Application of complement factor C3 as a therapeutic target for crystalline retinitis pigmentosa

Abstract

The present invention belongs to the technical field of ophthalmic disease treatment, and in particular, relates to the application of complement factor C3 as a therapeutic target for crystalline retinitis pigmentosa. The present invention proposes complement factor C3 as a therapeutic target for crystalline retinitis pigmentosa for the first time. By inhibiting the function of complement factor C3, the lipid metabolism disorder of crystalline retinitis pigmentosa cells and zebrafish models can be significantly improved, and the accumulation of lipid droplets can be slowed down, providing favorable evidence for the treatment of crystalline retinitis pigmentosa. Moreover, the treatment method of the present invention is simple and low in cost, and has significant advantages over gene therapy and cell therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of ophthalmic disease treatment, and in particular to the application of complement factor C3 as a target for the treatment of crystalline retinitis pigmentosa. Background Art

[0002] Bietti crystalline dystrophy (BCD) is a progressive retinitis pigmentosa disease characterized by retinal crystals and abnormal lipid metabolism and atrophy of the retinal pigment epithelium, and is inherited in an autosomal recessive manner. It has been reported worldwide and is more common in Asian populations, especially in Japan and China. Epidemiological surveys show that the frequency of the pathogenic gene is about 0.005, and the population prevalence is 1 / 24000. The clinical symptoms of BCD are progressive night blindness, decreased vision, visual field constriction, and finally gradual blindness. In the early stage of the lesion, there may be a large number of yellow-white deposits similar to crystalline particles, which are scattered in the posterior pole and mid-peripheral retina of both eyes, and are mainly located in the retinal pigment epithelium (RPE). As the disease progresses, it is accompanied by accumulation and atrophy of lipid droplets in the retinal pigment epithelium, choroidal capillary lesions, photoreceptor cell death, and ultimately blindness.

[0003] CYP4V2 (cytochrome P450 family 4 subfamily V member 2) gene is the only known BCD pathogenic gene. CYP4V2 is a member of cytochrome P450 family 4, which is mainly involved in the synthesis of long-chain and medium-chain fatty acids. Oxidation, together with mitochondrial and peroxisome β-oxidation, degrades lipids. Dysfunction of CYP4V2 in RPE cells leads to disruption of lipid metabolism homeostasis and accumulation of lipid droplets, which is the pathological basis of crystalline retinitis pigmentosa.

[0004] Crystalline retinitis pigmentosa seriously affects the health and quality of life of patients, but there are currently no effective treatment interventions for the disease in clinical practice at home and abroad. In recent years, research has made some breakthroughs in gene therapy, for example, by complementing the correct copy of the CYP4V2 gene, RPE cells can express CYP4V2 protein, but gene therapy is complex, the cost of treatment is huge, and there are potential biosafety risks. Drug therapy is a more important treatment direction for the disease, but there are currently no reports on drug treatments for crystalline retinitis pigmentosa. Therefore, finding therapeutic drugs that can effectively prevent or delay the progression of BCD has always been an important research topic in this field. Summary of the invention

[0005] In view of the above technical problems in the prior art, the present invention provides the application of complement factor C3 as a therapeutic target for crystalline retinitis pigmentosa. The present invention is specifically implemented by the following technical solutions:

[0006] The first aspect of the present invention provides the use of complement factor C3 as a therapeutic target in the preparation of a drug for preventing and / or treating crystalline retinitis pigmentosa.

[0007] The present invention observed the abnormal activation of the complement pathway in the crystalline retinitis pigmentosa cell model, and the expression level of complement factor C3 was significantly upregulated. The pathological changes of the cell model in which the function of complement factor C3 was inhibited by complement inhibitors were further analyzed. It was found that reducing the activity of complement factor C3 can improve the lipid metabolism disorder of cells and zebrafish models, slow down the accumulation of lipid droplets, and provide strong evidence for the prevention and / or treatment of crystalline retinitis pigmentosa. These results show that complement factor C3 activation promotes the occurrence and development of crystalline retinitis pigmentosa, and inhibits its function by downregulating the expression of complement factor C3 or inhibiting the activation of complement factor C3, which provides a new and effective means for the prevention and / or treatment of crystalline retinitis pigmentosa, and has great scientific significance and clinical application value.

[0008] Based on the same concept as above, the second aspect of the present invention also provides the use of an agent that inhibits the function of complement factor C3 in the preparation of a drug for preventing and / or treating crystalline retinitis pigmentosa.

[0009] Furthermore, the agent that inhibits the function of complement factor C3 includes at least one of an agent that reduces the expression level of complement factor C3 gene or an agent that inhibits the activation of complement factor C3 protein.

[0010] The aforementioned reagents are used as active ingredients of the drug to inhibit the transcription or translation process of the complement factor C3 gene, thereby reducing the protein's synthesis ability, or to act on the active site of the protein to inhibit the activation of the protein, thereby affecting the realization of the complement factor C3 function and playing a therapeutic role.

[0011] Furthermore, the agent for reducing the expression level of complement factor C3 gene is selected from at least one of interfering RNA (siRNA) targeting complement factor C3 gene, hairpin RNA (shRNA) or guide RNA (sgRNA) for gene knockout.

[0012] Furthermore, the agent that inhibits the activation of complement factor C3 protein is POT-4.

[0013] Furthermore, the concentration of POT-4 in the drug is 0.5-10 μM.

[0014] The present invention has the following beneficial effects:

[0015] The present invention proposes complement factor C3 as a therapeutic target for crystalline retinitis pigmentosa for the first time. By inhibiting the function of complement factor C3, lipid metabolism disorders in crystalline retinitis pigmentosa cells and zebrafish animal models can be significantly improved, and the accumulation of lipid droplets can be slowed down. In view of the fact that lipid metabolism disorders and lipid droplet accumulation in retinal pigment epithelial cells (RPE) are the pathological basis for crystalline retinitis pigmentosa, the present invention provides favorable evidence for the treatment of crystalline retinitis pigmentosa. The treatment method of the present invention is simple and low in cost, and has significant advantages over gene therapy and cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a confocal microscopic examination image of lipid metabolism in the crystalline retinitis pigmentosa cell model of Example 2 of the present invention;

[0018] Figure 2 In Example 3 of the present invention, the expression level of complement factor C3 in crystalline retinitis pigmentosa cells was detected by immunoblotting;

[0019] Figure 3 This is a cell viability curve diagram of the crystalline retinal pigment epithelial cell model treated with POT-4 for different time periods in Example 4 of the present invention;

[0020] Figure 4 This is a confocal microscopic examination of lipid metabolism in a crystalline retinal pigment epithelial cell model treated with the complement inhibitor POT-4 according to Example 4 of the present invention;

[0021] Figure 5 This is a confocal microscopic examination of lipid metabolism in wild-type and mutant zebrafish retinal sections treated with the complement inhibitor POT-4 according to Example 4 of the present invention;

[0022] Figure 6 This is a confocal microscopic examination of lipid metabolism in wild-type and mutant zebrafish retinal pigment epithelial cells after treatment with the complement inhibitor POT-4 according to Example 4 of the present invention. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0025] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0026] The meaning of the terms "include", "comprising", "containing", "having" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the result may be added. The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is regarded as including the following situations: (i) A, (ii) B and (iii) A and B.

[0027] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art.

[0028] The term "gene" refers to the entire nucleotide sequence required to produce a polypeptide chain or functional RNA. Therefore, gene expression includes transcription and the stable accumulation of coding RNA (mRNA) or functional RNA derived from the gene, and may also refer to the translation of mRNA into polypeptides or proteins. In the context of the present invention, the expression level of a gene includes the mRNA level at the gene transcription level or the protein level at the translation level. Therefore, unless otherwise specified, the CYP4V2 gene expression level may refer to both the mRNA level and the CYP4V2 protein level. Accordingly, the expression level of complement factor C3 may refer to both the mRNA level and the complement factor C3 protein level.

[0029] Furthermore, when complement factor C3 is used as a therapeutic target, it can act on the complement factor C3 gene to reduce the transcription of its mRNA, or it can act on mRNA to reduce protein translation, thereby reducing the synthesis of complement factor C3 protein, or it can directly act on the complement factor C3 protein to inhibit the activation of the protein, thereby affecting the activity or function of the protein.

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the technical principle of the present invention is described in detail below.

[0031] The present invention takes cytochrome P450 family 4subfamily V member 2 (CYP4V2), the pathogenic gene of bietti crystalline dystrophy (BCD), as the target, and constructs a retinal pigment epithelial cell (RPE) model with CYP4V2 gene knockout by using siRNA gene knockout technology. It is observed through liposome staining, Western blot (WB) and other experiments that abnormal CYP4V2 function will lead to the destruction of lipid metabolism homeostasis, which can well simulate the phenotype of bietti crystalline dystrophy (BCD). Later, based on the RPE cell model, the study found that the decreased expression level of CYP4V2 would lead to a significant increase in the protein expression level of complement factor C3 in the complement pathway. As a core component of the complement system, the increased expression level of complement factor C3 indicates the presence of abnormal activation of the complement pathway. Studies have shown that abnormal expression of complement factor C3 can lead to metabolic disorders of fatty acids and triglycerides, leading to the accumulation of triglycerides, which is consistent with the pathological manifestations of the RPE cell model with CYP4V2 functional deficiency. These results suggest that the activation of the complement pathway caused by BCD pathogenic gene defects may be the cellular basis for the occurrence of crystalline retinitis pigmentosa.

[0032] Based on the above findings, the present invention proposes for the first time to prevent and / or treat crystalline retinitis pigmentosa by inhibiting the activation of the cell complement system of patients with crystalline retinitis pigmentosa. The present invention uses complement factor C3 as a target, inhibits the function of complement factor C3 through the complement inhibitor POT-4, and finds that inhibiting the activation of complement factor C3 can significantly improve the lipid metabolism disorder of RPE cells, thereby delaying damage to retinal pigment epithelial cells, revealing the feasibility of inhibiting the activation of the complement system to improve the progression of BCD.

[0033] The above studies fully confirmed the relationship between complement system activation and the occurrence and development of BCD. The core complement factor C3 in the complement pathway can be used as a new therapeutic target for BCD disease. By downregulating the synthesis ability of complement factor C3 or reducing its protein activity or function by inhibiting the activation of complement factor C3, it is possible to prevent and / or treat crystalline retinitis pigmentosa. The potential of the complement inhibitor POT-4 as a therapeutic and / or preventive drug for crystalline retinitis pigmentosa has been confirmed, which contributes new ideas to the treatment of BCD disease.

[0034] POT-4 is a small molecule cyclic peptide drug that has the activity of binding to complement factor C3, preventing its active fragments C3a and C3b from being cleaved, thereby inhibiting the activation of complement factor C3 and achieving the inhibition of complement levels in retinal pigment epithelial cells. Its structural formula is shown below:

[0035]

[0036] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or the conditions recommended by the manufacturer. In addition, the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0037] Main experimental reagents: Liposome staining solution Nile Red (Nile Red) was purchased from Beijing SolarbioScience & Technology Co., Ltd., catalog number N8440; siRNA transfection reagent Lipofectamine TM 3000 was purchased from Invitrogen, catalog number L3000075; instant protein loading buffer Omni-Easy TM Purchased from Shanghai Epizyme Biomedical Technology Co., Ltd, catalog number LT101S; anti-ACTIN antibody purchased from Proteintech, catalog number 81115; anti-CYP4V2 antibody purchased from Abclonal, catalog number A6573; anti-complement factor C3 antibody purchased from Abcam, catalog number ab200999; anti-Rabbit-HRP secondary antibody purchased from Thermo scientific, catalog number 32260; CCK-8 detection kit purchased from Vazyme Biotech Co., Ltd, catalog number A311-01; complement inhibitor POT-4 purchased from MedChemExpress, catalog number AL-78898A.

[0038] Cells: Retinal pigment epithelial cells ARPE-19 are commonly used in retinal function research, purchased from the American Type Culture Collection (ATCC), and the cell number is ATCC CRL-2302. The culture medium used for the ARPE-19 cell line is DMEM / F12 medium + 10% FBS + 1% penicillin-streptomycin mixture, and the culture conditions are a 37°C incubator with a carbon dioxide concentration of 5%.

[0039] Gene: The CYP4V2 gene sequence can be found in NCBI accession number NM_207352.4, as shown below:

[0040]

[0041] The sequence of the small interfering RNA (siRNA) used to interfere with the expression of the CYP4V2 gene is as follows:

[0042] si-CYP4V2: 5'-ACAGAGATCCGAGATACTT-3' (see SEQ ID NO. 1).

[0043] Example 1 Construction of a crystalline retinitis pigmentosa cell model

[0044] A cell model of CYP4V2 deficiency, the pathogenic gene of crystalline retinitis pigmentosa, was established in retinal pigment epithelial cells ARPE-19 by RNA interference. The specific implementation is as follows:

[0045] Guangzhou RIBOBIO designed and purified three small interfering RNAs (siRNAs) targeting different targets of the human gene CYP4V2. The siRNA sequence targeting exon 10 had a better effect. The target sequence is as follows: si-CYP4V2: 5'-ACAGAGATCCGAGATACTT-3'. Non-specific sequence siNC was used as a negative control in the experiment. The synthesized si-CYP4V2 lyophilized powder was dissolved in RNAse-free H2O into a 20μmol / L mother solution and stored at -20℃. The working concentration in the experiment was 100pmol per well in a 12-well plate.

[0046] siRNA was transferred into cells using Lipofectamine TM 3000 reagent. ARPE-19 cells were plated in a 12-well plate at a certain density one day before transfection. When the cell confluence reached 30%-50%, siCYP4V2 suspension and Lipofectamine TM 3000 suspension were prepared with 50 μL Opti-MEM medium at a ratio of siRNA: Lipofectamine TM 3000 = 2:1, and the two suspensions were mixed and allowed to stand for 15 minutes (the negative control group was treated in the same way as above, and the same ratio of siNC suspension and Lipofectamine TM 3000 suspension were prepared, and the two suspensions were mixed and allowed to stand for 15 minutes). Then, 100 μL of the mixture was added to the ARPE-19 cells to be transfected, and after gently mixing, the cells were placed in a 37°C incubator for 72 hours.

[0047] Example 2 Pathological phenotype detection of crystalline retinitis pigmentosa cell model

[0048] To analyze the lipid metabolism of the cell model of crystalline retinitis pigmentosa, determine whether the CYP4V2 gene defect destroys the lipid metabolism ability of retinal pigment epithelial cells, and evaluate whether the cell model is successfully constructed.

[0049] Nile red staining was used to detect lipid metabolism disorders and lipid droplet accumulation in the above cell model. Nile red is a lipophilic fluorescent dye that emits fluorescent detection signals after binding to lipid substances, quickly detects lipid components in cells, and appears orange or red under a fluorescence microscope. It combines with anions and cations in cells or tissues through electrostatic interactions and hydration to form a stable conjugate to achieve the staining effect. The specific operation of Nile red staining is as follows: ARPE-19 cells are placed in a cell crawler in a 12-well plate. After transfection, the culture medium is discarded and washed with room temperature PBS for 5 minutes to wash away excess culture medium and cell impurities; 4% paraformaldehyde is added to cover the cells to fix the cells for 20 minutes; after washing with PBS, PBST containing 5% TritonX-100 is used to permeabilize the membrane for 15 minutes, which is conducive to Nile red entering the cells to mark lipid droplets; then 1μg / mL Nile red is used for staining at room temperature in the dark for 20 minutes (PBS configuration), and the cell nucleus is marked by DPAI; finally, it is washed with PBS 3 times, and the sealing agent is used to seal the slice after 15 minutes each time. After drying, the samples were observed under a confocal microscope.

[0050] Figure 1The figure shows the accumulation of lipid droplets in ARPE-19 cells observed by Nile Red staining before and after siRNA interference, where Nile Red is red dots, marking lipid droplet signals, and DAPI is blue signal, marking cell nuclei. Nile Red staining revealed that compared with the control group NC, the number of lipid droplets in the siCYP4V2 interference group increased and became larger, and a large number of lipid droplets accumulated around the cell nucleus, indicating that the defect of the CYP4V2 gene caused lipid metabolism disorders in the cells. This result is consistent with the cell and animal models of crystalline retinitis pigmentosa (see reference [1] Wang, Y., et al., A novel and efficient murine model of Bietti crystalline dystrophy. Dis Model Mech, 2022. 15(3). Reference [2] Gao, P., et al., Accumulation of Lipid Droplets in a Novel Bietti Crystalline Dystrophy Zebrafish Model With Impaired PPARα Pathway. Invest Ophthalmol Vis Sci, 2022. 63(5): p. 32. Reference [3] Dulken, BW, et al., Crystalline Hepatopathy Associated With Bietti Crystalline Dystrophy: A Striking Manifestation of Disordered Fatty Acid Metabolism. Am J Surg Pathol, 2024. Reference [4] Hata, M., et al., Reduction of lipid accumulation rescues Bietti's crystalline dystrophy phenotypes. Proc Natl Acad Sci USA, 2018.115(15): p.3936-3941. The performance is consistent with that in the literature [5] Zhang, Z., et al., PSCs Reveal PUFA-Provoked Mitochondrial Stress asa Central Node Potentiating RPE Degeneration in Bietti's Crystalline Dystrophy. Mol Ther, 2020.28(12): p.2642-2661.

[0051] Example 3 Detection of expression levels of complement pathway factors in a crystalline retinitis pigmentosa cell model

[0052] The levels of complement factor proteins in ARPE-19 cells in the CYP4V2 interference group and NC cells in the control group were detected by Western Blot (WB) experiments. The protein samples separated by PAGE were transferred to a solid phase carrier (such as nitrocellulose (NC) film). The solid phase carrier adsorbs the protein in the form of non-covalent bonds and can keep the type of polypeptides separated by electrophoresis and their biological activity unchanged.

[0053] Preparation of cell protein samples: Rinse the ARPE-19 cells interfered by CYP4V2 with pre-cooled PBS 3 times, each time for 5 minutes. Then add 100μL protein lysis buffer and lyse on ice for 5 minutes. Collect the cells, add Omni-EasyTM instant protein loading buffer, vortex mix, and boil in boiling water for 10 minutes. After cooling on ice for 3 minutes, centrifuge at 12000r / min for 3 minutes, and finally put the prepared protein sample into -20℃ for use.

[0054] The specific operation of WB is as follows: 1) Transfer: Use 7.5% SDS-PAGE separation gel to separate the proteins in the cell protein sample, and then transfer to the NC membrane; 2) Blocking: Put the NC membrane into TBST solution containing 5% skim milk powder and block it at room temperature for 2 hours on a horizontal shaker; 3) Primary antibody incubation: Discard the blocking solution, wash the blocking solution on the NC membrane with TBST buffer, then add the primary antibody dilution solution, and incubate it at 4°C on a horizontal shaker overnight. The primary antibodies are anti-CYP4V2 antibody, anti-C3 antibody and anti-ACTIN antibody, among which AC TIN is the internal reference protein; 4) Secondary antibody incubation: After the primary antibody incubation, the NC membrane was washed with TBST buffer for 5 minutes each time, and then anti-Rabbit-HRP secondary antibody diluted with TBST was added at a dilution ratio of 1:2000, and incubated at room temperature for 2 hours on a horizontal shaker; 5) Development: After the secondary antibody incubation, the secondary antibody dilution solution was discarded, and the NC membrane was washed with TBST buffer for 5 minutes each time. The prepared developer was evenly added to the position of the target protein and the development was started. The development results were analyzed by Quantify One software.

[0055] The results are as follows Figure 2 As shown in the figure, compared with the NC group, the protein expression level of CYP4V2 in ARPE-19 cells after CYP4V2 interference was significantly downregulated, while the protein expression level of complement factor C3 was significantly upregulated. Complement factor C3 is a central regulatory factor of the complement system. The upregulation of this protein indicates the upregulation of the complement pathway.

[0056] Example 4 Treatment with complement inhibitor POT-4 improves crystalline retinitis pigmentosa

[0057] Based on the above observation of obvious upregulation of complement factor C3 protein level in RPE cells of crystalline retinitis pigmentosa, this embodiment aims to use complement factor C3 as a therapeutic target to achieve improvement or treatment of BCD. Specifically, the complement inhibitor POT-4 was used to treat the crystalline retinitis pigmentosa cell model, and the effect of the complement inhibitor POT-4 on the viability of retinal pigment epithelial cells was analyzed, and its biological safety was observed. In addition, by detecting the improvement of lipid metabolism disorders and lipid droplet accumulation, the effect of inhibiting the expression of complement factor C3 on the improvement of crystalline retinitis pigmentosa was evaluated.

[0058] 4.1 Effects of complement inhibitor POT-4 treatment on cell viability

[0059] The effect of drugs on cell viability was detected by CCK-8 detection kit. The specific operation was as follows: ARPE-19 cells cultured to 90%-95% were digested, centrifuged and resuspended in an appropriate amount of complete medium. The resuspended cells were counted and 1×10 4 After inoculating cells / 0.1mL into a 96-well plate, the plate was placed in a cell culture incubator for culture. Inoculate 4 96-well plates, wait for 2 hours for the cells to adhere to the wall, add culture medium, dimethyl sulfoxide (DMSO) and POT-4 diluted with DMSO, respectively. The concentration of POT-4 is 2μM (consistent with the treatment concentration), and each treatment has 8 replicates. Collect samples at 0h, 24h, 48h and 72h of treatment, add 10μL of CCK8 detection solution to each well during detection, put it in an incubator at 37℃ and 5% CO2, incubate in the dark for 30min, and then detect the OD value at 450nm at 37℃. Cell viability is proportional to OD value.

[0060] Figure 3 The ARPE-19 cell viability curves at different treatment times are shown, with the horizontal axis representing time and the vertical axis representing OD value. The results showed that compared with the control group to which only the culture medium was added, the OD value of the cells treated with DMSO and 2 μM POT-4 drug did not change significantly, indicating that the drug dissolving agent DMSO and the drug did not affect cell viability.

[0061] 4.2 Effects of complement inhibitor POT-4 treatment on cellular lipid metabolism

[0062] The lipid droplet accumulation of the complement inhibitor-treated group in the POT-4-treated cell model was detected by Nile red staining. The specific operation was the same as above, and the POT-4 treatment time was 48h. The grouping was as follows: the NC group represented ARPE-19 cells interfered with by siNC without POT-4 treatment, the NC+POT-4 group represented ARPE-19 cells interfered with by siNC treated with POT-4, the siCYP4V2 group represented ARPE-19 cells interfered with by siCYP4V2 without POT-4 treatment, and the siCYP4V2+POT-4 group represented ARPE-19 cells interfered with by siCYP4V2 treated with POT-4.

[0063] The results are as follows Figure 4 As shown. It can be seen that compared with the NC group, the accumulation of lipid droplets in ARPE-19 cells after interfering with CYP4V2 was significantly upregulated, and after treatment with the complement inhibitor POT-4, the lipid droplets in ARPE-19 cells were significantly reduced and smaller, indicating that the complement inhibitor POT-4 can slow down the accumulation of lipid droplets and improve the lipid metabolism disorder in retinal pigment epithelial cells.

[0064] 4.3 Effects of complement inhibitor POT-4 treatment on lipid metabolism in the retina of zebrafish with crystalline retinitis pigmentosa

[0065] The lipid droplet accumulation in the zebrafish model treated with POT-4 and complement inhibitors was detected by Nile red staining. The specific operation is as follows:

[0066] First, the wild type (WT) and mutant (cyp4v7) of 2.5 mpf were cultured under the same conditions. - / - cyp4v8 - / - , which is a mutation of the CYP4V2 homologous gene) zebrafish (for details of zebrafish, please refer to the literature [2] Gao, P., et al., Accumulation of Lipid Droplets in a Novel Bietti Crystalline Dystrophy Zebrafish Model With Impaired PPARα Pathway. Invest Ophthalmol Vis Sci, 2022. 63 (5): p. 32.) The body weight was measured, and the zebrafish with determined body weight were treated with a dose of 50 mg / kg. The drug treatment was carried out by gavage, and the drug was added every other day for half a month. The grouping is as follows: the wt+dmso group represents the control group treated with DMSO by gavage, the wt+pot-4 group represents the control group treated with POT-4 by gavage, and the cyp4v7 - / - cyp4v8 - / -+DMSO group means the experimental group treated with DMSO by gavage, cyp4v7 - / - cyp4v8 - / - The +pot-4 group represents the experimental group treated with POT-4 by gavage. After the drug addition, the zebrafish genotype was first identified, and then the zebrafish were frozen and killed for sample collection. The specific experimental operation is as follows:

[0067] 4.3.1. Zebrafish retina cryosections

[0068] The genotyped zebrafish, whether drugged or not, were frozen on ice and killed. The eyes were gently removed with forceps and ophthalmic scissors and temporarily placed in 4% paraformaldehyde (PFA) for fixation at room temperature. The eyes were removed half an hour later and rinsed with PBS. Under a dissecting microscope, the surrounding excess tissue was cut off with ophthalmic scissors, and a few small holes were gently poked in the cornea with an injection needle to allow the fixative to fully infiltrate. After the treatment, the eyes were returned to 4% paraformaldehyde and fixed overnight at 4°C. The next day, the paraformaldehyde was discarded, rinsed twice with PBS for 10 minutes each time, and then fixed with 30% sucrose at room temperature for about 6 hours. The cryostat was turned on in advance, and the eyes were aligned with the marked opening of the tray according to the line at the black and white junction of the sclera. The eyes were embedded with OCT and placed in the cryostat for freezing, and then the tray was fixed to the blade and the slicing began. Cut slices of fixed thickness according to the experimental requirements, and start pasting when the optic nerve is cut. After slicing, equilibrate the slices at 37℃ for 1-2 hours and store them at -20℃.

[0069] 4.3.2. Retinal pigment epithelial cell peeling and tiling experiments

[0070] The genotyped zebrafish, whether drugged or not, were frozen on ice and killed. The eyeballs were immediately removed and fixed overnight at 4°C with 4% paraformaldehyde. Rinse three times with PBS, place under a dissecting microscope, and cut the cornea along the edge with scissors. Then, place in new 4% paraformaldehyde and fix overnight at 4°C. Rinse three times with PBS, and under a dissecting microscope, cut the optic cup into four petals with scissors. Gently press the outer sclera and choroid, which will be directly separated from the retinal pigment epithelial cell layer. Cut off the entire sclera and choroid at the optic nerve site, and you can see the exposed retinal pigment epithelial cells. Because they are single-layer cells, they can be directly attached to the outside of the retina for immunofluorescence.

[0071] 4.3.3 Nile red staining

[0072] Take out the retinal frozen sections from the -20℃ refrigerator, balance at room temperature for 20 minutes, and then rinse with distilled water to wipe off the excess OCT. First, use decolorization to remove the pigment in the retinal pigment epithelial cell tissue. Specific tissue section decolorization treatment: 3% H2O2 and 1% KOH solution are mixed and added to the tissue slide for 5 minutes, the reaction solution is poured out, and the slide is rinsed with PBS for 5 minutes. Nile red powder is prepared into a 1mg / mL mother solution with DMSO and stored at -20℃ away from light. Dilute it to 1000 times with PBS, add it to the tissue slide and stain it away from light for 20 minutes, discard the Nile red staining solution, and use DAPI to stain the nucleus for 10 minutes. After staining, wash the slide with PBS three times, each time for 15 minutes, and then seal it with a sealing agent, and then use a FV3000 confocal microscope to shoot.

[0073] The peeled retinal pigment epithelial cells were placed in a 12-well plate and rinsed three times with PBS for 5 minutes each time, and then the pigment in the retinal pigment epithelial cell tissue was removed by decolorization. Specific tissue decolorization treatment: 6% H2O2 and 2% KOH solution were mixed and added to the tissue for 10 minutes, the reaction solution was poured out, and the tissue was rinsed four times with the following liquid (PBS + 1% DMSO + 1% Tween-20 + 1% Triton X-100), each time for 10 minutes. Nile red powder was configured into a 1mg / mL mother solution with DMSO and stored at -20℃ away from light. Diluted to 1000 times with PBS, added to the tissue and stained for 20 minutes away from light, discarded the Nile red dye solution, and stained the nucleus with DAPI for 10 minutes. After the staining, the tissue was washed three times with PBS for 15 minutes each time, and the tissue was spread on a slide, sealed with a sealing agent, and then photographed with a FV3000 confocal microscope.

[0074] The results are as follows Figure 5-6 As shown, Figure 5 This is the result of Nile red staining of retinal sections. Figure 6 The figure shows the results of Nile red staining of the retinal pigment epithelial cell layer. It can be seen that compared with the zebrafish in the WT+DMSO group, cyp4v7 can be found in both retinal sections and Nile red staining of the retinal pigment epithelial cell layer. - / - cyp4v8 - / - There was obvious accumulation of lipid droplets near the retinal pigment epithelial cells in the zebrafish + DMSO group, while DMSO dissolved in the drug could not achieve the therapeutic effect. After treatment with the complement inhibitor POT-4, cyp4v7 - / - cyp4v8 - / -+pot-4 showed a significant decrease and reduction in lipid droplets, indicating that the complement inhibitor POT-4 can not only slow down the accumulation of lipid droplets in ARPE-19 cells and improve the lipid metabolism disorder in retinal pigment epithelial cells, but also slow down the accumulation of lipid droplets in zebrafish simulating crystalline retinitis pigmentosa and improve its lipid metabolism disorder.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of an agent that inhibits the function of complement factor C3 in the preparation of a drug for preventing and / or treating crystalline retinitis pigmentosa, characterized in that: The agent that inhibits the function of complement factor C3 is POT-4.

2. Use of the agent for inhibiting the function of complement factor C3 according to claim 1 in the preparation of a drug for preventing and / or treating crystalline retinitis pigmentosa, characterized in that: The concentration of POT-4 in the drug is 0.5-10 μM.

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