Application of zinc-ginsenoside coordination nano-enzyme in treatment of diabetic nephropathy

Abstract

The invention relates to the technical field of biological medicine, in particular to application of zinc-ginsenoside coordination nano-enzyme in treatment of diabetic nephropathy. The Zn (at) Gs-Re nano-enzyme provided by the invention not only enhances the stability and biological activity of ginsenoside Re, but also endows the ginsenoside Re with novel similar SOD / CAT enzyme activity, realizes multiple synergistic effects of resisting oxidation, resisting inflammation, resisting apoptosis, protecting mitochondria and improving renal functions, and shows remarkable superiority in the aspect of treating diabetic nephropathy and related complications.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of zinc-ginsenoside coordination nanozymes in the treatment of diabetic nephropathy. Background Technology

[0002] Diabetes is one of the fastest-growing diseases globally, with an estimated 693 million adults affected by the disease by 2045. Diabetic kidney disease (DKD) is one of the most common complications of diabetes and a leading cause of end-stage renal disease (ESRD). The pathogenesis of DKD is multifactorial, including changes in renal hemodynamics, inflammation, metabolic disorders, oxidative stress, and genetic factors. Clinically, DKD is primarily treated by controlling blood glucose and blood pressure. However, these treatments can only delay the onset and progression of DKD; they have not achieved satisfactory results in reversing kidney damage and dysfunction or preventing the development of ESRD.

[0003] In recent years, metal and metal oxide nanoparticles have been used as drugs to treat diseases including cancer and bacterial infections. Metal and metal oxide-based nanoparticles possess excellent optical, electrical, thermal, and magnetic properties, as well as good catalytic activity and adsorption capacity. They can exhibit activities such as catalase, oxidase, superoxide dismutase, and peroxidase, and are widely used in hydrogen storage, medicine, sensing, and catalysis. Traditional Chinese medicine (TCM) has received widespread attention for its advantages in treating diabetes and its complications. It plays an indispensable role in regulating immune responses, alleviating symptoms, reducing proteinuria, and controlling disease progression. Coordinating metal particles with active ingredients from TCM to form novel nanozymes shows broad prospects in disease diagnosis and treatment.

[0004] Ginseng, a traditional Chinese medicine with high medicinal value, has long been a focus of attention in both traditional Chinese and Western medicine. The active ingredients in ginseng include ginsenosides, polysaccharides, peptides, polyethynyl alcohols, and fatty acids. Among these, ginsenosides are crucial components responsible for many biochemical and pharmacological properties. Currently, nearly 200 ginsenosides have been isolated and identified from the roots, stems, leaves, buds, and fruits of ginseng. Re compounds are the most abundant, accounting for over 30% of the total ginsenoside content. Studies have shown that ginsenoside Re (Gs-Re) has significant renal protective effects and can be used as an antioxidant, anti-inflammatory, and anti-apoptotic agent. Zinc is essential for glucose metabolism and homeostasis, playing a vital regulatory role in the production, storage, and secretion of insulin. Summary of the Invention

[0005] This invention covers the following technical solutions: This invention relates to the application of zinc-ginsenoside coordination nanozymes (Zn@Gs-Re nanozymes) in the preparation of drugs for treating diabetic nephropathy.

[0006] The Zn@Gs-Re nanozyme provided by this invention not only enhances the stability and bioactivity of ginsenoside Re, but also endows it with novel SOD / CAT-like enzyme activity, achieving multiple synergistic effects of antioxidation, anti-inflammation, anti-apoptosis, mitochondrial protection, and improvement of renal function, showing significant superiority in the treatment of diabetic nephropathy and related complications. Attached Figure Description

[0007] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0008] Figure 1 Structural characterization data of Zn@Gs-Re nanozymes.

[0009] Figure 2 Intracellular ROS levels and dynamic changes in SV40-MES-13 cells under high glucose conditions.

[0010] Figure 3 : Detection of cytokine expression levels.

[0011] Figure 4 Annexin V / PI flow cytometry detection of cell apoptosis.

[0012] Figure 5 TUNEL staining to detect apoptosis.

[0013] Figure 6 : JC-1 staining to detect mitochondrial membrane potential.

[0014] Figure 7 MitoTracker Green staining results.

[0015] Figure 8 Results of intracellular MDA, SOD, and GSH levels.

[0016] Figure 9 Comparison of fasting blood glucose, urinary protein and serum creatinine levels in mice of different groups.

[0017] Figure 10 : Histopathological staining results of mouse kidney tissue.

[0018] Figure 11 Detection of biochemical indicators related to the biosafety of Zn@Gs-Re nanozymes.

[0019] Figure 12 Tissue staining of various organs in mice reveals inflammatory changes. Detailed Implementation

[0020] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0021] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0022] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, and immunology-related terms and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0023] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this invention, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0024] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0025] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0026] As used in this invention, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5%, of a given value or range. It also includes specific numbers, such as about 20 including 20.

[0027] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0028] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0029] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0030] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0031] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0032] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0033] All references to this invention are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this invention, the referenced documents are incorporated herein by reference in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the referenced technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this invention, this invention shall prevail or modifications shall be made adaptively according to the description in this invention.

[0034] This invention relates to the application of a zinc-ginsenoside coordination nanozyme in the preparation of a drug for treating diabetic nephropathy.

[0035] In some embodiments, the indications for the drug also include the treatment of diabetic complications.

[0036] In some embodiments, the diabetic complication is selected from diabetic cardiomyopathy, diabetic retinopathy, and diabetic neuropathy.

[0037] In some embodiments, the efficacy of the drug includes at least one of the following: It lowers blood glucose levels, reduces urinary protein levels, lowers serum creatinine levels, and improves renal tissue pathological damage.

[0038] Experimental data from the embodiments of the present invention show that zinc-ginsenoside coordination nanozymes (Zn@Gs-Re nanozymes) not only exhibit significant therapeutic effects in animal models of diabetic nephropathy, but also have a universal mechanism of action that can be extended to other diabetic complications, including diabetic cardiomyopathy, diabetic retinopathy, and diabetic neuropathy.

[0039] Firstly, regarding its antioxidant effects, experimental results show that the Zn@Gs-Re nanozyme can scavenge DPPH and ABTS free radicals, effectively reduce high glucose / AGEs-induced ROS levels in cell experiments, and significantly increase intracellular SOD and GSH levels while reducing MDA expression. Oxidative stress is widely considered a common pathological mechanism of diabetic complications, including myocardial damage, retinal ganglion cell death, and peripheral neuronal dysfunction. Therefore, the antioxidant results of this invention not only support its application in diabetic nephropathy but also reasonably extend to the prevention and treatment of diabetic cardiomyopathy, diabetic retinopathy, and diabetic neuropathy.

[0040] Secondly, regarding anti-inflammatory effects, the results of the embodiments of this invention show that Zn@Gs-Re nanozymes can significantly reduce the levels of AGEs-induced IL-6, TNF-α, and VEGF. Chronic inflammation plays an important role in diabetic cardiovascular complications, angiogenesis in diabetic retinopathy, and neuroinflammation in diabetic neuropathy. Therefore, the mechanism by which Zn@Gs-Re nanozymes significantly inhibit the expression of inflammatory factors can effectively support their application in diabetic complications.

[0041] Furthermore, regarding anti-apoptosis and mitochondrial protection, the results obtained from flow cytometry, TUNEL staining, and JC-1 membrane potential assays demonstrate that the Zn@Gs-Re nanozyme can effectively reduce apoptosis levels and significantly improve mitochondrial dysfunction under high glucose conditions. Mitochondrial damage, along with apoptosis, is a crucial pathological mechanism in diabetic complications, particularly cardiomyocyte apoptosis, retinal cell apoptosis, and neuronal damage. Therefore, the data from this invention further support its potential applications in diabetic cardiomyopathy, diabetic retinopathy, and diabetic neuropathy.

[0042] Finally, in animal experiments, this invention verified in a mouse model of diabetic nephropathy that the Zn@Gs-Re nanozyme can significantly reduce fasting blood glucose, urinary protein, and serum creatinine levels, and improve renal pathological damage. These results not only demonstrate the significant therapeutic effect of this nanozyme in protecting the kidneys, but also indirectly verify its universal therapeutic value for diabetes-related multi-organ damage through its comprehensive systemic antioxidant, anti-inflammatory, and anti-apoptotic effects.

[0043] In summary, the embodiments of the present invention fully demonstrate that the mechanism of action of Zn@Gs-Re nanozymes includes anti-oxidation, anti-inflammation, anti-apoptosis, and mitochondrial protection, which are prevalent in the pathogenesis of diabetic complications.

[0044] In some embodiments, the zinc-ginsenoside coordination nanozyme is formulated as a drug alone or in combination with other drugs.

[0045] In some embodiments, the inhibitor is combined with pharmaceutically acceptable excipients to form a pharmaceutical composition. The pharmaceutical composition can be formulated using methods known to those skilled in the art. For example, it can be administered parenterally as an injection of a sterile solution or suspension of water or a pharmaceutically acceptable solution other than water. For example, a suitable combination of pharmacologically acceptable carriers or mediators, specifically sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, mediators, preservatives, binders, etc. It is considered to be formulated by mixing in a unit dosage form required for generally accepted pharmaceutical administration. The amount of the active ingredient in these formulations is set to obtain an appropriate volume within the indicated range.

[0046] Sterile compositions for injection can be formulated using excipients such as distilled water for injection, following conventional formulation methods.

[0047] Examples of aqueous solutions for injection include isotonic solutions containing, for example, physiological saline, lactose, glucose, and other supplements (such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride). Appropriate dissolving aids can be used in combination, such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80(TM), HCO-50, etc.).

[0048] As an oily liquid, sesame oil and soybean oil can be examples, and benzyl benzoate and / or benzyl alcohol can be used in combination as a solubilizing agent. The composition can be mixed with buffers (e.g., phosphate buffer solutions and sodium acetate buffer solutions), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is typically packaged in appropriate ampoules.

[0049] The pharmaceutical composition is preferably administered via parenteral administration. For example, in some embodiments, the pharmaceutical composition is selected from one or more of the following forms: solution, injection, spray, nasal drops, aerosol, powder, tablet, capsule, and granule. For example, it can be administered systemically or locally, such as by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0050] In some embodiments, the zinc-ginsenoside coordination nanozyme is prepared by mixing zinc salt, ginsenoside Re, and a stabilizer in an organic solvent, stirring, and then dialysis for purification.

[0051] In some embodiments, the zinc salt is zinc chloride hexahydrate.

[0052] In some embodiments, the organic solvent is methanol.

[0053] In some embodiments, the stabilizer is polyvinylpyrrolidone (PVP).

[0054] In some implementations, the stirring reaction time is 1 to 5 hours.

[0055] In some embodiments, the zinc-ginsenoside coordination nanozyme has a particle size of 40 nm to 100 nm, such as 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm.

[0056] In some embodiments, the zeta potential of the zinc-ginsenoside coordination nanozyme is -10 mV to -20 mV.

[0057] In this invention, the terms "subject" and "patient" are used interchangeably and refer to a mammalian individual, including preferred humans and non-human primates, as well as common laboratory animals (such as rabbits, rats, mice, etc.). The term specifically refers to an individual to whom the compounds, compositions, or formulations of this invention may be administered. Suitable subjects can be determined through conventional screening methods and are typically individuals diagnosed with a relevant disease or pathological condition requiring treatment, relief, prevention, or improvement.

[0058] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0059] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0060] Example 1: Preparation and Characterization of Zn@Gs-Re Nanozymes 1. Preparation process of Zn@Gs-Re nanozymes (1) Place the vial, magnetic stir bar and EP tube in an autoclave and sterilize for 20 min for later use; (2) Solution A: 10 mg of zinc chloride hexahydrate was dissolved in 1 mL of methanol solution and shaken thoroughly to mix. (3) Solution B: Weigh 10 mg of baicalein and dissolve it in 1 mL of methanol solution, then sonicate for 5 min; (4) Solution C: Weigh 66 mg of PVP and dissolve it in 5 mL of methanol solution, stirring thoroughly. (5) Solution D: Add solution C dropwise to solution A and stir for 5 min; (6) Solution E: Add solution B dropwise to solution D and stir for 3 h; (7) Solution F: Place solution E in a dialysis bag and dialyze it overnight with sufficient 0.9% sodium chloride solution to obtain 10 mL of aqueous solution of zinc-ginsenoside coordination nanozyme (Zn@Gs-Re nanozyme) with a concentration of 1 mg / mL. Store at 4 ℃ for later use.

[0061] 2. Structural characterization data of Zn@Gs-Re nanozymes See Figure 1 . Figure 1 Image 'a' shows the transmission electron microscopy characterization of the Zn@Gs-Re nanozyme, revealing a size of approximately 50 nm. The Zeta potential, measured using a nanoparticle size analyzer, is approximately -6.759 mV for Re and approximately -17.583 mV for the Zn@Gs-Re nanozyme, indicating that the binding of Zn ions to ginsenoside Re enhances the stability of ginsenoside Re. Figure 1 (b) According to DLS analysis, the hydrated particle size of the Zn@Gs-Re nanozyme is approximately 70 nm. Figure 1 (c), which is basically consistent with the particle size observed by TEM.

[0062] like Figure 1 As shown in the figure, the electronic spectrum of the Zn@Gs-Re nanozyme detected Zn, C, and O elements, with corresponding peak values ​​of 975, 285, and 531 eV, respectively. Figure 1 (d); specifically reflected in the C 1s spectrum ( Figure 1The main peak at 284.8 eV corresponds to the carbon (CC) on the benzene ring of the Zn@Gs-Re nanozyme, while the shoulder peak at 288.9 eV corresponds to the OC=O bond. The Zn 3d energy spectrum of the Zn@Gs-Re nanozyme, after convolution, shows a single peak, with the peak at 1044.4 eV attributed to Zn. 2+ The oxidation state of the ions, and the peak at 1021.55 is attributed to Zn. 2+ Reduced state of ions ( Figure 1 (f)

[0063] Furthermore, Zn@Gs-Re nanozymes exhibit strong scavenging abilities against both DPPH and ABTS free radicals, and these abilities are positively correlated with the concentration of Zn@Gs-Re nanozymes. Figure 1 As shown in Figure 1, when the concentration of Zn@Gs-Re nanozyme is 100 µg / mL, the ABTS clearance rate is approximately 38%; when the concentration of Zn@Gs-Re nanozyme is 100 µg / mL, the DPPH clearance rate is approximately 36%. Figure 1 As shown in jk (n=3).

[0064] Example 2: Cellular level functional verification Cell culture experiments were divided into the following groups: ① Normal control group (DMEM / F12 medium), ② High glucose model group (DMEM / F12 medium, 200 μg / mL AGEs), ③ Gs-Re group (DMEM / F12 medium, 100 μg / mL Gs-Re), ④ Zn@Gs-Re nanozyme group (DMEM / F12 medium, 100 μg / mL Zn@Gs-Re nanozyme). The S treatment group was first treated with an appropriate concentration of Gs-Re and Zn@Gs-Re nanozyme for 4 h, and then, together with the model group, was treated with an appropriate concentration of AGEs for 24 h.

[0065] 1. Intracellular ROS levels The level and dynamic changes of ROS were analyzed using 2',7'-dichlorodihydroxyfluorescein diacetate (DCFH-DA) staining. Results are as follows: Figure 2As shown, compared with the control group, the ROS level in SV40-MES-13 cells treated with AGEs was significantly increased (p<0.001). When SV40-MES-13 cells were pretreated with Gs-Re and Zn@Gs-Re nanozymes, the ROS level in the cells was significantly decreased compared with the model group (p<0.05, p<0.005), and the therapeutic effect of Zn@Gs-Re nanozymes was the most significant between the two treatment groups (p<0.05). These results indicate that Zn@Gs-Re nanozymes can effectively reduce the ROS level in SV40-MES-13 cells under high glucose conditions.

[0066] 2. Comparison of the expression of cytokines such as TNF-α, IL-6, and VEGF in cell supernatant See Figure 3 As shown in the figure, compared with the normal group, the expression of IL-6 (P < 0.001), TNF-α (P < 0.001), and VEGF (P < 0.001) in the cell supernatant of the model group was significantly increased after the addition of AGEs. Compared with the model group, the expression of IL-6 (P < 0.01; P < 0.001), TNF-α (P < 0.005; P < 0.001), and VEGF (P < 0.01; P < 0.001) in the cell supernatant of the Gs-Re group and the Zn@ Gs-Re nanozyme group was significantly decreased, and the differences between the two groups were statistically significant. This indicates that Zn@ Gs-Re nanozyme has the most significant therapeutic effect, demonstrating that Zn@ Gs-Re nanozyme can effectively regulate the level of cellular inflammatory response, thereby alleviating the pathological damage associated with DKD.

[0067] 3. Flow cytometry detection of apoptosis levels See Figure 4 Apoptosis was detected using a combined Annexin V and PI labeling strategy. Flow cytometry results showed that, compared with the control group, the apoptosis level of SV40-MES-13 cells treated with AGEs was significantly increased (p<0.001). When SV40-MES-13 cells were pretreated with Gs-Re and Zn@Gs-Re nanozymes, the apoptosis level was significantly decreased compared with the model group (p<0.05, p<0.01), but there was no statistically significant difference between the two treatment groups (p>0.05). These results indicate that Zn@Gs-Re nanozymes can effectively reduce the apoptosis level of SV40-MES-13 cells under high glucose conditions.

[0068] 4. TUNEL staining to detect apoptosis levels Cytotoxicity was assessed using TUNEL staining. After incubation with the Zn@Gs-Re nanozyme, the survival rate of SV40-MES-13 cells was higher than that of the AGEs control group (…). Figure 5 This indicates that Zn@Gs-Re nanozymes have a protective effect against AGEs-induced damage.

[0069] 5. JC-1 staining Under normal physiological conditions, mitochondria retain a high membrane potential. JC-1 enters the mitochondria as a polymer, forming clusters with red fluorescence. After damage, the mitochondrial membrane potential decreases, and JC-1 exists in monomeric form, emitting strong green fluorescence, indicating increased mitochondrial membrane permeability and apoptosis. See also Figure 6 In the AGEs-treated group, red and green fluorescence were significantly lower and higher than those of normal SV40-MES-13 cells, respectively. Both Zn@Gs-Re nanozymes and Gs-Re treatment significantly rescued the fluorescence changes. Quantitative results showed that the green / red fluorescence ratio was lower in the Zn@Gs-Re nanozyme group compared to the AGEs group, indicating that Zn@Gs-Re nanozymes have a protective effect on mitochondria. Furthermore, the protective effect of Zn@Gs-Re nanozymes on mitochondria was significantly greater than that of the same concentration of Gs-Re.

[0070] 6 MitoTracker Green staining By labeling with MitoTracker Green, live cells were identified and their mitochondrial status was assessed, with results as follows: Figure 7 As shown in the figure, fluorescence staining results revealed a significant reduction in MitoTracker staining (green fluorescence) in AGEs-treated cells compared to normal SV40-MES-13 cells. Furthermore, Zn@Gs-Re nanozyme treatment significantly inhibited AGEs-induced reduction in MitoTracker staining. Gs-Re treatment also inhibited AGEs-induced reduction in MitoTracker staining, but its inhibitory effect was less than that of Zn@Gs-Re nanozyme.

[0071] 7. Levels of MDA, SOD and GSH in cells.

[0072] Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) are important indicators for assessing cellular ferroptosis. SOD is a key antioxidant enzyme that protects cells and tissues by processing excess reactive oxygen species (ROS). GSH is a potent endogenous antioxidant essential for maintaining intracellular redox balance. Excessive ROS leads to enhanced cellular lipid peroxidation, increased MDA expression, further damaging cells and tissues, and exacerbating inflammatory responses. The expression levels of MDA, SOD, and GSH in cells were examined. Results showed that treatment of SV40-MES-13 cells with AGEs significantly increased MDA expression and significantly decreased SOD and GSH expression. Pretreatment with Zn@Gs-Re nanozymes and Gs-Re resulted in decreased MDA expression and increased SOD and GSH expression, with the Zn@Gs-Re nanozyme showing the most significant therapeutic effect. Figure 8 These data demonstrate that Zn@Gs-Re nanozymes protect mitochondrial structure and function from oxidative damage. They also prove that the novel Zn@Gs-Re nanozymes exhibit superior SOD enzyme activity compared to Gs-Re.

[0073] Example 3: In vivo functional verification This experiment used male C57BL / 6J mice (8 weeks old, SPF grade) housed at a temperature of 20-25℃ and humidity of 40%-70%, with cyclical lighting (12 h light / 12 h dark). In this study, the normal control group received a standard diet, while the DKD group was fed a high-fat diet for four weeks before the experiment. Subsequently, streptozotocin (STZ) was administered intraperitoneally at a dose of 55 mg / kg for five days. In contrast, the normal control group received an equal dose of citrate-sodium citrate buffer via intraperitoneal injection. Blood glucose levels were measured in the tail vein of the mice within 72 hours after the last injection. A random blood glucose level exceeding 16.7 mmol / L indicated successful establishment of the diabetic model. Two weeks after injection, a significant increase in urinary protein indicated successful establishment of the DKD model. Successfully modeled C57BL / 6J mice were divided into a model group, a Gs-Re group, and a Zn@Gs-Re nanozyme group. Mice in the Gs-Re group were administered Gs-Re ultrafine powder solution (20 mg / kg) via intraperitoneal injection, mice in the Zn@Gs-Re nanozyme group were administered Zn@Gs-Re nanozyme solution (20 mg / kg) via intraperitoneal injection, and mice in the model group were administered an equal volume of PBS via intraperitoneal injection. Administered every two days for 7 weeks. Mice had free access to food and water during the experiment, and their body weight was measured weekly.

[0074] 1. Comparison of fasting blood glucose, urinary protein, and serum creatinine among the groups of mice The body weight of the model group mice showed a significant downward trend in the first two weeks. Treatment with Gs-Re and Zn@Gs-Re nanozymes alleviated symptoms compared to the model group. Treatment with Zn@Gs-Re nanozymes resulted in the most significant symptom reduction. Figure 9 (A)

[0075] (1) Blood glucose: Compared with the normal group, the fasting blood glucose of the model group mice was significantly increased, with a statistically significant difference (P < 0.001). Compared with the model group mice, the fasting blood glucose of the Gs-Re group (P < 0.01) and the Zn@Gs-Re nanozyme group (P < 0.001) was significantly reduced. Moreover, the therapeutic effect of Zn@Gs-Re nanozyme was the most significant between the two treatment groups (p < 0.05). See [link to relevant documentation]. Figure 9 Based on this, it can be concluded that the Zn@Gs-Re nanozyme has a significant hypoglycemic effect.

[0076] (2) Urinary protein: Compared with the normal group, the urinary protein level in the model group mice was significantly increased, with a statistically significant difference (P < 0.001). Compared with the model group mice, urinary protein was significantly reduced in the Gs-Re group (P < 0.01) and the Zn@Gs-Re nanozyme group (P < 0.001). Furthermore, the therapeutic effect of Zn@Gs-Re nanozyme was the most significant between the two treatment groups (p < 0.05). See [link to relevant documentation]. Figure 9 Based on this, it can be determined that Zn@Gs-Re nanozymes have a significant effect in reducing proteinuria.

[0077] (3) Serum creatinine: Compared with the normal group, the serum creatinine of mice in the model group was significantly increased, with a statistically significant difference (P < 0.001). Compared with the model group, serum creatinine was significantly decreased in the Gs-Re group (P > 0.05) and the Zn@Gs-Re nanozyme group (P < 0.01). Furthermore, the therapeutic effect of Zn@Gs-Re nanozyme was the most significant between the two treatment groups (p < 0.05). See [link to relevant documentation]. Figure 9 Based on this, it can be concluded that Zn@Gs-Re nanozymes can effectively protect kidney function.

[0078] 2. Comparison of histopathological changes in the kidney tissues of mice in different groups To evaluate the pathological effects of Zn@Gs-Re nanozyme on the kidney tissue of DKD mice, we used HE, PAS, and Masson staining to assess the pathological changes in the kidneys from different perspectives. Each group was marked with a red arrow to highlight the kidney structure. In the model group, the kidney tissue of mice showed glomerular hypertrophy, thickening of the glomerular or tubular basement membrane, mesangial proliferation, increased mesangial matrix, tubular vacuolar degeneration, brush border shedding of tubular epithelial cells, protein casts, and interstitial fibrosis. After Gs-Re treatment, tubular vacuolar degeneration and interstitial fibrosis were still observed in the kidney pathology. After Zn@Gs-Re nanozyme treatment, a small amount of tubular vacuolar degeneration and interstitial fibrosis were observed in the kidney pathology, but no tubular protein deposition was observed. Figure 10 These results highlight the effectiveness and superiority of using Zn@Gs-Re nanozymes to treat DKD.

[0079] 3. Biosafety assessment To investigate the biosafety of Zn@Gs-Re nanozymes, we collected major organ and blood samples from mice that had been intraperitoneally injected with PBS, Gs-Re (20 mg / kg), and Zn@Gs-Re nanozymes (20 mg / kg) at different time points. Pathological analysis and routine blood and biochemical tests were then performed on these samples. Figure 11 Hematological analysis included blood biochemistry and complete blood count (CBC). Blood biochemistry included eight liver function indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total protein (TP), serum globulin (GLO), serum bilirubin (TBIL), albumin / globulin ratio (A / G), and AST / ALT; and three kidney function indicators: blood urea nitrogen (BUN) / cretin (CRE), blood urea nitrogen (BUN), and creatinine (CRE). CBC analysis included white blood cell count (WBC), red blood cell count (RBC), neutrophil count (Neu), and lymphocyte count (Lym). The results showed that, compared with the PBS injection group, after intraperitoneal injection of Gs-Re (20 mg / kg) and Zn@Gs-Re nanozyme (20 mg / kg), there were no statistically significant differences in blood biochemistry indicators and CBC results between the mice and the control group (PBS group), indicating that Zn@Gs-Re nanozyme did not adversely affect liver and kidney function or the hematologic system in mice. Furthermore, no significant histological changes were observed in any of the mouse's organs, and no inflammation was observed. Figure 12 The above results indicate that the Zn@Gs-Re nanozyme has good biosafety.

[0080] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Application of zinc-ginsenoside coordination nanozyme in the preparation of drugs for the treatment of diabetic nephropathy.

2. According to claim 1, the indications for the drug also include the treatment of diabetic complications.

3. The application according to claim 2, wherein the diabetic complication is selected from diabetic cardiomyopathy, diabetic retinopathy, and diabetic neuropathy.

4. The application according to any one of claims 1 to 3, wherein the efficacy of the drug covers at least one of the following: It lowers blood glucose levels, reduces urinary protein levels, lowers serum creatinine levels, and improves renal tissue pathological damage.

5. According to claim 1, the zinc-ginsenoside coordination nanozyme can be used alone or in combination with other drugs.

6. The application according to claim 1, wherein the zinc-ginsenoside coordination nanozyme and pharmaceutically acceptable excipients constitute a pharmaceutical composition.

7. The application according to claim 6, wherein the form of the pharmaceutical composition is selected from one or more of the following: solution, injection, spray, nasal drops, aerosol, powder, tablet, capsule, and granule.

8. The application according to any one of claims 1 to 3, 5 to 7, wherein the zinc-ginsenoside coordination nanozyme is prepared by the following method: mixing zinc salt, ginsenoside Re and a stabilizer in an organic solvent, stirring and dialysis for purification.

9. The application according to any one of claims 1 to 3, 5 to 7, wherein the subject of the drug is a mammal.

10. The application according to claim 9, wherein the subject of the drug is a human.

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