Use of ophiopogonin A in the preparation of anti-tissue fibrosis drugs

Abstract

This invention discloses the application of daidzein A in the preparation of anti-fibrotic drugs. This invention is the first to propose and verify that daidzein A can effectively inhibit ferroptosis, restore renal cell activity, downregulate the expression of fibrosis-related proteins, alleviate collagen deposition, reduce kidney damage, and effectively curb the progression of renal fibrosis, providing a new option with clinical translational potential for the treatment of renal fibrosis.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and more particularly to the application of daidzein A in the preparation of anti-fibrotic drugs. Background Technology

[0002] Chronic kidney disease (CKD) is one of the most common long-term diseases worldwide, with its incidence and mortality rates rising in recent years, currently affecting approximately 10% of the global adult population. With the aging population, the prevalence of CKD is projected to continue to climb, potentially exceeding 10% in some regions globally by 2050 (G3-G5 stages). Adult CKD is characterized by a persistent decline in glomerular filtration rate or elevated urinary albumin excretion for more than 12 weeks. Its core pathological process is renal fibrosis, namely, the excessive accumulation of extracellular matrix proteins leading to tissue scarring and organ failure. The TGF-β / Smad signaling pathway plays a crucial driving role in this process, promoting fibroblast activation, epithelial-mesenchymal transition, and excessive deposition of matrix proteins such as collagen.

[0003] Currently, the main treatments for CKD include medication to slow disease progression and end-stage renal replacement therapy, including hemodialysis, peritoneal dialysis, and kidney transplantation. However, these treatments have significant limitations: dialysis cannot replace the kidney's endocrine function, and the 5-year survival rate on long-term dialysis is only about 50%; while kidney transplantation is the best treatment option, there is a severe shortage of donors, with only 10%–15% of end-stage patients having the opportunity to receive a transplant. Therefore, finding new drugs that can effectively reduce renal interstitial fibrosis remains extremely urgent.

[0004] Ophiopogonin A is a steroidal saponin natural product isolated from Ophiopogon japonicus. Studies have shown that it possesses antitumor activity, exerting its effects by inducing apoptosis and inhibiting cell proliferation. However, its role in kidney protection has not yet been reported. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for quasi-ophiopogonin A to alleviate the progression of tissue fibrosis by inhibiting ferroptosis and downregulating the expression of fibrosis-related proteins, and to apply this method in the preparation of anti-fibrosis therapeutic drugs.

[0006] Technical solution: The application of the daidzein A described in this invention in the preparation of anti-fibrotic drugs.

[0007] Preferably, the CAS number of the daidzein A is 2423917-90-0.

[0008] Preferably, the application is in the preparation of a drug that inhibits the expression of fibrosis-related proteins; more preferably, the fibrosis-related proteins include α-smooth muscle actin and / or type I collagen.

[0009] Preferably, the application is in the preparation of a drug that inhibits ferroptosis.

[0010] Preferably, the tissue fibrosis is treated with a drug for renal fibrosis.

[0011] Preferably, the drug contains daidzein A or its pharmaceutically acceptable salt, solvate, or hydrate as an active ingredient.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0013] Preferably, the dosage form of the drug includes capsules, granules, powders, tablets, microcapsules, injections, infusions, oral liquids, suspensions, patches, suppositories, microemulsions, liposomes, nanoparticles, and lyophilized powder injections.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention proposes and verifies for the first time that douchiside A can effectively inhibit ferroptosis, restore renal cell activity, downregulate the expression of fibrosis-related proteins, alleviate collagen deposition, reduce kidney damage, and effectively curb the progression of renal fibrosis, providing a new option with clinical translational potential for the treatment of renal fibrosis. Attached Figure Description

[0015] Figure 1 A statistical graph of organ coefficients of renal tissue in a mouse model of renal fibrosis after treatment with daidzein A. Figure 2 A statistical chart showing the results of renal function indicators in mice with renal fibrosis model after treatment with daidzein A. Figure 3 Representative H&E staining images of kidney tissue from a mouse model of renal fibrosis after treatment with daidzein A; Figure 4 Representative Masson staining images of kidney tissue from a mouse model of renal fibrosis after treatment with daidzein A. Figure 5 Representative PAS staining images of kidney tissue from a mouse model of renal fibrosis after treatment with douchidinoside A. Figure 6 Representative images of Sirius red staining of kidney tissue from a mouse model of renal fibrosis after treatment with daidzein A. Figure 7 Figure 1 shows the results of measuring the expression levels of biomarkers of renal tissue damage in a mouse model of renal fibrosis after treatment with daidzein A. Figure 8Representative immunohistochemical staining images of renal tissue damage biomarkers in a mouse model of renal fibrosis after treatment with douchidinoside A. Figure 9 Figure 1 shows the results of measuring the expression levels of fibrosis-related proteins in the renal tissue of a mouse model of renal fibrosis after treatment with douchiside A. Figure 10 Figure 1 shows the results of hydroxyproline level measurement in kidney tissue of mice with renal fibrosis model after treatment with daidzein A. Figure 11 Figure 1 shows the results of measuring the levels of iron death-related indicators in the renal tissue of mice with renal fibrosis model after treatment with daidzein A. Figure 12 Figure 1 shows the results of viability assay of HK2 cells, a renal fibrosis model, after treatment with daidzein A. Figure 13 Figure 1 shows the results of the proliferation activity assay of HK2 cells, a renal fibrosis model, after treatment with daidzein A. Figure 14 Figure 1 shows the results of measuring the expression levels of fibrosis-related proteins in HK2 cells, a renal fibrosis model, after treatment with daidzein A. Figure 15 Figure 1 shows the flow cytometry results of ROS levels in HK2 cells, a renal fibrosis model, after treatment with daidzein A. Figure 16 Figure 1 shows the results of lipid peroxidation analysis in HK2 cells, a renal fibrosis model, after treatment with daidzein A. Figure 17 The figure shows the results of measuring the levels of iron death-related indicators in HK2 cells, a renal fibrosis model, after treatment with daidzein A. Detailed Implementation

[0016] The technical solution of the present invention will be further described below.

[0017] Example 1: In vivo efficacy verification of Ophiopogonside A (OPA) in the treatment of renal fibrosis 1. Construction and drug treatment of animal models of renal fibrosis A mouse model of renal fibrosis was induced by unilateral ureteral ligation (UUO). Eight-week-old male C57BL / 6J mice, weighing 20-22 g, were purchased from the Experimental Animal Center of Nantong University and randomly divided into a sham-operated group (n=8) and a UUO group (n=24). In the UUO group, mice were anesthetized and fixed in a supine position on the operating table. After skin preparation and disinfection, a 1.5 cm longitudinal incision was made 1 cm below the costal margin on the left side of the back. The left kidney was located by dissecting the muscles and peritoneum layer by layer, and the upper segment of the ureter was freed and double-ligated with 4-0 silk suture. The incision was then sutured layer by layer, and routine postoperative anti-infection treatment was administered. In the sham-operated group, only the left ureter was dissected and exposed without ligation.

[0018] Fourteen days after model establishment, the UUO group mice were randomly divided into three groups (n=8 per group): KF+OPA group, KF+FIN group, and KF group. Drug administration was initiated simultaneously, denoted as day 0. Drugs were administered via gavage once daily. Mice in the KF+OPA group received 10 mg / kg OPA (purchased from MedChemExpress LLC., catalog number HY-N5146), mice in the KF+FIN group received 5 mg / kg Finerenone (FIN, purchased from MedChemExpress LLC., catalog number HY-111372), and mice in the KF group and sham-operated group received the same volume of saline. Treatment continued until day 15 (d15).

[0019] 2. Organ coefficient detection At the end of the experiment (d15), the mice were weighed and euthanized. The intact kidney tissue was removed, the surface moisture was blotted with filter paper, and the tissue was weighed. The kidney tissue coefficient was calculated according to the following formula: Kidney tissue coefficient (%) = wet weight of kidney tissue / mouse weight × 100.

[0020] The statistical results of the organ coefficient of mouse kidney tissue at the experimental endpoint are as follows: Figure 1 As shown, the renal tissue coefficient of mice in the Sham group was 0.78, the renal tissue coefficient of mice in the KF group decreased to 0.42, the renal tissue coefficient of mice in the KF+OPA group recovered to 0.66, and the renal tissue coefficient of mice treated with the positive drug FIN recovered to 0.76. This indicates that OPA drugs have a therapeutic effect on renal fibrosis.

[0021] 3. Kidney function test Mouse kidney tissue was collected and supplemented with BeyoLysis at a ratio of 10 mg tissue to 100 μL. TM Buffer A for Metabolic Assay (catalog number S0291S-1) was homogenized in an ice bath, centrifuged at 12000 ×g for 5 min at 4°C, and the supernatant was collected. The urinary creatinine content was measured using the Beyotime Amplex Red Creatinine Assay Kit (catalog number S0291S), and the urea nitrogen content was measured using the Beyotime Urea Assay Kit (catalog number S0574S). The results are as follows Figure 2As shown in the figure, the left image represents the urine creatinine test results, and the right image represents the blood urea nitrogen test results. In the Sham group mice, the urine creatinine level was 0.59 mg / dL, and the blood urea nitrogen level was 44.83 mg / dL. In the KF group mice, the urine creatinine level was 1.24 mg / dL, and the blood urea nitrogen level was 81 mg / dL, which were significantly higher than those in the normal group, indicating severe kidney damage. In the KF+OPA group mice, the urine creatinine level was 0.76 mg / dL, and the blood urea nitrogen level was 59.3 mg / dL. In mice treated with the positive drug FIN, the urine creatinine level was 0.71 mg / dL, and the blood urea nitrogen level was 52 mg / dL. This indicates that OPA treatment inhibited kidney damage and restored kidney function in mice, and its therapeutic effect was comparable to that of the positive drug treatment.

[0022] 4. Pathological evaluation of kidney tissue 4.1 H&E staining Mouse kidney tissues were fixed in 4% paraformaldehyde solution for 48 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 3 μm sections. After dewaxing and hydration, hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin and observed and images were acquired under a microscope.

[0023] The results are as follows Figure 3 As shown, in the Sham group mice, the glomeruli exhibited a clear structure, intact capillary walls, and orderly cell arrangement, with no obvious inflammatory cell infiltration. In contrast, compared to the Sham group mice, the KF group mice showed renal tubular dilation, disordered renal structure, loss of renal tubular cell nuclei, and nuclear condensation, indicating impaired renal function, along with a significant increase in inflammatory cell infiltration. After OPA treatment, HE staining showed that these symptoms in the renal tissue were alleviated, with an effect comparable to that of the positive drug FIN treatment group, suggesting that OPA can serve as a potential therapeutic agent for renal fibrosis.

[0024] 4.2 Masson staining The preliminary steps were the same as those for H&E staining. In the staining section, the collagen deposits in the liver tissue were stained using the Beyotime Masson Tricolor Staining Kit (catalog number C0189S). After mounting with neutral resin, the tissue was observed and images were acquired under a microscope.

[0025] The results are as follows Figure 4 As shown, compared with the Sham group mice, the KF group mice showed a higher level of collagen deposition in their kidney tissue (blue area). After OPA treatment, the blue area was significantly reduced, inhibiting collagen deposition. Its therapeutic effect was similar to that of the positive drug FIN treatment group, indicating that the symptoms of renal fibrosis in mice were initially improved with OPA treatment.

[0026] 4.3 Periodic acid-Schiff staining The preliminary steps were the same as those for H&E staining. The staining part was stained using the Beyotime periodic acid-Scheff (PAS) staining kit (catalog number C0142M). After mounting with neutral resin, the slides were observed and images were acquired under a microscope.

[0027] The results are as follows Figure 5 As shown, compared with the KF group mice, the KF+OPA group mice showed reduced renal tubular damage and preserved kidney structure, especially with a significant reduction in renal tubular dilation and epithelial flattening, while the integrity of the brush border was partially restored.

[0028] 4.4 Sirius Red Staining The preliminary steps were the same as those for H&E staining. The staining part was stained with Beyyun Sirius Red Staining Kit (catalog number C0190S). After mounting with neutral resin, the slides were observed and images were acquired under a microscope.

[0029] The results are as follows Figure 6 As shown, collagen deposition was widespread in the kidneys of mice in the KF group, and this condition was significantly improved after OPA treatment, with a therapeutic effect similar to that after treatment with the positive drug KIN.

[0030] 5. Determination of biomarkers for renal tissue damage Kidney Injury Molecule-1 (KIM1) and Neutrophil Gelatinase-Associated Lipocalin (NGAL) were selected for assay.

[0031] 5.1 Protein Expression Level Measurement Mouse kidney tissue was collected and ground in liquid nitrogen using a mortar and pestle. For every 100 mg of tissue, 1 mL of RIPA lysis buffer (Catalog No. WB3100) containing 0.1 volume of a mixture of cyprotease inhibitors (P001) and cyprotease inhibitors (P003) was added. The mixture was lysed at 4°C for 30 min, centrifuged at 12000 rpm for 30 min, and the supernatant was collected and added to 5× Loading buffer (Catalog No. WB2001) in a controlled ratio. The mixture was then boiled in a metal bath for 10 minutes. After 1 minute, SDS-PAGE electrophoresis was performed. After transfer and blocking, KIM1 primary antibody (Proteintech, catalog number 30948-1-AP) diluted 1:1000, NGAL primary antibody (Proteintech, catalog number 31721-1-AP) diluted 1:10000, or GAPDH primary antibody (Proteintech, catalog number 10494-1-AP) diluted 1:10000 were added and incubated overnight at 4°C. After rinsing, secondary antibody (Proteintech, catalog number SA00001-2) diluted 1:10000 was added and incubated at room temperature for 2 h. Finally, the chemiluminescence was developed and images were acquired using the New Semiconductor Ultrasensitive ECL Chemiluminescence Kit (catalog number P10100).

[0032] The results are as follows Figure 7 As shown, the protein levels of KIM1 and NGAL were significantly increased in the KF group mice, but significantly decreased after OPA treatment, with the reduction level being comparable to that in the positive drug treatment group.

[0033] 5.2 Immunohistochemical staining analysis Mouse kidney tissue was fixed in 4% paraformaldehyde solution for 48 h, graded dehydration, paraffin embedding, and cut into 4 μm sections. Antigen retrieval was performed at 95°C for 20 min using citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide solution for 10 min, followed by blocking with 5% bovine serum albumin solution for 1 h. The sections were incubated overnight at 4°C with either a 1:500 dilution of KIM1 primary antibody or a 1:500 dilution of NGAL primary antibody. After washing, the sections were incubated with a 1:500 dilution of horseradish peroxidase (HRP) conjugated with secondary antibody (Proteintech, catalog number SA00004-2) at room temperature for 1 h. Signal detection was performed using a Beyotime DAB chromogenic reagent kit (catalog number P0202), and cell nuclei were counterstained with hematoxylin. Images were acquired using an optical microscope, and relative protein levels were quantified.

[0034] The results are as follows Figure 8As shown, OPA can significantly inhibit the protein levels of KIM1 and NGAL, with an inhibitory effect similar to that of the positive control drug FIN. The above experiments further confirm that OPA has a significant therapeutic effect on renal fibrosis in mice.

[0035] 6. Measurement of expression levels of renal fibrosis-related proteins The supernatant of the aforementioned mouse kidney tissue protein extract was added to a 5× Loading buffer solution of NewSyneema, heated to boiling in a metal bath for 10 min, and then subjected to SDS-PAGE electrophoresis. After transfer and blocking, COL1A1 primary antibody (purchased from Proteintech, catalog number 67288-1-Ig) diluted 1:5000, α-SMA primary antibody (purchased from Proteintech, catalog number 14395-1-AP) diluted 1:5000, or GAPDH primary antibody diluted 1:10000 was added and incubated overnight at 4°C. After rinsing, secondary antibody (purchased from Proteintech, catalog number SA00001-2 or SA00001-1) diluted 1:10000 was added and incubated at room temperature for 2 h. Finally, the images were acquired and developed using the NewSyneema ultrasensitive ECL chemiluminescence kit.

[0036] The results are as follows Figure 9 As shown, compared with the kidney tissue of the Sham group mice, the protein levels of COL1A1 and α-SMA in the kidney tissue of the KF group mice were significantly increased. However, with the intervention of OPA, the levels of the above protein molecules were significantly inhibited. The treatment effect of OPA was comparable to that of the positive drug FIN, and there was no significant difference between OPA and the Sham group mice.

[0037] 7. Determination of hydroxyproline levels in kidney tissue Kidney tissue of 100 mg from mice was collected, ground in liquid nitrogen using a mortar and pestle, and then lysed with 1 mL of RIPA lysis buffer on ice for 30 min. After centrifugation at 12000 rpm for 30 min at 4 °C, the supernatant was collected. The hydroxyproline content in the kidney tissue was determined using the Solarbio Hydroxyproline Assay Kit (catalog number BC0250).

[0038] The results are as follows Figure 10 As shown, the level of hydroxyproline in the Sham group mice was 35.33 μg / mg; while in the KF model mice, the level of hydroxyproline was significantly increased, specifically 137.67 μg / mg; in the KF+OPA group mice, the level of hydroxyproline was significantly decreased, down to 58.33 μg / mg; similarly, in the KF+FIN group mice, the level of hydroxyproline decreased to 50.67 μg / mg. The above experiments indicate that OPA drugs can significantly alleviate the level of fibrosis in the renal tissue of KF group mice.

[0039] 8. Measurement of renal tissue ferroptosis-related indicators 100 mg of mouse kidney tissue was collected and 1 mL of pre-cooled physiological saline was added to prepare a tissue homogenate. After centrifugation at 12,000 rpm for 15 min, the supernatant was collected. The levels of malondialdehyde (MDA), glutathione (GSH), and 4-hydroxynonenal (4-HNE) in the supernatant were detected using the Beyotime Lipid Oxidation Detection Kit (Catalog No. S0131S), the Beyotime GSH and GSSG Detection Kit (Catalog No. S0053), and the Sangon Biotech 4-Hydroxynonenal ELISA Kit (Catalog No. D751041).

[0040] The results are as follows Figure 11 As shown, compared with the Sham group mice, the MDA and 4-HNE levels in the KF group mice were significantly increased, and the treatment with OPA and FIN could significantly reverse the increase in MDA and 4-HNE; at the same time, the GSH level in the KF group mice was significantly decreased, and this decrease could also be reversed by OPA and FIN.

[0041] Example 2: In vitro efficacy verification of daidzein A in the treatment of renal fibrosis 1. Construction of a renal fibrosis cell model Human renal cortical proximal tubular epithelial cells HK2 (purchased from Wuhan Pronosei Life Science Co., Ltd., catalog number CL-0109) were cultured in DMEM medium supplemented with 10% fetal bovine serum in a dedicated incubator at 37°C and 5% carbon dioxide. When the cell density reached 70%, TGFβ1 (purchased from Thermo Fisher Scientific Inc., catalog number 100-21-10UG) was added to the medium at a final concentration of 5 ng / mL for 24 h to construct a renal fibrosis-related cell model.

[0042] 2. Cell viability assay after OPA treatment Normal HK2 cells or TGFβ1-induced HK2 cells were seeded into 96-well plates. When the cell density reached 70%, the original medium was replaced with DMEM complete medium containing 1‰ DMSO for normal HK2 cells, and the original medium was replaced with DMEM complete medium containing 1‰ DMSO and a final concentration of 20 µM OPA or a final concentration of 1 µM FIN for TGFβ1-induced HK2 cells.

[0043] After 24 h of treatment, each well was replaced with 100 μL of DMEM basal medium containing 10% CCK8 reagent and incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader, and the relative level of cell viability was calculated.

[0044] The results are as follows Figure 12As shown, compared with normal HK2 cells (DMSO), the activity of HK2 cells induced by TGFβ1 (TGFβ1) decreased to 39.34% of that of normal HK2 cells, while the activity of cells treated with OPA (TGFβ1+OPA) recovered to 68.6% of that of normal HK2 cells. At the same time, the activity of cells treated with FIN positive drug (TGFβ1+FIN) recovered to 79.77%. The above experiments preliminarily indicate that OPA can significantly alleviate the symptoms of renal fibrosis.

[0045] 3. Determination of cell proliferation capacity after OPA treatment Normal HK2 cells or TGFβ1-induced HK2 cells were seeded into 96-well plates. When the cell density reached 70%, the original medium was replaced with DMEM complete medium containing 1‰ DMSO for normal HK2 cells, and the original medium was replaced with DMEM complete medium containing 1‰ DMSO and a final concentration of 20 µM OPA or a final concentration of 1 µM FIN for TGFβ1-induced HK2 cells.

[0046] Cells were collected 24 h after treatment, and 20 μL of EdU reagent (purchased from Novizan, catalog number A413-01) was added to each well. The cells were incubated at 37°C for 2 h, fixed with 4% paraformaldehyde solution at room temperature for 15 min, and then permeabilized with Beyotime immunostaining permeabilization solution (catalog number P0096) at room temperature for 20 min. Click reaction solution (purchased from Novizan, catalog number A413-01) was then added, and the cells were incubated in the dark for 30 min. The cells were then mounted with Beyotime anti-fluorescence quenching mounting solution (catalog number P0131), and the images were observed and acquired using a fluorescence microscope.

[0047] The results are as follows Figure 13 As shown, compared with normal HK2 cells, the EdU positivity rate of HK2 cells induced by TGFβ1 was significantly reduced, and cell activity was inhibited; while after treatment with OPA or FIN, the proportion of positive cells increased significantly, proving that OPA has a good restorative effect on the activity of renal fibrosis cells.

[0048] 4. Determination of expression levels of fibrosis-related proteins after OPA treatment Normal HK2 cells or TGFβ1-induced HK2 cells were seeded into 96-well plates. When the cell density reached 70%, the original medium was replaced with DMEM complete medium containing 1‰ DMSO for normal HK2 cells, and the original medium was replaced with DMEM complete medium containing 1‰ DMSO and a final concentration of 20 µM OPA or a final concentration of 1 µM FIN for TGFβ1-induced HK2 cells.

[0049] Cells were collected 24 h after treatment and lysed with RIPA lysis buffer containing 0.1 volume of RIPA protease inhibitor mixture and 0.1 volume of RIPA phosphatase inhibitor mixture at 4°C for 30 min. After centrifugation at 12,000 rpm at 4°C for 30 min, the supernatant was collected. RIPA 5× Loading buffer was then added, and the mixture was boiled in a metal bath for 10 min. SDS-PAGE electrophoresis was then performed. After transfer and blocking, α-SMA primary antibody (1:5000), COL1A1 primary antibody (1:5000), or GAPDH primary antibody (1:10000) were added and incubated overnight at 4°C. After rinsing, the cells were incubated with the corresponding species secondary antibody (1:10000) at room temperature for 2 h. Finally, the cells were developed and images were acquired using the RIPA ultrasensitive ECL chemiluminescence kit.

[0050] The results are as follows Figure 14 As shown, compared with normal HK2 cells, the protein levels of α-SMA and COL1A1 in HK2 cells induced by TGFβ1 were significantly increased, while OPA and FIN could significantly inhibit the expression levels of the above two proteins. This further confirms that OPA can significantly reduce fibrosis indicators and alleviate the symptoms of renal fibrosis.

[0051] 5. Measurement of reactive oxygen species (ROS) levels in cells after OPA treatment Normal HK2 cells or TGFβ1-induced HK2 cells were seeded into 6-well plates. When the cell density reached 70%, the original medium was replaced with DMEM complete medium containing 1‰ DMSO for normal HK2 cells, and the original medium was replaced with DMEM complete medium containing 1‰ DMSO and a final concentration of 20 µM OPA or a final concentration of 1 µM FIN for TGFβ1-induced HK2 cells.

[0052] Cells were collected 24 h after treatment and stained with the DCFH-DA probe (purchased from MedChemExpress LLC., catalog number HY-D0940) at a final concentration of 5 μM for 30 min at 37°C. Quantitative analysis was performed by flow cytometry.

[0053] The results are as follows Figure 15 As shown, compared with normal HK2 cells, the ROS level in HK2 cells induced by TGFβ1 was significantly increased, while OPA and FIN could inhibit the increase of ROS, reduce intracellular oxidative stress, and restore cell activity.

[0054] 6. Measurement of cellular lipid peroxidation levels after OPA treatment Normal HK2 cells or TGFβ1-induced HK2 cells were seeded into 6-well plates. When the cell density reached 70%, the original medium was replaced with DMEM complete medium containing 1‰ DMSO for normal HK2 cells, and the original medium was replaced with DMEM complete medium containing 1‰ DMSO and a final concentration of 20 µM OPA or a final concentration of 1 µM FIN for TGFβ1-induced HK2 cells.

[0055] Cells were collected 24 h after treatment and stained with BODIPY 581 / 591 C11 probe (purchased from MedChemExpress LLC., catalog number HY-D1301) at 37°C for 30 min. Intracellular lipid peroxidation level was detected by flow cytometry and analyzed.

[0056] The results are as follows Figure 16 As shown, consistent with the ROS trend, OPA can significantly inhibit the level of lipid peroxidation in HK2 cells induced by TGFβ1, and further restore cell activity.

[0057] 7. Determination of cell ferroptosis markers after OPA treatment Normal HK2 cells or TGFβ1-induced HK2 cells were seeded into 6-well plates. When the cell density reached 70%, the original medium was replaced with DMEM complete medium containing 1‰ DMSO for normal HK2 cells, and the original medium was replaced with DMEM complete medium containing 1‰ DMSO and a final concentration of 20 µM OPA or a final concentration of 1 µM FIN for TGFβ1-induced HK2 cells.

[0058] After 24 h of treatment, cell culture supernatant was collected, and the levels of MDA, GSH, and 4-hydroxynonenal in the supernatant were detected using the Beyotime Lipid Oxidation Detection Kit, the Beyotime GSH and GSSG Detection Kit, and the Sangon Biotech 4-Hydroxynonenic acid ELISA kit.

[0059] The results are as follows Figure 17 As shown, compared with normal HK2 cells, TGFβ1-induced HK2 cells contain high levels of MDA and 4-HNE, while OPA or FIN can inhibit the increase of the above indicators; at the same time, OPA can also partially restore the level of intracellular GSH. The above experiments confirm that OPA can inhibit the level of ferroptosis in cells, thereby restoring cell activity.

Claims

1. The application of daidzein A in the preparation of anti-fibrotic drugs.

2. The application according to claim 1, characterized in that, The CAS number of the daidzein A is 2423917-90-0.

3. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit the expression of fibrosis-related proteins.

4. The application according to claim 3, characterized in that, The fibrosis-related proteins include α-smooth muscle actin and / or type I collagen.

5. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit ferroptosis.

6. The application according to claims 1 to 5, characterized in that, The tissue fibrosis is renal fibrosis.

7. The application according to claim 1, characterized in that, The drug contains daidzein A or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include capsules, granules, powders, tablets, microcapsules, injections, infusions, oral liquids, suspensions, patches, suppositories, microemulsions, liposomes, nanoparticles, and lyophilized powder injections.

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