Application of quercetin in preparation of medicine for improving kidney injury caused by hyperuricemia

Abstract

The invention discloses an application of quercetin in preparation of a medicine for improving renal injury caused by hyperuricemia in the technical field of biological medicine, and experimental results show that the quercetin medicine can activate hepatocyte nuclear factor-alpha, enhance expression of an organic anion transporter OAT2 protein on a renal tubular epithelial cell substrate outer membrane, and improve the renal injury caused by hyperuricemia. The traditional Chinese medicine composition can promote blood vessels to actively secrete uric acid into renal tubular cavities so as to improve renal injury caused by hyperuricemia, and can be used for treating renal injury symptoms caused by primary or secondary hyperuricemia. Quercetin is applied to preparation of drugs, health care products and / or food for preventing and / or treating kidney injury diseases caused by hyperuricemia and has good application value and development prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of quercetin in preparing a drug for improving kidney damage caused by hyperuricemia. Background Art

[0002] Hyperuricemia is a common metabolic disease caused by impaired purine metabolism. Chronically elevated serum uric acid levels can lead to urate crystal deposition in the kidneys, inducing glomerular sclerosis, tubulointerstitial inflammation, and fibrosis, which can progress to hyperuricemic nephropathy (HN) and ultimately chronic renal failure. Currently, the core clinical treatment strategy for HN relies on lowering serum uric acid levels. Commonly used drugs include xanthine oxidase inhibitors (such as allopurinol and febuxostat) and uricosuric drugs (such as benzbromarone).

[0003] However, existing treatments have significant limitations: while xanthine oxidase inhibitors can inhibit uric acid production, they can cause severe skin allergic reactions and liver toxicity, and cannot reverse existing renal tissue damage. Uric acid excretion drugs reduce uric acid reabsorption by inhibiting the renal tubular URAT1 transporter, but are contraindicated in patients with renal insufficiency and may aggravate renal tubular urate crystal deposition. More importantly, existing drugs all target uric acid production or reabsorption, lack effective regulation of the active tubular uric acid secretion pathway, and cannot fully inhibit high uric acid-induced renal inflammation and fibrosis, resulting in insufficient renal protection.

[0004] Although quercetin (Qct), a natural flavonoid, has been reported to have potential uric acid-lowering activity, its specific protective mechanism against HN renal injury remains unclear. Currently, there is no evidence that quercetin promotes uric acid excretion by regulating the expression of organic anion transporters (such as the OAT family protein OAT2) in renal tubular epithelial cells (mTECs). Furthermore, there is a lack of research demonstrating that quercetin directly acts on transcription factors and regulates downstream target gene pathways, severely limiting its translational application to clinical treatment.

[0005] Therefore, it is urgent to reveal the deep molecular mechanism of quercetin in the treatment of HN, clarify its core target, and develop new therapeutic and health-care drugs with the dual effects of lowering uric acid and protecting the kidneys. Summary of the Invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide the use of quercetin in the preparation of a drug for improving kidney damage caused by hyperuricemia. The quercetin in the drug can reduce serum uric acid levels by activating hepatocyte nuclear factor-α (HNF1A protein) and upregulating the expression of OAT2 protein, inhibiting the occurrence of inflammation and renal fibrosis, and can be used for the symptoms of kidney damage caused by primary or secondary hyperuricemia. Quercetin is used to prepare drugs, health products and / or foods for preventing and / or treating kidney damage caused by hyperuricemia, and has great application value and development prospects.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows: use of quercetin in the preparation of a drug for improving kidney damage caused by hyperuricemia.

[0008] Further, the drug is used to bind to hepatocyte nuclear factor-alpha.

[0009] Furthermore, amino acid residues Gln98 and Ala15 of hepatocyte nuclear factor-α formed hydrogen bonds with the drug's carbocyclic carbonyl O atom and the B-ring 4′ oxygen atom, respectively;

[0010] Amino acid residues Gly31 and Arg52 of hepatocyte nuclear factor-α form carbon-hydrogen interactions with the oxygen atom at position 5 of the A ring and the oxygen atoms at positions 3′ and 4′ of the B ring, respectively;

[0011] The B and C rings are involved in π-σ and π-π interactions with amino acid residue Phe95 of hepatocyte nuclear factor-α.

[0012] Furthermore, hepatocyte nuclear factor-α was used to enrich the gene fragment of organic anion transporter OAT2.

[0013] Furthermore, the binding sequence in the promoter region of the organic anion transporter OAT2 gene fragment targeted and bound to hepatocyte nuclear factor-α.

[0014] Furthermore, the organic anion transporter OAT2 protein was used to reduce the expression of proinflammatory cytokines, including interleukin IL-1β, IL-6, and tumor necrosis factor TNF-α;

[0015] OAT2 is used to reduce the expression of fibrosis-related factors, including α-smooth muscle actin (α-SMA), type I collagen (Collagen-I), and fibronectin (Fn);

[0016] OAT2 is used to inhibit the phosphorylation and activation of NF-κB P65 protein;

[0017] OAT2 is used to reduce the expression of vimentin.

[0018] The experimental results show that quercetin drugs rely on OAT2 protein to play anti-inflammatory and anti-fibrosis effects and inhibit hyperuricemia kidney damage through a series of transcription factor activation and gene expression regulation.

[0019] Furthermore, hyperuricemia includes primary hyperuricemia and secondary hyperuricemia.

[0020] Experimental results show that in individuals with primary hyperuricemia, quercetin can significantly reduce serum uric acid, creatinine and urea nitrogen levels, while producing anti-inflammatory, anti-fibrotic and collagen deposition-clearing therapeutic effects, reducing the degree of renal damage.

[0021] In summary, quercetin can improve renal damage caused by hyperuricemia by activating hepatocyte nuclear factor-α, enhancing the expression of OAT2 on the basal membrane of renal tubular epithelial cells, and promoting active secretion of uric acid from blood vessels into the renal tubular lumen. Therefore, by activating the HNF1A-OAT2 interaction, it can lower serum uric acid levels, exert anti-inflammatory and anti-fibrotic therapeutic effects, and protect renal cell activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a bar graph showing the changes in intracellular uric acid levels in mTECs induced by uric acid under treatment with 16 μM quercetin.

[0023] Figure 2 The bar graph shows the changes in mRNA expression levels of pro-inflammatory cytokines in uric acid-induced mTECs cells under treatment with different concentrations of quercetin.

[0024] Figure 3 The bar graph shows the changes in mRNA expression levels of fibrosis-related factors in uric acid-induced mTECs cells under treatment with different concentrations of quercetin.

[0025] Figure 4 This is the result of identifying the expression level of OAT2 mRNA in mTECs cells overexpressing OAT2.

[0026] Figure 5 The bar graph shows the changes in mRNA expression levels of pro-inflammatory cytokines and fibrosis-related factors in mTECs cells overexpressing OAT2.

[0027] Figure 6 Figure 2 shows the results of Western blot detection of the phosphorylation level of P65 in mTECs cells overexpressing OAT2.

[0028] Figure 7 The bar graph shows the changes in the levels of P65 protein and α-SMA in mTECs cells overexpressing OAT2.

[0029] Figure 8Figure 2 shows the immunofluorescence detection results of vimentin in mTECs cells overexpressing OAT2.

[0030] Figure 9 The figure shows the results of immunofluorescence detection of α-SMA in mTECs cells overexpressing OAT2.

[0031] Figure 10 This is a bar graph showing the changes in mRNA expression levels of pro-inflammatory cytokines in the kidney tissue of a hyperuricemia mouse model treated with allopurinol and different concentrations of quercetin.

[0032] Figure 11 This is the result of transcriptome sequencing analysis of kidney tissue in a hyperuricemia mouse model.

[0033] Figure 12 This is a bar graph showing the changes in the expression levels of Slc22a7 gene and OAT2 in the kidney tissue of the hyperuricemia mouse model.

[0034] Figure 13 This is the qPRC identification result of OAT2 knockdown expression.

[0035] Figure 14 The bar graph shows the changes in mRNA expression levels of proinflammatory cytokines in AAV9-mediated renal tubule-targeted OAT2-overexpressing cells.

[0036] Figure 15 The figure shows the immunofluorescence staining results of the restoration of HNF1A expression level in mTECs cells after quercetin treatment.

[0037] Figure 16 This is a bar graph showing the changes in OAT2 mRNA expression in HNF1A-overexpressing mTECs cells.

[0038] Figure 17 This is an agarose gel electrophoresis diagram showing the binding relationship between the OAT2 gene fragment and the HNF1A protein.

[0039] Figure 18 Schematic diagram of the interaction between HNF1A and quercetin molecules.

[0040] Figure 19 This is a bar graph showing the changes in mRNA expression levels of pro-inflammatory cytokines in the kidney tissue of the spontaneous hyperuricemia mouse model under quercetin treatment.

[0041] Figure 20 The agarose gel electrophoresis diagram of the phosphorylation level of P65 in the kidney tissue of the spontaneous hyperuricemia mouse model under quercetin treatment.

[0042] Figure 21This is a bar graph showing the changes in P65 and α-SMA levels in the kidney tissue of a spontaneous hyperuricemia mouse model treated with quercetin.

[0043] Figure 22 Figure 2 shows the immunofluorescence detection results of α-SMA and vimentin in the kidney tissue of a spontaneous hyperuricemia mouse model treated with quercetin. DETAILED DESCRIPTION

[0044] The following is further detailed by a specific embodiment: In the specific examples, the raw materials and reagents used can be purchased from the market, and mouse renal tubular epithelial cells were kindly provided by Professor Lan Huiyao of the Chinese University of Hong Kong. Male C57BL / 6J mice (19-22 g) aged 6-8 weeks were provided by the Experimental Animal Center of Anhui Medical University. The mice were raised in a specific pathogen-free facility, and the environmental conditions were controlled at 40-70% humidity, 20-25°C temperature, 12-hour light / dark cycle, and free access to food and water. After the treatment, the animals were sacrificed by intraperitoneal injection of sodium pentobarbital (200 mg / kg) and the kidneys were collected. All operations were performed in accordance with the protocol approved by the Animal Ethics Committee of Anhui Medical University (LLSC20241881).

[0045] Example 1

[0046] The drug quercetin and OAT2 overexpression improve uric acid-induced mTECs cell inflammation and fibrosis.

[0047] In this example, mTECs were cultured in DMEM / F-12 medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. To establish an in vitro hyperuricemia model, mTECs were treated with uric acid. Specifically, the cells were first treated with different concentrations of quercetin (4, 8, and 16 μM) for 1 hour and then incubated with 15 mg / dL of uric acid for 24 hours. After treatment, the cells were harvested for subsequent testing.

[0048] Cell viability assay: Cells in the logarithmic growth phase were taken and the mTEC suspension was diluted with culture medium to the desired inoculation concentration. After 12 hours of incubation in an incubator, the culture medium was discarded and fresh culture medium containing varying concentrations of quercetin (1, 2, 4, 8, 16, 32, and 64 μM) was added. After 24 hours of drug stimulation, the culture medium was removed from each well and 20 μL of CCK-8 solution was added. The cells were incubated for an additional 1 to 4 hours. The absorbance of each well was measured at 450 nm using a microplate reader and the results were calculated according to the formula.

[0049] qPCR: Total RNA was extracted from cells using Trizol reagent. qPCR was performed using SYBR Green Supermix on a CFX96 Real-Time PCR Detection System.

[0050] Western blot analysis: Total protein was extracted from cultured cells using RIPA buffer. The extracted proteins were transferred to a PVDF membrane and blocked with 5% nonfat dry milk in TBST. After blocking, the membrane was washed three times with TBST and incubated with the primary antibody overnight at 4°C. The next day, the membrane was washed three times again and incubated with the secondary antibody for 1.5 hours at room temperature. Images were acquired using a Bio-Rad infrared imaging system, and the results were quantified using ImageJ software.

[0051] Immunofluorescence assay: Cells were seeded on glass slides in 24-well plates. mTECs were fixed with 4% paraformaldehyde, blocked with 5% bovine serum albumin, and then incubated with primary antibodies overnight at 4°C. After incubation with the corresponding secondary antibody for 1 hour, cell nuclei were counterstained with DAPI and observed and images were acquired under a fluorescence microscope.

[0052] The experimental results of this example show that: we used the CCK-8 assay to preliminarily quantitatively evaluate the effect of different concentrations of quercetin on the viability of mTECs. The results showed that at a concentration of 32 μM, quercetin significantly inhibited cell viability. Based on these findings, three concentration gradients of 4 μM, 8 μM and 16 μM were subsequently selected for further experimental studies. In the uric acid uptake experiment, it was observed that 16 μM concentration of quercetin significantly reduced the intracellular uric acid level (such as Figure 1 In addition, qPCR technology was used to detect the mRNA levels of pro-inflammatory cytokines interleukin IL-1β, IL-6 and TNF-α. The results showed that quercetin significantly reduced the expression of these pro-inflammatory cytokines in a dose-dependent manner and effectively inhibited the uric acid-induced inflammatory response (such as Figure 2 ). Immunofluorescence and Western blot results showed that quercetin inhibited uric acid-induced P65 phosphorylation and nuclear translocation. In addition, the mRNA levels of fibrosis-related factors α-SMA, Collagen-I, and Fn were also evaluated. The results showed that with the increase of quercetin dose, the levels of these fibrosis indicators decreased accordingly (such as Figure 3 The results of immunofluorescence staining and western blot were consistent with those of qPCR, indicating that quercetin can alleviate the inflammatory and fibrotic responses of mTECs induced by uric acid.

[0053] The effects of OAT2 protein overexpression on mTECs cells treated in vitro were further studied. The results showed that overexpression of OAT2 could significantly reduce the pro-inflammatory factors (such as Figure 4 、 Figure 5 Western blot analysis showed that OAT2 overexpression significantly reduced the phosphorylation level of P65 (e.g. Figure 6 、 Figure 7Immunofluorescence assays showed that OAT2 overexpression could effectively block the nuclear translocation of P65, suggesting that it has an inhibitory effect on the NF-κB signaling pathway. In the fibrosis phenotype analysis, western blot and immunofluorescence assays found that OAT2 overexpression could significantly downregulate the expression of α-SMA and vimentin (e.g. Figure 8 、 Figure 9 ), confirming that OAT2 can effectively inhibit the uric acid-induced fibrosis of mTECs. In summary, OAT2 overexpression can significantly alleviate the uric acid-induced inflammatory response and fibrotic damage of renal tubular epithelial cells.

[0054] Example 2

[0055] The improving effect of quercetin in the drug on renal inflammation and fibrosis in a hyperuricemia mouse model and its mechanism of action.

[0056] In this example, 6-8 week old male C57BL / 6J mice (19-22 g) provided by the Experimental Animal Center of Anhui Medical University were randomly divided into 7 groups (n=6 / group) after one week of acclimatization: negative control (NC), quercetin (Qct), HN, HN + quercetin (Qct, 15 mg / kg), HN + quercetin (Qct, 30 mg / kg), HN + quercetin (Qct, 60 mg / kg), and positive control HN + allopurinol (AP, 10 mg / kg). HN was induced by daily oral administration of potassium oxonate (2100 mg / kg) + adenine (140 mg / kg) suspension (dissolved in 0.5% sodium carboxymethylcellulose, CMC-Na). The quercetin treatment group was given quercetin dissolved in CMC-Na, and the NC group was given an equal volume of CMC-Na. After 21 days, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (200 mg / kg) for endpoint sampling.

[0057] AAV9-mediated tubular-targeted OAT2 overexpression: 6-8 week-old male C57BL / 6J mice (19-22 g) provided by the Experimental Animal Center of Anhui Medical University were randomly divided into four groups after acclimation for 7 days. Tubule-targeted AAV9 vectors carrying the control and OAT2 transgenes controlled by the Ksp-cadherin promoter were injected via the tail vein. After induction of the HN model, mice were divided into AAV-control (Ctrl), AAV-OAT2, HN+AAV-control, and HN+AAV-OAT2 groups.

[0058] Biochemical markers: Blood samples were collected from mice to determine serum uric acid, creatinine, and blood urea nitrogen levels. Uric acid, creatinine, and blood urea nitrogen levels were determined according to the kit instructions.

[0059] Histopathological section staining: Mouse tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections according to conventional procedures. To assess histological damage, sections were dewaxed and rehydrated with graded ethanol, followed by HE, Masson's, and Sirius red staining.

[0060] RNA sequencing: Kidney samples of mice in the HN group (n=3) and HN+quercetin (Qct, 60 mg / kg) group (n=3) were sequenced and analyzed by Wuhan Fraser Genetics.

[0061] ChIP-PCR: Upon collection of mTECs, protein-DNA binding was fixed with 1% formaldehyde cross-linking, followed by sonication. A portion of the chromatin was extracted and purified (input), while another portion was incubated with an antibody against the target protein and enriched using magnetic beads. After cross-linking, the DNA was purified (immunoprecipitation group), while another portion of the chromatin was enriched with an IgG antibody (IgG group). OAT2 enrichment in these samples was assessed by qPCR.

[0062] Cell transfection: OAT2 siRNA (si-OAT2), OAT2 full-length expression plasmid (OE-OAT2) and HNF1A full-length expression plasmid (OE-HNF1A) provided by Genetix (China) were transfected by Lipofectamine TM 3000 were transfected into mTECs cells. 24 hours after transfection, the transfected cells were treated with urea for 48 hours, and finally mTECs were collected for subsequent experiments.

[0063] Molecular docking: To explore the possible binding mechanism between quercetin and HNF1A, molecular docking was performed using Discovery Studio 2017R2. The three-dimensional structure of HNF1A was obtained from the Protein Data Bank (ID: 1F93).

[0064] Thermal migration assay: mTEC total protein was extracted and divided equally into two aliquots. One aliquot was incubated with DMSO, and the other with quercetin (16 μM) at room temperature for 2 hours. Each lysate was further divided into seven equal aliquots and heated at 37, 42, 47, 52, 57, 62, 67, and 72°C for 3 minutes, respectively. All protein samples were loaded onto SDS-PAGE gels for western blot analysis.

[0065] Drug affinity target stability: mTECs were lysed at low temperature using lysis buffer. The lysate was incubated with DMSO or quercetin (16 μM) at room temperature for 2 hours. The lysate was then treated with a gradient of pronase (0, 25, 50, and 125 ng) and digested at room temperature for 10 minutes. The reaction was terminated with a pre-chilled protease inhibitor cocktail and boiled at 100°C for 10 minutes. Samples were loaded onto SDS-PAGE gels for western blot analysis.

[0066] The experimental results of this embodiment show that: according to the experimental modeling scheme, blood and tissue samples of mice were collected. Monitoring of weight changes during modeling showed that quercetin and allopurinol can effectively promote weight recovery. Histological evaluation showed that at a dose of 60 mg / kg quercetin, there was no obvious tissue damage in the main organs of mice. Compared with the model group, quercetin and allopurinol significantly improved glomerular degeneration, renal interstitial inflammation, edema and vacuolar degeneration. Determination of serum uric acid levels showed a decrease with increasing quercetin dose. In addition, measurements of serum creatinine and blood urea nitrogen levels showed that quercetin and allopurinol help to improve renal damage. Quercetin and allopurinol significantly reduced the mRNA expression levels of inflammatory mediators (including IL-1β, IL-6 and TNFα) and the phosphorylation level of P65 protein (such as Figure 10 In summary, quercetin significantly reduced the inflammatory response in HN mice, and this effect was dose-dependent. Sirius red and Masson staining of mouse kidney tissue showed that quercetin and allopurinol significantly reduced uric acid-induced renal fibrosis. α-SMA, Col-1, and FnmRNA levels were significantly decreased after quercetin and allopurinol treatment. Quercetin treatment significantly reduced the expression of α-SMA and vimentin in the kidney tissue of HN mice. In summary, quercetin can alleviate hyperuricemia-induced renal damage and exhibit significant anti-fibrotic effects in a dose-dependent manner.

[0067] To further investigate the molecular mechanism of quercetin in treating HN, we performed transcriptome sequencing analysis on the kidney tissues of mice in the model group and the high-dose quercetin treatment group. The sequencing results showed that 2592 genes showed significant expression changes under quercetin treatment, of which 1599 genes were upregulated and 993 genes were downregulated. Through analysis, we identified the top 30 genes with the most significant changes, among which the significant upregulation of the Slc22a7 gene attracted our special attention (e.g. Figure 11 ). The Slc22a7 gene encodes OAT2, the second member of the OAT organic anion transporter family to be discovered. Similar to OAT1 and OAT3, OAT2 plays a vital role in the active secretion of a variety of endogenous and exogenous substances, including urate. OAT2 dysregulation has been shown to increase the risk of hyperuricemia. Given that the Slc22a7 gene was significantly upregulated under the influence of quercetin, we hypothesized that OAT2 may be a key molecule for the therapeutic effects of quercetin. To verify this hypothesis, we used qPCR technology to measure the mRNA level of OAT2, and the results were consistent with the transcriptome sequencing results, showing that the OAT2 mRNA level was significantly increased after quercetin treatment (such as Figure 12In addition, western blot analysis and immunofluorescence staining confirmed the upregulation of OAT2 protein expression, and in vitro cell experiments also confirmed these results. In summary, we believe that OAT2 may be a potential target for quercetin in the treatment of HN.

[0068] Small interfering RNA technology was used to specifically knock down OAT2 in a cell model. We successfully established a cell model with reduced OAT2 expression and verified its knockdown efficiency using qPCR technology. The results showed that in OAT2 knockdown cells, even after quercetin treatment, the mRNA expression levels of proinflammatory factors IL-1β, IL-6, TNF-α and fibrosis-related factors α-SMA, CollagenI, and Fn were significantly higher than those in the non-knockdown control group (such as Figure 13 ), and the results of western blot and immunofluorescence staining were consistent. In cells with OAT2 knockdown, quercetin treatment no longer effectively inhibited P65 phosphorylation, α-SMA, and vimentin expression and localization. Our results suggest that quercetin may exert protective and repairing effects on renal injury in a manner dependent on OAT2 expression.

[0069] To investigate the direct relationship between OAT2 and HN, we used an adeno-associated virus expressing an AAV9 renal tubule-specific promoter to specifically overexpress OAT2 in the renal tubules of mice. The body weight of mice overexpressing OAT2 was significantly restored, and HE staining showed that the pathological changes in the kidneys of HN model mice, such as glomerulosclerosis, interstitial inflammation, edema, and vacuolar degeneration, were significantly alleviated after overexpression of OAT2. OAT2 overexpression significantly reduced serum uric acid, creatinine, and blood urea nitrogen levels. In addition, the mRNA levels of pro-inflammatory cytokines IL-β, IL-6, and TNF-α were also significantly reduced (e.g., Figure 14 ). Further experiments revealed that OAT2 overexpression inhibited the phosphorylation and activation of P65. Next, we investigated the role of OAT2 overexpression in hyperuricemia-induced renal fibrosis. The results showed that OAT2 overexpression had an inhibitory effect on renal fibrosis. These results demonstrate that OAT2 overexpression significantly reduces renal inflammation and fibrosis in hyperuricemia-induced HN mice.

[0070] Considering that quercetin treatment increased the expression of OAT2, the specific regulatory mechanism is not very clear. We used the knocktf, chip atlas, and gtrd databases to predict transcription factors that may regulate OAT2. The intersection of the prediction results of the three databases showed HNF1A and Crebbp, and then we verified the levels of these two genes in the HN model. qPCR results showed that the HNF1A mRNA level in the quercetin-treated group was significantly higher than that in the control model group, while there was no significant difference in the Crebbp mRNA level. We detected the expression of HNF1A in tissues by immunohistochemical analysis, and the results showed that HN treatment significantly reduced the expression of HNF1A, while quercetin treatment reversed this inhibition and restored the expression level of HNF1A (as shown in Figure 2). Figure 15 ), and the subsequent mTECs immunofluorescence staining results further confirmed the restorative effect of quercetin on HNF1A expression.

[0071] To further investigate the potential link between HNF1A and OAT2, we determined whether HNF1A directly regulates OAT2 activity at the transcriptional level. First, we constructed an HNF1A overexpression plasmid and successfully overexpressed HNF1A in mTECs. We found that OAT2 expression was also increased in the HNF1A overexpression group (e.g., Figure 16 ). The JASPAR database was searched to predict the complementary binding sequence of the HNF1A targeting motif in the promoter region of the OAT2 gene. The predicted binding sequence "5′-TCCCCTTGATCTTG-3′" (score 12.273825) is the targeting motif of HNF1A in the OAT2 gene. We performed ChIP-PCR analysis on mTECs, and the results showed that the OAT2 gene fragment was enriched on the HNF1A protein, and the degree of enrichment was proportional to the expression level of HNF1A. This was further supported by the subsequent agarose gel images (e.g. Figure 17 ). These findings demonstrate the transcriptional regulatory function of HNF1A on OAT2. In addition, it was found that quercetin significantly upregulated the expression of HNF1A in uric acid-stimulated mTECs. To explore the mechanism of altered HNF1A expression, we performed molecular docking, and the results showed that the carbonyl O atom of the C ring and the 4′-position O atom of the B ring of quercetin formed hydrogen bonds with HNF1A amino acid residues Gln98 and Ala15, respectively; the O atom at the 5-position of the A ring and the O atoms at the 3′ and 4′-positions of the B ring of quercetin formed carbon-hydrogen interactions with HNF1A amino acid residues Gly31 and Arg52, respectively; the B and C rings of quercetin also participated in the π-σ and π-π interactions of the HNF1A amino acid residue Phe95, which suggests that HNF1A may be a direct target of quercetin (such as Figure 18Cell-based thermal shift assays (CETSA) further confirmed that quercetin treatment significantly increased the thermal stability of HNF1A. Drug target experiments also showed that increasing the concentration of pronase enhanced quercetin's resistance to pronase digestion. Together, these results further confirm that HNF1A is a direct target of quercetin and suggest that quercetin improves renal function in the HN model by activating the HNF1A-OAT2 transcriptional pathway.

[0072] Example 3

[0073] Quercetin in the drug improves renal inflammation and fibrosis in primary hyperuricemia mice (GLUT9- / - mice).

[0074] The primary hyperuricemia mice used in this example are GLUT9 systemic knockout (KO) mice. The plasma uric acid level in primary hyperuricemia mice is significantly increased and early-onset nephropathy occurs. Kidney structural abnormalities can be detected as early as 2 weeks of age, confirming its suitability for the study of hyperuricemia-related nephropathy. We used GLUT9KO mice (C57BL / 6 background) constructed by Saiye Bio (Suzhou) and identified their genotypes by agarose gel electrophoresis. Mice were randomly divided into four groups: WT, WT+quercetin (Qct), KO, and KO+quercetin (Qct).

[0075] The experimental results of this example show that a primary hyperuricemia mouse model was successfully established using gene knockout technology, and the experimental design process is described in detail. The observation results showed that the untreated homozygous mice gained the least weight, while the weight gain of the homozygous mice treated with quercetin was significantly restored. The renal pathological changes in untreated homozygous mice, such as glomerulosclerosis, interstitial inflammation, edema and vacuolar degeneration, were significantly alleviated after quercetin treatment. Quercetin treatment significantly reduced serum uric acid, creatinine and blood urea nitrogen levels. At the same time, the mRNA expression levels of inflammatory factors IL-β, IL-6 and TNF-α were also significantly reduced (such as Figure 19 ), the phosphorylation of P65 was significantly inhibited (e.g. Figure 20 ). Taken together, these results suggest that quercetin may play an important role in the treatment of HN by regulating inflammatory responses and improving renal function. There was obvious collagen deposition in the kidney tissue of homozygous mice, which tended to normalize after quercetin treatment. qPCR detection results showed that quercetin treatment significantly reduced the expression levels of α-SMA, Collagen-I and Fn mRNA, which was consistent with the changes in α-SMA protein expression detected by western blot (such as Figure 21 Finally, immunofluorescence staining of mouse kidney tissue showed that the expression of α-SMA and vimentin in the kidney tissue of HN mice was significant, and showed a downward trend after quercetin treatment (e.g. Figure 22 ).

[0076] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0077] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Application of quercetin in the preparation of drugs for improving renal damage caused by hyperuricemia.

2. The use according to claim 1, characterized in that: The drug is used to bind to hepatocyte nuclear factor-alpha.

3. The use according to claim 2, characterized in that: The amino acid residues Gln98 and Ala15 of hepatocyte nuclear factor-α form hydrogen bonds with the drug's carbocyclic carbonyl O atom and the 4′ oxygen atom of the B ring, respectively; Amino acid residues Gly31 and Arg52 of hepatocyte nuclear factor-α form carbon-hydrogen interactions with the oxygen atom at position 5 of the A ring and the oxygen atoms at positions 3′ and 4′ of the B ring, respectively; The B and C rings are involved in π-σ and π-π interactions with amino acid residue Phe95 of hepatocyte nuclear factor-α.

4. The use according to claim 3, characterized in that: Hepatocyte nuclear factor-α was used to enrich the gene fragment of the organic anion transporter OAT2.

5. The use according to claim 4, characterized in that: The binding sequence in the promoter region of the organic anion transporter OAT2 gene fragment targets and binds to hepatocyte nuclear factor-α.

6. The use according to claim 5, characterized in that: The organic anion transporter OAT2 is used to reduce the expression of proinflammatory cytokines, including interleukin IL-1β, IL-6, and tumor necrosis factor TNF-α; OAT2 is used to reduce the expression of fibrosis-related factors, including α-smooth muscle actin, type I collagen, and fibronectin; OAT2 is used to inhibit the phosphorylation and activation of NF-κB P65 protein; OAT2 is used to reduce the expression of vimentin.

7. The use according to claim 1, characterized in that: Hyperuricemia includes primary hyperuricemia and secondary hyperuricemia.

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