Application of Y27632 in the preparation of drugs that promote repair and / or regeneration after kidney injury

Abstract

This invention relates to the application of Y27632 in the preparation of drugs that promote the repair and / or regeneration of kidney injury, and pertains to the field of pharmaceutical technology. The application of Y27632 in the preparation of drugs that promote the repair and / or regeneration of kidney injury. This invention demonstrates for the first time the significant effect of Y27632 in promoting the repair and regeneration of acute kidney injury in mice, providing strong experimental evidence for the development of novel drugs for the repair and / or regeneration of kidney injury. This invention provides a new and effective means of treating kidney injury, such as acute kidney injury, by using the ROCK signaling pathway inhibitor Y27632. Experiments of this invention confirm that Y27632 can effectively promote the repair and regeneration of kidney injury by inhibiting the ROCK signaling pathway, upregulating SOX-9 expression, activating the endogenous repair mechanism of the kidney, reducing the expression of damage markers, alleviating kidney pathological damage, relieving renal fibrosis, and salvaging kidney function. This has significant clinical application value and broad market prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of Y27632 in the preparation of a medicament that promotes the repair and / or regeneration of kidney injury. Background Technology

[0002] The kidneys are vital excretory organs in the human body, responsible for removing metabolic waste and regulating electrolyte balance, among other important physiological functions. For example, acute kidney injury is a common clinical problem with a complex pathogenesis involving multiple factors such as ischemia, toxins, and immunity. It often leads to a rapid decline in kidney function and can be life-threatening in severe cases.

[0003] Currently, clinical treatments for kidney injury, such as acute kidney injury, are limited, mainly focusing on supportive care and symptomatic treatment, lacking drugs that can effectively promote kidney repair and regeneration. Therefore, developing a drug that can promote kidney repair and regeneration after injury is of significant clinical importance. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this invention provides an application of Y27632 in the preparation of a drug that promotes repair and / or regeneration after kidney injury. The technical solution is as follows:

[0005] Application of Y27632 in the preparation of drugs that promote repair and / or regeneration after kidney injury.

[0006] Optionally, the Y27632 reduces the expression of the kidney injury marker KIM-1.

[0007] Optionally, the Y27632 can reduce pathological damage to the kidneys, alleviate renal fibrosis, and salvage kidney function.

[0008] Optionally, the kidney injury is acute kidney injury.

[0009] Optionally, the chemical formula of Y27632 is as follows:

[0010] .

[0011] A drug that promotes repair and / or regeneration after kidney injury, said drug comprising: Y27632.

[0012] Optionally, the Y27632 reduces the expression of the kidney injury marker KIM-1.

[0013] Optionally, the Y27632 can reduce pathological damage to the kidneys, alleviate renal fibrosis, and salvage kidney function.

[0014] Optionally, the kidney injury is acute kidney injury.

[0015] Optionally, the chemical formula of Y27632 is as follows:

[0016] .

[0017] The beneficial effects of the technical solutions provided by the embodiments of the present invention include at least the following:

[0018] Through relevant experimental design and results, this invention demonstrates for the first time the significant effect of Y27632 in promoting repair and regeneration after acute kidney injury in mice, providing strong experimental evidence for the development of novel drugs for kidney injury repair and / or regeneration.

[0019] This invention provides a novel and effective treatment for kidney injury, such as acute kidney injury, by using the ROCK signaling pathway inhibitor Y27632. Experiments have demonstrated that Y27632 can effectively promote kidney repair and regeneration after injury by inhibiting the ROCK signaling pathway, upregulating SOX-9 expression, activating endogenous renal repair mechanisms, reducing the expression of damage markers, alleviating renal pathological damage, relieving renal fibrosis, and salvaging renal function. This has significant clinical application value and broad market prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0021] Figure 1 This is a graph showing the upregulation of SOX-9 expression in the kidneys of patients with acute kidney injury by Y27632, as provided in Embodiment 1 of the present invention; where blue represents DAPI and red represents SOX-9.

[0022] Figure 2 This is a graph showing how Y27632 reduces the expression of the acute kidney injury marker KIM-1, as provided in Embodiment 1 of the present invention; where blue represents DAPI, green represents the proximal tubular marker LTL, and red represents the acute kidney injury marker KIM-1.

[0023] Figure 3 This is a graph showing the detection results of serum creatinine provided in Embodiment 1 of the present invention;

[0024] Figure 4 This is a graph showing the detection results of urea nitrogen provided in Embodiment 1 of the present invention;

[0025] Figure 5 This is an image of the HE staining results provided in Embodiment 1 of the present invention;

[0026] Figure 6 This is a diagram of the Masson trichrome staining results provided in Embodiment 1 of the present invention;

[0027] Figure 7 This is a COL-1 staining result image provided in Embodiment 1 of the present invention; wherein, blue represents dapi and green represents COL-1;

[0028] Figure 8 This is a Western blotting image of Y27632 inhibiting ROCK1 expression and upregulating SOX-9 expression, provided in Embodiment 1 of the present invention. Detailed Implementation

[0029] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0030] The purpose of this invention is to provide a drug application that utilizes the small molecule compound Y27632, an inhibitor of the ROCK signaling pathway, to promote the repair and / or regeneration of the kidney after injury. By inhibiting the ROCK signaling pathway, upregulating SOX-9 expression, activating the endogenous repair mechanism of the kidney, reducing the expression of the injury marker KIM-1, alleviating kidney pathological damage, relieving renal fibrosis, and salvaging kidney function, the invention aims to promote the repair and regeneration of the kidney after injury.

[0031] The chemical structural formula of Y27632 (Y-27632) is as follows:

[0032] .

[0033] The specific information for Y27632 is shown in Table 1 below:

[0034] Table 1

[0035]

[0036] Example 1

[0037] (a) Experimental materials

[0038] 1. Laboratory animals: Healthy adult male mice (C57BL / 6), weighing approximately 20-25g, were selected and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0039] 2. Drug: ROCK signaling pathway inhibitor Y27632, purchased from MCE, catalog number Y-27632.

[0040] 3. Reagents for detecting the following indicators: Antibodies for detecting SOX-9, KIM-1, and COL-1 expression (SOX-9 primary antibody, Abcam, catalog number: ab185966, species: rabbit); KIM-1 primary antibody, Abcam, catalog number: ab133973, species: rabbit; COL-1 primary antibody, Santa Cruz, catalog number: sc-59772, species: mouse; GAPDH primary antibody, Beyotime Biotechnology, catalog number: AF1186, 1:5000; ROCK1 primary antibody, Qinke Biotechnology, catalog number: AF3895, 1:2000).

[0041] Fluorescent secondary antibody against rabbit primary antibody Alexa Fluor 594 Goat Anti-Rabbit IgG (H+L), nvitrogen, catalog number: A21207;

[0042] (Alexa Fluor 488 Goat Anti-Mouse IgG (H+L), Invitrogen, catalog number: A21202), a fluorescent secondary antibody against mouse primary antibodies.

[0043] Serum creatinine and blood urea nitrogen tests used to assess renal function were performed using a fully automated biochemical analyzer, Vitalab Selectra E, Netherlands;

[0044] The hematoxylin-eosin (HE) staining reagent used for kidney pathology examination was purchased from Solarbio, catalog number G1004.

[0045] Masson's trichrome staining reagent was purchased from Solarbio, product number G1340.

[0046] (II) Construction of an acute kidney injury model

[0047] An acute kidney injury model was established using the classic ischemia-reperfusion injury method. C57BL / 6 mice were anesthetized, and both kidneys were surgically exposed. The renal pedicles were clamped for 28 minutes, then released to restore renal perfusion, thus establishing the acute kidney injury model.

[0048] (III) Drug intervention

[0049] 1. Grouping: Mice were divided into three groups: a healthy control group (NC), a model control group (MC), and a Y27632 intervention group (Y27632-M).

[0050] 2. Administration method: Mice in the Y27632 intervention group were given 10 mg / kg of Y27632 via intraperitoneal injection after renal ischemia-reperfusion injury, while the healthy control group and the model control group were given the same amount of physiological saline.

[0051] 3. Dosage frequency: Once daily for 3 consecutive days.

[0052] (iv) Detection indicators and methods

[0053] 1. Acute phase injury repair and regeneration indicators SOX-9 and KIM-1 (sample taken on day 4 after modeling):

[0054] (1.1) Detection of SOX-9 and KIM-1 expression: Mouse kidney tissue was taken and the expression of SOX-9 and KIM-1 in the kidney tissue was detected by immunofluorescence and other methods. The expression level of each indicator was evaluated by observing the number of positive cells.

[0055] The detection method is as follows: (immunofluorescence assay)

[0056] (1.1.1) Preparation of frozen slices

[0057] Material collection: On day 4 / 28 of modeling, mice were sacrificed, and both kidneys were harvested and fixed overnight in 4% paraformaldehyde.

[0058] Dehydration: Remove the kidney tissue block, absorb the surface liquid with filter paper, and place it in a sucrose solution (10%-30%) for gradient dehydration.

[0059] Embedding: Use filter paper to absorb the liquid on the surface of the tissue block. Place the tissue block with the largest cross section facing down in the center of the frozen section embedding cassette. Then slowly add frozen section embedding medium (OCT) until the entire tissue block is submerged. Float the embedding cassette on liquid nitrogen. When the OCT has completely solidified and turned white, remove the embedding cassette. Wrap the OCT tissue block in aluminum foil (mark the tissue information on the foil beforehand) and quickly transfer it to a -80°C freezer for later use.

[0060] Sectioning: Before sectioning, pre-cool the microtome to -20°C. Add a small amount of OCT to the sample holder, then quickly place the tissue block on the still-unsolidified OCT. After the OCT embedding medium has completely solidified, fix the sample holder onto the microtome. Set the initial section thickness to 15μm and section continuously. After exposing the complete kidney outline, adjust the section thickness to 6μm and section continuously. Adsorb the tissue sections onto anti-detachment glass slides, mark them, and store them in a -80°C freezer for later use.

[0061] (1.1.2) Immunofluorescence staining

[0062] Rewarming: Remove the frozen sections and allow them to warm to room temperature for 30 minutes.

[0063] Washing: Wash with PBS buffer for 5 minutes, repeat 3 times.

[0064] Membrane breakage treatment: 0.5% Triton X-100 for 10 minutes to break the membrane.

[0065] Washing: Wash with PBS buffer for 5 minutes, repeat 3 times.

[0066] Blocking: Dry the PBS buffer, add 40 μL of 10% goat serum, and block at room temperature for 1 hour.

[0067] Primary antibody incubation: Dry the goat serum, prepare the target antibody at an appropriate concentration with 10% goat serum, place it in a humidified chamber, and incubate overnight at 4°C.

[0068] Washing: Rinse with PBS solution for 5 minutes, repeat 3 times.

[0069] Secondary antibody incubation: Aspirate the residual liquid, prepare the corresponding species' fluorescent secondary antibody with PBS, and incubate in a humidified chamber for 2 hours (avoiding light throughout).

[0070] Washing: Rinse with PBS solution for 5 minutes, repeat 3 times.

[0071] Mounting: Blot dry any residual liquid and mount the slide with a DAPI-containing anti-fluorescence quenching mounting medium.

[0072] Observation: Observe, photograph, and evaluate under a fluorescence microscope.

[0073] (1.2) Serum renal function test: Blood samples were collected from mice, and serum creatinine, blood urea nitrogen and other indicators were detected by a biochemical analyzer to assess the recovery of renal function.

[0074] The detection method is as follows: (fully automated biochemical analyzer)

[0075] (1.2.1) Sample collection:

[0076] Blood was collected from the eyes of mice on day 4 of mouse modeling.

[0077] Fasting is usually required when collecting blood samples.

[0078] Avoid eating high-protein foods, as they may affect the test results.

[0079] Avoid strenuous exercise to prevent excessive muscle breakdown and the production of creatinine, which can interfere with the accuracy of serum creatinine levels.

[0080] (1.2.2) Sample processing:

[0081] After standing at room temperature for half an hour, centrifuge at 15,000 rpm for 10 minutes.

[0082] 200 μL of serum was collected to detect serum creatinine and uremic nitrogen, which was performed using a fully automated biochemical analyzer.

[0083] (1.3) Kidney pathological examination: HE staining of kidney tissue was performed to observe pathological changes in kidney tissue, such as degeneration, necrosis and shedding of renal tubular epithelial cells, glomerular atrophy, etc., and to assess the degree of kidney pathological damage.

[0084] The specific method is as follows:

[0085] (1.3.1) Preparation of paraffin sections

[0086] Sample collection: After routine disinfection and anesthesia, the kidneys of both sides of the mice were removed and immediately fixed overnight with 4% paraformaldehyde solution.

[0087] Sample collection time: Samples were collected on the 3rd and 28th day after modeling for HE / PAS and Masson staining.

[0088] Tissue dehydration and transparency:

[0089] Tissue dehydration:

[0090] 70% ethanol: 30 minutes;

[0091] 80% ethanol: 30 minutes;

[0092] 90% ethanol: 30 minutes;

[0093] 95% ethanol I: 30 minutes;

[0094] 95% ethanol II: 30 minutes;

[0095] Anhydrous ethanol I: 60 minutes;

[0096] Anhydrous ethanol II: 60 minutes;

[0097] Organizational transparency:

[0098] Xylene I: 15 minutes;

[0099] Xylene II: 15 minutes;

[0100] Wax impregnation and embedding:

[0101] After the clearing process is completed, the xylene on the surface of the remaining tissue block is dried and then immersed in liquid paraffin at 65°C for 1-2 hours.

[0102] After thorough paraffin impregnation, place the tissue block with its largest cross-section facing down at the bottom of the iron embedding mold. Pour paraffin wax to submerge the tissue block, adjust its position to be centered in the mold, and place it at freezing point for rapid cooling and fixation. Then, place the embedding cassette on top of the tissue block and pour in an appropriate amount of paraffin wax to ensure tight adhesion between the tissue and the embedding cassette. Transfer to a freezing stage to cool. After the paraffin wax has fully solidified, remove the mold, trim the wax block, and store at room temperature.

[0103] (1.3.2) Slicing, rinsing, and baking:

[0104] Before slicing, place the wax block in ice water for 30 minutes to moisten it and make it easier to cut.

[0105] Install the blade and adjust it to the appropriate position and angle, fix the paraffin block, set the initial section thickness to 15 μm, and quickly and continuously slice until the complete kidney structure is exposed.

[0106] Adjust the section thickness to 4μm (note the need for humidification), slice continuously and place them in a spreader (preheated to 42℃) for full development.

[0107] Using a tissue-resistant slide, lift the flattened section and try to center it on the slide. Gently tap off any water, then transfer it to a slide oven and bake for 3-6 hours (preheat the slide oven to 65°C).

[0108] Transfer the slide to the slide box and store at room temperature for later use.

[0109] (1.3.3) HE staining

[0110] Dewaxing and rehydration:

[0111] Tissue dewaxing:

[0112] Xylene I: 10 minutes;

[0113] Xylene II: 10 minutes;

[0114] Organ rehydration:

[0115] Anhydrous ethanol I: 5 minutes;

[0116] Anhydrous ethanol II: 5 minutes;

[0117] 95% ethanol: 5 minutes;

[0118] 90% ethanol: 5 minutes;

[0119] 85% ethanol: 5 minutes;

[0120] 80% ethanol: 5 minutes;

[0121] 75% ethanol: 5 minutes;

[0122] Rinse with tap water for 5 minutes, then soak in distilled water for 5 minutes.

[0123] Staining with hematoxylin and violet solution: 5 minutes, then rinse with tap water.

[0124] 0.5% hydrochloric acid alcohol differentiation: 2-5 seconds.

[0125] Ammonia solution turns blue: 5 minutes.

[0126] Staining with eosin solution: 3-5 minutes.

[0127] Rinse with distilled water for 5 seconds.

[0128] Dehydrated, transparent:

[0129] Tissue dehydration:

[0130] 75% ethanol: 1 second;

[0131] 80% ethanol: 1 second;

[0132] 85% ethanol: 1 second;

[0133] 90% ethanol: 1 second;

[0134] 95% ethanol: 1 second;

[0135] Anhydrous ethanol I: 1 second;

[0136] Anhydrous ethanol II: 1 second;

[0137] Organizational transparency:

[0138] Xylene I: 15 minutes;

[0139] Xylene II: 15 minutes;

[0140] Neutral resin sealing: xylene air-dried in a fume hood.

[0141] Observation: Observe the staining under an inverted phase contrast microscope.

[0142] 2. Chronic fibrosis markers (sample taken on day 28 post-modeling):

[0143] (2.1) Masson trichrome staining: Masson trichrome staining was performed on kidney tissue to observe the deposition of collagen fibers and assess the degree of renal fibrosis.

[0144] The specific method is as follows:

[0145] (2.1.1) Preparation of paraffin sections: Same as HE staining;

[0146] (2.1.2) Sectioning, slide preparation, and slide copying: Same as HE staining;

[0147] (2.1.3) Masson's trichrome staining

[0148] Dewaxing and rehydration:

[0149] Tissue dewaxing:

[0150] Xylene I: 10 minutes;

[0151] Xylene II: 10 minutes;

[0152] Organ rehydration:

[0153] Anhydrous ethanol I: 5 minutes;

[0154] Anhydrous ethanol II: 5 minutes;

[0155] 95% ethanol I: 5 minutes;

[0156] 95% Ethanol II: 5 minutes;

[0157] 70% ethanol: 5 minutes;

[0158] Staining with Weigert's iron violet solution: Stain for 5-10 minutes.

[0159] Acidic ethanol differentiation: 5-15 seconds, rinse with running water for 3 minutes.

[0160] Masson's blue solution staining: stain for 3-5 minutes, then rinse with running water for 3 minutes.

[0161] Staining with a combination of Pomfret and magenta: Drip staining for 5-10 minutes.

[0162] Staining with weak acid working solution: Stain for 1 minute.

[0163] Staining with phosphomolybdic acid solution: Stain for 1-2 minutes.

[0164] Staining with weak acid working solution: Stain for 1 minute.

[0165] Aniline blue solution staining: Stain for 1-2 minutes.

[0166] Dehydrated, transparent:

[0167] Tissue dehydration:

[0168] 70% ethanol: 5 minutes;

[0169] 80% ethanol: 5 minutes;

[0170] 90% ethanol: 5 minutes;

[0171] 95% ethanol I: 5 minutes;

[0172] 95% Ethanol II: 5 minutes;

[0173] Anhydrous ethanol I: 5 minutes;

[0174] Anhydrous ethanol II: 5 minutes;

[0175] Organizational transparency:

[0176] Xylene I: 15 minutes;

[0177] Xylene II: 15 minutes;

[0178] Neutral resin sealing: xylene air-dried in a fume hood.

[0179] Observation: Observe the staining under an inverted phase contrast microscope.

[0180] (2.2) COL-1 expression detection: Mouse kidney tissue was collected, and the expression of COL-1 in the kidney tissue was detected by immunofluorescence and other methods. The expression level of each indicator was evaluated by observing the positive area. The detection method was the same as that for SOX-9 and KIM-1.

[0181] 3. Mechanism Investigation: Western Blot Method

[0182] (3.1) Extraction of total protein from kidney tissue:

[0183] Prepare protein lysis buffer: Prepare sufficient protein lysis buffer with a RIPA:PMSF ratio of 100:1.

[0184] Sample collection: Take out the kidney tissue frozen in liquid nitrogen, freeze and thaw it on ice, and weigh 50 mg into a 1.5 mL EP tube.

[0185] Lysis: Add 500 μL of protein lysis buffer, and use sterilized ophthalmic scissors to thoroughly mince the tissue before adding another 500 μL of protein lysis buffer. Lyse on ice for 30 minutes, vortexing once every 5 minutes to ensure complete lysis.

[0186] Centrifugation: Transfer the EP tube to a centrifuge that has been pre-cooled to 4°C, centrifuge at 12,000 rpm for 15 minutes, collect the supernatant and label it.

[0187] (3.2) Protein sample concentration determination and denaturation:

[0188] Concentration determination: A standard curve was constructed and sample concentration was determined. 3 μL of sample protein solution was added to 27 μL of distilled water to prepare the protein dilution buffer. BCA working solution was prepared at a ratio of A:B = 50:1. 10 μL of protein standard solution and 10 μL of sample protein dilution buffer were added to a 96-well plate, followed by 200 μL of BCA working solution. The plate was incubated at 37°C for 30 minutes. The absorbance at 450 nm was read using a microplate reader to plot the standard curve, thereby calculating the concentration of the protein to be tested.

[0189] Denaturation: Add an appropriate amount of loading denaturation buffer 5xSDS to the remaining sample protein solution (sample protein solution: 5xSDS = 3:1), place it in boiling water above 95℃ for 5-10 minutes to fully denature it, and after denaturation, quickly place the protein in liquid nitrogen for rapid freezing and store it in a freezer at -80℃ for later use.

[0190] (3.3) SDS-PAGE gel electrophoresis:

[0191] Gel preparation: Prepare separating and stacking gels according to the molecular weight of the target protein. See Tables 2 and 3 for specific formulations.

[0192] Table 2 SDS-PAGE separating gel formulation

[0193]

[0194]

[0195] Table 3 SDS-PAGE Stacking Gel Formulation

[0196]

[0197] Protein loading: After the gel solidifies, fix the gel plate onto the electrophoresis clamp and place it in the electrophoresis tank. Add freshly prepared 1xRunning buffer to the inner tank until overflowing, and add recycled 1xRunning buffer to the outer tank. Evenly pull out the electrophoresis gel combs and load proteins according to the principle of equal mass (20-50 μg). Then, adjust the loading volume to 20 μL with 1xSDS solution. Add 3 μL of protein marker to each of the comb wells on both sides to determine the location of the target protein band.

[0198] Electrophoresis: Connect the corresponding electrodes, set the initial constant voltage to 80V to start electrophoresis, and when the sample reaches the separating gel, switch to a constant voltage of 120V to continue electrophoresis until bromophenol blue is observed to be close to the bottom of the glass plate. Then turn off the power and stop electrophoresis. The whole process takes about 1.5 hours.

[0199] (3.4) Transfer:

[0200] Prepare the transfer buffer: Prepare a 1x transfer buffer containing 20% ​​methanol and pre-cool it at 4°C for later use.

[0201] Materials preparation: Open the transfer clamp, moisten the sponge and filter paper inside with 1x transfer buffer, immerse the PVDF membrane in formaldehyde for 1 minute to activate it, and place it in the transfer solution for later use.

[0202] Transfer procedure: Remove the electrophoresis gel, peel off one side of the glass plate, use a scraper to remove the stacking gel, free the separating gel, and place it in the center of the filter paper on the negative electrode side. Then, place the activated PVDF membrane on top of the gel (be careful to remove air bubbles), and cover it layer by layer with filter paper and sponge. Close the transfer clamp and fix it in the transfer tank. Add sufficient 1x transfer buffer to the transfer tank and place the entire transfer tank in ice water to prevent overheating. Connect the electrodes, set a constant voltage of 100V, and transfer for 2 hours.

[0203] (3.5) Enclosure and Incubation

[0204] Blocking: After the transfer, the PVDF membrane was transferred to a 5% skim milk blocking solution (the blocking solution was prepared with 1x TBST solution) and blocked on a shaker at low speed for 2 hours.

[0205] Incubation of primary antibody:

[0206] Membrane removal: After sealing, remove the PVDF membrane, cut appropriate strips according to the position of the target strip, and mark them.

[0207] Incubation: Then place the strip in the appropriate primary antibody dilution solution (prepare the primary antibody dilution solution according to the instructions) and incubate overnight at 4°C.

[0208] Incubation of secondary antibodies:

[0209] Washing: After primary antibody incubation, the bands were washed three times with 1x TBST solution for 10 minutes each time.

[0210] Incubation: Then place the strips in the corresponding species' secondary antibody dilution solution (dilution concentration as per the instructions) and incubate at room temperature for 2 hours.

[0211] exposure:

[0212] Washing: After the secondary antibody incubation, the bands were washed three times with 1xTBST solution for 10 minutes each time.

[0213] Prepare the luminescent solution: Mix equal volumes of solution A and solution B to prepare the ECL luminescent solution (avoid light).

[0214] Exposure: The PVDF film is exposed on a gel imaging system.

[0215] Analysis: ImageJ software can be used to perform grayscale analysis on protein expression.

[0216] (V) Experimental Results

[0217] 1. Acute phase injury repair and regeneration indicators

[0218] (1.1) Results of SOX-9 and KIM-1 expression detection:

[0219] The results of SOX-9 expression detection can be found in [link to relevant documentation]. Figure 1 ,from Figure 1 As can be seen, Sox9 begins to rise 24 hours after AKI, and Y27632 can further increase Sox9.

[0220] The KIM-1 expression detection results are shown below. Figure 2 ,from Figure 2As can be seen, blue represents the cell nucleus (DAPI), green represents the proximal renal tubular epithelial cell marker (LTL), and red represents the acute kidney injury marker (KIM-1). On the third day after AKI, the renal injury markers were significantly reduced after treatment with Y27632.

[0221] (1.2) Serum renal function test results:

[0222] Serum creatinine and blood urea nitrogen test results are as follows Figure 3 and Figure 4 As shown. From Figure 3 and Figure 4 The test results show that serum renal function in the model group deteriorated sharply on days 1-3 and gradually decreased on day 7. The Y27632 intervention group can effectively alleviate the deterioration of renal function.

[0223] (1.3) Kidney pathology examination results:

[0224] HE staining was performed on kidney tissue to observe pathological changes. The staining results are as follows: Figure 5 As shown. From Figure 5 As can be seen, the control group had normal kidney structure without damage; the model group showed severe kidney pathological damage, mainly manifested as: disappearance of the brush border of the proximal renal tubules, degeneration, necrosis and shedding of renal tubular epithelial cells, dilation or atrophy of the renal tubular lumen, formation of a large number of casts, and a small number of inflammatory cells infiltrating the renal interstitium; the Y27632 intervention group showed significant repair effect, with occasional new rearrangement phenomenon.

[0225] 2. Chronic fibrosis marker results

[0226] (2.1) Results of Masson trichrome staining

[0227] Masson's trichrome staining was performed on kidney tissue to observe collagen fiber deposition. The staining results are as follows: Figure 6 As shown. From Figure 6 As can be seen, collagen fibers appear blue in Masson staining, while other tissue components appear red or pink. No significant collagen deposition was observed in the control group, while the model group showed more collagen fiber deposition and a higher degree of fibrosis. In the Y27632 intervention group, the blue collagen fiber deposition was significantly reduced, indicating a lower degree of fibrosis.

[0228] (2.2) Results of COL-1 staining

[0229] COL-1 staining was performed on kidney tissue to assess the degree of renal fibrosis. The staining results are as follows: Figure 7 As shown. From Figure 7 As can be seen, the results are consistent with those of masson staining.

[0230] 3. The impact of Y27632 on the ROCK signal path

[0231] On the third day after modeling, Western blot analysis was performed on kidney tissue to assess the effect of Y27632 on the ROCK pathway. The results are as follows: Figure 8 As shown. From Figure 8 As can be seen, Y27632 can significantly inhibit the ROCK signaling pathway and upregulate SOX-9 expression.

[0232] In summary, the results show that the Y27632 intervention group can inhibit the ROCK signaling pathway, upregulate SOX-9 expression, activate the endogenous repair mechanism of the kidney, reduce the expression of damage markers, alleviate kidney pathological damage, relieve renal fibrosis, salvage kidney function, and promote repair and regeneration after acute kidney injury.

[0233] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of Y27632 in the manufacture of a medicament for promoting regeneration after acute kidney injury, characterized in that, The chemical formula of the Y27632 is as follows:

2. Use according to claim 1, characterized in that, The Y27632 reduces the expression of a kidney injury marker KIM-1, up-regulates SOX-9 expression, and activates an endogenous repair mechanism in the kidney.

3. Use according to claim 1, characterized in that, The Y27632 reduces kidney pathological injury, relieves kidney fibrosis, and rescues kidney function.

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