Pyrazole compound for treating renal fibrosis and application thereof

Abstract

The invention relates to a pyrazole compound and application thereof in preparation of a medicine for treating renal fibrosis. The micromolecule AK compound can relieve kidney interstitial collagen deposition, obviously reduce the expression level of fibrosis indexes FN, Col1a1 and alpha-SMA, effectively inhibit kidney fibrosis and delay or / and reverse the disease course of chronic kidney diseases, and has no obvious toxic or side effect. Therefore, the AK compound has a wide clinical application prospect as a renal fibrosis therapeutic agent.

Description

Technical Field

[0001] The 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 increase in the number of aging people and patients with diabetes, obesity and hypertension, chronic kidney disease (CKD) has shown an upward trend year by year. According to incomplete statistics, there are 850 million people suffering from CKD worldwide. If CKD is not treated promptly and effectively, it may 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 progressive chronic kidney diseases, with renal tissue structural remodeling and renal function loss caused by excessive deposition of extracellular matrix (ECM) as the main manifestations. It is generally believed that excessive activation of renal fibrosis is the main cause of irreversible loss of renal function. Blocking the activation of the renin-angiotensin-aldosterone system (RAAS) is a clinical treatment method that can delay the progression of renal fibrosis. Currently, the RAAS system blocking drugs used in clinical practice mainly include angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB), both of which are the first-line recommended drugs for CKD patients with proteinuria. Many randomized controlled clinical trials have shown that RAAS system antagonists can effectively delay renal function loss and reduce mortality in CKD patients while controlling blood pressure and urine protein. However, the use of ACEI and ARB can easily lead to hyperkalemia and acute renal failure in patients with mid- to late-stage CKD, and there are restrictions on their use in pregnant women, patients with renal artery stenosis, and non-hypertensive people, and they can only partially delay the progression of renal fibrosis. With the rapid increase in the prevalence of CKD in recent years, it is particularly urgent to develop safer and more effective anti-renal fibrosis drugs, so it is of great clinical significance to find kidney-protective drugs that can reduce renal fibrosis and delay the progression of chronic kidney disease. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies in the prior art and provide a new 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, wherein the pharmaceutical composition comprises the compound of formula (I) and a pharmaceutically acceptable excipient.

[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 drug 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 dosage of the compound of formula (I) is 10-50 mg / kg body weight. More preferably, the single dosage 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 drug 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 dosage of the compound of formula (I) is 10-50 mg / kg body weight. More preferably, the single dosage of the compound of formula (I) is 40 mg / kg body weight.

[0021] The fifth aspect of the present invention provides the 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 dosage of the compound of formula (I) is 10-50 mg / kg body weight. More preferably, the single dosage 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 index, wherein the renal tubular epithelial cell fibrosis index 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 index 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 dosage of the compound of formula (I) is 10-50 mg / kg body weight. More preferably, the single dosage of the compound of formula (I) is 40 mg / kg body weight.

[0030] Beneficial effects of the present invention:

[0031] The inventor unexpectedly found that the AK compound of the present invention can reduce the expression levels of fibrosis indicators FN, Col1a1 and α-SMA in renal fibrosis animal models and renal tubular epithelial cells, improve renal tissue pathology of renal fibrosis animals, inhibit renal interstitial collagen deposition, effectively reduce renal fibrosis, delay or / and reverse the course of chronic kidney disease, and has no obvious 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 compounds inhibited the fibrotic response of renal tubular epithelial cells induced by repeated hypoxia and reoxygenation;

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

[0035] Figure 4 The immunofluorescence image is of α-SMA expression in the fibrosis response of renal tubular epithelial cells induced by repeated hypoxia and reoxygenation 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 graph showing that AK compounds inhibit TGF-β1-induced fibrosis in renal tubular epithelial cells;

[0038] Figure 7 The immunofluorescence image of α-SMA expression in the fibrosis response of renal tubular epithelial cells induced by AK compounds to TGF-β1;

[0039] Figure 8 The effect of AK compound on the levels of serum creatinine and urea nitrogen in mouse serum is shown in Figure 2. Figure 8 A is urea nitrogen, Figure 8 B is blood creatinine;

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

[0041] Fig.10 This is the result of staining the heart, liver, spleen, lung and kidney tissues of mice in the simple drug group using a hematoxylin and eosin (H&E) staining kit;

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

[0043] Fig.12 The Western Blot diagram of Col1a1 and α-SMA W in the mouse renal fibrosis model after administration of different concentrations of AK compounds;

[0044] Fig.13 The fibrosis indicators FN, Col1a1 and α-SMAReal-time PCR in the mouse renal fibrosis model after administration of different concentrations of AK compounds;

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

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

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

[0048] The compound 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 of 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 methods

[0051] 1.1. CCK8 method

[0052] Human renal tubular epithelial cells (HK2) were inoculated in 96-well plates at a density of about 4000 cells / well. After the cells adhered to the wall, the culture medium was aspirated and replaced with AK solutions containing different concentrations (the concentrations of AK were 5, 10, 20, 40, 80, 160, 320, and 640 μM, respectively). The culture medium without AK was used as the normal control group (NC). After 24 hours of continuous 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 450nm using an ELISA instrument, and the results were recorded. Cell viability was plotted against the 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 / well were divided into normal group (Normoxia), simple AK group (AK 16μM), repeated hypoxia-reoxygenation model group (HR), AK low-dose group (HR+AK 4μM), AK medium-dose group (HR+AK 8μM) and AK high-dose group (HR+AK 16μM). The HR model specifically used 0.5% low-glucose medium for 9 hours of hypoxia and then reoxygenation for 3 hours, repeating hypoxia and reoxygenation 3 times. After the end of hypoxia and reoxygenation, PBS was washed three times, cells were collected, total protein was extracted, and the expression of fibrosis indicators Col1a1 and α-SMA protein was detected by Western Blot method and quantitative analysis was performed. The experiment was repeated 3 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 / well were divided into normal group (Normoxia), simple AK group (AK 16μM), repeated hypoxia and reoxygenation model group (HR), AK low-dose group (HR+AK 4μM), AK medium-dose group (HR+AK 8μM) and AK high-dose group (HR+AK 16μM). The HR model specifically used 0.5% low-glucose medium for 9 hours of hypoxia and then reoxygenation for 3 hours, repeating hypoxia and reoxygenation for 3 times. After the end of hypoxia and reoxygenation, PBS was washed three times, the cells were collected, RNA was extracted, reverse transcribed, and amplified. The experiment was repeated 3 times for each group.

[0057] 1.4 Immunofluorescence

[0058] HK2 cells in the logarithmic growth phase were inoculated in a 6-well plate with a glass slide at a density of approximately 1.0×10 5 Cells / ml 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), 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 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, added primary antibody, washed off after 24 hours of incubation, added fluorescent secondary antibody, washed off after 1.5 hours of incubation in dark, added DAPI staining solution, incubated in dark for 10 minutes, added anti-fluorescence quencher, sealed, observed with a fluorescence inverted microscope, and filmed. The experiment was repeated 3 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 AK compounds had no obvious toxicity.

[0061] Western Blot results Figure 2 As shown (using β-actin as an internal reference), the protein expression levels of fibrosis indicators Col1a1 and α-SMA were significantly inhibited in HK2 cells treated with AK compounds after repeated hypoxia and reoxygenation, 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 shown in 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-reoxygenation treatment. This phenomenon was improved 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-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 of renal tubular epithelial cells in vitro

[0065] 1. Test methods

[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 normal group (NC), AK 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) for 24 hours, and then starved for 12 hours with serum-free medium. Stimulation and drugs were added respectively. Culture was continued for 24 hours. Washed three times with PBS, cells were collected, total protein was extracted, and the expression levels of fibrosis indicators Col1a1 and α-SMA protein were detected by Western Blot method and quantitative analysis was performed. The experiment was repeated 3 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 / well were divided into normal group (NC), group with AK alone (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). After incubation for 24 hours, the cells were starved for 12 hours with serum-free medium and then stimulated and drugs were added. The cells were cultured for 24 hours. The cells were washed three times with PBS, and the RNA was extracted, reverse transcribed, and amplified. The experiment was repeated 3 times for each group.

[0070] 1.3 Immunofluorescence

[0071] HK2 cells in the logarithmic growth phase were inoculated in a 6-well plate with a glass slide at a 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), incubated for 24 hours, starved with serum-free medium for 12 hours, then added with stimulation and drugs, 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 off the primary antibody, add fluorescent secondary antibody, incubate in the dark for 1.5 hours and then wash off 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 3 times for each group.

[0072] 2. Test results

[0073] Western Blot results Figure 5 As shown in the figure, 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 showed that the high-dose group had the most obvious inhibitory effect.

[0074] Real-time PCR results are as follows Figure 6 As shown, AK compounds 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 caused by repeated hypoxia and reoxygenation.

[0077] Experimental Example 3: Effects of AK compounds on the heart, liver, spleen, lung and kidney tissues of untreated mice

[0078] 1. Test methods

[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). All mice had free access to food and water under a 12-hour light / 12-hour dark cycle.

[0080] Construction of simple AK mouse model: Six mice were treated with drugs by gavage every day, with an interval of 24 hours between each gavage. The dosage of AK compound was 40 mg / kg body weight. Serum samples and kidney tissues were collected under anesthesia after 14 days, and the levels of creatinine and urea nitrogen in the serum of the animal model were detected according to the instructions of the serum creatinine, urea nitrogen, alanine aminotransferase and aspartate aminotransferase kits (purchased from Nanjing Jiancheng Bioengineering Institute). The heart, liver, spleen, lung and kidney were embedded and processed for HE staining.

[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 (442μmol / L)

[0084] Note: Dilution factor K = (sample amount + enzyme solution A volume) / (sample amount + 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 get ALT activity (Karmen's unit) * 0.482 * N

[0093] Among them, 0.482 is the conversion of Karman's unit to U / L; N is the dilution multiple 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) = Substitute into the standard curve to get AST activity (Karmen's unit) * 0.482 * N

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

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

[0101] The heart, liver, spleen, lung and kidney tissues of mice were embedded in paraffin and then sliced. The slices were placed in a 65°C oven for 2 hours and then placed in xylene and gradient ethanol for dewaxing. After dewaxing, the nuclei were stained with hematoxylin and the floating color was washed off with distilled water. Differentiation was performed in 1% hydrochloric acid alcohol differentiation solution for 30 seconds. The cytoplasm was stained with eosin, and then the floating color was washed off with distilled water, dehydrated with gradient ethanol, and sealed after xylene was transparent.

[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 A and 8B. As can be seen from the figure, there is no significant difference in urea nitrogen and creatinine in the serum of mice in the simple AK group and the normal control group, indicating that AK compounds have no toxicity to the kidneys.

[0104] The results of liver function indicators blood alanine aminotransferase and blood aspartate aminotransferase were as follows Fig. 9 As shown in A and 9B. As can be seen from the figure, there is no significant difference in the serum levels of alanine aminotransferase and aspartate aminotransferase between the simple AK group and the normal control group, indicating that the AK compound has no toxicity to the liver.

[0105] HE staining Fig.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] The mouse kidney tissue was embedded in paraffin and then sliced. The slices were placed in a 65°C oven for 2 hours and then dewaxed in xylene and gradient ethanol. After hematoxylin staining for 5 minutes, the slices were stained with Ponceau acid fuchsin solution for 10 minutes, treated with phosphomolybdic acid aqueous solution for about 5 minutes, counterstained with aniline blue solution for 5 minutes, acidified with 1% glacial acetic acid for 1 minute, dehydrated with gradient ethanol, and sealed.

[0109] 2. Test results

[0110] Masson staining Fig.11 As shown in the figure, after 14 days of bilateral renal ischemia-reperfusion in mice, renal fibrosis occurred. The main manifestation was a large amount of collagen deposition in the renal interstitium, while 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 improvement effect on the renal fibrosis pathology of IRI mice.

[0111] Experimental Example 5: Effect of AK compound on renal fibrosis in IR model mice in vivo

[0112] 1. Test methods

[0113] 1.1. Western Blot method

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

[0115] 1.2 Real-time PCR

[0116] Weigh the kidney tissue, add Trizol lysis buffer at a mass volume ratio of 600 μL / 15 mg, grind thoroughly, transfer to an EP tube, and lyse at 4°C for 30 min. The whole process is performed on ice. For subsequent steps, refer to the cell test 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, indicators 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, and the high-dose group had the most significant effect (see Fig.12 ).

[0119] Real-time PCR was used to measure the mRNA expression level of fibrosis in renal tissue. Fig.13It can be seen that the mRNA expression levels of fibrosis indicators α-SMA, FN, Col1a1 and Col3a1 in the renal tissue of mice in the IRI group increased significantly, causing renal fibrosis in mice; and different concentrations of AK compounds inhibited the mRNA levels of fibrosis indicators α-SMA, FN, Col1a1 and Col3a1 in the IRI model to varying degrees, thereby inhibiting the progression of renal fibrosis in mice caused by bilateral renal ischemia-reperfusion, and the high-dose group had the most significant inhibitory effect. In summary, the experiment proved that AK compounds can play an important role in renal fibrosis diseases. Therefore, this drug is expected to become a key drug for the prevention and treatment of chronic kidney disease.

[0120] The above experimental examples are preferred implementation modes of the present invention, but the implementation modes 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A compound for preventing or treating renal fibrosis, characterized in that: The compound structure is shown in formula (I):

2. A pharmaceutical composition for preventing or treating renal fibrosis, characterized in that: The pharmaceutical composition comprises the compound of formula (I) as claimed in claim 1 and pharmaceutically acceptable excipients.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition further comprises other drugs for treating renal fibrosis.

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

5. The pharmaceutical composition according to claim 2, characterized in that The dosage form of the pharmaceutical composition is tablet, capsule, granule, pill, oral solution, injection, patch, gel or ointment.

6. Use of the compound of formula (I) according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 5 in the preparation of a medicament for preventing or treating renal fibrosis.

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

8. Use of the compound of formula (I) according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 5 in the preparation of a medicament for treating chronic kidney disease.

9. Use of the compound of formula (I) according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 5 in the preparation of a medicament for treating renal tubular epithelial cell fibrosis.

10. Use of the compound of formula (I) according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 5 in the preparation of a drug for reducing the expression of renal tubular epithelial cell fibrosis indicators, characterized in that: The renal tubular epithelial cell fibrosis index is selected from one or more of fibronectin, type I collagen and α-smooth muscle actin.

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