CN114272363A - Application of inducible nitric oxide synthase in preparation of medicine for treating acute high intraocular pressure early blood retinal barrier injury - Google Patents
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Abstract
The invention belongs to the field of biological medicines, and particularly relates to application of inducible nitric oxide synthase in preparation of a medicine for treating acute high intraocular pressure early blood retinal barrier injury. The invention is established through a rat acute ocular hypertension model, the expression of iNOS is positioned by fluorescence, the iNOS expression is mainly expressed in a retinal nerve fiber layer, an ganglion cell layer and an inner nuclear layer and is co-expressed with a vascular endothelial cell marker CD31, the iNOS expression is up-regulated in early stages (3h and 6h) after acute ocular hypertension is known through an immunoblotting result, then the iNOS and ZO-1 expression are down-regulated, and the iNOS and ZO-1 expression are down-regulated through the intervention effect comparison of an iNOS specific inhibitor, EB leakage is increased, which shows that the iNOS has a protective effect on early-stage blood retinal barrier injury of the ocular hypertension, can be used for treating the early-stage blood retinal barrier injury of the acute ocular hypertension, and provides a new idea for the early-stage treatment of the acute ocular hypertension.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of inducible nitric oxide synthase in preparation of a medicine for treating acute high intraocular pressure early blood retinal barrier injury.
Background
Glaucoma is the second most irreversible blinding eye disease in the world. In recent years, much research has focused on retinal ganglion cell death. The survival of retinal nerve cells is closely related to the surrounding microenvironment. The Blood Retinal Barrier (BRB) is critical to maintaining retinal microenvironment homeostasis. Structurally, a BRB consists of two distinct barriers: an outer brb (obrb), consisting of retinal pigment epithelial cells, that regulates transport between the choroidal capillaries and the retina; the inner layer, BRB (iBRB), regulates transport between retinal capillaries. The local microenvironment of Retinal Ganglion Cells (RGCs) is largely protected by the iBRB. The iBRB is not an absolute barrier, as substances in the blood can pass through it by two different mechanisms, vesicle-mediated transport (transcellular) and paracellular transport. Paracellular transport is strictly dependent on Tight Junctions (TJs), such as claudins and Zonula Occludens (ZO); adhesive Junctions (AJ), such as vascular endothelial cadherin (VE cadherin); and Gap Junctions (GJs), such as connexin 43 (Cx 43). Studies have shown that ZO-1 is an indicator of BRB integrity, and that loss or reduction of ZO-1 is associated with an increase in barrier permeability.
The BRB can be damaged due to various reasons, such as ischemia, abnormal carbohydrate metabolism, release of inflammatory cytokines, mechanical traction of vitreous body and retina anterior membrane, etc., so that intravascular liquid and macromolecular substances leak out of cells to form retinal edema, and the commonly used therapeutic drugs at present comprise: the glucocorticoid medicaments GCs such as triamcinolone acetonide and the anti-vascular endothelial growth factor medicaments such as ranibizumab are injected into the vitreous body, wherein the GCs promote the expression of tight junction protein by regulating the acidification of the Jianmi junction protein plum, enhance the barrier function or stabilize BRB, or reach inflammatory factors through anti-inflammatory effect, but the GCs have large side effect in the whole body application, and only can adopt a local administration mode including vitreous injection and perisphere injection treatment. There is therefore a need to develop drugs for damage to the blood retinal barrier.
Disclosure of Invention
In order to solve the technical problems, the invention prepares a rat acute ocular hypertension model, then carries out immunofluorescence positioning inducible nitric oxide synthase iNOS expression, carries out immunoblotting detection on iNOS and tight junction protein ZO-1 expression changes, carries out intravenous injection on rats with 3% Evans Blue (EB), detects EB distribution in a retinal sheet, uses a spectrophotometer to quantitatively detect BRB damage, carries out interference on iNOS specific inhibitor 1400W, then detects iNOS, ZO-1 expression and BRB leakage again, based on the above experimental results, it was found that the expression of iNOS mainly appears in the early stage of the acute ocular hypertension model, then the expression of iNOS and ZO-1 is reduced, and the iNOS specific inhibitor is given to the iNOS specific inhibitor group, EB leakage amount is obviously larger than that of the group without the iNOS specific inhibitor in the early stage of injury, and the iNOS is known to play a role in protecting BRB injury by being up-regulated in the early stage of acute ocular hypertension.
Based on the above findings, the present invention aims to provide the use of inducible nitric oxide synthase for the preparation of a medicament for treating acute ocular hypertension and early stage blood retinal barrier injury.
Based on the same inventive concept, the embodiment of the invention also provides application of the inducible nitric oxide synthase activator in preparing a medicament for treating the early damage of the blood retinal barrier of acute high intraocular pressure.
The embodiment of the invention also provides a pharmaceutical composition for treating acute high intraocular pressure early blood retinal barrier injury, which comprises inducible nitric oxide synthase.
Further, the pharmaceutical composition further comprises at least one of pharmaceutically acceptable carriers, excipients, diluents, adjuvants and vehicles.
Has the advantages that:
the invention is established by a rat acute ocular hypertension model, the expression of iNOS is positioned by fluorescence, the iNOS is mainly expressed in a retinal nerve fiber layer, an ganglion cell layer and an inner nuclear layer and is co-expressed with a vascular endothelial cell marker CD31, the iNOS expression is up-regulated in early stages (3h and 6h) after acute ocular hypertension and then the iNOS and ZO-1 expression are down-regulated by immunoblotting results, and the iNOS and ZO-1 expression are down-regulated and EB leakage is increased by comparing the intervention effect of an iNOS specific inhibitor, so that the iNOS has a protection effect on early-stage blood retinal barrier injury of the ocular hypertension, can be used for treating the early-stage blood retinal barrier injury of the acute ocular hypertension, and provides a new thought for early-stage treatment of the acute ocular hypertension.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the results of immunofluorescence double staining of retinas of normal control group, 6h and 12h groups provided in the examples of the present invention;
FIG. 2 is a graph showing the changes in iNOS and ZO-1 protein expression at different time points after acute ocular hypertension in rats according to an embodiment of the present invention;
FIG. 3 shows the amount of BRB leakage at different time points after acute ocular hypertension in rats according to the present invention;
FIG. 4 shows the western blot analysis of different groups of rat retinas for the expression of iNOS and ZO-1;
FIG. 5 is a graph showing retinal leaks in different groups of rats provided by an embodiment of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments, but the scope of the present invention is not limited to the following specific embodiments.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
The present invention follows the principles of the declaration of helsinki and the use of animal statements in ophthalmic and vision studies by alvor animals.
In the embodiment of the invention, the experimental animals are included in well-developed and healthy SD rats with the standard of body weight of 200g-250g, and the male and female are not limited. Animals were purchased from slagochada laboratory animals limited, Hunan, and Yushi, Haikou, Biotech, Inc. During the experiment, rats were housed under standard conditions, 2-3 rats per cage, and provided standard food and water. Animal certification number: 1107262011000875, 430727211101955725, 430727211102090613.
In this example, the expression of iNOS was first localized by immunofluorescence, the change in expression of its protein after ocular hypertension was detected by immunoblotting, the damage of the blood-retinal barrier in the retina at various time periods of acute ocular hypertension was quantitatively detected by EB, and then the action of iNOS in the damage of the blood-retinal barrier was investigated by using the iNOS-specific inhibitor 1400W. 9 rats for immunofluorescence double-label staining are respectively a control group, a 6h group and a 12h group; the immunoblotting and EB quantitative detection respectively use 21 rats, randomly divide the animals into a control group, 3h, 6h, 12h, 1d, 2d and 3d groups, and each group comprises 3 rats; the immunoblots for dry prognosis given 1400w (medchemeexpress) as a specific inhibitor of iNOS were treated with 12 rats, 24 rats for EB detection, and the animals were randomly divided into a control group (Cont), a normal + inhibitor group (Cont + Inh), a 6h group (6h), and a 6h + inhibitor group (6h + Inh), 3 rats per group. The rats in the normal + inhibitor group are 1400W of 1mg/100g prepared by 10% DMSO and 90% (20% SBE-beta-CD in saline) for intraperitoneal injection before 1d of material taking; in the 6h + inhibitor group, 1mg/100g of 1400W prepared by 10% DMSO and 90% (20% SBE-beta-CD in saline) is injected in the abdominal cavity 1d before the acute ocular hypertension model is built; rats in the control group and the 6h group were injected with an equal volume of 0.9% normal saline intraperitoneally.
Establishing an acute ocular hypertension model of a rat:
3-5ml/kg of 10% chloral hydrate solution is used for carrying out intraperitoneal injection on the rat to enable the rat to be anesthetized, the tail of the rat is pinched after a plurality of minutes to judge whether the rat is well anesthetized, and the anesthesia is finished if no reaction exists. The rats were placed on a Jiangwan type I locator (second department of military medical university), the iron block was hung on the upper teeth of the rats, and the four limbs were tied up and fixed with cotton thread. The eye drops of the chloromycetin are successively dropped on the eyes of rats for bacteriostasis, the eye drops of the proparacaine hydrochloride (Aierkaein company, imported drug registration number: H20160133) are used as an ocular analgesic, and the eye drops of the compound tropicamide (Yongguan pharmaceutical, national standard of medicine: H20066782) are used as mydriatic. After mydriasis is finished, the infusion apparatus is adjusted to enable the liquid drops of the needle head to slowly drip out, the needle is inserted from the junction of the cornea and the conjunctiva to the anterior chamber of the eye, and no liquid flows out of the eye, namely the needle insertion is successful. After the needle insertion is successful, the scalp needle is fixed. The physiological saline bottle is moved upwards 0.3m every 2min until 1.5m, and the time is started for 1 h. In order to prevent the rat from breaking loose, 0.1-0.2ml of chloral and an ocular analgesic can be supplemented at 30 min. Moving down 0.3m every 2min after 1h until the mouse is parallel, taking the needle in parallel after stopping for 2min, and completing modeling. After the rats are kept flat for 10min, the rats are placed in a cage after eye drops of chloramphenicol and eye drops.
Preparing tissues:
making an acute ocular hypertension model of the rat, anesthetizing the rat after the rat survives for a corresponding time point, perfusing the rat with 200ml of normal saline preheated to 37 ℃ by a water bath, and then performing fast perfusion by 200ml of 4% paraformaldehyde, namely performing slow perfusion by 200ml of 4% paraformaldehyde. Then dissecting the eyes, removing cornea, crystalline lens and vitreous body, and making the eyeball into a cup. Dehydrating with gradient sucrose solution, embedding with OCT frozen section embedding medium (Japanese cherry blossom SAKURA), slicing on full-automatic freezing microtome (U.S. Thermo) with thickness of 10 μm, standing at room temperature for 30min, and storing in 4 deg.C refrigerator after the sample adheres firmly. Samples used as immunoblots were anesthetized immediately after making a rat acute ocular hypertension model and allowing the rat to survive the corresponding time point, eyes were dissected, corneas, lenses, and vitreous were removed, and retinas were scraped off, placed in a cryovial and snap frozen in liquid nitrogen, and stored at-80 ℃ for later use.
And (3) performing immunofluorescence detection:
the sections were removed from the freezer, placed at room temperature until no beads were present on the sections, blocked with 5% donkey serum in 1 XPBS and 0.3% Triton X-100, and incubated overnight in a freezer at 4 ℃. After washing with 3X 5min PBS, the sample was incubated with secondary antibody at 37 ℃ for 1h in the dark. After washing with PBS for 3X 5min, the sections were mounted with an anti-fluorescence quencher containing DAPI. The images were observed and photographed respectively under a laser confocal microscope (olympus FV 1000). The results are shown in FIG. 1. The immunofluorescence double staining result shows that iNOS is expressed in retinas of a normal control group and a 6h group, and is not expressed in the 12h group, and the iNOS is mainly expressed on two sides of a retinal nerve fiber layer, a ganglion cell layer and an inner nuclear layer; co-expressed with the vascular endothelial cell marker CD31 and not with the retinal ganglion cell marker NeuN.
Westernblot detection:
after extracting the retinal protein, 60 mul of protein is subpackaged and the protein content is measured by using a BCA protein detection kit (Thermo), and the rest protein is subpackaged and placed in a refrigerator at the temperature of 80 ℃ below zero for standby. SDS-PAGE electrophoresis is carried out according to the loading amount of 40 mu g protein per hole, the voltage is firstly set to be 80V, the buffer solution to be loaded is run to the boundary of the separation gel and the concentration gel for about 25min, and then 120V electrophoresis separation is carried out for 60 min. After completion of electrophoresis, the protein was transferred to a nitrocellulose membrane at 330mA for 90 min. The membrane is placed in 5% skimmed milk powder prepared by TBST and sealed for 2h at normal temperature, and then immediately washed by TBST for 3 × 10min after sealing. The membranes were then incubated with the corresponding specific primary antibody in TBST overnight at 4 ℃. The membrane was again washed with TBST for 3 × 10min, incubated with diluted secondary antibody at room temperature for 90min to detect antibody-antigen complexes, and protein bands were visualized using the hypersensitivity ECL chemiluminescence kit (cloudy day). Beta-actin was used as a standard protein, and iNOS and ZO-1 were detection proteins. The iNOS or ZO-1 protein level was expressed by calculating the ratio of the iNOS or ZO-1 to the gray scale value of the standard protein using ImageJ. The results are shown in fig. 2, after acute ocular hypertension, rat retinas at different time points are taken to carry out Western blot to detect the expression of iNOS and ZO-1, and the results show that iNOS begins to be increased 3h after the acute ocular hypertension, reaches the peak 6h (P <0.001), and is not expressed basically in the last 12h, 24h, 48h and 72 h; ZO-1 expression was not different at 3h and 6h compared to the normal group, whereas expression was down-regulated at 12h, 24h, 48h and 72h (P < 0.001).
EB detection of BRB Damage:
four groups of rats, Cont + Inh, 6h, 6h + Inh, were anesthetized by abdominal injection of 10% chloral hydrate prior to sacrifice and 3% Evans blue (4.5mg/100g) was injected into the greater saphenous vein (Sigma, E2129), and after injection, it was seen that the eyes and toes of the rats turned blue. When reaching the corresponding time node, the eyeball is immediately taken out on ice, the cornea is cut off, the lens and the vitreous body are dug out, the eyeball is placed in 4 percent paraformaldehyde in a dark condition and fixed for 30 minutes, then the retina is taken out and is observed and photographed by a fluorescence microscope (Olympus IX 51). EB quantification was performed by diluting 1. mu.l Evans blue (2%) with 1ml of mephtalamine 1000-fold at a concentration of 20 ng/. mu.l, then diluting it in half for 7 times in sequence and blank mephtalamine tubes for a total of 8 standard tubes for the preparation of standard EB in mephtalamine curves. The retina of the eyeball is taken out, naturally dried and weighed, and then mixed with 160 mu l of formamide respectively in an EP tube to be incubated for 24 hours in a constant temperature box at 60 ℃. The extract was then centrifuged at 15294g at high speed for 30min at 4 ℃. The optical density values of the standard tube and sample tube supernatants were measured with a spectrophotometer at a wavelength of 620 nm.
Taking rat retinas of Cont, Cont + Inh, 6h, 6h + Inh groups to carry out western blot to detect the expression of iNOS and ZO-1, wherein the result shows that the iNOS expression of the 6h group is up-regulated, and the iNOS is not expressed basically in the control + inhibitor group and the 6h + inhibitor group; ZO-1 expression did not change significantly in the control + inhibitor group and the 6h + inhibitor group, and the decrease was significant in the 6h + inhibitor group, with the results shown in FIG. 4. Injecting Evans blue into rats in Cont, Cont + Inh, 6h and 6h + Inh groups, taking rat retinas to carry out tablet paving and Evans blue quantitative detection on BRB leakage conditions, wherein the tablet paving result shows that no red EB fluorescent spots are seen outside blood vessels of a control group, after an inhibitor is added, the red EB fluorescent spots are seen outside the blood vessels, a small amount of red fluorescent spots are seen in the 6h groups, and after the inhibitor is added, the red EB fluorescent spots (possibly formed by fusing the EB red fluorescent spots) of the blood vessels are seen. The quantitative result shows that the EB leakage of the control and inhibitor group is increased, and the leakage of the 6h and inhibitor group is obviously increased (P <0.001), and the specific result is shown in a detailed chart of figure 5.
From the above experiments and results, it was found that iNOS is mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer after acute ocular hypertension, and is co-expressed with the vascular endothelial cell marker CD 31. In the early stage (3h, 6h) after acute ocular hypertension, iNOS expression was up-regulated, and then iNOS and ZO-1 expression were down-regulated. EB quantification results showed significant BRB damage after acute ocular hypertension, but at 6h, there was little EB leakage. According to the result data of the inhibitor group, it is known that the expression of iNOS and ZO-1 is down-regulated, and the EB leakage amount is obviously increased compared with that before the inhibitor is added, so that the early up-regulation of iNOS after acute ocular hypertension can play a role in protecting BRB injury.
The foregoing is a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should be construed as the protection scope of the present invention.
Claims (4)
1. Use of inducible nitric oxide synthase in the manufacture of a medicament for the treatment of acute ocular hypertension in early stages of blood retinal barrier injury.
2. Application of an activator of inducible nitric oxide synthase in preparing a medicament for treating acute high intraocular pressure early blood retinal barrier injury.
3. A pharmaceutical composition for treating acute ocular hypertension in the early stage of blood retinal barrier injury, said pharmaceutical composition comprising an inducible nitric oxide synthase or an inducible nitric oxide synthase activator.
4. The pharmaceutical composition for treating early stage blood retinal barrier injury of acute ocular hypertension according to claim 3, wherein the pharmaceutical composition further comprises at least one of pharmaceutically acceptable carriers, excipients, diluents, adjuvants and vehicles.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20070112076A1 (en) * | 2005-11-16 | 2007-05-17 | Southern Illinois University | Methods and materials for treating retinopathy |
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| US20070112076A1 (en) * | 2005-11-16 | 2007-05-17 | Southern Illinois University | Methods and materials for treating retinopathy |
| CN101062407A (en) * | 2006-04-29 | 2007-10-31 | 中国科学院上海生命科学研究院 | Function of erythropoietin in the preventing and treating of retinal injury |
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