An integrated network pharmacology and RNA-seq approach for exploring the renal protection of quercetin on attenuating Ang II- induced cells apoptosis

doi:10.21203/rs.3.rs-2553212/v1

Download PDF

Article

An integrated network pharmacology and RNA-seq approach for exploring the renal protection of quercetin on attenuating Ang II- induced cells apoptosis

https://doi.org/10.21203/rs.3.rs-2553212/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Quercetin exerts antihypertensive effects, while its role on hypertensive renal injury remain unknown. Network pharmacology analysis identified multiple potential candidate targets (including TP53, Bcl-2 and BaX) and enriched signaling pathways (including apoptosis and p53 signaling pathway). Hematoxylin and eosin staining revealed that quercetin treatment reduced the pathological changes in renal tissues of Ang II infused mice. RNA sequencing identified quercetin treatment significantly reversed 464 DETs and enriched several signaling pathway (including apoptosis and p53 pathways). Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling staining and Annexin V staining revealed that quercetin treatment reduced cell apoptosis in renal tissues of Ang II-infused mice and in NRK-52E cells stimulated with Ang II. Furthermore, immunohistochemistry and western-blotting indicated that quercetin treatment alleviated the upregulation of p53, BaX, cleaved-caspase-9, and cleaved-caspase-3 protein expression and the downregulation of Bcl-2 protein expression in both renal tissue of Ang II infused mice and NRK-52E cells stimulated with Ang II stimulation. Moreover, the molecular docking results indicated potential binding activity between quercetin-TP53. Quercetin treatment significantly attenuated hypertensive renal injury and cell apoptosis in renal tissues of Ang II-induced mice and Ang II stimulated NERK-52E cell, and by targeting p53 may be one of the underlying mechanisms.

Biological sciences/Drug discovery/Pharmacology

Health sciences/Diseases/Cardiovascular diseases

Health sciences/Diseases/Kidney diseases

Biological sciences/Drug discovery

Health sciences/Diseases

Elevated blood pressure is a major cause of death, responsible for approximately 9.4 million deaths annually worldwide¹. It also contributes significantly to preclinical and clinical damage to target organs, including the renal, and the damage usually manifests as other cardiovascular disease and impaired renal function². Renal is one of the most important organs implied in the development of hypertension, and play most important role in the maintaining of blood pressure and renal function³. In spite of advances in understanding the pathogenesis of hypertensive nephropathy, the current diagnostic and therapeutic approaches are still not effective in reducing hypertensive renal dysfunction⁴. Therefore, this situation raised an urgent request to identify novel treatment approaches for preventing hypertensive renal damage.

Hypertension is a key pathogenic factor that contributes to the progression of renal failure⁵. Chronic hypertension causes renal damage, including tubular interstitial fibrosis, vasculature, and glomerular sclerosis⁶. The hypertensive renal injury has been implicated due to several factors, including oxidation stress, stress of endoplasmic recticulum, dysfunction of mitochondria, inflammation and renin-angiotensin systems (RASS) activities^7–10. Among them, abnormal renin angiotensin aldosterone system (RAAS) activation is an important set of processes that modulates numerous pathophysiological and physiological processes in renal disease^11–13.

The RAAS is found in many organs and tissues, including cardiac muscle cells, vascular smooth muscle, and kidneys, and it plays a role in controlling specific organ activities¹⁴. With increase of renin levels rise in the blood, angiotensinogen (Ang) is produced in the liver and is broken down to angiotensin I (Ang I), which further stimulates the secretion of epinephrine and norepinephrine¹⁵. In lungs, Ang I is transformed into Ang II with the help of angiotensin converting enzymes¹⁶. Ang II is well known and most essential mediating partner of the RAAS¹⁷. Several researches shown that the intrarenal renin-angiotensin system (RAS) exhibits a meaningful influence on tubular cell proliferation, apoptosis, and renaissance the renal damage^18–21. Due to the essential role of tubular cell apoptosis contributing renal function, the inhibition of RAAS is an important approach to attenuate hypertension induced renal cell apoptosis²².

Flavonoids are considered potentially useful substances for the treatment and prevention for a number of diseases, including cardiovascular disease^23–24. Quercetin is an organic and significant flavonoid substance found in both fruits and vegetables²⁵. Numerous researches have indicated that quercetin is effective in the treatment of cardiovascular diseases, like hypertension^26–30. Moreover, studies suggest that quercetin has also been reported to have a therapeutic effect on renal function and tissue damage^31–32. However, the possible function of quercetin in hypertensive renal damage and its underling mechanism remained unclear. For the exploration of protective underlying potential targets and molecular mechanisms antihypertensive role of quercetin, we investigated its effect on Ang II- infused mice and stimulated NRK-52E cells, as well as an integrated network pharmacology and RNA sequencing, aiming to develop a novel therapeutic approach for the prevention and treatment of hypertension using quercetin.

Materials. Dulbecco’s Modified Eagle’s Medium (DMEM) (Cat No. C11995500BT), trypsin–ethylenediaminetetraacetic acid (EDTA) (Cat No. 25200072), fetal bovine serum (FBS) (Cat No. 10091148), and bicinchoninic acid (BCA) (Cat No. 23227) protein assay were obtained from Thermo Fisher Scientific (Waltham, MA, USA). TUNEL Apoptosis Detection Kit I POD (Cat No. MK1025) was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA). Antigen repair solution (Cat No. MVS-0066) and UltraSensitive™ SP (mouse/rabbit) immunohistochemistry (IHC) (Cat No. KIT-9720) and DAB (Cat No. DAB-0031) kits were purchased from Maixin Biotechnology (Fuzhou, Fujian, China). Annexin V- AbFluorTM647 Apoptosis Detection Kit (Cat No. KTA0004), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat No. Abp57259) antibody, and Cell Counting Kit-8 (CCK8) (Cat No. KTA1020) were purchased from Abbkine (Wuhan, Hubei, China). Polyvinylidene fluoride (PVDF) membranes (Cat No. IPVH00010) was obtained Millipore (Billireca, MA, USA). Antibodies against p53 (Cat No. 10442-1-AP) and Bcl-2 (Cat No. 12789-1-AP) were acquired Proteintech (Rosemont, IL, USA). Antibodies against BaX (Cat No. 2772), cleaved-caspase-3 (Cat No. 9662), and cleaved-caspase-9 (Cat No. 9508) and secondary antibodies anti-mouse (Cat No. 7076) and anti-rabbit (Cat No. 7074) were obtained from Cell Signaling Technology (CST; Danvers, MA, USA). Angiotensin II (Ang II) (Cat No. ab120183) was acquired Abcam (Cambridge Science Park, Cambridge, UK). Quercetin (Cat No. Q4951-10G) was purchased from Sigma (St. Louis, MO, USA) with > 95% purity (HPLC).

Retrieval of quercetin and hypertensive renal injury targets. Hypertensive renal injury related targets were collected from the Genecards (https://www.GeneCards.org/) database³³. Further, DisGeNET (https://www.disgenet.org/) database³⁴ was employed for the extraction of targets via “kidney injury (KI) terms”. Quercetin targets were collected using Traditional Chinese Medicine Systems Pharmacology database (TCMSP, https://old.tcmsp-e.com/tcmsp.php)³⁵ and Traditional Chinese Medicine database (TCMIP, http://www.tcmip.cn/TCMIP/index.php/)³⁶.

Construction and analysis Protein-Protein Interactions (PPIs). To identify the potential hub targets of common genes between quercetin and hypertensive renal injury, the selected targets were introduced into the search tool for the retrieval of interacting genes (STRING) network platform (https://cn.string-db.org/, Version 11.5), followed by construction the PPI network. The interaction network of “quercetin- hypertensive renal injury -target” was further processed to realize visualization and screen out the core targets by using the software of Cytoscape (Version 3.8.2).

Functional annotation and pathways enrichment analyses. Based on the common genes, database of Database for Annotation Visualization and Integration Discovery (DAVID) was used to identify the enriched functional processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways^37–38.

Molecular docking. As one of the most enriched apoptosis-associated target, TP53 was selected as candidate potential target of quercetin, which was performed by molecular docking. The 3D protein structures of TP53 (PDB: 8F2I)³⁹ was retrieved from the protein databank (PDB)⁴⁰. Moreover, pocket sites of TP53 was identified by implementing Prankweb online server. The 2D structures of quercetin was retrieved from the PubChem database⁴¹. Then, the energy of quercetin structures was minimized by using the Chem3D program (Version 20.0.0.41). Quercetin and protein structure were optimized and converted into PDBQT format for docking using AutoDockTools (Version 1.5.7), and the interaction between proteins and quercetin were visualized by PyMOL program (Version 2.5.4). The 2D- interacting residues of proteins to quercetin molecule were obtain by using Proteins Plus online server.

Animal experiments. Animal welfare and experimental protocols were performed in accordance with the ARRIVE guidelines and were applied in strict accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. The animal experimental protocol of this research was approved by the Animal Use Committee of the Fujian University of Traditional Chinese Medicine (Fuzhou, Fujian, China; Ethical code: FJTCM IACUC 2022112). Male C57BL/6 mice (weight, 28 ± 1 g; age, 8 weeks) were obtained from SLAC Laboratory Animal Technology Co., Ltd. (Shanghai, China). All animals were subjected to a 12 h light dark cycle under specific pathogen free conditions at constant temperature (24 ± 2°C) and relative humidity (50–60%). The mice were randomly divided into four groups (n = 6 each group): Control, Ang II, Ang II + Quercetin-L, and Ang II + Quercetin-H groups. The mice in the Ang II, Ang II + Quercetin-L, and Ang II + Quercetin-H groups were infused with Ang II (500 ng/kg/min), and those in the control group were infused with saline for 4 weeks by subcutaneously implanting micro osmotic pumps. The mice in the Ang II + Quercetin-L and Ang II + Quercetin-H groups intragastrically administrated with quercetin daily (2.5 mg/kg/day and 5 mg/kg/day, respectively; 200 µL for each mouse), and those in the control and Ang II groups were intragastrically administrated with equal volumes of double distilled water for 4 weeks.

hmatoxylin-eosin (H&E) staining. The collected renal tissues were fixed at room temperature for 48 h using a 4% paraformaldehyde solution, followed by dehydrated via an alcohol gradient and embedded into paraffin. Then tissues were sectioned into 4-µm thick slices and stained with hematoxylin for 1 min and eosin for 2 sec at room temperature. The stained sliced tissues were imaged with optical microscope using 400x magnification (Leica DM4000B, Wetzlar, Germany).

RNA sequencing. The collected renal tissues were stored in RNA later® (Takara, Beijing, China) for 1 h at room temperature and then was stored at -20°C for prolonged time. RNA sequencing was performed by the Capital Bio Technology (Beijing, China). Briefly, the NEBNext Ultra RNA Library Prep Kit for Illumina was used to prepare a sequencing library (NEB, Ipswich, MA, USA) as per the guidelines of according to the manufacturer, and oligo(dT) beads were used to purify mRNA from total RNA. Total RNA concentration and quality were determined using the Qubit 3.0 and Agilent 2100. Bioanalyzers were used for the determination of quantity and quality of extracted total RNA. Then, library was appropriately quantified using the KAPA quantitative kit (KAPA Biosystem, Wilmington, MA, USA). As stated in earlier studies, raw data were processed and differential expressed transcripts were identified⁴². Moreover, analyses of gene ontology (GO) processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed to identify enriched functional processes and pathways.

Immunohistochemical (IHC) analysis. IHC was used to detect the protein expression of p53, BaX, Bcl-2, cleaved-caspase-3, and cleaved-caspase-9 in renal tissues of mice from each group. Briefly, the renal tissues were cut into 4-µm sections. After rehydration with gradient ethanol, the tissues were treated with a 0.01-M citrate-based antigen retrieval solution for 10 min at a high temperature, and then incubated with the primary antibodies for p53 (1:200), BaX (1:100), Bcl-2 (1:200), cleaved caspase-3 (1:200), or cleaved caspase-9 (1:200) at 4°C overnight. After washed with PBS, slides were incubated with secondary antibody coated with biotinylated beads, and the conjugated with streptavidin (horseradish peroxidase-labeled). These slides were stained with diaminobenzidine as chromogen and suspended with diluted Harris hematoxylin. The images were taken using optical microscope (Leica DM4000B, Wetzlar, Germany) at a magnification of 400x.

Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The renal tissues were sliced into 4-µm thick sections and staining was performed via TUNEL kit, as directed by the producer. Briefly, after that sections were deparaffinized and were incubated with Proteinase K for 15 min at 37°C, and then washed with 1× Tris-buffered saline (TBS) for three times. Slides were coated with Labeling Reaction Mix at 37°C for 2 h, then rinsed thrice with 1× TBS and incubated with block solution, followed by incubation of diaminobenzidine at room temperature for 30 min and diluted Harris hematoxylin. The images were taken under an optical microscope (Leica, Wetzlar, Germany) at 400x magnification. Image J was used to identify the percentage of positive area relative to the total area.

Cell culture and quercetin treatment. NRK-52E cells were obtained from BeNa (Beijing, China). And was seeded into DMEM contains 10% FBS, 100-µg/mL streptomycin, and 100-units/mL penicillin at 37°C with 5% CO₂ (humified air). NRK-52E cells was stimulated with Ang II (1µM) and were treated with or without quercetin (20 µM and 40 µM) for 48 h. The cells in the control group were treated with an equal volume of dimethyl sulfoxide for 48 h.

Cell viability analysis. The NRK-52E cells were seeded in 96-well plates in suspension (100 µL) at a density of 2 × 10⁴ cells/mL overnight at 37°C and 5% CO₂, they were incubated with indicated concentrations of quercetin for 48 h. At the end of treatment, CCK-8 reagent (10 µL) was supplemented into each well and incubated in dark for additional 2 h at 37°C. A microplate reader (Multiskan FC; Thermo Fisher Scientific) was used to observe absorbance at 450 nm. The viability of untreated cells was considered at 100%.

Annexin V staining. Annexin V apoptosis kit with propidium iodide (PI) was used to identify cellular apoptosis. The NRK-52E cells (8 × 10⁴ cells/well) were seeded into six-well plates and incubated for 24 h, and then starved with DMEM serum free media for 12 h and were then treated with Ang II (1 µM) and without or with quercetin (20 µM or 40 µM) in minimal medium (0.5% FBS) for 24 h. After treatment, cells were washed using PBS and collected with trypsin (without EDTA). The centrifuged supernatant was discarded and pellet propagated in 1x binding buffer (5 µL annexin V-fluorescein isothiocyanate and 2 µL PI) and then incubated for 15 min. Fluorescence-activated cell sorting (BD, Franklin Lakes, NJ, USA) analysis was used to identify and quantify cell apoptosis.

Western blotting. The collected cells were lysed with cell lysis buffer contains protease inhibitor and phenylmethylsulfonyl fluoride (PMSF) on ice for 30 min. The lysed cells were centrifuged at 14,000 g for 20 min at 4°C.The protein concentration was determined using BCA kit. The total 50 µg of protein from each group was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, and were blocked using 5% non-fat milk in TBST (TBS + 0.1% Tween) at 37ºC for 2 h. Furthermore, the membranes were incubated with primary antibodies, including rabbit anti-BaX (1:1000), Bcl-2 (1:1000), p53 (1:1000), cleaved-caspase-3 (1:1000), GAPDH (1:5000), or mouse anti- cleaved-caspase-9 (1:1000) 4ºC overnight, followed by incubation of secondary anti-mouse/rabbit IgG (1:5000) for 2 h at room temperature and determination of protein expression by advanced chemiluminescence detection ChemiDoc XRS and Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Image J was used to calculate the optical density of each protein band.

Statistical analysis. The results were presented as the mean value ± standard deviation (SD). One-way analysis of variance was used to compare the differences among three or more groups when the data were normally distributed, followed by Bonferroni analysis when the variance was chi-square, or Games Howell analysis when the variance was not chi-square. Statistical significance was assigned to differences with p values < 0.05. The Statistical Package for the Social Sciences, version 25.0 (IBM Corp., Chicago, IL, USA) was used for all statistical analyses.

Identification of potential targets and pathways of quercetin against hypertensive renal injury based on pharmacology network analysis. The intersection of targets from different databases was represented into Circos diagram. Gene searches were performed using these terms, KI, hypertensive renal injury and quercetin using TCMSP and TCMIP (Fig. 1A). The overlapping of gene analysis identified 31 common genes (Fig. 1B). Moreover, by performing STRING platform and Cytoscape (3.8.2) software, we constructed the PPI of common genes and identified multiple potential crucial genes, including TP53, Bcl-2, and BaX, etc (Fig. 1C).

The GO functional enrichment analysis of the 31 common genes was used to identify related biological processes, cellular components, and molecular functions using (P Value cutoff 0.05). In term of biological processes, several processes, including positive regulation of apoptotic process, response to drug, and neuron apoptotic process were significantly enriched (Fig. 1D). The analysis of cellular components also identified multiple enriched components, including Bcl-2 family protein complex, extracellular region, andclathrin-coated endocytic vesicle membrane (Fig. 1E). In addition, the category of molecular functions identified multiple functions, including cytokine activity and enzyme binding (Fig. 1F). Moreover, KEGG pathway enrichment analysis by the DAVID database (P Value cutoff 0.05) revealed that the key target genes had a close relationship with HIF-1 signaling pathway, PI3K-AKT signaling pathway, and p53 signaling pathway and so on (Fig. 1G).

Identification of DETs in the renal tissues of Ang II-induced mice using RNA sequencing. The current study further verified the therapeutic effect of quercetin on hypertensive renal injury based on network pharmacology, and H&E staining observed obvious glomerular atrophy, atrophy of epithelial cells, and tubular dilatation in the renal tissues of the Ang II infused mice, which were mitigated after quercetin treatment (Fig. 2A). Thus, this result suggests the renal protection of quercetin in Ang II infused mice, which encouraged us to further explore its potential underlying mechanisms.

To further investigate the underlying protective mechanism of quercetin against hypertensive renal injury, RNA-sequencing was used to recognize DETs in kidney tissues among different groups (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE207102). As compare to control 1,438 transcripts were up regulated significantly in Ang II-infused renal tissues of mice and 1,334 transcripts downregulated, as denoted in Fig. 2B and 2C (left panel), while quercetin treatment increased the expression of 1,247 transcripts and decreased the expression of 1,198 transcripts, especially compared to Ang II group, as shown in Fig. 2B and 2C (right panel). The integrated comparative analysis between two groups (Ang II vs. Control and Ang II + Quercetin vs. Ang II) revealed 143 downregulated transcripts (Fig. 2D, left panel) and 321 upregulated transcripts (Fig. 2D, right panel) in the Ang II group were reversed after quercetin treatment.

Functional processes and KEGG pathways enrichment analysis from the renal tissues of Ang II induced mice models. These DETs were categories into three groups; biological processes, cellular components and molecular functions. Figure 3A (left panel) showed the most enriched GO evaluation based on DETs between control and Ang II-induced groups. Further, these DETs were enriched into several cellular components into cytosolic ribosome, membrane-bound organelle, and ribosomal subunit. Moreover, DETs were enriched into numerous biological process associated with the cellular protein metabolic process, expression of genes, as well as translation. These DETs were substantially enriched into multiple molecular functions, including structural constituent of ribosome, binding, and structural molecule activity, and etc. However, the GO enrichment assessment compared DETs between the Ang II + quercetin and Ang II groups were performed and showed several significant functions. Cellular component analysis showed that DETs were significantly enriched in the membrane-bound organelle, intracellular organelle part, and organelle part, as shown in Fig. 3B (middle panel). Biological processes analysis indicated that DETs were associated with the cellular nitrogen compound metabolic process, biological adhesion, and cellular process. The molecular function analysis for these DETs included enzyme activator activity, binding, and nucleoside-triphosphatase regulator activity. As shown in Fig. 3C (right panel), the integrated comparative analysis between two (Ang II vs. Control and Ang II + Quercetin vs. Ang II) groups revealed that 67.08% of cellular components (including chromatin and Bcl-2 family protein complex), 65.47% of biological processes (including extrinsic apoptotic signaling pathway in absence of ligand and positive regulation of apoptotic process), and 61.7% of molecular functions (including damaged DNA binding and p53 binding) were enriched.

Several KEGG pathways were enriched from compared DETs of both control and Ang II groups into numerous signaling pathways, like the MAPK, TNF, and p53 signaling pathways and apoptosis (Fig. 3D). Moreover, the compared DETs of Ang II and Ang II + Quercetin groups were enriched into PI3K-AKT, MAPK, apoptosis and so on (Fig. 3E). Notably, in the comparative integrated analysis (Control vs. Ang II and Ang II vs. Ang II + Quercetin) showed that p53 signaling pathway as well as apoptosis were enriched (Fig. 3F), which motivated us to investigate the regulatory effects of quercetin on the apoptosis of cells and p53 pathway activation in the renal tissues of Ang II-induced mice.

Quercetin treatment reduces cell apoptosis in renal tissues of Ang II infused mice. TUNEL-stained cells indicated the increased percentage of positive cells in the renal tissues of Ang II-induced mice, (Fig. 4A and 4B), whereas a decrease in this percentage was observed after quercetin treatment (Fig. 4A and 4B), validating that quercetin reduces apoptosis in the renal tissues of Ang II-induced mice.

IHC analysis indicated that Ang II infusion downregulated anti-apoptotic protein Bcl-2 expression (Fig. 4C and 4D) and upregulated the pro-apoptotic BaX (Fig. 4E and 4F), cleaved-caspase-9 (Fig. 4G and 4H), and cleaved-caspase-3 (Fig. 4I and 4J) proteins expression, whereas, quercetin treatment were reversed the above proteins expression (Figure.4C-J).

Quercetin treatment inhibits apoptosis of Ang II-stimulated NRK-52E cells. In vitro study, we found that cells viability was not affected up to 60 µM concentration doses of quercetin (Supplementary Fig. 1). Therefore, 20 µM and 40 µM of quercetin were used for the following in vitro studies. Consistently, Annexin V-PI staining indicated that cell apoptosis was substantially increased with Ang II stimulation and reduced post quercetin treatment (Fig. 5A and 5B). Western blotting confirmed the pro-apoptotic protein levels of BaX, cleaved-caspase-9, and cleaved-caspase-3 were highly upregulated in Ang II-stimulated NRK-52E cells, whereas downregulation of the anti-apoptotic protein Bcl-2 expression was determined (Fig. 5C-F), which thus substantially increased the expression ratio of BaX/ Bcl-2 (Fig. 5D). As expected, quercetin treatment reversed these effects (Fig. 5C-F).

Quercetin exhibits target potential to p53 and reduces Ang II induced p53 pathway activation in vivo and in vitro. To further determine the mechanism of quercetin in reducing Ang II induced renal cell apoptosis. The comparative integrated analysis (KEGG pathways were enriched by RNA sequencing and by network pharmacology) showed that also p53 signaling pathway as well apoptosis were enriched (Fig. 6A). Moreover, the TP53 was identified as one of lead potential targets and was found abundantly enriched into most GO categories and pathways by network pharmacology. Therefore, TP53 was considered renal cells apoptosis potential target and docked with quercetin in order to provide a deeper insight into the binding interactions of the inhibitor (quercetin) at the active sites. It is generally believed that the lower the docking energy between ligands and proteins, the stronger their bond. The results analysis showed that the docking binding energy − 6.12 kcal/mol was determined between TP53 and quercetin (Fig. 6B), suggesting that quercetin might be one of the potential inhibitor molecules for renal cells apoptosis.

Moreover, IHC analysis indicated that p53 expression was substantially elevated in renal tissues of Ang II infused mice, but was downregulated after quercetin treatment (Fig. 6C and 6D). In vitro study showed that, western blotting confirmed the p53 protein expression was highly upregulated in Ang II-stimulated NRK-52E cells, whereas, quercetin treatment reversed these effects (Fig. 6E and 6F).

Chronic hypertension is severely associated with damages to target organs, including the kidneys⁴³. Several studies indicated the antihypertensive and multiple-target-organ protective effects of quercetin^44–49. However, the pharmacological effects of quercetin in hypertension induced renal damage is remained unknown. This study showed that quercetin treatment decreases apoptosis of renal cells, mediated several signaling pathways (such as p53 pathway) in Ang II-infused renal injury in both in vivo and in vitro studies.

Although several antihypertensive medicines have been widely used for treating hypertension, many patients with hypertension still ultimately suffer from hypertensive renal damage⁴. Thus, it is immediately required to investigate and progress effective approaches for treating hypertensive renal injury. Natural compounds may provide a chance for enriching the treatment of hypertensive target organ injury. Quercetin is a major flavonoid component found in several fruits, vegetables, red wine, and tea⁵⁰. Quercetin exerts anti-inflammatory and antihypertensive effects on patients with hypertension and on different models of hypertension^{25, 47, 51–54}. Furthermore, quercetin exhibits vascular and cardiac protection against hypertension^{44, 48–49}.

Network pharmacology integrates diseases and drugs into the biomolecular network, which focuses on the interactions between drugs and their targets and point out the way for the discovery of novel drug molecules. Our current study identified the overlapping gene analysis was performed and 31 common genes were found between quercetin and hypertensive renal injury targets. In addition, STRING database and Cytoscape software were used for analysis and obtained potential gene targets, including EGFR, XDH, TP53, BaX, THF, IL6, and Bcl-2 with different binding potential with quercetin, among them, TP53, BaX, TNF, IL6 and Bcl-2 have been reported to be involved in hypertension^55–57. GO and KEGG analysis showed that the therapeutic effects of quercetin on hypertensive renal injury involves multiple signaling pathways (including apoptosis and p53 signaling pathway), therefore, we investigated whether quercetin treatment employs its functional role on hypertensive renal injury via this pathway. However, other enriched signaling pathways, including PI3K/AKT signaling pathway, HIF-1 signaling pathway have been widely reported to play vital functional activities in renal injury disease^58–61, which needs to be further verified in our future study.

In addition, this study indicated that quercetin treatment meaningfully reduces Ang II-induced renal pathological changes, indicating that quercetin prevents the injury of target organs (such as renal damage). However, renal damage must be studied at advance level via determining renal failure (including proteinuria expression) or using other animal models of hypertension. High-resolution, high-throughput analysis of the data may reveal the unique molecular mechanism of the disease and the therapeutic target⁵². RNA-sequencing was to analyze the expression of DETs, followed by GO and KEGG enrichment to identify the complex molecular mechanisms of quercetin in curing Ang II-induced renal injury. DETs analysis revealed upregulated 321 and downregulated 143 transcripts in the kidney tissue of Ang II mice were reversed after quercetin treatment. Among them, HIF-1α and MDM2 have been described to perform a vital role in renal injury^53–54, these may be targets of quercetin to reduce hypertensive renal injury. However, these identified genes have closely linked to hypertensive renal injury must be explored in our subsequent scientific work.

The predicted target genes and their functional analysis help to reveal the detailed mechanism of quercetin in treating Ang II-induced renal injury. Thus, based on the DETs in the comparisons between the Ang II and control groups and between the Ang II + Quercetin and Ang II groups, we performed GO analysis involving several biological processes, such as Bcl-2 family protein complex, positive regulation of the apoptotic process, transporter activity, and damaged DNA binding, which may play a role in hypertensive renal injury. Through enrichment analysis of the KEGG pathways, we found that multiple signaling pathways, such as the necrosis factor-kappa B (NF-κB) and PI3K-AKT signaling pathways, were meaningfully enriched in both compared groups. Furthermore, integrated analysis between the two compared both groups were enriched into apoptosis and the p53 signaling pathway. Hence, we investigated that quercetin may exerts its purposeful effect on hypertensive renal injury through this pathway.

Tubular cell apoptosis, which ultimately leads to loss of tubular mass and renal atrophy, is an important manifestation of the progression of tubulointerstitial renal fibrosis⁶². As constant with an earlier study, Ang II substantially induced cells to undergo renal tissue apoptosis and NRK-52E cells²², which were reduced post quercetin treatment, suggesting the potential role of quercetin in reducing renal injury. However, the underlying mechanisms remain unclear. The role of quercetin in fibrosis should also be studied in forthcoming reports.

p53 is a transcription factor and performs a vital function in the control of cell apoptosis⁶³. Many reports indicated the inhibition of p53 reduces kidney injury in vivo^64–67. Elevated p53 levels can induce apoptosis by either effectively triggering the pro-apoptotic protein BaX or reducing the anti-apoptotic Bcl-2 protein^68–69. The protein family of Bcl-2 is immersed in mitochondrial apoptosis, which contains the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein BaX⁷⁰. In NRK-52E cells Ang II causes BaX to be upregulated while Bcl-2 is downregulated²². Caspase, a cytoplasmic cysteine aspartic protease, is a primary determinant in the mechanism of cellular apoptosis through preferential cleavage and activation of caspase-9 and caspase-3⁷¹. Consistently, we studied that quercetin substantially reduced the Ang II-induced BaX, cleaved-caspase-9, and cleaved-caspase-3 and Bcl-2 down regulation.

Molecular docking is the most frequently employed strategy for evaluating protein-ligand interactions, and we employed the AutoDockTools software to scrutinize the probable binding affinity. Finally, molecular docking analysis revealed the direct quercetin targets, including TP53, which was apoptosis related genes. These results showed that quercetin has a best activity, and it can dock well with renal injury targets, which is consistent with the results of previous study showing that quercetin is an effective drug for the treatment of renal injury⁷². Consistently, we studied that quercetin substantially reduced the expression of p53 protein in Ang II infused mice and Ang II-stimulated NRK-52E cells. Such studies showed that suppressing the Ang II-induced activation of the p53 signaling pathway, it might be one of the underlying mechanisms of quercetin in reducing apoptosis cells in renal hypertension. However, the use of the inhibitors of the p53 pathway or p53 transgenic mice used for the validation of anti-apoptotic role of quercetin in upcoming scientific work. Moreover, blocking the activity of quercetin affects the direct interaction between quercetin and p53 must be evaluated in future studies. In addition, the mechanism of quercetin in regulating other signaling pathways and DETs must be examined at next level.

Quercetin treatment substantially attenuates renal tubular epithelial apoptosis cells in vivo and in vitro, by targeting p53 may be considered as a potential mechanism that could provide a novel treatment initiative for protection of renal injury induced hypertension.

Ang II, angiotensin II; BP, biological process; BaX, Bcl2-Associated X; BCA, bicinchoninic acid; CC, cellular components; CCK8, cell counting kit-8; DETs, expressed transcripts; dd H₂O, double distilled water; DMEM, dulbecco's modified eagle's medium; DBP, diastolic blood pressure; ECL, electrochemiluminescence; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GO, gene ontology; H&E, hmatoxylin-eosin; KEGG, Kyoto Encyclopedia of Genes and Genomes; KI, kidney injury; MAP, mean arterial pressure; MF, molecular function; PVDF, polyvinylidene fluoride; RASS, renin-angiotensin systems; RAAS, renin‑angiotensin‑aldosterone system; RIPA, radioimmunoprecipitation assay; RNA-seq, RNA sequencing; RTEC, renal tubular epithelial cells ; SBP, systolic blood pressure; SDS-PAGE , sodium dodecyl sulfate polyacrylamide gel electrophoresis ; TUNEL, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling.

Data Accessibility of Sequence Data

The RNA sequencing data used in present study are deposited at NCBI Gene Expression Omnibus (GEO) under the accession numbers GSE207102; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE207102.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

AS and JP conceived and designed the experiments. JL, YL, SH, LL, and XZ performed the animal experiments. YW, YL, and JL conducted H&E staining. JL, MW, and FA conducted TUNEL staining. XZ, QD, LL, and YW performed cell culture. XZ, JW, and SH performed Annexin V staining. XZ and SH conducted Western blotting. XZ, JW, QD, FA, and MW analyzed the data. XZ and CF drafted the manuscript, which was revised by AS and JP. All authors provided critical contributions to the manuscript and approved the submitted version of the manuscript.

Acknowledgements

This study was sponsored by the National Natural Science Foundation of China (82074363; U22A20372), the Science and Technology Major Project of Fujian Province (2019YZ014004), the Young Elite Scientists Sponsorship Program by China Association of Chinese Medicine (2021-QNRC2-B19), the Scientific Research Foundation for the Top Youth Talents of Fujian University of Traditional Chinese Medicine (XQB202202), This study was sponsored by the Development Fund of Chen Keji Integrative Medicine (CKJ2020003).

Lim, S. S. et al. A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2224–2260. https://doi.org/10.1016/S0140-6736(12)61766-8 (2012).
Mensah, G. A. Hypertension and target organ damage: Don’t believe everything you think. Ethn. Dis. 26, 275–278. https://doi.org/10.18865/ed.26.3.275 (2016).
Ruiz-Hurtado, G. & Ruilope, L. M. Microvascular injury and the kidney in hypertension. Hipertens. Riesgo. Vasc. 35, 24–29. https://doi.org/10.1016/j.hipert.2017.03.002 (2018).
Dong, Z. et al. Mechanism of herbal medicine on hypertensive nephropathy (review). Mol. Med. Rep. 23, 234. https://doi.org/10.3892/mmr.2021.11873 (2021).
Bakris, G. L. & Ritz, E. The message for world kidney day 2009: hypertension and kidney disease: a marriage that should be prevented. J. Hypertens. 27, 666–669. https://doi.org/10.1097/HJH.0b013e328327706a (2009).
Mennuni, S. et al. Hypertension and kidneys: unraveling complex molecular mechanisms underlying hypertensive renal damage. J. Hum. Hypertens. 28, 74–79. https://doi.org/10.1038/jhh.2013.55 (2014).
Linkermann, A. et al. Regulated cell death in AKI. J. Am. Soc. Nephrol. 25, 2689–2701. https://doi.org/10.1681/ASN.2014030262 (2014).
Padda, R. S., Shi, Y., Lo, C. S., Zhang, S. L. & Chan, J. S. D. Angiotensin-(1–7): A novel peptide to treat hypertension and nephropathy in diabetes? J. Diabetes Metab. 6, 2155–2156. https://doi.org/10.4172/2155-6156.1000615 (2015).
Gai, Z., Gui, T., Hiller, C. & Kullak-Ublick, G. A. Farnesoid X receptor protects against kidney injury in uninephrectomized obese mice. J. Biol. Chem. 291, 2397–2411. https://doi.org/10.1074/jbc.M115.694323 (2016).
Miloradović, Z. et al. Angiotensin 2 type 1 receptor blockade different affects postishemic kidney injury in normotensive and hypertensive rats. J. Physiol. Biochem. 72, 813–820. https://doi.org/10.1007/s13105-016-0514-4 (2016).
Benigni, A. et al. Blocking angiotensin II synthesis/activity preserves glomerular nephrin in rats with severe nephrosis. J. Am. Soc. Nephrol. 12, 941–948. https://doi.org/10.1681/ASN.V125941 (2001).
Suzuki, K. et al. Angiotensin II type 1 and type 2 receptors play opposite roles in regulating the barrier function of kidney glomerular capillary wall. Am. J. Pathol. 170, 1841–1853. https://doi.org/10.2353/ajpath.2007.060484 (2007).
Zhou, L. & Liu, Y. Wnt/β-catenin signaling and renin-angiotensin system in chronic kidney disease. Curr. Opin. Nephrol. Hypertens. 25, 100–106. https://doi.org/10.1097/MNH.0000000000000205 (2016).
Albashir, A. A. D. Renin-angiotensin-aldosterone system (RAAS) inhibitors and coronavirus disease 2019 (COVID19). South. Med. J. 114, 51–56. https://doi.org/10.14423/SMJ.0000000000001200 (2021).
Matsusaka, T. et al. Liver angiotensinogen is the primary source of renal angiotensin II. J. Am. Soc. Nephrol. 23, 1181–1189. https://doi.org/10.1681/ASN.2011121159 (2012).
Bargagli, E. et al. Metabolic dysregulation in idiopathic pulmonary fibrosis. Int. J. Mol. Sci. 21, 5663. https://doi.org/10.3390/ijms21165663 (2020).
Sztechman, D., Czarzasta, K., Cudnoch-Jedrzejewska, A., Szczepanska-Sadowska, E. & Zera, T. Aldosterone and mineralocorticoid receptors in regulation of the cardiovascular system and pathological remodelling of the heart and arteries. J. Physiol. Pharmacol. 69. https://doi.org/10.26402/jpp.2018.6.01 (2018).
Zhang, S. L., Guo, J., Moini, B. & Ingelfinger, J. R. Angiotensin II stimulates Pax-2 in rat kidney proximal tubular cells: impact on proliferation and apoptosis. Kidney Int. 66, 2181–2192. https://doi.org/10.1111/j.1523-1755.2004.66008.x (2004).
Kim, C. S. et al. Angiotensin-(1–7) attenuates kidney injury due to obstructive nephropathy in rats. PLoS One 10, e0142664. https://doi.org/10.1371/journal.pone.0142664 (2015).
Zhu, H. et al. Increased apoptosis in the paraventricular nucleus mediated by AT1R/Ras/ERK1/2 signaling results in sympathetic hyperactivity and renovascular hypertension in rats after kidney injury. Front. Physiol. 8, 41. https://doi.org/10.3389/fphys.2017.00041 (2017).
Choi, H. S. et al. Angiotensin-[1–7] attenuates kidney injury in experimental alport syndrome. Sci. Rep. 10, 4225. https://doi.org/10.1038/s41598-020-61250-5 (2020).
Ning, W. B. et al. Fluorofenidone inhibits ang II-induced apoptosis of renal tubular cells through blockage of the Fas/FasL pathway. Int. Immunopharmacol. 11, 1327–1332. https://doi.org/10.1016/j.intimp.2011.04.016 (2011).
Martinsen, A., Dessy, C. & Morel, N. Regulation of calcium channels in smooth muscle: new insights into the role of myosin light chain kinase. Channels (Austin) 8, 402–413. https://doi.org/10.4161/19336950.2014.950537 (2014).
Lovegrove, J. A., Stainer, A. & Hobbs, D. A. Role of flavonoids and nitrates in cardiovascular health. Proc. Nutr. Soc. 1–13. https://doi.org/10.1017/S0029665116002871 (2017).
Edwards, R. L. et al. Quercetin reduces blood pressure in hypertensive subjects. J. Nutr. 137, 2405–2411. https://doi.org/10.1093/jn/137.11.2405 (2007).
Ożarowski, M. et al. Pharmacological effect of quercetin in hypertension and its potential application in pregnancy-induced hypertension: review of in vitro, in vivo, and clinical studies. Evid. Based Complement. Alternat. Med. 2018, 7421489. https://doi.org/10.1155/2018/7421489 (2018).
Chen, W. J. et al. Quercetin attenuates cardiac hypertrophy by inhibiting mitochondrial dysfunction through SIRT3/PARP-1 pathway. Front. Pharmacol. 12, 739615. http://doi.org/10.3389/fphar.2021.739615 (2021).
Oyagbemi, A. A. et al. Quercetin attenuates hypertension induced by sodium fluoride via reduction in oxidative stress and modulation of HSP 70/ERK/PPARγ signaling pathways. Biofactors. 44, 465–479. http://doi.org/10.1002/biof.1445 (2018).
Wang, L., Tan, A., An, X., Xia, Y. & Xie, Y. Quercetin dihydrate inhibition of cardiac fibrosis induced by angiotensin II in vivo and in vitro. Biomed. Pharmacother. 127, 110205. https://doi.org/10.1016/j.biopha.2020.110205 (2020).
Wang, D. et al. Quercetin inhibits angiotensin II-induced vascular smooth muscle cell proliferation and activation of JAK2/STAT3 pathway: A target based networking pharmacology approach. Front. Pharmacol. 13, 1002363. http://doi.org/10.3389/fphar.2022.1002363 (2022).
Chen, B. L. et al. Quercetin attenuates renal ischemia/reperfusion injury via an activation of AMP-activated protein kinase-regulated autophagy pathway. J. Nutr. Biochem. 25, 1226–1234. http://doi.org/10.1016/j.jnutbio.2014.05.013 (2014).
Erboga, M., Aktas, C., Erboga, Z. F., Donmez, Y. B. & Gurel, A. Quercetin ameliorates methotrexate-induced renal damage, apoptosis and oxidative stress in rats. Ren. Fail. 37, 1492–1497. http://doi.org/10.3109/0886022X.2015.1074521 (2015).
Safran, M. et al. The GeneCards Suite. Practical Guide to Life Science Databases 27–56. https://doi.org/10.1007/978-981-16-5812-9_2 (2022).
Piñero, J. et al. The DisGeNET knowledge platform for disease genomics: 2019 update. Nucl. Acids Res. 48, D845-D855. http://doi.org/10.1093/nar/gkz1021 (2019).
Ru, J. et al. TCMSP: a database of systems pharmacology for drug discovery from herbal medicines. J. Cheminform. 6, 13. http://doi.org/10.1186/1758-2946-6-13 (2014).
Xu, H. Y. et al. ETCM: an encyclopaedia of traditional Chinese medicine. Nucleic. Acids. Res. 47, D976-D982. http://doi.org/10.1093/nar/gky987 (2019).
Sherman, B. T. et al. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic. Acids. Res. 50, W216-W221. http://doi.org/10.1093/nar/gkac194 (2022).
Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57. http://doi.org/10.1038/nprot.2008.211 (2009).
Solares, M. J., et al. High-resolution imaging of human cancer proteins using microprocessor materials. Chembiochem. 23, e202200310. http://doi.org/10.1002/cbic.202200310 (2022).
Berman, H. M., Henrick, K. & Nakamura, H. Announcing the worldwide protein data bank. Nat. Struct. Biol. 10, 980. http://doi.org/10.1038/nsb1203-980 (2003).
Wang, Y. et al. PubChem: a public information system for analyzing bioactivities of small molecules. Nucleic. Acids. Res. 37, W623-W633. http://doi.org/10.1093/nar/gkp456 (2009).
Wu, M. et al. Qingda granule attenuates angiotensin II-Induced blood pressure and inhibits Ca²⁺/ERK signaling pathway. Front. Pharmacol. 12 688877. https://doi.org/10.3389/fphar.2021.688877 (2021).
Unger, T. et al. 2020 international society of hypertension global hypertension practice guidelines. Hypertension 75, 1334–1357. https://doi.org/10.1161/HYPERTENSIONAHA.120.15026 (2020).
Chen, W. J. et al. Quercetin attenuates cardiac hypertrophy by inhibiting mitochondrial dysfunction through SIRT3/PARP-1 pathway. Front. Pharmacol. 12, 739615. https://doi.org/10.3389/fphar.2021.739615 (2021).
Elbarbry, F., Abdelkawy, K., Moshirian, N. & Abdel-Megied, A. M. The antihypertensive effect of quercetin in young spontaneously hypertensive rats; role of arachidonic acid metabolism. Int. J. Mol. Sci. 21, 6554. https://doi.org/10.3390/ijms21186554 (2020).
Carlstrom, J. et al. A quercetin supplemented diet does not prevent cardiovascular complications in spontaneously hypertensive rats. J. Nutr. 137, 628–633. https://doi.org/10.1093/jn/137.3.628 (2007).
Duarte, J. et al. Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br. J. Pharmacol. 133, 117–124. https://doi.org/10.1038/sj.bjp.0704064 (2001).
Pérez-Vizcaíno, F. et al. Endothelium-independent vasodilator effects of the flavonoid quercetin and its methylated metabolites in rat conductance and resistance arteries. J. Pharmacol. Exp. Ther. 302, 66–72. https://doi.org/10.1124/jpet.302.1.66 (2002).
Chen, C. K. & Pace-Asciak, C. R. Vasorelaxing activity of resveratrol and quercetin in isolated rat aorta. Gen. Pharmacol. 27, 363–366. https://doi.org/10.1016/0306-3623(95)02001-2 (1996).
Russo, G. L. Ins and outs of dietary phytochemicals in cancer chemoprevention. Biochem. Pharmacol. 74, 533–544. https://doi.org/10.1016/j.bcp.2007.02.014 (2007).
Serban, M.C. et al. Effects of quercetin on blood pressure: a systematic review and meta-analysis of randomized controlled trials. J. Am. Heart. Assoc. 5, e002713. https://doi.org/10.1161/JAHA.115.002713 (2016).
Kumar, G. et al. Transcriptomic validation of the protective effects of aqueous bark extract of terminalia arjuna (roxb.) on isoproterenol-induced cardiac hypertrophy in rats. Front. Pharmacol. 10, 1443. https://doi.org/10.3389/fphar.2019.01443 (2019).
Duan, X. & Qin, G. Notch inhibitor mitigates renal ischemia–reperfusion injury in diabetic rats. Mol. Med. Rep. 21, 583–588. https://doi.org/10.3892/mmr.2019.10857 (2020).
Luo, X., Zhang, L., Han, G. D., Lu, P. & Zhang, Y. MDM2 inhibition improves cisplatin-induced renal injury in mice via inactivation of Notch/hes1 signaling pathway. Hum. Exp. Toxicol. 40, 369–379. https://doi.org/10.1177/0960327120952158 (2021).
Long, L. et al. Qingda granule attenuates angiotensin II-induced renal apoptosis and activation of the p53 pathway. Front. Pharmacol. 12, 770863. https://doi.org/10.3389/fphar.2021.770863 (2022).
Banaszak, B., Świętochowska, E., Banaszak, P. & Ziora, K. Endothelin-1 (ET-1), N-terminal fragment of pro-atrial natriuretic peptide (NTpro-ANP), and tumour necrosis factor alpha (TNF-α) in children with primary hypertension and hypertension of renal origin. Endokrynol. Pol. 70, 37–42. https://doi.org/10.5603/EP.a2018.0079 (2019).
Das, S. et al. A novel angiotensin II-induced long noncoding RNA giver regulates oxidative stress, inflammation, and proliferation in vascular smooth muscle cells. Circ. Res. 123, 1298–1312. https://doi.org/10.1161/CIRCRESAHA.118.313207 (2018).
Fu, Z. J. et al. HIF-1α-BNIP3-mediated mitophagy in tubular cells protects against renal ischemia/reperfusion injury. Redox. Biol. 36, 101671. https://doi.org/10.1016/j.redox.2020.101671 (2020).
He, X., Cantrell, A. C., Williams, Q. A., Chen, J. X. & Zeng, H. TIGAR deficiency sensitizes angiotensin-II-induced renal fibrosis and glomerular injury. Physiol. Rep. 10, e15234. https://doi.org/10.14814/phy2.15234 (2022).
Liu, C. et al. Gastrin attenuates renal ischemia/reperfusion injury by a PI3K/Akt/Bad-mediated anti-apoptosis signaling. Frot. Pharmacol. 11, 540479. https://doi.org/10.3389/fphar.2020.540479 (2020).
Ma, S. K. et al. Activation of the renal PI3K/Akt/mTOR signaling pathway in a DOCA-salt model of hypertension. Chonnam. Med. J. 48, 150–154. https://doi.org/10.4068/cmj.2012.48.3.150 (2012).
Hauser, P. & Oberbauer, R. Tubular apoptosis in the pathophysiology of renal disease. Wien. Klin. Wochenschr. 114, 671–677. (2002).
Livesey, K. M. et al. p53/HMGB1 complexes regulate autophagy and apoptosis. Cancer Res. 72, 1996–2005. https://doi.org/10.1158/0008-5472.CAN-11-2291 (2012).
Molitoris, B. A. et al. siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury. J. Am. Soc. Nephrol. 20, 1754–1764. https://doi.org/10.1681/ASN.2008111204 (2009).
Zhou, L. et al. Activation of p53 promotes renal injury in acute aristolochic acid nephropathy. J. Am. Soc. Nephrol. 21, 31–41. https://doi.org/10.1681/ASN.2008111133 (2010).
Ying, Y., Kim, J., Westphal, S. N., Long, K. E. & Padanilam, B. J. Targeted deletion of p53 in the proximal tubule prevents ischemic renal injury. J. Am. Soc. Nephrol. 25, 2707–2716. https://doi.org/10.1681/ASN.2013121270 (2014).
Zhang, D. et al. Tubular p53 regulates multiple genes to mediate AKI. J. Am. Soc. Nephrol. 25, 2278–2289. https://doi.org/10.1681/ASN.2013080902 (2014).
Moll, U. M., Wolff, S., Speidel, D. & Deppert, W. Transcription-independent pro-apoptotic functions of p53. Curr. Opin. Cell. Biol. 17, 631–636. https://doi.org/10.1016/j.ceb.2005.09.007 (2005).
Heo, K. S. et al. PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation. J. Cell. Biol. 193, 867–884. https://doi.org/10.1083/jcb.201010051 (2011).
Garner, T. P., Lopez, A., Reyna, D. E., Spitz, A. Z. & Gavathiotis, E. Progress in targeting the BCL-2 family of proteins. Curr. Opin. Chem. Biol. 39, 133–142. https://doi.org/10.1016/j.cbpa.2017.06.014 (2017).
Parrish, A. B., Freel, C. D. & Kornbluth, S. Cellular mechanisms controlling caspase activation and function. Cold. Spring Harb. Perspect. Biol. 5, a008672. https://doi.org/10.1101/cshperspect.a008672 (2013).
Li, F. et al. Quercetin regulates inflammation, oxidative stress, apoptosis, and mitochondrial structure and function in H9C2 cells by promoting PVT1 expression. Acta. Histochem. 123, 151819. https://doi.org/10.1016/j.acthis.2021.151819 (2021).

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

An integrated network pharmacology and RNA-seq approach for exploring the renal protection of quercetin on attenuating Ang II- induced cells apoptosis

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

Data Accessibility of Sequence Data

Conflict of Interest

Author Contributions

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1