Application of phloretin in preparation of medicine for preventing or treating renal ischemia-reperfusion injury

Abstract

The invention provides application of phloretin in preparation of a medicine for preventing or treating renal ischemia-reperfusion injury, and belongs to the technical field of medical application. According to the application, research finds that the phloretin can up-regulate the protein expression level of PAK3 and reduce the apoptosis level under the conditions of renal ischemia reperfusion or hypoxia reoxygenation. On the basis, the phloretin or the pharmaceutically acceptable salt, ester or solvate thereof not only can be used as an activator of PAK3, but also can effectively inhibit cell apoptosis induced by renal ischemia reperfusion or hypoxia reoxygenation, so that a relatively good prevention and treatment effect on renal ischemia reperfusion injury caused by cell apoptosis is achieved. The application not only provides a new application for phloretin, but also provides a new drug choice for activation of PAK3, inhibition of apoptosis and renal ischemia-reperfusion injury, and has important guiding significance for prevention and treatment of renal ischemia-reperfusion injury.

Description

Technical Field

[0001] The invention relates to the technical field of medical application, and in particular to application of phloretin in preparing medicine for preventing or treating renal ischemia-reperfusion injury. Background Art

[0002] Renal ischemia-reperfusion injury (RIRI) refers to a pathological process in which renal ischemia and reperfusion for a period of time not only fail to restore renal function, but aggravate renal dysfunction and structural damage. It is a common cause of acute kidney injury (AKI, a clinical syndrome caused by rapid decline of renal function in a short period of time due to multiple causes), and often occurs in clinical situations such as renal transplantation, renal artery occlusion, sepsis, and abuse of nephrotoxic drugs. Currently, there is no specific drug for the treatment of RIRI. Therefore, finding drugs to prevent and treat renal ischemia-reperfusion injury is still a challenging problem in the field of renal medicine.

[0003] The pathogenesis of renal ischemia-reperfusion injury is complex. The damage induced by renal ischemia-reperfusion includes many different types such as apoptosis, inflammation, and ferroptosis. The pathogenic mechanisms and treatment strategies of different types are different. Among them, apoptosis is an important factor causing renal ischemia-reperfusion injury. During renal ischemia-reperfusion, ischemia will lead to a decrease in oxygen supply, while reperfusion will increase the oxygen supply. The hypoxia-reoxygenation environment caused by ischemia-reperfusion will cause abnormal mitochondrial energy metabolism and increased consumption, acidosis and ion imbalance, and then tubular cell apoptosis will occur, leading to renal ischemia-reperfusion injury. Therefore, effectively inhibiting or alleviating apoptosis induced by ischemia-reperfusion or hypoxia-reoxygenation is of great significance for preventing or treating renal ischemia-reperfusion injury and acute kidney injury caused by apoptosis.

[0004] Phloretin (PHL) is a dihydrochalcone compound with multiple pharmacological activities such as anti-inflammatory, anti-fibrotic, anti-oxidative stress, hypoglycemic, and cardiovascular protection. However, there is currently no relevant use of phloretin in the prevention or treatment of renal ischemia-reperfusion injury. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present application provides the use of phloretin in the preparation of a drug for preventing or treating renal ischemia-reperfusion injury. The present application confirms through research that phloretin can upregulate the protein expression level of PAK3 and reduce the level of cell apoptosis under renal ischemia-reperfusion or hypoxia-reoxygenation conditions, which is of great significance for the prevention and treatment of renal ischemia-reperfusion injury.

[0006] In a first aspect, the embodiments of the present application provide the use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury.

[0007] In some embodiments, the renal ischemia-reperfusion injury is renal injury caused by renal ischemia-reperfusion-induced cell apoptosis.

[0008] In some embodiments, the use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof includes:

[0009] Reduces serum creatinine and urea nitrogen levels;

[0010] and / or, reducing tubular dilatation and inflammatory cell infiltration;

[0011] and / or, activation of PAK3.

[0012] In some embodiments, the effect of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof is dose-dependent.

[0013] In some embodiments, the dosage of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof is 1 to 20 mg / kg calculated as phloretin.

[0014] In some embodiments, the drug for preventing or treating renal ischemia-reperfusion injury is a preparation comprising phloretin or a pharmaceutically acceptable salt, ester or solvate thereof as an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0015] In some embodiments, the dosage form of the preparation is a liquid preparation, a solid preparation or a semisolid preparation.

[0016] In a second aspect, the embodiments of the present application provide the use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a drug for inhibiting apoptosis of renal tubular epithelial cells induced by renal ischemia-reperfusion.

[0017] In a third aspect, the embodiments of the present application provide the use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a drug for inhibiting hypoxia-reoxygenation-induced apoptosis of renal tubular epithelial cells.

[0018] In a fourth aspect, the embodiments of the present application provide the use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a PAK3 activator.

[0019] The beneficial effects of this application are:

[0020] This application confirms through research that in a mouse model of renal ischemia-reperfusion injury, phloretin can upregulate the protein expression level of PAK3, reduce the level of apoptosis, and effectively reduce the levels of creatinine and urea nitrogen in mouse serum, and improve renal pathological changes, which has a good therapeutic effect on renal ischemia-reperfusion injury. Moreover, in the NRK-52E cell model of hypoxia-reoxygenation, phloretin can also upregulate the protein expression level of PAK3 and reduce the level of apoptosis. Based on this, phloretin or its pharmaceutically acceptable salt, ester or solvate can not only be used as an activator of PAK3, but also can effectively inhibit apoptosis induced by renal ischemia-reperfusion or hypoxia-reoxygenation, and then play a good prevention and treatment effect on renal ischemia-reperfusion injury caused by apoptosis. This application not only provides a new use for phloretin, but also provides a new drug selection for the activation of PAK3, the inhibition of apoptosis and renal ischemia-reperfusion injury, which has important guiding significance for the prevention and treatment of renal ischemia-reperfusion injury.

[0021] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings used in the present application. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 This is a comparison chart of the detection results of the PAK3 protein expression level in the kidneys of mice in each group in the examples of this application;

[0024] Figure 2 This is a comparison chart of the detection results of the PAK3 protein expression level in NRK-52E cells of each group in the examples of this application;

[0025] Figure 3 This is a comparison chart of the detection results of Bax and Bcl-2 protein expression levels in the kidneys of mice in each group in the examples of this application;

[0026] Figure 4 This is a comparison chart of the detection results of Bax and Bcl-2 protein expression levels in NRK-52E cells of various groups in the examples of this application;

[0027] Figure 5 This is a comparison chart of the apoptosis levels of NRK-52E cells in each group in the examples of this application;

[0028] Figure 6 This is a comparison chart of creatinine and urea nitrogen levels in the serum of mice in each group in the examples of this application;

[0029] Figure 7 This is a comparison of HE staining of mouse kidneys in each group in the examples of this application. DETAILED DESCRIPTION

[0030] The following embodiments of the technical solution of the present application are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0032] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0033] p21-activated kinases (PAKs) are members of the serine / threonine protein kinase family and are involved in various signal cascades on the cell surface and in the cell, thereby affecting physiological processes such as cell cycle, proliferation, transformation, metabolism, and inflammation. PAKs include two major categories, PAK1-3 and PAK4-6. The former can be directly activated by binding to cell division cycle protein 42 and Ras-related C3 botulinum toxin substrates through the GTPase-binding domain, generating kinase activity, thereby regulating a variety of important cell physiological and pathological processes. Among them, PAK3 may be involved in the regulation of cell apoptosis. Since renal ischemia-reperfusion or hypoxia-reoxygenation can induce cell apoptosis, leading to renal damage, if the level of cell apoptosis can be regulated by regulating the expression level of PAK3, it will have important guiding significance for the prevention and treatment of renal ischemia-reperfusion injury.

[0034] The present application found in the study that phloretin can upregulate the protein expression level of PAK3 and reduce the level of cell apoptosis under renal ischemia-reperfusion or hypoxia-reoxygenation conditions. Based on this, phloretin can be used to inhibit renal ischemia-reperfusion or hypoxia-reoxygenation-induced cell apoptosis, and can be used to prevent and treat renal ischemia-reperfusion injury caused by cell apoptosis.

[0035] Specifically, the present application provides an application of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury. Wherein, the renal ischemia-reperfusion injury may include renal injury caused by apoptosis induced by renal ischemia-reperfusion. Phloretin can effectively inhibit apoptosis of renal tubular epithelial cells and alleviate renal ischemia-reperfusion injury by activating PAK3, reducing serum creatinine and urea nitrogen levels, alleviating tubular dilatation and inflammatory cell infiltration.

[0036] Furthermore, the effect of phloretin or its pharmaceutically acceptable salt, ester or solvate is dose-dependent.

[0037] In some embodiments of the present application, the dosage of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof is 1 to 20 mg / kg in terms of phloretin.

[0038] In some embodiments of the present application, the drug for preventing or treating renal ischemia-reperfusion injury is a preparation prepared by using phloretin or its pharmaceutically acceptable salt, ester or solvate as an active ingredient and adding a pharmaceutically acceptable carrier or excipient, wherein the preparation is in the form of a liquid preparation, a solid preparation or a semi-solid preparation.

[0039] According to the use of phloretin in activating PAK3 and inhibiting apoptosis, the present application examples also provide the following uses of phloretin or its pharmaceutically acceptable salts, esters or solvates:

[0040] 1. Application in the preparation of drugs for inhibiting apoptosis of renal tubular epithelial cells induced by renal ischemia-reperfusion;

[0041] 2. Application in the preparation of drugs for inhibiting apoptosis of renal tubular epithelial cells induced by hypoxia and reoxygenation;

[0042] 3. Application in the preparation of PAK3 activators.

[0043] Through the above-mentioned method, the present application not only provides a new use for phloretin, but also provides a new drug option for the activation of PAK3, the inhibition of cell apoptosis and renal ischemia-reperfusion injury, which has important guiding significance for the prevention and treatment of renal ischemia-reperfusion injury.

[0044] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

[0045] Example

[0046] In order to verify the actual effect of phloretin, the present application established a mouse model of renal ischemia-reperfusion injury and a rat NRK-52E cell line hypoxia-reoxygenation model, and verified the effect of phloretin in each model, including the following experiments:

[0047] Experiment 1: Establishment of renal ischemia-reperfusion injury (RIRI) mouse model, drug administration and material collection methods

[0048] SPF male C57BL / 6J mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 7 weeks old, weighing 18-22g) were randomly divided into a sham operation group and a RIRI model group. The sham operation group was further divided into a sham operation control group (Sham NS) and a sham operation drug administration group (dosage of 120 mg / kg / d, expressed as Sham PHL-120) according to whether the drug was administered. The RIRI model group was further divided into a RIRI model control group (RIRIPHL-0), a RIRI+PHL-30 group (dosage of 30 mg / kg / d), a RIRI+PHL-60 group (dosage of 60 mg / kg / d), and a RIRI+PHL-120 group (dosage of 120 mg / kg / d) according to whether the drug was administered and the dosage, with 8 mice in each group.

[0049] The method for establishing the RIRI model includes: injecting 1% sodium pentobarbital (50 mg / kg) into the mouse intraperitoneally, fixing the mouse on the operating table with its abdomen facing upward after anesthesia, cutting the cortex and muscle layer in sequence along the midline of the abdomen, exposing the right renal pedicle, and performing right nephrectomy, exposing the left kidney, clamping the renal artery with an artery clamp and starting the timing, the kidney turning purple-black is ischemia, the artery clamp is released after 30 minutes, and the kidney returns to a pink color, which means reperfusion is successful, the renal tissue is put back in place, the mouse abdominal wound is sutured layer by layer, and the model is established after reperfusion for 24 hours. The sham operation group did not undergo ischemia treatment, only right kidney removal and suturing were performed, and other operations were the same as the model group.

[0050] The administration method of PHL includes: suspending phloretin powder in normal saline, shearing with a handheld homogenizer until there are no large phloretin particles, ultrasonicating phloretin solutions of different concentrations for 30 minutes every day and fully mixing, and administering according to the dosage requirements of each group. The sham operation control group and the RIRI model control group were intragastrically administered with the corresponding volume of normal saline at 0.4mL / 20g for 14 consecutive days.

[0051] On the 14th day, the RIRI mouse model was established and reperfused for 24 hours. The eyeballs were removed to collect blood, which was centrifuged at 4000 rpm and 4°C for 15 min. The serum was collected and stored at -80°C for testing. After blood collection, the mice were killed by cervical dislocation, and the left kidney of the mice was removed on ice. The left kidney was cut in cross section, half of the kidney tissue was quickly placed at -80°C, and part of the kidney tissue was fixed in 4% paraformaldehyde.

[0052] Experiment 2: Establishment of Hypoxia / Reoxygenation (H / R) NRK-52E Cell Model and Drug Administration

[0053] (1) Inoculation of cells: Set up a blank control group (Ctrl NS), a blank drug group (dose of 50 μmol / L, expressed as Ctrl PHL-50), an H / R model control group (H / R PHL-0), an H / R+PHL-2 group (dose of 2 μmol / L), an H / R+PHL-10 group (dose of 10 μmol / L), and an H / R+PHL-50 group (dose of 50 μmol / L). Take a 6 cm cell culture dish (Corning, catalog number 430166), add 5 mL of cell suspension with a density of 0.8×105 / mL to each well, and place it in a cell culture incubator for 12 h.

[0054] (2) Serum starvation and phloretin intervention: The cells were grown to about 60% fusion and washed three times with room temperature PBS (Beijing BioDee Biotechnology Co., Ltd., Catalog No.: BL302A). The blank control group and H / R model control group were replaced with 5 mL of serum-free high-glucose DMEM medium (high-glucose DMEM culture medium: Beijing BioDee Biotechnology Co., Ltd., Catalog No.: BL304A; L-glutamine: Beijing BioDee Biotechnology Co., Ltd; 1% penicillin-streptomycin: Beijing BioDee Biotechnology Co., Ltd., Catalog No.: BL505A). The blank treatment group was added with 5 mL of serum-free high-glucose DMEM medium containing 50 μmol / L PHL, and the H / R+PHL-2 group, H / R+PHL-10 group, and H / R+PHL-50 group were replaced with 5 mL of serum-free high-glucose DMEM medium containing different concentrations of PHL, respectively. All cells were placed in a CO2 cell culture incubator (Thermo Fisher Scientific, Catalog No.: 3111) and cultured for 12 h.

[0055] (3) Hypoxia: The blank control group and the blank drug group were washed three times with PBS, replaced with a low-glucose serum-free medium (low-glucose DMEM culture medium: HyClone, SH30021.01; L-glutamine: Beijing BioDee Biotechnology Co., Ltd; penicillin-streptomycin: Beijing Lanjieke Technology Co., Ltd., catalog number: BL505A), and cultured in a conventional cell culture incubator for 24 h; the H / R+PHL-2 group, H / R+PHL-10 group, and H / R+PHL-50 group were discarded the original culture medium, washed three times with PBS, replaced with a serum-free low-glucose medium, and placed in an anaerobic incubator (37°C, 94% N 2 , 5% CO 2 , 1% O 2 ) for 24 h.

[0056] (4) Reoxygenation: The culture medium was discarded from all groups, the cells were washed three times with PBS, replaced with high-glucose complete culture medium, and cultured in a conventional cell culture incubator for 6 h.

[0057] Experiment 3: Detection of PAK3 expression levels in samples from Experiments 1 and 2

[0058] The method for testing tissue samples in Experiment 1 and Experiment 2 was the same, specifically including: adding 1000 μL lysis solution (prepared from 10 μL Protease Inhibitor Cocktail (CST, Catalog No.: #5871) and 990 μL RRIPA Buffer (CST, Catalog No.: #9806)) to every 100 mg of tissue. Quickly chop and homogenize the tissue on ice for 3 times, stopping for 10 seconds each time, then sonicate 10 times, and place in an ice bath for 30 minutes. Centrifuge at 12000g, 4℃ for 15 minutes, and take the supernatant into a new EP tube (Solarbio, Catalog No.: #YA1030) to obtain the total tissue protein product. Dilute the protein sample 10 times with ultrapure water, and determine the protein concentration by BCA method. Distilled water was used to adjust the protein concentration of each group, and 2× loading buffer (prepared by SDS, Solarbio, Catalog No.: S8010; Tris, Solarbio, Catalog No.: T8060; Propylene glycol, Tianjin Tianli Chemical Reagent Co., Ltd.; Bromophenol blue, Solarbio, Catalog No.: B8120) (containing 2% β-Mercaptoethanol, AMRESCO, Catalog No.: #M0482) was added and mixed, sealed with a sealing film (PARAFILM, Catalog No.: PM-996), boiled in boiling water for 10 minutes, and then ice-bathed for 5 minutes. The samples were divided into smaller portions according to the protein concentration and the amount of protein required for each electrophoresis, and stored at -80°C. 20-30 μg tissue protein samples were taken for gel electrophoresis, transfer, blocking, primary antibody (PAK, Abcam, catalog number: Ab40808; β-actin, CST, catalog number: 4970S), membrane washing, secondary antibody (Goat Anti-RabbitIgG H&L (HRP), Ab97051), membrane washing, ECL ultrasensitive chemiluminescent solution (Beijing Pulilai Gene Technology Co., Ltd., catalog number: P1050) color development, and optical density analysis using ImageQuant LAS 4000 gel imaging system (GE ImageQuant, LAS4000). The results are shown in Figure 1-2 shown.

[0059] in, Figure 1 This is a comparison chart of the test results of the PAK3 protein expression level in the kidneys of each group in Experiment 1. Figure 2 The following is a comparison chart of the detection results of the PAK3 protein expression levels in NRK-52E cells of each group in Experiment 2. Figure 1 It can be seen that compared with the sham operation group, the expression of PAK3 in the renal tissue of mice in the RIRI model control group was significantly reduced, while different doses of PHL were helpful to increase its expression level, and the higher the dose, the more obvious the improvement effect, indicating that phloretin can increase the expression level of PAK3 in the renal tissue of mice in the RIRI model. Figure 2Similar changes were also shown in the results, indicating that phloretin can also increase the expression level of PAK3 in mouse renal tubular epithelial cells in the H / R model.

[0060] Therefore, phloretin can increase the protein expression level of PAK3 in both the RIRI mouse model and the NRK-52E cell model and can be used as a PAK3 activator.

[0061] Experiment 4: Detection of Bax and Bcl-2 expression levels in samples from Experiments 1 and 2

[0062] Bax and Bcl-2 are both apoptosis-related genes, of which Bax is a pro-apoptotic gene and Bcl-2 is an apoptosis-inhibiting gene. The detection method of Bax and Bcl-2 expression levels is basically the same as that in Experiment 3, and will not be repeated here. The detection results are shown in Figure 3-4 shown.

[0063] in, Figure 3 This is a comparison chart of the test results of Bax and Bcl-2 protein expression levels in the kidneys of each group in Experiment 1. Figure 4 The figure is a comparison chart of the detection results of Bax and Bcl-2 protein expression levels in NRK-52E cells of each group in Experiment 2. Figure 3 It can be seen that compared with the sham operation group, the expression of Bax in the renal tissue of mice in the RIRI model control group was significantly increased, and the expression of Bcl-2 was significantly decreased, indicating a high degree of cell apoptosis. Different doses of PHL can help reverse the expression of the corresponding proteins, thereby reducing the expression level of Bax and increasing the expression level of Bcl-2, effectively inhibiting cell apoptosis. The higher the dose of PHL, the more obvious the inhibitory effect, indicating that phloretin can effectively inhibit cell apoptosis in the renal tissue of mice in the RIRI model. Figure 2 Similar changes were also shown in the results of the experiment, indicating that phloretin can also effectively inhibit the apoptosis of rat renal tubular epithelial cells in the H / R model.

[0064] Therefore, phloretin can inhibit renal tubular epithelial cell apoptosis induced by renal ischemia-reperfusion or hypoxia-reoxygenation, and phloretin or its pharmaceutically acceptable salts, esters or solvates can be used to prepare drugs for inhibiting renal tubular epithelial cell apoptosis induced by renal ischemia-reperfusion and / or hypoxia-reoxygenation.

[0065] Experiment 5: Detection of cell apoptosis levels in samples from Experiment 2

[0066] To further detect the level of cell apoptosis, the cells in Experiment 2 were collected by digestion with EDTA-free trypsin (biosharp, BL526A). After washing, 1× Binding Buffer (BD, 556547) was added to resuspend the cells and adjust the cell density. The cells were stained with Annexin V-FITC / PI apoptosis detection kit (BD, 556547) and detected by flow cytometry (Beckman Coulter). The results are shown in Figure 5 shown. Figure 5 In the table, ns means no statistical difference, ## means the significance level is P<0.01, ###, *** means the significance level is P<0.001.

[0067] Depend on Figure 5 It can be seen that the number of apoptotic cells in the H / R model control group was significantly increased compared with the blank control group, and different doses of PHL could reduce the apoptosis level, which further confirmed that phloretin can effectively reduce the apoptosis level of NRK-52E cells induced by H / R.

[0068] Experiment 6: Detection of creatinine and uric acid nitrogen levels in the serum of mice in Experiment 1

[0069] Kits (creatinine detection kit, Nanjing Jiancheng Bioengineering Institute, catalog number: C011-2-1; urea nitrogen detection kit, Nanjing Jiancheng Bioengineering Institute, catalog number: C013-2-1) were used to detect the levels of creatinine and urea nitrogen in mouse serum. The detection process was carried out according to the instructions of the kit. The results are shown in the figure. Figure 6 shown. Figure 6 In the table, ns means no statistical difference, * means the significance level is P<0.05, ** means the significance level is P<0.01, ###, *** means the significance level is P<0.001.

[0070] Depend on Figure 6 It can be seen that compared with the sham operation group, the levels of creatinine and urea nitrogen in the serum of mice in the RIRI model control group were significantly increased, indicating that renal ischemia-reperfusion caused significant renal damage in mice, and phloretin can effectively reduce the levels of creatinine and urea nitrogen in serum, and the higher the dose of phloretin, the greater the decrease in creatinine and urea nitrogen levels, indicating that phloretin can effectively improve renal ischemia-reperfusion injury. Based on this, phloretin or its pharmaceutically acceptable salts, esters or solvates can be used to prepare drugs for preventing or treating renal ischemia-reperfusion injury.

[0071] Experiment 7: Detection of renal pathology in mice from Experiment 1

[0072] The kidney tissue fixed in 4% paraformaldehyde in Experiment 1 was immersed in 4% paraformaldehyde for overnight fixation, rinsed with running water for 24 hours, and then dehydrated with ethanol gradient, 70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, 100% ethanol for 1 hour, twice, xylene transparent for 40 minutes, and embedded with a tissue embedding machine (Wuhan Hairui Technology Co., Ltd., HBM-200LF). The tissue was cut into 5 μm thick paraffin sections using a tissue ultrathin slicer (Leica, RM2245), and the sections were dewaxed: xylene for 15 minutes twice, 100% ethanol for 5 minutes, 80% ethanol for 5 minutes, 75% ethanol for 5 minutes, and distilled water for 5 minutes. Hematoxylin staining solution (Zhuhai Beso Biotechnology Co., Ltd., BA4097) was used for staining for 6 minutes, ddH 2 O for 30 seconds, 1% hydrochloric acid for 10 seconds, phosphate buffered saline (PBS) (Beijing Lanjieke Technology Co., Ltd., BL302A) for 3 minutes, ddHO 2 The slides were rinsed in PBS for 30 seconds, then stained with eosin staining solution (Zhuhai Beso Biotechnology Co., Ltd., BA4098) for 2 minutes, rinsed in PBS for 3 times, and then transparentized: anhydrous ethanol for 5 minutes, xylene for 5 minutes, and xylene for 5 minutes. Neutral gum (Servicebio, G1404) was added to the slides and sealed. The sections were observed under an optical microscope and photographed. The results are shown in Figure 7 .

[0073] Depend on Figure 7 It can be seen that compared with the sham operation group, tubulointerstitial damage occurred in the renal tissue of mice in the RIRI model control group, manifested as tubular dilatation and inflammatory cell infiltration (black arrows indicate inflammatory cell infiltration, and red arrows indicate tubular dilatation), and the addition of phloretin can significantly alleviate the above pathological changes, further proving that phloretin can effectively improve the inflammation and renal pathological changes induced by renal ischemia-reperfusion, which is of great significance for the prevention and treatment of renal ischemia-reperfusion.

[0074] In summary, the present application provides the application of phloretin in the preparation of a drug for preventing or treating renal ischemia-reperfusion injury, which belongs to the field of medical application technology. The present application has found through research that phloretin can up-regulate the protein expression level of PAK3 under renal ischemia-reperfusion or hypoxia-reoxygenation conditions and reduce the level of apoptosis. Based on this, phloretin or its pharmaceutically acceptable salt, ester or solvate can not only be used as an activator of PAK3, but also can effectively inhibit apoptosis induced by renal ischemia-reperfusion or hypoxia-reoxygenation, and then play a better preventive and therapeutic effect on renal ischemia-reperfusion injury caused by apoptosis. The present application not only provides a new use for phloretin, but also provides a new drug selection for the activation of PAK3, the inhibition of apoptosis and renal ischemia-reperfusion injury, which has important guiding significance for the prevention and treatment of renal ischemia-reperfusion injury.

[0075] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury.

2. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof according to claim 1 in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury, characterized in that: The renal ischemia-reperfusion injury is renal injury caused by cell apoptosis induced by renal ischemia-reperfusion.

3. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof according to claim 1 in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury, characterized in that: The uses of the phloretin or its pharmaceutically acceptable salt, ester or solvate include: Reduces serum creatinine and urea nitrogen levels; and / or, reducing tubular dilatation and inflammatory cell infiltration; and / or, activation of PAK3.

4. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof according to claim 1 in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury, characterized in that: The effect of phloretin or its pharmaceutically acceptable salt, ester or solvate is dose-dependent.

5. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof according to claim 4 in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury, characterized in that: The dosage of the phloretin or its pharmaceutically acceptable salt, ester or solvate is 1 to 20 mg / kg based on phloretin.

6. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof according to claim 1 in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury, characterized in that: The medicine for preventing or treating renal ischemia-reperfusion injury is a preparation prepared by taking phloretin or its pharmaceutically acceptable salt, ester or solvate as active ingredient and adding pharmaceutically acceptable carrier or excipient.

7. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof according to claim 6 in the preparation of a medicament for preventing or treating renal ischemia-reperfusion injury, characterized in that: The dosage form of the preparation is a liquid preparation, a solid preparation or a semisolid preparation.

8. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a medicament for inhibiting apoptosis of renal tubular epithelial cells induced by renal ischemia-reperfusion.

9. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a medicament for inhibiting apoptosis of renal tubular epithelial cells induced by hypoxia-reoxygenation.

10. Use of phloretin or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a PAK3 activator.

Images