Sophocarpine and application of sophocarpine in inhibition of formation of subretinal fibrosis

Abstract

The invention relates to the technical field of biological medicine, in particular to sophocarpine and application of sophocarpine in inhibiting formation of subretinal fibrosis, and the sophocarpine comprises the following steps: selecting 7-week-old male C57BL / 6J mice, adaptively feeding the mice for 1 week, and performing 12-hour illumination / 12-hour dark circulation at the environment temperature of 22-25 DEG C; the method comprises the following steps of: performing intraperitoneal injection of thiobarbital sodium on a C57 mouse fed for 1 week for anesthesia, realizing muscle relaxation by using hypnotic, and anesthetizing ocular surface by using 0.4% tetracaine and 0.5% tropicamide mydriasis; a mouse is randomly divided into a control eye and an experiment eye, and 1 microliter of PBS containing 1.5% of DMSO is injected into the control eye to serve as a carrier for control. The invention finds that injection of sophocarpine in vitreous cavity can inhibit RPE cell epithelial-mesenchymal transition, can be used for preventing or treating age-related macular degeneration subretinal fibrosis, may become a new medicine for treating subretinal fibrosis, has important value for inhibiting EMT of RPE cells, can also inhibit EMT of other cells, and can be used for preventing or treating age-related macular degeneration subretinal fibrosis and treating age-related macular degeneration subretinal fibrosis. Therefore, the method has potential application value on diseases caused by cell EMT.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a sophocarpine and application thereof in inhibiting subretinal fibrosis formation. BACKGROUND

[0002] Age-related macular degeneration (AMD) is one of the main causes of severe vision loss in the elderly, and subretinal fibrosis formation is an important cause of blindness. The pathogenesis of AMD is complex, and the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be one of the important factors leading to the onset of AMD. The transformation of RPE cells into myofibroblasts after EMT is the main participant of subretinal fibrosis.

[0003] AMD is a common cause of irreversible vision loss in the elderly, and neovascular age-related macular degeneration (nAMD) is characterized by the presence of macular neovascularization (MNV, formerly known as choroidal neovascularization, CNV) that can break through Bruch's membrane in the macular region. It is an important cause of vision impairment and even blindness in the elderly. Subretinal fibrosis (SRF) is the terminal pathological stage of nAMD and the most common natural outcome of MNV. It can cause irreversible central vision loss due to damage to photoreceptors, retinal pigment epithelial cells (RPE) and choroidal capillaries. Therefore, a sophocarpine and application thereof in inhibiting subretinal fibrosis formation are needed to solve these problems. SUMMARY

[0004] The present application aims to provide a sophocarpine and application thereof in inhibiting subretinal fibrosis formation to solve the problems presented in the background.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solution: a sophocarpine and application thereof in inhibiting subretinal fibrosis formation, comprising the following steps: S1, selecting 7-week-old male C57BL / 6J mice, and adaptively feeding for 1 week at an environmental temperature of 22-25℃, humidity of 50%-60%, and a 12-hour light / 12-hour dark cycle; S2, the C57 mice fed for 1 week are subjected to intraperitoneal injection of sodium thiopental for anesthesia, muscle relaxation is achieved using pentobarbital, and the ocular surface is anesthetized using 0.4% tetracaine and the pupils are dilated using 0.5% tropicamide; S3, the mice are randomly divided into control eyes and experimental eyes, the control eyes are injected with 1 microliter of PBS containing 1.5% DMSO as a carrier control, and the experimental eyes are subjected to intravitreal injection of 1 μL of sophocarpine at a concentration of 100 μM-400 μM; S4, on the second day after injection, laser photocoagulation is performed on all mice (control eyes and experimental eyes), four photocoagulation spots are made per eye to induce subretinal fibrosis; S5, on the seventh day: after the pupils of the mice are diffused, electrodes are connected, the corneal electrode is placed on the mouse cornea, the reference electrode is placed subcutaneously between the two ears of the mouse, and the ground electrode is implanted subcutaneously in the tail of the mouse; on the fourteenth day: the above visual function detection steps are repeated, the a-wave and b-wave amplitude data are collected again, the data on the seventh day are compared, and the changes in visual function over time are observed; on the twenty-first day: the last visual function detection is performed, the dynamic changes in visual function during the entire experimental period are analyzed, and the long-term effects of sophocarpine on visual function are evaluated; S6, after the visual function detection on the twenty-first day is completed, the mice are sacrificed, the cornea, iris, and lens of the eyeball are removed, the remaining optic cup tissue of the eyeball is placed in a 4% PFA solution, and then dehydration, transparency, wax immersion, and embedding treatment are performed to make retinal tissue sections for immunofluorescence staining: the optic cup tissue is washed three times with PBS, each time for 5 minutes, the pathological changes of the retinal tissue are observed, the extent and range of subretinal fibrosis are analyzed, and the relationship between the changes in visual function and subretinal fibrosis is explored; S7, the immunofluorescence staining results of IB4, collagen I, fibronectin, alpha-SMA, and RPE65 are quantitatively or qualitatively analyzed using ImageJ software to analyze the correlation between the extent of subretinal fibrosis and changes in visual function.

[0006] As a preferred technical solution of the present application, in the step S1, during the adaptive feeding period, the mice are checked for health every day, including the mental state, diet, hair color, and activity of the mice, and if any abnormal mice are found, they are promptly removed from the experiment.

[0007] As a preferred technical solution of the present application, when the step S2 is performed, the operator needs to strictly follow the aseptic operation principle, wear sterile gloves and a mask, and the syringes, needles, and other related instruments used need to be strictly sterilized to prevent infection of the mouse eyes and affect the experimental results.

[0008] As a preferred technical scheme of the present application, the injection of sophoridine in step S3 is divided into a low-concentration sophoridine group: 1 μL of 100 μM sophoridine is injected into the vitreous cavity of the experimental eye; a medium-concentration sophoridine group: 1 μL of 200 μM sophoridine is injected into the vitreous cavity of the experimental eye; and a high-concentration sophoridine group: 1 μL of 400 μM sophoridine is injected into the vitreous cavity of the experimental eye.

[0009] As a preferred technical scheme of the present application, the laser photocoagulation in step S4 uses a 532 nm laser photocoagulation instrument, and the parameters are set as a power of 200 mW, a spot diameter of 50 μm, and an exposure time of 0.1 s.

[0010] As a preferred technical scheme of the present application, the electrode implantation in step S5 is performed in accordance with the operation guide of the Kanghuasaiming electrophysiological instrument, and a mouse is stimulated with a light intensity of 6.325e-2 cd x s / m2, signals are collected, the amplitudes of a waves and b waves are analyzed, and the preliminary changes of visual function are recorded.

[0011] As a preferred technical scheme of the present application, the PBS washing in step S6 is performed for three times, each time for 5 minutes, and then, the optic cup tissue is added into a blocking solution for blocking non-specific binding sites at room temperature for 2 hours, and various primary antibody solutions (including alpha-smooth muscle actin (alpha-SMA), collagen I, and RPE65) are prepared according to the proportion and added into the corresponding optic cup tissue samples.

[0012] Compared with the prior art, the present application has the following beneficial effects: The present application is an application of sophoridine in inhibition of subretinal fibrosis, and it is found that intravitreal injection of sophoridine can inhibit the epithelial mesenchymal transition of RPE cells, and can be used for preventing or treating age-related macular degeneration subretinal fibrosis, and can become a new drug for treating subretinal fibrosis, and has important application value in inhibition of EMT of RPE cells, and can also inhibit EMT of other cells, thereby having potential application value in diseases caused by cell EMT. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical schemes in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below, and obviously, the drawings in the following description can only be some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0014] Figure 1 is a schematic diagram of the influence of sophoridine with different concentrations on the function of the mouse retina according to the present application; Figure 2 is a schematic diagram of the effect of sophoridine on neovascularization and subretinal fibrosis in laser-induced 7d CNV mice according to the present application; Figure 3 is a schematic diagram of the effect of sophoridine on RPE cell EMT and subretinal fibrosis in laser-induced 7d CNV mice according to the present application; Figure 4 is a schematic diagram of the effect of sophoridine on neovascularization and subretinal fibrosis in laser-induced 28d CNV mice according to the present application; Figure 5 is a schematic diagram of the effect of sophoridine on RPE cell EMT and subretinal fibrosis in laser-induced 28d CNV mice according to the present application; Figure 6 is a schematic diagram of the effect of sophoridine on reducing TGF-β1 -induced EMT marker immunofluorescence staining of iPSC-RPE cells according to the present application. DETAILED DESCRIPTION

[0015] Further description of the application will be made in conjunction with the accompanying drawings and specific embodiments: Example 1

[0016] Reference Figure 1 To illustrate Example 1, a 7-week-old male C57BL / 6J mouse was selected, and different concentrations of sophoridine (100-400 μM) were injected into the vitreous cavity. The C57 mouse was anesthetized by intraperitoneal injection of sodium thiopental, muscle relaxation was achieved using dexamethasone, and the eye surface was anesthetized using 0.4% tetracaine and mydriasis was performed using 0.5% tropicamide. After the mouse was anesthetized, a channel was created in the sclera into the vitreous cavity using a 1 ml syringe needle at the lower edge of the corneoscleral limbus under a body microscope. Through this channel, a Hamilton hand-held sampling needle was used to penetrate the sclera to inject 1 microliter of sophoridine solution (dissolved in DMSO) into the vitreous cavity of the posterior segment of the eye, so that the final dose in the eye was 100 μM, 200 μM, 400 μM, and the control eye was injected with 1 microliter of PBS containing 1.5% DMSO as a carrier control. After the C57 mouse was dark adapted for 12 to 16 hours, it was anesthetized by intraperitoneal injection of sodium thiopental, muscle relaxation was achieved using dexamethasone, the eye surface was anesthetized using 0.4% tetracaine, and mydriasis was performed using 0.5% tropicamide. After the pupil of the mouse was dilated, the electrodes were connected, the corneal electrode was placed on the mouse cornea, the reference electrode was placed subcutaneously between the two ears of the mouse, the ground electrode was implanted subcutaneously in the tail of the mouse, and the mouse was stimulated using a light intensity of 6.325e-2 cd x s / m^2 according to the operating guidelines of the Kanghuaer Ming electrophysiological instrument. The a-wave and b-wave amplitudes were analyzed by collecting signals to detect and analyze visual function, and the recording process was completed in a completely dark environment using only red light illumination. In this embodiment, the injected sophoridine is divided into three groups: a low-concentration sophoridine group (1 μL of 100 μM sophoridine injected intravitreally into the experimental eye), a medium-concentration sophoridine group (1 μL of 200 μM sophoridine injected intravitreally into the experimental eye), and a high-concentration sophoridine group (1 μL of 400 μM sophoridine injected intravitreally into the experimental eye). Example 2

[0017] refer to Figures 2-5Example 2 further illustrates Example 1, including the use of 7-week-old male C57BL / 6J mice. A laser-induced mouse CNV model was established using a previously reported protocol. In short, mice were generally anesthetized by intraperitoneal injection of 0.2 mL of 2% sodium pentobarbital, and pupils were dilated by intraocular drops of 1% tropicamide. Four or fifteen laser spots (laser power: 120 mW; duration: 100 ms; size: 50 μm) were uniformly induced around the optic disc of each mouse using a 532 nm laser photocoagulation system via a slit-lamp delivery system, resulting in rupture of the RPE and basement membrane, thus successfully establishing a mouse CNV model for subsequent immunofluorescence or Western blot analysis. On the same day as laser induction or on day 21, 1 μL of sophoridine solution (dissolved in DMSO) was injected intravitreally into the mouse, resulting in a final intraocular dose of 200 μmol / L. The control eye was injected with 1 μL containing 1.5% sodium phthalate. Using DMSO and PBS as a control, mice were anesthetized by intraperitoneal injection of 0.2 ml of 2% sodium pentobarbital solution on days 7 or 28 after the establishment of the CNV mouse model. The mice were fixed in a supine position on a foam board, and the thoracic cavity was opened using ophthalmic scissors to fully expose the heart. A syringe containing 10 ml of PBS was injected into the left ventricle of the mouse via an external blood collection needle. Immediately after PBS injection, the syringe was cut open at the right atrial appendage to allow PBS to enter the mouse's blood vessels for perfusion. Subsequently, the syringe was replaced with one containing 10 ml of 4% PFA to continue perfusion and fix the various tissues and organs of the mouse. Dissection and fixation: After perfusion, a 2-minute waiting period was allowed for full fixation. The eyeball was completely dissected using ophthalmic curved forceps and scissors and fixed in a 4% PFA fixative for 20 minutes. Under a stereomicroscope, the connective tissue and vitrectomy membrane around the eyeball were removed as much as possible. A circumferential incision was made along the limbus to remove the cornea, iris, and lens. The remaining optic cup tissue was placed in 4% PFA solution for immunofluorescence staining. The optic cup tissue was washed three times with PBS for 5 minutes each time. Then, the optic cup tissue was permeabilized in permeabilization solution at room temperature for 3 hours. It was washed three times with PBS for 5 minutes each time. Subsequently, the optic cup tissue was added to blocking solution and blocked at room temperature for 2 hours to block non-specific binding sites. Various primary antibody solutions (including alpha-smoothmuscle actin (α-SMA), type I collagen, and RPE65) were prepared according to the manufacturer's instructions and added to the corresponding optic cup tissue samples. The samples were incubated overnight at 4°C. The next day, the samples were washed five times with PBS for 8 minutes each time. Then, the prepared secondary antibody, such as fluorescently labeled anti-rabbit / mouse IgG solution or vascular endothelial cell probe isolectin B4 (IB4), was added, and the samples were incubated at room temperature in the dark for another 2 hours.The sample was washed five times with PBS, 8 minutes each time. The optic cup tissue sample was placed under a microscope, cut into four pieces along the blood vessel course, and laid flat on a glass slide. 10 μL of mounting medium was added, and a clear coverslip was placed over the sample to complete the mounting process. After standing for 12 hours, the RBCC tissue sample could be observed and images acquired under a Leica confocal fluorescence microscope. Quantitative measurements of CNV and subretinal fibrosis area were performed. ImageJ software was used to quantitatively or qualitatively analyze the expression levels and distribution of IB4, collagen I, fibronectin, α-SMA, and RPE65 based on immunofluorescence staining results, and to measure the area of ​​CNV and subretinal fibrosis region. In short, the images were converted to 8-bit format. Standardization was performed by using proportional calibration and establishing appropriate thresholds, including the effective fluorescence signal for quantitative region measurement. In this embodiment, intravitreal injection of sophoridine on days 7 and 21 after laser-induced CNV in mice significantly reduced the area of ​​CNV, subretinal fibrosis, and RPE cell EMT in laser-induced mice. Specifically, this was manifested as a reduction in the positive areas of type I collagen (collagen I) and isolectin B4 (IB4) immunomarkers, as well as a significant reduction in the area of ​​co-localized immunostaining of α-smooth muscle actin (α-SMA) and RPE65. Example 3

[0018] refer to Figure 6 Example 3 further illustrates Example 1, including iPSC-RPE cells being seeded on a culture plate, treated with 200 μM sophoridine, and simultaneously induced to undergo EMT with 10 ng / mL TGF-β1. After 8 days, the cells were fixed with 4% formaldehyde, and EMT protein markers were detected by immunofluorescence.

[0019] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A sophoridine and its application in inhibiting the formation of subretinal fibrosis, characterized in that, Includes the following steps: S1. Seven-week-old male C57BL / 6J mice were selected and acclimatized for one week under an ambient temperature of 22-25℃ and a humidity of 50%-60%, with a 12-hour light / 12-hour dark cycle. S2. C57 mice that had been fed for one week were anesthetized by intraperitoneal injection of sodium thiobarbital, muscle relaxation was achieved by using sedative, and the ocular surface was anesthetized by 0.4% tetracaine and the pupils were dilated by 0.5% tropicamide. S3. Mice were randomly divided into control eyes and experimental eyes. The control eyes were injected with 1 μL of PBS containing 1.5% DMSO as a carrier control. The experimental eyes were injected intravitreally with 1 μL of sophoridine at a concentration of 100 μM-400 μM. S4. On the second day after injection, all mice (control eyes and experimental eyes) were subjected to laser photocoagulation, with four photocoagulation spots created in each eye to induce subretinal fibrosis. S5, Day 7: After the mouse pupils dilate, connect the electrodes, place the corneal electrode on the mouse cornea, place the reference electrode subcutaneously between the mouse's ears, and implant the ground electrode subcutaneously in the mouse's tail; Day 14: Repeat the above visual function testing steps, collect a-wave and b-wave amplitude data again, compare with the data on Day 7, and observe the changes in visual function over time; Day 21: Conduct the final visual function test, analyze the dynamic changes in visual function throughout the experimental period, and evaluate the long-term effects of sophoridine on visual function; S6. After visual function testing on day 21, mice were euthanized, and the cornea, iris, and lens of the eyeball were removed. The remaining optic cup tissue was placed in 4% PFA solution, and then dehydrated, cleared, paraffin-embedded, and embedded to prepare retinal tissue sections for immunofluorescence staining. The optic cup tissue was washed three times with PBS for 5 minutes each time. The pathological changes of the retinal tissue were observed, the degree and extent of subretinal fibrosis were analyzed, and its relationship with changes in visual function was explored. S7. Using ImageJ software, the immunofluorescence staining results of IB4, collagen I, fibronectin, α-SMA and RPE65 were used to perform quantitative or qualitative analysis on the expression level and distribution of each protein, and CNV and the area of ​​subretinal fibrosis region were measured to analyze the correlation between the degree of subretinal fibrosis and changes in visual function.

2. The sophoridine according to claim 1 and its application in inhibiting the formation of subretinal fibrosis, characterized in that, In step S1, during the adaptation period, the mice are subjected to a health check every day. The check includes the mice's mental state, diet, fur color and activity level. If any abnormal mice are found, they are removed from the experiment in a timely manner.

3. The sophoridine according to claim 2 and its application in inhibiting the formation of subretinal fibrosis, characterized in that, When performing anesthesia and pupil dilation in step S2, the operator must strictly adhere to aseptic techniques, wear sterile gloves and masks, and ensure that all syringes, needles, and other related instruments used undergo strict disinfection and sterilization to prevent eye infections in mice from affecting the experimental results.

4. The sophoridine according to claim 1 and its application in inhibiting the formation of subretinal fibrosis, characterized in that, In step S3, the injected sophoridine is divided into three groups: a low-concentration sophoridine group (1 μL of 100 μM sophoridine injected intravitreally into the experimental eye), a medium-concentration sophoridine group (1 μL of 200 μM sophoridine injected intravitreally into the experimental eye), and a high-concentration sophoridine group (1 μL of 400 μM sophoridine injected intravitreally into the experimental eye).

5. The sophoridine according to claim 1 and its application in inhibiting the formation of subretinal fibrosis, characterized in that, In step S4, the laser photocoagulation uses a 532nm laser photocoagulation instrument with parameters set to power 200mW, spot diameter 50μm, and exposure time 0.1s.

6. The sophoridine according to claim 1 and its application in inhibiting the formation of subretinal fibrosis, characterized in that, In step S5, electrode implantation is performed according to the operating guidelines of the Kanghua Ruiming electrophysiological instrument. Mice are stimulated with light intensity of 6.325e-2cd×s / m², signals are collected and the amplitudes of a and b waves are analyzed, and preliminary changes in visual function are recorded.

7. The sophoridine according to claim 1 and its application in inhibiting the formation of subretinal fibrosis, characterized in that, In step S6, the tissue was washed three times with PBS for 5 minutes each time. Then, the optic cup tissue was added to the blocking solution and blocked at room temperature for 2 hours to block non-specific binding sites. Various primary antibody solutions (including alpha-smooth muscle actin (α-SMA), type I collagen, and RPE65) were prepared according to the ratio and added to the corresponding optic cup tissue samples.

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