Active peptide for inhibiting ferroptosis and application thereof

Abstract

The application belongs to the technical field of biotechnology, and provides an active peptide for inhibiting ferroptosis and application thereof, and an amino acid sequence of the active peptide is shown as SEQ ID No. 1. The active peptide for inhibiting ferroptosis provided by the application is derived from a walnut, has high activity and no obvious side effects compared with currently reported ferroptosis inhibitors; the biological activity peptide is verified to have anti-inflammatory activity through in-vitro and animal model tests, can significantly down-regulate cisplatin-induced iron overload, reduce the number of ferroptosis mitochondria, improve AKI and related metabolic indicators, and can be applied to preparation of a medicine for preventing or treating AKI.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an active peptide that inhibits ferroptosis and its applications. Background Technology

[0002] Acute kidney injury (AKI), a clinical syndrome characterized by rapid deterioration of renal function, has a high incidence in intensive care unit patients. AKI not only leads to prolonged hospital stays and increased medical costs but is also closely related to the development of chronic kidney disease and end-stage renal disease, seriously threatening patients' quality of life and survival. AKI can be caused by a variety of factors, including chemical drugs, toxins, ischemia, infection, and surgical trauma. Drug-related nephrotoxicity is one of the main causes of AKI, including factors such as antibiotics (e.g., aminoglycosides), anticancer drugs (e.g., cisplatin), analgesics (e.g., nonsteroidal anti-inflammatory drugs), and immunosuppressants (e.g., cyclosporine). Currently, clinical treatment for AKI is mainly supportive intervention, including correcting hypovolemia, discontinuing nephrotoxic drugs, and renal replacement therapy. However, these methods only temporarily relieve symptoms and cannot fundamentally stop the progression of kidney damage. Therefore, developing novel improvement strategies for AKI has become an urgent problem to be solved.

[0003] Ferroprelation, a form of iron-dependent programmed cell death, is essentially caused by the abnormal accumulation of phospholipid peroxides leading to cell membrane structural collapse. Its core mechanisms involve decreased glutathione peroxidase 4 (GPX4) activity, increased iron ion levels, and the accumulation of lipid peroxidation products. Ferroprelation is closely linked to various diseases, such as neurodegenerative diseases and cardiovascular diseases. In a cisplatin-induced AKI model, ferroptosis levels in renal tubular epithelial cells are significantly elevated. Ferroprelation inhibitors can effectively improve renal function by inhibiting lipid peroxidation and regulating iron metabolism-related genes (such as TFR1 and FPN1). Currently reported ferroptosis inhibitors are mainly derived from synthetic compounds, with fewer reports on naturally derived ferroptosis inhibitors. Summary of the Invention

[0004] The purpose of this invention is to provide an active peptide that inhibits ferroptosis, thereby addressing the problems mentioned in the background section.

[0005] The present invention is implemented as follows: an active peptide that inhibits ferroptosis, wherein the amino acid sequence of the active peptide is Phe-Gly-Met-Pro-Pro-Cys-Leu (FGMPPCL), as shown in SEQ ID No. 1.

[0006] Another objective of this invention is to provide the application of an active peptide that inhibits ferroptosis in the preparation of ferroptosis inhibitors.

[0007] Another objective of this invention is to provide the application of an active peptide that inhibits ferroptosis in the preparation of drugs for the prevention or treatment of AKI.

[0008] The present invention provides an active peptide for inhibiting ferroptosis, which is derived from walnut protein hydrolysate. Compared with currently reported ferroptosis inhibitors, it has high activity and no obvious side effects.

[0009] In vitro and animal model experiments have verified that this bioactive peptide has anti-inflammatory activity, can significantly downregulate cisplatin-induced iron overload, reduce the number of iron-dead mitochondria, and improve AKI and related metabolic indicators. It can be used to prepare drugs for the prevention or treatment of AKI. Attached Figure Description

[0010] Figure 1 This is a schematic diagram illustrating the effect of the active peptide FGMPPCL provided in Example 2 of the present invention on tumor necrosis factor-α (TNF-α).

[0011] Figure 2 This is a schematic diagram illustrating the effect of the active peptide FGMPPCL provided in Example 2 of the present invention on interleukin-6 (IL-6);

[0012] Figure 3 This is a schematic diagram illustrating the effect of the active peptide FGMPPCL provided in Example 2 of the present invention on interferon-γ (IFN-γ);

[0013] Figure 4 This is a graph showing the iron content analysis of mouse kidney tissue provided in Example 3 of the present invention;

[0014] Figure 5 This is a representative image of protein immunoblotting provided in Example 4 of the present invention;

[0015] Figure 6 This is the quantitative analysis of FTH1 protein by immunoblotting provided in Example 4 of the present invention;

[0016] Figure 7 This is the quantitative analysis of GPX4 protein immunoblotting provided in Example 4 of the present invention;

[0017] Figure 8 This is the quantitative analysis of xCT protein immunoblotting provided in Example 4 of the present invention;

[0018] Figure 9 The transmission electron microscopy analysis results of the ultrastructure of mouse kidney tissue provided in Example 5 of the present invention are shown in Figure 5 (a is the result of the control group, b is the result of the model group, and c is the result of the FGMPPCL intervention group).

[0019] Figure 10This is a schematic diagram illustrating the effect of the active peptide FGMPPCL provided in Example 6 of the present invention on the serum biochemical index urea nitrogen (BUN) in mice.

[0020] Figure 11 This is a schematic diagram illustrating the effect of the active peptide FGMPPCL provided in Example 6 of the present invention on the serum biochemical index creatinine (Scr) in mice.

[0021] Figure 12 The image shows a representative HE staining of mouse kidneys provided in Example 7 of this invention (a is the result of the control group, b is the result of the model group, and c is the result of the FGMPPCL intervention group; where black represents glomeruli and yellow represents renal tubules).

[0022] Figure 13 This is a representative immunofluorescence image provided in Example 8 of the present invention;

[0023] Figure 14 This is the KIM-1 immunofluorescence quantitative analysis provided in Example 8 of the present invention;

[0024] Figure 15 This is the NGAL immunofluorescence quantitative analysis provided in Example 8 of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0026] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0027] Example 1: Solid-phase synthesis method of active peptide FGMPPCL, comprising the following steps:

[0028] Solid-phase synthesis: Fmoc-Leu-Wang Resin was soaked and swollen in dichloromethane (DCM) and then dried. A decapping solution was added to remove the Fmoc protecting group. After washing, Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Gly-OH, and Fmoc-Phe-OH were added sequentially for condensation reactions. After each condensation step, the mixture was thoroughly washed. After adding the last amino acid (Fmoc-Phe-OH), the N-terminal Fmoc protecting group was removed again by the decapping solution and the mixture was washed.

[0029] Cutting and Purification: Trifluoroacetic acid (TFA) cutting solution was added to the resin for cutting reaction. The lysate was filtered out, and the resin was washed with a small amount of TFA. The filtrates were combined, and the resulting crude peptide solution was concentrated. The precipitate was precipitated with diethyl ether, centrifuged, and dried to obtain crude product. A small amount of crude product was taken for mass spectrometry (MS) analysis to confirm the target molecular weight. About 1 mg of crude product was completely dissolved, and the retention time of the target peptide main peak was determined using analytical HPLC (mobile phase A: 0.1% TFA / H2O, B: 0.1% TFA / ACN) at a gradient of 10-100% B. Based on this retention time, the gradient program of preparative HPLC (Beijing Innovation Tongheng CXTH-3000 liquid chromatography system) was set. The remaining crude product was dissolved, filtered, and injected. The equilibrated preparative system (mobile phase A: H2O, B: ACN) was started, and the correct target peptide main peak fraction was collected according to the set gradient. The qualified fractions were combined, and most of the acetonitrile was removed by rotary evaporation under reduced pressure. The remaining aqueous solution was freeze-dried to obtain the purified target peptide FGMPPCL.

[0030] Example 2: Determination of the anti-inflammatory activity of the active peptide FGMPPCL:

[0031] The experimental groups included three categories: control, model, and active peptide intervention. PC12 cell suspension was administered at a concentration of 1×10⁻⁶ cells / mL. 5Cells were seeded at a concentration of 100 μM / mL into six-well plates (2 mL per well) and incubated at 37°C with 5% CO2 for 24 h. The control group received 1640 complete medium. The model group was cultured in 1640 complete medium for 24 h, then replaced with a solution containing 1 μg / mL LPS-1640 for another 24 h. The active peptide intervention group was cultured in 100 μM active peptide 1640 complete medium for 24 h, then replaced with 1640 solution containing 1 μg / mL LPS for another 24 h. After cell culture, supernatant samples were collected from each well and centrifuged at 12000 r / min for 10 minutes. Cell debris was removed, and the operation of the rat interleukin (IL-6), interferon-gamma (INF-γ), and tumor necrosis factor (TNF-α) ELISA kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.) was strictly followed. The concentration levels of pro-inflammatory mediators (TNF-α, IL-6, and INF-γ) were quantitatively detected. Standard wells and sample wells were set up. 50 μL of different concentrations of standard solution were added to each standard well. 10 μL of the sample to be tested was added to each sample well, followed by 40 μL of sample diluent. No diluent was added to the blank wells. Except for the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each of the standard and sample wells. The reaction wells were sealed with sealing film and incubated at 37°C for 60 min. The liquid was discarded, and the plates were patted dry on absorbent paper. Each well was filled with washing buffer, allowed to stand for 1 min, the washing buffer was discarded, and the plates were patted dry on absorbent paper. This washing process was repeated 5 times. 50 μL each of substrate A and B were added to each well. μL, incubate at 37℃ in the dark for 15 min, add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm wavelength within 15 min.

[0032] The results are as follows Figures 1 to 3 As shown, when the concentration of the active peptide FGMPPCL was 100 µM, it significantly reduced the content of inflammatory factors in LPS-induced PC12 cells. The content of TNF-α decreased from 167.61 pg / mL to 79.96 pg / mL, the content of IL-6 decreased from 154.38 pg / mL to 91.75 pg / mL, and the content of IFN-γ decreased from 258.04 pg / mL to 192.25 pg / mL.

[0033] Example 3: Determination of iron ion content reduction by active peptide FGMPPCL:

[0034] Animal research was approved by the Ethics Committee of Jilin Agricultural University and followed the European Commission's Guidelines on the Care and Use of Laboratory Animals. Male C57BL / 6J mice (18-20 g) were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China). The mice were housed at the Experimental Animal Center of Jilin Agricultural University at a constant temperature (20 ± 2℃) with 12 hours of light / dark conditions and free access to food and water.

[0035] After acclimatizing to the indoor environment for one week, the mice were randomly divided into three groups (n=6): (I) Control group: administered physiological saline by gavage daily for 28 consecutive days; (II) Model group: administered physiological saline by gavage daily, and injected with cisplatin at a concentration of 20 mg / kg on the 25th day. The animals were sacrificed 72 h after injection; (III) Active peptide intervention group: administered FGMPPCL solution by gavage daily at a concentration of 80 mg / kg, and injected with cisplatin at a concentration of 20 mg / kg on the 25th day. The animals were sacrificed 72 h after injection.

[0036] Strictly follow the procedure of the rat iron content (ferriazine colorimetric method) detection kit (purchased from Edison Biotechnology Co., Ltd.). Take about 0.1g of kidney tissue, add 1 mL of extraction solution, homogenize on ice, centrifuge at 12000 rpm for 5 min at 4℃, take the supernatant and place it on ice for testing.

[0037] Preheat the microplate reader for 30 minutes and set the wavelength to 562 nm. Thaw all reagents to room temperature and divide them into three groups: test tubes, standard tubes, and blank tubes. For samples: add 120 μL to the test tubes, and do not add any to the standard or blank tubes; for standards: add 120 μL to the standard tubes, and do not add any to the test or blank tubes; for distilled water: add 120 μL to the blank tubes, and do not add any to the test or standard tubes; for reagent one: add 260 μL to each test, standard, and blank tube; for reagent two: add 20 μL to each test, standard, and blank tube.

[0038] After adding the sample, mix thoroughly and let stand at room temperature for 15 min. If the solution is turbid, centrifuge at 3000 rpm for 5 min, take 200 μL of supernatant to a 96-well plate, and measure the absorbance A at a wavelength of 562 nm (if the absorbance A of the measuring tube is greater than 0.8, the sample supernatant can be diluted with distilled water, and the dilution factor D can be substituted into the calculation formula). This absorbance is used in subsequent experimental steps such as calculating the content of the target component in the sample.

[0039] Calculated based on organizational quality:

[0040] ;

[0041] In the formula, D is the dilution factor; W is the sample mass. To measure the absorbance of the tube; The absorbance of the blank tube;

[0042] The results are as follows Figure 4 As shown, the iron content in the model group (cisplatin-treated group) was 32.60 ± 1.73 μg / g, which was significantly higher than the iron content in the control group (26.42 ± 0.70 μg / g). P <0.05), while the iron content of the FGMPPCL group was 20.71 ± 2.76 μg / g ( P The iron ion content was significantly lower than that in the model group (<0.05), indicating that FGMPPCL has the ability to reduce iron ion content.

[0043] Example 4: Determination of the ability of the active peptide FGMPPCL to regulate ferroptosis core protein:

[0044] The experimental animals were grouped and treated as in Example 3. Then, 30 mg of kidney tissue was rapidly collected on ice and 300 μL of RIPA lysis buffer containing the protease inhibitor PMSF (1:100) was added. The mixture was rapidly ground on ice for 20 min, followed by centrifugation at 12000 r / min for 10 min at 4°C. The supernatant was collected into EP tubes, and the protein concentration was determined using the BCA method. The specific procedure for Western blotting is as follows:

[0045] Electrophoresis: Based on the molecular weight of the target protein, prepare a separating gel with a concentration of 8-12%, run at a constant voltage of 80 V for 20 min, then adjust to 120 V for 90 min or 250 V for 30 min;

[0046] Cut the gel according to the molecular weight of the target protein. Cut the 0.45 μm PVDF into strips according to the size of the gel strip. Activate in 100% anhydrous ethanol for 10 s and then put it into the wet transfer solution. Place the strips in the order of "black clip - transfer sponge - gel - PVDF membrane - transfer sponge - white clip" from bottom to top to avoid air bubbles. Place the strips in the wet transfer tank and set the transfer time and current according to the molecular weight according to the instructions.

[0047] After the transfer was completed, the PVDF membrane was placed in the antibody incubation box and washed three times with TBST for 10 minutes each time. After washing, the membrane was placed in 5% skim milk and blocked at room temperature for 60 minutes.

[0048] After blocking, the PVDF membrane was washed three times with TBST for 5 minutes each time. The primary antibody was diluted to different concentrations with 3% skim milk powder according to the antibody instructions and incubated with the PVDF membrane at 4°C overnight.

[0049] The following day, the PVDF membrane was washed three times with TBST for 5 min each time, then HRP-labeled IgG secondary antibody was added and incubated at room temperature for 60 min; subsequently, it was washed three times with TBST for 5 min each time.

[0050] ECL color development: ECL luminescent solution was developed and photographed in a gel imaging system, and ImageJ software was used for strip grayscale analysis.

[0051] The results are as follows Figures 5 to 8 As shown, compared with the blank group, cisplatin treatment can significantly downregulate the expression level of ferroptosis-related proteins. P <0.05); the relative expression levels of FTH1, GPX4, and xCT proteins in the model group decreased to 1.59 ± 0.07, 1.60 ± 0.13, and 2.57 ± 0.36, respectively, suggesting that cisplatin induced ferroptosis, while the expression of all three proteins was significantly upregulated after FGMPPCL intervention. P <0.05), with FTH1 increasing to 2.18 ± 0.31, GPX4 increasing to 2.39 ± 0.21, and xCT increasing to 3.93 ± 0.22, indicating that the expression levels of the three proteins were significantly restored after FGMPPCL intervention.

[0052] Example 5: Active peptide FGMPPCL improves cellular ferroptosis morphology:

[0053] The experimental animals were grouped and treated as in Example 3. Subsequently, the kidneys of euthanized mice were rapidly removed on ice and placed in 1.5 mL centrifuge tubes. The samples were fixed overnight at 4°C in 2.5% glutaraldehyde fixative, ensuring complete immersion. After fixation, the glutaraldehyde solution was discarded, and the samples were thoroughly washed three times with 0.1 M phosphate buffer (pH 7.0), each wash lasting approximately 15 minutes. The samples were then transferred to 1% osmium tetroxide solution for a second fixation of 1-2 hours, followed by three washes with phosphate buffer. Dehydration was performed using a gradient ethanol method, sequentially passing the samples through 30%, 50%, 70%, 80%, 90%, and 95% ethanol solutions, each concentration for 15 minutes, followed by a final treatment with anhydrous ethanol for 20 minutes. The dehydrated samples were then transferred to pure acetone for 20 minutes to prepare for the subsequent permeation step. The permeation process was conducted in stages: first, a 1:1 mixture of acetone and embedding agent was used for 1 hour, followed by a 3:1 mixture for 3 hours. h, finally transition to pure embedding medium for overnight infiltration. After complete infiltration, the sample is embedded and placed in a 70℃ oven for polymerization overnight. The embedded sample is sectioned on a LEICA EM UC7 ultramicrotome to obtain ultrathin sections of 70-90 nm. After double staining with lead citrate and uranium acetate (50% ethanol saturated solution) for 5-10 min each, the tissue ultrastructure can be observed under a transmission electron microscope.

[0054] The results are as follows Figure 9As shown, the mitochondria in the control group exhibited a typical double-membrane structure, with uniform distribution and tight arrangement, and good membrane integrity. In contrast, the model group showed a large number of ferroptotic mitochondria, with the disappearance, breakage and reduction of mitochondrial cristae, a decrease in mitochondrial volume and an increase in membrane density, and shrinkage of the nuclear membrane. In the FGMPPCL intervention group, a significant reduction in ferroptotic mitochondria was observed, the nuclear membrane returned to normal, and the mitochondria were evenly distributed, indicating that FGMPPCL has the ability to improve the morphology of ferroptosis.

[0055] Example 6: The effect of active peptide FGMPPCL on AKI determination:

[0056] The experimental animals were grouped and treated as in Example 3. Then, mouse kidney tissue was quickly removed on ice, rinsed with pre-cooled physiological saline, and stored at -80°C for later use. Samples were prepared according to the instructions of the blood urea nitrogen (BUN) kit and creatinine (CRE) assay kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.) to measure the urea nitrogen and creatinine content in mouse kidney tissue.

[0057] Creatinine content determination:

[0058] Classification by aperture: Determination (T), Standard (S), Blank (B);

[0059] Additives and corresponding volumes: Sample (6 μL for assay wells only), Reagent 3: Standard (6 μL for standard wells only), Double-distilled water (6 μL for blank wells only), Reagent 1: Enzyme solution A (180 μL for all three wells), Reagent 2: Enzyme solution B (60 μL for all three wells);

[0060] Incubation and detection: Incubate at 37℃ for 5 min, and measure absorbance A1 at 546 nm wavelength; after adding reagent II, incubate under the same conditions and measure A2. Calculate using the formula ΔA = A2 - K*A1.

[0061] The formula for calculating K is as follows:

[0062] ;

[0063] For the formula used to calculate creatinine content:

[0064] ;

[0065] in, The meaning is the concentration of the standard, which is 442 μmol / L. The absorbance difference of well (T) after sample addition, incubation, and other operations is calculated using a formula. The absorbance difference between the blank well (B) and the incubation well (B) is calculated using a formula.

[0066] Determination of urea nitrogen content:

[0067] Blank tube: Add 0.02 mL distilled water and 0.25 mL buffer enzyme solution; Standard tube: Add 0.02 mL 10 mmol / L BUN standard working solution and 0.25 mL buffer enzyme solution; Test tube: Add 0.02 mL test sample and 0.25 mL buffer enzyme solution. Mix the blank tube, standard tube, and test tube thoroughly after adding the above samples, place them in a 37°C environment, and incubate them in a water bath for 10 min. Add 1 mL phenol colorimetric reagent and 1 mL alkaline sodium hypochlorite to the blank tube, standard tube, and test tube, respectively. Mix the tubes thoroughly again, place them in a 37°C environment, and incubate them in a water bath for 10 min. Then, measure the absorbance at a wavelength of 640 nm.

[0068] ;

[0069] In the formula: N is the dilution factor of the sample before testing, which is 1 for undiluted samples;

[0070] The results are as follows Figure 10 , 11 As shown, the serum urea nitrogen (BUN) and creatinine (Scr) levels in the cisplatin-induced acute kidney injury model mice were significantly elevated, reaching 14.25 mmol / L and 50.71 μmol / L, respectively, while the corresponding levels in the control group were 10.15 mmol / L and 29.66 μmol / L, respectively. The results confirm that cisplatin successfully induced an acute kidney injury model, accompanied by significant impairment of glomerular filtration function and metabolic waste excretion. FGMPPCL intervention can significantly reduce the BUN and Scr levels in the model mice (11.44 ± 0.53 mmol / L and 31.00 ± 1.46 μmol / L, respectively).

[0071] Example 7: Analysis of the structural morphology of glomeruli, renal tubules, and interstitium improved by the active peptide FGMPPCL:

[0072] The experimental animals were grouped and treated as in Example 3. Subsequently, mouse kidney tissue samples were rapidly collected on ice and fixed at a ratio of 1:20 (tissue:fixative) for 48 h. Then, they were subjected to gradient dehydration, paraffin impregnation and embedding to prepare 3 μm thick sections for subsequent histological staining.

[0073] Mouse kidney tissue fixation: After the mice were sacrificed, the kidney tissue was quickly removed on ice to avoid excessive force that could cause kidney rupture. After the operation, excess blood was washed off with physiological saline and the tissue was immediately placed in 4% paraformaldehyde, with the volume of the paraformaldehyde solution being 20 times the volume of the kidney tissue.

[0074] After fixation, the kidney tissue samples were placed in a labeled embedding cassette and then subjected to gradient processing in a pre-programmed dehydrator. The program settings were as follows: 3 h 4% paraformaldehyde → 1 h 75% ethanol → 1 h 85% ethanol → 1 h 95% ethanol → 1 h 95% ethanol → 1 h 100% ethanol → 45 min xylene → 1 h paraffin → 1 h paraffin → 2 h paraffin.

[0075] The biological tissue embedding machine needs to be preheated to completely melt the paraffin. Place the dehydrated sample in the center of the mold and slowly pour in the molten paraffin (be careful to avoid the formation of air bubbles). Immediately transfer it to a -20℃ freezing platform for rapid solidification. After the wax block has completely solidified, carefully remove the mold to obtain the formed tissue wax block, and store it at room temperature for later use.

[0076] Set the rotary microtome to 3 μm thickness for continuous sectioning. Hold the section edge with tweezers and float it on the surface of 37℃ warm water. After the section naturally flattens, use a glass slide to pick it up, tilt it at 45° to drain excess water, melt the wax in a 60℃ slide oven, and then store it at room temperature.

[0077] Tissue sections were dewaxed in xylene, then rehydrated with a gradient of ethanol solutions. Next, the sections were stained with hematoxylin for 3-5 minutes, differentiated with 1% hydrochloric acid alcohol, and then blued back in running water to make the cell nuclei appear clearly blue. After that, the cytoplasm was stained with eosin for 1-2 minutes to make it appear red. Finally, the sections were dehydrated, cleared, and mounted with neutral resin to allow observation of the staining of the cell nuclei and cytoplasm under a microscope.

[0078] The results are as follows Figure 12 As shown, the glomeruli in the model group exhibited significant congestion and vacuolation, with numerous vacuoles appearing in the cytoplasm of the renal tubular epithelial cells. The basement membrane was ruptured and accompanied by interstitial edema. After FGMPPCL intervention, the degree of glomerular vacuolation was significantly reduced, the basement membrane structure was completely repaired, and the interstitial edema disappeared, indicating that FGMPPCL has the ability to improve the morphology of structures such as glomeruli, renal tubules, and interstitium.

[0079] Example 8: Active peptide FGMPPCL improves cisplatin-induced renal tubular injury:

[0080] Paraffin sections prepared in Example 7 were dewaxed, treated with xylene, dehydrated in a gradient of different concentrations of ethanol, washed multiple times with distilled water, placed in EDTA antigen retrieval solution, and microwaved for antigen retrieval. After natural cooling, the sections were washed three times with PBS for 10 min each time, blocked with BSA for 30 min, the blocking solution was discarded, and primary antibody was added directly and incubated overnight at 4°C. The sections were washed three times with PBS for 5 min each time, and incubated with HRP secondary antibody at room temperature in the dark for 60 min. After three PBS washes, Cy3 fluorescence enhancer was added, and cell nuclei were counterstained with DAPI for 5 min. After multiple PBS washes, the sections were mounted with an anti-fluorescence quencher. Images were acquired under a Nikon ECLIPSE Ti-SR microscope. DAPI was excited at 330–380 nm and emitted at 420 nm; Cy3 was excited at 510–560 nm and emitted at 590 nm.

[0081] The results are as follows Figure 13-15 As shown, the relative expression level of KIM-1 in the model group was 1.26 ± 0.04, which was significantly higher than that in the control group (1.00 ± 0.11). P <0.05), after FGMPPCL intervention, KIM-1 expression level recovered to 0.98 ± 0.10 ( P <0.05); Furthermore, the relative expression level of NGAL in the model group (5.43 ± 0.60) was significantly higher than that in the control group (1.00 ± 0.21), while FGMPPCL pretreatment significantly inhibited the upregulation of NGAL, reducing its expression level to 3.61 ± 0.34 ( P <0.05), indicating that FGMPPCL can effectively reverse cisplatin-induced renal tubular injury.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An active peptide that inhibits ferroptosis, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID No.

1.

2. The use of the active peptide for inhibiting ferroptosis as described in claim 1 in the preparation of drugs for the prevention or treatment of AKI, characterized in that, The AKI mentioned is caused by cisplatin.

Images