Use of 2,4-dihydroxybenzaldehyde in preparing medicine for preventing and / or treating renal injury

Abstract

The present invention belongs to the field of drug application, and specifically relates to the use of 2,4-dihydroxybenzaldehyde in preparing a drug for preventing and / or treating renal injury. The present invention finds through experiments that 2,4-dihydroxybenzaldehyde can specifically inhibit cisplatin-induced renal tissue ferroptosis, thereby completely preventing renal injury, reducing blood creatinine, blood urea nitrogen levels, and kidney coefficient, and reducing the weight loss that often occurs in cancer patients during cisplatin chemotherapy. The effect is better than protocatechuic aldehyde at the same dose, and the safety is high, and it has practical promotion and application value.

Description

Technical Field

[0001] The invention belongs to the field of drug application, and specifically relates to use of 2,4-dihydroxybenzaldehyde in preparing drugs for preventing and / or treating kidney damage. Background Art

[0002] Malignant tumors are a type of disease caused by the uncontrolled proliferation of abnormal cells with invasive and metastatic properties. The harm is that it quickly destroys normal tissue structures, affects organ function, and can spread throughout the body through the lymphatic or blood system, leading to the death of patients. Cisplatin is a platinum compound widely used in the treatment of malignant tumors. Its unique anticancer mechanism makes it one of the core drugs for the treatment of various solid tumors, including testicular cancer, ovarian cancer, bladder cancer, head and neck squamous cell carcinoma, and certain types of lung cancer and gastric cancer. Cisplatin has a significant anticancer effect in tumor treatment, but its side effects are also significant, especially nephrotoxicity, which has become the main limiting factor in clinical use. The nephrotoxicity caused by cisplatin is mainly manifested as acute tubular damage, and its mechanism includes the accumulation of drugs in renal tubular cells leading to oxidative stress, mitochondrial dysfunction, and activation of inflammatory response and apoptosis. This toxicity can cause problems such as increased blood creatinine and blood urea nitrogen, decreased urine volume, electrolyte disorders, and in severe cases, it can develop into acute renal failure. In addition, the nephrotoxicity of cisplatin is dose-dependent, and repeated use may lead to irreversible renal damage. Because the exact molecular mechanism of cisplatin-induced acute kidney injury has not been fully elucidated, the prevention and treatment strategies for this injury are still very limited.

[0003] 2,4-Dihydroxybenzaldehyde is a natural compound from the macrocarpa serrata. It is in the form of white needle-shaped crystals. Its molecular formula is C7H6O3, its CAS number is 95-01-2, and its structural formula is: , has strong antioxidant and antibacterial activity. In the pharmaceutical field, 2,4-dihydroxybenzaldehyde is often used as an organic synthesis intermediate and can be used to prepare a variety of drugs, such as anticancer drugs, antiviral drugs, anti-hepatitis drugs, etc. At present, there is no report on the application of 2,4-dihydroxybenzaldehyde in kidney injury, especially cisplatin-induced acute kidney injury. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of 2,4-dihydroxybenzaldehyde in preparing a medicament for preventing and / or treating kidney damage.

[0005] Furthermore, the drug is a drug for preventing and / or treating acute kidney injury or chronic kidney injury.

[0006] Furthermore, the drug is a drug for preventing and / or treating acute kidney injury induced by chemical drugs; the acute kidney injury induced by chemical drugs includes acute kidney injury caused by cisplatin.

[0007] Furthermore, the drug is a drug that inhibits elevated levels of lipid peroxidation in renal cells.

[0008] Furthermore, the drug is a drug that inhibits the increase of ferrous ion content in renal cells.

[0009] Furthermore, the drug is a drug that inhibits the increase of active oxygen content in renal cells.

[0010] Furthermore, the drug is a drug that reduces ferroptosis-like structural damage in renal cells.

[0011] Furthermore, the drug is a drug for reducing blood creatinine and / or blood urea nitrogen levels.

[0012] Furthermore, the drug is a drug for reducing renal index and / or improving weight loss.

[0013] Furthermore, the drug is a drug that increases the GPX4 protein level in kidney tissue and / or reduces the iron content in kidney tissue.

[0014] Furthermore, the drug is a preparation prepared with 2,4-dihydroxybenzaldehyde as an active ingredient and pharmaceutically acceptable excipients; the preparation includes an oral preparation or an injection.

[0015] The use of 2,4-dihydroxybenzaldehyde in the preparation of drugs for preventing and / or treating renal injury of the present invention has focused on the fact that previous studies have mainly focused on cisplatin-induced apoptosis and necrosis of renal tubular cells, but specific inhibitors of apoptosis and necrosis have failed to completely prevent or reverse the phenomenon of acute renal injury caused by cisplatin in clinical practice. Considering that there are other forms of cell death mechanisms that may be involved in the occurrence of renal injury, according to existing studies, ferroptosis plays an important role in the pathological process of cisplatin-induced acute renal injury, which is characterized by iron-dependent lipid peroxidation and cell membrane damage, and inhibition of ferroptosis has a renal protective effect. Therefore, taking this as a breakthrough, through a large number of cell and animal experimental screening, it was found that 2,4-dihydroxybenzaldehyde can inhibit the increase of renal cell lipid peroxidation level, ferrous ion content and reactive oxygen content, and reduce renal cell ferroptosis-like structural damage, thereby completely preventing renal injury, reducing blood creatinine, blood urea nitrogen levels, and kidney coefficient, and reducing the weight loss that often occurs in cancer patients during cisplatin chemotherapy, and the effect is better than protocatechuic aldehyde at the same dose. Toxicity tests show that the cytotoxicity of 2,4-dihydroxybenzaldehyde is far less than that of protocatechuic aldehyde, which has a similar chemical structure. Its clinical value in improving renal injury, especially cisplatin-induced acute renal injury, is incomparable to that of protocatechuic aldehyde, and it has practical value for promotion and application.

[0016] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0017] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows that 2,4-dihydroxybenzaldehyde has an inhibitory effect on cell ferroptosis caused by cisplatin and erastin; a: The effect of erastin treatment for 48 hours on the cell viability of HK-2 cells. b: The effect of cisplatin treatment for 48 hours on the cell viability of HK-2 cells; c: The effect of 2,4-dihydroxybenzaldehyde treatment for 48 hours on the cell viability of HK-2 cells; d: The effect of 2,4-dihydroxybenzaldehyde combined with erastin treatment for 24 hours on the cell viability of HK-2 cells; e: The effect of 2,4-dihydroxybenzaldehyde combined with cisplatin treatment for 24 hours on the cell viability of HK-2 cells;

[0019] Figure 2 The figure shows that 2,4-dihydroxybenzaldehyde has an inhibitory effect on the increase of cellular reactive oxygen content caused by cisplatin and erastin; a: The effect of 2,4-dihydroxybenzaldehyde on the changes of reactive oxygen in HK-2 cells induced by cisplatin; b: The effect of 2,4-dihydroxybenzaldehyde on the changes of reactive oxygen in HK-2 cells induced by erastin;

[0020] Figure 3 The figure shows that 2,4-dihydroxybenzaldehyde has an inhibitory effect on the increase of ferrous ion content in cells caused by cisplatin and erastin; a: The effect of 2,4-dihydroxybenzaldehyde on the change of ferrous ion in HK-2 cells induced by cisplatin; b: The effect of 2,4-dihydroxybenzaldehyde on the change of ferrous ion in HK-2 cells induced by erastin;

[0021] Figure 4 The figure shows that 2,4-dihydroxybenzaldehyde has an inhibitory effect on the increase in cellular lipid peroxidation levels caused by cisplatin and erastin; a: The effect of 2,4-dihydroxybenzaldehyde on the changes in lipid peroxidation levels in HK-2 cells induced by cisplatin; b: The effect of 2,4-dihydroxybenzaldehyde on the changes in lipid peroxidation levels in HK-2 cells induced by erastin;

[0022] Figure 5The diagram shows the improvement effect of 2,4-dihydroxybenzaldehyde on the cell morphological structure damage caused by cisplatin and erastin; a: the effect of 2,4-dihydroxybenzaldehyde on the cell morphological structure damaged by cisplatin in HK-2 cells, b: the effect of 2,4-dihydroxybenzaldehyde on the cell morphological structure damaged by erastin in HK-2 cells;

[0023] Figure 6 This is a diagram showing that 2,4-dihydroxybenzaldehyde alleviates acute kidney injury caused by cisplatin; a: Mouse blood creatinine detection, b: Mouse blood urea nitrogen detection, c: Recording the changes in mouse body weight during treatment; d: Quantification of the kidney coefficient of each group of mice (kidney coefficient = kidney weight / body weight);

[0024] Figure 7 This is a picture showing that 2,4-dihydroxybenzaldehyde alleviates the pathological damage caused by cisplatin; HE and PAS staining were used to examine the pathological changes in the kidneys of mice;

[0025] Figure 8 The figure shows that 2,4-dihydroxybenzaldehyde can inhibit ferroptosis in acute kidney injury caused by cisplatin; a: Upper: Immunohistochemistry to detect the effect of dihydroxybenzaldehyde on GPX4 protein expression in kidney tissue; Lower: Enhanced Prussian blue staining to examine the effect of dihydroxybenzaldehyde on iron content in kidney tissue, b: Statistics of GPX4 protein expression levels in each group C: Statistics of iron content in each group;

[0026] Fig. 9 Figure 2 shows that 2,4-dihydroxybenzaldehyde can activate the Nrf2 signaling pathway by targeting Keap1; ab: Surface plasmon resonance experiment (SPR) verifies the binding affinity of 2,4-dihydroxybenzaldehyde to Keap1, c: Microscale thermophoresis (MST) technology verifies the binding affinity of 2,4-dihydroxybenzaldehyde to Keap1, de: Immunohistochemistry detects the expression of Nrf2 protein in kidney tissue;

[0027] Fig.10 The figure shows that the cytotoxicity of 2,4-dihydroxybenzaldehyde is much lower than that of protocatechuic aldehyde; a: IC50 value of 2,4-dihydroxybenzaldehyde; b: IC50 value of protocatechuic aldehyde;

[0028] Fig.11 The figure shows that the same dose of 2,4-dihydroxybenzaldehyde is more effective than protocatechuic aldehyde in alleviating acute kidney injury; a: blood creatinine test; b: blood urea nitrogen test. DETAILED DESCRIPTION

[0029] The raw materials, equipment and reagents used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0030] Example 1 Study on the Effect of 2,4-Dihydroxybenzaldehyde on Kidneys

[0031] 1. Inhibitory effect of 2,4-dihydroxybenzaldehyde on ferroptosis induced by cisplatin and erastin

[0032] 1.1 Experimental Methods

[0033] 1) 1000 renal epithelial HK-2 cells were seeded in a 96-well cell culture plate and cultured in a carbon dioxide incubator for 24 h.

[0034] 2) Different concentrations of ferroptosis inducers, such as erastin, cisplatin, and 2,4-dihydroxybenzaldehyde, were added to the cells. The cells were treated with either drug alone for 48 h or with a combination of drugs for 24 h. DMSO was added to the control group.

[0035] 3) After drug treatment, add 10 μL of CCK8 reagent to each well and incubate at 37°C for 90 min.

[0036] 4) Measure the absorbance at 450 nm and calculate the cell viability.

[0037] 1.2 Experimental results: Figure 1 As shown, ferroptosis inducers erastin and cisplatin can inhibit the viability of HK-2 cells in a concentration-dependent manner, 2,4-dihydroxybenzaldehyde has no obvious cytotoxicity to HK-2, and the combined treatment of HK-2 cells with 2,4-dihydroxybenzaldehyde and cisplatin or erastin can enhance the cell viability of HK-2.

[0038] 1.3 Experimental conclusion: 2,4-dihydroxybenzaldehyde has an inhibitory effect on cell ferroptosis caused by cisplatin and erastin, an inducer of ferroptosis.

[0039] 2. Inhibitory effect of 2,4-dihydroxybenzaldehyde on the increase of cellular reactive oxygen species caused by cisplatin and erastin, an inducer of ferroptosis

[0040] 2.1 Experimental Methods

[0041] 1) 10,000 renal epithelial HK-2 cells were seeded in a 6-well cell plate and cultured in a carbon dioxide incubator for 24 h.

[0042] 2) Different concentrations of erastin (10 μM), cisplatin (10 μM) and 2,4-dihydroxybenzaldehyde (250 μM) were added to the cells for 24 h, and DMSO was added to the control group.

[0043] 3) After drug treatment, remove the cell culture medium, add 2 μL of DCFH-DA reagent to each well, and incubate at 37°C for 20 min.

[0044] 4) Remove the cell supernatant, wash twice with PBS, add trypsin, incubate for 1 min, then discard the trypsin, add 500 μL DMEM complete medium, gently blow the cells, resuspend and collect the cell suspension.

[0045] 5) Pass the stained cells through a cell strainer and analyze the samples using a flow cytometer. Use FlowJo software to analyze the resulting data.

[0046] 2.2 Experimental Results

[0047] like Figure 2 As shown, both ferroptosis inducers erastin and cisplatin can increase the content of reactive oxygen species in HK-2 cells. In the combination group, the content of reactive oxygen species was significantly decreased compared with that in the erastin and cisplatin group.

[0048] 2.3 Experimental conclusion: 2,4-Dihydroxybenzaldehyde has an inhibitory effect on the increase in cellular reactive oxygen species caused by cisplatin and the ferroptosis inducer erastin.

[0049] 3. Inhibitory effect of 2,4-dihydroxybenzaldehyde on the increase of cellular ferrous ion content caused by cisplatin and erastin, an inducer of ferroptosis

[0050] 3.1 Experimental methods:

[0051] 1) 10,000 renal epithelial HK-2 cells were seeded in a 6-well cell plate and cultured in a carbon dioxide incubator for 24 h.

[0052] 2) Different concentrations of erastin (10 μM), cisplatin (10 μM) and 2,4-dihydroxybenzaldehyde (250 μM) were added to the cells for 24 h, and DMSO was added to the control group.

[0053] 3) Discard the cell culture supernatant, add PBS to wash twice, then add trypsin (250 μL), stop the reaction with serum medium (1 mL), and transfer 1.25 ml of the cell suspension to a microcentrifuge tube.

[0054] 4) Centrifuge the cell suspension at 1,500 rpm for 3 minutes.

[0055] 5) Discard the supernatant, add HBSS (1 ml) to the microcentrifuge tube, and resuspend using a pipette.

[0056] 6) Centrifuge the cell suspension at 1,500 rpm for 3 minutes and discard the supernatant.

[0057] 7) Add 1 μmol / L FerroOrange (300 μL) in MEM (serum-free medium) to the cells.

[0058] 8) Incubate the cells in a 37°C incubator for 15-30 minutes.

[0059] 9) Pass the stained cells through a cell strainer and analyze the sample using a flow cytometer. Use FlowJo software to analyze the resulting data.

[0060] 3.2 Experimental Results

[0061] like Figure 3 As shown, both erastin and cisplatin, ferroptosis inducers, can increase the content of ferrous ions in HK-2 cells. In the combination group, the content of ferrous ions was significantly decreased compared with that in the erastin and cisplatin group.

[0062] 3.3 Experimental Conclusion

[0063] 2,4-Dihydroxybenzaldehyde has an inhibitory effect on the increase of cellular ferrous ion content caused by cisplatin and the ferroptosis inducer erastin.

[0064] 4. Inhibitory effect of 2,4-dihydroxybenzaldehyde on the elevated cellular lipid peroxidation levels caused by cisplatin and erastin, an inducer of ferroptosis

[0065] 4.1 Experimental Methods

[0066] 1) 10,000 renal epithelial HK-2 cells were seeded in a 6-well cell plate and cultured in a carbon dioxide incubator for 24 h.

[0067] 2) Different concentrations of erastin (10 μM), cisplatin (10 μM) and 2,4-dihydroxybenzaldehyde (250 μM) were added to the cells for 24 h, and DMSO was added to the control group.

[0068] 3) Discard the cell culture supernatant, add PBS to wash twice, then add trypsin (250 μL), stop the reaction with serum medium (1 mL), and transfer 1.25 ml of the cell suspension to a microcentrifuge tube.

[0069] The culture medium was removed and the cells were washed twice with culture medium.

[0070] 4) Remove the supernatant, add the prepared lipid peroxidation probe (BDP 581 / 591 C11) working solution, and incubate in a 37°C, 5% CO2 incubator for 30 min.

[0071] 5) Remove the supernatant, wash twice with HBSS, and resuspend in HBSS again.

[0072] 6) Pass the stained cells through a cell strainer and analyze the samples using a flow cytometer. Use FlowJo software to analyze the resulting data.

[0073] 4.2 Experimental Results

[0074] like Figure 4 As shown, both ferroptosis inducers erastin and cisplatin could increase the level of lipid peroxidation in HK-2 cells. In the combination group, the level of lipid peroxidation was significantly decreased compared with that in the erastin and cisplatin groups.

[0075] 4.3 Experimental Conclusion

[0076] 2,4-Dihydroxybenzaldehyde has an inhibitory effect on the increased level of cellular lipid peroxidation caused by cisplatin and the ferroptosis inducer erastin.

[0077] 5. 2,4-Dihydroxybenzaldehyde alleviates the cell ferroptosis-like structural damage caused by cisplatin and ferroptosis inducer erastin

[0078] 5.1 Experimental methods:

[0079] 1) HK-2 cells were cultured at 2×10 per well 6 The cells were seeded in a 60 mm dish and cultured for 24 hours.

[0080] 2) Different concentrations of erastin (10 μM), cisplatin (10 μM) and 2,4-dihydroxybenzaldehyde (250 μM) were added to the cells for 24 h, and DMSO was added to the control group.

[0081] 3) Discard the cell culture supernatant, add PBS to wash twice, then add trypsin (250 μL), stop the reaction with serum medium (1 mL), and transfer the cell suspension to a microcentrifuge tube.

[0082] 4) Remove the culture medium and wash the cells 2-3 times with pre-cooled PBS (pH 7.4). Add 2.5% glutaraldehyde solution (dissolved in 0.1 M phosphate buffer, pH 7.4) to fix the cells at 4°C for 2-4 hours. After fixation, wash again with 0.1 M phosphate buffer for 3 times, 10 minutes each time.

[0083] 5) Post-fix with 1% osmium oxide (OsO4) solution at 4°C for 1-2 hours to enhance the contrast of membrane structure. After post-fixation, wash with 0.1 M phosphate buffer three times, 10 minutes each time.

[0084] 6) Replace ethanol with propylene oxide twice, 10 minutes each time.

[0085] 7) Dehydrate with gradient concentrations of ethanol (30%, 50%, 70%, 90%, 100%) for 15 minutes each time. Wash twice with 100% ethanol to ensure thorough dehydration.

[0086] 8) Replace ethanol with propylene oxide twice, 10 minutes each time.

[0087] 9) Soak the cells in epoxy embedding solution (such as Epon 812) and stir at room temperature for 4-6 hours to ensure sufficient penetration.

[0088] The samples were placed in fresh epoxy resin embedding solution, transferred to embedding molds, and placed in a 60 °C oven for polymerization for 48 h.

[0089] 10) Use an ultramicrotome to cut ultrathin sections of 70-90 nm and transfer them to a copper or nickel mesh (about 3 mm in diameter).

[0090] 11) Stain with 2% uranyl acetate for 20-30 minutes, and then stain with 0.2% lead citrate for 5-10 minutes.

[0091] After dyeing, wash with deionized water and dry for later use.

[0092] 12) Observe the sample using a transmission electron microscope.

[0093] 5.2 Experimental Results

[0094] like Figure 5 As shown, electron microscopy observation showed that mitochondrial damage in cells treated with erastin and cisplatin was characterized by mitochondrial fragmentation, disappearance of mitochondrial cristae, and shortening and rounding of mitochondria. It is worth noting that 2,4-dihydroxybenzaldehyde treatment significantly reduced the occurrence of the above-mentioned mitochondrial damage.

[0095] 5.3 Experimental Conclusion

[0096] 2,4-Dihydroxybenzaldehyde alleviates the cell ferroptosis-like structural damage caused by cisplatin and the ferroptosis inducer erastin, thereby improving the cell morphology and structure.

[0097] 6. Effect of 2,4-dihydroxybenzaldehyde on alleviating acute kidney injury caused by cisplatin

[0098] 6.1 Experimental Methods

[0099] 1) Male KM mice (32-35 g) were acclimatized for 1 week under standard conditions, including temperature (22 ± 2°C), humidity (50 ± 5%), and photoperiod (12 h light / 12 h dark).

[0100] 2) The mice were randomly divided into four groups (10 mice in each group). The blank control group was treated with normal saline only, and the cisplatin group was treated with a single injection of cisplatin (25 mg / kg, intraperitoneal injection) and normal saline injection for three consecutive days (once every 24 hours, intraperitoneal injection). The cisplatin and 2,4-dihydroxybenzaldehyde combination group was treated with a single injection of cisplatin (25 mg / kg, intraperitoneal injection) and 2,4-dihydroxybenzaldehyde (once every 24 hours, 100 mg / kg, intraperitoneal injection) for three consecutive days. The 2,4-dihydroxybenzaldehyde group was treated with 2,4-dihydroxybenzaldehyde (once every 24 hours, 100 mg / kg, intraperitoneal injection) for three consecutive days.

[0101] 3) Observe the weight changes of mice daily.

[0102] 4) 72 hours after injection, blood was collected from the eye socket for testing of serum creatinine and blood urea nitrogen.

[0103] 5) Kidney tissues were removed, weighed, collected and used for subsequent pathology (HE staining and PAS staining), immunohistochemistry and enhanced Prussian blue detection.

[0104] 6) HE (Hematoxylin and Eosin) staining is a commonly used histopathological staining method, mainly used to observe morphological changes in tissues. First, remove the mouse kidney, fix it in 4% paraformaldehyde solution, fix it at room temperature for 24 hours, and perform routine dehydration after fixation. Then, embed the tissue sample in paraffin and cut it into 4μm thick slices in paraffin. Dewax the slices with xylene twice, each for 5-10 minutes, and then dehydrate them with gradient concentrations of alcohol (100%, 95%, 90%, 80%, 70%), each for 5 minutes. After that, wash the slices with distilled water to remove alcohol residues. Then, soak the slices in hematoxylin solution, stain for 5-10 minutes, and rinse with running water for 5-10 minutes after staining until the staining solution is rinsed clean. Then, soak the slices in eosin solution, stain for 2-5 minutes, and rinse with running water after staining. After staining, dehydrate with 100% alcohol twice, 5 minutes each time, and then wash with xylene twice, 5 minutes each time. Finally, use neutral resin to seal the slides, observe the sections after the sealing agent solidifies, and use a microscope for pathological analysis.

[0105] 7) PAS (Periodic Acid-Schiff) staining is a staining method commonly used to detect glycogen, glycoproteins and certain types of extracellular matrix (such as basement membrane) in tissues. The experimental method is as follows: the methods of sample fixation, dehydration, paraffin embedding and sectioning are the same as the HE experimental method. Next, soak the sections in 0.5% potassium permanganate solution to oxidize the aldehyde groups in the tissue for 10-15 minutes, and then wash with running water for 5 minutes after oxidation to remove excess oxidants. Then, soak the sections in a staining solution containing Schiff reagent for 15-30 minutes, and then wash with running water for 5-10 minutes after staining to remove excess staining solution. Next, dehydrate with gradient concentrations of alcohol (70%, 90%, 100%) for 5 minutes each time, and then wash with xylene twice, each for 5 minutes. Finally, use neutral resin to seal the sections, and observe the sections after the sealing agent solidifies. PAS staining can significantly show the red or purple-red reaction of glycogen and glycoproteins, and is often used to study structures related to carbohydrates in renal tubules, liver, heart and other tissues.

[0106] 6.2 Experimental Results

[0107] like Figure 6 As shown in the figure, after acute kidney injury caused by cisplatin, blood creatinine, blood urea nitrogen content and kidney coefficient increased significantly, and body weight decreased significantly. After treatment with 2,4-dihydroxybenzaldehyde, the levels of blood creatinine and blood urea nitrogen in mice with acute kidney injury were reduced, the kidney coefficient was reduced, and the weight loss was alleviated.

[0108] like Figure 7 As shown, neutrophil infiltration, tubular dilatation, tubular cast formation, tubular degeneration and necrosis occurred in the cisplatin group, while in the combination group, these pathological conditions were alleviated by 2,4-dihydroxybenzaldehyde treatment.

[0109] 6.3 Experimental conclusion: 2,4-dihydroxybenzaldehyde can alleviate acute kidney injury caused by cisplatin.

[0110] 7. Effect of 2,4-dihydroxybenzaldehyde on inhibiting ferroptosis in acute kidney injury induced by cisplatin

[0111] 7.1 Experimental Methods

[0112] 1) Immunohistochemistry (IHC) staining is used to detect the expression of GPX4 protein in tissues. The experimental method is as follows: Sample fixation, dehydration, and paraffin embedding methods are the same as those in 6.1. In order to remove endogenous peroxidase activity in tissues, use 3% H2O2 solution to block the sections for 10-15 minutes. Then, the sections are subjected to antigen retrieval, usually heated in a microwave oven with sodium citrate buffer (pH 6.0) for 10-20 minutes, and washed with PBS three times after cooling, each for 5 minutes. Next, add 5% bovine serum albumin (BSA) and block at room temperature for 30 minutes. After blocking, add primary antibody (GPX4 antibody, usually diluted 1:100-1:200) on the sections and incubate overnight at 4°C. On the next day, wash with PBS three times for 5 minutes each, then add secondary antibody (such as HRP-labeled rabbit anti-mouse IgG secondary antibody, diluted 1:1000) and incubate at room temperature for 30-60 minutes. Wash with PBS three times, use DAB colorimetric solution for color development, and observe the color reaction. Finally, the color development reaction was terminated with distilled water, and gradient alcohol dehydration (70%, 80%, 90%, 100%) was performed for 5 minutes each, followed by washing with xylene and sealing. The expression of GPX4 protein in tissues was observed under a microscope, and the positive signal was brown-yellow.

[0113] 2) Enhanced Prussian Blue staining is a staining method commonly used to detect iron deposition in tissues. The experimental method is as follows: The sample fixation, dehydration, paraffin embedding, and sectioning methods are the same as the experimental method in 6.1. Then, the sections are immersed in a 2% potassium perchlorate (or potassium ferrocyanide) solution and stained for 30 minutes, which will cause the iron ions in the tissue to react with potassium ferrocyanide to form Prussian blue. After staining, wash thoroughly with distilled water for 5-10 minutes to remove excess staining solution. Next, use a contrast staining method for enhancement. The common method is to immerse the sections in a 0.5% sea buckthorn red stain solution for background staining for 5 minutes, and then wash again with distilled water. Then, the sections are dehydrated through a gradient alcohol (70%, 80%, 90%, 100%) for 5 minutes each, and finally washed twice with xylene for 5 minutes each. Finally, the sections are sealed with a neutral resin and the sections are observed after the sealing agent solidifies. The iron deposition area stained with Prussian blue will appear blue, and the positive area is the site of iron deposition. This method is widely used to detect iron deposition in tissues, especially in tissues such as the liver, heart, and spleen, to help evaluate abnormal iron metabolism and pathological changes in related diseases.

[0114] 7.2 Experimental Results

[0115] like Figure 8As shown, in the acutely injured renal tissue of the cisplatin group, GPX4 protein expression was decreased and iron content was increased, while in the renal tissue after 2,4-dihydroxybenzaldehyde treatment, GPX4 protein expression was increased and iron content was decreased.

[0116] 7.3 Experimental conclusion: 2,4-Dihydroxybenzaldehyde can inhibit ferroptosis in acute kidney injury caused by cisplatin.

[0117] 8. Mechanism of 2,4-dihydroxybenzaldehyde activating Nrf2 signaling pathway by targeting Keap1

[0118] 8.1 Experimental Methods

[0119] 1) Surface plasmon resonance (SPR) was used to verify the binding affinity of 2,4-dihydroxybenzaldehyde to Keap1. The binding affinity was evaluated using a Biacore T-200 SPR instrument. First, recombinant Keap1 or BRD4 protein was coupled to the surface of a CM5 carboxymethyl dextran chip. The chip surface was activated using the EDC / NHS cross-linking method, and the protein was dissolved in a suitable buffer for coupling. After the coupling was completed, the chip was washed with a decontamination buffer to remove uncoupled proteins. Then, the compound was dissolved in PBS (containing 0.5% DMSO) at different concentrations (usually in the range of 10^-9M to 10^-6M), the compound solution was gradually injected, and its binding behavior with the coupled protein was monitored. After each injection, it was washed with an elution buffer (such as PBS) and the binding and dissociation processes were recorded. The data were analyzed using the Biacore T-200 Evaluation State Model software to calculate the equilibrium dissociation constant (KD). The binding affinity of the compound was obtained based on the shape of the binding and dissociation curves, and a lower KD value represents a stronger binding affinity.

[0120] 2) The interaction between 2,4-dihydroxybenzaldehyde and Keap1 protein was analyzed by microscale thermophoresis (MST). First, recombinant Keap1 protein was labeled with RED-NHS dye (MO-L018, NanoTemper, Germany) according to the manufacturer's instructions. Then, 200 nM of His-tagged recombinant Keap1 protein was conjugated with RED-Tris-NTA and incubated with a two-fold dilution of 2,4-dihydroxybenzaldehyde (ranging from 750 μM to 0.046 μM) in binding buffer at room temperature for 30 min. Microvolume thermophoresis was performed on a Premium MonolithTM NT.115 microvolume thermophoresis instrument (NanoTemper, Germany) with an LED power of 40% and an MST power of medium intensity. After the experiment, the thermophoresis data were analyzed using MO software to evaluate the binding affinity of 2,4-dihydroxybenzaldehyde derivatives to Keap1 protein.

[0121] 3) Except for selecting Nrf2 as the antibody for immunohistochemistry, other experimental methods are the same as those in 7.1 Experimental Methods.

[0122] 8.2 Experimental results: Fig. 9 As shown, 2,4-dihydroxybenzaldehyde can directly bind to Keap1, and 2,4-dihydroxybenzaldehyde increases the expression of Nrf2 protein in acutely injured renal tissue.

[0123] 8.3 Experimental conclusion: 2,4-dihydroxybenzaldehyde can activate the Nrf2 signaling pathway by targeting Keap1, thereby improving renal damage.

[0124] 9. Cytotoxicity test of 2,4-dihydroxybenzaldehyde

[0125] Protocatechuic aldehyde and 2,4-dihydroxybenzaldehyde have similar chemical structures, the difference is that the hydroxyl group is located in a different position on the benzene ring. Studies have shown that protocatechuic aldehyde can prevent acute kidney injury induced by cyclophosphamide (CTX), but protocatechuic aldehyde itself has toxic side effects, such as reports that it has significant cardiotoxicity. Therefore, the cytotoxicity of 2,4-dihydroxybenzaldehyde was investigated using protocatechuic aldehyde, which has a similar chemical structure, as a control.

[0126] 9.1 Experimental methods:

[0127] 1) 1000 renal epithelial HCT116 cells were seeded in a 96-well cell culture plate and cultured in a carbon dioxide incubator for 24 h.

[0128] 2) Different concentrations of 2,4-dihydroxybenzaldehyde and protocatechuic aldehyde were added to the cells for 48 hours, and DMSO was added to the control group.

[0129] 3) After drug treatment, add 10 μL of CCK8 reagent to each well and incubate at 37°C for 90 min.

[0130] 4) Measure the absorbance at 450 nm and calculate the IC50 value.

[0131] 9.2 Experimental results: Fig.10 As shown, based on the data of CCK-8 cytotoxicity assay, the IC50 values ​​were calculated. The IC50 value of 2,4-dihydroxybenzaldehyde was 2229 μM, and the IC50 value of protocatechuic aldehyde was 134.3 μM.

[0132] 9.3 Experimental conclusion: Compared with protocatechuic aldehyde, the cytotoxicity of 2,4-dihydroxybenzaldehyde is much lower than that of protocatechuic aldehyde. This indicates that in terms of improving renal injury, 2,4-dihydroxybenzaldehyde has a greater safety advantage than protocatechuic aldehyde.

[0133] 10. Evaluation of the effect of 2,4-dihydroxybenzaldehyde on improving acute kidney injury caused by cisplatin

[0134] Studies have shown that protocatechuic aldehyde can prevent acute kidney injury induced by cyclophosphamide (CTX). 2,4-dihydroxybenzaldehyde is less toxic than protocatechuic aldehyde. If the efficacy of 2,4-dihydroxybenzaldehyde in improving acute kidney injury caused by cisplatin is also reduced accordingly, its clinical application value will be greatly reduced. Therefore, under the same experimental conditions, the therapeutic effect of protocatechuic aldehyde on acute kidney injury caused by cisplatin was tested, as follows:

[0135] 10.1 Experimental Methods

[0136] 1) Male KM mice (32-35 g) were acclimatized for 1 week under standard conditions, including temperature (22 ± 2°C), humidity (50 ± 5%), and photoperiod (12 h light / 12 h dark).

[0137] 2) The mice were randomly divided into four groups (6 mice in each group). The blank control group was treated with normal saline only, and the cisplatin group was treated with a single injection of cisplatin (25 mg / kg, intraperitoneal injection) and normal saline injection for three consecutive days (once every 24 hours, intraperitoneal injection). The cisplatin and protocatechuic aldehyde combination group was treated with a single injection of cisplatin (25 mg / kg, intraperitoneal injection) and protocatechuic aldehyde injection for three consecutive days (once every 24 hours, 100 mg / kg, intraperitoneal injection). The protocatechuic aldehyde group was treated with protocatechuic aldehyde injection for three consecutive days (once every 24 hours, 100 mg / kg, intraperitoneal injection).

[0138] 3) 72 hours after injection, blood was collected from the eye socket for testing of serum creatinine and blood urea nitrogen.

[0139] 10.2 Experimental Results

[0140] like Fig.11 As shown in the figure, after acute renal injury caused by cisplatin, the levels of blood creatinine and blood urea nitrogen increased; compared with the cisplatin group, after treatment with protocatechuic aldehyde at a therapeutic dose of 100 mg / kg, the levels of blood creatinine and blood urea nitrogen in mice showed a slight downward trend, but there was no statistical significance compared with the cisplatin group. What is more noteworthy is that the levels of blood creatinine (average value: 95.15μM) and blood urea nitrogen (average value: 32.57 mM) in the protocatechuic aldehyde group were higher than those in the 2,4-dihydroxybenzaldehyde group (average value: 42.25μM) and blood urea nitrogen (average value: 19.65 mM). Figure 6 ).

[0141] 10.3 Experimental conclusion: The structure of protocatechuic aldehyde is similar to that of 2,4-dihydroxybenzaldehyde, but the same dose of 2,4-dihydroxybenzaldehyde is better than protocatechuic aldehyde in relieving acute kidney injury and has lower toxicity than protocatechuic aldehyde. Its clinical application value is incomparable to that of protocatechuic aldehyde.

[0142] In summary, the present invention has found through a large number of cell and animal experimental screening that 2,4-dihydroxybenzaldehyde can inhibit the increase of renal cell lipid peroxidation level, ferrous ion content and reactive oxygen content, reduce renal cell ferroptosis-like structural damage, thereby completely preventing renal damage, reducing blood creatinine, blood urea nitrogen levels, and kidney coefficient, and reducing the weight loss that often occurs in cancer patients during chemotherapy. The effect is better than protocatechuic aldehyde at the same dose. Toxicity tests show that the cytotoxicity of 2,4-dihydroxybenzaldehyde is much less than that of protocatechuic aldehyde, which has a similar chemical structure. In terms of improving renal injury, especially cisplatin-induced acute renal injury, the clinical value is incomparable to protocatechuic aldehyde, and it has practical promotion and application value.

Claims

1. Use of 2,4-dihydroxybenzaldehyde in the preparation of a drug for preventing and / or treating acute kidney injury caused by cisplatin.

2. The use according to claim 1, characterized in that: The medicine is a preparation prepared by taking 2,4-dihydroxybenzaldehyde as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

3. The use according to claim 2, characterized in that: The preparation is an oral preparation or an injection.

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