Application of flavonoid metabolite for improving tacrolimus-induced chronic renal injury

Abstract

The invention relates to the technical field of medicines, in particular to application of a flavonoid metabolite for improving tacrolimus-induced chronic renal injury. The invention provides application of a flavonoid metabolite, namely quercetin-7-O-beta-glucuronide, in an abelmoschus esculentus capsule to improvement of chronic renal injury induced by tacrolimus, and particularly provides application of the flavonoid metabolite, namely quercetin-7-O-beta-glucuronide in the abelmoschus esculentus capsule. The research shows that the quincetin-7-O-beta-glucuronide has relatively strong biological activities of resisting inflammation, resisting oxidation, resisting fibrosis and the like, and plays an important role in preventing and treating the chronic renal injury caused by tacrolimus.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to an application of flavonoid metabolites in Huangkui capsules to improve tacrolimus-induced chronic renal injury. Background Art

[0002] Chronic kidney damage (CKD) is a common and serious health problem, with an increasing prevalence worldwide. The causes of CKD are complex, including diabetes, hypertension, chronic glomerulonephritis, etc. It progresses slowly, but can eventually lead to renal failure, seriously affecting the patient's quality of life. Currently, the treatment of CKD mainly focuses on delaying disease progression and preventing complications. However, the effects of existing treatments are limited and are often accompanied by more adverse reactions.

[0003] Tacrolimus (TAC) is an immunosuppressant widely used in organ transplant patients to prevent graft rejection by inhibiting T cell activity. However, long-term use of tacrolimus can lead to nephrotoxicity, manifested as chronic kidney damage symptoms such as decreased renal function and proteinuria. This nephrotoxicity seriously limits the clinical application of tacrolimus. Therefore, finding effective methods to reduce or prevent tacrolimus-induced renal damage is of great clinical significance.

[0004] Abelmoschus manihot is a traditional Chinese medicine. Its main active ingredients include a variety of flavonoids, which have multiple biological activities such as anti-inflammatory and antioxidant. Abelmoschus manihot capsule, a drug made from Abelmoschus manihot extract, has been widely used in the treatment of diseases such as chronic nephritis. In the body, the flavonoids in Abelmoschus manihot capsule can be metabolized to produce a variety of products, among which quercetin-7-O-β-glucuronide is one of the main metabolites.

[0005] Studies have shown that quercetin-7-O-β-glucuronide has strong anti-inflammatory, antioxidant and anti-fibrotic biological activities. It works through multiple signaling pathways, including inhibiting the NF-κB pathway, reducing oxidative stress, and reducing the release of inflammatory factors. However, there are no reports on the possible role of quercetin-7-O-β-glucuronide in preventing and treating tacrolimus-induced chronic renal injury.

[0006] The inventors have been committed to the analysis and identification of the metabolites of Huangkui Capsules in vivo, and have completed in vitro activity experiments on the flavonoid metabolites of Huangkui Capsules. Through in vitro activity experiments, flavonoid metabolites with active effects were screened out. Summary of the invention

[0007] The object of the present invention is to provide an application of quercetin-7-O-β-glucuronide, a flavonoid metabolite in Huangkui capsule, in improving tacrolimus-induced chronic renal injury.

[0008] The first aspect of the present invention provides the use of quercetin-7-O-β-glucuronide in the preparation of a drug for preventing and treating tacrolimus-induced chronic renal injury.

[0009] Furthermore, in the application, quercetin-7-O-β-glucuronide significantly improves the cell viability in tacrolimus-induced chronic renal injury.

[0010] Furthermore, in the application, quercetin-7-O-β-glucuronide significantly antagonizes the expression of GSH, SOD, CAT, MDA and Fe in cells with tacrolimus-induced chronic renal injury. 2+ Abnormal changes in levels.

[0011] Furthermore, in the application, quercetin-7-O-β-glucuronide improves the mitochondrial morphological and structural damage in cells with tacrolimus-induced chronic renal injury.

[0012] Furthermore, in the application described above, quercetin-7-O-β-glucuronide can participate in the System Xc- / GSH / GPX4 signaling pathway to correct abnormal glutathione metabolism caused by TAC, inhibit ferroptosis, and exert the therapeutic effect of improving tacrolimus-induced chronic renal injury.

[0013] The second aspect of the present invention provides the use of quercetin-7-O-β-glucuronide in the preparation of a drug for improving abnormal glutathione metabolism caused by tacrolimus.

[0014] The third aspect of the present invention provides the use of quercetin-7-O-β-glucuronide in the preparation of a drug for inhibiting tacrolimus-induced ferroptosis.

[0015] The advantages of the present invention are:

[0016] The present invention provides the use of quercetin-7-O-β-glucuronide, a flavonoid metabolite in Huangkui capsule, to improve tacrolimus-induced chronic kidney injury. The experimental results of the present invention show that quercetin-7-O-β-glucuronide can participate in the System Xc- / GSH / GPX4 signaling pathway to correct abnormal glutathione metabolism caused by TAC, inhibit ferroptosis, and play an important role in improving the efficacy of TAC chronic kidney injury, and in preventing and treating chronic kidney injury caused by tacrolimus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 .Cell viability graph.

[0018] Figure 2 . Graph of oxidative stress indicators in each group.

[0019] Figure 3 .Fe in each group 2+ Horizontal graph.

[0020] Figure 4 .Morphological structure diagram of mitochondria in each group.

[0021] Figure 5 .Molecular docking diagram.

[0022] Figure 6 .Molecular dynamics simulation diagram. DETAILED DESCRIPTION

[0023] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0024] Example:

[0025] 1 Experiment and Materials

[0026] 1.1 Materials and Equipment

[0027] DMEM / F-12 culture medium (MA0214) and cell proliferation and toxicity detection kit (MA-0218) were purchased from Dalian Meilun Biotechnology Co., Ltd., antibiotics-antimycotics (triple antibodies) were purchased from Shanghai McLean Biochemical Co., Ltd., Cryosure-DMSO was purchased from WAK CHEMIE, Germany, fetal bovine serum and 0.25% trypsin cell digestion solution were purchased from Shanghai Beyotime Biotechnology Co., Ltd., and standard substances Tacrolimus (TAC), Quercetin-7-O-β-glucuronide, and Ferrostatin-1 were purchased from MedChemExpress, USA.

[0028] Automatic cell counter ( II FL, USA), transmission electron microscope (HT7800, Japan), ultrathin slicer (Leica UC7, Shanghai Leica Instrument Co., Ltd.), diamond microtome (Ultra45°, Shanghai Leica Instrument Co., Ltd.), inverted microscope (DMi 1, Shanghai Leica Instrument Co., Ltd.), multifunctional microplate reader ( 3, United States).

[0029] 1.2 Cell grouping

[0030] The HK-2 cell experiment was divided into 4 groups. The control group (CON group) cultured the cells normally without any treatment; the model group (TAC group) cultured the cells normally for 24 hours, then added TAC and continued to culture for 24 hours; the treatment group (Quercetin-7-O-β-glucuronide group) cultured the cells normally for 24 hours, then added TAC and Quercetin-7-O-β-glucuronide and continued to culture for 24 hours; the positive drug group (Ferrostatin-1 group) cultured the cells normally for 24 hours, then added TAC and Ferrostatin-1 and continued to culture for 24 hours.

[0031] 1.3 Cell viability assay

[0032] After the cells in the logarithmic growth phase were digested with trypsin solution, the cells were collected and resuspended in DMEM / F-12 complete medium. The cell number was counted using an automatic cell counter and 2×10 4 The cells were inoculated at a density of 1000 cells / well in a 96-well plate and cultured for 24 hours. After the cells were completely attached to the wall, Quercetin-7-O-β-glucuronide (10 μM, 20 μM, 40 μM) and Ferrostatin-1 (1 μM) were added respectively, and the cells in the 96-well plate were treated according to the treatment methods of each group. Six replicate wells were set up in each group. After the drug was applied for 24 hours, all the liquid in each well was discarded, and a DMEM / F-12 culture medium solution containing 10% CCK-8 reagent was added. The cells were incubated in a light-proof incubator at 37°C and 5% CO2 for 2 hours. The absorbance value (A) of each well was detected at 450 nm using a full-wavelength microplate reader, and the cell survival rate was calculated according to the calculation formula.

[0033]

[0034] 1.4 Cellular GSH, SOD, CAT, MDA, Fe 2+ Level determination

[0035] Cell culture was performed according to conventional methods. When HK-2 cells grew to about 90% fusion, the cells were digested and collected, and the cells were counted. The cells were diluted to an appropriate cell concentration and the cells were plated at 4×105 The cells were inoculated at a density of 100 cells / well in a 6-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours. After the cells grew and merged to about 80%-90%, they were treated according to the grouping and cultured for 24 hours. The cell suspensions of each group were collected, and the cell suspensions were treated according to the instructions of the kit. The OD values ​​were measured at the corresponding absorbance using a full-wavelength microplate reader, and the values ​​of various indicators were calculated according to the formula.

[0036] 1.5 Mitochondrial morphology observation

[0037] Cell culture was performed according to conventional methods. When HK-2 cells grew to about 90% fusion, the cells were digested and collected, and the cells were counted. The cells were diluted to an appropriate cell concentration and the cells were plated at 1×10 6 The cells were inoculated in a culture dish and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours. When the cells grew and merged to about 80%-90%, they were treated according to the grouping and cultured for 24 hours. The old culture medium was discarded, and the cells were washed with PBS 2-3 times. The cell suspension was digested and collected. The cells were centrifuged at 1000rpm for 4 minutes, and the supernatant was discarded. 2.5% glutaraldehyde electron microscope fixative was slowly added to the tube; it was left to stand in a refrigerator at 4°C for 24 hours. The next day, it was washed with PBS, centrifuged in a centrifuge, the supernatant was discarded, and agar was used for pre-embedding. Then, 1% osmium acid prepared with 0.1M phosphate buffer PB (pH7.4) was fixed at room temperature in the dark for 2 hours, and 0.1M phosphate buffer PB (pH7.4) was used to repeat the rinsing 3 times, with an interval of 15 minutes each time. It was placed at room temperature and dehydrated with different gradient alcohols, each gradient was maintained for 20 minutes, and then dehydrated twice with 100% acetone, each time for 20 minutes. Different ratios of acetone-812 embedding agents were used for penetration embedding, and sliced ​​into thin slices with a microtome. After light-blocking staining with 2% uranyl acetate and staining with 2.6% lead citrate solution to avoid carbon dioxide, the slices were washed, blotted, and dried overnight. Finally, a transmission electron microscope was used to collect image information for analysis.

[0038] 1.6 Molecular docking

[0039] In the AutoDock Tool 1.5.7 software, select the compound .pdbqt format as the ligand molecule, select the protein .pdbqt format as the receptor protein, set the Grid box size to just cover the receptor protein, save the Grid box parameter information in .gpf format, select the genetic algorithm, select 50 docking times, adjust the final parameters and perform molecular docking, select the optimal binding conformation to analyze the interaction, and use Pymol and Discocery studio software to visualize the results and generate a protein-ligand interaction diagram.

[0040] 1.7 Molecular dynamics

[0041] Desmond / Maestro noncommercial version 2022.1 was selected as the dynamics simulation software, OPLS_2005 molecular mechanics force field was applied, and TIP3P water model was used to add TIP3P to the complex system. The system was immersed in a By adding Na + / Cl ions to neutralize the pH value of the system. + / Cl ions were added to neutralize the pH value of the system. After minimization and relaxation of the system, molecular dynamics simulations were performed for 100 ns in an isothermal and isobaric ensemble at 300 K and 1 bar. The trajectory coordinates were recorded every 100 ps. Molecular dynamics analysis was performed using Desmond's simulation interaction diagram. Root mean square deviation (RMSD), root mean square fluctuation (RMSF) and hydrogen bonding were used to analyze structural changes and dynamic behaviors.

[0042] 2 Results

[0043] 2.1 Cell viability

[0044] The results are as follows Figure 1 As shown, compared with the CON group, the cell survival rate in the TAC group was significantly decreased (P < 0.001); compared with the TAC group, the quercetin-7-O-β-glucuronide group was able to improve the viability of TAC-injured HK-2 cells in a dose-dependent manner.

[0045] 2.2 Oxidative stress indicators

[0046] The results are as follows Figure 2 As shown in the data, compared with the CON group, the MDA level in the TAC group was significantly increased (P < 0.001), and the GSH, SOD, and CAT levels were significantly decreased (P < 0.001); compared with the TAC group, the MDA, GSH, SOD, and CAT levels in the quercetin-7-O-β-glucuronide group and the Fer-1 group were significantly restored (P < 0.001).

[0047] 2.3Fe 2+ Level determination

[0048] The experimental results are as follows Figure 3 As shown, compared with the CON group, the Fe 2+ Compared with the TAC group, quercetin-7-O-β-glucuronide and Fer-1 could reduce Fe 2+The results showed that HK-2 cells injured by TAC had abnormal iron metabolism, and quercetin-7-O-β-glucuronide had an inhibitory effect on the abnormal iron metabolism caused by TAC.

[0049] 2.4 Mitochondrial morphology

[0050] like Figure 4 The results showed that the mitochondrial morphology of HK-2 cells in the CON group was normal, the mitochondrial cristae were neatly arranged, and no obvious damage was observed. Compared with the CON group, the mitochondria in the TAC group underwent ultrastructural changes such as outer membrane rupture, morphological shrinkage, and a significant increase in focal vacuoles, which were consistent with the mitochondrial morphological changes during ferroptosis; compared with the TAC group, the effect of quercetin-7-O-β-glucuronide was equivalent to that of Fer-1, and electron microscopy showed that the mitochondrial outer membrane was relatively intact and the increase in membrane density was mild, which was the closest to the CON group.

[0051] 2.5 Molecular docking

[0052] like Figure 5 As shown, quercetin-7-O-β-glucuronide can spontaneously bind to SLC7A11, GPX4, and SLC3A2, and the binding is tight (<-7kcal / mol, 1Kcal=4.2kJ), indicating that quercetin-7-O-β-glucuronide binds stably to SLC7A11, GPX4, and SLC3A2, and suitable spatial complementarity is formed between the ligand and the receptor. Among them, quercetin-7-O-β-glucuronide formed a hydrogen bond with ILE57 in SLC7A11, and the binding energy of the protein-small molecule complex was -9.5 kcal / mol; quercetin-7-O-β-glucuronide formed a hydrophobic force with LYS47 in GPX4, and the binding energy of the protein-small molecule complex was -7.3 kcal / mol; quercetin-7-O-β-glucuronide formed a hydrophobic force with ASP398 of SLC3A2, and the binding energy of the protein-small molecule complex was -8.7 kcal / mol.

[0053] 2.6 Molecular dynamics

[0054] Based on the high-performance computer system to simulate the trajectory within 100ns, and combined with the root mean square deviation (RMSD) system to determine the binding stability of small molecules and proteins, when the RMSD difference span does not exceed When , it indicates that the binding between the ligand and the receptor is stable and the system has reached a stable state. Figure 6As shown in the figure, the SLC7A11-quercetin-7-O-β-glucuronide and GPX4-quercetin-7-O-β-glucuronide systems were stable from the beginning of the simulation, and the SLC3A2-quercetin-7-O-β-glucuronide system was also stable after 20ns. The above results suggest that quercetin-7-O-β-glucuronide can stably bind to SLC7A11, SLC3A2, and GPX4.

[0055] 3 Conclusion

[0056] Quercetin-7-O-β-glucuronide can significantly improve the viability of HK-2 cells damaged by TAC. In addition, quercetin-7-O-β-glucuronide can also significantly inhibit the expression of GSH, SOD, CAT, MDA and Fe in HK-2 cells damaged by TAC. 2+ The results of transmission electron microscopy showed that quercetin-7-O-β-glucuronide could improve the morphological and structural damage of mitochondria in HK-2 cells damaged by TAC; molecular docking and molecular dynamics experiments showed that the complex system composed of quercetin-7-O-β-glucuronide and SLC7A11, SLC3A2 and GPX4 proteins was in a stable state. Based on the above results, the present invention believes that quercetin-7-O-β-glucuronide can participate in the System Xc- / GSH / GPX4 signaling pathway to correct the abnormal glutathione metabolism caused by TAC, inhibit ferroptosis, and play a role in improving the therapeutic effect of TAC chronic kidney injury.

[0057] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of quercetin-7-O-β-glucuronide in the preparation of drugs for preventing and treating tacrolimus-induced chronic renal injury.

2. The use of quercetin-7-O-β-glucuronide according to claim 1 in the preparation of a drug for preventing and treating tacrolimus-induced chronic renal injury, characterized in that: Quercetin-7-O-β-glucuronide significantly improves cell viability in tacrolimus-induced chronic renal injury.

3. The use of quercetin-7-O-β-glucuronide according to claim 1 in the preparation of a drug for preventing and treating tacrolimus-induced chronic renal injury, characterized in that: Quercetin-7-O-β-glucuronide significantly antagonized the expression of GSH, SOD, CAT, MDA and Fe in cells with tacrolimus-induced chronic renal injury 2+ Abnormal changes in levels.

4. The use of quercetin-7-O-β-glucuronide according to claim 1 in the preparation of a drug for preventing and treating tacrolimus-induced chronic renal injury, characterized in that: Quercetin-7-O-β-glucuronide improves mitochondrial morphological and structural damage in cells with tacrolimus-induced chronic renal injury.

5. The use of quercetin-7-O-β-glucuronide according to claim 1 in the preparation of a drug for preventing and treating tacrolimus-induced chronic renal injury, characterized in that: Quercetin-7-O-β-glucuronide participates in the SystemXc- / GSH / GPX4 signaling pathway to correct TAC-induced abnormal glutathione metabolism, inhibit ferroptosis, and play an therapeutic effect in improving tacrolimus-induced chronic renal injury.

6. Application of quercetin-7-O-β-glucuronide in the preparation of drugs for improving abnormal glutathione metabolism caused by tacrolimus.

7. Use of quercetin-7-O-β-glucuronide in the preparation of drugs for inhibiting tacrolimus-induced ferroptosis.

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