CN114099683A - Application of NOX2 specific inhibitor in preparation of retinal degeneration medicines - Google Patents

Abstract

Translated fromChinese

本发明涉及NOX2特异性抑制剂在制备视网膜变性药物中的应用，属于生物技术领域。本发明提供了NOX2特异性抑制剂在制备治疗和/或预防视网膜变性的药物中的应用；研究发现，与遗传性视网膜变性动物模型rd1小鼠相比，NOX2基因缺陷rd1小鼠模型视网膜外核层感光细胞丢失明显延迟，小胶质细胞活化显著抑制，小胶质细胞中NOX2表达明显减少，并且，研究发现，与rd1小鼠模型相比，体内注射NOX2特异性抑制剂的rd1小鼠模型视网膜外核层厚度明显增加，小胶质细胞活化明显抑制，小胶质细胞中NOX2表达明显减少，另外，与香荚兰乙酮(apocynin)相比，NOX2特异性抑制剂作用机制更明确、特异性更强。

The invention relates to the application of NOX2 specific inhibitor in the preparation of retinal degeneration medicine, and belongs to the field of biotechnology. The invention provides the application of NOX2 specific inhibitor in the preparation of medicine for treating and/or preventing retinal degeneration; research finds that compared with rd1 mouse, an animal model of hereditary retinal degeneration, NOX2 gene-deficient rd1 mouse model retinal outer nucleus Laminar photoreceptor cell loss was significantly delayed, microglia activation was significantly inhibited, NOX2 expression in microglia was significantly reduced, and, compared to the rd1 mouse model, it was found that the rd1 mouse model injected with a NOX2-specific inhibitor in vivo The thickness of the outer nuclear layer of the retina was significantly increased, the activation of microglia was significantly inhibited, and the expression of NOX2 in microglia was significantly reduced. more specific.

Description

Application of NOX2 specific inhibitor in preparation of retinal degeneration medicines

Technical Field

The invention relates to application of a NOX2 specific inhibitor in preparation of a retinal degeneration medicine, and belongs to the technical field of biology.

Background

Primary Retinitis Pigmentosa (RP) is a group of inherited eye diseases that cause progressive photoreceptor apoptosis, with an incidence of 1/3000-4000, and has led to blindness in more than 150 million adults worldwide. Although gene and stem cell therapies have made some progress in the treatment of RP, their wide clinical application is not yet practical due to the complexity of the RP phenotype or the potential problems of the technology itself, and therefore, the study of common pathological mechanisms secondary to genetic variation is still very important. Even if the pathogenic cause (gene mutation) of the pathological changes cannot be radically changed, the loss of the photoreceptor cells can be delayed by taking the common pathogenic mechanism core link as a target point to carry out drug treatment, the blindness time of a patient is delayed, and the life quality of the patient is improved.

Numerous research results have shown that chronic neuronal inflammation can lead to the development of a variety of degenerative diseases of the Central Nervous System (CNS). Microglia are natural host cells in the CNS and, upon activation, produce a variety of neuronal virulence factors that induce cell death. The retina is an extended brain tissue. Inflammatory responses local to RP have been considered as a secondary event of genetic variation leading to photoreceptor cell death. However, there is increasing evidence that chronic inflammatory responses may lead to degeneration of the rod cone cells, and that the level of retinal inflammation is inversely related to its visual function in patients with RP. In an animal model of RP experiments, previous studies have shown that microglia migrate out into the outer nuclear layer of the retina, activating and releasing a large amount of the neuronal virulence factor TNF-alpha, before apoptosis occurs. Inhibition of microglial activation may reduce photoreceptor cell loss. Therefore, the microglia are not only bystanders of photoreceptor cell apoptosis, but also participate in the initiation and the persistence process of retinal degeneration more remarkably, and are important pathogenic pathways leading to the final apoptosis of photoreceptor cells. At this stage, the mechanism of microglial activation in RP is not well understood.

Recent CNS studies have shown that NADPH oxidase (nicotinic amide dehydrogenase) activation is an important mechanism for the activation of microglia. Although a variety of pathological factors exert toxic effects through microglial activation, the production of Reactive Oxygen Species (ROS) inside and outside the cell is a common fate and feature of most neuronal degenerative diseases. There are generally three main sources of ROS in the macrophage system: oxidation processes of peroxidases in cells, cell membrane surface NOXs or mitochondria. Among these, NOX2 is a major source of extracellular ROS production by microglia upon stimulation. Of the 7 isomers of NOX (including NOX1-5, DUOX1, and DUOX2), NOX2 is found primarily in microglia and macrophages. NOx2 is the decomposition of oxygen into O^2-The membrane-bound enzyme of (1). The enzyme complex is dormant in quiescent phagocytes and is activated upon stimulation. In resting phagocytes, the cytoplasmic subunit of NOX2 (P47)^phox、P67^phox、P40^phoxAnd Rac2) are activated and translocated to the cell membrane, and subunits in the cell membrane (gp 91)^phox、P22^phox) Combined to produce O^2-Is activated enzyme state. Activation of NOX2 in microglia produces toxic effects on neuronal cells through two pathways. On the one hand, neuronal toxicity is directly generated by the production of extracellular ROS; on the other hand, intracellular ROS signaling (redox signaling) is initiated. Intracellular ROS can be used as a second messenger to regulate a plurality of downstream signal molecules, including mitogen-activated protein kinase (MAPK), NF-kB and the like, so as to promote the generation of a large number of inflammatory or neuronal toxic factors, including TNF-alpha, IL-1 beta, IL-6, iNOS and the like. And ROS activate pre-apoptotic signaling pathway MAPK, such as SAPK/JNK, ERK1/2 and P38MARK, P53 can directly induce apoptosis.

Oxidative stress is an important biological process in the development of RP. The oxidation reactions of macromolecular substances such as lipids, proteins and nucleic acids are significantly enhanced in various RP animal models. Studies by the Campochiro team have shown that oxidative damage is a major causative factor in cone apoptosis following hereditary retinal degeneration rod cell death. They thought that rod death due to genetic variation in various animal models of RP caused hyperoxia in the outer retina, increased oxygen levels caused progressive oxidative damage to cones, and NADPH oxidase was the major source of ROS production during cone degeneration in rd1 mice and Q344ter transgenic mice. It is well known that rod loss represents a more early stage of retinal degeneration than cone apoptosis, and that the study of the apoptotic mechanism at this stage would facilitate early intervention in the disease, delaying the onset of cone apoptosis that ultimately affects vision. Research on ROS generation and NOX2 activation in the rod cell apoptosis process shows that NOX2 in microglia is obviously activated and ROS generation is increased in the retinal degeneration process of rd1 mice, and the time and space relation is obviously parallel to that of rod cell loss and apoptosis; and, systemic use of the NOX2 inhibitor, acetovanillone (apocynin), reduces ROS production and delays loss of rod cells. Thus, the hypothesis can be put forward: during the process of hereditary retinal degeneration, NOX2 activation may play a central pathogenic role in microglia-mediated rod apoptosis, and apocynin may be considered a drug that may have a promising prospect for treating hereditary retinal degeneration.

However, recently, several studies have indicated that apocynin is not a specific inhibitor of NOX2 or other NOXs, and that Its mitigating effect on neuronal apoptosis is likely based on Its oxidative clearance activity, rather than NADPH Oxidase inhibitory activity (see in particular the references "Augsburger, F., et al. pharmaceutical characterization of the seven human NOX isoformes and the third inhibitor. Redox biol. 2019; 26: 101272." and "Chory, M.and L.Leloup. the NADPH oxidant Family and Its inhibitors. endogenous Redox. 2020; 33(5): 332. 353."). This not only presents a challenge to the above-mentioned hypothesis that NOX2 activation may play a central pathogenic role in microglia-mediated rod apoptosis during hereditary retinal degeneration, but also severely hinders the commercial development and clinical application of apocynin in the treatment of neuronal degenerative diseases, including retinal degeneration. The literature "Source, S., K.H.Krause, and V.Jaquet.targeting NOx enzymes in the central nervous system: thermal opportunities. cell Mol Life Sci.2012; 2387: "2387-. There is a great need to understand the pathogenic mechanism of NOX2 activation in microglia to find new specific targeted drugs for the treatment and/or prevention of retinal degeneration and thereby slow down the progression of retinal degeneration and ultimately treat retinal degeneration.

Disclosure of Invention

In order to solve the above problems, the present invention provides the use of a specific inhibitor of NOX2 in the manufacture of a medicament for the treatment and/or prevention of retinal degeneration.

In one embodiment of the invention, the retinal degeneration is hereditary retinal degeneration.

In one embodiment of the invention, the specific inhibitor of NOX2 is one or more of gp91phox-tat (NOX2ds-tat), GSK-2795039(CAS 1415925-18-6) or VAs2870(CAS 722456-31-7).

In one embodiment of the invention, the NOX2 specific inhibitor is gp91phox-tat or GSK-2795039.

The present invention also provides a medicament for treating and/or preventing retinal degeneration, which comprises an active ingredient; the active component is a specific inhibitor of NOX 2.

In one embodiment of the invention, the NOX2 specific inhibitor is one or more of gp91phox-tat, GSK-2795039, or Vas 2870.

In one embodiment of the invention, the NOX2 specific inhibitor is gp91phox-tat or GSK-2795039.

In one embodiment of the present invention, the medicament further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment of the invention, the drug carrier comprises one or more of a microcapsule, a microsphere, a nanoparticle, or a liposome.

In one embodiment of the present invention, the pharmaceutical excipients comprise one or more of solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, or release retardants.

In one embodiment of the present invention, the pharmaceutical formulation is powder, tablet, granule, capsule, solution, emulsion, suspension or injection.

The invention also provides application of the NOX2 gene as a therapeutic target of a medicament for treating and/or preventing retinal degeneration.

In one embodiment of the invention, the retinal degeneration is hereditary retinal degeneration.

The technical scheme of the invention has the following advantages:

the invention provides the use of a specific inhibitor of NOX2 in the manufacture of a medicament for the treatment and/or prevention of retinal degeneration; research shows that compared with an rd1 mouse which is an animal model of hereditary retinal degeneration, the loss of photoreceptor cells of the outer nuclear layer of the retina of an rd1 mouse model with NOX2 gene deficiency is obviously delayed, the activation of microglia is obviously inhibited, and the expression of NOX2 in the microglia is obviously reduced, and research shows that compared with an rd1 mouse model, the thickness of the outer nuclear layer of the retina of an rd1 mouse model injected with an in vivo NOX2 specific inhibitor is obviously increased, the activation of the microglia is obviously inhibited, and the expression of the NOX2 in the microglia is obviously reduced, and in addition, compared with apocynin, the action mechanism of the NOX2 specific inhibitor is more definite and the specificity is stronger, so the NOX2 specific inhibitor has a great application prospect in preparing medicines for treating and/or preventing retinal degeneration.

Drawings

FIG. 1: mating flow schematic of the rd1 mouse model with NOX2 gene deficiency.

FIG. 2: the identification result of the genotype of the F1 mouse NOX 2. Wherein, the wild type is a 240bp band; the homozygous type is a 195bp strip; the heterozygote is two bands of 240bp and 195 bp; 002365 is NOX2 gene code.

FIG. 3: the identification result of the genotype of the F2 mouse NOX 2. Wherein, the wild type is a 240bp band; the homozygous type is a 195bp strip; the heterozygote is two bands of 240bp and 195 bp; 002365 is NOX2 gene code.

FIG. 4: and identifying the Pde6b genotype of the F2 mouse. Wherein, the wild type is a 400bp band; the homozygote type is a 550bp strip; the heterozygote is a band of 550bp and a band of 400 b; the Pde6b gene is the rd1 mouse variant gene (allelic variant).

FIG. 5: the identification result of the genotype of the F3 mouse NOX 2. Wherein, the wild type is a 240bp band; the homozygous type is a 195bp strip; the heterozygote is two bands of 240bp and 195 bp; 002365 is NOX2 gene code.

FIG. 6: loss of photoreceptor cells from the outer nuclear layer of the retina in different mice.

FIG. 7: apoptosis of photoreceptor cells in the outer nuclear layer of retina in different mice.

FIG. 8: different mouse retinas NOX2 major subunit gp91^phoxProtein expression and microglial activation.

FIG. 9: loss of photoreceptor cells from the outer nuclear layer of the retina in rd1 mice (intravitreal DMSO injection).

FIG. 10: loss of photoreceptor cells from the outer nuclear layer of the retina in rd1 mice (intravitreal DMSO + VAS 2870).

FIG. 11: loss of photoreceptor cells from the outer nuclear layer of the retina of rd1 mice (i.p. PBS).

FIG. 12: loss of photoreceptor cells from the outer nuclear layer of the retina of rd1 mice (i.p. PBS + gp91 phox-tat).

FIG. 13: loss of photoreceptor cells from the outer nuclear layer of the retina of rd1 mice (i.p. PBS + GSK-2795039).

FIG. 14: rd1 mouse visionOmentum NOX2 major subunit gp91^phoxProtein expression and microglial activation (i.p. PBS).

FIG. 15: rd1 mouse retina NOX2 major subunit gp91^phoxProtein expression and microglial activation (i.p. PBS + gp91 phox-tat).

FIG. 16: rd1 mouse retina NOX2 major subunit gp91^phoxProtein expression and microglial activation (i.p. PBS + GSK-2795039).

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Experimental example 1: validation of NOX2 gene as therapeutic target for drugs for treating and/or preventing hereditary retinal degeneration

1.NOX2 knockout (NOX2KO) mice are crossed with rd1 mice to obtain F1 generation mice

Four male NOX2 gene knockout mice with the genotype gp91phox-/Y rd1W/W (namely B6.129S-Cybb knockout of NOX2 gene on X chromosome)^tm1DinA/J mouse, purchased from Jackson laboratories, JAX coded 002365) was crossed with eight female rd1 mice (purchased from Beijing Wintorituximab laboratory technologies, Inc.) of genotype gp91phox +/+ rd1+/+ to give male-crossed F1 mice of genotype gp91phox +/Y rd1W/+ and female-crossed F1 mice of genotype gp91phox +/-rd1w +.

To verify the correctness of the genotype of the F1 generation mice, the genome of the F1 generation mice was PCR-amplified using the primers in Table 1, and the amplification products were analyzed by gel electrophoresis, the analysis results of which are shown in FIG. 2.

As shown in FIG. 2, the genotype of the male-crossed F1 mouse was Gp91phox +/Y rdW/+, and the genotype of the female-crossed F1 mouse was Gp91phox +/rdW +, and F1 mouse was successfully prepared.

Primer sequences of Table 1002365

Note: 002365 genotype judgment criteria:

mutant (homozygous) ═ 195bp

Heterozygate (hybrid) 195bp and 240bp

Wild type 240bp

2. Mating F1 mouse to obtain F2 mouse

Male-hybridizing F1-generation mice, which have a genotype of gp91phox +/Y rd1W/+ and female-hybridizing F1-generation mice, which have a genotype of gp91phox +/-rd1w +, were crossed to obtain F2-generation mice (48 mice in total, numbered #61 to #108, respectively).

In order to screen mice with correct genotypes, the genome of the F2 mouse was PCR-amplified using the primers in tables 1 and 2, and the amplification products were analyzed by gel electrophoresis, and the results are shown in FIGS. 3 to 4. And classifying the F2 mouse according to the genotypes of gp91phox and Pde6b, wherein the classification results are shown in tables 3-4.

According to the classification results, #67, #81, #85 with the genotype gp91phox-/Y rd1+/+ were selected as male-hybridizing F2-generation mice, and #76, #79, #102 with the genotype gp91phox +/-rd1+/+ were selected as female-hybridizing F2-generation mice.

Primer sequences of Pde6b in Table 2

Note: judgment standard of Pde6b genotype:

mutant (homozygous) ═ 500bp

Heterozygate (wild) 550bp and 400bp

Wild type 400bp

Classification of genotypes in Table 3002365

TABLE 4 Classification of the Pde6b genotypes

3. Mating F2 mouse to obtain F3 mouse

Male-crossed F2-generation mice with the genotype gp91phox-/Y rd1+/+ and female-crossed F2-generation mice with the genotype gp91phox +/-rd1+/+ were crossed to obtain F3-generation mice (18 mice in total, and the numbers are #126 to # 143).

In order to screen mice with the correct genotype, the genome of the F3 mouse was PCR-amplified using the primers shown in Table 1, and the amplification products were analyzed by gel electrophoresis, and the results of the analysis are shown in FIG. 5. The F3 mice were also classified according to the gp91phox genotype, and the classification results are shown in Table 5.

According to the classification results, #126, #138, #139 and #140 with the genotype gp91phox-/Y rd1+/+ were selected as male-hybrid F3-generation mice, and #129, #131, #132 and #142 with the genotype gp91phox +/-rd1+/+ were selected as female-hybrid F3-generation mice.

Male hybrid F2 generation mice and male hybrid F3 generation mice with the genotype of gp91phox-/Y rd1+/+ are double homozygous male NOX2 gene defect rd1 mouse models; the female hybrid F3 mouse with the genotype gp91phox-/-rd1+/+ is a double homozygous female NOX2 gene-deficient rd1 mouse model. The male NOX2 gene-deficient rd1 mouse model and the female NOX2 gene-deficient rd1 mouse model can be propagated by crossing.

Classification of genotypes in Table 5002365

Genotype(s)	Number (#126 to #143)
		Hybrid	#	126#129#131#132#138#139#140#142
Wild plant	#128#130#133#141#143
		Homozygous for	#127#134#135#136#137

4. Retinal histocytology characterization of rd1 mouse model with NOX2 gene deficiency

4.1 Experimental reagent and instrument

Hoechst33258 fluorescent dye (Sigma Alorich, usa); sakura frozen section embedding medium (McMormick, USA); TUNEL method apoptosis detection kit (NOREZHA, a national product); DAPI containing anti-quench block tablets (Sigma Alorich, usa); mouse anti-mouse gp91^phoxMonoclonal antibodies (BD corporation, usa); rat anti-mouse CD11b antibody (burle, usa); FITC-labeled anti-rat IgG, TRITC-labeled anti-mouse fluorescent secondary antibody (Invitrogen, usa); model CM1850 quick-frozen sample preparation microtome, model DM4000B optical microscope, Confocal laser Confocal microscope (SPE), LAS X photographic system (Leica, Germany).

4.2 Experimental methods

4.2.1 retinal tissue sections

C57BL/6N mice (purchased from Beijing Wittingle laboratory animal technology, Inc.), NOX2 knockout mice and rd1 mice (day old 14d) are used as control group mice, and the RD1 mouse model (day old 14d) with NOX2 gene deficiency obtained in the step 3 is used as experimental group mice; killing each group of mice by excessive anesthesia of chloral hydrate, quickly removing eyeballs, quickly placing the mice into an OCT embedding medium, quickly quenching the mice in liquid nitrogen, and storing the mice in a refrigerator at minus 80 ℃ for later use; when in use, frozen sections of 7 mu m are made through the optic disc and the sawtooth edge, and 12 eyes are made on 6 mice in each group; the obtained mouse eyeball frozen section is naturally dried, fixed by 4% (w/v, g/100mL) paraformaldehyde at room temperature for 15min, rinsed 3 times by 0.1mol/L PBS buffer solution (pH7.4) for 5min each time, stained by hematoxylin for 20min, differentiated by 1% (v/v) hydrochloric acid ethanol for 20s, soaked in tap water for 20min, stained by eosin for 2min, dehydrated by gradient ethanol, transparent by xylene, sealed by neutral gum, observed under an optical microscope and photographed. 3 sections are selected from each eyeball, the retinas at the same position of the posterior pole part are selected from each section for photographing, and the cell layer number of each group of outer nuclear layers is compared. The results of the experiment are shown in FIG. 6.

4.2.2 retinal photoreceptor apoptosis assay

Adopting a TUNEL cell apoptosis in-situ detection kit, naturally drying the frozen section of the mouse eyeball obtained in the step 4.2.1, fixing the frozen section with 4% (w/v, g/100mL) of paraformaldehyde at room temperature for 15min, rinsing with 0.1mol/L of PBS buffer solution (pH7.4) for 3 times, 5min each time, digesting for 15min at 37 ℃ by protease K, washing TBS, dripping FITC fluorescent labeled detection solution, incubating for 2h in a wet box at 37 ℃ in the dark, washing the TBS, sealing by using a DAPI fluorescent quenching-preventing sealing agent, and observing and photographing by using a fluorescent microscope at a wavelength of 488 nm. Green fluorescence signal was TUNEL positive cells. The results of the experiment are shown in FIG. 7.

4.2.3、gp91^phoxImmunofluorescent staining and co-localization with CD11b

gp91^phoxIs the main subunit of NOX2, and the CD11b monoclonal antibody labels microglia. And the two immunofluorescent stains are co-located to observe the expression of the NOX2 in the microglia. Naturally airing the frozen section of the mouse eyeball obtained in the step 4.2.1, fixing the frozen section by cold acetone for 10min, rinsing the frozen section by 0.1mol/L PBS buffer solution (pH7.4) for 3 times, each time for 5min, sealing the normal sheep serum working solution, incubating the frozen section for 10min at room temperature (25 ℃), discarding serum, and not washing; incubation of two Primary antibodies (gp 91) from different species^phoxMouse-derived, CD11 b-derived, rat-derived), incubation at 4 ℃After 30min of rewarming over night (16h), rinsing by 0.1mol/L PBS buffer (pH7.4), incubating corresponding FITC and TRITC (1: 600, v/v), incubating at room temperature (25 ℃) for 1h, rinsing by 0.1mol/L PBS buffer (pH7.4) for 3 times, 5min each time; the fluorescent quenching sealing piece containing DAPI fluorescence-resistant sealing piece agent is used for sealing pieces, FITC green fluorescence is observed under the wavelength of 488nm of a fluorescence microscope, TRITC red fluorescence is observed under the wavelength of 532nm, and photographing is carried out. The results of the experiment are shown in FIG. 8.

The experimental results of the above experiments were statistically analyzed using SPSS 20.0 statistical software. The data were normally distributed by Shapiro-Wilk test and expressed as mean + -SD. Differences in retinal outer nuclear layer thickness and percentage of TUNEL cells were compared between different groups using one-way variance (ANOVA) analysis, and LSD-t test was used for pairwise comparisons between groups. P <0.05 is statistically significant for the differences.

4.3, results of the experiment

As can be seen from FIG. 6, the retinas of the C57BL/6N mice and NOX2 gene-deficient mice are complete in structure, the cells of the inner and outer nuclear layers are regularly and densely arranged, and the average thicknesses of the outer nuclear layer are 54.44 +/-2.33 μm and 52.96 +/-1.31 μm respectively; the retina outer nuclear layer of an rd1 mouse of the same age is obviously thinned, the thickness of the outer nuclear layer is 21.45 +/-1.33 mu m, the number of cell nuclei is obviously reduced, the arrangement is sparse and disordered, and the shapes are different; compared with rd1 mice, the retina outer nuclear layer thickness of the Nox2 gene-deficient rd1 mice is obviously increased (36.18 +/-2.59 mu m, t is 8.770, p is 0.001), and the inner nuclear layer cells and the outer nuclear layer cells are regularly and densely arranged. This result indicates that NOX2 is significantly delayed by the loss of defective rd1 mouse photoreceptor cells.

As can be seen from FIG. 7, TUNEL positive cells were occasionally observed in the outer nuclear layer of retina of C57BL/6N mice and NOX2 gene-deficient mice (0.17. + -. 0.07% and 0.08. + -. 0.03%, respectively); compared with C57BL/6N mice and NOX2 gene-deficient mice, the number of TUNEL cells in the outer nuclear layer of rd1 mice is obviously increased (5.37 +/-0.75 percent) (t is 19.645, and P is less than 0.01); compared with rd1 mice, the thickness of the retina outer nuclear layer of the rd1 mice with NOX2 gene defect is increased, and the number of TUNEL cells is obviously reduced (1.5 +/-0.3%) (t is 8.42, and P is less than 0.01). This result further indicates that NOX2 is significantly delayed by the loss of defective rd1 mouse photoreceptor cells.

As can be seen from FIG. 8, in C57BL/6N miceGp91 is only occasionally seen in the inner retina (from the inner limiting membrane to the outer plexiform layer of the retina)^phoxPositive cells and branched CD11b positive microglia; similar positions of the retina in NOX2 gene-deficient mice also show gp91^phoxPositive cells and a small number of branched CD11b positive stained cells; in the above control mice, part of gp91^phoxExpressed in CD11b microglia; rd1 mouse retina gp91 in comparison to the control mice described above^phoxThe expression quantity is obviously increased, the staining is aggravated, part of the microglia invades towards the outer nuclear layer, the quantity of CD11b positive microglia is obviously increased, the cell nucleus is enlarged, part of the microglia is in an amoeba shape, most of the microglia infiltrates towards the outer nuclear layer, the visual rod layer and the visual cone layer, and part of the microglia in the outer plexiform layer and the outer nuclear layer obviously express gp91^phox(ii) a Compared with rd1 mice, the retina outer nuclear layer of the rd1 mice with NOX2 gene defect is obviously thickened, gp91^phoxThe number of protein and CD11b positive cells was significantly reduced, with the outer nuclear layer evident. The results show that NOX2 is remarkably inhibited by the activation of the defective rd1 mouse microglia, and the expression of NOX2 in the microglia is remarkably reduced.

The results prove that the phenotype of the rd1 mouse with the defect of NOX2 is correct, the construction of the rd1 mouse with the defect of NOX2 is successful, and the activation of NOX2 in microglia is definitely verified to be a pathogenic mechanism of hereditary retinal degeneration, so that the method provides a basis for screening and researching drugs which have more definite action mechanisms, stronger specificity, and better market and clinical application prospects and are used for treating and/or preventing retinal degeneration, and has great application prospects.

Experimental example 2: validation of specific inhibitors of NOX2 as a medicament for the treatment and/or prevention of hereditary retinal degeneration

1. Experimental methods

1.1 retina tissue slice (VAS2870)

Rd1 mice (9 d day old) were injected intravitreally with a PI-100 microinjector with 0.5. mu.g of VAS2870 (purchased from Sigma under product number SML2967) dissolved in 1. mu.L of 10% (w/v, g/100mL) DMSO to give a final effective concentration in the vitreous cavity of 50. mu.g/mL, and the control group was injected with the same dose of 10% DMSO to the contralateral eye. On the 5 th day after injection (apoptosis peak period), an rd1 mouse is killed by excessive chloral hydrate anesthesia, the eyeball is rapidly removed, and the mouse is placed in an OCT embedding medium and rapidly placed in liquid nitrogen for quenching, and is stored in a refrigerator at minus 80 ℃ for standby; when in use, frozen sections of 7 mu m are made through the optic disc and the sawtooth edge, and 12 eyes are made on 6 mice in each group; the obtained mouse eyeball frozen section is naturally dried, fixed by 4% (w/v, g/100mL) paraformaldehyde at room temperature for 15min, rinsed 3 times by 0.1mol/L PBS buffer solution (pH7.4) for 5min each time, stained by hematoxylin for 20min, differentiated by 1% (v/v) hydrochloric acid ethanol for 20s, soaked in tap water for 20min, stained by eosin for 2min, dehydrated by gradient ethanol, transparent by xylene, sealed by neutral gum, observed under an optical microscope and photographed. Each eyeball selected 3 slices, each slice selected the retina at the same position of the posterior pole for photographing and comparing the thickness of the outer nuclear layer of each group. The experimental results are shown in FIGS. 9 to 10.

1.2 retinal tissue sections (gp91phox-tat, GSK-2795039)

18 rd1 mice (9 d of age) were randomly divided into three groups, namely a PBS control group, a gp91phox-tat test group and a GSK-2795039 test group. After completion of the grouping, 50. mu.g of gp91phox-tat (purchased from Sigma) dissolved in 50. mu.L of 0.1mol/L PBS buffer (pH7.4) was intraperitoneally injected into gp91phox-tat test group rd1 mice, 50. mu.g of GSK-2795039 (purchased from MCE) dissolved in 100. mu.L of 0.1mol/L PBS buffer (pH7.4) was intraperitoneally injected into GSK-2795039 test group mice, and an equivalent dose of 0.1mol/L PBS buffer (pH7.4) was intraperitoneally injected into PBS control group mice, and the injections were continued for 5 days. After 5 days of injection, the rd1 mouse is killed by excessive anesthesia of chloral hydrate, the eyeball is rapidly removed, and the OCT embedding medium is rapidly placed in liquid nitrogen for quenching and is stored in a refrigerator at minus 80 ℃ for standby; when in use, frozen sections of 7 mu m are made through the optic disc and the sawtooth edge, and 12 eyes are made on 6 mice in each group; the obtained mouse eyeball frozen section is naturally dried, fixed by 4% (w/v, g/100mL) paraformaldehyde at room temperature for 15min, rinsed 3 times by 0.1mol/L PBS buffer solution (pH7.4) for 5min each time, stained by hematoxylin for 20min, differentiated by 1% (v/v) hydrochloric acid ethanol for 20s, soaked in tap water for 20min, stained by eosin for 2min, dehydrated by gradient ethanol, transparent by xylene, sealed by neutral gum, observed under an optical microscope and photographed. Each eyeball selected 3 slices, each slice selected the retina at the same position of the posterior pole for photographing and comparing the thickness of the outer nuclear layer of each group. The experimental results are shown in FIGS. 11 to 13.

1.3、gp91^phoxImmunofluorescent staining and co-localization with CD11b

gp91^phoxIs the main subunit of NOX2, and the CD11b monoclonal antibody labels microglia. And the two immunofluorescent stains are co-located to observe the expression of the NOX2 in the microglia. Naturally airing the frozen section of the mouse eyeball obtained in the step 1.2, fixing the frozen section with cold acetone for 10min, rinsing the frozen section with 0.1mol/L PBS buffer solution (pH7.4) for 3 times, 5min each time, sealing the frozen section with normal sheep serum working solution, incubating the frozen section at room temperature (25 ℃) for 10min, removing serum, and not washing; incubation of two Primary antibodies (gp 91) from different species^phoxMouse-derived, CD11 b-derived, rat-derived), incubated overnight (16h) at 4 ℃, rewarmed for 30min the next day, rinsed with 0.1mol/L PBS buffer (pH7.4), and incubated with FITC and TRITC corresponding thereto (1: 600, v/v), incubating for 1h at room temperature (25 ℃), and rinsing for 3 times, each time for 5min, with 0.1mol/L PBS buffer (pH 7.4); the fluorescent quenching sealing piece containing DAPI fluorescence-resistant sealing piece agent is used for sealing pieces, FITC green fluorescence is observed under the wavelength of 488nm of a fluorescence microscope, TRITC red fluorescence is observed under the wavelength of 532nm, and photographing is carried out. The experimental results are shown in FIGS. 14 to 16.

The experimental results of the above experiments were statistically analyzed using SPSS 20.0 statistical software. The data were normally distributed by Shapiro-Wilk test and expressed as mean + -SD. And comparing the difference of the thicknesses of the outer nuclear layers of the retinas of the mice at the same positions of the dosing group and the control group by using a t test. P <0.05 is statistically significant for the differences.

2. Results of the experiment

As shown in FIGS. 9-10, the vitreous chamber thickness of the VAS2870 outer nuclear layer (22.33. + -. 1.42) of the rd1 mouse was not significantly different from that of the DMSO-injected control eye (20.16. + -. 2.08) (P > 0.05).

From FIGS. 11-13, it can be seen that the intraperitoneal injection of gp91phox-tat and GSK2795039 significantly increased the thickness of the outer nuclear layer of rd1 mice (37.10. + -. 1.67 and 38.23. + -. 2.02, respectively, with P <0.05), compared to the control group (PBS injection) (23.08. + -. 1.23), i.e., photoreceptor cell loss was significantly delayed.

From FIGS. 14 to 16, it is clear that the activation of gp91phox-tat and GSK2795039 CD11b positive microglia cells injected intraperitoneally and the expression of gp91phox in the cells were significantly reduced compared to the control group (PBS).

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

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Claims (10)

1. Use of a specific inhibitor of NOX2 in the manufacture of a medicament for the treatment and/or prevention of retinal degeneration.
2. 2. The use of claim 1, wherein the NOX2 specific inhibitor is one or more of gp91phox-tat, GSK-2795039, or Vas 2870.
3. 3. The use of claim 1 or claim 2, wherein the NOX2 specific inhibitor is gp91phox-tat or GSK-2795039.
4. 4. A medicament for the treatment and/or prevention of retinal degeneration, characterized in that it comprises an active ingredient; the active component is a specific inhibitor of NOX 2.
5. 5. The medicament of claim 4, wherein the specific inhibitor of NOX2 is one or more of gp91phox-tat, GSK-2795039, or VAs 2870.
6. 6. The medicament of claim 4 or 5, wherein the specific inhibitor of NOX2 is gp91phox-tat or GSK-2795039.
7. 7. The medicament according to any one of claims 4 to 6, further comprising a pharmaceutical carrier and/or a pharmaceutical excipient.
8. 8. The drug of claim 7, wherein the drug carrier comprises one or more of a microcapsule, a microsphere, a nanoparticle, or a liposome.
9. 9. The pharmaceutical composition of claim 7, wherein the pharmaceutical excipients comprise one or more of solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, encapsulation agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, or release retardants.
10. Use of the NOX2 gene as a therapeutic target for a medicament for the treatment and/or prevention of retinal degeneration.

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