Application of TGR5 agonist in preparation of medicine for treating retinopathy of premature infants

Abstract

The invention discloses an application of a TGR5 agonist in preparation of a medicine for treating retinopathy (ROP) of a premature infant. The method comprises the following steps: firstly, establishing an ROP animal and cell model to prove the correlation between a TGR5 functional state and ROP; then, on the cellular level, a TGR5 agonist or TGR5 siRNA is used for treating cells for an experiment, and it is proved that the TGR5 agonist can inhibit proliferation, migration, tubulation and other functions of retina endothelial cells; in tissue and animal levels, a TGR5 agonist or inhibitor is given to a model mouse for an experiment, and it is proved that the TGR5 agonist can inhibit ROP pathological characteristics such as arterial tortuosity, vein dilatation, retinal vascular abnormal proliferation and bleeding. By integrating cell, tissue and animal level experiment results, the TGR5 agonist has an application prospect of being developed into the medicine for treating ROP.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a TGR5 agonist in the preparation of a drug for treating retinopathy of prematurity. Background Art

[0002] Retinopathy of Prematurity (ROP) refers to a disease in which the avascular retina of a premature infant develops fibrovascular hemangioma hyperplasia, contraction, and further causes traction retinal detachment and blindness. Due to the progress of modern life support systems such as thermostatic incubators and supplementary oxygen therapy technologies, the survival rate of premature infants with small gestational ages and low birth weights has increased significantly. However, at the same time, the incidence of ROP has also increased significantly. It is the main cause of blindness in children worldwide and is one of the key indicators for the prognosis of the quality of life of extremely low birth weight infants.

[0003] Currently, the standard treatment for severe ROP patients is laser ablation of the avascular area of the peripheral retina. Due to the lack of unified and optimal treatment parameters such as laser density, problems such as insufficient clinical treatment and over-treatment are prominent; common complications include retinal burns, choroidal hemorrhage, exudative retinal detachment, vitreous hemorrhage, endophthalmitis, anterior segment ischemia, cataract, and elevated intraocular pressure. Another currently widely used ROP treatment method is intravitreal injection of anti-VEGF drugs. However, the effect of anti-VEGF antibodies is powerful, and after intravitreal injection, they enter the blood circulation. Whether it will have an adverse effect on the development of the brain and lung structures of children is still unclear; repeated intravitreal injections will increase the risks of infection, endophthalmitis, increased intraocular pressure, and vitreous hemorrhage, and the significance and safety of the treatment remain to be evaluated; the price of this drug is relatively high, increasing the economic burden on the families of children.

[0004] Currently, there is no report on the G protein-coupled bile acid receptor (Takeda G-protein-coupled receptor 5, TGR5) related to the treatment of ROP. Summary of the Invention

[0005] The object of the present invention is to provide the application of a TGR5 agonist in the preparation of a drug for treating retinopathy of prematurity. The TGR5 agonist can inhibit the angiogenesis of ROP, inhibit the retinal inflammatory reaction of ROP, and improve the ROP lesion, thereby providing a new application of the TGR5 agonist.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: the application of a TGR5 agonist in the preparation of a drug for treating retinopathy of prematurity.

[0007] Further, the TGR5 agonist is INT-777.

[0008] Furthermore, the drug is formulated into a dosage form suitable for oral, intravenous, intraperitoneal, transdermal, ophthalmic, nasal, subcutaneous, intramuscular or rectal administration.

[0009] Furthermore, the TGR5 agonist can inhibit ROP neovascularization, inhibit ROP retinal inflammatory response, and improve ROP lesions.

[0010] Furthermore, the drug is prepared or administered in combination with any active or inactive ingredient.

[0011] Advantages of the present invention:

[0012] Experiments on a model of retinopathy of prematurity (ROP) cells in the present invention show that at the cellular level, the TGR5 agonist can effectively inhibit the functions of retinal endothelial cell proliferation, migration and tube formation.

[0013] Experiments on a model of retinopathy of prematurity (ROP) rats in the present invention show that at the tissue and animal levels, the TGR5 agonist can inhibit ROP pathological features such as arterial tortuosity, venous dilation, abnormal proliferation and bleeding of retinal blood vessels, and inflammatory response.

[0014] Based on the experimental results at the cellular, tissue and animal levels, the TGR5 agonist has the application prospect of being developed into a drug for treating ROP. Description of the Drawings

[0015] Figure 1 It is a color fundus photograph of neonatal rats in Example 1.

[0016] Figure 2 On the left is an EB staining photograph of the retinal tissue of neonatal rats in Example 2; on the right is a quantitative statistical analysis chart of the percentage of the lesion area in the total retinal area in the photograph of Example 2.

[0017] Figure 3 It is a statistical analysis chart of the mRNA levels of pro-inflammatory factors detected by qRT-PCR experiment on the retinal tissue of neonatal rats in Example 2.

[0018] Figure 4 It is a photograph of cell immunofluorescence staining of Ki67 cell proliferation experiment in Example 3.

[0019] Figure 5 It is a microscopic photograph of cells in the scratch experiment in Example 3.

[0020] Figure 6 It is a microscopic photograph of cells in the tube formation experiment in Example 3. Detailed Description of the Invention

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] Example 1: Animal level

[0023] I. Experimental materials

[0024] Construct a ROP animal model: Put the mother rats and their pups into an oxygen chamber within 4 hours after parturition, and start the hypoxia-hyperoxia exposure process (the volume fraction of O2 alternates between 50% and 10% every 24 hours). After 14 days, take out the mother rats and pups and place them in a normoxic environment for 6 days. Keep the normal lighting cycle and standard room temperature (22°C). Weigh them on the 7th and 20th days respectively, and inject drugs into the vitreous cavity of the pups' eyes on the 14th day. On the 20th day, euthanize the pups by injecting a lethal dose of sodium pentobarbital. According to the experimental needs, some eyeballs are embedded with O.C.T. glue or paraffin after fixation and other procedures, and further sectioned with a cryostat or a paraffin microtome. The cryosections are stored in a -80°C ultra-low temperature freezer, and the paraffin sections are stored at room temperature. Some eyeballs are taken out the eye cups and placed in EP tubes and stored in a -80°C ultra-low temperature freezer.

[0025] II. Experimental methods

[0026] In vivo fundus photography of ROP neonatal rats: Prepare anesthetic 3.5% chloral hydrate: 3.5 g chloral hydrate + 100 ml ddH2O. Anesthetize the rats intraperitoneally with 0.1 ml / 10 g of 3.5% chloral hydrate, cut the whiskers, dilate the pupils, and apply ofloxacin eye ointment. Place the rats on the stage, use a mouse retinal imaging microscope system (PhoenixMICRON®IV imaging system, USA) and its built-in StreamPix software, wipe the lens with a cotton swab, move the eyeball parallel to the lens (note to keep the optic disc in the center of the field of view), and adjust the knob after approaching until the fundus is clear. Take fundus photos. Apply levofloxacin eye drops to both eyes to prevent infection. Monitor the rats after they wake up and put them back into the cage.

[0027] III. Experimental results

[0028] As Figure 1 , representative color fundus photographs of neonatal rats. Compared with the control group, the vitreous of the neonatal rats in the model group was turbid, the fundus arteries were tortuous, the veins were dilated, the normal vascular distribution decreased, the blood vessels were occluded, the peripheral retinal blood vessels showed a "brush-like" appearance, and there were multiple clusters of neovascularization (yellow arrows) and hemorrhage (blue arrows); after activating TGR5, the fundus vascular manifestations improved. It is proved that TGR5 agonists can improve various ROP fundus lesions.

[0029] Example 2: Organizational level

[0030] I. Experimental materials

[0031] Preparation of ROP neonatal rat retinal flat mounts: Rat neonatal retinal section flat mounts: The neonatal rats were sacrificed, and their eyeballs were removed. The eyeballs were fixed in 4% PFA dissolved in 1×PBS (pH 7.4) for 1 - 2 h. Retinal flat mount: It was carried out in a covered petri dish suitable for holding the eyes. Under a dissecting microscope, 1 - 2 mL of PBS was added. The peribulbar fat and connective tissue near the ciliary body were dissected to expose the sclera, leaving a small segment of the optic nerve bulge. The cornea was clamped with micro toothed forceps, and a circular continuous incision was made along the circumference with a curved tip micro scissors. The cornea, iris, and lens were discarded. Four equally spaced radial relaxation incisions were made with scissors, and the eye cup was flattened. The sclera - choroid was removed, leaving only the retina. The retinal flat mount was made on a glass slide.

[0032] II. Experimental methods

[0033] 1. EB histochemical staining of rat retinal flat mounts:

[0034] Evans Blue tail vein injection and fundus fluorescein angiography in neonatal rats: Prepare EB: 30 g / L. Inject 0.3 mL / rat of EB via the tail vein. Circulate for 2 - 3 h. The rats were sacrificed, and the eyeballs were removed and fixed in formaldehyde for half an hour in the dark. The retinal flat mounts were made. The coverslips were sealed with a fluorescence quenching inhibitor and placed in a moist chamber in the dark. Photographs were taken with a fluorescence microscope in the cell room.

[0035] 2. qRT - PCR:

[0036] (1) The PCR primers and siRNA fragment sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown in Table 1.

[0037] Table 1

[0038]

[0039] (2) Loading samples: Prepare the reaction system: 5 μL SYBR Green PCR preMIX, 2 μL ddH 2 O / DEPC water, 1 μL primer, 2 μL template DNA.

[0040] (3) Film application, centrifugation (1000 r, 3 - 5 min).

[0041] (4) Set the qPCR reaction program and process the data.

[0042] III. Experimental results

[0043] 1. TGR5 agonist inhibits ROP retinal neovascularization

[0044] As Figure 2 , the EB staining photographs of the retinas of neonatal rats. Blue: neovascularization and its hemorrhage area, *: P < 0.05 vs. OIR group, n = 3, scale bar: 500 μm. In the OIR group, obvious neovascularization and hemorrhage were observed in the peripheral retina, manifested as local clustered enhancement of fluorescence signals in the periphery, uneven internal signals, and fluorescein leakage at the edges. After using INT-777, the above pathological manifestations improved significantly. The results of area quantification analysis showed that the percentage of the lesion area of neovascularization and its hemorrhage in the retinal area decreased significantly. It was proved that the TGR5 agonist could inhibit the neovascularization of ROP and improve the ROP lesions.

[0045] 2. TGR5 agonist inhibits the retinal inflammatory response in ROP

[0046] As Figure 3 , TGR5 activation downregulates the levels of inflammatory factors in the ROP state. The mRNA levels of pro-inflammatory factors in the retinas of each group were detected by qRT-PCR in the retinal tissues of neonatal rats. n = 3 rats / group. One-way ANOVA or Kruskal-wallis H test. #: P < 0.05 vs. Ctrl group, *: P < 0.05 vs. OIR / HP group. The results showed that compared with the control group (Ctrl group), the levels of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 in the model group (OIR / HP + PBS group) were significantly upregulated and could be inhibited by INT-777 (OIR / HP + INT-777 group). It was proved that the TGR5 agonist could inhibit the retinal inflammatory response in ROP and improve the ROP lesions.

[0047] Example 3: At the cellular level

[0048] I. Experimental materials

[0049] 1. Construction of a hypoxic model of retinal microvascular endothelial cells induced by CoCl 2 RMECs were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells after two passages were used for the experiment. CoCl2 (200 μM) was added to the cell culture medium to induce a cell hypoxic model, or TNF-α (10 ng / mL) was used to simulate the vascular damage caused by inflammation in the late stage of ROP.

[0050] DMEM culture medium, supplemented with 10% fetal bovine serum, penicillin 100 U / mL and streptomycin 100 μg / mL, and then RMECs were cultured. The cells after two passages were used for the experiment. CoCl2 (200 μM) was added to the cell culture medium to induce a cell hypoxic model, or TNF-α (10 ng / mL) was used to simulate the vascular damage caused by inflammation in the late stage of ROP.

[0051] 2. siRNA transfection:

[0052] Six-well plate RMECs cells, initial transfection siRNA concentration 100 nM. Transfect 24 h after cell passage, administer drugs and establish models to the cells 24 - 48 h after transfection, detect 48 h - 72 h after transfection, and verify the transfection efficiency by western blotting. Use the siRNA sequences designed by RiboBio Company, as shown in Table 2.

[0053] Table 2

[0054]

[0055] Add 120 μl Optv-MEM and 10 μl siRNA to each tube, mix gently to dilute the siRNA. Add 12 μl Lipo-2000 to each tube, pipette gently to mix, flick gently after closing the lid, and incubate at room temperature for 15 min to prepare the transfection complex. Replace the cell culture medium in the six-well plate with 1858 μl of medium (without FBS, - / -). Add the transfection complex to the medium in the six-well plate and mix gently. Detect after placing in the incubator for 24 - 72 h.

[0056] II. Experimental methods

[0057] 1. Ki67 cell proliferation experiment:

[0058] (1) Fixation: Fix with 4% paraformaldehyde for one hour, aspirate and discard, wash with PBS.

[0059] (2) Permeabilization: Prepare 0.25% Tritox-100 permeabilization solution with PBS, soak for 10 minutes, aspirate and discard, wash with PBS.

[0060] (3) Blocking: Add 5% BSA, incubate in a wet box for one hour, aspirate and do not wash.

[0061] (4) Primary antibody incubation: Add diluted rabbit anti-Ki67, incubate overnight at 4 °C. Wash with PBS after rewarming.

[0062] (5) Secondary antibody incubation: Incubate for one hour, wash with PBS.

[0063] (6) Stain with DAPI: Stain for 8 minutes, wash with PBS.

[0064] (7) Observation: Drop anti-fluorescence quenching agent, observe and take pictures with a confocal microscope.

[0065] 2. Scratch assay:

[0066] (1) Inoculate cell suspension in a six-well plate.

[0067] (3) After the cells adhere, use a 20-μl sterile pipette tip to make horizontal and vertical scratches, keep the pipette tip vertical without tilting, and use the same force each time.

[0068] (4) Wash the cells with PBS three times to remove the scraped cells, and add serum-free medium to reduce the false positive results caused by cell proliferation on migration.

[0069] (5) Place it in the cell culture incubator for culture. Sampling and photographing are carried out at the time points of 0 hour and 24 hours.

[0070] 3. Tube formation assay:

[0071] (1) About 2 hours before the experiment, dissolve the aliquoted Matrigel (150 μl / tube) on an ice pack at 4°C. Pre-cool the 96-well plate and 200 μl pipette tips at -20°C.

[0072] (2) Operate on an ice box, pipette about 45 - 50 μl of Matrigel into the 96-well plate, and place it in the incubator for gelation for 30 min.

[0073] (3) Cells can be transfected in advance as needed; passage the cells.

[0074] (4) Prepare the sample-added cell suspension: Take 100 μl of cell suspension and try to dilute it 10-fold, measure the cell density after dilution, and calculate the dilution density required for the target density of 10^4 / well (10 x 10^4 / mL) according to the ratio and formally dilute the cell suspension.

[0075] (5) Add drugs: Divide 200 μl of cell suspension into each centrifuge tube of each group. Add CoCl2 (working concentration 200 μM) to the suspension in the model group, and add INT-777 (working concentration 20 μM) to the cell suspension containing CoCl2 in the treatment group.

[0076] (6) Add 100 μl of cell suspension to each well.

[0077] (7) Place it in the cell culture incubator for culture, and take pictures under the microscope the next day.

[0078] III. Experimental results

[0079] As Figure 4 , Ki67 cell proliferation experiment. Ki67 immunofluorescence staining, green: Ki67; blue: DAPI, cell nucleus. Scale bar = 20 μm. The Ki67 cell proliferation experiment detects the proliferation function of retinal microvascular endothelial cells.

[0080] As Figure 5 , Scratch assay. Magnification: 20X. The scratch assay detects the migration function of retinal microvascular endothelial cells.

[0081] As Figure 6 , Tube formation assay. Scale bar = 200 μm. The tube formation assay detects the tube formation function of retinal microvascular endothelial cells.

[0082] The above experimental results show that under the hypoxic state of ROP, the proliferation, migration and tube formation abilities of endothelial cells after siTGR5 transfection are enhanced, leading to neovascular proliferation; after using the TGR5 agonist INT-777, the proliferation, migration and tube formation functions of endothelial cells are inhibited, which can reduce the abnormal neovascular proliferation in the retina and improve the ROP lesion.

[0083] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of TGR5 agonists in the preparation of drugs for the treatment of retinopathy of prematurity.

2. The use according to claim 1, characterized in that: The TGR5 agonist is INT-777.

3. The use according to claim 1, characterized in that: The drug contains a therapeutically effective amount of a TGR5 agonist; the drug is prepared into a dosage form suitable for oral, intravenous, intraperitoneal, transdermal, ocular, nasal, subcutaneous, intramuscular or rectal administration.

4. The use according to claim 1, characterized in that: The TGR5 agonist can inhibit ROP neovascularization, inhibit ROP retinal inflammatory response, and improve ROP lesions.

5. The use according to claim 1, characterized in that: The medicament is prepared or co-administered with any active or inactive ingredient.

Images