A pyrazole compound for treating renal fibrosis and its application

Abstract

The present invention relates to a pyrazole compound and its use in the preparation of a drug for treating renal fibrosis. The small molecule AK compound of the present invention can reduce renal interstitial collagen deposition, significantly reduce the expression levels of fibrosis indicators FN, Col1a1, and α-SMA, effectively inhibit renal fibrosis, and delay and / or reverse the course of chronic kidney disease without obvious toxic side effects. Therefore, the AK compound of the present invention has broad clinical application prospects as a therapeutic agent for renal fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to a pyrazole compound for treating renal fibrosis. Background Art

[0002] In recent years, with the increasing number of aging individuals and those suffering from diabetes, obesity, and hypertension, chronic kidney disease (CKD) has been on the rise. According to incomplete statistics, 850 million people worldwide suffer from CKD. If CKD is not treated promptly and effectively, it can eventually progress to end-stage renal disease (ESRD), requiring long-term renal replacement therapy or kidney transplantation.

[0003] Renal fibrosis is a common pathological feature of all advanced chronic kidney diseases, primarily characterized by renal tissue remodeling and loss of renal function due to excessive deposition of the extracellular matrix (ECM). It is generally believed that overactivation of the renal fibrotic response is the primary cause of irreversible renal function loss. Blocking the activation of the renin-angiotensin-aldosterone system (RAAS) is a clinical treatment approach that can slow the progression of renal fibrosis. Currently, RAAS blockade drugs used in clinical practice primarily include angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs), both of which are recommended first-line treatments for CKD patients with proteinuria. Multiple randomized controlled clinical trials have demonstrated that RAAS blockers can effectively delay renal function loss and reduce mortality in CKD patients while controlling blood pressure and proteinuria. However, the use of ACE inhibitors and ARBs can easily lead to hyperkalemia and acute renal failure in patients with mid- to late-stage CKD. Their use is limited in pregnant women, those with renal artery stenosis, and those without hypertension. Furthermore, they can only partially slow the progression of renal fibrosis. With the rapid increase in the prevalence of CKD in recent years, the development of safer and more effective anti-renal fibrosis drugs is urgent. Therefore, the search for renal-protective drugs that can reduce renal fibrosis and delay the progression of chronic kidney disease is of great clinical significance. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies in the prior art and to provide a novel pyrazole compound for treating renal fibrosis.

[0005] Based on the above findings, the technical solutions proposed by the present invention to solve the above technical problems are as follows.

[0006] The first aspect of the present invention provides a compound for preventing or treating renal fibrosis, wherein the compound structure is shown in formula (I):

[0007]

[0008] The second aspect of the present invention provides a pharmaceutical composition for preventing or treating renal fibrosis, which comprises the compound of formula (I) and pharmaceutically acceptable excipients.

[0009] Preferably, the pharmaceutical composition further comprises other drugs for treating renal fibrosis.

[0010] Preferably, the pharmaceutically acceptable excipient is selected from one or more of fillers, disintegrants, binders, lubricants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH regulators, osmotic pressure regulators, surfactants, coating materials, antioxidants, flavoring agents, and colorants.

[0011] Preferably, the pharmaceutical composition is in the form of tablets, capsules, granules, pills, oral liquids, injections, patches, gels or ointments.

[0012] Preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 1-20 μM. More preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 10-20 μM. Further preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 16 μM.

[0013] The third aspect of the present invention provides use of the compound of formula (I) or the pharmaceutical composition in the preparation of a medicament for preventing or treating renal fibrosis.

[0014] Preferably, the renal fibrosis is renal fibrosis caused by renal ischemia-reperfusion.

[0015] Preferably, the concentration of the compound of formula (I) in the drug is 1-20 μM. More preferably, the concentration of the compound of formula (I) in the drug is 10-20 μM. Further preferably, the concentration of the compound of formula (I) in the drug is 16 μM.

[0016] Preferably, the single dose of the compound of formula (I) is 10-50 mg / kg body weight. More preferably, the single dose of the compound of formula (I) is 40 mg / kg body weight.

[0017] The fourth aspect of the present invention provides use of the compound of formula (I) or the pharmaceutical composition in the preparation of a medicament for treating chronic kidney disease.

[0018] Preferably, the chronic kidney disease is a chronic kidney disease with renal fibrosis as a pathological feature.

[0019] Preferably, the concentration of the compound of formula (I) in the drug is 1-20 μM. More preferably, the concentration of the compound of formula (I) in the drug is 10-20 μM. Further preferably, the concentration of the compound of formula (I) in the drug is 16 μM.

[0020] Preferably, the single dose of the compound of formula (I) is 10-50 mg / kg body weight. More preferably, the single dose of the compound of formula (I) is 40 mg / kg body weight.

[0021] The fifth aspect of the present invention provides use of the compound of formula (I) or the pharmaceutical composition in the preparation of a drug for treating renal tubular epithelial cell fibrosis.

[0022] Preferably, the renal tubular epithelial cells are human renal tubular epithelial cells HK2.

[0023] Preferably, the renal tubular epithelial cell fibrosis is TGF-β1-induced renal tubular epithelial cell fibrosis.

[0024] Preferably, the concentration of the compound of formula (I) in the drug is 1-20 μM. More preferably, the concentration of the compound of formula (I) in the drug is 10-20 μM. Further preferably, the concentration of the compound of formula (I) in the drug is 16 μM.

[0025] Preferably, the single dose of the compound of formula (I) is 10-50 mg / kg body weight. More preferably, the single dose of the compound of formula (I) is 40 mg / kg body weight.

[0026] The sixth aspect of the present invention provides the use of the above-mentioned compound of formula (I) or the above-mentioned pharmaceutical composition in the preparation of a drug for reducing the expression level of a renal tubular epithelial cell fibrosis indicator, wherein the renal tubular epithelial cell fibrosis indicator is selected from one or more of fibronectin (FN), type I collagen (Col1a1) and α-smooth muscle actin (α-SMA).

[0027] Preferably, the reducing the expression level of renal tubular epithelial cell fibrosis indicators is to simultaneously reduce the expression levels of fibronectin (FN), type I collagen (Col1a1) and α-smooth muscle actin (α-SMA).

[0028] Preferably, the concentration of the compound of formula (I) in the drug is 1-20 μM. More preferably, the concentration of the compound of formula (I) in the drug is 10-20 μM. Further preferably, the concentration of the compound of formula (I) in the drug is 16 μM.

[0029] Preferably, the single dose of the compound of formula (I) is 10-50 mg / kg body weight. More preferably, the single dose of the compound of formula (I) is 40 mg / kg body weight.

[0030] Beneficial effects of the present invention:

[0031] The inventors unexpectedly discovered that the AK compound of the present invention can reduce the expression levels of fibrosis markers FN, Col1a1, and α-SMA in animal models of renal fibrosis and in renal tubular epithelial cells, improve renal tissue pathology in animals with renal fibrosis, inhibit renal interstitial collagen deposition, effectively alleviate renal fibrosis, and delay and / or reverse the course of chronic kidney disease, all without significant toxic side effects. Therefore, the AK compound of the present invention has broad clinical application prospects as a therapeutic agent for renal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison of the effects of different concentrations of AK compounds on HK2 cell viability;

[0033] Figure 2 Western Blot image showing that AK compound inhibits the fibrotic response of renal tubular epithelial cells induced by repeated hypoxia and reoxygenation;

[0034] Figure 3 Real-time PCR images of AK compounds inhibiting the fibrosis response of renal tubular epithelial cells induced by repeated hypoxia and reoxygenation;

[0035] Figure 4 The immunofluorescence image shows the expression of α-SMA in renal tubular epithelial cells in response to repeated hypoxia-reoxygenation induced by AK compounds;

[0036] Figure 5 Western Blot image showing that AK compounds inhibit TGF-β1-induced fibrosis in renal tubular epithelial cells;

[0037] Figure 6 Real-time PCR images of AK compounds inhibiting TGF-β1-induced fibrosis in renal tubular epithelial cells;

[0038] Figure 7 The immunofluorescence images show the expression of α-SMA in the fibrosis response of renal tubular epithelial cells induced by AK compounds;

[0039] Figure 8 The figure shows the effect of AK compound on the levels of blood creatinine and urea nitrogen in mouse serum. Figure 8 A is urea nitrogen, Figure 8 B is blood creatinine;

[0040] Figure 9 The effect of AK compounds on ALT and AST levels in mouse serum is shown in Figure 2. Figure 9 A is alanine aminotransferase, Figure 9 B is aspartate aminotransferase;

[0041] Figure 10 The results of staining the heart, liver, spleen, lung and kidney tissues of mice in the simple drug group using the hematoxylin and eosin (H&E) staining kit;

[0042] Figure 11 The staining image of renal interstitial collagen deposition in the mouse renal fibrosis model after administration of different concentrations of AK compounds;

[0043] Figure 12 The Western Blot images of Col1a1 and α-SMAW in the mouse renal fibrosis model after administration of different concentrations of AK compounds;

[0044] Figure 13 Figure 2 is the fibrosis index FN, Col1a1 and α-SMAReal-time PCR graph in the mouse renal fibrosis model after administration of different concentrations of AK compounds;

[0045] Figure 14 is the nuclear magnetic resonance spectrum of the AK compound of the present invention;

[0046] Figure 15 is the mass spectrum of compound AK of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below with reference to experimental examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the significance symbols of P values ​​in the experimental examples and accompanying drawings of this application are defined as follows: *P<0.05, **P<0.01, ***P<0.001.

[0048] Compounds of formula (I) used in the test examples of the present invention Purchased from Shanghai Taoshu Biotechnology Co., Ltd., product number AK-968 / 36977265, referred to as "AK compound" in the test examples in the manual, and its structure confirmation spectrum is as follows Figure 14-15 shown.

[0049] Experimental Example 1: Inhibitory effect of AK compound on renal tubular epithelial cell fibrosis induced by repeated hypoxia-reoxygenation in vitro

[0050] 1. Test method

[0051] 1.1. CCK8 method

[0052] Human renal tubular epithelial cells (HK2) were seeded in 96-well plates at a seeding density of approximately 4000 cells / well. After the cells adhered, the culture medium was aspirated and replaced with AK solution containing different concentrations (AK concentrations were 5, 10, 20, 40, 80, 160, 320, and 640 μM). A culture medium without AK was used as the normal control group (NC). After 24 hours of culture, 10 μL of CCK8 solution was added to each well. After 2 hours of CCK8 solution, the OD value of each well was measured at 450 nm using a microplate reader and the results were recorded. Cell viability was plotted against dose. Cell viability = (OD value of cells in the experimental group - OD value of cells in the blank group) / (OD value of cells in the control group - OD value of cells in the blank group) × 100%.

[0053] 1.2 Western Blot

[0054] HK2 cells in the logarithmic growth phase were seeded in 6-well plates at a seeding density of approximately 1.0 × 10 5 Cells were divided into normal group (Normoxia), AK alone group (AK 16μM), repeated hypoxia-reoxygenation model group (HR), low-dose AK group (HR+AK 4μM), medium-dose AK group (HR+AK 8μM), and high-dose AK group (HR+AK 16μM). The HR model specifically used 0.5% low-glucose medium for 9 hours of hypoxia followed by 3 hours of reoxygenation, repeating the hypoxia-reoxygenation cycle three times. After the hypoxia-reoxygenation cycle, cells were washed three times with PBS, and total protein was extracted. The expression of fibrosis indicators Col1a1 and α-SMA proteins was detected by Western Blot and quantitatively analyzed. The experiment was repeated three times for each group.

[0055] 1.3 Real-time PCR

[0056] HK2 cells were seeded in 12-well plates at a density of approximately 0.5 × 10 5 Cells were divided into normal control group (Normoxia), AK alone group (AK 16μM), repeated hypoxia-reoxygenation model group (HR), low-dose AK group (HR + AK 4μM), medium-dose AK group (HR + AK 8μM), and high-dose AK group (HR + AK 16μM). The HR model used 0.5% low-glucose medium for 9 hours of hypoxia followed by 3 hours of reoxygenation. After the hypoxia-reoxygenation period, cells were washed three times with PBS, and RNA was collected, reverse transcribed, and amplified. The experiment was repeated three times for each group.

[0057] 1.4 Immunofluorescence

[0058] HK2 cells in the logarithmic growth phase were seeded in a 6-well plate with a glass slide at a seeding density of approximately 1.0 × 10 5 Cells were divided into normal group (Normoxia), group with AK alone (AK 16μM), repeated hypoxia-reoxygenation model group (HR), low-dose AK group (HR+AK 4μM), medium-dose AK group (HR+AK 8μM), and high-dose AK group (HR+AK 16μM). The cells were incubated for 24 hours, starved with serum-free medium for 12 hours, and then replaced with 0.5% low-glucose medium for hypoxia-reoxygenation. After the end of hypoxia-reoxygenation, the cells were washed three times with PBS, fixed with paraformaldehyde for 10 minutes, washed three times with PBS, blocked with 10% BSA for 0.5 hours, washed three times with PBS, and then the primary antibody was added. After incubation for 24 hours, the primary antibody was washed away, and the fluorescent secondary antibody was added. After incubation in the dark for 1.5 hours, the secondary antibody was washed away, and DAPI staining solution was added for 10 minutes. Anti-fluorescence quencher was added, the slides were mounted, and the slides were observed under an inverted fluorescence microscope. The experiment was repeated three times for each group.

[0059] 2. Test results

[0060] The results of CCK-8 assay are as follows Figure 1 As shown, at concentrations of 4-640 μM, there was no significant difference in cell survival rate between the AK group and the normal control group (NC), indicating that the AK compound had no obvious toxicity.

[0061] Western Blot results are as follows Figure 2 As shown in the results (using β-actin as an internal reference), the protein expression levels of fibrosis indicators Col1a1 and α-SMA in HK2 cells treated with repeated hypoxia and reoxygenation were significantly inhibited after treatment with AK compounds, and the effect was most obvious in the high-dose group, suggesting that AK compounds can inhibit the cell fibrosis response caused by repeated hypoxia and reoxygenation.

[0062] Real-time PCR results are as follows Figure 3 As shown in the data, the mRNA levels of FN, Col1a1 and α-SMA in the model group were significantly upregulated after repeated hypoxia and reoxygenation treatment. This phenomenon was improved after treatment with AK compound, and the effect was most obvious in the high-dose group, suggesting that AK compound can inhibit the cell fibrosis response caused by repeated hypoxia and reoxygenation.

[0063] Immunofluorescence results Figure 4 As shown, AK compounds significantly reduced the expression of α-SMA in HK2 cells induced by repeated hypoxia and reoxygenation in a concentration-dependent manner, suggesting that AK compounds can effectively inhibit the cell fibrosis response caused by repeated hypoxia and reoxygenation.

[0064] Experimental Example 2: Inhibitory effect of AK compound on TGF-β1-induced fibrosis in renal tubular epithelial cells in vitro

[0065] 1. Test method

[0066] 1.1、Western Blot

[0067] HK2 cells in the logarithmic growth phase were seeded in 6-well plates at a seeding density of approximately 1.0 × 10 5 Cells were divided into a normal group (NC), a group with AK alone (AK 16μM), a model group (TGF-β 120ng / ml), a low-dose AK group (TGF-β 120ng / ml + AK 4μM), a medium-dose AK group (TGF-β 120ng / ml + AK 8μM), and a high-dose AK group (TGF-β 120ng / ml + AK 16μM). The cells were incubated for 24 hours, starved for 12 hours with serum-free medium, and then stimulated with the drug. Cultures were continued for another 24 hours. The cells were washed three times with PBS, harvested, and total protein was extracted. The expression levels of the fibrosis markers Col1a1 and α-SMA were analyzed by Western blotting and quantified. The experiment was repeated three times for each group.

[0068] 1.2 Real-time PCR

[0069] HK2 cells were seeded in 12-well plates at a density of approximately 0.5 × 10 5 Cells were divided into a normal group (NC), an AK alone group (AK 16μM), a model group (TGF-β 120ng / ml), a low-dose AK group (TGF-β 120ng / ml + AK 4μM), a medium-dose AK group (TGF-β 120ng / ml + AK 8μM), and a high-dose AK group (TGF-β 120ng / ml + AK 16μM). Cells were incubated for 24 hours, starved for 12 hours with serum-free medium, and then stimulated with the drug. Cultures were continued for another 24 hours. Cells were washed three times with PBS, and RNA was harvested, reverse transcribed, and amplified. The experiment was repeated three times for each group.

[0070] 1.3 Immunofluorescence

[0071] HK2 cells in the logarithmic growth phase were seeded in a 6-well plate with a glass slide at a seeding density of approximately 1.0 × 10 5The cells were divided into normal group (NC), AK alone group (AK 16μM), model group (TGF-β120ng / ml), low-dose AK group (TGF-β120ng / ml+AK 4μM), medium-dose AK group (TGF-β120ng / ml+AK 8μM) and high-dose AK group (TGF-β120ng / ml+AK 16μM), and incubated for 24 hours. After starvation with serum-free medium for 12 hours, stimulation and drugs were added and cultured for another 24 hours. Wash three times with PBS, fix with paraformaldehyde for 10 minutes, wash three times with PBS, add 10% BSA to block for 0.5 hours, wash three times with PBS, add primary antibody, incubate for 24 hours and then wash away the primary antibody, add fluorescent secondary antibody, incubate in the dark for 1.5 hours and then wash away the secondary antibody, add DAPI staining solution and incubate in the dark for 10 minutes, add anti-fluorescence quencher, seal the slides, observe under a fluorescence inverted microscope, and take pictures. Repeat the experiment three times for each group.

[0072] 2. Test results

[0073] Western Blot results are as follows Figure 5 As shown in the results, AK compound significantly downregulated the expression levels of fibrotic proteins Col1a1 and α-SMA in HK2 cells induced by TGF-β1. Comparison of the experimental groups revealed that the high-dose group had the most significant inhibitory effect.

[0074] Real-time PCR results are as follows Figure 6 As shown, AK compound significantly downregulated the expression levels of Col1a1 and FN, the cell fibrosis indicators induced by TGF-β1, and the inhibitory effect was most obvious in the high-dose group.

[0075] Immunofluorescence results Figure 7 As shown, AK compound significantly reduced the expression of α-SMA in HK2 cells induced by TGF-β1, and the inhibitory effect was best at high doses, suggesting that AK compound can effectively inhibit the cell fibrosis response caused by TGF-β1.

[0076] The above results suggest that AK compounds can effectively inhibit TGF-β1 and cell fibrosis response caused by repeated hypoxia and reoxygenation.

[0077] Experimental Example 3: Effects of AK Compound on Heart, Liver, Spleen, Lung, and Kidney Tissues in Untreated Mice

[0078] 1. Test method

[0079] Healthy male C57BL / 6J mice (20-22 g) aged 6-8 weeks were selected and housed in a pathogen-free environment (23±2°C and 55±5% humidity) with free access to food and water under a 12-hour light / 12-hour dark cycle.

[0080] To establish a simple AK mouse model, six mice were gavaged daily with the drug, 24 hours apart, at a dose of 40 mg / kg body weight. Serum and kidney tissue samples were collected under anesthesia 14 days later. Serum creatinine and urea nitrogen levels were measured using assays for creatinine, urea nitrogen, alanine aminotransferase, and aspartate aminotransferase (AST) according to the manufacturer's instructions. Heart, liver, spleen, lung, and kidney samples were harvested, embedded, and stained with hematoxylin and eosin.

[0081] (1) The method for determining creatinine is as follows:

[0082]

[0083] Creatinine content (μmol / L) = [(Assay A2-K*Assay A1)-(Blank A2-K*Blank A1)] / [(Standard A2-K*Standard A1)-(Blank A2-K*Blank)]*Standard concentration (442 μmol / L)

[0084] Note: Dilution factor K = (sample volume + enzyme solution A volume) / (sample volume + enzyme solution A volume + enzyme solution B volume) = 186 / 246

[0085] (2) Urea nitrogen test method is as follows:

[0086]

[0087]

[0088] Urea nitrogen content (mmol / L) = (measured OD value - blank measured value) / (standard OD value - blank OD value) * standard concentration (10mmol / L) * sample dilution multiple before testing

[0089] (3) The test method for alanine aminotransferase is as follows:

[0090]

[0091] Calculation method for serum (plasma) and other liquid samples:

[0092] ALT activity (U / L) = Substitute into the standard curve to obtain ALT activity (Karmen's unit) * 0.482 * N

[0093] Where, 0.482 is the conversion from Karman's unit to U / L; N is the dilution factor of the sample before testing.

[0094] (4) The test method for aspartate aminotransferase is as follows:

[0095]

[0096]

[0097] Calculation method for serum (plasma) and other liquid samples:

[0098] AST activity (U / L) = AST activity (Karmen's unit) * 0.482 * N

[0099] Where, 0.482 is the conversion from Karman's unit to U / L; N is the dilution factor of the sample before testing.

[0100] (5) HE staining method is as follows:

[0101] Mouse heart, liver, spleen, lung, and kidney tissues were paraffin-embedded and then sectioned. The sections were placed in a 65°C oven for 2 hours and then dewaxed in xylene and then graded ethanol. After dewaxing, cell nuclei were stained with hematoxylin, and excess stain was removed by washing with distilled water. The sections were then differentiated in 1% hydrochloric acid-alcohol solution for 30 seconds. The cytoplasm was stained with eosin, and excess stain was removed by washing with distilled water. The sections were dehydrated with graded ethanol, and then transparentized with xylene before mounting.

[0102] 2. Test results

[0103] The results of renal function indicators blood urea nitrogen and blood creatinine are as follows Figure 8 As shown in Figures A and 8B, there was no significant difference in the levels of urea nitrogen and creatinine in the serum of mice in the AK group alone and the normal control group, indicating that the AK compound had no toxicity to the kidneys.

[0104] The results of liver function indicators blood alanine aminotransferase and blood aspartate aminotransferase were as follows: Figure 9 As shown in Figures A and 9B, there was no significant difference in serum alanine aminotransferase and aspartate aminotransferase between the AK group and the normal control group, indicating that the AK compound had no toxicity to the liver.

[0105] HE staining Figure 10 As shown, there was no abnormal pathological structure in the heart, liver, spleen, lung and kidney tissues of mice in the simple AK group, and there was no significant difference in the pathological state of the heart, liver, spleen, lung and kidney tissues of mice in the normal control group, indicating that AK at this dose had no obvious toxicity to other organs.

[0106] Experimental Example 4: Effect of Compound AK on Renal Tissue Pathology in IR Model Mice

[0107] 1. Test method (MASSON method)

[0108] Mouse kidney tissue was paraffin-embedded and then sectioned. The sections were placed in a 65°C oven for 2 hours and then dewaxed in xylene and then graded ethanol. After hematoxylin staining for 5 minutes, the sections were stained with Ponceau acid fuchsin solution for 10 minutes, treated with phosphomolybdic acid aqueous solution for approximately 5 minutes, counterstained with aniline blue solution for 5 minutes, acidified with 1% glacial acetic acid for 1 minute, dehydrated with graded ethanol, and mounted.

[0109] 2. Test results

[0110] MASSON staining Figure 11 As shown in the results, 14 days after bilateral renal ischemia-reperfusion in mice, renal fibrosis occurred. This was mainly manifested by the deposition of a large amount of collagen in the renal interstitium. However, the renal interstitial collagen deposition in mice in the IRI+AK (40 mg / kg) group was significantly reduced, indicating that AK compounds have a significant effect on the pathology of renal fibrosis in IRI mice.

[0111] Experimental Example 5: Effect of AK Compound on Renal Tissue Fibrosis in IR Model Mice

[0112] 1. Test method

[0113] 1.1. Western Blot method

[0114] Weigh kidney tissue and add protein lysis buffer at a mass-to-volume ratio of 14 μL / mg. After thorough grinding, transfer the tissue to an EP tube and lyse on a shaker at 4°C for 30 min. Operate on ice throughout the entire process. For subsequent steps, refer to the Western Blot method for cell experiments.

[0115] 1.2 Real-time PCR

[0116] Weigh the kidney tissue and add Trizol lysis buffer at a mass-to-volume ratio of 600 μL / 15 mg. After thorough grinding, transfer to an EP tube and lyse at 4°C for 30 min. Operate on ice throughout the entire process. For subsequent steps, refer to the cell-based real-time PCR method in Experimental Example 1.

[0117] 2. Test results

[0118] Western Blot results showed that the levels of Col1a1 and α-SMA proteins, markers of fibrosis, increased significantly 14 days after bilateral renal ischemia-reperfusion, while different concentrations of AK reduced the expression of these proteins to varying degrees, with the high-dose group showing the most significant reduction effect (see Figure 12 ).

[0119] Real-time PCR was used to measure the mRNA expression level of fibrosis in renal tissue. Figure 13As can be seen, the mRNA expression levels of fibrosis markers α-SMA, FN, Col1a1, and Col3a1 in the renal tissues of mice in the IRI group were significantly increased, leading to the development of renal fibrosis in the mice. Different concentrations of the AK compound inhibited the mRNA levels of fibrosis markers α-SMA, FN, Col1a1, and Col3a1 in the IRI model to varying degrees, thereby inhibiting the progression of renal fibrosis in mice induced by bilateral renal ischemia-reperfusion, with the high-dose group showing the most significant inhibitory effect. In summary, these experiments demonstrate that the AK compound plays an important role in renal fibrosis. Therefore, this drug has the potential to become a key therapeutic approach for the prevention and treatment of chronic kidney disease.

[0120] The above experimental examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above experimental examples. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for preventing or treating renal fibrosis, characterized in that: The pharmaceutical composition comprises a compound of formula (I) and pharmaceutically acceptable excipients. The structure of the compound of formula (I) is shown below: （I）。 2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises other drugs for treating renal fibrosis.

3. The pharmaceutical composition according to claim 2, characterized in that The other drugs for treating renal fibrosis are selected from one or more of pirfenidone, nintedanib, aliskiren, pentoxifylline, canagliflozin, and atrasentan.

4. The pharmaceutical composition according to claim 1, characterized in that The dosage form of the pharmaceutical composition is tablets, capsules, granules, pills, oral liquids, injections, patches, gels or ointments.

5. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for preventing or treating renal fibrosis.

6. The use according to claim 5, characterized in that The renal fibrosis is renal fibrosis caused by renal ischemia-reperfusion.

7. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for treating chronic kidney disease.

8. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for treating renal tubular epithelial cell fibrosis.

9. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for reducing the expression of renal tubular epithelial cell fibrosis indicators, characterized in that: The renal tubular epithelial cell fibrosis indicator is selected from one or more of fibronectin, type I collagen and α-smooth muscle actin.

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