Use of compounds of the taxifolin class for the preparation of a medicament for the treatment of chronic kidney disease

Abstract

The application belongs to the field of biological medicine, and particularly relates to the use of mulberroside A compounds in the preparation of drugs for treating chronic kidney disease. The mulberroside A compounds can inhibit the TGF-beta signal pathway, reduce the expression of fibrosis-related genes and proteins, reduce the fibrosis area, and alleviate the damage to the renal tubules, thereby treating renal fibrosis of chronic kidney disease, providing a new choice for clinically treating renal fibrosis of chronic kidney disease, and having a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to the use of mulberrypeptide compounds in the preparation of a drug for treating chronic kidney disease. BACKGROUND

[0002] Chronic kidney disease (CKD) refers to a class of kidney diseases with persistent damage to kidney structure or function. According to the national survey in 2018-2019, the prevalence rate of CKD among Chinese adults is as high as 8.2%. If there is no appropriate medical intervention, CKD can develop into renal failure, endangering the life of the patient. Clinically, CKD is mainly treated symptomatically, including the control of blood sugar, blood pressure, blood lipids, etc., and there is no specific therapeutic drug. Renal fibrosis refers to the pathological phenotype of excessive accumulation of extracellular matrix in the renal tubulointerstitium and glomerulus. The fibrotic tissue can affect the function and survival of the renal parenchymal cells in the spatial structure and cause damage to the overall kidney function. Studies have shown that renal fibrosis is one of the core pathological features of CKD, and improvement of renal fibrosis can alleviate the damaged kidney function. Therefore, anti-renal fibrosis is an important strategy for the treatment of CKD.

[0003] TGF-β signal is the core signal for regulating organ fibrosis. The classic TGF-β signal starts from the sequential binding of extracellular TGF-β1 / 2 / 3 factor and cell membrane receptors TGFBR2 and TGFBR1, and then phosphorylates intracellular effector proteins Smad2 and Smad3 (also transcription factors). After Smad2 / 3 forms a complex with Smad4, it translocates into the nucleus to regulate the expression of genes related to the fibrosis process. In various types of CKD kidney tissues, TGF-β signal shows excessive activation. In CKD animal models, inhibition of TGF-β signal through genetic modification or drug intervention can significantly reduce renal fibrosis and improve the damaged kidney function. Therefore, the development of anti-renal fibrosis drugs targeting TGF-β signal is an important way for the development of new drugs for the treatment of CKD.

[0004] Traditional Chinese medicine mulberry bark (Cortex Mori) refers to the dried root bark of Morus alba L. of Moraceae, which is cold in nature, sweet and pungent in taste, and belongs to the lung meridian. It has the effects of relieving lung and asthma, promoting water and reducing swelling, and is often used to treat symptoms such as lung heat cough and asthma, edema and oliguria. Mulberry bark also has therapeutic effects on obesity-related metabolic diseases, such as lowering blood sugar, blood, and improving insulin resistance. In the patent application (Mulberry bark extract for treating chronic kidney disease and its preparation method and use, CN202410256216.8), it is reported that mulberry bark has significant anti-TGF-β signal drug activity and can be used to improve CKD-related renal fibrosis. However, the active pharmaceutical ingredients in mulberry bark with anti-TGF-β signal activity and the activity of improving CKD renal fibrosis are still unknown.

[0005] Moracin compounds are a series of natural products extracted from Moraceae plants, which have a wide range of biological activities, such as anti-tumor, antibacterial, antioxidant, etc., but there is no relevant report on the treatment of chronic kidney disease. SUMMARY

[0006] In view of the problems in the prior art, the application provides use of moracin compounds in preparation of a medicine for treating chronic kidney disease.

[0007] The use of the compound shown in formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopically labeled compound thereof, or a crystal form thereof, or a prodrug thereof in preparation of a medicine for preventing and / or treating kidney disease;

[0008]

[0009] wherein ring A is selected from a substituted or unsubstituted 5-10 membered heterocyclic ring, and the heteroatom is selected from oxygen, sulfur and nitrogen; and the substituent is selected from hydroxyl, C1-C5 alkyl and C1-C5 alcohol group.

[0010] Preferably, the compound has a structural formula as shown in formula II.

[0011]

[0012] Preferably, the compound has a structural formula as shown in formula III.

[0013]

[0014] Preferably, the kidney disease is chronic kidney disease.

[0015] Preferably, the chronic kidney disease is chronic kidney disease of renal fibrosis.

[0016] Preferably, the medicine can reduce the expression of fibrosis genes.

[0017] Preferably, the fibrosis gene is selected from fibronectin, Col1a1 and alpha-SMA.

[0018] Preferably, the medicine is used for inhibiting TGF-beta signal activity.

[0019] Preferably, the medicine can reduce the damage of renal tubules.

[0020] The application also provides a medicine composition for preventing and / or treating kidney diseases, which is a preparation prepared from an active ingredient or a main ingredient of an active part of mulberry bark in which a compound shown in formula I, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer, or a geometric isomer, or an isotope label, or a crystal form, or a prodrug thereof is used, and pharmaceutically acceptable adjuvants; the structure of the compound shown in formula I is:

[0021]

[0022] wherein ring A is selected from a substituted or unsubstituted 5-10 membered heterocyclic ring, and the heteroatom is selected from oxygen, sulfur and nitrogen; and the substituent is selected from a hydroxyl group, a C1-C5 alkyl group and a C1-C5 alcohol group.

[0023] The chemical structural formula of morusin O is The chemical structural formula of morusin P is

[0024] The morusin compound (morusin O and morusin P) can inhibit TGF-β signal, reduce the expression of fibrosis-related genes and proteins, reduce the fibrosis area and relieve the tubular injury caused by UUO, thereby treating chronic kidney disease and renal fibrosis, providing a new choice for clinically treating chronic kidney disease and renal fibrosis, and having a good application prospect.

[0025] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above technical idea of the application.

[0026] The above content of the application is further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the above subject matter of the application is limited to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1Inhibition of TGF-β signaling activity in TCMK1 cells by mulberroside O / P. A-B. CCK8 assay to test the effect of different concentrations of mulberroside O and P on the viability of TCMK1 cells. The dotted line in the figure indicates the 90% cell viability interval. C. TCMK1 cells were pretreated with different concentrations of mulberroside O for 12 hours, and then stimulated with 5 ng / mL TGF-β1 for 30 min. Western blot analysis of Smad2, Smad3 and their phosphorylation levels. D-G. Quantitative analysis of the corresponding protein bands in C. H. TCMK1 cells were pretreated with different concentrations of mulberroside P for 12 hours, and then stimulated with 5 ng / mL TGF-β1 for 30 min. Western blot analysis of Smad2, Smad3 and their phosphorylation levels. I-L. Quantitative analysis of the corresponding protein bands in H.

[0028] Figure 2 Inhibition of renal fibrosis by mulberroside O in UUO mice. A. HE and Masson staining to analyze the structure of kidney tissue and the distribution of collagen (blue), scale bar is 100 μm. B. Tubular injury score based on HE staining. C. Quantitative analysis of fibrosis area based on Masson staining. D-F. RT-PCR analysis of fibronectin, Col1a1, α-SMA mRNA expression in kidney tissue. G. Western blot analysis of fibrosis proteins fibronectin, Collagen I, α-SMA protein expression. H-J. Quantitative analysis of the corresponding protein bands in G. Each point in the statistical chart represents an independent animal specimen. **p<0.01 and ***p<0.001 Vs. Sham. # p<0.05, ## p<0.01 and ### p<0.001 Vs. UUO.

[0029] Figure 3 Inhibition of renal fibrosis by mulberroside P in UUO mice. A. HE and Masson staining to analyze the structure of kidney tissue and the distribution of collagen (blue), scale bar is 100 μm. B. Tubular injury score based on HE staining. C. Quantitative analysis of fibrosis area based on Masson staining. D-F. RT-PCR analysis of fibronectin, Col1a1, α-SMA mRNA expression in kidney tissue. G. Western blot analysis of fibrosis proteins fibronectin, Collagen I, α-SMA protein expression. H-J. Quantitative analysis of the corresponding protein bands in G. Each point in the statistical chart represents an independent animal specimen. **p<0.01 and ***p<0.001 Vs. Sham. # p<0.05,## p<0.01 and ### p<0.001 Vs.UUO. DETAILED DESCRIPTION

[0030] In the following examples and experimental examples, reagents and materials not specifically mentioned are commercially available.

[0031] Example 1 Morin O and Morin P can inhibit TGF-β signaling activity

[0032] I. Experimental Methods

[0033] 1. CCK8 cytotoxicity experiment

[0034] Mouse renal tubular epithelial TCMK1 cells were seeded in 96-well plates, and the culture medium was DMEM (high sugar) + 10% FBS (fetal bovine serum), and the culture conditions were 37°C, 5% CO2 and 100% humidity. When the cell density was close to 80-90%, different concentrations (0.78 μg / mL, 1.56 μg / mL, 3.13 μg / mL, 6.25 μg / mL, 12.25 μg / mL, 25 μg / mL) of morin O and morin P were added to 0.5% FBS for 24 hours. Then, 10% CCK8 reagent (Shanghai Biotech, item number: E606335) was added to the serum-free medium for 2 hours, and the absorbance at 450 nm wavelength was measured by a multifunctional enzyme marker, and the cell viability was calculated.

[0035] 2. Morin O and Morin P anti-TGF-β signaling activity cell treatment

[0036] Mouse renal tubular epithelial TCMK1 cells were seeded in 6-well plates, and when the cell density was close to 80-90%, different concentrations (1.56 μg / mL, 3.13 μg / mL, 6.25 μg / mL) of morin O and morin P were added to 0.5% FBS medium for 24 hours. Then, 5 ng / mL TGF-β1 was added for 30 min. The cells were collected, the protein was extracted, and the expression levels of Smad2, Smad3, p-Smad2, and p-Smad3 were checked by western blot, and β-actin was used as an internal reference. The antibodies used in western blot include rabbit anti-Smad2 (CST, item number 5339, USA, dilution ratio 1:1000), rabbit anti-Smad3 (CST, item number 9523, USA, dilution ratio 1:1000), rabbit anti-p-Smad2 (CST, item number 18338, USA, dilution ratio 1:1000), rabbit anti-p-Smad3 (Abeam, item number ab52903, USA, dilution ratio 1:1000), and mouse anti-β-actin (Abway, item number AB0011, China, dilution ratio 1:5000).

[0037] II. Experimental Results

[0038] Results are shown in Figure 1 As shown in Figure 1 (C-L) shows that pre-treatment of TCMK1 cells with 1.56, 3.13, 6.25 μg / mL moracin O and moracin P for 12 hours can dose-dependently inhibit the phosphorylation activation of Smad2 and Smad3 induced by transient (30 min) TGF-β1 stimulation, while the total protein levels of Smad2 and Smad3 are not affected. The results show that moracin O and moracin P can block the activation of the TGF-β signaling pathway by inhibiting the phosphorylation of Smad2 and Smad3, thereby improving impaired renal function and reducing renal fibrosis.

[0039] Example 2 Moracin O can be used to treat renal fibrosis in chronic kidney disease

[0040] I. Experimental Methods

[0041] 1. Construction of unilateral ureteral ligation (UUO) mouse model and drug treatment: 8-10 week old male C57BL / 6 mice were randomly divided into sham operation group (Sham), UUO model group, drug treatment low dose group (Moracin O-L) and drug treatment high dose group (Moracin O-H), 7-8 mice in each group. The UUO operation modeling process is as follows: after the mice were anesthetized with 40 mg / kg sodium pentobarbital, the back skin was shaved, and after disinfection, a 1 cm skin and body wall incision was made on the left kidney site, blunt forceps were used to free the ureter and perform surgical suture ligation, then the tissue was returned to its original position, and the body wall and skin tissue was sutured. The mice in the Sham group were subjected to the same surgical procedure, but the ureter was not ligated. All animals were given iodophor disinfection of the wound and intraperitoneal antibiotic injection to prevent infection after operation, and were placed in a warm environment for recovery. The mice in the drug treatment low dose group and the drug treatment high dose group were injected intraperitoneally with 20 mg / kg (L, injection volume 100 uL) and 40 mg / kg (H, injection volume 200 uL) of Moracin O (moracin O, MW 326.37, CAS No. 123702-97-6) respectively at 7 days after operation. The model group and Sham group mice were given solvent (corn oil) injection. At the end of the experiment, the mice were euthanized with 1 g / kg sodium pentobarbital, and the kidney tissue was collected for analysis of relevant indicators.

[0042] 2. Histopathological staining: Kidney paraffin-embedded tissues were cut into 4 pm thickness sections, and then deparaffinated with xylene and rehydrated with gradient alcohol. HE staining (Bi Yun Tian, C0105, China) and Masson staining (Nanjing Jiancheng, D026-1-3, China) were performed using the kits. The stained sections were mounted with neutral resin and observed under a upright white light microscope (Leica, DM500, Germany). Tubular injury score was performed using HE staining pictures. The sections were scored as follows: 0, normal; 1, less than 10% of cortex involvement; 2, 10%-25% of cortex involvement; 3, 25%-50% of cortex involvement; 4, 50%-75% of cortex involvement; 5, more than 75% of cortex involvement. Masson staining was directly analyzed by Image J software for the percentage of positive staining (collagen) area. At least 3 random fields were analyzed for each animal in each group.

[0043] 3. RT-PCR: RNA was extracted from kidney tissues using Trizol method. 1 pg of RNA was used to synthesize cDNA using M-MuLV reverse transcriptase (Shanghai Biogiga, B600005, China) with the following steps: 25 °C for 5 min; 42 °C for 60 min; 85 °C for 5 min. ChamQ Universal SYBR qPCR Master Mix (Norgen, Q711-02, China) was used for RT-PCR amplification. The relative expression of genes was calculated using 2 -△△Ct The primers used in RT-PCR included:

[0044] fibronectin, F: 5’-CTGGGACTGTACCTGCATCG-3’ (SEQ ID NO. 1),

[0045] R: 5’-CCTCCACTTGTCGCCAATCT-3’ (SEQ ID NO. 2);

[0046] Col1a1, F: 5’-ATCCAACGAGATCGAGCTCA-3’ (SEQ ID NO. 3),

[0047] R: 5’-AAGGGAGCCACATCGATGAT-3’ (SEQ ID NO. 4);

[0048] a-SMA, F: 5’-AGAGTTTTGTGCTGAGGTCCC-3’ (SEQ ID NO. 5),

[0049] R: 5'-GGAGCATCATCACCAGCGAA-3' (SEQ ID NO. 6);

[0050] β-actin, F: 5'-AGAGGGAAATCGTGCGTGAC-3' (SEQ ID NO. 7),

[0051] R: 5'-CAATAGTGATGACCTGGCCGT-3' (SEQ ID NO. 8).

[0052] 4. Western blot: kidney tissues were lysed and protein extracted using IP lysis buffer, and protein concentration was determined by Coomassie brilliant blue method. After denaturation and lysis of the protein by SDS loading buffer, 20 μg of protein per lane was loaded into 10% SDS-PAGE gel for electrophoresis. After electrophoresis, the proteins in the gel were transferred to PVDF membrane, blocked with 5% skim milk or 2.5% BSA (for detection of phosphorylated proteins) for 1 h, incubated with primary antibody at 4°C overnight, washed with TBST for 3 times, 5 min each time, and incubated with secondary antibody labeled with species-matched horseradish peroxidase (HRP) for 1 h, washed with TBST for 3 times, 5 min each time. Then signal exposure was performed using SuperPico ECL Chemiluminescence Kit (Nanjing Keygen Biotech Co., Ltd., E422-01, China), and signal collection was performed using ChemiScope 600EXp System (China). The Western blot band signals were quantitatively analyzed using ImageJ software. The antibodies used in the experiments of the present embodiment include goat anti-collagen I (SouthernBiothech, item number 1310-01, USA, dilution ratio 1:500), mouse anti-α-SMA (Boster, item number BM0002, China, dilution ratio 1:1000), rabbit anti-fibronectin (Abmart, item number T59537, China, dilution ratio 1:1000), and mouse anti-β-actin (Abway, item number AB0011, China, dilution ratio 1:5000).

[0053] II. Experimental results

[0054] The in vivo anti-renal fibrosis efficacy of morin O was analyzed in a UUO mouse model. HE staining showed that morin O intervention significantly improved the abnormal structure of UUO kidney tissues and reduced the tubular injury score. Masson staining showed that the extracellular collagen area of the kidney tissues of the morin O intervention mice was significantly reduced Figure 2A-C). Renal tissue RT-PCR analysis found that compared with the UUO model group, the expression level of fibronectin, Col1a1, and a-SMA mRNA in the renal tissue fibrosis gene was significantly inhibited by moracin O intervention Figure 2 D-F). Western blot analysis further confirmed that moracin O intervention reduced the expression levels of fibronectin, collagen I, and a-SMA fibrosis proteins Figure 2 G-J). The results showed that moracin O can treat chronic kidney disease renal fibrosis by reducing the expression of fibrosis-related genes and proteins, reducing the fibrosis area, and reducing the tubular damage caused by UUO.

[0055] Example 3 Moracin P can be used to treat chronic kidney disease renal fibrosis

[0056] I. Experimental method

[0057] 1. Unilateral ureteral ligation (UUO) mouse model construction and drug treatment: 8-10 week old male C57BL / 6 mice were randomly divided into sham operation group (Sham), UUO model group, drug treatment low dose group (Moracin P-L) and drug treatment high dose group (Moracin P-H), 7-8 in each group. The UUO operation modeling process is as follows: after the mice were anesthetized with 40mg / kg sodium pentobarbital, the back skin hair was shaved, and after disinfection, a 1cm skin and body wall incision was made at the left kidney site. Blunt forceps were used to free the ureter and perform surgical suture ligation, then the tissue was returned to its original position, and the body wall and skin tissue was sutured. The mice in the Sham group were subjected to the same surgical procedure, but the ureter was not ligated. All animals were given wound iodophor disinfection and intraperitoneal antibiotic injection after operation to prevent infection, and were placed in a warm environment for recovery. The mice in the drug treatment low dose group and the drug treatment high dose group were intraperitoneally injected with 20mg / kg (L, injection volume 100uL) and 40mg / kg (H, injection volume 200uL) of Moracin P (Moracin P, MW 326.37, CAS No. 102841-43-0) respectively at 7 days after operation. The model group and Sham group mice were given solvent (corn oil) injection. At the end of the experiment, the mice were euthanized with 1g / kg sodium pentobarbital, and the kidney tissue was collected for analysis of relevant indicators.

[0058] 2. Histopathological staining: Kidney paraffin-embedded tissues were cut into 4 pm thickness sections, and then deparaffmized with xylene and rehydrated with gradient alcohol. HE staining (Bi Yun Tian, C0105, China) and Masson staining (Nanjing Jiancheng, D026-1-3, China) were performed using the kits. The stained sections were mounted with neutral balsam and observed under a upright white light microscope (Leica, DM500, Germany). Tubular injury score was performed using HE staining pictures. The score was based on the presence of cell degeneration and necrosis, tubular dilation, cast deposition, brush border loss or necrosis, and was graded as follows: 0, normal; 1, less than 10% of the cortex involved; 2, 10-25% of the cortex involved; 3, 25-50% of the cortex involved; 4, 50-75% of the cortex involved; 5, more than 75% of the cortex involved. Masson staining was directly analyzed using Image J software to calculate the positive staining (collagen) area ratio. At least 3 randomly taken fields were analyzed for each animal in each group.

[0059] 3. RT-PCR: RNA was extracted from kidney tissues using Trizol method. 1 pg of RNA was used to synthesize cDNA using M-MuLV reverse transcriptase (Shanghai Biogiga, B600005, China) with the following steps: 25 °C for 5 min; 42 °C for 60 min; 85 °C for 5 min. ChamQ Universal SYBR qPCR Master Mix (Norgen, Q711-02, China) was used for RT-PCR amplification. The relative expression of genes between different treatment groups was calculated using 2 -△△Ct The primers used in RT-PCR were the same as in Example 2.

[0060] 4. Western blot: kidney tissues were lysed and protein extracted using IP lysis buffer, and protein concentration was determined using Coomassie brilliant blue method. After denaturation and lysis of the protein by SDS loading buffer, 20 μg of protein per lane was loaded onto a 10% SDS-PAGE gel for electrophoresis. After electrophoresis, the proteins in the gel were transferred to a PVDF membrane, blocked with 5% skim milk or 2.5% BSA (for detecting phosphorylated proteins) for 1 h, incubated with primary antibody at 4°C overnight, washed with TBST for 3 times, 5 min each time, and incubated with secondary antibody labeled with horseradish peroxidase (HRP) for 1 h, washed with TBST for 3 times, 5 min each time. Then, signal exposure was performed using SuperPico ECL Chemiluminescence Kit (Nanjing NorgenBiotek, E422-01, China), and signal collection was performed using ChemiScope 600EXp System (China). The Western blot band signals were quantitatively analyzed using ImageJ software. The antibodies used in this experiment were the same as those in Example 2.

[0061] II. Experimental results

[0062] The in vivo anti-renal fibrosis efficacy of morusin P was analyzed in a UUO mouse model. HE staining showed that morusin P intervention significantly improved the abnormal structure of UUO kidney tissues and reduced the tubular injury score. Masson staining showed that the extracellular collagen area of the kidney tissues of the morusin P intervention mice was significantly reduced Figure 3 A-C). RT-PCR analysis of kidney tissues found that compared with the UUO model group, the expression levels of fibronectin, Col1a1, and a-SMA mRNA in the kidney tissues of the morusin P intervention group were significantly inhibited Figure 3 D-F). Western blot analysis further confirmed that morusin P intervention reduced the expression levels of fibronectin, collagen I, and a-SMA Figure 3 G-J). The results showed that morusin P can treat chronic kidney disease renal fibrosis by reducing the expression of fibrosis-related genes and proteins, reducing the fibrosis area, and reducing the tubular injury caused by UUO.

[0063] In summary, the morusin compounds (morusin O and morusin P) of the present application can treat chronic kidney disease renal fibrosis by inhibiting TGF-β signaling, reducing the expression of fibrosis-related genes and proteins, reducing the fibrosis area, and reducing the tubular injury caused by UUO, thereby providing a new choice for drugs for treating chronic kidney disease renal fibrosis in clinical practice, and having good application prospects.

Claims

1. The use of a compound or a pharmaceutically acceptable salt thereof as a single active ingredient in the preparation of a medicament for the prevention and / or treatment of renal fibrosis in chronic kidney disease, characterized in that: The structural formula of the compound is shown in Formula II: Formula II.

2. The use of a compound or a pharmaceutically acceptable salt thereof as a single active ingredient in the preparation of a medicament for the prevention and / or treatment of renal fibrosis in chronic kidney disease, characterized in that: The structural formula of the compound is shown in Formula III: Formula III.

3. The use according to claim 1 or 2, characterized in that: The drug can reduce the expression of fibrosis genes.

4. The use according to claim 3, characterized in that: The fibrosis gene is selected from fibronectin, Col1a1, α-SMA .

5. The use according to claim 1 or 2, characterized in that: The drug is used to inhibit TGF-β signaling activity.

6. The use according to claim 1 or 2, characterized in that: The drug can reduce renal tubular damage.

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