Application of JAG-1 protein in preparation of medicine for treating enterovirus infection and repairing blood-brain barrier damage

Abstract

The invention relates to the technical field of biological medicine, in particular to application of JAG-1 protein in preparation of medicine for treating enterovirus infection and repairing blood-brain barrier damage. The Notch-1 signal channel ligand protein JAG-1 provided by the invention can be used for treating enterovirus infection and repairing the integrity and selective permeability of a blood brain barrier by activating a Notch-1 signal channel. According to the invention, the JAG-1 protein is used for reducing viral load in the brain, relieving neurological illness related to damage of the blood-brain barrier and performing targeted treatment on the neurological illness, so that the severe death rate is reduced, complications are prevented, the treatment effect is improved, the treatment risk is reduced, and the life quality of a patient is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of JAG-1 protein in preparing medicines for treating enterovirus infection and repairing blood-brain barrier damage. Background Art

[0002] Enterovirus infection is a widespread infectious disease worldwide, affecting both adults and children, but particularly children. Mild cases present with rashes, blisters, and generalized weakness on the extremities, while severe cases can cause systemic damage to vital organs (including the brain, heart, and liver), resulting in a poor prognosis and potentially fatal consequences. Enterovirus infection is widespread, and the resulting poliomyelitis and hand, foot, and mouth disease (HFMD) present complex and diverse clinical manifestations. Currently, prevention remains the primary approach for clinical prevention and control.

[0003] The polio vaccine is highly effective in preventing polio, but specific control methods for other enterovirus infections remain lacking. The EV71 vaccine is somewhat effective in preventing viral infections, but a significant number of children are infected with EV71, and there is no specific treatment. Currently, anti-enteroviral drugs primarily target a single strain of enterovirus, and a new broad-spectrum drug for the prevention and treatment of enterovirus infections is still lacking. Furthermore, new treatments for patients with severe neurological illness are urgently needed.

[0004] The blood-brain barrier (BBB) ​​is a major structural component that protects the central nervous system (CNS) from toxins, pathogens, and inflammation, maintaining CNS homeostasis. Components of the BBB include tight junctions, integrins, annexins, and lectins mediated by a multicellular system including vascular endothelial cells (BMECs), astrocytes, and pericytes, forming a highly selective barrier that prevents most blood components and pathogens from entering brain tissue. However, studies have shown that certain enterovirus infections can lead to BBB disruption. Some members of the human enterovirus family are neurotropic and are associated with a variety of clinical diseases associated with encephalitis. Based on sequencing, they are divided into 12 types: enterovirus A to L. Four of these enteroviruses (A to D) are widely distributed worldwide. The most famous neurotropic enteroviruses are poliovirus (PV) and a variety of other enteroviruses such as enterovirus 71 (EV71) and coxsackievirus B3 (CVB3). Studies have shown that they involve the central nervous system and cause a variety of neurological complications, such as encephalitis, meningitis and acute flaccid paralysis.

[0005] The Notch signaling pathway is an important cellular signaling pathway involved in regulating numerous biological processes, including cell proliferation, differentiation, apoptosis, cell fate determination, organ development, angiogenesis, and immune responses. The Notch signaling pathway is mediated through the interaction between the Notch receptor and its ligand. In the classic Notch signaling pathway, mature Notch receptors on the cell membrane are heterodimers, and the heterodimerization domain is cleaved in the Golgi apparatus (S1 cleavage). Generally, binding to the Notch receptor extracellular domain allows the ligand to initiate endocytosis. This endocytosis induces the receptor to change its conformation, exposing the enzymatic site for S2 cleavage. The receptor then undergoes S3 cleavage, transforming into its effector form: the Notch intracellular domain (NICD). The NICD is degraded in the cytoplasm or transported to the nucleus to regulate target gene transcription. However, current research targeting the Notch signaling pathway to prevent and treat enterovirus infection and repair blood-brain barrier disruption remains insufficient. Summary of the Invention

[0006] In view of this, the present invention proposes that the Notch-1 signaling pathway ligand protein JAG-1 can treat enteroviral infection by activating the Notch-1 signaling pathway, repair the integrity and selective permeability of the blood-brain barrier, reduce the viral load in the brain, alleviate severe neurological diseases related to blood-brain barrier destruction and provide targeted treatment for them, thereby reducing severe mortality, preventing complications, improving treatment effects, reducing treatment risks and improving patients' quality of life.

[0007] The technical solution of the present invention is achieved as follows:

[0008] In a first aspect, the present invention provides a use of a JAG-1 protein in the preparation of a medicament for treating enterovirus-infected diseases. The Uniprot database ID of the JAG-1 protein is P78504.

[0009] The JAG-1 protein is a Notch-1 ligand protein. The Notch1-Notch4 receptor is a transmembrane receptor that plays a central role in the activation of signaling pathways. Notch signals are initiated by Delta-like (DLL1-DLL4) or jagged (JAG1 and JAG2) ligands that bind to the Notch1-Notch4 receptors. JAG-1 is one of the ligands of the Notch receptor and interacts with the Notch receptor to initiate the signaling pathway, leading to proteolytic cleavage and translocation of the Notch intracellular domain (NICD) to the nucleus, and then inducing transcriptional activation.

[0010] JAG-1 contains 26 exons spanning over 36 kb, resulting in a 1218 amino acid protein consisting of a relatively small intracellular domain, a transmembrane domain, and a larger extracellular component. The extracellular portion of the protein includes four motifs necessary for normal protein function, including a 21-amino acid signal peptide, an N-terminal region, a 40-amino acid highly conserved DSL domain, followed by 16 epidermal growth factor-like repeats and a cysteine-rich region. The molecular formula is C 93 H 127 N 25 O 26 S3, structured as Figure 1 shown.

[0011] Furthermore, the drug is a preparation prepared with JAG-1 protein as the active ingredient and pharmaceutically acceptable excipients.

[0012] Furthermore, the preparation is an oral preparation or an injection.

[0013] Furthermore, the enterovirus includes at least one of enterovirus 71, coxsackievirus B3 and coxsackievirus B4.

[0014] In a second aspect, the present invention provides a use of JAG-1 protein in the preparation of a medicament for treating a disease causing blood-brain barrier damage, wherein the disease is a disease causing blood-brain barrier damage caused by enterovirus infection. The Uniprot database ID of the JAG-1 protein is P78504.

[0015] Furthermore, the drug is a preparation prepared with JAG-1 protein as the active ingredient and pharmaceutically acceptable excipients.

[0016] Furthermore, the preparation is an oral preparation or an injection.

[0017] Furthermore, the enterovirus includes at least one of enterovirus 71, coxsackievirus B3 and coxsackievirus B4.

[0018] Furthermore, the blood-brain barrier destruction includes at least one type of structural destruction, functional destruction, and inflammatory destruction of the blood-brain barrier; the diseases causing blood-brain barrier destruction include brain trauma, stroke, brain tumor, Alzheimer's disease, cerebral hemorrhage, cerebral hypoxia, cerebral edema, encephalitis, meningitis, multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), autoimmune encephalitis, viral encephalitis, vascular dementia, and Parkinson's disease.

[0019] The present invention has the following beneficial effects:

[0020] The present invention discloses the significant role of JAG-1 protein in activating Notch signaling and downstream junction protein expression in BMEC, and further confirms its effectiveness in inhibiting enterovirus invasion of BBB in vitro model.

[0021] Furthermore, in vivo experiments further confirmed the practical application value of JAG-1 protein in treating enteroviral infections and repairing blood-brain barrier damage. The results showed that injection of JAG-1 protein significantly reduced weight loss and mortality in infected suckling mice, effectively inhibited viral replication in the brain, and significantly reduced the permeability of the BBB in infected suckling mice, thereby restoring BBB integrity.

[0022] The JAG-1 protein proposed in this invention has significant beneficial effects in the preparation of drugs for treating enteroviral infections and repairing blood-brain barrier disruption. It not only alleviates the symptoms of severe neurological illnesses associated with blood-brain barrier disruption, enabling targeted treatment, reducing mortality from severe illness, and improving therapeutic efficacy, but also reduces treatment risks and significantly improves patients' quality of life. Therefore, this invention has broad application prospects in the medical field and significant social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the structural diagram of Notch-1 ligand protein JAG-1;

[0024] Figure 2 The results are the results of the detection of the effect of JAG-1 on Notch signaling and downstream junction protein expression in BMEC (human brain microvascular endothelial cells), where NC is the control, Notch1 is the Notch receptor, ZO-1, VE-cadherin, and β-catenin are junction proteins, and β-actin is the internal control;

[0025] Figure 3 The results are the results of the test on the effect of JAG-1 on enterovirus replication in intestinal cells, where: Mock is the control (no EV71 treatment), Notch-1 is the Notch receptor, VP1 is the EV71 surface antigen protein, and β-actin is the internal control;

[0026] Figure 4 The results show the effect of JAG-1 on the selective permeability of the blood-brain barrier in an in vitro blood-brain barrier model. HT29 EVs represent exosomes (EVs) derived from HT29 infected with EV71. From left to right in the figure, they correspond to the Mock group, EV71 group, HT29 EVs Mock group, HT29 EVs+EV71 group, and HT29 EVs+EV71+JAG-1 group, respectively.

[0027] Figure 5The results are from the test of the effect of JAG-1 on the integrity of the blood-brain barrier in an in vitro model of the blood-brain barrier;

[0028] Figure 6 The results are from testing the effect of JAG-1 on viral invasion in an in vitro model of the blood-brain barrier;

[0029] Figure 7 The results are the test results of the effect of JAG-1 on the body weight (i) and survival rate (ii) of suckling mice;

[0030] Figure 8 This is the result of testing the effect of JAG-1 on viral replication in the intestine and brain tissues of suckling mice;

[0031] Figure 9 This is the test result of the effect of JAG-1 on the pathological changes in the intestine and brain tissue of suckling mice;

[0032] Figure 10 This is the test result of the effect of JAG-1 on the brain permeability of neonatal mice. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] The present invention will be described in further detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0035] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions were used. The reagents used in the examples were all commercially available unless otherwise specified.

[0036] Experimental materials and reagents:

[0037] DMEM medium and fetal bovine serum (FBS) were purchased from GIBCO, DMSO from Sigma, CCK8 (CellCounting Kit-8) activity detection kit from Dojindo, Japan, crystal violet and low-melting-point agarose from Shanghai Biotech, EV71 VP1 antibody from Abnova, Taiwan, EV713C antibody from Wuhan Aibotek Biotechnology, HRP luminescent substrate reaction solution from Bio-Rad, sterile PBS solution from Hyclone, dsRNA mouse primary antibody from Scicons, Hungary, DAPI from Roche, and goat anti-mouse Cy3 fluorescent secondary antibody from Wuhan Mitaka Biotechnology. Human brain microvascular endothelial cells (BMEC), human colon cancer cells (HT29), and human embryonic rhabdomyosarcoma cells (RD) were purchased from the China Center for Type Culture Collection (CCTCC). JAG-1 human recombinant polypeptide was purchased from MCE (MedChemExpress), USA. C57BL / 6WT mice were purchased from the Guangdong Laboratory Animal Center and housed in individually ventilated cages under specific pathogen-free (SPF) conditions. The enterovirus 71 (EV71) strain (Xiangyang-Hubei-09) was derived from brain tissue of an infant who died of EV71 infection in Xiangyang City, Hubei Province, China. It was previously isolated by the State Key Laboratory of Virology, Wuhan University (GenBank: JN230523.1), and the viral stock was amplified in RD cells.

[0038] Experimental instruments:

[0039] The multi-label microplate reader and high-content cell analyzer were purchased from PerkinElmer, the cell culture incubator was from Thermofisher, the bioluminescence analyzer was purchased from Fujifilm, Japan, and the fluorescence confocal microscope was purchased from Olympus, Japan.

[0040] Example 1 Detection of Notch Signaling and Downstream Connector Protein Expression in BMECs by JAG-1

[0041] This example detects the effect of JAG-1 on the activation degree of Notch signaling in BMEC and the integrity of BMEC at the molecular level. BMEC is a human brain microvascular endothelial cell and is the main component cell of the blood-brain barrier (BBB). Notch1 is a Notch signaling receptor protein. The expression level of Notch1 indicates the activation degree of the Notch signaling pathway, and ZO-1, VE-cadherin, β-catenin) is related to the degree of tight junctions between BMEC cells. For example, zonula occludens (ZOs, including ZO-1) form tight junctions between BMECs, filling the gaps between cells, thereby limiting paracellular permeability. The expression and distribution of junction proteins such as ZO-1, VE-cadherin, and β-catenin are crucial for maintaining the integrity of the blood-brain barrier. Detecting the expression levels of these junction proteins can assess the integrity of BMEC, and the expression of these proteins also directly affects the selective permeability of BMEC.

[0042] In this example, BMECs were first treated with different concentration gradients of JAG-1, and then Notch1 was detected by Western blotting.

[0043] The expression levels of Notch signaling receptor, ZO-1, VE-cadherin, β-catenin (junction protein) and β-actin (internal reference) were measured to detect the effect of JAG-1 on Notch signaling and the expression of downstream junction proteins in BMECs.

[0044] 1.1 Experimental steps

[0045] BMEC cells were cultured at a rate of 5 × 10 5 Cells were seeded in 12-well cell culture plates using 10% FBSDMEM medium supplemented with 1% penicillin and streptomycin and cultured in a humidified incubator at 37°C and 5% CO2.

[0046] After BMECs adhered, they were treated with varying concentrations of JAG-1. JAG-1 was diluted in fresh 2% FBSDMEM medium to concentrations of 0, 0.5, 2.5, 5, and 10 μM and then added to adherent BMECs. BMECs were treated for 24 hours, and protein lysates were collected for immunoblotting to analyze Notch1 and connexin expression.

[0047] The specific steps of the protein immunoblotting detection are as follows:

[0048] (i) Discard the cell culture medium, rinse the cell sample with PBS buffer, digest the cells with 0.25% trypsin, resuspend the cells in a 1.5 mL EP tube, centrifuge at 3,000 g for 5 min, aspirate the supernatant, and pellet the cells. Gently pipette the cell pellet using RIPA cell lysis buffer containing 1% cocktail protease inhibitors. Incubate on ice for 30 min and disrupt the cell pellet using an ultrasonic cell disruptor at 30 Hz until the lysate is clear. Centrifuge the ultrasonicated sample at 12,000 g for 10 min at 4°C. Remove the supernatant and transfer it to a new 1.5 mL EP tube.

[0049] (ii) Determine the protein concentration of different protein samples using BCA protein quantification reagent. Prepare protein standards and BCA working solution. Measure the absorbance at wavelength A562 using a microplate reader. Plot a standard curve. Substitute the samples into the regression curve and calculate the protein concentration based on the sample volume. Take 40 μg of total protein from each sample and run SDS-PAGE protein gel at 100 V. Stop electrophoresis when the protein samples reach the bottom of the separation gel.

[0050] (iii) Prepare the transfer buffer and sandwich the gel, NC membrane, transfer filter paper, and filter cotton in the order of negative electrode-filter cotton-transfer filter paper-gel-NC membrane-transfer filter paper-filter cotton-positive electrode. Insert the gel into the transfer tank and transfer the membrane at a constant current of 200 mA for 1.5 h in a 4°C refrigerator.

[0051] (iv) After transfer, the membrane was cut to the desired size along the protein marker. A blocking buffer containing 10% skim milk powder was prepared in TBST buffer and blocked on a shaker at room temperature for 1 h. The skim milk was then washed off with TBST. The NC membrane was cut according to protein molecular weight and incubated with primary antibodies against Notch1, ZO-1, VE-cadherin, β-catenin, and the internal control β-actin, respectively, overnight at 4°C on a shaker.

[0052] (v) Discard the primary antibody and rinse the membrane twice with PBST buffer on a shaker for 10 minutes each. Prepare the secondary antibody at a dilution of 1:5000 in TBST buffer containing 2% nonfat dry milk and incubate the membrane at room temperature for 1 hour. Rinse the membrane five times with TBST buffer for 5 minutes each. Blot the membrane dry with filter paper and develop color with the luminescent substrate mixture for 5 minutes in the dark. Blot the membrane dry with filter paper and place it in a chemiluminescence imager for exposure and storage.

[0053] 1.2 Experimental Results

[0054] like Figure 2As shown in the results, JAG-1 treatment significantly upregulated the expression level of Notch-1 mRNA in BMEC cells, indicating that it can activate the Notch-1 signaling pathway at the transcriptional level and provide an upstream signal basis for barrier repair.

[0055] Example 2 Detection of the Effect of JAG-1 on Enterovirus Replication in Intestinal Cells

[0056] HT29 cells are human colon cancer cells. In this example, HT29 cells were treated with different concentration gradients of JAG-1. The experimental group (EV71) was treated with EV71 virus, while the mock group was not treated with EV71 virus. Protein lysates were then collected and immunoblotted to detect the expression levels of Notch-1 (Notch receptor), VP1 (EV71 surface antigen protein), and β-actin (internal control) to examine the effect of JAG-1 on enterovirus replication in intestinal cells.

[0057] 2.1 Experimental steps

[0058] HT29 cells were cultured at a rate of 5 × 10 5 Cells were seeded in 12-well cell culture plates using 10% FBSDMEM medium supplemented with 1% penicillin and streptomycin and cultured in a humidified incubator at 37°C and 5% CO2.

[0059] After HT29 cells adhered, JAG-1 (0, 10, 20, 50 μM) was added and treated for 2 h, followed by EV71 (MOI = 0.5) treatment for 24 h. Protein lysates were collected and immunoblotting (as described in the immunoblotting detection steps in Example 1) was used to detect the expression levels of Notch-1 and EV71VP1.

[0060] 2.2 Experimental Results

[0061] like Figure 3 As shown, treatment with JAG-1 at concentrations of 10-50 μM resulted in a corresponding increase in Notch1 expression, unaffected by EV71 infection. JAG-1 has a strong ability to activate the Notch1 signaling pathway and is not easily inhibited by enterovirus infection.

[0062] Example 3: Detection of the Selective Permeability of JAG-1 in an In Vitro Blood-Brain Barrier Model

[0063] BMEC is the main component cell of BBB. In the embodiment, the present invention uses BMEC cells to establish a blood-brain barrier (BBB) ​​in vitro model in a Transwell culture system, and BMEC in the chamber is co-cultured with the bottom cells (RD cells) in the lower chamber. There is no physical contact between the two cells. EV71-infected HT29-derived exosomes (EVs) were labeled with DiI dye. JAG-1 treated BMEC (the control group was not treated), and EVs (adsorbed DiI dye) and EV71, or only EV71, or only EVs (adsorbed DiI dye) were infected with BMEC cells (added to the upper chamber), and the fluorescence of the RD cells in the lower chamber was detected to detect the situation of EVs penetrating the blood-brain barrier in vitro model, so as to understand the effect of JAG-1 on its selective permeability in the blood-brain barrier in vitro model.

[0064] 3.1 Experimental steps

[0065] BMECs were digested from T75 cells using 0.25% trypsin, resuspended in complete medium, and counted. 1 × 10 5 The cells were plated on the chamber membrane (0.4 μm pore size) of a Transwell cell culture plate and cultured into a dense monolayer of cells. JAG-1 (50 μM) was then added and treated for 2 h.

[0066] Exosomes (EVs) from HT29 cells infected with EV71 were incubated with DiI dye for 30 minutes. EV71 or DiI-adsorbed EVs were added to the upper chamber of the in vitro BBB model for 24 hours. RD cells in the lower chamber were observed using a 20x objective lens under bright field and fluorescence (red) conditions, and images were captured and saved.

[0067] 3.2 Experimental Results

[0068] like Figure 4 As shown in the results, EV71 infection-derived exosomes significantly increased the permeability of the blood-brain barrier model, while JAG-1 could significantly reduce its permeability level, indicating that it has the function of alleviating barrier damage caused by exosome damage.

[0069] Example 4 Effect of JAG-1 on the integrity of the blood-brain barrier in vitro model

[0070] BMEC is the main component cell of BBB. In an embodiment, the present invention uses BMEC cells to establish a blood-brain barrier (BBB) ​​in vitro model in a Transwell culture system, and BMEC in the chamber is co-cultured with the bottom cells (RD cells) in the lower chamber. There is no physical contact between the two cells. JAG-1 treats BMEC (the control group is not treated). BMEC cells are infected with EVs and EV71, or only EV71, or only EVs (added to the upper chamber). FITC-labeled dextran is added to each chamber. The integrity of the BBB in vitro model is detected by detecting the fluorescence value of the RD cells in the lower chamber, thereby understanding the effect of JAG-1 on its integrity in the BBB in vitro model. Then qPCR detects VP1 mRNA in the lower layer RD cells to detect the invasion of EV71 and EVs into the BBB in vitro model, so as to understand the effect of JAG-1 on viral invasion in the BBB in vitro model.

[0071] 4.1 Experimental steps

[0072] BMECs were digested from T75 cells using 0.25% trypsin, resuspended in complete medium, and counted. 1 × 10 5 Cells were plated on the chamber membrane (0.4 μm pore size) of Transwell cell culture plates and cultured. Cells were counted and 5×10 5 RD cells were plated in the lower chamber. After the upper chamber was cultured into a dense monolayer of cells, JAG-1 (50 μM) was added and treated for 2 hours.

[0073] Exosomes (EVs) derived from EV71 or EV71-infected HT29 cells were added to the upper chamber of the in vitro BBB model for 24 hours. 10KFITC-labeled dextran was diluted to 10 mg / mL and 50 μL was added to each chamber, and the culture was continued for 2 hours.

[0074] After the culture is completed, 100 mL of the lower layer of culture medium was removed and added to a light-proof 96-well plate. The fluorescence value at 485 / 520 nm was read using a multifunctional microplate reader. The samples were repeated in triplicate, and the data were recorded and saved. The lower layer of cells was removed and mRNA was detected using the qPCR method.

[0075] The specific steps of qPCR detection of mRNA are as follows:

[0076] (i) Rinse the collected cells twice with PBS and transfer them to a 1.5 mL centrifuge tube. Add 1 mL of Trizol reagent to each sample, pipette thoroughly to evenly distribute the cells, and lyse the cells at room temperature for 10 min. Using an RNase-free pipette tip, pipette 200 μL of chloroform into the RNA sample. Shake vigorously on a shaker for 15 seconds. Let the tube stand at room temperature for 5 minutes. Transfer the tube to a high-speed refrigerated centrifuge and centrifuge at 13,000 g for 15 minutes at 4°C.

[0077] (ii) Transfer the supernatant to a new 1.5 mL RNase-free centrifuge tube, add an equal volume of isopropanol, mix by inversion, and let stand at room temperature for 10 minutes. Transfer the tube to a high-speed refrigerated centrifuge and centrifuge at 13,000 g for 10 minutes at 4°C. Decant the supernatant. Add 1 mL of 70% ethanol in DEPC water to the pellet, desalt it, and centrifuge at 13,000 g for 5 minutes at 4°C. Repeat twice. Discard the ethanol wash solution, air dry for 10-20 minutes, and dissolve the RNA in 40 μL of DEPC water. Aspirate 1 μL to determine the total RNA concentration, and prepare the remaining volume for reverse transcription.

[0078] (iii) 1 μg of RNA was taken from each tube and added to 50 μL with 10 μL 5X Reaction Buffer, 2 μL dNTPs (10 mM), 1 μL Ribonuclease Inhibitor, 2 μL random primers (10 nM), 1 μL M-MLV RT, and DEPC water for reverse transcription.

[0079] (iv) qPCR reaction was performed on the reverse transcribed cDNA using Roche LightCycler 480, using 2 -(ΔΔCt) The relative quantification of mRNA was calculated by the method.

[0080] 4.2 Experimental Results

[0081] like Figure 5 As shown in the results, EV71 and EVs destroyed the integrity of the BBB in vitro model. Under the action of JAG-1, the integrity of the BBB in vitro model was restored and the permeability was significantly reduced, indicating that JAG-1 may play a certain role in repairing the integrity of the BBB.

[0082] like Figure 6 As shown in the figure, no VP1 transcription was found in BMEC infected with EV71. VP1 was significantly transcribed in BMEC infected with EV71 and EVs. However, after JAG-1 treatment, the relative expression of VP1 mRNA was significantly reduced, indicating that the degree of viral replication was inhibited, indicating that JAG-1 enhanced the ability of the BBB in vitro model to prevent viral invasion. EV71-infected exosomes increase viral invasion, and JAG-1 slows this process by repairing the blood-brain barrier.

[0083] Example 5: Detection of the Effect of JAG-1 on Body Weight and Survival Rate of Suckling Mice Infected with EV71

[0084] In the examples, suckling mice were divided into four groups: an infected group injected with JAG-1, an infected control group injected with BSA, a non-infected group injected with JAG-1, and a non-infected control group injected with BSA. The weight and survival status of the suckling mice were continuously measured.

[0085] 6.1 Experimental steps

[0086] (1) Three-day-old C57BL / 6 suckling mice were divided into infection group and non-infection group, and EV71 was injected intraperitoneally (1×10 7 PFU (PFU, resuspended in 20 μL PBS) and an equal volume of PBS. Six and 24 hours after viral infection, mice were injected twice with equal amounts of BSA or 0.5 μg / g JAG-1 protein. Body weight and survival rate of the mice were measured and calculated over seven days.

[0087] (2) The body weight, clinical scores, and survival status of the suckling mice were measured and calculated for seven consecutive days, and the survival curve was calculated.

[0088] 6.2 Experimental Results

[0089] like Figure 7 As shown, compared to infected suckling mice not injected with JAG-1 protein, those injected with JAG-1 protein showed significantly improved weight loss and a significantly reduced mortality rate. These results indicate that EV71-infected suckling mice experience weight loss and decreased survival, while JAG-1 treatment mitigates weight loss and significantly prolongs survival, suggesting that it has the effect of slowing disease progression and improving survival in vivo.

[0090] Example 6 Effect of JAG-1 on Viral Replication in the Intestinal Tissue and Brain Tissue of Suckling Mice

[0091] In the example, suckling mice were divided into four groups: an infected group injected with JAG-1, an infected control group injected with BSA, a non-infected group injected with JAG-1, and a non-infected control group injected with BSA. Different tissues of the suckling mice were obtained and the relative quantification of viral TCID50 in different tissues was detected.

[0092] 7.1 Experimental steps

[0093] (1) Three-day-old C57BL / 6 suckling mice were divided into infection group and non-infection group, and EV71 was injected intraperitoneally (1×10 7 PFU, resuspended in 20 μL PBS) and an equal volume of PBS. At 6 h and 24 h after virus infection, an equal amount of BSA or 0.5 μg / g JAG-1 protein was injected twice.

[0094] (2) On the third day of infection, 4 suckling mice were selected from each group, and the following organs were dissected and separated: brain, liver, heart, lung, spleen, intestine and muscle, and weighed.

[0095] (3) Grind the tissue, resuspend it in 100 μL PBS, centrifuge it at 12,000 rpm at 4°C for 5 minutes, collect the supernatant, and dilute it 10-fold in series into sterile centrifuge tubes.

[0096] (4) Remove the 96-well plate containing RD cells from the CO2 incubator, discard the supernatant, wash twice with PBS, and add 100 μL of virus at different dilutions to each well. Repeat for each dilution in 8 wells. For the control group, replace the virus solution with 100 μL PBS, and then add 100 μL of fresh DMEM culture medium to each well, for a total volume of 200 μL.

[0097] (5) Observe the cell culture plate and continue culturing in a 37°C, 5% CO2 incubator. Observe the cell lesions daily with an inverted microscope for at least one week. Calculate the viral TCID using the Reed-Muench formula. 50 relative quantification.

[0098] 7.2 Experimental Results

[0099] like Figure 8 As shown, viral replication was significantly inhibited in the brain tissue of infected suckling mice injected with JAG-1 protein compared to the control group. However, this effect was not observed in the intestinal tissue. This suggests that JAG1 inhibits enterovirus replication in the brain but not in the intestinal tissue. This suggests that JAG-1 blocks viral replication in the brain by enhancing blood-brain barrier function.

[0100] Example 7 Effects of JAG-1 on Pathological Changes in Intestinal Tissue and Brain Tissue of Suckling Mice

[0101] In the examples, suckling mice were divided into four groups: an infected group injected with JAG-1, an infected control group injected with BSA, a non-infected group injected with JAG-1, and a non-infected control group injected with BSA. The pathological changes in the small intestine and brain of the suckling mice in each group were detected by sectioning.

[0102] 8.1 Experimental steps

[0103] (1) Three-day-old C57BL / 6 suckling mice were divided into infection group and non-infection group, and EV71 was injected intraperitoneally (1×10 7 PFU, resuspended in 20 μL PBS) and an equal volume of PBS. At 6 h and 24 h after virus infection, an equal amount of BSA or 0.5 μg / g JAG-1 protein was injected twice.

[0104] (2) On the third day of infection, four suckling mice were selected from each group, and the small intestine and brain were dissected and separated. The small intestine and brain were dehydrated once with 50%, 70%, 80%, and 95% ethanol, respectively, and dehydrated twice with anhydrous ethanol, each dehydration level for 1 hour. The brain was then transparentized with xylene for 2 hours and embedded in paraffin.

[0105] (3) Slice the wax block with a microtome, place it in secondary xylene for 20 min each, secondary anhydrous ethanol for 20 min each, 95% ethanol for 5 min, 70% ethanol for 5 min, 50% ethanol for 5 min, and finally wash with distilled water.

[0106] (4) Stain with hematoxylin solution for 15 minutes, then rinse with distilled water until blue. Transfer to 1% hydrochloric acid alcohol for differentiation for 30 seconds until the sections fade to light red.

[0107] (5) Re-stain with eosin solution for 3 minutes, wash with distilled water, dehydrate with 90% ethanol for 5 minutes each, dehydrate with anhydrous ethanol for 5 minutes each, and clear with xylene for 5 minutes each.

[0108] (6) After drying at room temperature, seal the slides with resin, observe under a microscope, take photos, and preserve them.

[0109] 8.2 Experimental Results

[0110] The results are as follows Figure 9 As shown in the figure, the viral load in the brain tissue of the JAG-1-treated group was significantly lower than that of the EV71 group, and the structure was clear and complete, indicating that JAG-1 can prevent the virus from crossing the BBB and entering the brain tissue by improving the structural integrity of the blood-brain barrier.

[0111] Example 8 Effect of JAG-1 on Brain Permeability in Suckling Rats

[0112] In the examples, the suckling mice were divided into four groups: an infected group injected with JAG-1, an infected control group injected with BSA, a non-infected group injected with JAG-1, and a non-infected control group injected with BSA. Each group of suckling mice was intraperitoneally injected with FITC-labeled dextran, and the FITC fluorescence leakage in the brain of each group of suckling mice was detected.

[0113] 9.1 Experimental steps

[0114] (1) Three-day-old C57BL / 6 suckling mice were divided into infection group and non-infection group, and EV71 was injected intraperitoneally (1×10 7 PFU, resuspended in 20 μL PBS) and an equal volume of PBS. At 6 h and 24 h after virus infection, an equal amount of BSA or 0.5 μg / g JAG-1 protein was injected twice.

[0115] (2) On the third day of infection, 4-5 suckling mice were selected from each group and injected intraperitoneally with FITC-Dextran (10 kDa). 90 minutes later, the brains were removed by dissection and homogenized with methylcobalamin at 37°C, 200 rpm, and incubated overnight.

[0116] (3) Low-speed centrifugation was performed to obtain the supernatant, and the multifunctional microplate reader was used to measure the ex / λ em = FITC fluorescence leakage value at 485 / 520 nm, statistically plotted.

[0117] 9.2 Experimental Results

[0118] The results are as follows Figure 10 As shown, the permeability of the brain tissue of infected rats in the JAG-1 protein injection group was significantly reduced compared with the control group, indicating that JAG-1 treatment restored the integrity of the BBB in the brain tissue of infected rats. The results show that JAG-1 can activate the Notch pathway in vivo and restore the expression of tight junction proteins, thereby strengthening the structure and function of the blood-brain barrier.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of JAG-1 protein in the preparation of a medicament for treating enterovirus-infected diseases, wherein the Uniprot database ID of the JAG-1 protein is P78504.

2. The use according to claim 1, characterized in that The medicine is a preparation prepared by taking JAG-1 protein as the active ingredient and adding pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that The preparation is an oral preparation or an injection.

4. The use according to claim 1, wherein The enterovirus comprises at least one of enterovirus 71, coxsackievirus B3 and coxsackievirus B4.

5. Use of JAG-1 protein in the preparation of a drug for treating diseases caused by blood-brain barrier damage, characterized in that: The disease is a disease in which the blood-brain barrier is destroyed due to enterovirus infection, and the Uniprot database ID of the JAG-1 protein is P78504.

6. The use according to claim 5, characterized in that The medicine is a preparation prepared by taking JAG-1 protein as the active ingredient and adding pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that The preparation is an oral preparation or an injection.

8. The use according to claim 5, characterized in that The enterovirus comprises at least one of enterovirus 71, coxsackievirus B3 and coxsackievirus B4.

9. The use according to claim 5, characterized in that The diseases causing blood-brain barrier damage include at least one of brain trauma, stroke, brain tumor, Alzheimer's disease, cerebral hemorrhage, cerebral hypoxia, cerebral edema, encephalitis, meningitis, multiple sclerosis, acute disseminated encephalomyelitis, autoimmune encephalitis, viral encephalitis, vascular dementia, and Parkinson's disease.

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