Application of THBS1 protein inhibitor in preparation of medicine for preventing and treating diabetic cataract and corresponding medicine

Abstract

The invention relates to the technical field of biological medicine, and provides application of a THBS1 protein inhibitor in preparation of a medicine for preventing and treating diabetic cataract, and the medicine plays a role by improving the pathological state of lens epithelial cells related to the diabetic cataract. Meanwhile, the invention further provides a medicine for preventing and treating diabetic cataract, and the medicine comprises the THBS1 protein inhibitor as an active ingredient. The application has the advantages that THBS1 is determined to be a key target for high glucose-induced lens epithelial cell apoptosis for the first time, the inhibitor of THBS1 can directly block a core link of disease progression, and compared with traditional drugs, the THBS1 has higher specificity and effectiveness, and specific accurate intervention on diabetic cataract is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of a THBS1 protein (Thrombospondin 1) inhibitor in preparation of a drug for preventing and treating diabetic cataract and a corresponding drug. BACKGROUND

[0002] Diabetic cataract is a common eye complication of diabetic patients, and is mainly characterized by accelerated lens opacity, which can lead to decreased vision and even blindness. Compared with ordinary senile cataract, diabetic cataract has an earlier onset and faster progression, and is often accompanied by other eye diseases, increasing the difficulty of treatment.

[0003] At present, the clinical treatment methods for diabetic cataract mainly include drug-assisted treatment and surgical treatment. Drug treatment mainly uses aldehyde reductase inhibitors (such as benzyldaraine) and antioxidants (such as glutathione), but can only slightly delay disease progression and cannot reverse lens opacity; surgical treatment (such as phacoemulsification combined with intraocular lens implantation) can improve vision, but has the problem of high risk of postoperative complications (such as macular edema and posterior capsule opacity), especially in diabetic patients, the incidence of complications is significantly higher than that in non-diabetic patients.

[0004] Hyperglycemia is the core inducer of diabetic cataract, which activates the polyol pathway, induces oxidative stress, promotes the accumulation of advanced glycation end products, and eventually leads to lens epithelial cell apoptosis and lens protein denaturation, resulting in opacity. However, the existing treatment methods do not precisely intervene in the key molecular mechanism of hyperglycemia-induced lens epithelial cell apoptosis, so it is urgent to find new specific targets and targeted drugs to fill the gap in clinical treatment. SUMMARY

[0005] The present application aims to overcome the defect of lack of specific target drug in the existing treatment of diabetic cataract, and provides application of a THBS1 protein inhibitor in preparation of a drug for preventing and treating diabetic cataract and a drug containing the inhibitor, which precisely intervenes in the mechanism of hyperglycemia-induced lens epithelial cell apoptosis, and realizes effective prevention and treatment of the disease.

[0006] The present application adopts the following technical solutions:

[0007] Application of a THBS1 protein inhibitor in preparation of a drug for preventing and treating diabetic cataract.

[0008] As one of the preferred modes of the present application, the drug plays a role by improving the pathological state of lens epithelial cells related to diabetic cataract.

[0009] As one of the preferred embodiments of the present invention, the pathological state includes abnormal apoptosis of lens epithelial cells; the THBS1 protein inhibitor can reduce the apoptosis level of lens epithelial cells, manifested by increasing the Bcl-2 / Bax ratio and decreasing the levels of Cleaved Caspase-3 / Caspase-3 and Cleaved Caspase-9 / Caspase-9.

[0010] As one of the preferred embodiments of the present invention, the pathological condition includes abnormal mitochondrial membrane potential; the THBS1 protein inhibitor can restore the mitochondrial membrane potential of lens epithelial cells.

[0011] As one of the preferred embodiments of the present invention, the lens epithelial cells include HLEpiCs cells.

[0012] As one of the preferred embodiments of the present invention, the THBS1 protein inhibitor includes THBS1 siRNA.

[0013] A drug for the prevention and treatment of diabetic cataracts, characterized in that it contains a THBS1 protein inhibitor as an active ingredient.

[0014] As one of the preferred embodiments of the present invention, the THBS1 protein inhibitor works by reducing the expression level of THBS1 protein and includes THBS1 siRNA.

[0015] As one of the preferred embodiments of the present invention, it also includes pharmaceutically acceptable excipients, said excipients including solvents, solubilizers, emulsifiers, binders, lubricants or preservatives.

[0016] As one of the preferred embodiments of the present invention, the dosage form of the drug is an injection, spray, ointment, eye drops, or ophthalmic ointment.

[0017] Design concept and principles:

[0018] This invention is based on the core pathological mechanism of diabetic cataracts—high glucose environment induces apoptosis and functional damage of lens epithelial cells—and achieves disease prevention and treatment by targeting and regulating the key pathogenic molecule THBS1.

[0019] High glucose levels, as an initiating factor in diabetic cataracts, can increase intracellular reactive oxygen species (ROS) levels, activate the transcription factor E2F1 to enter the nucleus, and thus promote the expression and secretion of THBS1 protein. Clinical samples and cell experiments of this invention have confirmed that THBS1 is highly expressed in lens tissues of diabetic cataract patients and in high-glucose cultured lens epithelial cells (HLEpiCs). This high expression exacerbates cell damage through two pathways: first, it directly induces apoptosis in lens epithelial cells, manifested as an imbalance in the ratio of the anti-apoptotic protein Bcl-2 to the pro-apoptotic protein Bax, and the activation of apoptosis-executing molecules Caspase-3 and Caspase-9; second, it disrupts mitochondrial function, leading to abnormal mitochondrial membrane potential, and mitochondrial dysfunction is a crucial upstream driver of apoptosis.

[0020] Based on the above mechanism, this invention designs a method to inhibit the expression or activity of THBS1 protein (e.g., using THBS1 siRNA) to block the downstream effects of the high glucose → ROS → E2F1 → THBS1 pathway, thereby reversing abnormal apoptosis of lens epithelial cells and mitochondrial dysfunction, ultimately achieving the goal of delaying or preventing the progression of diabetic cataracts. This design precisely targets key molecular nodes in the disease's development, overcoming the limitations of traditional therapies that only target symptoms or indirect pathological processes, and achieving specific intervention for diabetic cataracts.

[0021] The advantages of this invention compared to the prior art are:

[0022] (1) Highly targeted, directly addressing the core mechanisms

[0023] This invention is the first to clearly identify THBS1 as a key target for high glucose-induced apoptosis of lens epithelial cells, and its inhibitors can directly block the core link of disease progression, showing higher specificity and efficacy compared to traditional drugs (such as antioxidants).

[0024] (2) The experimental evidence is sufficient and highly reliable.

[0025] This invention validates the pathological role of THBS1 and the preventive and therapeutic effects of inhibiting THBS1 from multiple levels, from clinical samples (patient lens tissue) to cell models (HLEpiCs), with a complete chain of evidence;

[0026] (3) It has broad application prospects and high transformation value.

[0027] The drug formulations of this invention are diverse and can meet the needs of local ocular administration (such as eye drops) or systemic administration, making it particularly suitable for individualized treatment of diabetic patients; the preparation technology of inhibitors such as THBS1 siRNA is mature, which facilitates rapid clinical translation.

[0028] (4) Reduce surgical dependence and risks

[0029] This invention can reduce the need for surgery, lower the risk of postoperative complications in diabetic patients, and reduce vision loss and medical burden by delaying or reversing lens opacity. Attached Figure Description

[0030] Figure 1 The figures show the expression levels of THBS1, CD47, and CD36 in lens epithelial cells of patients with senile cataract (SC) and diabetic cataract (DC) in Example 1 (Figure a shows the immunohistochemical results of THBS1 in epithelial cells, and Figure d shows the corresponding expression level statistics; Figure b shows the immunohistochemical results of CD47 in epithelial cells, and Figure e shows the corresponding expression level statistics; Figure c shows the immunohistochemical results of CD36 in epithelial cells, and Figure f shows the corresponding expression level statistics; values ​​are expressed as mean ± SEM; n = 8; * indicates p < 0.05, ** indicates p < 0.01).

[0031] Figure 2 The figures show the expression levels of THBS1, CD47, and CD36 in HLEpiCs cultured in normal glucose (NG) and high glucose (HG) environments in Example 1 (Figure a shows the immunoblotting results of THBS1 protein, Figure d shows the corresponding expression level statistics; Figure b shows the immunoblotting results of CD47 protein, Figure e shows the corresponding expression level statistics; Figure c shows the immunoblotting results of CD36 protein, Figure f shows the corresponding expression level statistics; values ​​are expressed as mean ± SEM; n = 4-5; * indicates p < 0.05, ** indicates p < 0.01).

[0032] Figure 3 This is a graph showing the THBS1 secretion results in the supernatant of HLEpiCs after NG and HG culture, as indicated by enzyme-linked immunosorbent assay in Example 1 (in the graph, *** indicates p < 0.001);

[0033] Figure 4 The results of treatment with THBS1 siRNA and other agents under HG culture in Example 2 on Bcl-2 / Bax, Cleaved Caspase-9 / Caspase-9, and Cleaved Caspase-3 / Caspase-3 in HLEpiCs are shown in the figure (a-c are representative Western blot plots, d-f are corresponding statistical results; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001).

[0034] Figure 5The results of the effects of THBS1 siRNA treatment on the apoptosis rate of HLEpiCs under HG culture in Example 2 are shown in Figure 2 (Figure a shows the TUNEL staining results, and Figure b shows the corresponding statistical results; values ​​are expressed as mean ± SEM; n = 4; ** indicates p < 0.01).

[0035] Figure 6 The results of the effects of THBS1 siRNA and other treatments on mitochondrial membrane potential and ROS of HLEpiCs under HG culture in Example 3 are shown in Figure 3 (Figure a shows the mitochondrial membrane potential results, and Figure b shows the ROS results; values ​​are expressed as mean ± SEM; n = 4-5; **** indicates p < 0.0001).

[0036] Figure 7 The results show the expression levels of E2F1 and THBS1 in Example 3 (Figures a and c show the immunoblot images of E2F1 and THBS1 proteins and the corresponding expression level statistics after adding the E2F1 inhibitor HLM006474 to human lens epithelial cells; Figures b and d show the immunoblot images of E2F1 and THBS1 proteins and the corresponding expression level statistics after culturing human lens epithelial cells in normal glucose NG or high glucose HG medium with the addition of Tempol; values ​​are expressed as mean ± SEM; n = 4-5; * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001).

[0037] Figure 8 The results show the changes in the nuclear penetration rate of E2F1 under NG and HG culture in Example 3 (Figures a and b are representative immunofluorescence images, and Figure b is the corresponding statistical result; values ​​are expressed as mean ± SEM; n = 4; ** indicates p < 0.01). Detailed Implementation

[0038] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0039] Example 1: Expression changes of THBS1, CD36, and CD47 in diabetic cataract patients and in high glucose-induced lens epithelial cells.

[0040] 1. Experimental Materials and Methods

[0041] Clinical samples: Lens capsule epithelial tissue was collected from patients with senile cataract (SC) and diabetic cataract (DC) (all with written informed consent), with 30 patients in each group.

[0042] Cell line: Human lens epithelial cells (HLEpiCs, CP-H127, Procell Life Science & Technology, China).

[0043] Main reagents:

[0044] Culture media and additives: DMEM medium; fetal bovine serum (C04001, Bioind, Israel); penicillin / streptomycin (C0222, Beyotime Biotechnology, China);

[0045] Sugar concentration treatment: Normal sugar (NG) medium contains 5.5 mM glucose, and high sugar (HG) medium contains 25.6 mM glucose;

[0046] Antibodies: Anti-THBS1 (bs-2715R, Bioss, China); Anti-CD36 (YT5585, Immunoway); Anti-CD47 (YT5509, Immunoway); Anti-β-tubulin (AF7011, Affinity Biosciences, China);

[0047] Other reagents: 4% paraformaldehyde; 3% hydrogen peroxide; DAB colorimetric kit; RIPA buffer (P0013B, Beyotime Biotechnology, China); PVDF membrane (ISEQ00010, Millipore, USA); THBS1 enzyme-linked immunosorbent assay kit; Lipofectamine 3000 (L3000015, Thermo Fisher Scientific, USA).

[0048] Cell culture: HLEpiCs were cultured in a 37℃, 5% CO2 incubator in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Cells were passaged when confluence reached 70%–80%. The experiment was divided into an NG group (5.5 mM glucose) and an HG group (25.6 mM glucose), cultured for 1, 3, and 7 days respectively, with daily medium changes.

[0049] Immunohistochemical detection (clinical samples): Fresh tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned; sections were deparaffinized and rehydrated; incubated with 3% hydrogen peroxide for 10 minutes to remove endogenous peroxidase; blocked with 5% BSA, and anti-THBS1, CD36, or CD47 antibodies (all 1:200) were added and incubated overnight at 4°C; the next day, HRP-labeled secondary antibody was added and incubated at room temperature; DAB staining was performed, counterstained with hematoxylin, and the slides were dehydrated and mounted. The cytoplasm of the obtained positively stained cells was brown or yellow, and the slides were observed and photographed using an optical microscope.

[0050] Western blot analysis of proteins (cell samples): HLEpiCs from each group were collected, washed with PBS, lysed with RIPA buffer, and the supernatant was collected by centrifugation. Proteins were quantified using the BCA method, and denatured after adjusting the concentration. After SDS-PAGE electrophoresis, the samples were transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder, and anti-THBS1, CD36, CD47, and β-tubulin antibodies (all 1:1000) were added and incubated overnight at 4°C. After washing, HRP-labeled secondary antibody was added and the membranes were incubated at room temperature for 2 hours. The membranes were washed with PBST buffer. Protein signals were detected using an image reader.

[0051] Enzyme-linked immunosorbent assay (cell supernatant): After culturing HLEpiCs in NG or HG medium for 3 days, collect the supernatant and centrifuge; follow the kit instructions to dilute the standard, add the sample and incubate; add biotin-labeled primary antibody and HRP avidin in sequence, incubate and develop TMB color; add stop solution, measure the OD value at 450nm with an ELISA reader, and calculate the THBS1 concentration according to the standard curve.

[0052] 2. Experimental Results

[0053] Clinical samples: Immunohistochemistry showed that THBS1 ( ) was present in the lens epithelial cells of the DC group (diabetic cataract). Figure 1 a, d) and CD47 Figure 1 The percentage of positive cells (b, e) was significantly higher than that in the SC group (senile cataract), and the percentage of CD36 ( Figure 1 c and f) were significantly lower than those in the SC group (P<0.01).

[0054] Cell model: Western blot analysis showed that, compared with the NG group, the expression levels of HLEpiCs THBS1 and CD47 proteins in the HG group were significantly increased on day 7. Figure 2 a, d, b, e), while CD36 was significantly reduced ( Figure 2 c, f). Enzyme-linked immunosorbent assay (ELISA) revealed that HLEpiCs in the HG group secreted significantly more THBS1 than those in the NG group. Figure 3 ).

[0055] Example 2: The role of THBS1, CD36, and CD47 in high glucose-induced apoptosis of lens epithelial cells

[0056] 1. Experimental Materials and Methods

[0057] Cell treatment: HLEpiCs were seeded in 6-well plates and treated in groups when confluence reached 50%–60%: HG group (25.6 mM glucose), HG + Scrambled siRNA group (200 nM negative control), HG + THBS1 siRNA group (200 nM), HG + CD47 siRNA group (200 nM), and HG + CD36 inhibitor SSO group (50 μM). Transfection was performed using Lipofectamine 3000, followed by replacement with serum-containing HG medium, for a total treatment time of 48 hours. Scrambled siRNA served as the negative control; THBS1 siRNA was a THBS1 protein inhibitor (Sangon Biotech, China); CD47 siRNA was a CD47 protein inhibitor (ImmunoWay, China); and CD36 inhibitor SSO was a CD36 protein inhibitor (ImmunoWay, China).

[0058] Detection of apoptosis-related proteins by Western blotting: The detection antibodies included anti-Bcl-2 (12789-1-AP, Proteintech, China), anti-Bax (50599-2-Ig, Proteintech, China), anti-Caspase-3 (19677-1-AP, Proteintech, China), and anti-Caspase-9 (10380-1-AP, Proteintech, China). The procedure was the same as in Example 1. The ratios of Bcl-2 / Bax, Cleaved Caspase-3 / Caspase-3, and Cleaved Caspase-9 / Caspase-9 were calculated.

[0059] TUNEL assay for apoptosis: HLEpiCs were treated with the slides for 48 hours, then fixed and permeabilized; incubated with TUNEL assay solution, the nuclei were stained with DAB, and the slides were mounted; the percentage of apoptotic cells in the total number of cells was counted under a fluorescence microscope.

[0060] 2. Experimental Results

[0061] Apoptosis-related proteins: Compared with the HG+Scrambled siRNA group, the Bcl-2 / Bax ratio was significantly increased in the THBS1 siRNA, CD47 siRNA, and SSO treatment groups, while the Cleaved Caspase-3 / Caspase-3 and Cleaved Caspase-9 / Caspase-9 ratios were significantly decreased. Figure 4 (a~f).

[0062] TUNEL assay: The apoptosis rates in the THBS1 siRNA group, CD47 siRNA group, and SSO group were all significantly lower than those in the control group. Figure 5 a, b).

[0063] Example 3: The role of ROS / E2F1 in the regulation of THBS1 expression

[0064] 1. Experimental Materials and Methods

[0065] Cell treatment: Group 1 (ROS and mitochondrial function): HG group, HG+THBS1 siRNA group, HG+CD47 siRNA group, HG+SSO group, treated for 48 hours; Group 2: NG group, HG group, HG+Tempol (1mM, ROS inhibitor) group, cultured for 3 days; Group 3: HG group, HG+E2F1 inhibitor HLM006474 (10μM) group, cultured for 24 hours.

[0066] Reactive oxygen species (ROS) assay: After treatment with different groups, the culture medium was aspirated, and the cells were washed once with PBS. The fluorescent probe DCFH-DA was diluted 1:1000 in serum-free medium, and the cells were incubated at 37°C with 5% CO2 for 30 minutes. Excess fluorescent dye was washed away with serum-free cell culture medium, and the fluorescence intensity was checked using a Flexstation 3 at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0067] Mitochondrial membrane potential assay: After treatment in different groups, the culture medium was aspirated, cells were washed once with PBS, and the fluorescent probe Rhodamine 123 was diluted 1:1000 in staining buffer. Cells were then incubated at 37°C with 5% CO2 for 30 minutes. Excess fluorescent dye was washed away with serum-free cell culture medium, and fluorescence intensity was checked using a Flexstation 3 at an excitation wavelength of 507 nm and an emission wavelength of 529 nm.

[0068] Immunofluorescence detection of E2F1 nuclear translocation: After cell slides were fixed and permeabilized, they were blocked; anti-E2F1 antibody (sc-251, Santa Cruz Biotechnology, USA) was added and incubated; after washing, fluorescent secondary antibody was added, the nuclei were stained and the slides were mounted; the E2F1 nuclear translocation rate was calculated under a microscope.

[0069] Western blot detection of E2F1 and THBS1: The procedure was the same as in Example 1, and the detection antibodies were anti-E2F1 (sc-251, Santa Cruz Biotechnology, USA) and anti-THBS1 (bs-2715R, Bioss, China).

[0070] 2. Experimental Results

[0071] ROS and mitochondrial function: No significant changes in ROS levels were observed after treatment with THBS1 siRNA, CD47 siRNA, and SSO. Figure 6 b), but the mitochondrial membrane potential was restored ( Figure 6 a).

[0072] E2F1 regulation of THBS1: THBS1 expression was significantly reduced after treatment with HLM006474. Figure 7 a, c); Tempol treatment significantly reduced the expression of E2F1 and THBS1 in the HG group (a, c); Figure 7 b, d); Immunofluorescence showed that the E2F1 nuclear entry rate in the HG group was significantly higher than that in the NG group, and Tempol treatment could alleviate this behavior. Figure 8 a, b).

[0073] In summary, high glucose induces E2F1 nuclear translocation by increasing ROS, which promotes THBS1 expression. THBS1 exacerbates lens epithelial cell apoptosis through the CD47 / CD36 pathway (reducing the Bcl-2 / Bax ratio and activating the Caspase family), while inhibiting THBS1 can improve mitochondrial function and reduce apoptosis, suggesting that THBS1 is a potential therapeutic target for diabetic cataracts.

[0074] The above description is merely 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of THBS1 protein inhibitors in the preparation of drugs for the prevention and treatment of diabetic cataracts.

2. The application according to claim 1, characterized in that, The drug works by improving the pathological state of lens epithelial cells associated with diabetic cataracts.

3. The application according to claim 2, characterized in that, The pathological condition includes abnormal apoptosis of lens epithelial cells; the THBS1 protein inhibitor can reduce the apoptosis level of lens epithelial cells, manifested by increasing the Bcl-2 / Bax ratio and decreasing the levels of Cleaved Caspase-3 / Caspase-3 and Cleaved Caspase-9 / Caspase-9.

4. The application according to claim 2, characterized in that, The pathological condition includes abnormal mitochondrial membrane potential; the THBS1 protein inhibitor can restore the mitochondrial membrane potential of lens epithelial cells.

5. The application according to claim 2, characterized in that, The lens epithelial cells include HLEpiCs cells.

6. The application according to claim 1, characterized in that, The THBS1 protein inhibitor includes THBS1 siRNA.

7. A drug for preventing and treating diabetic cataracts, characterized in that, It contains a THBS1 protein inhibitor as an active ingredient.

8. The medicament according to claim 7, characterized in that, The THBS1 protein inhibitor works by reducing the expression level of the THBS1 protein and includes THBS1 siRNA.

9. The drug according to claim 7, characterized in that, It also contains pharmaceutically acceptable excipients, including solvents, solubilizers, emulsifiers, binders, lubricants, or preservatives.

10. The medicament according to claim 7, characterized in that, The dosage form of the drug is injection, spray, ointment, eye drops, or ophthalmic ointment.

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