CN114146175B

CN114146175B - Application of HVCN1 antibody in preparing medicine for treating nerve damage or nerve retrogressive disease

Info

Publication number: CN114146175B
Application number: CN202111251581.2A
Authority: CN
Inventors: 赵经纬; 汪帆; 武洋; 马晓茹; 董昭君
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2023-07-25
Anticipated expiration: 2041-10-22
Also published as: CN114146175A

Abstract

The application of the HVCN1 antibody in preparing a medicament for treating nerve injury or neurodegenerative diseases is provided, the HVCN1 antibody can enhance migration capability of microglial cells or macrophages so as to promote recruitment of microglial cells or macrophages to an injury area and phagocytose myelin fragments of the injury area, so that the elimination of myelin fragments is realized, a novel method is provided for eliminating myelin fragments, and the effect of treating myelin fragment accumulation diseases is fundamentally played; and the administration safety is good, the cost is reduced, and the compliance of patients is high.

Description

Application of HVCN1 antibody in preparing medicine for treating nerve damage or nerve retrogressive disease

Technical Field

The disclosure relates to the technical field of genetic engineering, in particular to application of an HVCN1 antibody in preparing a medicament for treating nerve injury or neurodegenerative diseases.

Background

Myelin debris accumulation is a pathological feature of various nerve injuries and neurodegenerative diseases such as multiple sclerosis, alzheimer's disease, huntington's disease and amyotrophic lateral sclerosis, and the removal of myelin debris is beneficial for the treatment of nerve injuries or neurodegenerative diseases.

Microglia or macrophages are able to clear myelin fragments, and the microglia or macrophages are first required to recruit microglia or macrophages around myelin fragments when they clear myelin fragments; currently, the antibody treatment of myelin sheath fragments promotes the phagocytosis of myelin sheath by microglia or macrophages, each of which has a limited ability to phagocytose myelin sheath fragments, and depletion of myelin sheath fragments results in microglia or macrophages activating and even dying. Thus, new methods for promoting myelin debris removal are needed.

Disclosure of Invention

In view of the above, the disclosure aims to provide an application of an HVCN1 antibody in preparing a medicament for treating nerve injury or neurodegenerative diseases.

Based on the above objects, the present disclosure provides an application of an HVCN1 antibody in preparing a medicament for treating nerve injury or neurodegenerative disease.

Optionally, the drug for treating nerve injury or neurodegenerative disease comprises a drug for treating myelin sheath-chip accumulation disease.

Optionally, the agent for treating myelin debris accumulation disease comprises an agent that promotes myelin debris removal.

Optionally, the agent that promotes myelin debris removal comprises an agent that enhances migration of microglia or macrophages to promote myelin debris removal.

Alternatively, the neurodegenerative disease comprises multiple sclerosis, alzheimer's disease, parkinson's syndrome, spinal cord lateral sclerosis, and Huntington's chorea.

Alternatively, the drug for treating nerve injury or neurodegenerative disease is a pharmaceutical composition comprising an HVCN1 antibody as the sole active ingredient or an HVCN1 antibody.

Optionally, the medicament for treating nerve injury or neurodegenerative disease comprises any pharmaceutically acceptable dosage form prepared by pharmaceutically acceptable auxiliary materials.

Optionally, the medicament for treating nerve injury or neurodegenerative disease comprises at least one of decoction, powder, pill, medicated wine, lozenge, colloid, tea, qu Ji, cake, lotion, stick, thread, strip, nail, moxibustion agent, ointment, pellet, liposome preparation, aerosol, injection, mixture, oral ampoule, tablet, capsule, dripping pill, emulsion, membrane and sponge.

From the above, it can be seen that the application of the HVCN1 antibody provided by the present disclosure in preparing a medicament for treating nerve injury or neurodegenerative disease can enhance migration ability of microglial cells or macrophages to promote recruitment of microglial cells or macrophages to an injury region, and engulf myelin fragments in the injury region, so as to remove myelin fragments, thereby providing a new method for removing myelin fragments and fundamentally playing a role in treating myelin fragment accumulation disease; and the administration safety is good, the cost is reduced, and the compliance of patients is high.

Drawings

In order to more clearly illustrate the technical solutions of the present disclosure or related art, the drawings required for the embodiments or related art description will be briefly described below, and it is apparent that the drawings in the following description are only embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort to those of ordinary skill in the art.

FIG. 1A is a schematic illustration of the in vivo myelin elimination experiment design in an in-brain HVCN1 antibody neutralization HVCN1 channel effect study on microglial elimination of myelin fragments;

FIG. 1B is a schematic diagram of immunofluorescent staining of an effect study of neutralizing HVCN1 channel with an HVCN1 antibody in brain on microglial cell to remove myelin debris;

FIG. 1C is a volume statistic of residual myelin sheath in the brain of mice;

FIG. 1D is a graph of cumulative fluorescence intensity statistics of residual myelin sheath in the brains of mice;

FIG. 2A is a schematic illustration of the in vivo myelin sheath removal assay design in a study of the effect of HVCN1 antibody injection on microglial cells;

FIG. 2B is a schematic diagram of immunofluorescence staining in an experiment of the effect of HVCN1 antibody injection on microglial cells;

FIG. 2C is a statistical plot of total cell and microglial count for the injured core region and periinjured region;

FIG. 3A is a schematic representation of immunofluorescence staining of whether myelin sheath removal after in vivo neutralization with HVCN1 antibodies is primarily mediated by microglial cells in a study;

FIG. 3B is a schematic representation of immunofluorescent staining of whether myelin removal after in vivo neutralization of HVCN1 antibodies is primarily mediated by microglia expressing HVCN1 channels in a study;

fig. 3C is a cytogram of HVCN1 expression in myelin damaged area after mice were injected intracerebrally with myelin.

Detailed Description

For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same.

It should be noted that unless otherwise defined, technical or scientific terms used in the embodiments of the present disclosure should be given the ordinary meaning as understood by one of ordinary skill in the art to which the present disclosure pertains. The terms "first," "second," and the like, as used in embodiments of the present disclosure, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

Microglia or macrophages are able to clear myelin debris, to investigate whether HVCN1 antibodies play a role in the process of myelin debris removal, the present disclosure investigated whether the overall effect of HVCN1 antibody neutralization HVCN1 channels on microglial clearance of myelin debris, the effect of HVCN1 antibody injection on microglial cells, and the potentiation of myelin clearance after HVCN1 antibody neutralization were mediated primarily by microglial cells expressing HVCN1 channels. The following will explain the specific experimental examples in detail.

Experimental example

The antibody names, sources, numbers and dilutions used for immunofluorescent staining in the following experiments are shown in table 1.

TABLE 1

The solutions used in the following experiments were configured as follows:

1% sodium pentobarbital solution: about 8mL ddH was added to 0.1g sodium pentobarbital and 0.09g NaCl ₂ O vortex dissolution with ddH ₂ O was constant to 10mL.

0.01M PBS: 8g NaCl, 0.2g KCl and 1.44g Na are taken ₂ HPO ₄ And 0.24g KH ₂ PO ₄ Dissolving in 800mL distilled water, adjusting pH to 7.4, adding water to 1L, sterilizing, and preserving at room temperature.

0.2M PB: at 800mL ddH ₂ 55.42g of Na was added to O ₂ HPO ₄ ·12H ₂ O and 7.04g NaH ₂ PO ₄ 2H2O, ddH was added after dissolution ₂ O is fixed to volume of 1L, pH is adjusted to 7.3, and the mixture is preserved at room temperature.

4% pfa: 400mL ddH ₂ Heating O to 65-70 ℃, adding 40g of paraformaldehyde while stirring, dropwise adding 5M NaOH until the paraformaldehyde is completely dissolved, cooling, filtering, adding 500mL of 0.2M PB, adjusting pH to 7.3,4 ℃ and preserving.

Sucrose solution: 10g, 20g and 30g of sucrose are respectively weighed, 50mL of 0.2M PB is added, and ddH is added after full stirring and dissolution ₂ O is fixed to 100mL to obtain 10%,20% and 30% sucrose solution.

2.5mM Tris-HCl: 2.5mL of 1M Tris-HCl (pH 7.4) was added to about 800mL of ddH ₂ In O, the pH is adjusted to 7.0, and ddH is added ₂ O is fixed to volume to 1L.

0.32M sucrose: 109.54g of sucrose was taken and dissolved by adding 700mL of 2.5mM Tris-HCl with sufficient stirring, and the volume was fixed to 1L by adding 2.5mM Tris-HCl.

0.85M sucrose: 290.96g of sucrose was taken and dissolved by adding 700mL of 2.5mM Tris-HCl with sufficient stirring, and the volume was fixed to 1L by adding 2.5mM Tris-HCl.

Triton X-100 dilution: mu.l of Triton X-100, available from Sigma under the designation T8787, was added to 100mL of PBS.

Sealing liquid: to 10mL Triton X-100 dilutions were added 500. Mu.L donkey serum.

Antibody dilution: to 10mL Triton X-100 dilutions were added 250. Mu.L donkey serum.

1. Experimental method

1.1 myelin extraction and fluorescent labelling

The female rats of 12 weeks old are anesthetized by intraperitoneal injection of 1% pentobarbital sodium with the injection dosage of 50mg/kg body weight, and then are perfused with 0.01M PBS for 2-3 min by heartThe scissors break the rat head, cut the skull, and take out the brain with forceps. The brain was transferred to a Dounce homogenization tube, and a pre-chilled 0.32M sucrose solution was added to homogenize. 25mL of a pre-chilled 0.85M sucrose solution was added to the ultracentrifuge tube, the upper layer was carefully added with homogenate, and centrifuged at 75000g for 30min at 4 ℃. Collecting myelin sheath of intermediate layer, and using ddH ₂ O was washed three times, then centrifuged once at 75000g for 15min at 4℃and twice at 12000g for 10min at 4 ℃. The concentration of the centrifuged myelin pellet was adjusted to 8mg protein/mL with 0.01M PBS, CFSE dye was added to a final concentration of 5. Mu.M, and incubated at 37℃for 30min; then 12000g was centrifuged for 10min, the supernatant was discarded, and after washing three times with 0.01M PBS, resuspended with 0.01M PBS and stored at-80 ℃.

1.2 experiments on myelin phagocytosis in brain

The CFSE labeled myelin sheath of 1.1 was boiled for 10min and immediately then cooled on ice to denature. Antibody was added to the denatured myelin to a final concentration of 1mg/mL, and myelin to a final concentration of 25mg/mL, to give an antibody-myelin mixture.

Adult mice were anesthetized with 50mg/kg body weight by intraperitoneal injection of sodium pentobarbital and their head levels were fixed on a stereotactic apparatus. Instruments such as small scissors, forceps and the like are soaked in 75% ethanol for sterilization, and the connective tissue on the surface of the skull is wiped off by using a sterile dry cotton ball, so that the operation area is fully exposed. With the injection needle moved back and forth and left and right, the top skull of the mouse is leveled, the Z axis error of bregma and bregma is less than or equal to 0.2mm, the bregma is opened by 1.5mm, and the site error at two sides is less than or equal to 0.2mm. The skull above the target site was drilled with a skull drill, and a microinjection needle was used to aspirate 1 μl of antibody-myelin sheath mixture, slowly needle down to the target site, with coordinates: anterior bregma 0.0mm, lateral opening 2.0mm, ventral 1.5mm. Stopping the needle for 5min, completing injection within 5min, stopping the needle for 5min, pulling out the needle for 0.2mm every 2min, and slowly and completely pulling out the needle after three times of needle pulling. The scalp is sutured, the mice are placed on an electric blanket for heat preservation, and the mice are transferred into a squirrel cage after waking up. Perfusion was used for 48h after injection.

1.3 perfusion sampling and slicing

The needle tube was fixed on a constant flow pump and the flow rate was adjusted, and the mice were anesthetized by intraperitoneal injection of pentobarbital sodium according to a weight of 50mg/kg and then fixed on a foam plate in a supine position. The thoracic cavity was cut off along the xiphoid process with a scissors and fully exposed, the needle was inserted into the left atrium, the sinus was cut off with a small scissors, the constant flow pump was turned on, and ice was infused through the heart with 0.01m PBS for 2-3 min, followed by continued ice infusion with 4% pfa for 8min. The head is broken by the scissors and the skull is cut off, the skull is broken off towards two sides by forceps, the brain is fully exposed, and the brain is fished out by the forceps. The brain is transferred to a new EP tube, fixed for 2 hours after 4% PFA is added, 10% sucrose is changed for dehydration, and after the tissue is settled, 20% sucrose and 30% sucrose are sequentially changed for continuous dehydration. Taking out the tissue, sucking the sucrose solution on the surface of the absorbent paper, wrapping a layer of OCT on the surface of the tissue, transferring the tissue into an embedding box, and adding the OCT until the top end of the tissue is not covered after the direction is set. Transferring the embedding box to-80 ℃ for quick freezing for 5min, taking out the tissue and OCT after OCT is solidified, transferring and fixing the tissue and OCT on a freezing head, slicing the tissue and OCT in a slicing machine, wherein the slicing thickness is 12 mu m, pasting the obtained tissue slice on a glass slide, standing at room temperature for 2h, and transferring to-80 ℃ for long-term storage.

1.4 tissue immunofluorescent staining

The tissue sections were left to stand at room temperature for 5min and dried, and then washed three times with 0.01M PBS for 5min each. Then transferring the tissue sections into a wet box, and sealing the tissue sections in 5% donkey serum for 2 hours at room temperature; the antibody was incubated overnight at 4℃and washed three times with PBS for 5min each. The secondary antibody was incubated at 4℃for 1h, washed three times with PBS for 5min each. DAPI was incubated at room temperature for 10min, washed three times with PBS for 5min each. The residual liquid around the tissue was gently sucked off, and the fluorosave sealing tablet was dropped, sealed, left at room temperature in the dark for 2 hours for curing, stored at-20 ℃ or photographed under a microscope.

1.5 data statistics

Data are shown as mean ± standard error. Data statistics were performed using Graphpad Prism software, using single factor anova in combination with post-hoc Dunnett's multiple comparisons, unpaired two-tailed t-test, or paired two-tailed t-test, with p <0.05 considered significant differences, depending on the situation.

1.6 study of the Effect of HVCN1 antibody in brain on neutralizing HVCN1 channel on microglial Remover myelin sheath fragment

One side of adult mice was injected with Rabbit anti-HVCN1 antibody and myelin, the other side was injected with equal volume concentrations of isotype IgG antibody and myelin as controls (FIG. 1A), the amount and method of injection was referenced 1.2, wherein the IgG antibody was Rabbit IgG, available from Yinqiao, under the designation CR 1. After 48h, the mice were perfusion-sectioned in the same manner as 1.3. Adopting DAPI to dye and seal the slice, wherein the method is the same as 1.4; the residual myelin was then photographed using a fluorescence microscope, the amount of residual myelin was compared, and one section was taken every 24-36 μm for immunofluorescent staining, and then subjected to a stereoscopic reconstruction, scale 100 μm (FIG. 1B), with the results shown in FIGS. 1C-1D. The results in the figures show that the HVCN1 antibody-injected side has significantly less residual myelin sheath than the control side; quantitative evaluation of the volume and cumulative fluorescence intensity of the residual myelin showed that the total volume of the residual myelin was significantly reduced on the side of HVCN1 antibody injection compared to the control group (fig. 1C); the cumulative fluorescence intensity of residual myelin in brain on the side of HVCN1 antibody injection was significantly lower than on the side of control antibody (fig. 1D); the statistical method of the results is the same as 1.5. The above results demonstrate that the injection of HVCN1 antibody side significantly reduced the amount of remyelination relative to the injection of control antibody side, demonstrating that neutralization of HVCN1 channels by HVCN1 antibodies in vivo promotes myelin debris clearance.

Research on influence of 1.7HVCN1 antibody injection on microglial cells

One side of adult mice was injected with Rabbit anti-HVCN1 antibody and myelin, the other side was injected with bulk concentrations of isotype IgG antibody and myelin as controls (FIG. 2A), the amount and method of injection was referenced to 1.2. After 48h, the mice were perfusion-sectioned in the same manner as 1.3. Immunofluorescence staining and sealing are carried out on the slice by adopting a first anti-Rat anti-CD68 antibody, a second anti-Cy 3-conjugated donkey anti-rate antibody and DAPI, and the method is the same as 1.4; then taking a picture of the myelin sheath by using a fluorescence microscope, and detecting the number of CD68 positive microglial cells around the myelin sheath; taking the remyelinated area and the area with a particularly high microglial density immediately adjacent to the remyelinated area as the lesion core area, the area with a significantly increased microglial density outside the core area as the peri-lesion area (fig. 2B), the neutralization of HVCN1 antibody in fig. 2B increases the density of CD68 positive microglial cells in the myelin-damaged core area (C) and the peri-lesion area (P); cells from the injured core region and the periinjured region were counted, and the results are shown in fig. 2C. Fig. 2C shows that DAPI density, i.e., total cell density, of the damaged core region and the peri-damaged region did not significantly differ; statistics of CD68 positive microglial cells shows that compared with a control antibody side, the density of CD68 positive microglial cells on the side of HVCN1 antibody injection is obviously improved in a damaged core area and a damaged peripheral area, and the proportion of the CD68 positive microglial cells in the damaged core area and the damaged peripheral area to total cells is also obviously improved; the statistical method of the results is the same as 1.5. The above results indicate that neutralization of HVCN1 channels by HVCN1 antibodies can promote microglial recruitment to the lesion in vivo.

1.8 in vivo studies on whether myelin removal after neutralization of HVCN1 antibodies was primarily mediated by microglia expressing HVCN1 channels

The adult mice were injected intracerebrally with the Rabbit anti-HVCN1 antibody and myelin sheath in an amount and method of injection referred to 1.2. After 48h, the mice were perfusion-sectioned in the same manner as 1.3. Immunofluorescence staining and sealing are carried out on the slice by adopting a primary anti-coat anti-Iba1 antibody, a secondary anti-Cy 3-conjugated donkey anti-coat antibody and DAPI, and the method is the same as 1.4; the myelin sheath chip was then photographed using a fluorescence microscope, and the number of microglia around the myelin sheath chip was examined, and the results are shown in fig. 3A. The results in fig. 3A show that after injecting the myelin-antibody mixture in the brain of mice, a large number of Iba 1-positive microglia are present around myelin fragments, and a large number of myelin fragments are detected in these microglia cells, indicating that microglia are still able to phagocytize myelin fragments after neutralizing HVCN1 channels in vivo with HVCN1 antibodies.

To further investigate the proportion of various cells in cells expressing HVCN1 protein around myelin fragments, CFSE-labeled myelin was injected into adult mouse brains, the injection amounts and methods were referenced 1.2. After 48h, the mice were perfusion-sectioned in the same manner as 1.3. The double labeling of HVCN1 protein and the surface markers of the main cell types in the brain were detected by immunofluorescent staining. Specifically, the microglial cells are subjected to immunofluorescence staining sealing sheets by using an anti-coat anti-Iba1 antibody, a secondary anti-Cy 3-conjugated donkey anti-coat antibody and DAPI, the oligodendrocyte cells are subjected to immunofluorescence staining sealing sheets by using an anti-coat anti-SOX10 antibody, a secondary anti-Cy 3-conjugated donkey anti-coat antibody and DAPI, the astrocyte cells are subjected to immunofluorescence staining sealing sheets by using an anti-Chicken anti-GFAP antibody, a secondary anti-Cy 3-conjugated donkey anti-Chicken antibody and DAPI, the neurons are subjected to immunofluorescence staining sealing sheets by using an anti-Guinea anti-NeuN antibody, a secondary anti-Cy 3-conjugated donkey anti-Guinea pig antibody and DAPI, and the HVCN1 protein is subjected to immunofluorescence staining sealing sheets by using an anti-Rabbit anti-HVCN1 antibody, a secondary anti-Alexa Fluor 647-conjugated donkey anti-rabit antibody and DAPI, and the same method as 1.4. The myelin sheath chip was photographed using a fluorescence microscope, and the results are shown in fig. 3B to 3C. The results in FIGS. 3B-3C show that cells around myelin sheath chips expressing HVCN1 protein are predominantly Iba1 positive microglia (FIG. 3B); of the cells expressing HVCN1 protein around myelin sheath chips, iba1 positive microglial cells account for up to 90%, SOX10 positive oligodendrocytes account for only about 10%, and GFAP positive astrocytes and NeuN positive neurons are almost absent (fig. 3C); the statistical method of the results is the same as 1.5. The above results indicate that cells expressing HVCN1 protein in cells surrounding myelin fragments are mainly microglia, demonstrating that HVCN1 antibodies exert a promoting myelin clearance by neutralizing HVCN1 channels on microglia.

The application of the HVCN1 antibody in preparing the medicament for treating the nerve injury or the neurodegenerative disease can enhance the migration capability of microglial cells or macrophages so as to promote the recruitment of microglial cells or macrophages to an injury area and phagocytose myelin fragments of the injury area, thereby realizing the elimination of myelin fragments, providing a new method for the elimination of myelin fragments and fundamentally playing a role in treating myelin fragment accumulation diseases; and the administration safety is good, the cost is reduced, and the compliance of patients is high.

It should be noted that the embodiments of the present disclosure may be further described in the following manner:

use of an HVCN1 antibody in the manufacture of a medicament for the treatment of nerve injury or neurodegenerative disease.

Optionally, the medicament for treating nerve injury or neurodegenerative disease comprises at least one of decoction, powder, pill, medicated wine, lozenge, colloid, tea, qu Ji, cake, lotion, stick, thread, strip, nail, moxibustion agent, ointment, pellet, liposome preparation, aerosol, injection, mixture, oral ampoule, tablet, capsule, dripping pill, emulsion, membrane and sponge. .

Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined under the idea of the present disclosure, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in details for the sake of brevity.

The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Accordingly, any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the embodiments of the disclosure, are intended to be included within the scope of the disclosure.

Claims

Use of an hvcn1 antibody in the manufacture of a medicament for treating myelin sheath element accumulation diseases.
2. The use according to claim 1, wherein the medicament for treating myelin debris accumulation disease comprises a medicament for promoting myelin debris removal.
3. The use of claim 2, wherein the agent that promotes myelin debris removal comprises an agent that enhances migration of microglia or macrophages to promote myelin debris removal.
4. The use according to any one of claims 1 to 3, wherein the medicament for treating myelin debris accumulation disease comprises an HVCN1 antibody as the only active ingredient or a pharmaceutical composition comprising an HVCN1 antibody.
5. The use according to claim 1, wherein the medicament for treating myelin sheath-chip accumulation disease comprises the use of pharmaceutically acceptable excipients.
6. The use according to claim 5, wherein the medicament for treating myelin sheath-chip accumulation disease comprises at least one of a liposome preparation, an aerosol, an injection, a capsule, a drop pill, an emulsion, a film, and a sponge.