Use of a lymphangiogenesis inducer

Abstract

The present invention discloses the use of a lymphangiogenesis inducer. The lymphangiogenesis inducer can be used to treat neurotropic virus infections in an individual, especially viral encephalitis and inflammation-related central nerve damage. The lymphangiogenesis inducer can promote the generation and function of dural lymphatics, enhance lymphatic circulation, block excessive central nerve inflammatory responses, etc., thereby enhancing the body's resistance to lethal central nervous system virus infections, reducing viral encephalitis, and reducing damage to nerve cells and central nerve tissues. The present invention also discloses the use of monoclonal antibody Enbrel in the preparation of a drug for treating neurotropic virus infections in an individual, especially applicable to individuals with VEGFR-3 mutations or impaired brain lymphatic function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to the use of lymphangiogenesis inducers, in particular to the use of a lymphangiogenesis inducer in the preparation of a drug for treating neurotropic virus infections in an individual. Among them, the lymphangiogenesis inducer can promote the generation of lymphatic vessels in the brain and central nervous system, and the lymphangiogenesis inducer includes multiple members of the vascular endothelial growth factor (VEGF) family, especially VEGF-C. Background Art

[0002] Infectious encephalitis is an inflammation of the brain parenchyma caused by various pathogen infections. It is a serious neurological disease that affects world public health security. If the inflammatory lesions of infectious encephalitis spread or become fibrotic, the damage and necrosis of brain cells are irreversible, which will affect the normal functions of cranial nerves and cerebral blood vessels and is not conducive to the recovery and prognosis of brain function. Therefore, timely and reasonable treatment should be carried out in the early stage of the course of infectious encephalitis.

[0003] In the United States, approximately 7 people per 100,000 population are hospitalized for encephalitis each year. 20 - 50% is attributed to viruses. Viral infections are the main cause of encephalitis in the central nervous system. Viral encephalitis occurs due to viral infection of the brain parenchyma, usually accompanied by meningitis, resulting in meningoencephalitis. The usual situation is an acute disease with a diffuse inflammatory process, severely affecting brain function, sometimes irreversibly. The result of the virus entering the central nervous system (CNS) is jointly determined by the host immune response and the neurovirulence of the infecting virus. Viral encephalitis causes acute inflammation of the brain parenchyma and is an important cause of human morbidity and mortality. An increasing variety of viruses can cause viral encephalitis. Herpesviruses and arboviruses are the most important pathogens in the world. Among them, herpes simplex virus is the most common cause of encephalitis in adults in Western countries. Herpes simplex virus (HSV) is a member of the Herpesvirinae subfamily of the Herpesviridae family. It can cause devastating central nervous system infections, such as herpes simplex viral encephalitis (HSE), which is fatal and can lead to significant neurological diseases (including behavioral disturbances and focal nerve damage). According to statistics, the global incidence of HSE is (1.5 - 7) people per 100,000 per year. With the application of antiviral drugs such as acyclovir, the fatality rate of HSE has dropped from 79% to less than 20%, but the disability rate of survivors is still as high as 58%, bringing a heavy burden to the patients' families and society. In addition, Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are neurotropic flaviviruses belonging to the Flaviviridae family. Japanese encephalitis (JE) caused by JEV is the most common encephalitis in many Asian countries, with an estimated 68,000 clinical cases and 10,000 - 15,000 related deaths each year. ZIKV led to a large-scale and expanding disease pandemic in the Americas from 2014 to 2015. It is an emerging flavivirus of increasing global importance and has been confirmed to cause severe acute infections, congenital microcephaly, and viral encephalitis.

[0004] Currently, there is no effective treatment for encephalitis, neurological symptoms, or death caused by neurotropic viruses. Immunomodulators can be used to treat encephalitis, either as an adjunct to antiviral drugs or as a single therapy when there is no effective antibacterial agent. Glucocorticoids are probably the most widely used drugs, and their effects are uncertain. Reports show that interferon treatment may have good effects on arbovirus infections caused by West Nile virus or St. Louis encephalitis virus, but in a randomized trial of Japanese encephalitis patients, interferon had no effect. Intravenous injection of immunoglobulin containing high-titer virus-specific antibodies also cannot change the prognosis of patients with West Nile virus encephalitis. Therefore, finding new drugs is of great significance for the treatment of viral encephalitis and resistance to neurotropic virus infections.

[0005] VEGF-C is a specific growth factor for lymphatic vessels. It mainly acts on lymphatic endothelial cells (LECs) through its receptor VEGFR-3, promoting their survival, growth and migration, thereby promoting lymphangiogenesis. In addition, VEGF-C can also act on VEGFR-3 and VEGFR-2 to promote angiogenesis and regulate vascular permeability. Recombinant VEGF-C and AAV-VEGF-C, which are VEGFR-3 ligands, are injected into the cerebrospinal fluid through the cisterna magna, and can reconstruct the meningeal lymphatic vessels at the confluence of the dura mater of the cerebral sinus and the sagittal sinus. Studies have confirmed that in experimental autoimmune encephalomyelitis (EAE), lymphangiogenesis dependent on the VEGF-C / VEGFR-3 pathway occurs in the lymphatic vessels near the cribriform plate, which helps to expel central nervous system-derived antigens and cells. Meningeal lymphangiogenesis dependent on VEGF-C has been shown to significantly enhance the effectiveness of anti-PD-1 / CTLA-4 checkpoint combination therapy in a brain tumor model. In addition, VEGF-C can regulate the macrophage-induced antibacterial infection response. Articles have confirmed that VEGF-C can inhibit the excessive inflammation and septic shock induced by TLR4 during bacterial infection. In addition, macrophages can utilize the VEGFR-3 / VEGF-C axis to enhance phagocytosis and bacterial clearance, while inhibiting inflammasome activation and macrophage apoptosis. However, its application in inducing meningeal lymphangiogenesis, enhancing the function of meningeal lymphatic vessels, inhibiting neurotropic virus infection, viral encephalitis and nerve injury has not been reported.

[0006] In addition, there are a large number of patients with weak lymphatic circulation in clinical practice, such as patients with tumors and atherosclerosis. It is difficult for them to resist neurotropic virus infection by enhancing lymphatic circulation. There is no report in the prior art on using TNF-α inhibitors to treat the inflammatory response and neuronal death after neurotropic virus infection. Summary of the Invention

[0007] The object of the present invention is to solve the defects of the prior art, and provide a use of a lymphangiogenesis inducer, especially a use of a lymphangiogenesis inducer in the preparation of a drug, for treating neurotropic virus infection in an individual. The lymphangiogenesis inducer is an effective drug for resisting neurotropic virus infection, treating viral encephalitis and nerve injury. The use of the lymphangiogenesis inducer in the preparation of drugs for resisting neurotropic virus infection and viral encephalitis has great clinical value and application prospects. The present invention also proposes to use the monoclonal antibody Enbrel targeting the TNF receptor in patients with neurotropic virus infection, viral encephalitis, inflammation-related central nervous system injury and diseases, especially those with VEGFR-3 mutation or cerebral lymphatic vessel dysfunction, to block the crosstalk between nerve cells, macrophages and microglia, effectively block the inflammatory response and neuronal death, and reduce the mortality rate.

[0008] On the one hand, the present invention provides the use of a lymphangiogenesis inducer in the preparation of a medicament for treating a neurotropic virus infection in an individual.

[0009] Preferably, the neurotropic virus infection includes an inflammation-related central nervous disease caused by a neurotropic virus infection, which includes administering a lymphangiogenesis inducer to the individual; the lymphangiogenesis inducer is used to promote lymphangiogenesis in the brain and / or central nervous system of the individual and enhance lymphatic vessel function.

[0010] Preferably, the neurotropic virus infection includes viral encephalitis caused by a neurotropic virus infection, which includes administering an effective dose of a lymphangiogenesis inducer to the individual.

[0011] In some specific embodiments, the lymphangiogenesis inducer includes VEGF-A, VEGF-B, VEGF-C or VEGF-D.

[0012] Preferably, the lymphangiogenesis inducer includes VEGF-C.

[0013] Preferably, the neurotropic virus includes, but is not limited to, rabies virus, Zika virus, Japanese encephalitis virus, herpes simplex virus, vesicular stomatitis virus.

[0014] Preferably, the medicament is an injection, and the lymphangiogenesis inducer is in the form of a protein.

[0015] Neurotropic virus infection can cause high expression of VEGF-C and VEGFR-3 in the brain. When human VEGF-C protein is slowly released in the central nervous system through the skull, it can induce meningeal lymphangiogenesis, enhance meningeal lymphatic vessel function, inhibit neurotropic virus infection in a mouse model, and prevent death caused by JEV. Manipulating meningeal lymphangiogenesis can effectively prevent and / or treat neurotropic virus infection, viral encephalitis, and nerve damage, etc.

[0016] On the other hand, the present invention also provides the use of a monoclonal antibody Enbrel in the preparation of a medicament for treating a neurotropic virus infection in an individual; the individual has a VEGFR-3 mutation or impaired brain lymphatic vessel function.

[0017] Preferably, the neurotropic virus infection includes an inflammation-related central nervous disease caused by a neurotropic virus infection, which includes administering an effective dose of monoclonal antibody Enbrel to the individual; the monoclonal antibody Enbrel is used to block excessive inflammation and nerve cell death.

[0018] Preferably, the neurotropic virus infection includes viral encephalitis caused by a neurotropic virus infection, which includes administering an effective dose of monoclonal antibody Enbrel to the individual.

[0019] In addition, the TNF receptor monoclonal antibody Enbrel injected intraperitoneally can reduce the death caused by JEV infection and reduce the expression of inflammatory genes and apoptotic genes in the brain.

[0020] Beneficial effects:

[0021] The present invention discloses the use of a lymphangiogenesis inducer and the monoclonal antibody Enbrel in the preparation of a medicament for treating individuals infected with neurotropic viruses. Infection with neurotropic viruses can cause high expression of VEGF-C and VEGFR-3 in the brain, activate VEGFR-3, and induce meningeal lymphangiogenesis. When a lymphangiogenesis inducer (i.e., VEGF-C) is injected, it can effectively inhibit the infection of neurotropic viruses and prevent death caused by the virus. Therefore, the present invention provides the application of manipulating meningeal lymphangiogenesis in preventing infections and neurological symptoms such as neurotropic virus infection, viral encephalitis, and nerve damage. By using human VEGF-C protein, the present invention can effectively induce meningeal lymphangiogenesis, enhance the function of meningeal lymphatics, inhibit the infection of neurotropic viruses, and play a protective role for the host.

[0022] In addition, VEGF-C can also inhibit the secretion of TNF-α and reduce apoptosis in the nervous system. Therefore, for individuals with VEGFR-3 mutations or impaired brain lymphatic function, administration of the monoclonal antibody Enbrel targeting the TNF receptor can effectively block the inflammatory response and neuronal death and reduce the mortality rate.

[0023] Other features and advantages of the present invention will be described in detail in the following specific implementation section. Brief Description of the Drawings

[0024] Figure 1 : High expression of inflammatory factors in the brain caused by neurotropic virus infection;

[0025] Figure 2 : High expression of VEGF-C and VEGFR-3 in the brain caused by neurotropic virus infection;

[0026] Figure 3 : VEGF-C induces meningeal lymphangiogenesis and enhances lymphatic function;

[0027] Figure 4 : Manipulating meningeal lymphangiogenesis reduces the viral load in the brains of mice infected with Japanese encephalitis virus;

[0028] Figure 5 : Manipulating meningeal lymphangiogenesis protects mice from lethal infection with Japanese encephalitis virus;

[0029] Figure 6 : Macrophage VEGF-C / VEGFR-3 signaling reduces neuronal apoptosis by inhibiting TNF-α secretion;

[0030] Figure 7 : Enbrel was given to reduce the expression of brain inflammation in mice infected with Japanese encephalitis virus;

[0031] Figure 8 : Enbrel was given to protect mice from lethal infection caused by Japanese encephalitis virus infection. Detailed implementation mode

[0032] The following will detail the implementation mode of this application through examples, so as to fully understand how this application uses technical means to solve technical problems and achieve the implementation process of technical effects and implement accordingly.

[0033] The raw materials and equipment used in this application, unless otherwise specified, are common raw materials and equipment in this field and are all from commercially available products. The methods used in this application, unless otherwise specified, are conventional methods in this field.

[0034] There are also many other implementable technical solutions in this application, which will not be listed one by one here. The technical solutions claimed in the claims of this application are all implementable.

[0035] "Comprising" or "including" is intended to mean that a composition (such as a medium) and a method include the recited elements, but do not exclude other elements. When used to define a composition and a method, "consisting essentially of" means excluding other elements that are of any significant importance for the purpose of the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not substantially affect the basic and novel features of the claimed application. "Consisting of" means excluding trace amounts of other components and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this application. Obtained by the preparation method disclosed in the patent.

[0036] The present invention relates to the application of strategies such as manipulating meningeal lymphangiogenesis, blocking excessive inflammation and nerve cell death in the prevention and / or treatment of neurotropic virus infection, viral encephalitis, inflammation-related central nervous system diseases or nerve injuries, etc.

[0037] The above-mentioned viruses can be various common virus types in the art, such as at least one of rabies virus, Zika virus, Japanese encephalitis virus, herpes simplex virus, vesicular stomatitis virus, and other neurotropic viruses. Among them, the above-mentioned subjects can be common mammals susceptible to virus infection, especially primates (such as humans or monkeys) or rodents (such as mice). Among them, the administration method can be intracranial administration or cisterna magna injection, etc. The administration dose can be a conventional dose (effective amount) in the art and can be determined according to various parameters, especially according to the age, weight, and sex of the subject. The present invention will be described in detail below through examples. In the following examples, room temperature refers to "25 °C"; human VEGF-C is purchased from PeproTech, product number 96-100-20C-20; Zika virus, Japanese encephalitis virus, herpes simplex virus type 1, vesicular stomatitis virus are from Wuhan Institute of Virology, Chinese Academy of Sciences; rabies virus is from Huazhong Agricultural University. Monoclonal antibody Enbrel (etanercept) is from Pfizer; Vegfr-3 fl / fl LyzM-Cre + / - Mice (labeled as Vegfr-3 DLBD / DLBD mice) were propagated. The mice were housed in a specific pathogen-free (SPF) animal house at the Wuhan Institute of Virology, Chinese Academy of Sciences. The apoptosis detection kit was purchased from BD Bioscience. Anti-Cleaved Caspase-3 antibody was purchased from CST, product number 9664. Anti-NeuN was purchased from Millipore, product number MAB377.

[0038] C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the mice were housed in the SPF animal house of the Wuhan Institute of Virology, Chinese Academy of Sciences; the animal infection experiments of Zika virus, Japanese encephalitis virus, herpes simplex virus type 1, and vesicular stomatitis virus were all completed in the Experimental Animal Center of the Wuhan Institute of Virology, Chinese Academy of Sciences, and the animal infection experiment of rabies virus was completed in the Experimental Animal Center of Huazhong Agricultural University, and the permission of the Ethics Committee of the Wuhan Institute of Virology, Chinese Academy of Sciences (batch numbers: NO.WIVA39201602, WIVA39201603, WIVA39201803, WIVA39201804) and the Ethics Committee of Huazhong Agricultural University (batch number: HZAUMO-2016-052) was obtained.

[0039] For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained by purchase.

[0040] Example 1: Neurotropic virus infection induces high expression of inflammatory factors in the brain

[0041] To detect the effect of neurotropic virus infection on the expression of inflammatory factors in the mouse brain. Five- to eight-week-old C57BL / 6 mice were infected with rabies virus (CVS-B2c strain) by intramuscular injection, Zika virus by intraperitoneal injection, Japanese encephalitis virus by intravenous injection, herpes simplex virus type 1 by corneal injection, or vesicular stomatitis virus by nasal mucosa infection. Seven to eight days after infection, mouse brain tissues were collected, and the expression levels of IL-6, IL-10, IL-1β, CCL5, and TNF-α were detected by ELISA or RT-qPCR. The experimental results showed that: compared with the control group (PBS), the mRNA levels of Il1b, Il6, Il10, Ccl5, and Tnfa in the brain tissues of mice infected with rabies virus were significantly increased ( Figure 1 A). Similarly, the mRNA levels of Il1b, Il6, Il10, and Tnfa in the brain tissues of mice infected with Zika virus were significantly increased ( Figure 1 B). The mRNA levels of Il6 and Ccl5 in the brain tissues of mice infected with herpes simplex virus type 1 were significantly increased (Figure 1C). The mRNA levels of Il6, Ccl5, and Tnfa in the brain tissues of mice infected with vesicular stomatitis virus were significantly increased ( Figure 1 D). Compared with the control group (PBS), the protein levels of IL-6 and TNF- in the brain tissues of mice infected with Japanese encephalitis virus were significantly increased ( Figure 1 E).

[0042] Example 2: Neurotropic virus infection induces high expression of VEGF-C and VEGFR-3 in the brain

[0043] To detect the effect of virus infection of the central nervous system on VEGFC and VEGFR-3. The levels of VEGF-C in the brains of mice after different virus infections were detected by ELISA. The results showed that, compared with the control group of mice, the levels of VEGF-C in the brains of mice infected with Japanese encephalitis virus, Zika virus, herpes simplex virus type 1, rabies virus, and vesicular stomatitis virus were significantly increased ( Figure 2 A-E). The expression levels of Vegfr3 in the brains of mice were detected by RT-qPCR. The results showed that, compared with the control group of mice, the levels of Vegfr3 in the brains of mice infected with Japanese encephalitis virus, Zika virus, and herpes simplex virus type 1 were significantly increased ( Figure 2 F-H).

[0044] Example 3: Human VEGF-C induces lymphangiogenesis in the mouse meninges and enhances the function of meningeal lymphatics

[0045] Since VEGF-C can act on lymphatic endothelial cells by activating VEGFR-3, thereby promoting lymphangiogenesis. The main function of meningeal lymphatics is to drain metabolic wastes in cerebrospinal fluid into the cervical lymph nodes. To detect the effect of human VEGF-C on the morphology and function of meningeal lymphatics, hydrogels prepared with 200 ng / ml human VEGF-C or Vehicle were covered on the thinned mouse skull. After 2 weeks, 200 ng / ml human VEGF-C or Vehicle solution was injected at the same position once every other day for a total of 3 times ( Figure 3 A). One week later, 5 μl of OVA-647 (ovalbumin, Alexa Fluor TM 647 conjugate, 2 mg / ml) was injected into the cisterna magna of the mouse cerebellum. At 30 min, 60 min, 120 min, and 180 min after injection, in vivo imaging of the ventral neck of the mouse was performed. Subsequently, the mouse was anesthetized, perfused through the heart, and meningeal tissues were taken to detect changes in meningeal lymphatics by immunofluorescence. The results showed that compared with the Vehicle group, the diameter of meningeal lymphatics in VEGF-C-administered mice was significantly increased ( Figure 3 B). At the same time, 60 min after OVA-647 injection, the fluorescence intensity of OVA-647 in the neck of VEGF-C-administered mice was significantly higher than that of the control group, indicating that VEGF-C administration significantly enhanced the downward drainage ability of meningeal lymphatics in mice ( Figure 3 C).

[0046] Example 4: Manipulating meningeal lymphangiogenesis reduces the viral load in the brains of Japanese encephalitis virus-infected mice

[0047] To detect the role of VEGF-C in the infection of neurotropic viruses. Hydrogels prepared with 200 ng / ml human VEGF-C or Vehicle were covered on the thinned mouse skull. After 2 weeks, 200 ng / ml human VEGF-C or Vehicle solution was injected at the same position once every other day for a total of 3 times, and then Japanese encephalitis virus (7.4x10^6 PFU / mouse) was injected via the tail vein ( Figure 4 A). Four days after infection, mouse brain tissues were taken and the viral load was detected by TCID 50 The results showed that compared with the Vehicle group, VEGF-C significantly reduced the viral load in the brain tissues of virus-infected mice ( Figure 4 B).

[0048] Example 5: Manipulating meningeal lymphangiogenesis protects mice from lethal infection with Japanese encephalitis virus

[0049] Hydrogels prepared with 200 ng / ml human VEGF-C or Vehicle were covered on the thinned mouse skull. After 2 weeks, 200 ng / ml human VEGF-C or Vehicle solution was injected at the same position once every other day for a total of 3 times. Subsequently, Japanese encephalitis virus (7.4x10^6 PFU / mouse) was injected via the tail vein ( Figure 4 A). The body weight changes and mortality of the mice were observed daily. The results showed that VEGF-C treatment could significantly enhance the survival ability of the mice ( Figure 5 ).

[0050] Example 6: Macrophage VEGF-C / VEGFR-3 signaling can also inhibit TNF-α secretion and reduce neuronal apoptosis

[0051] After JEV invades the CNS, it mainly proliferates massively in neuronal cells. Virally infected cells will release a variety of effector factors. Some of these factors directly play an antiviral role, and some will act as signal transduction molecules to activate or chemotax immune cells. However, the specific interaction between neurons and immune cells during this process is not very clear. Therefore, we used the conditioned medium of JEV-infected Neuro-2a cells (N2a, mouse neuroblastoma cells) (JEV-CM) to stimulate PEMs (peritoneal macrophages) to establish a simplified microenvironment model of the effect of virally infected neurons on macrophages. PEMs were pretreated with VEGF-C, and then the Tnfa mRNA level in PEMs supplemented with JEV-CM (JEV-CM was mixed with fresh cell culture medium at a ratio of 1:1) was detected. It was found that the transcription of Tnfa could be downregulated after adding VEGF-C ( Figure 6 A). Further confirmation in Vegfr-3 △LBD / △LBD PEMs showed that the secretion of TNF-α in JEV-CM-treated PEMs increased significantly after VEGFR-3 deletion ( Figure 6 B). To detect whether VEGFR-3 + macrophages can protect neurons from apoptosis by downregulating TNF-α under viral infection conditions, WT or Vegfr-3 △LBD / △LBD PEMs were co-cultured with JEV-infected N2a cells, and Annexin and PI staining flow cytometry was used to detect the apoptosis of N2a cells. The results showed that the apoptosis rate of N2a cells co-cultured with Vegfr-3 △LBD / △LBD PEMs was higher ( Figure 6 C). After the mice were challenged with the virus, the brain tissues were collected and cryosectioned for caspase-3 immunofluorescence staining. The immunofluorescence staining results showed that compared with WT mice, caspase-3 △LBD / △LBD in the brain tissue sections of JEV-infected Vegfr-3 +More cells( Figure 6 D). This indicates that the macrophage VEGF-C / VEGFR-3 signal can also inhibit TNF-α secretion and reduce neuronal apoptosis.

[0052] Example 7: Administration of Enbrel to reduce the expression of brain inflammation in mice infected with Japanese encephalitis virus

[0053] Mice were injected with JEV (7.4x10^6 PFU / mouse) via the tail vein. Starting from the first day of virus challenge, Enbrel (etanercept, a recombinant tumor necrosis factor receptor that blocks the binding of TNF-α to its receptor) or control saline (NS) was intraperitoneally injected into JEV-infected mice at a dose of 10 mg / kg every other day for a total of 3 injections. Compared with the NS control group, the mRNA levels of Tnfa, Il1b, and Ifng in the brains of Enbrel-treated mice were downregulated( Figure 7 ), indicating that blocking or inhibiting the TNF-α signal can reduce the level of neuroinflammation.

[0054] Example 8: Administration of Enbrel to protect mice from lethal infection caused by Japanese encephalitis virus

[0055] Mice were injected with JEV (7.4x10^6 PFU / mouse) via the tail vein. Starting from the first day of virus challenge, Enbrel (etanercept, a recombinant tumor necrosis factor receptor that blocks the binding of TNF-α to its receptor) or control saline (NS) was intraperitoneally injected into JEV-infected mice at a dose of 10 mg / kg every other day for a total of 3 injections. The body weight changes and mortality of the mice were observed daily. The results showed that compared with the NS control group, Enbrel treatment significantly enhanced the survival ability of the mice( Figure 8 ).

[0056] The specific steps of the experimental methods used in the above examples are as follows:

[0057] (1) RNA extraction, reverse transcription, and real-time quantitative PCR:

[0058] The brain tissue was homogenized and ground. A portion of the tissue was taken and lysed with 500 μl of Trizol, incubated at room temperature for 5 min, 100 μl of chloroform was added, vortexed vigorously for 30 s, incubated at room temperature for 10 min, centrifuged at 12,000 rpm at 4°C for 15 min. The upper aqueous phase (about 200 μl of the upper liquid) was added with an equal volume of isopropanol, incubated at room temperature for 10 min, centrifuged at 12,000 rpm at 4°C for 15 min, washed once with 800 μl of 75% ethanol, centrifuged at 12,000 rpm at 4°C for 5 min. The supernatant was removed, the liquid was aspirated with a small pipette tip, and the precipitate was air-dried for 5 - 10 min. After adding 20 μl of ddH2O (DEPC-treated) and dissolving and mixing, the RNA concentration was measured using Nanodrop2000. The RNA was stored at -80°C.

[0059] Reverse transcription. First, mix 1 μg of RNA and 100 ng of Oligo(dN6), and adjust the volume to 15 μl with DEPC water. Incubate at 70 °C for 10 min, then place on ice for 2 min. Then add the Mix solution (5 μl of reverse transcriptase buffer (5×), 1 μl of M-MLV reverse transcriptase, 1 μl of 10 mM dNTP, adjusted to 10 μl with DEPC water). Incubate at 37 °C for 1 h, and inactivate the reverse transcriptase activity at 72 °C for 10 min. Add 200 μl of deionized water to the reverse transcription product, which can be used as the cDNA template for the qPCR reaction.

[0060] qPCR reaction system (20 μl): 3.2 μl of deionized water, 10 μl of SYBR-Green Master Mix, 6 μl of diluted cDNA template, 0.8 μl of forward and reverse primer mixture (each 10 μM). The reaction program is as follows: 95 °C for 3 min; 95 °C for 10 sec, 60 °C for 30 sec, 72 °C for 30 sec, read fluorescence value, for a total of 40 cycles; 95 °C for 1 min; 55 °C for 1 min; melting at 55 °C - 98 °C, with the temperature increasing by 5 °C every 5 sec, and reading the fluorescence value simultaneously. Detect the relative content of mRNA in tissues through specific primers, and select Gapdh as the internal reference gene.

[0061] Table 1 PCR primer sequences

[0062]

[0063] (2) ELISA detection

[0064] The content of IL-6 and TNF-α in the supernatant of mouse brain tissue homogenate was detected by ELISA according to the following steps: the primary antibody was coated with coating solution (PBS containing 10mM NaHCO3; pH 9.0) in a 96-well plate at 4°C overnight, and each well was washed 5 times with 200μl washing solution (PBS+0.05% Tween), and then blocked with 200μl of 1% BSA at room temperature for 1h. The standard was diluted with 1% BSA in a gradient manner, and the sample was also diluted in a certain proportion as needed. The standard dilution solution and the diluted sample were added to each well at 100μl, and incubated at room temperature for 2h or 4°C overnight. Then add 200μl washing solution to each well and wash 5 times, then add 100μl diluted Streptavadin-coupled detection antibody to each well, incubate at room temperature for 2h, wash 5 times with 200μl washing solution, then add 100μl Biotin-HRP solution, incubate at room temperature in the dark for 2h, then add 200μl washing solution to each well and wash 7 times, add 100μl TMB substrate solution, incubate in the dark for about 15min, and add 100μl stop solution (2M H2SO4). Measure the absorbance at 450nm. Determine the linear equation based on the concentration of the standard and its OD value to calculate the content of cytokines in the sample.

[0065] VEGF-C was performed according to the manufacturer's manual, which is briefly described as follows: a. Dilute the standard sample gradient dilution and sample at a certain multiple, add 100 μL of the diluted sample or standard to be tested to each well, mix well and incubate at 37℃ for 40 minutes. b. Add 200 μL of washing solution to each well, wash 6 times, and print dry on paper. c. Dilute the first antibody solution with the same volume of deionized water, add 100 μL to each well and incubate at 37℃ for 20 minutes. d. Add 200 μL of washing solution to each well, wash 6 times, and print dry on paper. e. Add 100 μL of enzyme-labeled antibody to each well and incubate at 37℃ for 10 minutes. f. Add 200 μL of washing solution to each well, wash 6 times, and print dry on paper. g. Add 100 μL of TMB substrate solution to each well and incubate in the dark for 15 minutes. h. Add 100 μL of stop solution. i. Measure the absorbance at 450nm within 30 minutes. The linear equation was determined based on the standard and its OD value, and the content of cytokines in the sample was calculated.

[0066] (3)TCID 50 Experiment: Brain homogenate supernatant from mice infected with Japanese encephalitis virus was collected and then serially diluted 10 times. 100 μl of the diluted sample was added to a 96-well plate containing 70% density BHK-21 cells per well. The cell morphology was then observed, and cytopathology appeared after 5 days. The dilution that caused cytopathology in half of the culture medium was calculated using the Reed-Muench method.

[0067] (4) Immunofluorescence staining: The mice were perfused with PBS and 4% PFA through the heart. The skin and muscle at the skull of the mice were dissected, the mandible and the rostral maxilla of the skull were removed. Then the top of the skull was taken out with surgical scissors, and the skull connected to the meninges was fixed with 4% PFA at 4°C for 24 h. Then the dura mater was carefully dissected from the skull. The whole meninges were incubated in the blocking solution (PBS containing 0.5% Triton-x-100, 3% BSA and 2% fetal bovine serum) at room temperature for 1 h. The LYVE-1 antibody was diluted with PBS containing 3% BSA and 0.5% Triton-x-100, and incubated with the appropriately diluted primary antibody at 4°C overnight. Subsequently, it was washed 3 times with PBS, then incubated with the appropriately diluted fluorescently labeled secondary antibody at room temperature for 1 h, washed 1 time with PBS, incubated with DAPI (diluted 1:1000) at room temperature for 10 min, washed 3 times with PBS, and then the meninges were laid flat on the coverslip and sealed with an anti-quenching agent. Fluorescent images were obtained using a confocal microscope.

[0068] (5) In vivo imaging of mice: The ventral neck skin of the mice was depilated to prevent weakening of the fluorescence signal. Subsequently, the dorsal neck skin of the mice was incised, and 5 μl of OVA-647 (2 mg / ml) was injected into the cisterna magna of the mice at a rate of 1 μl / min. Then, the fluorescence intensity of OVA-647 in the neck was continuously measured for 3 h at different time points (30 min, 60 min, 120 min, 180 min) using an in vivo imaging system. The results were analyzed using Living Image 4.7.3.

[0069] (6) Delivery of human VEGF-C: Sterile methylcellulose (4000 cp, Sigma-Aldrich) and hyaluronic acid (1500 - 1800 KD, Sigma-Aldrich) were successively dissolved in sterile 0.1 M PBS and mixed overnight at 4°C to prepare a hydrogel pre-solution. The VEGF-C lyophilized powder (100 μg / ml) was resuspended with sterile PBS and then diluted to 2000 ng / ml with PBS containing 0.5% sterile methylcellulose. The diluted VEGF-C or Vehicle was mixed into the hydrogel pre-solution at a ratio of 1:10, loaded into a syringe, and gelled at 37°C. After the mice were anesthetized, the head was fixed, and a 2-cm-long incision was made on the scalp. The skull above the transverse sinus was thinned. 100 μl of the gel solution was injected onto the thinned skull surface, and the scalp was sutured. Two weeks later, 100 μl of PBS containing 200 ng / ml VEGF-C or Vehicle was injected at the thinned skull site once every other day for a total of 3 times.

[0070] (7) Method of monoclonal antibody administration: Mice were intraperitoneally injected with 10 mg / kg Enbrel (etanercept, a recombinant tumor necrosis factor receptor that blocks the binding of TNF-α to the receptor) or control normal saline (NS). Administration started after the mice were challenged with the virus, at an interval of one day, for a total of 3 times.

[0071] (8) Acquisition of PEM cells: For mice older than 6 weeks, wipe the abdomen of the mice with alcohol, and intraperitoneally inject 3 mL of broth culture medium. PEM can be obtained 3 days later. Decapitate the mice and soak them in 75% alcohol. Fix the mice on the operating board. Intraperitoneally inject 8 - 10 mL of complete DMEM medium into the mice using a 10 mL syringe. Massage the abdomen of the mice repeatedly with both hands several times. Cut open the abdominal skin of the mice to expose the peritoneal membrane. Aspirate the culture medium in the abdominal cavity with a 10 mL syringe and transfer it to a centrifuge tube (5 mL of complete DMEM was pre-loaded in the centrifuge tube and the cells were mixed with a pipette). Centrifuge at 1000 rpm for 5 min. Discard the supernatant. Depending on the number of red blood cells, add an appropriate amount (2 - 5 mL) of red blood cell lysate to remove red blood cells. Add an appropriate amount of complete DMEM to terminate the reaction of the red blood cell lysate. Filter the cell suspension through a 70 μm filter into a new centrifuge tube. Centrifuge at 1000 rpm for 5 min. Discard the supernatant. Resuspend the cells with complete DMEM medium (1 - 5 mL) and count. Adjust the cell density to 0.3 mln / mL and add 1 mL to each well of a 12-well plate.

[0072] (9) Co-culture of neurons and macrophages: WT or Vegfr-3 △LBD / △LBD After the PEMs were plated overnight, add 0.1 mln N2a cell line to each well, shake well. After the cells adhered, add 1 MOI of JEV virus solution. Detect the apoptosis of N2a 48 hours later.

[0073] (10) The apoptosis staining kit was purchased from BD Bioscience. Steps of apoptosis staining: Adjust the concentration of the cells to be detected to 10 6 cells / mL, take 200 μL, centrifuge at 1000 rpm for 5 min at 4°C. Wash twice with 1 mL of pre-cooled PBS, centrifuge at 1000 rpm for 5 min at 4°C. Resuspend the cells in 100 μL of binding buffer, add 2 μL of Annexin V-FITC (20 μL / mL), mix gently, and place in the dark on ice for 15 min. Transfer to a flow cytometry tube, add 400 μL of PBS, and add 1 μL of PI (50 μg / mL) to each sample immediately before loading onto the machine. Detect quickly 2 min later. At the same time, use the sample without adding Annexin V-FITC and PI as a negative control. Result determination: Use Annexin V as the horizontal axis and PI as the vertical axis; the upper left quadrant is mechanically damaged cells; the upper right is late apoptotic cells or necrotic cells; the lower left is negative normal cells; the lower right is early apoptotic cells.

[0074] (11) Immunofluorescence staining steps for brain frozen sections: Immerse the sample in 4% paraformaldehyde for 24 h to fix the sample tissue. Wash twice with PBS, placing it in PBS for about 1 - 2 h each time. Sediment with 30% sucrose until the sample tissue sinks to the bottom. If sectioning immediately, embed with embedding medium. If not, take the sample tissue out of 30% sucrose, try to remove the sucrose solution on its surface, and then place it at -80 °C; section at a thickness of 10 μm. Place the sections in a humid chamber, block with PBS containing 3% BSA for 60 min; incubate with the primary antibody overnight at 4 °C; wash 3 times with PBS, 5 min each time; incubate with the secondary antibody and DAPI at room temperature for 15 min; wash 3 times with PBS, 5 min each time; after mounting, observe the results with a fluorescence microscope and take pictures or store in the dark at -20 °C.

[0075] Statistical analysis

[0076] The inventor of the present invention performed statistical analysis on all data using GraphPad Prism 6 software. Unpaired t-test analysis was used for comparison between groups, and survival curve analysis

[0077] It was determined that p < 0.05 indicates statistical significance (*), p < 0.01 indicates significant statistical significance (**), and p < 0.001 indicates extremely significant statistical significance (***). In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms all fall within the scope defined by the appended claims of the present application.

[0078] The content not described in detail in the specification of the present application belongs to the common general knowledge of those skilled in the art.

[0079] As mentioned throughout the specification and claims, "comprising" is an open-ended term and should be interpreted as "comprising but not limited to". "Substantially" means within an acceptable error range, and those skilled in the art can solve the technical problem within a certain error range and basically achieve the technical effect.

[0080] It should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a commodity or system including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such commodity or system. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of another identical element in the commodity or system including the said element.

[0081] The foregoing description has shown and described several preferred embodiments of the present application. However, as previously mentioned, it should be understood that the present application is not limited to the forms disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and environments, and can be changed within the scope of the inventive concept described herein through the above teachings or the techniques or knowledge in the relevant field. Any changes and variations made by those skilled in the art without departing from the spirit and scope of the present application shall fall within the protection scope of the appended claims of the present application.

Claims

1. Use of a lymphangiogenesis inducer in the preparation of a medicament for treating a neurotropic virus infection in an individual; The lymphangiogenesis inducer is VEGF-C; The neurotropic viruses are rabies virus, Zika virus, Japanese encephalitis virus, herpes simplex virus, vesicular stomatitis virus.

2. The use according to claim 1, wherein The neurotropic virus infection includes an inflammation-related central nervous disease caused by a neurotropic virus infection, which includes administering a lymphangiogenesis inducer to the individual; the lymphangiogenesis inducer is used to promote lymphangiogenesis in the brain and / or central nervous system of the individual and enhance lymphatic vessel function.

3. The use according to claim 1, characterized in that, The neurotropic virus infection includes viral encephalitis caused by a neurotropic virus infection, which includes administering an effective dose of a lymphangiogenesis inducer to the individual.

4. The use according to claim 1, characterized in that, The medicament is an injection, and the lymphangiogenesis inducer is in the form of a protein.

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