Use of bisabolenone 2 in preparation of drugs for treating renal fibrosis

Abstract

The application discloses application of bisferenone 2 in preparation of a drug for treating renal fibrosis. The application first proposes and verifies that bisferenone 2 can effectively inhibit iron death of renal tubular epithelial cells to restore cell activity, delay epithelial-mesenchymal cell transformation, down-regulate expression of fibrosis-related proteins, relieve renal injury, effectively alleviate renal fibrosis progression, and has an excellent application prospect in clinical treatment of renal fibrosis.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, and more particularly to the application of a diasarone 2 in the preparation of a drug for treating renal fibrosis. Background Technology

[0002] Statistics show that the global prevalence of chronic kidney disease (CKD) is approximately 13.4%, making it a global public health challenge that seriously affects people's health. Its etiology is complex, and its onset is insidious, often progressing before patients are asymptomatic. Renal fibrosis is a common pathological pathway leading to end-stage CKD caused by various factors. It is characterized by abnormal proliferation of myofibroblasts in renal tissue. These cells highly express α-smooth muscle actin (α-SMA) and excessively produce extracellular matrix (ECM) such as collagen. The continuous deposition of ECM damages kidney structure, leading to an irreversible decline in glomerular filtration rate. Therefore, renal fibrosis is considered a key pathological predictor of renal function loss and a therapeutic target.

[0003] Current treatment strategies primarily include lifestyle interventions and the use of medications such as renin-angiotensin system (RAS) inhibitors and SGLT2 inhibitors to control blood pressure, blood sugar, and proteinuria, thereby slowing disease progression. However, even in patients receiving standard treatment, the risk of renal function decline and cardiovascular events remains, and the progression to end-stage renal disease cannot be completely prevented.

[0004] γ-Diasarone is a lignan natural product, primarily isolated from the traditional medicinal plant Acorus calamus (*Acorus calamus*). Acorus calamus The specific biological activities of the rhizome of ) have not been publicly reported. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for the application of bisasarone 2 in the preparation of drugs for the treatment of renal fibrosis, which synergistically alleviates the progression of renal fibrosis by inhibiting renal tubular epithelial cells, restoring the activity of renal tubular epithelial cells, delaying the epithelial-mesenchymal cell transformation, and downregulating the expression of fibrosis-related proteins.

[0006] Technical solution: The application of the bisasarone 2 described in this invention in the preparation of drugs for the treatment of renal fibrosis.

[0007] Preferably, the CAS number of the diasarone 2 is 80434-33-9.

[0008] Preferably, the application is in the preparation of a drug that inhibits ferroptosis in renal epithelial cells.

[0009] Preferably, the application is in the preparation of a drug for inhibiting the epithelial-mesenchymal transition of renal epithelial cells.

[0010] 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.

[0011] Preferably, the drug contains bisasarone 2 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 excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0013] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[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 asarone 2 can effectively inhibit ferroptosis of renal tubular epithelial cells to restore cell activity, delay the transformation of epithelial-mesenchymal cells while downregulating the expression of fibrosis-related proteins, reduce kidney damage and effectively alleviate the progression of renal fibrosis, and has excellent application prospects in the clinical treatment of renal fibrosis. Attached Figure Description

[0015] Figure 1 The graph shows the change in body weight in mice with renal fibrosis after treatment with cytosine 2. Figure 2 Representative images of kidney tissue at the endpoint of the experiment in a mouse model of renal fibrosis after treatment with bisasarone 2; Figure 3 A statistical graph of organ coefficients of renal tissue in a mouse model of renal fibrosis after treatment with asarone-2. Figure 4 Figure 1 shows the results of measuring the mRNA expression level of renal tissue damage biomarkers in a mouse model of renal fibrosis after treatment with asarone-2. Figure 5 Figure 1 shows the results of measuring the expression levels of renal tissue biomarkers of renal tissue injury in mice with renal fibrosis model after treatment with asarone-2. Figure 6 Immunohistochemical staining results of renal tissue damage biomarkers in a mouse model of renal fibrosis after treatment with cytosine 2; Figure 7 Figure 1 shows the quantitative analysis of immunohistochemical staining results of renal tissue injury biomarkers in a mouse model of renal fibrosis after treatment with cytosine 2. Figure 8 Figure 1 shows the results of renal function indicators in a mouse model of renal fibrosis after treatment with cytosine 2. Figure 9 Representative H&E staining images of kidney tissue from a mouse model of renal fibrosis after treatment with cytosine 2; Figure 10 Representative Masson staining images of kidney tissue from a mouse model of renal fibrosis after treatment with bisasarone 2; Figure 11 Figure 1 shows the mRNA expression levels of fibrosis-related proteins in the renal tissue of mice with renal fibrosis model after treatment with cytosine 2. Figure 12 Figure 1 shows the results of measuring the levels of inflammatory factors in the renal tissue of a mouse model of renal fibrosis after treatment with cytosine 2. Figure 13 Figure 1 shows the results of viability assay of HK2 cells, a renal fibrosis model, after treatment with asarone 2. 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 asarone 2. Figure 15 Figure 1 shows the flow cytometry results of ROS levels in HK2 cells, a renal fibrosis model, after treatment with asarone 2. Figure 16 Figure 1 shows the quantitative analysis of ROS levels in HK2 cells, a renal fibrosis model after treatment with asarone 2. Figure 17 Figure 1 shows the flow cytometry results of lipid peroxidation levels in HK2 cells, a renal fibrosis model, after treatment with bisasarone 2. Figure 18 Flow cytometry analysis of lipid peroxidation levels in HK2 cells, a renal fibrosis model, after treatment with asarone 2. Figure 19 Figure 1 shows the flow cytometry results of mitochondrial membrane potential analysis in HK2 cells, a renal fibrosis model, after treatment with asarone 2. Figure 20 A flow cytometry analysis of mitochondrial membrane potential in HK2 cells, a renal fibrosis model, after treatment with asarone 2. Figure 21 The figure shows the results of ferroptosis marker determination in HK2 cells, a renal fibrosis model, after treatment with bisasarone 2. Detailed Implementation

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

[0017] Example 1: In vivo efficacy verification of γ-Diasarone (DIA) 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). Ten-week-old female C57BL / 6J mice, weighing 20-22 g, were purchased from the Experimental Animal Center of Nantong University and randomly divided into a sham surgery group (n=10) and a UUO surgery group (n=20). In the UUO surgery group, mice were anesthetized and fixed in a supine position on the operating table. After dehairing and disinfection of the abdomen, a 2 cm midline incision was made, and the skin, subcutaneous tissue, and peritoneum were dissected layer by layer. An abdominal retractor was used to pull the abdominal cavity laterally, and the ureter was freed at the ureteropelvic junction. Two ligations were made at the lower pole of the left kidney with 3-0 silk sutures. After confirming no bleeding, the layers were sutured and the abdominal cavity closed. In the sham surgery group, only the left ureter was isolated and exposed, without ligation.

[0018] Fourteen days after model establishment, mice in the UUO surgery group were randomly divided into two groups (n=10 per group): the RF+DIA group and the RF group. Drug administration was initiated simultaneously, designated day 0. The drugs were administered via gavage once daily. Mice in the RF+DIA group received 10 mg / kg DIA (purchased from MedChemExpress LLC., catalog number HY-N3917), while mice in the Sham and RF groups received the same volume of saline. Treatment continued until day 10 (day 10).

[0019] 2. Weight monitoring Starting from the day after drug administration (d1), the body weight of each mouse was measured daily and statistical analysis was performed.

[0020] The results are as follows Figure 1 As shown, the weight of mice in the RF group was significantly reduced, while the weight of mice treated with DIA could be restored.

[0021] 3. Organ coefficient detection At the end of the experiment, 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.

[0022] Representative images of mouse kidney tissue at the experimental endpoint are shown below. Figure 2 As shown, compared with the Sham group mice, the kidney volume of the RF group mice was significantly smaller, while the kidney morphology was significantly improved after DIA treatment, and could be restored to the level of normal mice, which initially indicates that DIA has a significant therapeutic effect on renal fibrosis.

[0023] Statistical results of kidney tissue organ coefficients are as follows Figure 3As shown, the renal tissue coefficient of mice in the Sham group was 0.85, the renal tissue coefficient of mice in the RF group decreased to 0.52, and the renal tissue coefficient of mice treated with DIA recovered to 0.65, indicating that DIA drugs have the potential to treat renal fibrosis.

[0024] 4. Determination of biomarkers for renal tissue damage Kidney Injury Molecule-1 (KIM1) and Neutrophil Gelatinase-Associated Lipocalin (NGAL) were selected for assay.

[0025] 4.1 mRNA expression level determination Mouse kidney tissue was collected, homogenized in liquid nitrogen, and total RNA was extracted using the Novizan FreeZol Reagent Kit (catalog number R711-01). The RNA was then reverse transcribed into cDNA using the Novizan HiScript IV RT SuperMix for qPCR (+gDNA wiper) Kit (catalog number R423-01) with the primers shown below: KIM1 Upstream primer: 5'-tcaccctgtcacacttccat-3'; KIM1 Downstream primer: 5'-tgtaccgactgctcttctga-3'; NGAL Upstream primer: 5'-ggcaatgcggtccagaaaa-3'; NGAL :5'-agagtgaactggccagcc-3'; GAPDH Upstream primer: 5'-aacgaccccttcattgacct-3'; GAPDH Downstream primer: 5'-atgttagtggggtctcgctc-3'; qPCR detection was performed using Novizan ChamQ Universal SYBR qPCR Master Mix reagent (catalog number Q711-02), with reaction conditions of 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing extension for 30 s, for 35 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. GAPDH As an intrinsic parameter, the Ct value is calculated using the ΔΔCt method. KIM1 and NGAL The relative expression level of mRNA.

[0026] The results are as follows Figure 4 As shown, compared to the Sham group mice, the RF group mice had lower renal tissue density. KIM1 and NGALThe mRNA levels of the cells were significantly elevated, showing a significant difference. However, the levels of kidney injury factors were suppressed after DIA treatment, indicating that DIA can reduce kidney injury factors and has the function of restoring kidney function.

[0027] 4.2 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 containing 0.1 volume of a mixture of RIPA protease inhibitor and RIPA phosphatase inhibitor was added. Lysis was performed at 4°C for 30 min, followed by centrifugation at 12000 rpm for 30 min. The supernatant was collected and added to RIPA 5× Loading buffer (Catalog No. WB2001) in the appropriate 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).

[0028] The results are as follows Figure 5 As shown, the protein levels of KIM1 and NGAL were significantly increased in RF group mice, but gradually decreased after DIA treatment.

[0029] 4.3 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.

[0030] The results are as follows Figure 6 , 7 As shown, consistent with the results of Western blot analysis, DIA treatment significantly inhibited the protein levels of KIM1 and NGAL, further confirming that DIA has a significant therapeutic effect on renal fibrosis in mice.

[0031] 5. 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 No. 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 detected using the Beyotime Amplex Red Creatinine Assay Kit (Catalog No. S0291S), the uric acid content was detected using the Beyotime Amplex Red Uric Acid Assay Kit (Catalog No. S0232S), and the urea nitrogen content was detected using the Beyotime Urea Assay Kit (Catalog No. S0574S).

[0032] The results are as follows Figure 8 As shown, in the Sham group mice, the levels of urinary creatinine were 32.3 µmol / L, urea nitrogen was 4.8 nmol / L, and uric acid was 99.2 µmol / L. In the RF group mice, the levels of urinary creatinine were 79.8 µmol / L, urea nitrogen was 15.9 nmol / L, and uric acid was 277.1 µmol / L. These significantly elevated levels indicate substantial damage to renal function in the RF group mice. After DIA treatment, the levels of urinary creatinine were 49.7 µmol / L, urea nitrogen was 8.3 nmol / L, and uric acid was 197.3 µmol / L. These levels were significantly lower than in the RF group mice, indicating that DIA can effectively restore renal function.

[0033] 6. Pathological evaluation of kidney tissue 6.1 H&E staining Mouse lung tissue was collected and fixed in 4% paraformaldehyde solution for 48 h, then dehydrated in a gradient manner, embedded in paraffin, and cut into 3 μm sections. After dewaxing and hydration, the sections were stained with hematoxylin and eosin (H&E), mounted with neutral resin, and observed and images were acquired under a microscope.

[0034] The results are as follows Figure 9As shown, compared to the kidney tissue of the Sham group mice, the RF group mice exhibited significant variations in glomerular volume, widened mesangial area, increased mesangial matrix, collapsed glomerular capillary loops, adhesion of the Bowman's capsule wall, and increased inflammatory cell infiltration (increased proportion of blue). These indicators were alleviated after DIA treatment, suggesting that DIA can serve as a potential treatment for renal fibrosis.

[0035] 6.2 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.

[0036] The results are as follows Figure 10 As shown, compared with the Sham group mice, the RF group mice showed a higher level of collagen deposition in their kidney tissue (blue area). After DIA treatment, the blue area was significantly reduced, inhibiting collagen deposition, which indicates that the symptoms of renal fibrosis were initially improved.

[0037] 7. Measurement of relevant indicators of kidney tissue 7.1 Determination of mRNA expression levels of renal fibrosis-related proteins Mouse kidney tissue was collected, homogenized in liquid nitrogen, and total RNA was extracted using the Novizan FreeZol Reagent Kit (catalog number R711-01). The RNA was then reverse transcribed into cDNA using the Novizan HiScript IV RT SuperMix for qPCR (+gDNA wiper) Kit (catalog number R423-01) with the primers shown below: COL1A1 Upstream primer: 5'-tggtttcgacttcagcttcc-3', COL1A1 Downstream primer: 5'-atgttctcgatctgctggct-3'; FN1 Upstream primer: 5'-tacccttccacaccccaatc-3', FN1 Downstream primer: 5'-gcaggtatggtcttggccta-3'; ACTA2 Upstream primer: 5'-gaagctgataaacgtgggct-3', ACTA2 Downstream primer: 5'-tgctttatggctggaattggt-3'; GAPDH Upstream primer: 5'-gtgaaggtcggtgtgaacg-3'; GAPDHDownstream primer: 5'-tgccgtgagtggagtcatac-3'; qPCR detection was performed using Novizan ChamQ Universal SYBR qPCR Master Mix reagent (catalog number Q711-02), with reaction conditions of 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing extension for 30 s, for 35 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. GAPDH As an intrinsic parameter, the Ct value is calculated using the ΔΔCt method. COL1A1 , FN1 , ACTA2 The relative expression level of mRNA.

[0038] The results are as follows Figure 11 As shown, compared with the kidney tissue of the Sham group mice, the mRNA levels of the above-mentioned fibrotic molecules in the kidney tissue of the RF group mice were significantly increased. However, with DIA treatment, the levels of the above-mentioned fibrotic molecules were inhibited, and there was no significant difference between the two groups.

[0039] 7.2 Measurement of the expression level of inflammatory factors in renal tissue Take 100 mg of mouse kidney tissue, add 1 mL of pre-cooled physiological saline to prepare tissue homogenate, centrifuge at 12000 rpm for 15 min and collect the supernatant.

[0040] Cytokine levels in renal tissue were measured using the Elletet mouse interleukin-1β (IL-1β) enzyme-linked immunosorbent assay kit (catalog number E-EL-M0037), the Elletet mouse tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay kit (catalog number E-EL-M3063), and the Elletet mouse interleukin-6 (IL-6) enzyme-linked immunosorbent assay kit (catalog number E-EL-M0044).

[0041] The results are as follows Figure 12 As shown, in the Sham group mice, the levels of IL-1β were 32.2 ng / L, TNF-α were 400.9 ng / L, and IL-6 were 41.7 pg / mL; while in the RF group mice, the levels of the above pro-inflammatory cytokines were significantly increased, specifically IL-1β at 79.7 ng / L, TNF-α at 886.4 ng / L, and IL-6 at 145.3 pg / mL; in DIA-treated mice, the levels of IL-1β were 50.3 ng / L, TNF-α at 565.3 ng / L, and IL-6 at 78.7 pg / mL, indicating that DIA can significantly inhibit the levels of pro-inflammatory cytokines in the renal tissue of mice with renal fibrosis, thereby alleviating the level of renal fibrosis.

[0042] Example 2: In vitro efficacy verification of bisasarone 2 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 Pronosai Life Science Technology 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 Wuhan Pronosai Life Science Technology Co., Ltd., catalog number PCK091) was added to the medium at a final concentration of 5 ng / mL for 24 h to construct a renal fibrosis-related cell model.

[0043] 2. Cell viability assay after DIA 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 or DIA at a final concentration of 20 µM for TGFβ1-induced HK2 cells.

[0044] 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.

[0045] The results are as follows Figure 13 As shown, compared with normal HK2 cells (DMSO, TGFβ1- DIA-), the activity of TGFβ1-induced cells (TGFβ1, TGFβ1+ DIA-) was significantly reduced (to 32.7% of the control group), while the activity of cells (TGFβ1+DIA, TGFβ1+ DIA+) after DIA treatment was restored to 87.3% of the control group. The above experiments preliminarily indicate that DIA can alleviate the symptoms of renal fibrosis.

[0046] 3. Determination of expression levels of fibrosis-related proteins after DIA 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 or DIA at a final concentration of 20 µM for TGFβ1-induced HK2 cells.

[0047] Cells were collected 24 h after treatment and lysed using RIPA lysis buffer (WB3100) containing 0.1 volume of cyprotease inhibitor mixture (P001) and 0.1 volume of cyprotease inhibitor mixture (P003) at 4°C for 30 min. The supernatant was collected after centrifugation at 12000 rpm for 30 min at 4°C. Then, 5× loading buffer was added according to the specified ratio, and the mixture was boiled in a metal bath for 10 minutes. After a period of time, SDS-PAGE electrophoresis was performed. After transfer and blocking, α-SMA primary antibody (Proteintech, catalog number 14395-1-AP) diluted 1:5000, COL1A1 primary antibody (Proteintech, catalog number 67288-1-Ig) diluted 1:5000, 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 NewSymex ultrasensitive ECL chemiluminescence kit.

[0048] 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 DIA could significantly inhibit the protein levels of the above two indicators, which further confirms that DIA can significantly reduce fibrosis indicators.

[0049] 4. Measurement of reactive oxygen species (ROS) levels in cells after DIA 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 or DIA at a final concentration of 20 µM for TGFβ1-induced HK2 cells.

[0050] 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.

[0051] The results are as follows Figure 15 , 16As shown, compared with normal HK2 cells, the ROS level of HK2 cells induced by TGFβ1 was significantly increased, while DIA could inhibit the increase of ROS and reduce intracellular oxidative stress.

[0052] 5. Measurement of cellular lipid peroxidation levels after DIA 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 or DIA at a final concentration of 20 µM for TGFβ1-induced HK2 cells.

[0053] 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.

[0054] The results are as follows Figure 17 , 18 As shown, consistent with the ROS trend, DIA can significantly inhibit the level of intracellular lipid peroxidation induced by TGFβ1 and restore cell activity.

[0055] 6. Measurement of mitochondrial membrane potential in cells after DIA 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 or DIA at a final concentration of 20 µM for TGFβ1-induced HK2 cells.

[0056] Cells were collected 24 h after treatment and incubated in PBS buffer containing 10 µg / mL JC-1 (purchased from MedChemExpress LLC., catalog number: HY-15534) at 37°C in the dark for 30 min. Fluorescence signals were detected by flow cytometry. The excitation wavelength of JC-1 monomer (green fluorescence) was set to 488 nm and the emission wavelength was detected at 525 / 50 nm. The emission wavelength of JC-1 multimer (red fluorescence) was detected at 590 / 40 nm.

[0057] The results are as follows Figure 19 , 20As shown, the JC-1 monomer / JC-1 multimer ratio in normal HK2 cells was 4.5, while the ratio in TGFβ1-induced cells was 1.5. After DIA treatment, the JC-1 monomer / JC-1 multimer ratio recovered to 2.4. This experiment preliminarily indicates that, compared with TGFβ1-induced cells, DIA can significantly increase the JC-1 monomer / JC-1 multimer ratio, increase mitochondrial membrane potential, restore mitochondrial function, and thus improve cell viability.

[0058] 7. Determination of ferroptosis markers after DIA 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 or DIA at a final concentration of 20 µM for TGFβ1-induced HK2 cells.

[0059] After 24 h of treatment, cell culture supernatant was collected. The levels of malondialdehyde (MDA) and ferrous ions (Fe2+) in the supernatant were detected using the Beyotime Lipid Oxidation Detection Kit (Catalog No. S0131S), the Elite Cell Ferrous Ion Fluorescence Assay Kit (Catalog No. E-BC-F101), and the Sangon Biotech 4-Hydroxynonenoic Acid ELISA Kit (Catalog No. D751041). 2+ The levels of 4-hydroxynonenal (4-HNE).

[0060] The results are as follows Figure 21 As shown, compared to normal HK2 cells, TGFβ1-induced cells contain high levels of MDA and Fe. 2+ And 4-HNE, however, DIA suppressed the levels of the above indicators.

[0061] The above experiments confirm that DIA can inhibit TGFβ1-induced ferroptosis, thereby restoring cell viability.

Claims

1. The application of a diasarone 2 in the preparation of a drug for treating renal fibrosis.

2. The application according to claim 1, characterized in that, The CAS number of the aforementioned bisasarone 2 is 80434-33-9.

3. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit ferroptosis in renal epithelial cells.

4. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit the epithelial-mesenchymal transition of renal epithelial cells.

5. 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.

6. The application according to claim 5, characterized in that, The fibrosis-related proteins include α-smooth muscle actin and / or type I collagen.

7. The application according to claim 1, characterized in that, The drug contains bisasarone 2 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: excipients, diluents, lubricants, glidants, 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 tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

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