A, in the EAE animal model, there was no difference in the pathological scores of mice after intraperitoneal injection of bumetanide every other day (Two-Way ANOVA Repeated Measures, n = 5 / group); Figure 6 In B, in the EAE animal model, intraperitoneal injection of ZT-1a every other day significantly reduced the pathological scores of mice (Two-Way ANOVA Repeated Measures, n = 5 / group); Figure 6 C, 6D, ZT-1a was intraperitoneally injected the next day after EAE induction, and HE staining showed that the infiltration of spinal cord immune cells was reduced (Unpaired T test, n = 5 / group); Figure 6 In E and 6F, intraperitoneal injection of ZT-1a the day after EAE induction significantly improved the degree of spinal cord demyelination (Unpaired Ttest, n = 5 / group); Figure 6 G, 6H, ZT-1a was injected intraperitoneally every other day after EAE induction, CD4 + T lymphocyte infiltration in the spinal cord was reduced (Unpaired T test, n = 5 / group); Figure 6 In I and 6J, ZT-1a was injected intraperitoneally every other day after EAE induction, and CD4 + The infiltration of T lymphocytes in the choroid plexus was reduced (Unpaired T test, n = 5 / group); Figure 6 In K and 6L, intraperitoneal injection of ZT-1a the day after EAE induction significantly inhibited the activation of spinal macrophages and microglia (Unpaired T test, n = 5 / group); Figure 6 In M and 6N, ZT-1a was injected intraperitoneally the next day after EAE induction. 3D reconstruction showed that the activation of spinal microglia was inhibited, which was manifested by a decrease in cell body diameter, an increase in the number of cell synaptic branches, and a lengthening of the branches (Unpaired T test, n = 30 / group); (ns = P > 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001);

[0022] Figure 7 This is a result diagram of immune cells in the cerebrospinal fluid and spinal cord of mice after injection of ZT-1a in Example 5 of the present invention; wherein, Figure 7 In A, ZT-1a or control solvent was injected intraperitoneally after EAE induction, and CD4 + T cells, IFN-γ + T cells and IL-17 + T cell count; Figure 7 In B, 7C, and 7D, intraperitoneal injection of ZT-1a after EAE induction reduced the number of CD4 + T cells, IFN-γ+ T cells and IL-17 + T cell count; Figure 7 In E, 7F, and 7G, intraperitoneal injection of ZT-1a after EAE induction reduced CD4 + T cells, IFN-γ + T cells and IL-17 + T cell number; (UnpairedT test, *P<0.05, **P<0.01, ***P<0.001, n=5 / group);

[0023] Figure 8 This is a result diagram of the effect of targeted inhibition of choroid plexus MMP signaling on EAE pathological manifestations in Example 6 of the present invention; wherein, Figure 8 In A, targeted inhibition of MMP2, MMP9, and MMP2 / 9 at the choroid plexus can effectively reduce the pathological score of EAE (Two-Way ANOVARepeated Measures, n=5 / group); Figure 8 In B and 8C, inhibition of MMP2, MMP9, and MMP2 / 9 at the choroid plexus significantly inhibited the activation of spinal macrophages and microglia (One-Way ANOVA, n = 5 / group); Figure 8 In D and 8E, inhibition of MMP2, MMP9, and MMP2 / 9 at the choroid plexus improved the degree of spinal cord demyelination (One-Way ANOVA, n = 5 / group); Figure 8 In F and 8G, HE staining showed that the infiltration of spinal cord immune cells was reduced in the choroid plexus with inhibition of MMP2, MMP9, and MMP2 / 9 (One-Way ANOVA, n=5 / group); (*P<0.05, **P<0.01, ***P<0.001, ###P<0.001);

[0024] Fig. 9 7 is a graph showing the effect of choroid plexus-specific overexpression of SPAK (SPAK-OE) on EAE pathology in Example 1 of the present invention; wherein, Fig. 9 In A, choroid plexus-specific overexpression of SPAK aggravated EAE pathological scores (Two-Way ANOVARepeated Measures, n=5 / group); Fig. 9 B, 9C, choroid plexus-specific overexpression of SPAK promoted the activation of spinal macrophages and microglia (Unpaired T test, n = 5 / group); Fig. 9 D, 9E, choroid plexus-specific overexpression of SPAK aggravated spinal cord demyelination (Unpaired T test, n = 5 / group); Fig. 9In F and 9G, HE staining showed that the infiltration of spinal cord immune cells was aggravated after SPAK was specifically overexpressed in the posterior choroid plexus (Unpaired T test, n=5 / group); (*P<0.05, **P<0.01, ***P<0.001, #P<0.05). DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to the accompanying drawings.

[0026] Studies have reported that under the pathological conditions of experimental autoimmune myelitis (EAE), the "first wave" of immune cells first infiltrates the central nervous system through the choroid plexus and mediates the subsequent migration of immune cells through the blood-brain barrier to the central nervous system. Other studies have shown that the expression of SPAK and NKCC1 in choroid plexus epithelial cells far exceeds that of other cell types. SPAK is an important link between regulating inflammatory signals and cerebrospinal fluid. Activating SPAK can lead to increased infiltration of immune cells in the choroid plexus.

[0027] Example 1 Variation characteristics of SPAK, p-SPAK, and p-NKCC1 in different phases of EAE

[0028] 1. Experimental methods

[0029] First, C57BL / 6 mice were modeled with EAE. At six time points, namely 0, 4, 8, 12, 16, and 20 days, the mice were perfused with PBS and samples were collected for immunofluorescence staining of SPAK, p-SPAK, and p-NKCC1. This experiment mainly used wild-type C57BL / 6 female mice, weighing 22-25g and aged 8-10 weeks, purchased from Beijing Spaif Experimental Animal Center and raised in the Experimental Animal Center of Wenzhou Medical University. The breeding temperature was maintained at (24±0.5℃), the humidity was maintained at (60±2%), and the 12h light-dark cycle was followed. The animals involved in the experiment were completely in accordance with the "Regulations on the Administration of Experimental Animals" of the State Science and Technology Commission, and the experimental animal ethics regulations of the Animal Use Ethics Committee of Wenzhou Medical University were followed.

[0030] Among them, the methods for experimental autoimmune encephalomyelitis (EAE) modeling and pathological scoring are as follows:

[0031] (1) Preparation of MOG antigen emulsion: 4 mg of myelin oligodendrocyte glycoprotein (MOG 35-55 , Anaspec) was dissolved in 2 mL of phosphate buffered saline (PBS) to prepare an aqueous solution, and 16 mg of human Mycobacterium tuberculosis (H 37Ra) was dissolved in 2 mL of incomplete Freund's adjuvant to prepare a complete Freund's adjuvant oil solution, and the water and oil solutions were mixed thoroughly at a ratio of 1:1 and then vortexed in an ultra-high speed homogenizer for 15 min under ice bath conditions to prepare a MOG antigen emulsion;

[0032] (2) EAE modeling in mice: After the mice were anesthetized with 3.6% chloral hydrate, a total dose of 10 mL / kg of MOG antigen emulsion was subcutaneously injected at four points on the neck, back, and tail; pertussis toxin (PT, Biological Laboratories Inc.) with a concentration of 500 ng / mL was intraperitoneally injected at 0 h and 48 h after injection of MOG antigen emulsion, with an injection dose of 5 mg / kg;

[0033] (3) Neurological deficit score: Weaver's 15-point method was used for scoring. The specific scoring criteria are as follows:

[0034] Tail: 0 points for normal movement, 1 point for hemiplegia of the tail, 2 points for complete paralysis of the tail;

[0035] Limbs: 0 points for normal movement, 1 point for gait change, 2 points for mild paresis, and 3 points for complete paresis;

[0036] The sum of the scores of the limbs and tail was recorded as the neurological deficit score of the mouse on that day, and death was recorded as 15 points.

[0037] Among them, the method of immunofluorescence staining involved in the present invention is as follows:

[0038] The steps for staining the choroid plexus spread are as follows:

[0039] ① Choroid plexus sample preparation: After the mouse was perfused with PBS, the choroid plexus tissue was quickly removed under a microscope and fixed in 4% PFA for 1 hour;

[0040] ② Wash the fixed choroid plexus in PBS three times, 5 min each time;

[0041] ③ Blocking: Place the choroid plexus in blocking solution and incubate at room temperature for 2 hours;

[0042] ④ Primary antibody incubation: Place the choroid plexus in the primary antibody solution and incubate overnight at room temperature. After incubation, wash with PBS 5 times, 5 minutes each time;

[0043] ⑤ Secondary antibody incubation: Place the choroid plexus in fluorescent secondary antibody solution and incubate at room temperature for 2 hours in the dark (Note: all subsequent steps must be performed in the dark). After the incubation, wash with PBS 5 times, 5 minutes each time;

[0044] ⑥DAPI staining: Place the choroid plexus in DAPI staining solution and incubate at room temperature for 10 minutes away from light. After incubation, wash with PBS 3 times, 5 minutes each time.

[0045] ⑦ Sealing: Spread the choroid plexus on a glass slide under a microscope, wipe off excess water, dry in a dark place, add anti-fade agent, and seal with a coverslip.

[0046] The steps for staining frozen sections are as follows:

[0047] ① Preparation of frozen section samples: After perfusing mice with PBS and 4% PFA, the whole brain and spinal cord were removed and soaked in 4% PFA overnight; the tissue was placed in 30% sucrose solution for dehydration for 2-3 days; the tissue was embedded in OCT glue and frozen, and then coronal sections were cut using a cryostat, with a section thickness of 30 μm for brain and 40 μm for spinal cord, which were attached to a slide and dried at room temperature overnight;

[0048] ② Wash the tissue in PBS for 3 times, 5 min each time;

[0049] ③ Blocking: After washing, wipe off excess water, draw a closed circle around the tissue with a tissue pen, add blocking solution, and incubate in a wet box at room temperature for 2 hours;

[0050] ④ Primary antibody incubation: add primary antibody solution, incubate at room temperature overnight, and wash with PBS 5 times, 5 min / time;

[0051] ⑤ Secondary antibody incubation: add fluorescent secondary antibody solution, incubate at room temperature for 2 hours away from light, and wash with PBS 5 times after incubation, 5 minutes each time;

[0052] ⑥DAPI nuclear staining: add DAPI staining solution, incubate at room temperature in the dark for 10 minutes, wash with PBS 3 times, 5 minutes each time, wipe off excess water, dry in the dark, add anti-fade agent, and seal with coverslip.

[0053] The steps for staining paraffin sections are as follows:

[0054] ① Preparation of paraffin section samples: After perfusing mice with PBS and 4% PFA, the whole brain and spinal cord were removed and immersed in paraffin fixative overnight; after dehydration in an automatic dehydrator, they were immersed in wax and embedded; coronal sections were made with a paraffin slicer, with a section thickness of 10 μm for brain and 7 μm for spinal cord, and the sections were attached to glass slides and dried at room temperature overnight;

[0055] ② Drying: Place the paraffin slices on the slice rack and bake in a 60℃ oven for 1.5 to 2 hours;

[0056] ③ Dewaxing and rehydration: soak in xylene twice, 10 min / time; soak in anhydrous ethanol twice, 5 min / time; soak in 95%, 85%, 75%, 50% alcohol, distilled water and PBS for 5 min each;

[0057] ④ Antigen repair: Soak the slices in sodium citrate solution (PH=6.0), use a microwave oven to boil on high heat, then keep it on medium heat for 7 minutes, stop heating and let it cool naturally, then soak and wash in PBS for 3 times, 5 minutes each time;

[0058] Subsequent operation steps, including blocking, primary antibody incubation, secondary antibody incubation, and DAPI nuclear staining are consistent with frozen section staining ③ to ⑥.

[0059] (II) Experimental results

[0060] like Figure 1 As shown in Figure 2, SPAK, p-SPAK, and p-NKCC1 proteins were evenly distributed at the cell edges in the lateral ventricles, and during the pathological process of EAE, SPAK (see Figure 1 A, 1B), p-SPAK (see Figure 1 C, 1D), p-NKCC1 (see Figure 1 The expression levels of E and F) proteins in the choroid plexus were increased, indicating the importance of the SPAK-NKCC1 signaling pathway in the pathological process of EAE. This further proves that the choroid plexus is an important immune infiltration regulatory site in the pathological process of EAE.

[0061] Example 2 Virus-specific knockdown and targeted inhibition of SPAK levels in the choroid plexus

[0062] 1. Experimental methods

[0063] To further study the function of SPAK in the choroid plexus, we used intracerebroventricular injection of SPAK knockdown virus (SPAK-Kd, rAAV2 / 5-U6-shRNA (SPAK)-CMV-EGFP) to achieve specific knockdown of SPAK in the choroid plexus, thereby achieving the purpose of targeted inhibition of choroid plexus SPAK signals; at the same time, SPAK immunofluorescence staining was performed on the choroid plexus of mice after SPAK specific knockdown.

[0064] Among them, the method of unilateral intracerebroventricular injection of SPAK knockdown virus and control virus (rAAV2 / 5-U6-Scramble-CMV-EGFP) in mice is as follows:

[0065] ① Anesthesia and fixation: Anesthetize the mice with 3.6% chloral hydrate at 10 mg / kg intraperitoneally, remove the hair on the mouse skull; fix the bilateral ear bars of the brain stereotaxic instrument in the external auditory canal of the mouse, fix the mouse incisors to the incisor fixator at the front end, and apply erythromycin eye ointment to the eyes;

[0066] ② Exposure field of view: Disinfect the scalp with 75% alcohol, cut the skin along the midline between the two ears, fully expose the skull, and make the anterior and posterior fontanelles clearly visible under the microscope;

[0067] ③ Adjust the injection plane: Position the needle tip on the surface of the anterior fontanelle, return the X, Y, and Z axis coordinates to zero, use the anterior fontanelle position as the reference point, and move 2.5mm left and right. When the needle tip is positioned at two points of the skull, the difference in the Z axis values ​​on both sides is within 0.05mm, which is considered to be leveled left and right. Otherwise, adjust the height of the ear bars on both sides to level the left and right. Front and back leveling is leveled by leveling the anterior fontanelle point with the posterior fontanelle point, and the method is the same as before.

[0068] ④ Positioning: With the bregma as the origin of coordinates, the injection site of the lateral ventricle is: posterior to the bregma (AP) -0.4mm, lateral to the bregma (ML) ±1.10mm, depth (DV) -2.10mm. This experiment performed unilateral lateral ventricle injection;

[0069] ⑤ Drilling the skull hole: After the needle tip touches the skull plane, mark the injection point and use a handheld drill with a diameter of 0.5 mm to drill the skull to fully expose the brain tissue;

[0070] ⑥Injection of virus: Before inserting the needle, empty the air that may be in the needle tip, insert the 10μL Hamilton microsyringe to 2mm below the skull, and inject 2μL of virus at a rate of 0.4μL / min. After completion, leave it in place for 5 minutes and slowly withdraw the needle tip;

[0071] ⑦ Postoperative treatment: Suture the mouse's head skin with medical needle and thread and disinfect it, then place the mouse on a constant temperature electric blanket to rewarm it until it wakes up, and then transfer the mouse to a cage for breeding after it wakes up.

[0072] In addition, the virus was injected into the lateral ventricle of mice again, and choroid plexus samples were collected 2 weeks later for real-time fluorescence quantitative PCR detection.

[0073] The method of real-time fluorescence quantitative PCR is as follows:

[0074] ① After the mice were perfused with PBS, the choroid plexus tissue was quickly removed under a microscope and placed in a 1.5 mL centrifuge tube, and then quickly frozen in liquid nitrogen for 30 seconds. 500 μL of Trizol extract was added, and the tube was oscillated 6 times with a cryo-grinder, 1 minute per time, and then placed on ice for 5 minutes.

[0075] ② Add 100 μL of chloroform, vortex thoroughly for 15 seconds to mix, and place on ice for 10 minutes;

[0076] ③ Centrifuge at 4℃13000rpm for 15min, carefully transfer the supernatant to a new 1.5mL centrifuge tube, add an equal volume of isopropanol, mix gently, and place on ice for 10min;

[0077] ④ Centrifuge at 4℃13000rpm for 5-10min, discard the supernatant, and white flocculent precipitate will be visible. Immediately resuspend the RNA precipitate with 500μL 75% alcohol and gently shake the tube to wet the tube wall;

[0078] ⑤ Centrifuge at 8000 rpm at 4°C for 5 min, discard the supernatant, and dry in a clean fume hood for 30 min;

[0079] ⑥ Add 15 μL DEPC water to dissolve the RNA precipitate and measure the RNA concentration using NanoDrop;

[0080] ⑦ Take 1 μg of RNA in total and reverse transcribe it into cDNA according to the set RNA reverse transcription program. Use SYBR Green method and prepare quantitative PCR reaction system according to the instructions, a total of 20 μL, spot it in a 96-well plate, place it in the PCR instrument, set the amplification temperature according to the instructions, and analyze the quantitative results after running.

[0081] ⑧ In this study, the PPIA gene stably expressed in the choroid plexus was selected as an internal reference, and Q-PCR primers were designed using the Primer Bank website. The sequences of all gene primers are shown in Table 1. The real-time fluorescence quantitative PCR in other embodiments was performed with reference to this embodiment.

[0082] Table 1 Primer sequences

[0083]

[0084]

[0085] ⑨Perform the fluorescence quantitative PCR experiment according to the set program. The PCR cycle program is shown in Table 2:

[0086] Table 2 Q-PCR program

[0087] program set up Cycle times 1 95℃30s 1 time 2 95℃5s, 60℃30s 40 times 3 95℃15s, 60℃60s, 95℃15s 1 time

[0088] ⑩According to C t The relative fluorescence quantification (RQ) was calculated.

[0089] (II) Experimental results

[0090] The results are as follows Figure 2 As shown, after unilateral intracerebroventricular injection of the SPAK knockdown virus carrying a green fluorescent marker for 2 weeks, the choroid plexus of the lateral ventricle and the third ventricle expressed green fluorescent protein (see Figure 2 A). The results of SPAK immunofluorescence staining in the choroid plexus showed that the red fluorescence intensity of the knockdown group was significantly reduced compared with the control virus injected into the lateral ventricle (see Figure 2 A, 2B), verified the feasibility of SPAK specific knockdown technology.

[0091] In addition, the real-time fluorescence quantitative PCR test of choroid plexus samples obtained 2 weeks later found that the mRNA level of SPAK gene was reduced (see Figure 2C), which once again demonstrated that we have successfully established the choroid plexus SPAK-specific knockdown technology.

[0092] Example 3 Targeted inhibition of choroid plexus SPAK signaling to improve disease occurrence

[0093] 1. Experimental methods

[0094] After achieving specific knockdown of SPAK in the choroid plexus, we first injected control virus and SPAK knockdown virus into the lateral ventricle of mice, and then established EAE model 2 weeks later. At the peak of EAE pathological score, choroid plexus and spinal cord tissues were obtained for staining, and we used immunofluorescence staining to detect whether the degree of T lymphocyte immune infiltration was affected. In addition, we also performed immunofluorescence staining on spinal cord tissues.

[0095] In addition, we used flow cytometry to detect the changes in the number and types of immune cells infiltrating into the cerebrospinal fluid and spinal cord.

[0096] In the present invention, the steps of hematoxylin-eosin (HE) staining of spinal cord tissue are as follows:

[0097] ① Place the prepared spinal cord paraffin block on a paraffin slicer, then cut into tissues with a thickness of 7 μm, attach the wax slices to a glass slide, dry the slices, dewax and rehydrate, perform antigen repair, and wipe off excess water after washing;

[0098] ② Nuclear staining: Place the slices in hematoxylin solution, make sure the solution fully covers the tissue, leave for 5 minutes, and wash with running water for 1 minute;

[0099] ③ Differentiation: Place the slices in 1% hydrochloric acid alcohol for differentiation. When the color of the dye becomes lighter, take them out immediately, wash them with running water, and dry the surface moisture of the slices.

[0100] ④Restaining: Place the sections in eosin staining solution for 3 minutes;

[0101] ⑤ Transparent sealing: Place the slices in special color separation liquid for color separation: anhydrous ethanol for 2 minutes, anhydrous ethanol for 2 minutes, 90% alcohol for 2 minutes, 80% alcohol for 2 minutes, 70% alcohol for 2 minutes, xylene I for 5 minutes, xylene II for 10 minutes. Take the slices out of xylene and dry them, then use neutral gum to seal the slices.

[0102] Among them, the method of extracting cerebrospinal fluid is as follows:

[0103] ① Anesthesia and fixation: Anesthetize the mice with 3.6% chloral hydrate at 10 mg / kg intraperitoneally, remove the hair on the back of the head of the mice; fix the mice on the stereotaxic apparatus and adjust the position so that the head and body are at 135 degrees;

[0104] ② Exposure field: Disinfect the skin with 75% alcohol, cut along the midline between the two ears, and then bluntly separate the connective tissue under the surface skin. Then gently cut the surface muscles along the midline white line of the brain, separate the middle muscles to both sides, and use fine forceps under the microscope to gently peel off the last layer of muscles outside the skull to fully expose the cerebellomedullary cistern, and dry the area with a cotton swab;

[0105] ③ Use a microelectrode pulling instrument to pull a capillary glass tube with a diameter of 0.5 mm to make its head sharp, and puncture the blood vessels on both sides or between the blood vessels of the cerebellomedullary cistern to collect cerebrospinal fluid. Initially, the liquid level in the capillary glass tube rises rapidly. When the liquid level stops rising, remove the capillary glass tube and transfer the liquid to a centrifuge tube with a 1 mL syringe; collect about 5-10 μL of cerebrospinal fluid from each mouse;

[0106] ④The collected cerebrospinal fluid was placed on ice for later use.

[0107] Among them, the flow cytometry method is as follows:

[0108] ① After the mouse is perfused with PBS, the spinal cord is removed and a spinal cord single cell suspension is prepared; 5-10 μl of cerebrospinal fluid collected from the medullary cisterna magna is taken, the specific volume is marked, and the cerebrospinal fluid cells are treated with pre-cooled PBS;

[0109] ② Add FVS780 (565388, BD Horizon), CD3-FITC (11-0032-82, Invitrogen), CD4-APC (2376140, Invitrogen), and CD196-AF647 (129804, Biolegend) antibodies, mix thoroughly, and incubate at 4°C in the dark for 30 min;

[0110] ③After centrifugation, add PBS containing 1% BSA to promote cell resuspension;

[0111] ④Evaluate the stained cells individually using flow cytometry.

[0112] (II) Experimental results

[0113] like Figure 3 As shown in A, compared with the control group, the disease severity in the SPAK knockdown virus-treated group was significantly alleviated, and the neurological deficit score was significantly reduced.

[0114] HE staining results Figure 3 As shown in B and 3C, HE staining of the spinal cord showed that the pathological changes of inflammatory cells were significantly alleviated; the results of immunofluorescence staining were as follows Figure 3As shown in D and 3E (rabbit anti-MBP antibody, mouse anti-NF-H antibody, Cell Signaling Technology; anti-rabbit 488 secondary antibody, anti-mouse 594 secondary antibody, Invitrogen), immunofluorescence staining showed that the degree of demyelination in the spinal cord was greatly improved.

[0115] The results of immunofluorescence staining of T lymphocytes showed that SPAK knockdown significantly reduced CD4 + T lymphocytes in the spinal cord (see Figure 3 F, 3G (rat anti-CD4 antibody, anti-rat 594 secondary antibody, Invitrogen)), choroid plexus (see Figure 3 H, 3I (rat anti-CD4 antibody, anti-rat 594 secondary antibody, Invitrogen)) number of infiltrates.

[0116] The results of immunofluorescence staining of spinal cord tissue showed that SPAK knockdown significantly inhibited the activation of spinal cord macrophages and microglia (see Figure 3 J, 3K (rat anti-CD68 antibody, Bio-Rad; rabbit anti-Iba1 antibody, Wako; anti-rat 594 secondary antibody, anti-rabbit 488 secondary antibody, Invitrogen)), 3D reconstruction shows that microglia are activated after EAE induction, which is manifested by enlarged cell body diameter, reduced number of cell synaptic branches, and shortened branch length, but SPAK knockdown inhibits its activation (see Figure 3 L, 3M (rabbit anti-Iba1 antibody, Wako; anti-rabbit 488 secondary antibody, Invitrogen)). This indicates that targeted inhibition of choroid plexus SPAK signaling can effectively reduce the accumulation of immune cells in the choroid plexus and spinal cord, and significantly improve the severity of EAE.

[0117] Flow cytometry analysis showed that the number of immune cells in the SPAK knockdown virus treatment group was significantly reduced (see Figure 4 A~G). This indicates again that targeted inhibition of choroid plexus SPAK protein can improve the degree of EAE immune infiltration.

[0118] Tight junctions play a key role in maintaining epithelial cell polarity and barrier integrity, among which Occludin (OCLN), Claudin, and ZO proteins are the main proteins that make up tight junctions. Matrix metalloproteinases (MMPs) are involved in regulating the homeostasis of the extracellular matrix and can increase the permeability of the blood-brain barrier. Studies have shown that in the nervous system, MMP-2 / 9 can damage the blood-brain barrier and promote inflammation and microglial activation.

[0119] Example 4 Targeted inhibition of choroid plexus SPAK signaling affects tight junction proteins and matrix metalloproteinases in choroid plexus epithelial cells

[0120] 1. Experimental methods

[0121] To further study how SPAK affects the junction status and cell-matrix status of choroid plexus epithelial cells, we used Q-PCR to detect the expression levels of SPAK-related tight junction proteins (OCLN, ZO-1, Claudin1 / 3 / 5) and matrix metalloproteinases (MMPs, such as MMP2 and MMP9) in the choroid plexus.

[0122] The experiment further used immunofluorescence staining to detect the expression levels and cellular localization of OCLN and MMP2 / 9 proteins.

[0123] (II) Experimental results

[0124] The results of tight junction protein and matrix metalloproteinase expression levels showed that compared with the control virus injected into the lateral ventricle, the mRNA expression levels of OCLN, ZO-1 and Claudin1 / 3 / 5 in the choroid plexus were significantly increased after the SPAK knockdown virus was injected into the lateral ventricle, while the mRNA expression level of MMP2 / 9 was significantly decreased ( Figure 5 A~5G).

[0125] The results of immunofluorescence staining showed that OCLN and MMP2 / 9 proteins were distributed on the matrix side of the choroid plexus epithelial cells; under physiological conditions, compared with the untreated group or the control virus group injected into the lateral ventricle, the expression level of OCLN protein in the choroid plexus of the SPAK knockdown virus group was increased, while the expression level of MMP2 / 9 protein was decreased ( Figure 5 H~5M); under EAE pathological conditions, the expression level of OCLN protein in the choroid plexus was decreased compared with the untreated group after intracerebroventricular injection of the control virus, while the expression level of MMP2 / 9 protein was increased. However, the expression level of OCLN protein in the choroid plexus was increased compared with the control virus after intracerebroventricular injection of the SPAK knockdown virus, while the expression level of MMP2 / 9 protein was the opposite ( Figure 5 N~5S), indicating that targeted inhibition of choroid plexus SPAK signaling can partially reverse the effect of EAE on OCLN and MMP protein expression.

[0126] Bumetanide is a loop diuretic that can inhibit NKCC1 at low concentrations and has been used clinically. However, due to its high ionization, it is not easy to cross the blood-brain barrier after systemic administration, which limits its application. ZT-1a is a selective SPAK kinase inhibitor that inhibits NKCC1 by phosphorylation. Studies have shown that it can reduce brain edema, protect brain damage, and improve neurological function by reducing SPAK.

[0127] Example 5 Effect of SPAK-NKCC1 antagonist on EAE pathological damage

[0128] 1. Experimental methods

[0129] Intraperitoneal injection of SPAK, NKCC1 antagonist ZT-1a, and bumetanide

[0130] ① Preparation of ZT-1a: weigh 1.5 mg ZT-1a into a 5 mL centrifuge tube, dissolve in 360 μL DMSO, add an equal volume of castor oil, mix thoroughly, then add 1680 μL PBS solution, mix thoroughly again to make a final concentration of 0.625 mg / mL ZT-1a; inject ZT-1a intraperitoneally at the same time point every day according to the weight of the mouse, with an injection volume of 5 mg / kg;

[0131] ② Preparation of bumetanide: Weigh 1.5 mg of bumetanide into a 5 mL centrifuge tube, dissolve in 375 μL of DMSO, add 2025 μL of saline, mix thoroughly, and prepare a final concentration of 0.625 mg / mL of bumetanide; inject bumetanide intraperitoneally at the same time every day according to the weight of the mouse, with an injection volume of 5 mg / kg.

[0132] After confirming the protective effect of ZT-1a on EAE, we took choroid plexus and spinal cord tissues for staining at the peak of EAE pathological score. At the same time, we used immunofluorescence staining to detect whether the degree of T lymphocyte immune infiltration was affected and also performed immunofluorescence staining on spinal cord tissue.

[0133] In addition, we used flow cytometry to detect the changes in the number and types of immune cells infiltrating into the cerebrospinal fluid and spinal cord.

[0134] (II) Experimental results

[0135] The results showed that intraperitoneal administration of bumetanide every other day after EAE induction (starting from day 0) did not improve the pathological damage of mice, and there was no significant difference in the neurological deficit score ( Figure 6 A). This further indicates that drugs need to pass through the blood-brain barrier to work on EAE. After EAE induction, intraperitoneal injection of the SPAK antagonist ZT-1a was found to improve the pathological damage of mice by intraperitoneal administration every other day (starting from day 0). There was a significant difference in the neurological deficit scores between the two groups ( Figure 6 B).

[0136] HE staining of the spinal cord showed that the pathological changes of inflammatory cells were significantly alleviated ( Figure 6 C, 6D), immunofluorescence staining showed that the degree of demyelination was greatly improved ( Figure 6 E, 6F (rabbit anti-MBP antibody, mouse anti-NF-H antibody, Cell Signaling Technology; anti-rabbit 488 secondary antibody, anti-mouse 594 secondary antibody, Invitrogen)).

[0137] The results of immunofluorescence staining of T lymphocytes showed that ZT-1a significantly reduced the number of CD4 + T lymphocytes in the spinal cord ( Figure 6 G, 6H (rat anti-CD4 antibody, anti-rat 594 secondary antibody, Invitrogen)), choroid plexus ( Figure 6 I, 6J (rat anti-CD4 antibody, anti-rat 594 secondary antibody, Invitrogen)) number of infiltrates.

[0138] The results of immunofluorescence staining of spinal cord tissue showed that ZT-1a significantly inhibited the activation of spinal cord macrophages and microglia ( Figure 6 K, 6L (rat anti-CD68 antibody, Bio-Rad; rabbit anti-Iba1 antibody, Wako; anti-rat 594 secondary antibody, anti-rabbit 488 secondary antibody, Invitrogen)). The 3D reconstruction of microglia showed that compared with the control solvent group, the diameter of microglia cell bodies became smaller, the number of cell synaptic branches increased, and the length of branches became longer after ZT-1a administration ( Figure 6 M, 6N (rabbit anti-Iba1 antibody, Wako; anti-rabbit 488 secondary antibody, Invitrogen)). This indicates that ZT-1a can effectively reduce the accumulation of immune cells in the choroid plexus and spinal cord by inhibiting SPAK signaling, and significantly improve the severity of EAE.

[0139] Flow cytometry analysis showed that the number of immune cells in the ZT-1a treatment group was significantly reduced ( Figure 7 A~7G). This again shows that ZT-1a can reverse the pathological degree of EAE.

[0140] Example 6 Targeted inhibition of choroid plexus MMP signaling has a protective effect on EAE

[0141] 1. Experimental methods

[0142] We have previously shown that targeted inhibition of choroid plexus SPAK has an inhibitory effect on MMP2 and MMP9. To further study the effects of MMP2 and MMP9 on EAE, we injected control virus (rAAV2 / 5-U6-Scramble-CMV-EGFP), MMP2 knockdown virus (MMP2-Kd, rAAV2 / 5-CMV-MMP2-Kd), MMP9 knockdown virus (MMP9-Kd, rAAV2 / 5-CMV-MMP9-Kd) and MMP2 / 9 double knockdown virus (MMP2 / 9-Kd, rAAV2 / 5-CMV-MMP2 / 9-Kd) into the lateral ventricle of mice to achieve the purpose of targeted inhibition of choroid plexus MMP2, MMP9, and MMP2 / 9. EAE models were established 2 weeks later. Spinal cord tissues were taken for immunofluorescence staining at the peak of EAE pathological scores.

[0143] (II) Experimental results

[0144] The results showed that compared with the control virus injected into the lateral ventricle, the severity of the disease in the group treated with MMP2 knockdown virus, MMP9 knockdown virus, and MMP2 / 9 double knockdown virus injected into the lateral ventricle was greatly reduced, and the neurological deficit score was significantly reduced. In addition, the onset time of the double knockdown virus treatment group was delayed and the degree of disease improvement was the most obvious ( Figure 8 A).

[0145] At the peak of EAE pathological score, spinal cord tissue was obtained for immunofluorescence staining. The results showed that intracerebroventricular injection of MMP2 knockdown virus, MMP9 knockdown virus, and MMP2 / 9 double knockdown virus significantly inhibited the activation of spinal cord macrophages and microglia ( Figure 8 B, 8C), the degree of spinal cord demyelination was greatly improved ( Figure 8 D, 8E).

[0146] HE staining of the spinal cord showed that the pathological changes of inflammatory cells were significantly alleviated ( Figure 8 F, 8G). This indicates that targeted inhibition of choroid plexus MMP signaling can effectively reduce the accumulation of immune cells in the spinal cord and significantly improve the severity of EAE.

[0147] Example 7 Choroid plexus-specific overexpression of SPAK aggravates EAE pathology

[0148] 1. Experimental methods

[0149] We have confirmed that targeted inhibition of choroid plexus SPAK can improve EAE pathology. To further determine the role of SPAK signaling, we induced SPAK overexpression in the choroid plexus to reversely demonstrate the effect of SPAK on EAE. We injected control virus and SPAK overexpression virus (SPAK-OE, rAAV2 / 5-CMV-SPAK-OE) into the lateral ventricle of mice to achieve the purpose of choroid plexus-specific overexpression of SPAK, and EAE models were established 2 weeks later.

[0150] (II) Experimental results

[0151] The results showed that compared with the control virus injected into the lateral ventricle, the severity of the disease in the group treated with SPAK overexpression virus was aggravated ( Fig. 9 A). Spinal cord tissue was taken for immunofluorescence staining at the peak of EAE pathological score. The results showed that overexpression of SPAK in the choroid plexus significantly aggravated the activation of spinal macrophages and microglia ( Fig. 9 B, 9C), aggravating the degree of spinal cord demyelination ( Fig. 9 D, 9E). HE staining of the spinal cord showed an increase in inflammatory cell pathological changes ( Fig. 9F, 9G). This indicates that choroid plexus-specific overexpression of SPAK can further enhance the accumulation of immune cells in the spinal cord and aggravate the severity of EAE.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for treating multiple sclerosis, characterized in that: The pharmaceutical composition comprises at least one of an inhibitor of SPAK kinase, a down-regulator that reduces the expression of SPAK kinase, a down-regulator that reduces the expression of MMP2, and a down-regulator that reduces the expression of MMP9.

2. The use according to claim 1, characterized in that: The inhibitors of SPAK kinase include ZT-1a.

3. The use according to claim 2, characterized in that: The administration method of ZT-1a is intraperitoneal injection.

4. The use according to claim 1, characterized in that: The pharmaceutical composition includes a down-regulator that reduces the expression of MMP2 and a down-regulator that reduces the expression of MMP9.

5. The use according to claim 1, characterized in that: The down-regulator for reducing the expression of SPAK kinase includes at least one of active peptides, proteins, oligonucleotides and small molecule compounds that inhibit the expression of SPAK kinase.

6. The use according to claim 1, characterized in that: The down-regulator for reducing the expression of MMP2 includes at least one of active peptides, proteins, oligonucleotides and small molecule compounds that inhibit the expression of MMP2.

7. The use according to claim 1, characterized in that: The down-regulator for reducing the expression of MMP9 kinase includes at least one of active peptides, proteins, oligonucleotides and small molecule compounds that inhibit the expression of MMP9.

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