Use of recombinant hirudin in the preparation of a medicament for the treatment of cataracts and / or diabetic retinopathy

Abstract

This invention discloses the application of recombinant hirudin in the preparation of drugs for treating cataracts and / or diabetic retinopathy, belonging to the field of biomedical technology. This invention, through the construction of four models—STZ-induced diabetic cataract, high-glucose cataract, H2O2 oxidative cataract, and UV-induced cataract—as well as a high-fat combined with STZ-induced type 2 diabetic retinopathy rat model, demonstrates for the first time that recombinant hirudin, administered via eye drops, can effectively reduce lens opacity and repair retinal structural damage. Mechanistic studies show that recombinant hirudin exerts a multi-target synergistic protective effect by upregulating GSH and SOD activity and downregulating MDA content to inhibit oxidative stress, regulating the Bcl-2 / BAX / Caspase-3 pathway to inhibit apoptosis, and downregulating VEGF and Ang-1 expression to inhibit pathological angiogenesis. This invention provides a novel non-invasive treatment strategy for cataracts and diabetic retinopathy, possessing promising clinical translational prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the use of recombinant hirudin in the preparation of drugs for treating cataracts and / or diabetic retinopathy. Background Technology

[0002] Cataracts and diabetic retinopathy (DR) are leading causes of vision impairment and blindness worldwide. With the aging global population and the continued rise in diabetes prevalence, the burden of these two diseases is increasing. Cataracts are characterized by clouding caused by the denaturation and aggregation of lens proteins, while diabetic retinopathy involves progressive damage, leakage, ischemia, and subsequent neurodegenerative changes in the retinal microvessels. Although their pathological focuses differ, mounting evidence suggests they share key molecular pathological pathways, particularly oxidative stress damage and apoptosis, providing a starting point for developing drugs with multi-target interventions.

[0003] Currently, clinical treatment options for these two eye diseases have significant limitations. The only effective treatment for cataracts is surgical removal of the cloudy lens and implantation of an artificial lens; however, this is an invasive procedure with risks such as infection and posterior capsule opacification, and it cannot fundamentally inhibit the biological process of lens aging. For diabetic retinopathy, intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is currently the first-line treatment, effectively reducing macular edema; however, there are risks of poor response in some patients, the need for repeated injections, and potential cardiovascular risks. Therefore, exploring innovative drugs that can target the shared pathological mechanisms mentioned above and have multi-target protective effects has become an urgent need in research on this disease.

[0004] Hirudin is the main active ingredient of the traditional Chinese medicine leech, possessing the effects of breaking up blood stasis, removing blood stasis, eliminating scars, and promoting menstruation. Modern pharmacological studies have shown that hirudin is the most potent natural specific thrombin inhibitor known to date. Since natural hirudin exists only in the salivary glands of leeches and its content is extremely limited, with the rise of genetic engineering technology, recombinant hirudin (rH) can now be produced using recombinant DNA technology. The advent of recombinant hirudin not only solves the problems of limited natural product sources and batch-to-batch variations but also offers higher purity and consistency. Its mechanism of action is to directly and irreversibly bind to the active site of thrombin, effectively blocking all its enzymatic functions, including fibrinogen cleavage and activation of protease-activated receptors (PARs). Compared with indirect anticoagulants such as heparin, recombinant hirudin is independent of antithrombin III, is not inhibited by platelet factor 4, and effectively inhibits thrombin bound to fibrin.

[0005] Currently, recombinant hirudin is mainly used in anticoagulation and antithrombotic therapy. While its therapeutic potential in non-thrombotic diseases (such as anti-tumor metastasis and anti-fibrosis) has shown initial promise, systematic pharmacodynamic studies and mechanistic explorations are lacking in its application to ophthalmology, particularly for cataracts and diabetic retinopathy, to investigate its protective effects through multiple targets such as anti-oxidation, anti-apoptosis, and inhibition of pathological angiogenesis. Given the pivotal role of thrombin in oxidative stress, apoptosis, and pathological angiogenesis, blocking the thrombin-PAR signaling axis with recombinant hirudin holds promise for providing a novel, non-invasive, multi-target treatment strategy for these two common blinding eye diseases. Therefore, in-depth research into the specific roles and molecular mechanisms of recombinant hirudin in cataracts and retinopathy has significant clinical translational value and practical implications for the development of novel ophthalmic drugs. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of recombinant hirudin in the preparation of drugs for treating cataracts and / or diabetic retinopathy by administering it via eye drops.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] Application of recombinant hirudin in the preparation of drugs for the treatment of cataracts and / or diabetic retinopathy.

[0009] Furthermore, the drug is administered via eye drops.

[0010] Furthermore, the cataract is selected from diabetic cataract, hyperglycemic cataract, oxidative cataract and / or ultraviolet-damaged cataract.

[0011] Furthermore, the drug exerts its therapeutic effect on cataracts by inhibiting oxidative stress and / or inhibiting apoptosis.

[0012] Furthermore, the inhibition of oxidative stress is manifested by upregulating GSH content and / or SOD activity, and / or downregulating MDA content; the inhibition of apoptosis is manifested by upregulating the expression of the anti-apoptotic protein Bcl-2, downregulating the expression of the pro-apoptotic protein BAX, and / or inhibiting the activation of Caspase-3.

[0013] Furthermore, the drug exerts its therapeutic effect on diabetic retinopathy by inhibiting pathological angiogenesis and / or inhibiting apoptosis.

[0014] Furthermore, the inhibition of pathological angiogenesis is manifested by downregulating the expression of vascular endothelial growth factor and / or angiopoietin Ang-1; the inhibition of apoptosis is manifested by upregulating the expression of the anti-apoptotic protein Bcl-2 and / or inhibiting the activation of Caspase-3.

[0015] On the other hand, the present invention also provides a pharmaceutical composition for treating cataracts and / or diabetic retinopathy, comprising an effective amount of recombinant hirudin and pharmaceutically acceptable excipients.

[0016] Furthermore, the pharmaceutical composition is in the form of eye drops.

[0017] Furthermore, the concentration of recombinant hirudin in the eye drops is 0.25 mg / mL to 1 mg / mL.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) This invention provides the application of recombinant hirudin in the preparation of drugs for the prevention and treatment of cataract diseases. Through a type 2 diabetic rat model induced by high-fat combined with STZ, it is confirmed that recombinant hirudin can effectively improve the pathological damage of diabetic retinopathy through eye drops, providing a new drug option for the treatment of this disease.

[0020] (2) This invention systematically elucidates the molecular mechanism by which recombinant hirudin exerts multi-target protective effects by blocking thrombin-PAR signaling from three dimensions: oxidative stress (GSH, SOD, MDA), mitochondrial apoptosis pathway (Bcl-2 / BAX / Caspase-3), and pathological angiogenesis (VEGF, Ang-1).

[0021] (3) The present invention constructed four cataract models: diabetic, hyperglycemic, oxidative and ultraviolet damage, and verified the broad spectrum and universality of the protective effect of recombinant hirudin on the lens.

[0022] (4) This invention is the first to demonstrate that recombinant hirudin can be effectively treated by non-invasive administration via eye drops, avoiding the risks of intravitreal injection. At the same time, it achieves multi-target synergistic intervention, making up for the shortcomings of existing single-target drugs, and has good prospects for clinical translation and industrialization. Attached Figure Description

[0023] Figure 1 The change in body weight in STZ-induced diabetic cataract rats under rH treatment;

[0024] Figure 2 Changes in fasting blood glucose in STZ-induced diabetic cataract rats under rH treatment;

[0025] Figure 3 The changes in lens opacity in STZ-induced diabetic cataract rats under the action of recombinant hirudin;

[0026] Figures 4-6The changes in GSH, SOD, and MDA levels in the lens of STZ-induced diabetic cataract rats under the action of recombinant hirudin. * p<0.05, ** p<0.01;

[0027] Figures 7-9 The changes in serum GSH, SOD, and MDA levels in STZ-induced diabetic cataract rats under the action of recombinant hirudin. * p<0.05, ** p<0.01;

[0028] Figure 10 The changes in the expression levels of BAX, Bcl-2 and Caspase-3 in the lens of STZ-induced diabetic cataract rats under the action of recombinant hirudin;

[0029] Figures 11-13 The changes in GSH, SOD, and MDA content in the lens of patients with hyperglycemic cataracts under the influence of recombinant hirudin. * p<0.05, ** p<0.01;

[0030] Figure 14 The changes in the expression levels of BAX, Bcl-2, and Caspase-3 in the lens of patients with high-glycemic cataracts under the action of recombinant hirudin;

[0031] Figures 15-17 The changes in GSH, SOD, and MDA content in the lens of oxidative cataract patients under the action of recombinant hirudin. * p<0.05, ** p<0.01;

[0032] Figure 18 The changes in the expression levels of BAX, Bcl-2, and Caspase-3 in the lens of oxidative cataract patients under the action of recombinant hirudin;

[0033] Figures 19-21 The changes in GSH, SOD, and MDA content in the lens of ultraviolet-damaged cataracts under the influence of recombinant hirudin. * p<0.05, ** p<0.01;

[0034] Figure 22 The changes in the expression levels of BAX, Bcl-2, and Caspase-3 in the lens of ultraviolet-damaged cataracts under the action of recombinant hirudin;

[0035] Figure 23 The change in body weight in STZ-induced diabetic retinopathy rats under the action of recombinant hirudin;

[0036] Figure 24Changes in fasting blood glucose in STZ-induced diabetic retinopathy rats under the action of recombinant hirudin;

[0037] Figure 25 The results of H&E staining of the retina in rats with diabetic retinopathy under the action of recombinant hirudin;

[0038] Figure 26 This study investigated the changes in the expression levels of VEGF, Ang-1, Bcl-2, and Caspase-3 in the retina of diabetic retinopathy rats under the influence of recombinant hirudin. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0040] I. Experimental Materials

[0041] 1. Experimental animals: Male SD rats aged 4-6 weeks were purchased from Liaoning Changsheng Biotechnology Co., Ltd. by the Animal Experiment Center of Dalian Medical University. Experimental animal production license number: SCXK (Liaoning) 2025-0001. Mice were housed in a specific pathogen-free (SPF) environment. Animal husbandry and use complied with the regulations for the management of laboratory animals, and all operations were approved by the Animal Ethics Committee of Dalian Medical University.

[0042] 2. Test drug: Recombinant hirudin (rH), provided by Zhuhai Tianjian Hemu Biopharmaceutical Co., Ltd.

[0043] 3. Modeling reagent: streptozotocin (STZ), purchased from MCE Company.

[0044] II. Study on the improvement of STZ-induced diabetic cataracts in rats by recombinant hirudin

[0045] (1) Establishment of STZ-induced diabetic cataract model in rats

[0046] SD rats were intraperitoneally injected with STZ at a dose of 65 mg / kg. Rats with a persistent blood glucose level above 16.7 mmol / L 72 hours after injection were considered to have successfully developed a diabetic cataract model. After two weeks of continuous feeding, the lens opacity of the rats was observed using a slit-lamp microscope. The results showed that most rats exhibited significant lens opacity two weeks after successful induction of diabetes, indicating a successful establishment of the diabetic cataract model. Individuals without cataracts were discarded for subsequent experiments.

[0047] All qualified model rats and healthy control rats were systematically divided into five groups for subsequent studies:

[0048] ① Blank control group: Con;

[0049] ② Blank + rH (1 mg / mL) group: Con + rH (1 mg / mL);

[0050] ③ Diabetic cataract model group: Mod;

[0051] ④ Model + rH (0.25 mg / mL) group: Mod + rH (0.25 mg / mL);

[0052] ⑤ Model + rH (1 mg / mL) group: Mod + rH (1 mg / mL).

[0053] All animals in each group received rH via eye drops for 8 weeks. There were no unexpected animal deaths or interruptions in the administration during the entire experiment.

[0054] (2) Establishment of an in vitro model of hyperglycemic cataract in rats

[0055] Lenses were harvested from SD rats after euthanasia with overdose. Clear lenses were selected and cultured in vitro in a medium containing 35.5 mM glucose to establish a high-glucose-induced cataract model. Compared with lenses cultured in blank medium, the high-glucose culture group showed significantly increased lens opacity, demonstrating the effectiveness of the in vitro model.

[0056] The group settings were consistent with those in in vivo animal experiments. The lenses of each group were co-incubated with the corresponding concentration of rH culture medium for 7 days to complete the drug administration intervention.

[0057] (3) Slit lamp examination of the degree of lens opacity in SD rats

[0058] Before the examination, rats were anesthetized with isoflurane inhalation and their pupils were fully dilated. During the examination, the rats were fixed in a lateral recumbent position, and a slit light beam was projected onto the cornea at an angle of approximately 45° to form an optical section of the lens, and experimental images were obtained by taking pictures.

[0059] (4) Biochemical index detection

[0060] Using the detection kit provided by Beyotime Biotechnology Co., Ltd., three oxidative stress markers—glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA)—were detected in lens tissue and rat serum, respectively.

[0061] (5) Western blotting detection

[0062] Total protein was extracted from the lens and rat serum using IP lysis buffer on ice. The protein supernatant was collected by low-temperature high-speed centrifugation. A portion of the supernatant was used for BCA protein quantification. The remaining supernatant was added to 5× Loading Buffer and denatured at 100°C for 10 min. After cooling to room temperature, it was aliquoted and stored.

[0063] SDS-PAGE gels of different concentrations were prepared according to protein molecular weight. After solidification, samples were grouped and loaded according to the protein quantification results, and markers were added to the outer lanes. Stacking gel electrophoresis was performed at a constant 80 V for 30 min, and separating gel electrophoresis was performed at a constant 100 V for 1 h. The gel containing the target protein was cut and placed on a methanol-activated 0.45 µm PVDF membrane, and the membrane was transferred at a constant current of 300 mA. Subsequently, the PVDF membrane was blocked in 5% skim milk for 2 h at room temperature, and then washed three times with TTBS solution. The PVDF membrane was then placed in diluted primary antibody solution and incubated overnight at 4°C. The next day, the PVDF membrane was washed and placed in diluted secondary antibody solution, and incubated at 37°C for 1 h. After three TTBS washes, ECL chemiluminescence reagent was added, and protein band images were acquired using a chemiluminescence imaging system.

[0064] (6) Experimental results

[0065] ① rH can alleviate STZ-induced diabetic cataracts in rats

[0066] Changes in body weight and fasting blood glucose in STZ-induced diabetic cataract rats under rH treatment are as follows: Figure 1 and Figure 2 As shown in the figure, compared with the control group (Con), the fasting blood glucose level of rats in the model group was significantly increased, and their weight gain was significantly inhibited. Notably, compared with the model group, there were no statistically significant changes in blood glucose and body weight in any of the rH-treated groups. These results indicate that, at the dosage and duration of administration used in this invention, rH did not cause additional interference with the body's basal metabolic homeostasis, further confirming the drug's high in vivo safety and low toxicity.

[0067] Slit-lamp examination results as follows Figure 3 As shown in the figure, the model group rats had the most severe lens opacity. After intervention with recombinant hirudin eye drops, the lens opacity scores of both the low-dose and high-dose groups decreased significantly, with the 1 mg / mL high-dose group showing a more prominent improvement, proving that recombinant hirudin can effectively delay the progression of diabetic cataracts.

[0068] The results of biochemical tests are as follows Figures 4-9 As shown, where Figures 4-6 The changes in the contents of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in the lens tissue are shown respectively. Figures 7-9The figures show the changes in the levels of GSH, SOD, and MDA in rat serum. As can be seen from the figures, the levels of the antioxidant GSH and SOD activity in the model group were significantly decreased, while the content of the lipid peroxidation product MDA was significantly increased. After rH administration, the oxidative stress status of each treatment group was improved, specifically manifested in varying degrees of recovery in GSH and SOD levels and a decrease in MDA content.

[0069] Western blotting results are as follows Figure 10 As shown, in the diabetic model group, the expression of the pro-apoptotic protein BAX in the lens was upregulated, while the expression of the anti-apoptotic protein Bcl-2 was downregulated. rH intervention could reverse the expression trend of this protein, increase the Bcl-2 / BAX ratio, inhibit Caspase-3 activation, and reduce lens epithelial cell apoptosis by regulating the apoptosis pathway.

[0070] ②rH can alleviate hyperglycemic cataracts in rats

[0071] Results of lens biochemical index testing in an in vitro model of hyperglycemic cataract: Figures 11-13 As shown, the redox state of the lens in the model group was unbalanced, specifically manifested as a significant decrease in GSH content and SOD activity, while MDA content increased significantly. After rH administration, this trend was effectively reversed, with the treated group showing a recovery in GSH and SOD levels and a dose-dependent decrease in MDA content.

[0072] In the analysis of apoptosis pathways, the results of Western blotting are as follows: Figure 14 As shown, a high-glucose environment can upregulate the expression of the pro-apoptotic protein BAX and downregulate the expression of the anti-apoptotic protein Bcl-2, while activating Caspase-3. After rH treatment, the Bcl-2 / BAX ratio in lens tissue was significantly increased, and the protein activation level of Caspase-3 was inhibited, suggesting that rH can alleviate high-glucose-induced apoptosis of lens epithelial cells by regulating Bcl-2 / BAX / Caspase-3.

[0073] III. Anti-oxidative cataract effect of rH in rats

[0074] (1) Establishment of a rat model of oxidative cataract

[0075] Healthy SD rats were euthanized under anesthesia, and intact lenses were isolated. Transparent lenses were selected and cultured in vitro. The rat lenses were then cultured in an in vitro culture medium with a final concentration of 0.2 μmol / L H₂O₂ added for 24 h to establish an in vitro model of oxidative stress-induced cataracts. The grouping scheme was consistent with the previous description; each group's lenses were co-incubated with the corresponding concentration of recombinant hirudin for 24 h for intervention.

[0076] (2) Detection method

[0077] The procedures for biochemical index detection and Western blotting detection of apoptosis proteins are exactly the same as those described above.

[0078] (3) Experimental results

[0079] Regarding oxidative stress indicators, biochemical test results are as follows: Figures 15-17 As shown, compared with the control group, the model lens treated with H2O2 showed severe oxidative damage: the content of antioxidant markers GSH and SOD activity decreased significantly, while the content of lipid peroxidation product MDA increased sharply. After rH treatment, the oxidative damage in the high-dose group was effectively alleviated, specifically manifested as a significant recovery in GSH content and SOD activity, while the MDA content decreased significantly.

[0080] Regarding the apoptosis signaling pathway, Western blotting results are as follows: Figure 18 As shown, H2O2-induced oxidative stress significantly upregulates the expression of the pro-apoptotic protein BAX and downregulates the expression of the anti-apoptotic protein Bcl-2, accompanied by the activation of Caspase-3. rH intervention effectively reversed this trend, significantly increasing the Bcl-2 / BAX protein expression ratio and inhibiting Caspase-3 activation, indicating that rH can effectively inhibit oxidative stress-induced apoptosis of lens epithelial cells by regulating the Bcl-2 / BAX / Caspase-3 ratio.

[0081] IV. rH's effect on preventing UV-induced cataracts

[0082] (1) Establishment of an in vitro model of ultraviolet-induced cataract in rats

[0083] After euthanizing healthy SD rats under anesthesia, intact lenses were isolated, and transparent lenses were selected and cultured in vitro with the liquid level 1 mm above the lens. A 250 W high-pressure mercury lamp was used as the ultraviolet light source, and the lenses were continuously irradiated at a distance of 50 cm above the lens for 30 min to establish an in vitro model of ultraviolet-induced cataract. After successfully establishing the ultraviolet-induced cataract model, the grouping and drug administration / incubation conditions remained the same as described above. Lenses from each group were co-incubated with the corresponding concentration of recombinant hirudin for 24 h for intervention.

[0084] (2) Detection method

[0085] The methods for detecting biochemical indicators and Western blotting of apoptosis proteins are the same as those described above.

[0086] (3) Experimental results

[0087] Regarding oxidative stress indicators, the biochemical test results are as follows: Figures 19-21As shown, the lens in the ultraviolet model group exhibited typical oxidative damage characteristics: GSH content and SOD activity were significantly inhibited, while MDA content was significantly increased. After rH intervention, the redox state of the high-dose administration group was effectively improved, as evidenced by a dose-dependent recovery of GSH content and SOD activity, and a corresponding decrease in MDA content.

[0088] Regarding the apoptosis signaling pathway, the results of Western blotting are as follows: Figure 22 As shown, ultraviolet (UV) irradiation significantly induces apoptosis in lens cells, specifically by upregulating the expression of the pro-apoptotic protein BAX and downregulating the expression of the anti-apoptotic protein Bcl-2, while also significantly increasing the activation level of Caspase-3. rH intervention effectively reversed this process, significantly increasing the Bcl-2 / BAX protein expression ratio and inhibiting Caspase-3 activation, thus confirming that rH can effectively inhibit UV-induced apoptosis of lens epithelial cells by regulating the Bcl-2 / BAX / Caspase-3 ratio.

[0089] V. Anti-diabetic retinopathy effect of rH

[0090] (1) Establishment of STZ-induced diabetic retinopathy model in rats

[0091] After SD rats were fed a high-fat diet for 4 weeks, type 2 diabetes was induced by a single intraperitoneal injection of 40 mg / kg STZ. After successful modeling, the rats were divided into 5 groups according to the same grouping criteria as described above. Each group was given the corresponding concentration of rH by eye drops for 8 consecutive weeks.

[0092] (2) Morphological examination of retinal tissue by H&E staining

[0093] The fixed retinal tissue was dehydrated in ethanol solutions of increasing concentrations, then permeated with xylene solution, embedded in paraffin, and finally prepared into 5 µm sections. After dewaxing with xylene solution, ethanol solutions of decreasing concentrations, and triple-distilled water, the sections were stained with hematoxylin and eosin (H&E) dye, and photographed under an upright optical microscope to observe the morphology and arrangement of cells in each layer.

[0094] (3) Detection method

[0095] The Western blot assay was the same as described above, and the assay indicators included vascular endothelial growth factor (VEGF), angiopoietin Ang-1, apoptosis-related protein Bcl-2, and Caspase-3.

[0096] (4) Experimental results

[0097] ① rH does not affect blood glucose and body weight in diabetic rats

[0098] Changes in body weight and fasting blood glucose in STZ-induced diabetic retinopathy rats under rH treatment are as follows: Figure 23 and Figure 24 As shown, after successful induction of type 2 diabetes by high-fat + STZ, there were no statistically significant differences in fasting blood glucose and body weight between the recombinant hirudin administration groups and the model group, demonstrating that topical ophthalmic administration does not interfere with systemic glucose metabolism and has high safety.

[0099] ② Results of H&E staining of the retina in diabetic retinopathy rats under the action of recombinant hirudin are as follows: Figure 25 As shown, the model group rats exhibited typical pathological changes in their retinal structure, including disordered arrangement of ganglion cells and a decrease in the number of cells in the nuclear layer. In contrast, the rH treatment group, especially the high-dose group, showed a more orderly arrangement of retinal layers, intact cell morphology, and significantly reduced pathological damage.

[0100] At the molecular mechanism level, the results of Western blotting are as follows: Figure 26 As shown, the expression of vascular endothelial growth factor (VEGF) and Ang-1 was significantly upregulated in the retinal tissue of the model group, the activation level of Caspase-3 protein was significantly increased, while the expression of the anti-apoptotic protein Bcl-2 was significantly inhibited. After rH intervention, these conditions were effectively reversed: rH dose-dependently downregulated the expression of VEGF and Ang-1, while simultaneously upregulating the expression level of Bcl-2 and inhibiting Caspase-3 activation. This indicates that rH may exert a comprehensive protective effect on retinal tissue by simultaneously inhibiting both abnormal angiogenesis and apoptosis pathways.

[0101] VI. Conclusion

[0102] This invention is the first to demonstrate that recombinant hirudin (rH) can be used to prepare ophthalmic drugs for the treatment of cataracts and diabetic retinopathy, and its efficacy and mechanism of action are verified through a variety of in vivo and in vitro pathological models.

[0103] This invention constructs four pathogenic models related to cataracts: an STZ-induced diabetic cataract rat model, a high-glucose in vitro lens model, a H2O2 oxidative damage lens model, and an ultraviolet radiation-induced lens damage model. The experiments uniformly set up a blank control group, a drug-only control group, a disease model group, a low-dose rH intervention group (0.25 mg / mL), and a high-dose rH intervention group (1 mg / mL), using an eye drop / in vitro co-incubation method for drug administration. Results showed that rH intervention did not affect blood glucose or body weight in rats, demonstrating good in vivo safety; slit-lamp observation showed that rH dose-dependently reduced lens opacity; biochemical tests confirmed that rH significantly upregulated the lens and serum antioxidant markers GSH and SOD, downregulated the lipid peroxidation product MDA, and alleviated oxidative stress damage; Western blotting results indicated that rH upregulated the anti-apoptotic protein Bcl-2, downregulated the pro-apoptotic protein BAX, and inhibited Caspase-3 activation, reducing lens epithelial cell apoptosis by regulating the mitochondrial apoptosis pathway. All four cataract models described above reproduced this pharmacodynamic pattern, demonstrating that rH's lens protection effect does not depend on its blood sugar lowering effect, but rather stems from its direct intervention in the common terminal pathway of cataracts: oxidative stress and apoptosis.

[0104] A type 2 diabetic rat model of diabetic retinopathy was established using a high-fat diet combined with STZ, followed by continuous rH eye drops for 8 weeks. H&E retinal tissue staining showed disordered retinal ganglion cells and a reduction in nuclear layer cells in the model rats. rH administration could repair the retinal layered structure, with significant pathological improvement in the high-dose group. Western blotting analysis indicated that rH dose-dependently downregulated the expression of retinal pro-angiogenic factors VEGF and Ang-1, while upregulating Bcl-2 and inhibiting Caspase-3 activation, simultaneously blocking pathological angiogenesis and retinal neuron apoptosis. Mechanistically, rH, as a direct irreversible thrombin inhibitor, can upstream block the thrombin-PAR signaling pathway, complementing the mechanism of existing anti-VEGF drugs in clinical practice. It also has the additional advantages of inhibiting tissue fibrosis and reducing ocular inflammation, overcoming the limitations of single-target drug treatment for diabetic retinopathy.

[0105] The recombinant hirudin used in this invention is prepared through genetic engineering, overcoming the shortcomings of scarce natural hirudin raw materials and large batch-to-batch variations, ensuring stable purity. The non-invasive ophthalmic administration method avoids the surgical risks of intravitreal injections such as endophthalmitis and retinal detachment, significantly improving patient compliance. Compared to existing clinical protocols such as cataract surgery and intravitreal anti-VEGF injections, recombinant hirudin achieves multi-target synergistic protection, simultaneously intervening in multiple pathological processes including oxidative damage, apoptosis, vascular leakage, abnormal neoplasia, inflammation, and fibrosis. This provides a novel non-invasive treatment option for cataracts and diabetic retinopathy, possessing extremely high clinical translational value.

[0106] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. Application of recombinant hirudin in the preparation of drugs for the treatment of cataracts and / or diabetic retinopathy.

2. The application according to claim 1, characterized in that, The drug is administered via eye drops.

3. The application according to claim 1, characterized in that, The cataracts are selected from diabetic cataracts, hyperglycemic cataracts, oxidative cataracts, and / or ultraviolet-damaged cataracts.

4. The application according to claim 1, characterized in that, The drug exerts its therapeutic effect on cataracts by inhibiting oxidative stress and / or inhibiting apoptosis.

5. The application according to claim 4, characterized in that, The inhibition of oxidative stress is manifested by upregulating GSH content and / or SOD activity, and / or downregulating MDA content; the inhibition of apoptosis is manifested by upregulating the expression of the anti-apoptotic protein Bcl-2, downregulating the expression of the pro-apoptotic protein BAX, and / or inhibiting the activation of Caspase-3.

6. The application according to claim 1, characterized in that, The drug exerts its therapeutic effect on diabetic retinopathy by inhibiting pathological angiogenesis and / or inhibiting cell apoptosis.

7. The application according to claim 6, characterized in that, The inhibition of pathological angiogenesis is manifested by downregulating the expression of vascular endothelial growth factor and / or angiopoietin Ang-1; the inhibition of apoptosis is manifested by upregulating the expression of the anti-apoptotic protein Bcl-2 and / or inhibiting the activation of Caspase-3.

8. A pharmaceutical composition for treating cataracts and / or diabetic retinopathy, characterized in that, It contains an effective amount of recombinant hirudin and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is an eye drop formulation.

10. The pharmaceutical composition according to claim 9, characterized in that, The concentration of recombinant hirudin in the eye drops is 0.25 mg / mL to 1 mg / mL.

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