Use of diosgenin in the preparation of a medicament for preventing or treating acute kidney injury

Abstract

The application of diosgenin in the preparation of drugs for the prevention or treatment of acute kidney injury falls within the pharmaceutical field. Specifically, this invention discloses that diosgenin, by directly binding to the PIK3CA protein, regulates the PI3K / Akt-NF-κB pathway, inhibiting inflammatory responses, apoptosis, and pyroptosis in renal tubular epithelial cells, thereby alleviating kidney damage. Cellular experiments show that diosgenin at concentrations of 0.1–5 μM can significantly protect renal tubular epithelial cells; animal experiments show that doses of 1–20 mg / kg can significantly improve renal function and reduce pathological damage in mice with acute kidney injury. This invention clarifies a complete chain of evidence from "compound—target—pathway—efficacy," providing a new drug candidate for the treatment of acute kidney injury and offering a paradigm for the mechanistic study of active ingredients in traditional Chinese medicine, contributing to the transformation of traditional Chinese medicine from empirical medicine to evidence-based and molecular medicine.

Description

Technical Field

[0001] This invention relates to the field of acute kidney injury technology, and in particular to therapeutic targets and novel drug candidates for acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a critical clinical syndrome characterized by a rapid decline in kidney function over a short period of time, with both its incidence and mortality rates showing an increasing trend year by year. Globally, approximately 13.5 million people are diagnosed with AKI each year, of whom about 1.7 million die. In my country, between 1.4 million and 2.9 million patients are hospitalized for AKI annually, with an in-hospital mortality rate as high as 12.4%. Currently, clinical treatment of AKI mainly involves supportive measures, including fluid management and renal replacement therapy, which can alleviate the condition to some extent, but specific therapeutic drugs are still lacking.

[0003] In recent years, traditional Chinese medicine (TCM) and its active ingredients have shown potential in the treatment of acute kidney injury (AKI). For example, Xuebijing injection (composed of extracts of safflower, red peony root, chuanxiong rhizome, angelica root, and danshen root) has been approved in my country for the treatment of sepsis and its complications, and clinical studies have shown that it has an adjunctive therapeutic effect on sepsis-related AKI. The chuanxiong-huangqi compound has been reported to reduce inflammatory factor levels in clinical studies of patients with ischemic AKI. Active ingredients of TCM such as baicalin have also been systematically reviewed and considered to have potential value in the treatment of AKI; related studies have summarized their mechanisms of action and current status of clinical translation.

[0004] However, the following obvious shortcomings still exist: 1. Lack of specific treatment drugs: At present, the clinical treatment of AKI is mainly supportive measures. There are no specific drugs on the market that target the key pathological aspects of AKI, and the prognosis of patients is poor.

[0005] 2. The mechanisms of action of active ingredients in traditional Chinese medicine are unclear: Most studies on the treatment of AKI with active ingredients in traditional Chinese medicine are still at the level of describing apparent pharmacological effects, with unclear molecular targets and ambiguous mechanisms of action. For example, although some studies have reported the renal protective effects of ingredients such as quercetin and baicalin, their direct targets and downstream signaling pathways have not been clearly elucidated, which seriously restricts their clinical translation and application.

[0006] 3. Lack of low-dose, high-efficiency TCM monomer candidates: Existing research on active TCM ingredients often requires high doses to observe renal protective effects, potentially leading to toxic side effects and high medication costs. Currently, there are no reported TCM monomers that can take effect at extremely low doses and possess both clear targets and multi-pathway regulatory advantages.

[0007] In summary, developing a therapeutic agent for AKI with a clear target, well-defined mechanism, low-dose high efficacy, and good safety profile has significant clinical value and urgent market demand. This invention addresses the aforementioned shortcomings. Summary of the Invention

[0008] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide the application of diosgenin in the preparation of drugs for the prevention or treatment of acute kidney injury (AKI). Specifically, existing clinical treatments for AKI lack specific drugs, the molecular targets and mechanisms of action of active ingredients in traditional Chinese medicine (TCM) for treating AKI are still unclear, and there are very limited candidate monomers that can intervene in the pathological process of AKI with low doses and high efficacy. Therefore, this invention provides the application of diosgenin in the preparation of drugs for the prevention or treatment of AKI, and by revealing its complete mechanism of binding to PIK3CA protein and regulating the PI3K / Akt-NF-κB signaling pathway, it establishes an evidence chain of "compound-target-pathway-pharmacological effect," providing a new technical solution for AKI drug development and an example for the mechanism research of active ingredients in TCM, which helps to promote the transformation of TCM from empirical medicine to evidence-based medicine and molecular medicine.

[0009] (II) Technical Solution To achieve the above objectives, the present invention provides the use of diosgenin or its pharmaceutically acceptable salts, esters, prodrugs and / or derivatives as a preparation of medicaments for the prevention and / or treatment of acute kidney injury.

[0010] In one embodiment, the diosgenin is applied at a dose of 1.0~20 mg / kg, for example 1.0~10 mg / kg, 1.0~5 mg / kg, or 1.0~2.5 mg / kg.

[0011] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) A therapeutically effective amount of diosgenin or its pharmaceutically acceptable salts, esters, prodrugs and / or derivatives; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0012] In one embodiment, the active ingredient in the pharmaceutical composition is diosgenin.

[0013] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0014] In one embodiment, the dosage form of the pharmaceutical preparation includes an oral preparation or an injectable preparation.

[0015] In one embodiment, the oral formulation includes tablets, capsules, granules, or oral liquids.

[0016] In one embodiment, the injectable includes a powder for injection or an injectable solution.

[0017] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0018] In one embodiment, the pharmaceutical product is a vial or box.

[0019] In another aspect, the present invention also provides the above-described pharmaceutical compositions, pharmaceutical preparations and / or pharmaceutical products for use in the preparation of drugs for the prevention and / or treatment of acute kidney injury.

[0020] In one embodiment, the acute kidney injury is induced by ischemia-reperfusion (I / R) or cisplatin.

[0021] (III) Beneficial Effects This invention provides the use of diosgenin in the preparation of drugs for the prevention or treatment of acute kidney injury. Compared with the prior art, it has the following beneficial effects: 1. This invention demonstrates through in vitro cell experiments and in vivo animal models that diosgenin can significantly reduce renal tubular epithelial cell damage and improve renal function and histopathological damage in mice with acute kidney injury.

[0022] 2. This invention reveals for the first time that diosgenin exerts anti-inflammatory, anti-apoptotic, and anti-pyroptosis renal protective effects by directly binding to the PIK3CA protein and inhibiting the abnormal activation of the PI3K / Akt-NF-κB signaling pathway, thus forming a complete chain of evidence of "compound-target-pathway-efficacy".

[0023] 3. Diosgenin is derived from the traditional Chinese medicine Polygonatum sibiricum, which is both food and medicine. It has the characteristics of high efficacy at low doses (e.g., 1 mg / kg) and good safety, and has the potential to be developed into a drug for the prevention or treatment of acute kidney injury. It provides an example for the study of the mechanism of active ingredients in traditional Chinese medicine. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a graph showing the results of detecting serum CREA and BUN levels in mice; Figure 2 This is a diagram showing the H&E and PAS staining results of pathological damage in mouse kidney tissue. Figure 3This is a graph showing the results of qRT-PCR detection of inflammatory factors in mouse kidney tissue; Figure 4 This is a graph showing the results of Western blot analysis of proteins related to apoptosis and pyroptosis. Figure 5 This is a diagram showing the H&E and PAS staining results of the toxic effects of diosgenin on major organs and tissues. Figure 6 This is a graph showing the results of HK-2 cell viability testing; Figure 7 This is a diagram showing the TUNEL staining results of HK-2 cell apoptosis. Figure 8 This is a graph showing the qRT-PCR detection results of inflammatory factors in HK-2 cells; Figure 9 This is a CETSA assay result of diosgenin binding to PIK3CA protein; Figure 10 This is a molecular dynamics simulation diagram of the binding of diosgenin to PIK3CA protein; Figure 11 This is a Western blot result of HK-2 cell apoptosis and pyroptosis-related proteins. Figure 12 This is a Western blot result of the PI3K / Akt / NF-κB pathway. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Terms and Definitions As used herein, the term "diosgenin" refers to diosgenin, a natural steroidal saponin compound. It is primarily found in the rhizomes of plants in the Dioscoreaceae family (such as Polygonatum sibiricum, Dioscorea opposita, Dioscorea nipponica, and Curcuma longa), and is a key raw material for the synthesis of various steroidal hormone drugs (such as birth control pills and cortisone). Diosgenin can be prepared using methods known in the art, including but not limited to: acid hydrolysis, microbial fermentation, ultrasound-assisted extraction, microwave-assisted extraction, enzymatic hydrolysis, aqueous two-phase extraction, and supercritical fluid extraction.

[0028] As used herein, the term "inhibitor" refers to a substance capable of reducing the level or activity of PIK3CA protein. In this invention, the inhibitor is diosgenin. This invention demonstrates that diosgenin regulates the downstream PI3K / Akt-NF-κB signaling pathway by directly binding to and inhibiting the activity of PIK3CA protein. The diosgenin disclosed in this invention, as a PIK3CA inhibitor, can be used to prevent or treat acute kidney injury and related symptoms.

[0029] As used herein, the term "pharmaceutical composition" refers to a composition comprising diosgenin formulated with one or more pharmaceutically acceptable carriers.

[0030] The formulation of a pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, powders, syrups, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0031] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0032] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0033] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0034] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0035] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0036] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0037] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0039] 1. Reagents: Diosgenin (purity ≥99.35%): purchased from MedChemExpress (MCE), prepared as a stock solution with dimethyl sulfoxide (DMSO), diluted with culture medium to the final concentration (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0 μM) for cell experiments, and diluted with physiological saline or a suitable solvent to the required concentration for animal experiments.

[0040] PIK3CA (PI3K-p110) recombinant protein, anti-PIK3CA antibody, anti-p-Akt, anti-t-Akt, anti-p-NF-κB p65, anti-NF-κB p65, anti-GAPDH and other antibodies: purchased from Proteintech Group, Inc. and Immunoway Biotechnology Co., LTD.

[0041] Cell culture media, fetal bovine serum, trypsin, and other cell culture reagents were purchased from VivaCell, Beyotime Biotechnology, etc.

[0042] Apoptosis detection kit (Annexin V-FITC / PI) and pyroptosis-related protein (GSDMD, Caspase-1) detection kit were purchased from Proteintech Group, Inc. and Immunoway Biotechnology Co., LTD.

[0043] Kidney function indicator test kits (serum creatinine Scr, blood urea nitrogen BUN): purchased from Nanjing Jiancheng Bioengineering Institute.

[0044] Primers and antibodies for inflammatory factor detection (IL-6, TNF-α, IL-1β): purchased from General Biotechnology (Anhui) Co., Ltd. and Proteintech Group, Inc.

[0045] 2. Cells and Animals: Cells: Human renal tubular epithelial cells (HK-2 cell line), purchased from Wuhan Procell Life Science & Technology Co., Ltd.

[0046] Animals: SPF-grade male C57BL / 6 mice, weighing 20-25g, 8-10 weeks old, were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (Certificate No.: 20250706Abzz01050000052). They were housed in a constant temperature (22±2℃), constant humidity (50%±5%), 12h light / 12h dark environment with free access to food and water. All animal experiments were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (Anhui Provincial Hospital) (Approval No.: [2025-N(A)-221]).

[0047] Example 1: Establishment of an acute kidney injury (AKI) mouse model: Model 1 (IRI): Ischemia-reperfusion induced AKI. Mice were anesthetized by inhalation of ether, and a midline incision was made in the back. The bilateral renal pedicles were separated, and the bilateral renal pedicles were clamped with non-invasive arterial clamps for 42 min. After releasing the arterial clamps and restoring blood flow perfusion for 24 h, the mice were sacrificed.

[0048] Model 2 (Cis): Cisplatin-induced AKI. Mice were injected intraperitoneally with cisplatin (20 mg / kg) and sacrificed 72 h later.

[0049] Example 2: Group Treatment The AKI mouse models prepared in Example 1 were randomly divided into the following groups (8-10 mice per group): Sham surgery group: only open the abdomen without clamping the renal pedicle or injecting saline.

[0050] AKI Model Group (Model): Operate according to the method of Model 1 or Model 2 in Example 1.

[0051] Diosgenin low, medium and high dose groups (1, 10 and 20 mg / kg): Before model establishment (sham surgery group, prophylactic administration), diosgenin 10 mg / kg was administered intraperitoneally or by gavage once a day for 3 consecutive days. Alternatively, after the model is established (therapeutic administration), diosgenin 1, 10, or 20 mg / kg is administered intraperitoneally or orally once daily for 3 consecutive days.

[0052] Positive control group: After the AKI model group was established, positive control drugs, such as curcumin, were administered at 20 mg / kg once daily for 3 consecutive days.

[0053] Example 3: Verification of therapeutic effect: 1. Sample collection: After administration, 24-hour urine was collected from mice; after anesthesia, blood was collected by enucleation and centrifugation to separate serum; after euthanasia, both kidneys were removed, a portion of which was fixed in 4% paraformaldehyde for pathological staining, and the remainder was frozen at -80℃ for molecular biological detection.

[0054] Separate samples from the heart, liver, spleen, and lungs were fixed in 4% paraformaldehyde for pathological staining to assess the toxicity of diosgenin dosage to organs and tissues.

[0055] 2. Testing: 2.1 Kidney function tests: Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured using a biochemical assay kit.

[0056] The results are as follows Figure 1 As shown, in IRI-AKI ( Figure 1 (A) and cisplatin-AKI ( Figure 1 In model B), serum creatinine (CREA) and blood urea nitrogen (BUN) levels were significantly elevated in the model group mice (P<0.01), indicating severe renal impairment. Compared with the model group, the levels of CREA and BUN in the diosgenin (1, 10, and 20 mg / kg) treatment groups were significantly decreased, and the differences were statistically significant (P<0.001 or P<0.01). The positive control group also showed some improvement (P<0.01 or P<0.05).

[0057] 2.2. Kidney tissue pathological examination: Kidney tissue was fixed, dehydrated, embedded in paraffin, and sectioned (4 μm thick) for further processing. HE staining: Observe the extent of renal tubular damage, such as tubular dilation, loss of brush border, cell necrosis, and cast formation. Score the damage area (0-4 points).

[0058] PAS staining: to observe the renal tubular basement membrane and glycogen deposition.

[0059] The results are as follows Figure 2 As shown, H&E staining and PAS staining revealed extensive necrosis of renal tubular epithelial cells, loss of brush border, luminal dilation, and formation of protein casts in the model group, indicating significant renal tubular damage and glycogen deposition. In the diosgenin treatment group, renal tissue damage was significantly reduced, the renal tubular structure remained relatively intact, and the damage was significantly decreased.

[0060] 2.3 Detection of inflammatory factors in renal tissue: RNA was extracted from kidney tissue using the Trizol method, and the levels of IL-6, TNF-α, and IL-1β were detected by qRT-PCR.

[0061] The results are as follows Figure 3As shown, the mRNA levels of IL-6, TNF-α, and IL-1β in the kidney tissue of the model group were significantly increased (P<0.01). The mRNA levels of inflammatory factors were significantly decreased in each diosgenin dosage group (P<0.05 or P<0.01).

[0062] 2.4 Detection of pyroptosis-related proteins in kidney tissue: Western blot was used to detect the expression of GSDMD, Caspase-1, and IL-1β proteins in kidney tissue.

[0063] The results are as follows Figure 4 As shown, the expression of GSDMD-N, Cleaved-Caspase-1, IL-1β, and Cleaved-Caspase-3 proteins was significantly upregulated in the model group. Treatment with diosgenin significantly reduced the expression of these apoptosis- and pyroptosis-related proteins.

[0064] 2.5 Detection of the PI3K / Akt-NF-κB pathway in kidney tissue: Western blot analysis was used to detect the expression of proteins such as p-Akt, t-Akt, p-NF-κB p65, and NF-κB p65.

[0065] The results are as follows Figure 5 As shown, H&E staining and PAS staining revealed varying degrees of damage and glycogen deposition in the heart, liver, lungs, and spleen of the model group. The diosgenin-treated group showed no significant damage or glycogen deposition compared to the normal control group.

[0066] Example 4 Cell Experiment: 1. Cell culture: HK-2 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and incubated at 37°C in a 5% CO2 incubator. When the cells reached 80%–90% confluence, they were passaged using 0.25% trypsin. Cells in the logarithmic growth phase were used for experiments.

[0067] 2. Cell grouping: Normal control group (Control); AKI model group (Model): Renal tubular epithelial cell injury was induced by cisplatin 10 μM (CIS) or hypoxia (1% O2, 5% CO2, 94% N2, 37°C, 9h) reoxygenation stimulation for 3h (H / R, IRI); Diosgenin: In addition to the normal control or AKI model, 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10, 25, and 50 μM diosgenin were added for 2 h for pretreatment, and then co-cultured with the induction conditions for 24 h. Positive control group: Based on the AKI model, 20 μM curcumin was added for 2 h of treatment, and then co-cultured with the induction conditions for 24 h.

[0068] Each group has 3 replicate wells, and the experiment is repeated 3 times.

[0069] 3. Indicator Testing: 3.1 Cell viability assay (CCK-8 assay): Follow the instructions for the CCK-8 reagent kit. After treatment, add 10 μL of CCK-8 solution to each well and incubate at 37°C for 1-2 hours. Measure the absorbance (OD value) at 450 nm using a microplate reader. Calculate cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0070] The results are as follows Figure 6 As shown, compared with the normal control group, the cell viability of the model group decreased significantly to about 50%~60% (P<0.001). Compared with the model group, the cell viability of the diosgenin (0.1, 1.0, 2.5 μM) treatment groups was significantly increased (P<0.05 or P<0.01).

[0071] 3.2 Apoptosis Detection (TUNEL Assay): Apoptosis in kidney tissue or HK-2 cells was detected using the TUNEL apoptosis detection kit (Abbkine, Wuhan, China). Proteinase K permeabilization, TUNEL reaction incubation, and DAPI counterstaining were performed according to the manufacturer's instructions. The proportion of TUNEL-positive cells was observed and counted under a fluorescence microscope.

[0072] The results are as follows Figure 7 As shown, the apoptosis rate in the model group was significantly increased. Treatment with different concentrations of diosgenin significantly reduced cell apoptosis.

[0073] 3.3. Detection of pyroptosis: Western blot was used to detect the expression levels of pyroptosis-related proteins (GSDMD, Caspase-1, IL-1β).

[0074] The results are as follows Figure 11 As shown, the expression of GSDMD-N, Cleaved-Caspase-1, IL-1β, and Cleaved-Caspase-3 proteins was significantly upregulated in the model group. After treatment with diosgenin, the expression of all the above-mentioned pyroptosis-related proteins was significantly downregulated.

[0075] 3.4. Detection of inflammatory factors (qRT-PCR): Cultured cells were collected, total RNA was extracted using the Trizol method, and the levels of IL-6, TNF-α, and IL-1β were detected.

[0076] The results are as follows Figure 8 As shown, the mRNA levels of IL-6, TNF-α, and IL-1β in the model group cells were significantly increased (P<0.001). The mRNA levels of inflammatory factors in each diosgenin concentration group were significantly decreased (P<0.05 or P<0.01).

[0077] 3.5 Target binding verification (CETSA or surface plasmon resonance SPR): CETSA: Cells were treated with diosgenin, then subjected to gradient heating, lysis and centrifugation to collect the supernatant. The amount of soluble PIK3CA protein was then detected by Western Blot and a thermal melting curve was fitted to compare the differences in Tm values ​​between the two groups.

[0078] The results are as follows Figure 9 As shown, there are significant differences in the presence or absence of diosgenin treatment in PIK3CA protein within the denaturation temperature range (from room temperature to 62°C), that is, diosgenin treatment causes significant thermal transfer of PIK3CA protein in HK-2 cells.

[0079] SPR: The recombinant PIK3CA protein was immobilized on a chip and passed through different concentrations of diosgenin to calculate the binding affinity (KD value).

[0080] like Figure 10 As shown, molecular dynamics simulation results indicate that the binding of diosgenin to PIK3CA protein is stable in molecular dynamics simulations and may possess biological activity.

[0081] 3.6 Signal Path Detection (Western blot): Total protein was extracted from cells in each group, and protein concentration was determined using the BCA method. After SDS-PAGE electrophoresis, the membrane was transferred to a PVDF membrane, blocked with 5% skim milk for 1 h, and then incubated overnight at 4°C with primary antibodies (p-Akt, t-Akt, p-NF-κB p65, NF-κB p65, PIK3CA, etc.). After washing with TBST, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 h, followed by ECL imaging. The relative expression level of the target protein was calculated using GAPDH as an internal control.

[0082] The results are as follows Figure 12As shown, the p-Akt / Akt ratio was significantly decreased and the p-NF-κB p65 / NF-κB p65 ratio was significantly increased in the model group, indicating that the PI3K / Akt signaling pathway was inhibited and NF-κB signaling was abnormally activated. After diosgenin treatment, the p-Akt / Akt ratio significantly increased and the p-NF-κB p65 / NF-κB p65 ratio significantly decreased. Total Akt and total NF-κB p65 expression showed no significant changes.

[0083] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of diosgenin, its pharmaceutically acceptable salts, esters, prodrugs, derivatives and / or pharmaceutical compositions containing it in the preparation of medicaments for the prevention and / or treatment of acute kidney injury.

2. The application according to claim 1, characterized in that, The drug is an oral preparation or an injection; the oral preparation is selected from one or more of tablets, capsules, granules, and oral liquids; the injection is selected from one or more of powder for injection and injection solutions.

3. The application according to claim 2, characterized in that, The dosage of diosgenin is 1.0~20 mg / kg.

4. A pharmaceutical composition, characterized in that, include: (1) Diosgenin or its pharmaceutically acceptable salts, esters, prodrugs and / or derivatives; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition further comprises at least one other drug for treating acute kidney injury, wherein the other drug for treating acute kidney injury is selected from one or more of anti-inflammatory drugs, antioxidants, and adjunctive renal replacement therapy drugs.

6. A pharmaceutical preparation, characterized in that, Includes the pharmaceutical composition according to claim 4 or 5.

7. A pharmaceutical product, characterized in that, Includes the pharmaceutical preparation described in claim 6.

8. The pharmaceutical composition of claim 4 or 5, the pharmaceutical formulation of claim 6, and / or the pharmaceutical product of claim 7, as used in the preparation of a medicament for the prevention and / or treatment of acute kidney injury.

9. The application according to claim 1 or 8, characterized in that, The acute kidney injury includes ischemia-reperfusion induced acute kidney injury and / or cisplatin induced acute kidney injury.

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