Use of neomycin in the preparation of a medicament for the treatment of acute kidney injury

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of neomycin in the preparation of drugs for treating acute kidney injury. Through extensive experimental research, this invention has discovered that pre-nebulized inhaled neomycin can effectively reduce the degree of kidney damage in mice during ischemia-reperfusion through a specific mechanism. This provides new ideas and methods for the prevention and treatment of acute kidney injury caused by ischemia-reperfusion, thus offering direction for the development of drugs to treat acute kidney injury caused by ischemia-reperfusion and potentially overcoming the shortcomings of existing technologies in this field.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of neomycin in the preparation of a drug for treating acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a rapidly progressing form of kidney failure with high morbidity and mortality, becoming an increasingly serious global health problem. Ischemia-reperfusion injury (IRI) is one of the leading causes of AKI, commonly occurring in clinical situations such as kidney transplantation, cardiovascular surgery, cardiopulmonary bypass, and severe limb trauma. In kidney transplantation, renal ischemia is unavoidable during the retrieval and transplantation process, and subsequent reperfusion triggers inflammatory responses and oxidative stress, leading to kidney damage. Cardiovascular surgery and cardiopulmonary bypass can also cause renal ischemia-reperfusion injury due to hemodynamic changes and microthrombus formation. Studies have shown that poor renal repair after AKI can lead to chronic inflammation, ultimately resulting in serious consequences such as chronic kidney disease, renal fibrosis, and end-stage renal disease. However, current clinical treatments for AKI are limited, and alleviating renal inflammation after AKI has become the most important challenge in protecting kidney function after trauma, infection, and major surgery. Therefore, exploring the molecular mechanisms of AKI development and finding effective therapeutic drugs and strategies for AKI are urgent clinical needs.

[0003] Currently, there is a lack of specific and effective treatments for acute kidney injury (AKI) caused by ischemia-reperfusion injury in clinical practice. Treatment mainly focuses on the following aspects: ① Drug therapy: While existing drugs can scavenge free radicals and inhibit inflammatory responses to some extent, their effects are limited and they have side effects, making it difficult to fundamentally solve the kidney damage problem. Antioxidants can scavenge some free radicals, but they cannot effectively inhibit inflammatory responses and cell damage in the kidneys; anti-inflammatory drugs, while inhibiting inflammatory responses, may interfere with normal kidney physiological functions. Furthermore, administration is often invasive, such as intravenous injection. ② Renal replacement therapy: Although it can temporarily replace kidney function, it suffers from high treatment costs, limited resources, and the inability to directly repair damaged kidney tissue. ③ Preventive measures: Existing preventive measures cannot completely avoid AKI. For example, patients are given protective agents before surgery, but the timing, dosage, and effectiveness of their use are uncertain, and the efficacy varies greatly among individual patients. In high-risk surgeries such as kidney transplantation, some measures are taken intraoperatively to reduce ischemia-reperfusion injury, but the incidence of postoperative AKI remains high, and it requires a high level of surgical skill from the surgeon.

[0004] Furthermore, the pathogenesis of AKI is not fully understood with current technology, which to some extent limits the development of effective treatments. Although it is clear that ischemia-reperfusion injury involves multiple mechanisms such as oxidative stress, inflammatory response, and apoptosis, the interactions and specific details between these mechanisms remain unclear, posing challenges to the development of targeted treatments. Summary of the Invention

[0005] To address the problems in the prior art, the present invention provides the use of neomycin in the preparation of medicaments for the prevention and / or treatment of acute kidney injury, thereby enabling effective prevention and / or treatment of acute kidney injury caused by ischemia-reperfusion.

[0006] Therefore, the present invention provides the following technical solution.

[0007] The first aspect of the present invention provides the use of neomycin in the preparation of a medicament for the prevention and / or treatment of acute kidney injury.

[0008] In a preferred embodiment of the present invention, the acute kidney injury is an acute kidney injury caused by ischemia-reperfusion.

[0009] In a preferred embodiment of the present invention, the acute kidney injury caused by ischemia-reperfusion is caused by kidney transplantation, cardiovascular surgery, extracorporeal circulation or severe trauma.

[0010] In a preferred embodiment of the present invention, the route of administration of the drug is nebulized inhalation.

[0011] In a preferred embodiment of the present invention, the nebulized inhalation is performed through the mouth and nose, allowing the drug to act directly on the respiratory tract and lung microenvironment.

[0012] In a preferred embodiment of the present invention, the drug exerts its effect of preventing and / or treating acute kidney injury caused by ischemia-reperfusion by regulating the lung microenvironment.

[0013] In a preferred embodiment of the present invention, the regulation of the lung microenvironment includes regulating the lung flora, lung immune cells, or lung inflammatory mediators.

[0014] In a preferred embodiment of the invention, the drug is used to prevent and / or treat acute kidney injury caused by ischemia-reperfusion injury, while alleviating acute lung injury secondary to acute kidney injury.

[0015] In a preferred embodiment of the invention, the drug is used to achieve one or more biological effects selected from the group consisting of: (a) Reduce serum creatinine levels after acute kidney injury; (b) Reduce serum urea nitrogen levels after acute kidney injury; (c) Reduces the transcriptional level of kidney injury molecule-1 in kidney tissue; (d) Reduce the transcriptional level of neutrophil gelatinase-associated lipotransferase in kidney tissue; (e) Inhibits the expression of pro-inflammatory cytokines in kidney tissue; (f) Reduce swelling, vacuolar degeneration, or brush border shedding of renal tubular epithelial cells; (g) Reduce renal tubular injury score; (h) Reduce alveolar septal thickening, interstitial edema, or inflammatory cell infiltration.

[0016] In a preferred embodiment of the present invention, the pro-inflammatory cytokines are selected from one or more of tumor necrosis factor-α, interleukin-1β, and interleukin-6.

[0017] In a preferred embodiment of the present invention, the drug is a drug containing neomycin as the sole active ingredient.

[0018] In a preferred embodiment of the present invention, the drug is a combination drug comprising neomycin and other drugs for the prevention and / or treatment of acute kidney injury as active ingredients.

[0019] In a preferred embodiment of the present invention, the dosage of neomycin is 0-5 mg / kg body weight per day.

[0020] In a preferred embodiment of the invention, the neomycin is administered at a dose of 1-2 mg / kg body weight per day.

[0021] In a preferred embodiment of the invention, the drug has no significant nephrotoxic effect at the administered dose.

[0022] In a preferred embodiment of the present invention, the absence of significant toxic effects is manifested by no significant difference in serum creatinine levels and serum urea nitrogen levels compared with normal controls, and / or no obvious pathological changes observed in renal histology.

[0023] A second aspect of the invention provides a pharmaceutical composition for the prevention and / or treatment of acute kidney injury, comprising an effective amount of neomycin and a pharmaceutically acceptable carrier or excipient.

[0024] In a preferred embodiment of the present invention, the acute kidney injury is an acute kidney injury caused by ischemia-reperfusion.

[0025] In a preferred embodiment of the present invention, the acute kidney injury caused by ischemia-reperfusion is caused by kidney transplantation, cardiovascular surgery, extracorporeal circulation or severe trauma.

[0026] In a preferred embodiment of the present invention, the dosage form of the pharmaceutical composition is a nebulized inhaler.

[0027] In a preferred embodiment of the present invention, the nebulized inhalant is a solution-type, suspension-type, or powder-type nebulized inhalant.

[0028] In a preferred embodiment of the present invention, the pharmaceutical composition is in unit dose form, each unit dose containing 0.1-50 mg of neomycin.

[0029] In a preferred embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, excipient, or excipient, wherein the excipient is selected from one or more of osmotic pressure regulators, pH regulators, stabilizers, and preservatives.

[0030] In a preferred embodiment of the invention, the pharmaceutical composition further comprises other drugs for the prevention and / or treatment of acute kidney injury as active ingredients.

[0031] A third aspect of the invention provides a method for preventing and / or treating acute kidney injury induced by ischemia-reperfusion, the method comprising administering an effective amount of neomycin to a subject in need.

[0032] In a preferred embodiment of the present invention, the acute kidney injury caused by ischemia-reperfusion is caused by kidney transplantation, cardiovascular surgery, extracorporeal circulation or severe trauma.

[0033] In a preferred embodiment of the invention, the application is performed by nebulized inhalation.

[0034] In a preferred embodiment of the present invention, the nebulized inhalation is performed through the mouth and nose, allowing neomycin to act directly on the respiratory tract and lung microenvironment.

[0035] In a preferred embodiment of the invention, the method further includes simultaneously alleviating acute lung injury secondary to acute kidney injury.

[0036] In a preferred embodiment of the present invention, the method achieves one or more biological effects selected from the group consisting of: (a) Reduce serum creatinine levels in subjects; (b) Reduce serum urea nitrogen levels in subjects; (c) Reduced the transcriptional level of kidney injury molecule-1 in the kidney tissue of the subjects; (d) Reduced the transcriptional level of neutrophil gelatinase-associated lipotransferase in the kidney tissue of the subjects; (e) Inhibit the expression of pro-inflammatory cytokines in the kidney tissue of the subjects; (f) Reduce renal tubular injury in subjects; (g) Reduce acute lung injury secondary to acute kidney injury in subjects.

[0037] In a preferred embodiment of the present invention, the pro-inflammatory cytokines are selected from one or more of tumor necrosis factor-α, interleukin-1β, and interleukin-6.

[0038] In a preferred embodiment of the present invention, the dosage of neomycin is 0.1-5 mg / kg body weight per day.

[0039] In a preferred embodiment of the invention, the neomycin is administered at a dose of 1-2 mg / kg body weight per day.

[0040] In a preferred embodiment of the present invention, the subject is a mammal, preferably a human.

[0041] A fourth aspect of the invention provides the use of neomycin in the preparation of a medicament for alleviating acute lung injury secondary to ischemia-reperfusion-induced acute kidney injury.

[0042] In a preferred embodiment of the present invention, the route of administration of the drug is nebulized inhalation.

[0043] A fifth aspect of the present invention provides a nebulized inhaler containing an effective amount of neomycin for the prevention and / or treatment of acute kidney injury.

[0044] In a preferred embodiment of the present invention, the acute kidney injury is an acute kidney injury caused by ischemia-reperfusion.

[0045] In a preferred embodiment of the present invention, the concentration of neomycin in the nebulized inhalant is 0.1-5 mg / mL.

[0046] In a preferred embodiment of the present invention, the concentration of neomycin in the nebulized inhalant is 0.5-2 mg / mL.

[0047] A sixth aspect of the present invention provides a kit comprising: (a) A pharmaceutical composition containing neomycin; (b) Nebulizer inhalation device; (c) Instructions for use that instruct the user to use the drug composition by nebulization to prevent and / or treat acute kidney injury caused by ischemia-reperfusion.

[0048] This invention proposes an innovative treatment strategy to alleviate acute kidney injury induced by ischemia-reperfusion in mice through pre-nebulized inhalation of neomycin. Neomycin, a commonly used antibiotic, has wide applications in antibacterial therapy, but its potential in preventing and treating renal ischemia-reperfusion injury has not been fully explored and utilized. Through extensive experimental research, this invention has discovered that pre-nebulized inhalation of neomycin can effectively reduce the degree of kidney damage in mice during ischemia-reperfusion through a specific mechanism, providing a new approach and method for the prevention and treatment of acute kidney injury induced by ischemia-reperfusion, and is expected to overcome the shortcomings of existing technologies in this field. Specifically, this invention has the following beneficial effects: (1) Innovative drug administration method: The non-invasive drug administration method of nebulization inhalation is adopted. Compared with invasive methods such as intravenous injection, the patient compliance is higher, the operation is simpler, and the damage to the patient's body is less.

[0049] (2) Strong targeting: Nebulized inhalation can precisely regulate the respiratory tract and lung microenvironment, avoid the interference of drugs on the microenvironment of other organs, and reduce the risk of complications caused by the imbalance of the microenvironment of other organs.

[0050] (3) Significant therapeutic effect: Animal experiments have shown that pre-nebulized neomycin can significantly reduce serum creatinine and blood urea nitrogen levels in IRI mice, downregulate the expression of kidney injury markers and pro-inflammatory factors, and alleviate pathological damage to the renal tubules and lungs. The effect is better than other administration methods such as gavage and intravenous injection.

[0051] (4) High safety: It was verified that at the nebulization dose used in this invention, neomycin had no significant effect on the renal function of mice, and no obvious pathological changes were observed in the renal histology, indicating good biosafety.

[0052] (5) Low cost and easy to implement: Compared with renal replacement therapy, the treatment plan of the present invention is low cost, the nebulization inhalation operation is simple, and it is easy to promote in clinical practice.

[0053] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0054] Figure 1The changes in renal function indicators (CRE, BUN) and histopathological scores after nebulization with different antibiotics are shown. Among them, A is a schematic diagram of the treatment process of mouse models in different antibiotic administration groups; BC is the assessment of plasma CRE and BUN in each group after renal IRI; DE is the expression of Lcn2 and Kim-1 in each group; FH is the qRT-PCR results of TNF-α, IL-1β and IL-6 in different treatment groups; I is a representative image (scale bar of 50 μm) of H&E stained sections of the kidneys in each group and the renal tissue damage score; J is a representative image (scale bar of 100 μm) of H&E stained sections of the lungs in each group and the measurement of alveolar wall thickness.

[0055] Figure 2 The changes in renal function indicators (CRE, BUN) and histopathological scores under different neomycin administration routes are shown. Among them, A is a schematic diagram of the treatment process of mouse models in different neomycin administration groups; BC is the assessment of plasma CRE and BUN in each group after IRI; DE is the expression of Lcn2 and Kim-1 in the kidneys of each group; FH is the qRT-PCR results of TNF-α, IL-1β and IL-6 in different treatment groups; I is a representative image of kidney H&E stained sections (scale bar is 50 μm) and renal tissue damage score; J is a representative image of lung H&E stained sections (scale bar is 100 μm) and alveolar wall thickness measurement.

[0056] Figure 3 The nephrotoxicity assessment of different concentrations of neomycin nebulization is shown; where A represents the CRE and BUN detection in different groups of plasma; and C represents representative images (scale bar 50 μm) of H&E stained sections and renal tissue damage scores. Detailed Implementation

[0057] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0058] Example 1: Detection of the therapeutic effect of different antibiotics administered via nebulization on AKI To evaluate the effects of antibiotics on acute kidney injury (IRI), this study selectively intervened in the lung microenvironment of mice using different antibiotic nebulization strategies. Mice were treated with ampicillin (A), vancomycin (V), metronidazole (M), neomycin (N), or a quadruple antibiotic (Abx) via nebulization for 7 days, followed by IRI modeling. The specific steps included: 1. Construction of a mouse model of acute kidney injury (AKI) After at least 7 days of acclimatization, 6-8 week old male C57BL / 6J mice (purchased from Huachuang Xinno Pharmaceutical Technology Co., Ltd., animal license number SYXK2021(Su)-0007) were anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution and placed on a 37°C heated surgical board. A 1.5-2 cm incision was made along the midline of the abdomen using surgical scissors, and the bilateral renal vessels were carefully dissected under a microscope. The bilateral renal pedicles were clamped with non-traumatic microvascular clamps for 25 minutes; the renal pedicles changed from bright red to dark purple, indicating successful ischemia. After the clamps were removed, the kidneys returned to bright red, confirming reperfusion. After confirming no bleeding or organ damage, the abdomen was closed, the kidneys were gently repositioned, and the incision was sutured in layers to obtain the AKI model mouse.

[0059] Sham-operated group: The difference between this group of mice and the AKI model is that the mice only exposed the renal blood vessels without clamping them.

[0060] After the surgery, the mice in each group were kept warm at room temperature and their survival was monitored.

[0061] 2. Experimental treatment Experimental plan as follows Figure 1 As shown in Figure A. First, the model mice were divided into the following groups: AKI group, neomycin group (AKI + N group), metronidazole group (AKI + M group), ampicillin group (AKI + A group), vancomycin group (AKI + V group), and quadruple antibiotic group (AKI + Abx group), with 8 mice in each group. A sham-operated group (Sham group), consisting of 8 mice, served as the control group. Then, the mice in each group were treated as follows: Neomycin group (AKI + N group): Male C57BL / 6J mice were nebulized for 7 days before reperfusion, with 50 μL of 1 ~ 2 mg / kg neomycin solution (solvent is physiological saline) nebulized daily, followed by ischemia-reperfusion for 48 h (i.e., according to the AKI construction procedure). Metronidazole group (AKI + M group): Male C57BL / 6J mice were nebulized for 7 days before reperfusion, with 50 μL of 1 ~ 2 mg / kg metronidazole solution (solvent is physiological saline) nebulized daily, followed by ischemia-reperfusion for 48 h (i.e., according to the AKI construction procedure). Ampicillin group (AKI + A group): Male C57BL / 6J mice were nebulized for 7 days before reperfusion, with 50 μL of 1 ~ 2 mg / kg ampicillin solution (solvent is physiological saline) nebulized daily, followed by ischemia-reperfusion for 48 h (i.e., according to the AKI construction procedure). Vancomycin group (AKI + V group): Male C57BL / 6J mice were nebulized for 7 days before reperfusion, with 50 μL of 1 ~ 2 mg / kg vancomycin solution (solvent is physiological saline) nebulized daily, followed by ischemia-reperfusion for 48 h (i.e., according to the AKI construction procedure). The quadruple antibiotic group (AKI + Abx group): Male C57BL / 6J mice were nebulized for 7 days before reperfusion, and 50 μL of 1 ~ 2 mg / kg quadruple antibiotic (neomycin + metronidazole + ampicillin + vancomycin mixed in equal doses) solution (solvent is physiological saline) was nebulized daily, followed by ischemia-reperfusion for 48 h (i.e., according to the AKI construction procedure); Sham group: Male C57BL / 6J mice were nebulized for 7 days before reperfusion, receiving 50 μL of physiological saline daily, followed by ischemia-reperfusion for 48 hours (i.e., following the constructed sham surgery procedure). AKI group: Male C57BL / 6J mice were nebulized for 7 days before reperfusion, receiving 50 μL of physiological saline daily, followed by ischemia-reperfusion for 48 hours (i.e., following the AKI construction procedure).

[0062] After completing the above procedures, the mice in each group were sacrificed for tissue sampling and evaluation of renal function and histology.

[0063] 3. Evaluation of renal function and histology 3.1 Serum biochemical indicators Blood was collected from the retroorbital venous plexus, allowed to stand for 30 min, and then centrifuged at 3500 rpm for 10 min to collect the supernatant. Serum creatinine (CRE) and blood urea nitrogen (BUN) levels were measured using the creatine oxidase method and the urease method, respectively. Results are shown below. Figure 1 B-1C.

[0064] like Figure 1 As shown in B-1C, CRE (B) and BUN (C) in the AKI group increased to 6-10 times that of the sham surgery group (Sham group); compared with the AKI group, the neomycin nebulization group (AKI + N group) had the most significant effect, namely: CRE decreased by 53-63% and BUN decreased by 45-61% (P<0.05), indicating a significant reduction in renal function damage.

[0065] 3.2 Molecular expression of kidney injury Renal cortex was harvested, and total RNA was extracted using the Trizol method. After reverse transcription, the mRNA levels of neutrophil gelatinase-associated lipid transporter 2 (Lcn2) and kidney injury molecule-1 (Kim-1) were detected by SYBR Green qRT-PCR. β-actin was used as an internal control, and the relative expression levels were calculated using the 2^(-ΔΔCt) method. Results are shown below. Figure 1D-1E.

[0066] like Figure 1 As shown in D-1E, compared with the Sham group, the levels of Kim-1 (D) and Lcn2 (E) in the AKI group increased by 88 times and 151 times, respectively. Compared with the AKI group, the AKI + N group showed the most significant effect, with Kim-1 (D) decreasing from 88 times that of AKI to 30 times and Lcn2 (E) decreasing from 151 times that of AKI to 60 times (P<0.01).

[0067] 3.3 Detection of inflammatory signaling pathways The method was the same as step 2.2 above, detecting the mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), respectively. Results are shown below. Figure 1 FH.

[0068] like Figure 1 As shown in FH, compared with the Sham group, the AKI group showed significantly increased levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) mRNA. Compared with the AKI group, the AKI + N group showed the most significant effect, with TNF-α (F), IL-1β (G), and IL-6 (H) transcription levels decreasing by 66%, 57%, and 42%, respectively (P<0.05).

[0069] 3.4 Histopathology The left kidney was harvested, fixed with 4% (w / v) paraformaldehyde for 24 h, embedded in paraffin, and sectioned to a thickness of 5 μm. Hematoxylin and eosin (HE) staining was performed. The Paller score was used for blinded scoring: 0 points for no damage, and 7 points for the most severe. Results are shown below. Figure 1 I-1J.

[0070] like Figure 1 As shown in I-1J, the AKI group score was 5.9±0.3, with visible swelling of renal tubular epithelial cells, brush border detachment, and cast formation. In contrast, the AKI + N group score decreased to 2.2±0.1 (P<0.001) (I). H&E staining of the lungs showed that, except for the AKI + N group, all other antibiotic groups treated with IRI exhibited alveolar septal thickening, interstitial edema, and inflammatory cell infiltration, suggesting that the N group could alleviate acute lung injury secondary to AKI (J).

[0071] The above results indicate that nasal drops of cytosine can significantly alleviate IRI-induced renal dysfunction, inhibit renal inflammatory response, and reduce renal and secondary lung damage.

[0072] Example 2: Detection of the therapeutic effect of neomycin on AKI by different administration routes To rule out the possibility that the therapeutic effect of neomycin might be caused by some neomycin spilling from the airway into the gastrointestinal tract or being absorbed into the bloodstream and altering the microenvironment of other organs, this embodiment sets up a neomycin nebulization inhalation group (AKI + N), a neomycin gavage group (AKI + N (ig)), and a neomycin tail vein injection group (AKI + N (iv)) to compare the differences in relieving AKI among the three administration methods. The specific steps include: 1. Construction of AKI model mice The steps for constructing the mouse kidney AKI model are the same as in Example 1, specifically: After at least 7 days of acclimatization, 6-8 week old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution and placed on a 37°C heated surgical board. A 1.5-2 cm incision was made along the midline of the abdomen using surgical scissors, and the bilateral renal vessels were carefully dissected under a microscope. The bilateral renal pedicles were clamped with non-traumatic microvascular forceps for 25 minutes; the renal pedicles changed from bright red to dark purple, indicating successful ischemia. After the clamps were removed, the kidneys returned to bright red, confirming reperfusion. After confirming no bleeding or organ damage, the abdomen was closed, the kidneys were gently repositioned, and the incision was sutured in layers, resulting in the AKI model mouse.

[0073] Sham-operated group: The difference between this group of mice and the AKI model is that the mice only exposed the renal blood vessels without clamping them.

[0074] After the surgery, the mice in each group were kept warm at room temperature and their survival was monitored.

[0075] 2. Experimental treatment Experimental plan as follows Figure 2 As shown in Figure A. First, the model mice were divided into the following groups: AKI group, nebulization group (N group), gavage group (N(ig) group), and tail vein injection group (N(iv) group), with 8 mice in each group. A sham-operated group (Sham group), consisting of 8 mice, served as the control group. Then, the mice in each group were treated as follows: Nebulization group (N group): Male C57BL / 6J mice were nebulized for 7 days before reperfusion, with 50 μL of 1-2 mg / kg neomycin solution (solvent is physiological saline) nebulized daily, followed by ischemia-reperfusion for 48 h (i.e., according to the AKI construction procedure). Gavage group (N(ig) group): Male C57BL / 6J mice were gavaged for 7 days before reperfusion, and 50 μL of 1 ~ 2 mg / kg neomycin solution (solvent is physiological saline) was administered daily by gavage, followed by ischemia-reperfusion for 48 h (i.e., according to the AKI construction procedure). Tail vein injection group (N(iv) group): Male C57BL / 6J mice were treated with tail vein injection for 7 days before reperfusion, and 50 μL of 1 ~ 2 mg / kg neomycin solution (solvent is physiological saline) was injected into the tail vein daily, followed by ischemia-reperfusion for 48 h (i.e., according to the AKI construction procedure). Sham group: Male C57BL / 6J mice were nebulized for 7 days before reperfusion, receiving 50 μL of physiological saline daily, followed by ischemia-reperfusion for 48 hours (i.e., following the constructed sham surgery procedure). AKI group: Male C57BL / 6J mice were nebulized for 7 days before reperfusion, receiving 50 μL of physiological saline daily, followed by ischemia-reperfusion for 48 hours (i.e., following the AKI construction procedure).

[0076] After completing the above procedures, the mice in each group were sacrificed for tissue sampling and evaluation of renal function and histology.

[0077] 3. Evaluation of renal function and histology 3.1 Serum biochemical indicators The detection method was the same as in Example 1: blood was collected from the retroorbital venous plexus, allowed to stand for 30 minutes, and then centrifuged at 3500 rpm for 10 minutes to collect the supernatant. Serum creatinine (CRE) and blood urea nitrogen (BUN) levels were measured using the creatine oxidase method and the urease method, respectively. Results are shown below. Figure 2 B-2C.

[0078] like Figure 2 As shown in B-2C, CRE (B) and BUN (C) in the I / R model group (AKI group) were 1.3-4 times higher than those in the sham operation group (Sham group); compared with the AKI group, CRE in the neomycin nebulization group (N group) decreased by 40-80% and BUN decreased by 36-57% (P<0.01), while no significant differences were found in CRE and BUN in the other administration groups (gavage group and tail vein injection group).

[0079] 3.2 Molecular expression of kidney injury Renal cortex was harvested, and total RNA was extracted using the Trizol method. After reverse transcription, the mRNA levels of neutrophil gelatinase-associated lipid transporter 2 (Lcn2) and kidney injury molecule-1 (Kim-1) were detected by SYBR Green qRT-PCR. β-actin was used as an internal control, and the relative expression levels were calculated using the 2^(-ΔΔCt) method. Results are shown below. Figure 2 D-2E.

[0080] like Figure 2As shown in D-2E, compared with the Sham group, Kim-1 (D) and Lcn2 (E) in the AKI group increased by 56 times and 470 times, respectively. Compared with the AKI group, the neomycin nebulization group showed the most significant effect, with Kim-1 (D) decreasing from 56 times that of AKI to 18 times and Lcn2 (E) decreasing from 470 times that of AKI to 60 times (P<0.01). No significant differences were observed in the other administration groups (gavage group and tail vein injection group).

[0081] 3.3 Detection of inflammatory signaling pathways The method was the same as step 3.2 above, detecting the mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), respectively. Results are shown below. Figure 2 FH.

[0082] like Figure 2 As shown in FH, compared with the Sham group, the AKI group showed significantly increased levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) mRNA. Compared with the AKI group, the AKI + N group showed the most significant effect, with TNF-α (F), IL-1β (G), and IL-6 (H) transcription levels decreasing by 68%, 58%, and 43%, respectively (P < 0.01).

[0083] 3.4 Histopathology The left kidney was harvested, fixed with 4% (w / v) paraformaldehyde for 24 h, embedded in paraffin, and sectioned to a thickness of 5 μm. Hematoxylin and eosin (HE) staining was performed. The Paller score was used for blinded scoring: 0 points for no damage, and 7 points for the most severe. Results are shown below. Figure 2 I-2J.

[0084] like Figure 2 As shown in I-2J, the AKI group score was 5.8±0.2, with visible renal tubular epithelial cell swelling, brush border detachment, and cast formation. The neomycin nebulization group (N group) score decreased to 2.2±0.1 (P<0.001), while other administration methods did not show significant improvement (I). H&E staining of the lungs showed that, compared with the AKI group, gavage group, and tail vein treatment group, the renal tubules exhibited extensive dilation, vacuolar degeneration, and brush border detachment. The neomycin nebulization group showed significant relief of the above lesions and a decrease in the renal tubular necrosis score. At the same time, all groups after IRI treatment, except for the neomycin nebulization group, showed interstitial space dilation, increased cell density, and interstitial edema, accompanied by significant alveolar wall thickening, suggesting that neomycin nebulization can alleviate severe lung injury and inflammation secondary to AKI (J).

[0085] The above results indicate that neomycin exerts its protective effect on the kidneys and lungs after IRI by directly regulating the lung microenvironment rather than through systemic absorption.

[0086] Example 3: Nephrotoxicity assessment of neomycin nebulization dose Given the potential nephrotoxicity of neomycin at high doses, this example further evaluated the effects of intranasal instillation of different concentrations of neomycin (0, 0.125, 0.25, 0.5, 1 mg / mL) for 7 days on renal function in mice. The specific steps included: 1. Establishment of mouse models of neomycin nebulization at different concentrations After at least 7 days of acclimatization, 6-8 week old male C57BL / 6J mice were nebulized for 7 consecutive days with different concentrations of neomycin, administered daily at 0-2 mg / kg. The control group received an equal volume of physiological saline. All mice were sacrificed at the end of the 7-day nebulization period for renal function and histological evaluation.

[0087] 2. Evaluation of renal function and histology 2.1 Serum biochemical indicators Blood was collected from the retroorbital venous plexus, allowed to stand for 30 minutes, and then centrifuged at 3500 rpm for 10 minutes to collect the supernatant. Serum creatinine (CRE) and blood urea nitrogen (BUN) levels were measured using the creatine oxidase method and the urease method, respectively. Results are shown below. Figure 3 A-3B.

[0088] like Figure 3 As shown in A-3B, no significant differences were observed in CRE (A) and BUN (B) among the 0~2 mg / kg neomycin nebulization groups.

[0089] 2.2 Histopathology The left kidney was harvested, fixed with 4% (w / v) paraformaldehyde for 24 h, embedded in paraffin, and sectioned to a thickness of 5 μm. Hematoxylin and eosin (HE) staining was performed. The Paller score was used for blinded scoring: 0 points for no damage, and 7 points for the most severe. Results are shown below. Figure 3 C.

[0090] like Figure 3 As shown in Figure C, no significant pathological changes were observed in the kidney histology of the 0–2 mg / kg neomycin nebulization group.

[0091] The above results indicate that the neomycin dosage and administration route used in this invention have good renal safety, and neomycin can alleviate IRI-induced kidney damage by nebulization at a renal-safe dose.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The use of neomycin in the preparation of drugs for the prevention and / or treatment of acute kidney injury.

2. The application according to claim 1, characterized in that, The acute kidney injury mentioned refers to acute kidney injury caused by ischemia-reperfusion.

3. The application according to claim 1, characterized in that, The drug is administered via nebulized inhalation.

4. The application according to any one of claims 1-3, characterized in that, The drug is a drug containing neomycin as its sole active ingredient.

5. The application according to any one of claims 1-3, characterized in that, The drug is a combination drug containing neomycin and other drugs for the prevention and / or treatment of acute kidney injury as active ingredients.

6. The application according to any one of claims 1-3, characterized in that, The dosage of the neomycin is 0.1-5 mg / kg body weight per day.

7. A pharmaceutical composition for the prevention and / or treatment of acute kidney injury, characterized in that, It contains an effective amount of neomycin and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that, The acute kidney injury mentioned refers to acute kidney injury caused by ischemia-reperfusion.

9. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is a nebulized inhaler.

10. The pharmaceutical composition according to claim 7, characterized in that, The nebulized inhalant is a solution-type, suspension-type, or powder-type nebulized inhalant.

11. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, excipient, or excipient, wherein the excipient is selected from one or more of osmotic pressure regulators, pH regulators, stabilizers, and preservatives.

12. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also contains other drugs for the prevention and / or treatment of acute kidney injury as active ingredients.

13. A nebulized inhalant, characterized in that, The nebulized inhalant contains an effective amount of neomycin for the prevention and / or treatment of acute kidney injury; The acute kidney injury mentioned is acute kidney injury caused by ischemia-reperfusion. The concentration of neomycin in the nebulized inhaler is 0.1-5 mg / mL.

14. A reagent kit, characterized in that, The kit contains: (a) A pharmaceutical composition containing neomycin; (b) Nebulizer inhalation device; (c) Instructions for use that instruct the user to use the drug composition by nebulization to prevent and / or treat acute kidney injury caused by ischemia-reperfusion.

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