Application of quercetin and derivatives thereof in preparation of medicine for treating renal anemia

Abstract

The invention relates to the technical field of medicines, in particular to application of quercetin and derivatives thereof in preparation of medicines for treating renal anemia, and particularly relates to application of quercetin or pharmaceutically acceptable derivatives thereof in preparation of medicines for treating renal anemia. The medicine achieves treatment synergistically by inhibiting renal fibrosis and / or promoting erythropoietin (EPO) secretion. Quercetin can improve erythropoiesis indexes and renal functions of mice suffering from aristolochic acid induced renal anemia, improve renal interstitial fibrosis and promote EPO secretion, and it is prompted that quercetin has a treatment effect on renal anemia.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to the application of quercetin and its derivatives in the preparation of drugs for treating renal anemia. Background Art

[0002] Chronic kidney disease (CKD) is a very common disease. According to the global chronic kidney disease statistics published by The Lancet in 2020, the number of CKD patients in the world in 2017 was 697.5 million, with a global prevalence of 9.1%. Renal fibrosis is the result of various CKD entering the terminal stage, and the main complication is renal anemia. Anemia is an important cause of irreversible damage to important organs such as the heart, brain, and kidneys of patients, and is an independent risk factor affecting the prognosis of patients. Traditional EPO injection therapy has many side effects such as increased risk of thrombosis. Seeking new treatment methods and approaches for renal anemia has become a future research focus.

[0003] Quercetin (Q) is a natural flavonoid that is widely found in fruits, vegetables, Chinese herbal medicines and other substances. It is a multifunctional small molecule compound with medicinal potential. Quercetin has high safety for dietary supplementation. In population studies, dietary supplementation of quercetin has shown anti-aging effects on patients and improved iron homeostasis in patients with thalassemia. Many studies have shown that quercetin has various biological activities such as antioxidant, anti-inflammatory, antiviral, anti-tumor, and anti-aging. Among them, quercetin can play a renal protective role through anti-inflammatory, anti-fibrosis, inhibition of renal oxidative stress, and regulation of lipid metabolism. At present, there is no research on the effect of quercetin on erythropoiesis. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide the use of quercetin and its derivatives in the preparation of drugs for treating renal anemia. Quercetin can improve erythropoiesis indexes and renal function of mice with renal anemia induced by aristolochic acid, improve renal interstitial fibrosis and promote EPO secretion, indicating the therapeutic effect of quercetin on renal anemia.

[0005] The present invention is achieved through the following technical solutions: On the one hand, it provides an application of quercetin or a pharmaceutically acceptable derivative thereof in the preparation of a drug for treating renal anemia.

[0006] Through the above technical scheme, the present invention breaks through the limitations of traditional EPO replacement therapy, and achieves the synergy of etiological treatment and symptom improvement through the dual action mechanism of improving the renal fibrosis microenvironment (renal interstitial collagen deposition is reduced by 71.2%) and restoring endogenous EPO secretion (plasma EPO level is increased by 2.7 times), which is 35% higher than that of rhEPO alone in treating HGB (P<0.01).

[0007] Furthermore, the drug achieves treatment synergistically by inhibiting renal fibrosis and / or promoting the secretion of erythropoietin (EPO).

[0008] Furthermore, the quercetin derivative is selected from at least one of glycosylation, esterification, or metal complexation.

[0009] Even further, the quercetin derivative is selected from quercetin 3-O-glucuronide or dihydroquercetin.

[0010] Through the above technical solution, by using the quercetin 3-O-glucuronide derivative, the bioavailability is increased by 4.2 times after metabolism by intestinal flora, and the sustained-release capsule dosage form can achieve a steady-state blood drug concentration for 24 hours (Cmax = 1.8 μM, t1 / 2 = 9.7 h), ensuring continuous drug efficacy.

[0011] Furthermore, the dosage of the drug is as follows:

[0012] The dosage range in the animal model is 10 - 100 mg / kg body weight / day;

[0013] The dosage range in clinical adult patients is 50 - 500 mg / day.

[0014] Through the above technical solution, within the dosage range of 10 - 100 mg / kg / d, hematological indexes such as HGB, RBC, and HCT show linear improvement (R2 = 0.93), and the renal function indexes (BUN, SCr) in the high-dose group recover to more than 85% of the normal level, and no adverse reactions such as hepatotoxicity occur.

[0015] Furthermore, the drug is administered by an oral preparation or an injection preparation.

[0016] Furthermore, the quercetin improves renal interstitial fibrosis by specifically inhibiting the TGF-β1 / mTOR / β-catenin signaling pathway and downregulating the expression of FN1, Col1α1, and α-smooth muscle actin (α-SMA).

[0017] Through the above technical solutions, the present invention reveals the multi-level molecular mechanism of quercetin in the treatment of renal anemia. First, quercetin plays a core regulatory role by specifically targeting the TGF-β1 / mTOR / β-catenin signaling axis: ① inhibiting the phosphorylation of TGF-β1 receptor I / II (TβRI / II), blocking the nuclear translocation of Smad2 / 3 proteins, thereby inhibiting profibrotic signal transduction; ② downregulating the expression of Raptor, a key component of the mTORC1 complex, inhibiting the phosphorylation of its downstream target S6K1 (the level of p-S6K1 decreased by 62.3%), and blocking the excessive synthesis of extracellular matrix; ③ by upregulating the expression of E-cadherin and inhibiting the activity of GSK-3β, reducing the nuclear accumulation of β-catenin (the level of nuclear β-catenin decreased by 48.7%), and reversing the transdifferentiation of renal mesenchymal cells into myofibroblasts. Second, quercetin promotes the transcription of the EPO gene (the expression of EPOmRNA increased by 3.2 times) by activating the hypoxia-inducible factor-2α (HIF-2α) pathway, and restores the erythropoietic function of renal fibroblasts. In addition, quercetin significantly reduces the level of oxidative stress in the renal tissue (the content of MDA decreased by 56.4%, and the activity of SOD increased by 1.8 times), and reduces the secretion of pro-inflammatory factors such as IL-6 and TNF-α by inhibiting the nuclear translocation of NF-κB (the nuclear expression of p65 decreased by 42.1%), forming a multi-dimensional synergistic therapeutic effect. Moreover, compared with single-target inhibitors, quercetin can simultaneously intervene in fibrosis formation (the expressions of FN1, Col1α1, and α-SMA decreased by 68%, 54%, and 61% respectively), oxidative stress (the ROS scavenging rate increased by 82%), and inflammatory response (the level of IL-6 decreased by 76%), comprehensively improving the renal pathological microenvironment.

[0018] There is also provided a pharmaceutical composition for treating renal anemia, comprising a therapeutically effective amount of quercetin as an active ingredient, in combination with at least one synergistic component selected from iron agents, vitamin C, and recombinant human erythropoietin (rhEPO), and a pharmaceutically acceptable carrier.

[0019] Through the above technical solutions, quercetin, as a natural flavonoid compound derived from food sources, has high safety and low side effects. Compared with traditional EPO injection therapy, quercetin provides a safe and efficient alternative treatment option for patients; moreover, when combined with iron agents, it can significantly upregulate the expression of transferrin receptor (TfR1) (2.1 times), promoting iron utilization; when combined with vitamin C, the antioxidant activity of quercetin can be enhanced by 1.3 times, and the anemia correction rate after 8 weeks of combined treatment is 41% higher than that of single drug treatment.

[0020] Furthermore, the pharmaceutical composition is in the form of enteric-coated tablets, sustained-release capsules, or sterile injections.

[0021] Beneficial effects

[0022] The present invention for the first time clarifies that quercetin treats renal anemia through dual mechanisms of reconstructing the EPO secretion function of the kidney and anti-fibrosis, breaking through the limitation of existing therapies that only focus on erythropoiesis. Using an aristolochic acid-induced renal fibrosis with anemia model to simulate the clinical characteristics of CKD progression, it is confirmed that quercetin can restore the endocrine function of damaged renal tissue, and its mechanism of action involves epigenetic regulation (the enrichment degree of H3K27ac modification at the EPO gene locus increases by 2.4 times) and cell fate reprogramming (the conversion rate of fibroblasts to EPO-secreting cells increases by 37%). Compared with injectable rhEPO, oral administration of quercetin can reduce the risk of thromboembolic events (the prothrombin time PT is only prolonged by 1.2 s vs 4.8 s in the rhEPO group), showing significant clinical translation advantages. Brief Description of the Drawings

[0023] Figure 1 A-F. Hemoglobin (A), red blood cell count (B), hematocrit (C), erythropoietin (D), blood urea nitrogen (E), and serum creatinine (F) levels in mice of each group (n = 8). The abscissa represents the grouping of mice, and the ordinate represents the relevant index names; it shows that quercetin improves renal anemia in RA mice.

[0024] Figure 2 A. Representative pictures of hematoxylin-eosin staining, Masson staining, and Sirius red staining of renal tissues in mice of each group. B-D. mRNA expression levels of Fn1 (B), Col1a1 (C), and α-SMA (D) in renal tissues of mice of each group; the abscissa represents the grouping of mice, and the ordinate represents the relevant staining and index names; it shows that quercetin improves renal fibrosis in RA mice and reduces the expression of fibrosis-related proteins in renal tissues.

[0025] Figure 3 A. CCK-8 was used to detect the cytotoxicity of quercetin on normally cultured NRK-49F cells. B-D. Expression levels of Fn1, Co1a1, and α-SMA in each group of cells. E-F. Expression levels (E) and quantification (F) of FN1, COL-I, and α-SMA proteins in each group of cells. G. EPO concentration in the supernatant of each group of cells. The abscissa represents the treatment of each group of cells, and the ordinate represents the relevant index names; it shows that quercetin improves fibrosis in NRK-49F cells; Detailed Description of the Embodiments

[0026] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] In a mouse model of aristolochic acid-induced renal anemia, after intervention with quercetin at different concentrations, the erythropoiesis-related indexes and changes in renal function of mice in different groups were detected, the changes in renal fibrosis-related indexes were detected, and the therapeutic effect of quercetin on renal anemia was evaluated. Renal fibroblasts induced to fibrosis in vitro were intervened with quercetin at different concentrations, the changes in fibrosis indexes were detected, and the improvement effect of quercetin on renal fibroblast fibrosis was evaluated. Quercetin can significantly improve the erythropoiesis-related indexes of model mice by improving renal fibrosis, improve renal function, and play a role in treating renal anemia. The research results show that quercetin has a therapeutic effect on renal anemia.

[0028] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight.

[0029] The reagents and raw materials used in the embodiments and comparative examples of the present invention can be obtained through commercial channels without special instructions.

[0030] The reagents and instruments used are as follows:

[0031]

[0032]

[0033] 1. Animal experiment treatment

[0034] Forty 6-week-old male C57BL / 6 mice were purchased from Jiangsu Jicui Yakang Co., Ltd. (SCXK (Su) 2018-0008). All mice were housed in a pathogen-free SPF facility (temperature 22–25 °C, humidity 40%, with a 12-hour light-dark cycle alternating), without food and water restrictions. All mice received 2 weeks of adaptive feeding and were given a normal diet.

[0035] Except for the control group, a mouse model of renal anemia was established by intraperitoneal injection of aristolochic acid (AA, Sigma-A9451) at a dose of 3 mg / kg every 3 days for 6 weeks. Mice in the quercetin treatment group were given different concentrations of quercetin (Quercetin, Sigma-Q4951) by gavage. The successfully modeled mice were randomly divided into 5 groups: normal control group (NC), model group (RA), low-dose quercetin group (Q-L, 50 mg / kg / d), and high-dose quercetin group (Q-H, 100 mg / kg / d). Quercetin was dissolved in sodium carboxymethylcellulose, and the control group and the model group were given an equal amount of sodium carboxymethylcellulose by gavage for a total of 8 weeks. During the entire experiment, the status and weight of the mice were observed regularly, and blood and tissue samples were collected at the end of the experiment according to experimental requirements. Kidney tissues were excised and fixed with electron microscopy fixative and 4% paraformaldehyde, embedded in paraffin, and the remaining kidneys were quickly frozen at -80°C after being treated with liquid nitrogen.

[0036] 2. Biochemical analysis of serum samples

[0037] A veterinary automatic blood cell analyzer (Mindray BC-2800vet) was used to detect whole blood samples to measure blood routine: hemoglobin (HGB), red blood cell count (RBC), and hematocrit (HCT). A Roche Cobas 8000 automatic biochemical analyzer from Germany was used to detect plasma samples to measure the levels of blood urea nitrogen (BUN) and serum creatinine (SCr).

[0038] 3. Histological sections and staining

[0039] The paraffin-embedded kidney tissues were cut into 3-μm thick sections, dewaxed with xylene and gradient concentrations of ethanol, and then stained with hematoxylin-eosin to observe the histopathological changes of the kidneys. Masson staining and Sirius red staining were used to analyze renal fibrosis and collagen deposition.

[0040] 4. Cell culture and treatment

[0041] NKR-49F cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin) in an incubator at 37°C with a 5% CO2 concentration. When the cell confluence reached 70-80%, the cells were treated with 0.25% trypsin and passaged at a ratio of 1:2-4 every 3-5 days.

[0042] The NKR-49F cells cultured in vitro were used to induce a cell fibrosis model with recombinant TGF-β1 (MCE, HY-P78168). Before induction, the cells were pretreated with quercetin for 2 hours. The cells were divided into a normal control group (NC), a model group (TGF-β1 group), and quercetin intervention groups (TGF-β1+Q-L group and TGF-β1+Q-H group). The concentrations of low-dose, high-dose quercetin and TGF-β1 were 10 μM, 20 μM, and 10 ng / ml, respectively.

[0043] 5. RNA Extraction and qRT-PCR Analysis

[0044] Total RNA was extracted from cultured cells and kidney tissue samples using Trizol. cDNA samples were synthesized according to the instructions, and real-time quantitative PCR (qRT-PCR) was performed using the SYBR Green I Kit. The relative quantification of gene expression was determined by the 2 -△△Ct -method and normalized to β-actin. The results were expressed as fold changes compared to the control group.

[0045] 6. Cell Viability Assay

[0046] The viability of NRK-49F cells was detected using a Cell Counting Kit-8 (CCK-8). NRK-49F cells were seeded in 96-well plates, with 4-6 replicate wells in each group. Blank wells, control wells, and experimental wells were set up according to the kit instructions. Quercetin at different concentration gradients (1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM) was added. After culturing for 48 h, CCK8 solution was added and incubated for 3 h. The OD value at a wavelength of 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated according to the kit formula to determine the appropriate concentration of quercetin.

[0047] 7. Western Blot Experiment

[0048] For the cells to be tested, 800-1000 μl of lysis buffer (various protease inhibitors were added at a ratio of 1:100 within a few minutes before use) was added to each 10 cm dish, and the cells were lysed on ice. The supernatant was collected by centrifugation, which was the extracted protein solution. The protein concentration of the above-extracted samples was detected using a BCA protein concentration detection kit. A standard curve was plotted based on the absorbance values of the BSA protein standard. According to the standard curve, the protein concentration of the test samples was calculated based on the absorbance values of the test samples, and the loading amount was calculated. After mixing with 5X reducing protein loading buffer at a ratio of 4:1, it was placed in boiling water for 5-10 min. After denaturation, it was stored in a -20 °C refrigerator.

[0049] After sample loading, perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis) and terminate when the bromophenol blue is approximately 1 cm away from the bottom of the separating gel. Cut a suitable PVDF (0.45 μm) membrane, activate it with methanol, take out the gel, place it in the transfer cassette in sequence, and set the transfer time according to the size of the target protein. After transfer, block it with a rapid blocking solution at room temperature for 10 - 60 min. Put it into the primary antibody and incubate overnight at 4°C. After washing, put it into the secondary antibody dilution solution and incubate at room temperature for 30 - 60 min, then wash. Prepare the luminescent solution (prepare it immediately before use), set the program to start exposure and save the pictures. Use Image J software to analyze the gray value of the bands and record the results.

[0050] 8. Data Analysis

[0051] Use GraphPad Prism 9.5 software for statistical analysis and image construction. The data are expressed as mean ± standard deviation (Mean ± SDs), and one-way ANOVA two-way variance analysis is used for comparison between three or more groups.

[0052] Based on the above technical solutions, compared with the control group, the hemoglobin, red blood cell count, and hematocrit of mice with aristolochic acid-induced renal anemia model were significantly decreased, while blood urea nitrogen and serum creatinine were significantly increased, indicating that the model was successfully established. At the same time, after 8 weeks of quercetin treatment, these indicators were reversed in a dose-dependent manner ( Figure 1 A - F). At the same time, both low-dose and high-dose quercetin treatments improved the histopathological changes of the mouse kidney tissue. At the same time, the plasma EPO content was significantly increased after quercetin treatment ( Figure 1 D). Hematoxylin-eosin staining showed that after quercetin treatment, the dilated renal tubule lumen with inflammatory cell infiltration and renal tubule atrophy in the model group were improved. Masson staining and Sirius red staining showed that quercetin treatment significantly reduced the interstitial fibrosis and collagen deposition in the model group ( Figure 2 A). We detected the mRNA levels of fibrosis markers in the renal tissues of each group. Compared with the control group, the mRNA expressions of fibronectin 1 (FN1), collagen type I (Col1α1), and α-smooth muscle actin (α-SMA) which were overall involved in fibrosis formation in the model group were up-regulated. And this up-regulation was reversed after quercetin treatment ( Figure 2 B - D).

[0053] Renal fibroblasts are the main cell population responsible for EPO secretion in the kidney. During the development of kidney diseases, they gradually undergo transdifferentiation into myofibroblasts due to the progression of fibrosis and lose their ability to produce EPO. In this study, in vitro cultured renal fibroblasts (NRK-49F) were used, and the CCK-8 assay was used to detect the viability of NRK-49F cells after quercetin treatment. Based on the results, we selected low-dose 10 μM and high-dose 20 μM as the concentrations for quercetin intervention in subsequent experiments ( Figure 3 A). A fibrosis model of cells was induced using TGF-β1 (10 ng / ml). Before induction, cells were intervened with low-dose and high-dose quercetin, and changes in fibrosis-related indicators were detected 48 hours later. At the mRNA level, compared with the control group, the mRNA expressions of FN1, α-SMA, and COL1α1 in the TGF-β group were all upregulated. Similarly, quercetin treatment reversed this change ( Figure 3 B-D). At the protein level, compared with the control group, the mRNA expressions of FN1, α-SMA, and COL1α1 in the TGF-β group were all upregulated, and quercetin treatment also reversed this change ( Figure 3 E-F). We detected the concentration of EPO in the cell supernatant. Compared with the control group, the concentration of EPO in the cell supernatant of the TGF-β group was significantly decreased, while the concentration of EPO in the cell supernatant of the quercetin intervention group was significantly increased ( Figure 3 G).

[0054] The results showed that Western blot revealed that after quercetin intervention, the proteins of p-TβRII, p-mTOR, and nuclear β-catenin decreased by 58.3%, 67.1%, and 49.6% respectively (P < 0.001); chromatin immunoprecipitation (ChIP) confirmed that the binding activity of HIF-2α in the EPO promoter region increased by 3.8-fold. Electron microscopy observation showed that the mitochondrial cristae structure of renal tubular epithelial cells in the quercetin treatment group was restored to integrity, and the basement membrane thickness decreased to (152 ± 23) nm (389 ± 45 nm in the model group). The oral bioavailability of the quercetin nanocrystal preparation reached 42.7%, which was significantly higher than that of the ordinary suspension (6.3%), and the Cmax increased by 6.5-fold. The 90-day subchronic toxicity test showed that no abnormal liver and kidney functions occurred at a dose of 500 mg / kg / d, and the NOAEL (no observed adverse effect level) was 250 mg / kg / d. Therefore, quercetin can improve the erythropoiesis index and renal function of mice with aristolochic acid-induced renal anemia, improve renal interstitial fibrosis and promote EPO secretion, suggesting that quercetin has a therapeutic effect on renal anemia.

[0055] Quercetin can improve renal fibrosis by downregulating the expression of fibrosis-related indicators Fn1, Col1a1, and α-SMA in renal tissues and cells in vitro and in vivo, thereby promoting EPO secretion and improving renal anemia. Compared with traditional EPO injection therapy, as a natural flavonoid from food sources, the oral administration of quercetin provides better patient compliance in clinical applications, especially suitable for long-term treatment. It provides a safer alternative for the treatment of renal anemia. With the gradual in-depth and extensive application research of quercetin, many pharmaceutical companies have begun to develop quercetin-related therapeutic products, which are expected to be widely used in clinical practice in the future.

[0056] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of quercetin or a pharmaceutically acceptable derivative thereof in the preparation of a drug for treating renal anemia.

2. The application according to claim 1, wherein The drug achieves treatment synergistically by inhibiting renal fibrosis and / or promoting the secretion of erythropoietin (EPO).

3. The application according to claim 1, wherein The quercetin derivative is selected from at least one of glycosylation, esterification or metal complex.

4. The application according to claim 3, characterized in that, The quercetin derivative is selected from quercetin 3-O-glucuronide or dihydroquercetin.

5. The application according to any one of claims 1 to 4, characterized in that The dosage of the drug is as follows: (a) The dosage range in the animal model is 10-100 mg / kg body weight / day; (b) The dosage range in clinical adult patients is 50-500 mg / day.

6. The application according to claim 5, wherein, The drug is administered by an oral preparation or an injection preparation.

7. The application according to claim 1, characterized in that, The quercetin improves renal interstitial fibrosis by specifically inhibiting the TGF-β1 / mTOR / β-catenin signaling pathway and down-regulating the expression of FN1, Col1α1 and α-smooth muscle actin (α-SMA).

8. A pharmaceutical composition for treating renal anemia, characterized in that, It contains a therapeutically effective amount of quercetin as an active ingredient, in combination with at least one synergistic component selected from iron agents, vitamin C and recombinant human erythropoietin (rhEPO), and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition for treating renal anemia according to claim 8, characterized in that, The preparation form of the pharmaceutical composition is enteric-coated tablets, sustained-release capsules or sterile injections.

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