Abstract

   Diabetic nephropathy (DN), a leading cause of end-stage renal disease,
   remains a formidable challenge in diabetes management due to the
   complex nature of its pathogenesis, particularly the
   epithelial-mesenchymal transition (EMT) process. Our innovative study
   leverages network pharmacology to explore the therapeutic potentials of
   Myricetin, a natural flavonoid, focusing on its effects against NOX4, a
   critical mediator in DN progression. This investigation marks a
   pioneering approach by integrating network pharmacology to predict and
   elucidate the inhibitory relationship between Myricetin and NOX4.
   Utilizing a high-fat diet/streptozotocin (HFD/STZ) induced DN mouse
   model, we delved into the effects of Myricetin on renal EMT processes.
   Through network pharmacology analyses coupled with molecular docking
   studies, we identified and confirmed Myricetin's binding efficacy to
   NOX4. Extensive in vitro and in vivo experiments further established
   Myricetin's significant impact on mitigating EMT by modulating the
   NOX4-NF-κB-Snail signaling pathway. Results from our research
   demonstrated notable improvements in renal function and reductions in
   tissue fibrosis among treated HFD/STZ mice. By curtailing NOX4
   expression, Myricetin effectively reduced reactive oxygen species (ROS)
   production, thereby inhibiting NF-κB activation and subsequent Snail
   expression, crucial steps in the EMT pathway. Supported by both
   theoretical predictions and empirical validations, this study unveils
   the mechanism underlying Myricetin's modulation of EMT in DN through
   disrupting the NOX4-NF-κB-Snail axis. These findings not only
   contribute a new therapeutic avenue for DN treatment but also
   underscore the utility of network pharmacology in advancing drug
   discovery processes.

   Keywords: Myricetin, Diabetic nephropathy, Epithelial-mesenchymal
   transition, NOX4, Network pharmacology

Graphical abstract

   [37]Image 1
   [38]Open in a new tab

Highlights

     * •
       Myricetin effectively reduces fibrosis and improves renal function
       in diabetic nephropathy (DN).
     * •
       Myricetin is identified as a novel Nox4 inhibitor, offering new
       therapeutic possibilities for DN.
     * •
       Myricetin modulates the NOX4/NF-κB/Snail signaling axis, reducing
       oxidative stress and inflammation in DN.

1. Introduction

   Diabetic nephropathy (DN) represents one of the most severe
   complications associated with diabetes mellitus, leading to a
   significant increase in the risk of end-stage renal disease (ESRD)
   globally [[39]1,[40]2]. As the prevalence of diabetes continues to
   surge, DN emerges as a critical public health challenge [[41]3],
   underscoring the urgent need for innovative therapeutic strategies. The
   pathogenesis of DN is multifaceted, involving complex interactions
   between metabolic, hemodynamic, and inflammatory pathways [[42]4].
   Central to the progression of DN is the phenomenon of
   epithelial-mesenchymal transition (EMT) in renal tubular cells [[43]5],
   a process whereby epithelial cells lose their characteristic polarity
   and adhesion, acquiring mesenchymal features that contribute to the
   fibrosis and sclerosis of renal tissue [[44]6].

   The activation of the EMT process in DN is influenced by various
   factors, including hyperglycemia-induced oxidative stress,
   inflammation, and the dysregulation of signaling pathways such as
   TGF-β, Wnt/β-catenin, and Notch [[45][7], [46][8], [47][9]]. Among
   these, the role of oxidative stress mediated by NADPH oxidase 4 (NOX4)
   and the resultant reactive oxygen species (ROS) production is pivotal
   [[48]10]. ROS not only directly damages cellular components [[49]11]
   but also acts as a signaling molecule that activates downstream
   pathways, including NF-κB, culminating in the transcriptional
   activation of genes that promote EMT, such as Snail [[50]12]. Snail, a
   key transcriptional regulator [[51]13], orchestrates the repression of
   epithelial marker genes like E-cadherin and the induction of
   mesenchymal markers, facilitating the EMT process and renal
   fibrogenesis [[52]14].

   Despite the advances in understanding the molecular mechanisms
   underlying DN, the translation of this knowledge into effective
   therapeutic interventions has been limited. Cur-rent treatments
   primarily focus on controlling blood glucose levels and managing
   hypertension [[53]15] but fail to directly address the underlying
   cellular and molecular changes, such as EMT, driving the progression of
   DN.

   In this context, flavonoids, a diverse group of polyphenolic compounds
   with potent an-tioxidative, anti-inflammatory, and antifibrotic
   properties [[54]16,[55]17], have attracted attention for their
   potential therapeutic benefits in chronic diseases, including DN
   [[56]18]. Myricetin, a naturally occurring flavonoid, has been widely
   studied for its health benefits, encompassing anti-tumor,
   antioxidative, and antiviral activities [[57][19], [58][20], [59][21]].
   However, the specific impact of Myricetin on the EMT process in DN,
   particularly through the modulation of the NOX4-ROS–NF–κB-Snail axis,
   remains largely unexplored.

   This study leverages network pharmacology to identify the potential
   inhibitory effect of Myricetin on NOX4, a novel finding that sheds
   light on the molecular basis of its therapeutic action in DN. Through
   molecular docking and extensive in vitro and in vivo experiments, we
   aim to validate the role of Myricetin in modulating the
   NOX4-ROS–NF–κB-Snail signaling axis, offering new insights into its
   potential as a therapeutic agent for halting the progression of DN. By
   elucidating the mechanistic pathways through which Myricetin exerts its
   effects, this research contributes to the broader effort of developing
   targeted and effective treatments for diabetic nephropathy.

2. Materials and methods

2.1. Cell culture and chemicals

   HK-2 human proximal tubular epithelial cells were obtained from Procell
   [[60][22], [61][23], [62][24]] (CL-0109; Wuhan, China) and grown in
   DF12 (DMEM/F12 1:1 mixture) culture medium (Hyclone, UT, USA)
   containing 10 % fetal bovine serum (FBS; Hyclone) in a regular CO[2]
   incubator at 37 ^◦C. HK-2 cells were used in all experiments described
   here, which were authenticated by STR profiling conducted on April 28,
   2024 and tested negative for mycoplasma.

   Following subculturing and the subsequent attainment of an approximate
   80 % confluence, the cells were subjected to treatment. The
   experimental design entailed culturing the cells with glucose at two
   distinct concentrations: 5.5 mmol/L, serving as the control condition
   (CT), and 35 mmol/L, representing the high-glucose environment (HG,
   model group). In addition to these conditions, a treatment group was
   established, wherein cells were exposed to a combination of high
   glucose and Myricetin (Myricetin + HG) for a period of 24 h.

   Myricetin, procured with a purity exceeding 95 % from Topscience Co.,
   Ltd. (Shanghai, China), was dissolved in dimethyl sulfoxide (DMSO) and
   administered to the culture at concentrations of 40 μM or 60 μM. This
   strategic approach allowed for the investigation of cellular dynamics
   under normoglycemic conditions, hyperglycemic stress, and the
   mitigating influence of Myricetin within a hyperglycemic setting,
   providing insights into the potential therapeutic efficacy of Myricetin
   in counteracting the deleterious effects of elevated glucose levels.

2.2. Animal model and experimental design

   Animal housing and initial procedures: Male C57BL/6J mice (4 weeks
   old), a widely recognized model for type 2 diabetes research, were
   maintained under specific pathogen free (SPF) conditions. The mice were
   kept at an ambient temperature ranging from 18 to 23 °C, with a
   relative humidity of 40–70 %. They were provided with a standard clean
   diet and water. All animal experiments were conducted in accordance
   with established animal welfare guidelines and complied with all
   regulations. This study was reviewed and approved by the Animal Ethics
   Committee of Southern Medical University with the approval number:
   SMUL2022007.

   Diabetic model induction: The mice were subjected to a high-fat diet
   (HFD, with 60 % of kilocalories from fat) for a duration of one month.
   This was followed by intraperitoneal injections of streptozotocin (STZ;
   40 mg/kg dissolved in 0.1 mol/L citrate buffer, pH adjusted to 4.5)
   administered daily for five consecutive days. Successful induction of
   the diabetic nephropathy (DN) phase was confirmed in mice that
   consistently displayed fasting blood glucose levels exceeding
   11.1 mmol/L over three days, accompanied by positive 24-h urine protein
   tests.

   Grouping and treatment: Subsequent to the model confirmation, the mice
   were randomized into four distinct groups using a random number table
   method, each comprising six mice:

   Control Group (Con): Healthy mice without any signs of DN.

   Diabetic Nephropathy Group: Mice with STZ/HFD-induced diabetes, given
   equivalent volumes of double-distilled water as placebo.

   Myricetin-Low Dose Group: DN mice administered Myricetin at a dose of
   200 mg/kg/day.

   Myricetin-High Dose Group: DN mice administered Myricetin at a dose of
   300 mg/kg/day.

   All treatments were dispensed intragastrically on a daily regimen
   spanning 11 weeks.

   Tissue collection and preservation: Post 11 weeks of treatment, mice
   were anesthetized using pentobarbital sodium. Blood samples and kidney
   tissues were promptly harvested. A fraction of the kidney tissue
   samples was preserved in 4 % paraformaldehyde, suitable for
   histological evaluations. The remaining kidney tissue was snap-frozen
   and stored at −80^◦C, awaiting subsequent biochemical assessments.

2.3. Histological examination of kidney tissues

   Tissue preparation: Kidney tissues embedded in paraffin were sectioned
   to a thickness of 4 μm. Subsequent to deparaffinization, sections were
   prepared for various histological staining procedures.

   Staining processes: Hematoxylin and Eosin (H&E) Staining; Periodic Acid
   Schiff (PAS) Staining; Sirius Red Staining; Masson's Trichrome
   Staining. All staining kits were sourced from Beijing Solarbio Science
   & Technology Co. Ltd.

   Microscopic evaluation: Blinded microscopic analysis was executed to
   ensure unbiased results. Random fields from each slide were imaged
   using an OLYMPUS microscope (Japan).

2.4. Biochemical analysis of renal function markers

   The levels of 24H-Urine Protein, Serum Creatinine (SCr) and Blood Urea
   Nitrogen (BUN) were quantitatively analyzed using commercial assay kits
   sourced from Nanjing Jiancheng (Jiangsu, China). The assays were
   meticulously executed as per the detailed guidelines and protocols
   provided by the manufacturer, ensuring accuracy and reproducibility in
   the results.

2.5. Potential targets identification of myricetin, epithelial-mesenchymal
transition (EMT) and diabetic nephropathy (DN)

   Pharmacological target prediction of myricetin database: Myricetin's
   potential biological targets were predicted using the
   SwissTargetPrediction database (available at
   [63]http://www.swisstargetprediction.ch/[64]www.swisstargetprediction.c
   h/). The molecular structure of Myricetin was inputted into the
   SwissTargetPrediction platform. The database rendered its predictions
   based on the structural similarity of Myricetin to known ligands,
   proposing a list of prospective protein targets that Myricetin might
   interact with.

   Identification of targets pertinent to DN and EMT Database: To discern
   the targets specifically relevant to Diabetic Nephropathy (DN) and
   Epithelial-mesenchymal Transition (EMT), the GeneCards disease database
   was employed (accessible at [65]https://www.genecards.org/). Keywords
   "Diabetic Nephropathy (DN)" and "Epithelial-mesenchymal Transition
   (EMT)" were utilized to systematically comb through the GeneCards
   database. This integrative database consolidates gene-centric
   information derived from a myriad of sources, encapsulating both
   annotated and predicted human genes.

2.6. Construction of component-target-disease (CTD) and protein-protein
interaction (PPI) networks

   Target overlap analysis for drug and disease: To discern the potential
   targets through which Myricetin might alleviate EMT and subsequently
   treat DN, we utilized the jvenn tool
   ([66]https://jvenn.toulouse.inrae.fr/app/example.html). This
   facilitated the identification of shared targets between Myricetin and
   both DN and EMT.

   Construction of PPI network for overlapping targets: With the
   shortlisted disease targets in hand, we employed the STRING database
   ([67]http://string-db.org) to construct the Protein-Protein Interaction
   (PPI) network. In this, we set the organism as ‘Homo sapiens’ and the
   minimum required interaction score was medium confidence. For enhanced
   visualization and analysis, the results from STRING, saved in "tsv"
   format, were imported into Cytoscape 3.9.1. This approach led to the
   creation of a network termed "MYR-DN-EMT-target". It's noteworthy that
   within this network, the size and color of each target were modulated
   based on its Degree Centrality (DC) value, as calculated by the CytoNCA
   plugin in Cytoscape 3.9.1. These DC values provided insights into the
   relative importance of each target within the overall network.

2.7. Functional enrichment analyses

   GO and KEGG pathway analysis: Gene Ontology (GO) analysis and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
   were performed on the shared genes of Myricetin, DN and EMT using the
   Metascape online analysis platform
   ([68]https://metascape.org/gp/index.html#/main/step1). The GO
   enrichment analysis included cellular components (CC), molecular
   functions (MF) and biological processes (BP). The results of the first
   nineteen pathways from KEGG enrichment analysis were visualized using
   an online bioinformatics visualization platform
   ([69]http://www.bioinformatics.com.cn/), and the data were presented
   via information bubble plots. Statistically significant results were
   identified using a threshold of P < 0.05.

   Disease-Target-Pathway network construction: To construct the
   disease-target-pathway network, the top 20 pathways from the previous
   analysis and their corresponding 24 key targets were imported into
   Cytoscape 3.9.1 software. The visualization of the network was based on
   the BC (betweenness) values calculated using the CytoNCA plugin.

2.8. Molecular docking

   AutoDock Vina and Pymol software were used for molecular docking. The
   NOX4 crystal structure (PDB:[70]2ETR) was downloaded from the PDB
   ([71]https://www.rcsb.org), and the molecular structures were
   downloaded from Pubchem ([72]https://pubchem.ncbi.nlm.nih.gov/).

2.9. Western blotting

   The tissues of the renal cortex or HK2 cells were lysed with lysis
   buffer a protease inhibitor cocktail and a phosphate inhibitor cocktail
   (Sigma-Aldrich). The quantification of total protein content was
   determined using the bicinchoninic acid (BCA) protein assay kit (Thermo
   Fisher Scientific). The protein samples were subjected to sodium
   dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and
   subsequently transferred onto a polyvinylidene difluoride membrane
   (Bio-Rad, Hercules, CA, USA). To block non-specific binding, the
   membrane was treated with 5 % milk at room temperature for 2 h,
   followed by overnight incubation with specific primary antibodies: NOX4
   Polyclonal antibody (14347- 1-AP; Proteintech), E-cadherin Antibody
   (G-10) (sc-8426; Santa Cruz Biotechnology), Snail (C15D3) Rabbit mAb
   (3879T; CellSignalingTechnology), Vimentin Antibody (AF7013; Affinity),
   Beta Actin Monoclonal antibody (66009-1-Ig; Proteintech), NF-κB p65
   (D14E12) XP® Rabbit mAb (8242T; CellSignalingTechnology), Phospho–NF–κB
   p65 (Ser536) (93H1) Rabbit mAb (3033T; CellSignalingTechnology), GAPDH
   Antibody (AF7021; Affinity). After washing with TBS-T for three times,
   the membrane was incubated with corresponding secondary antibodies
   (1:10000) at 4 ^◦C for 2 h. The signals were detected with the enhanced
   chemiluminescence (ECL) method via the FluorChem E™ system
   (ProteinSimple, San Francisco, CA, USA).

2.10. Statistical analysis

   The statistical analyses were conducted using GraphPad Prism version
   9.4.0 (GraphPad Software Inc, San Diego, CA, USA). One-way or Two-way
   analysis of variance (ANOVA) and appropriate post hoc test were used to
   compare multiple groups. The Mean ± SD was used to represent the
   results. Significance was defined as *p < 0.05, **p < 0.01,
   ***p < 0.001, ****p < 0.0001 in all statistical analyses.

2.11. Immunohistochemical and immunocytochemical staining

   Mouse kidneys were prepared for paraffin embedding according to
   conventional tech-niques after being fixed in 4 % PFA (Biosharp Life
   Science, China) at 4 ^◦C for at least 24 h. Paraffin sections were
   prepared at 4-μm-thickness and deparaffinized and hydrated in graded
   ethanol series. Antigen retrieval was performed on the tissue sections
   by microwaving tissue sections in 10 mmol/L sodium citrate buffer (pH
   6.0) for 10 min. Endogenous peroxidase was inhibited by incubating for
   10 min with 3 % hydrogen peroxide. Then, sections were blocked with 5 %
   normal goat serum solution for 1 h at RT. Primary antibodies were
   applied to the sections and left on them at 4^◦C overnight. The
   appropriate secondary antibodies were applied to the sections and
   incubated. Diaminobenzidine (DAB) (ZSGB-BIO, China) was used as the
   chromogen. Sections were lightly counterstained with hematoxylin and
   were dehydrated and cover slipped. Image acquisition was performed on
   the KF-PRO-005-EX scanner.

   For immunocytochemical staining of NOX4 and NF-κB, HK2
   (2 × 10^4 cells/well) were seeded in a 4-well chamber, cells were
   treated with 35 μM glucose in the presence or absence of 10 μM MYR for
   24 h. Then cells were fixed, permeabilized, blocked, and incubated
   overnight at 4^◦C with NOX4/NF-κB, and subsequently incubated with an
   anti-rabbit Alexa Fluor 488 secondary antibody (A11010; Invitrogen).
   The nuclei were stained with DAPI, mounted, and observed under a FV3000
   confocal scanning microscope.

2.12. ROS determination

   Reactive oxygen species (ROS) in tissue sections were detected using
   Dihydroethidium (DHE, Beyotime, S0063). This ROS-sensitive red
   fluorescent dye was applied to the tissue sections following standard
   protocols. After appropriate incubation, the sections were examined
   under a fluorescence microscope (Nikon Eclipse C1). The intensity and
   localization of the red fluorescence emitted by DHE were used to assess
   ROS levels in the tissues.

2.13. MDA determination

   An appropriate amount of mouse kidney tissue was taken and nine times
   the volume of saline was added and then the tissue was ground into a
   homogenate. The supernatant of each tissue was collected after
   centrifugation and its content was measured using Malondialdehyde (MDA)
   assay kit (A003-1-1; Nanjing, China).

3. Results

3.1. Myricetin enhances kidney function and suppresses renal fibrosis in
STZ/HFD mice-induced diabetic mice

   To probe the potential therapeutic efficacy of MYR in ameliorating
   renal impairment in diabetic nephropathy (DN), we instituted high-fat
   diet/streptozotocin (HFD/STZ) paradigm to emulate DN in mice. Following
   this, the mice received oral doses of either MYR (at concentrations of
   200 or 300 mg/kg) or a control vehicle for a duration of 11 weeks
   ([73]Fig. 1 A).

Fig. 1.

   [74]Fig. 1
   [75]Open in a new tab

   Protective efficacy of myricetin in STZ/HFD-induced renal injury.

   A. Schematic representation of the Myricetin administration protocol in
   STZ/HFD mice is illustrated. B. Evaluation of the impact of Myricetin
   treatment over 11 weeks on the body mass of mice. C–F. Assessment of
   fasting blood glucose, blood urea nitrogen, serum creatinine, and 24-h
   urinary albumin levels post 11 weeks of Myricetin intervention. G.
   Histological examination of renal tissues post Myricetin treatment,
   visualized through hematoxylin and eosin (H&E), periodic acid-Schiff
   (PAS), and Masson's trichrome staining. (×200 magnification,
   Bar = 40 μm) The red arrows: renal interstitial fibrosis; the green
   arrows: swollen tubular epithelial cells and vacuolar degeneration; the
   black arrows: lymphocytic or plasma cell infiltration in the renal
   interstitium. Statistical representation is depicted as mean ± standard
   deviation (n = 5/group); significance denoted by #p < 0.05, ##p < 0.01,
   ###p < 0.001, and ####p < 0.0001, ns = no significant, versus control
   (Ctrl); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = no
   significant, versus diabetic nephropathy (DN).

   Weight assessments across cohorts revealed no significant deviations,
   attesting to Myricetin's safety ([76]Fig. 1 B). A granular analysis of
   fasting blood glucose (FBG) and renal function evinced pronounced
   alterations in salient markers—embracing urine protein metrics, serum
   creatinine (SCr), and blood urea nitrogen (BUN) values—within the
   purview of the HFD/STZ ensemble ([77]Fig. 1C–F). Contrarily, these
   metrics exhibited significant remedia-tion in a dose-responsive
   paradigm within the Myricetin-administered cohorts. Histopathol-ogy,
   using H&E staining, highlighted Myricetin's counteractive effects on
   hyperglycemia-induced renal anomalies ([78]Fig. 1 G). Periodic
   Acid-Schiff (PAS) staining indicated increased glycogen in the renal
   cortex of the model group, predominantly within the tubular basement
   and renal interstitium ([79]Fig. 1 G). This was reduced with Myricetin
   intervention. Sirius red and Masson's trichrome staining revealed
   exacerbated renal fibrosis in diabetic mice, indicating increased
   collagen in the renal interstices. However, Myricetin administration
   moderated this collagen accumulation ([80]Fig. 1 G). In essence,
   findings indicate Myricetin's potential in counteracting
   diabetic-induced renal fibrosis.

3.2. Myricetin alleviates renal tubular epithelial-mesenchymal transition in
vivo

   Building on the foundational results of Myricetin's renal protective
   effects, we delved into its specific impact on the process of
   epithelial-mesenchymal transition (EMT) within renal tubular cells—a
   critical event in the progression of renal fibrosis in diabetic
   nephropathy. Through quantitative RT-PCR, we observed a significant
   upregulation of E-cadherin, an epithelial marker typically suppressed
   in diabetic nephropathy, alongside a downregulation of mesenchymal
   markers Snail and Vimentin, in a dose-dependent manner following
   Myricetin treatment ([81]Fig. 2 A).

Fig. 2.

   [82]Fig. 2
   [83]Open in a new tab

   Mitigation of epithelial-mesenchymal transition in renal tubule cells
   by myricetin In vivo.

   A. RT-PCR quantification of mRNA levels of E-cadherin, Vimentin, and
   Snail in renal tissues (n = 3). B–C. Verification of E-cadherin,
   Vimentin, and Snail protein expression in renal tissues via Western
   blot analysis (n = 3), with β-actin serving as the internal standard.
   D-E. Immunohistochemical analysis of E-cadherin, Vimentin, and Snail in
   renal tissues. (×200 magnification, Scale bar = 60 μM) Data presented
   as means ± SD, (n = 3/group). Significance indicated by #p < 0.05,
   ##p < 0.01, ###p < 0.001, and ####p < 0.0001, ns = no significant,
   versus control (Ctrl); *p < 0.05, **p < 0.01, ***p < 0.001,
   ****p < 0.0001, ns = no significant, versus diabetic nephropathy (DN).

   Further reinforcing these observations, Western blot analysis ([84]Fig.
   2B and C) provided additional evidence of the dose-responsive effect of
   Myricetin on these EMT markers. The protein expression levels of
   E-cadherin increased while snail and vimentin decreased, aligning with
   the qRT-PCR findings.

   Immunofluorescence staining ([85]Fig. 2D and E) offered visual
   confirmation of these molecular alterations. In renal tubules affected
   by DN, a marked reduction in E-cadherin expression was evident, which
   was effectively reversed post-Myricetin intervention. Similarly, the
   elevated expression levels of snail and vimentin in DN conditions were
   substantially diminished in response to Myricetin, further validating
   its therapeutic efficacy.

3.3. DN-component-EMT target and PPI network analysis

   To elucidate the underlying molecular targets and pathways through
   which Myricetin exerts its anti-EMT effects, we employed a network
   pharmacology approach. Utilizing the SwissTargetPrediction database, we
   initially identified 100 potential Myricetin targets. To discern
   targets specifically relevant to DN and EMT, we employed the GeneCards
   disease database. This led to the integration of drug targets with
   disease targets through a Venn diagram approach. Ultimately, 35 targets
   specifically associated with the treatment of EMT in DN by Myricetin
   were selected ([86]Fig. 3 A). Constructing a Protein-Protein
   Interaction (PPI) network from these targets illuminated the intricate
   web of potential interactions modulated by Myricetin, with GO and KEGG
   enrichment analyses pinpointing ROS-related signaling pathways among
   the most significantly affected ([87]Fig. 3B–D).

Fig. 3.

   [88]Fig. 3
   [89]Open in a new tab

   Network pharmacology analysis and target identification for
   DN-Component-EMT Interface.

   A. Correlation of DN with Myricetin and EMT target genes illustrated.
   B. Shared PPI network among DN, Myricetin, and EMT delineated. C.
   Enriched GO Terms across Biological Processes (BP), Cellular Component
   (CC), and Molecular Function (MF) for Myricetin's Potential Targets. D.
   Visualization of predominant KEGG pathways via bubble maps,
   highlighting Myricetin's action spectrum.

3.4. Myricetin inhibits ROS in DN mice

   Given the pivotal role of reactive oxygen species (ROS) in the
   pathogenesis of diabetic nephropathy and its contribution to EMT, we
   quantitatively assessed the impact of Myricetin on ROS production in
   kidney tissues from DN mice. A notable reduction in ROS and MDA levels
   was observed in a dose-dependent manner in Myricetin-treated groups
   compared to diabetic controls ([90]Fig. 4 A and 4 D). Molecular docking
   studies provided structural insights into the interaction between
   Myricetin and NOX4, a key enzyme involved in ROS generation, suggesting
   a direct inhibitory effect of Myricetin on NOX4 activity ([91]Fig. 4B
   and C).

Fig. 4.

   [92]Fig. 4
   [93]Open in a new tab

   Myricetin's suppressive effect on reactive oxygen species in DN mice.

   A. Visualization of ROS in renal tissue sections through fluorescent
   staining, with ROS intensity mirrored by red fluorescence and nuclei
   depicted in blue via DAPI staining. (Scale bar = 100 μm) B.
   Construction of a target-pathway map, featuring top KEGG pathways and
   key targets, with visual adjustments based on betweenness centrality
   (BC) values. C. Illustration of Myricetin's interaction with NOX4,
   depicting the ligand and protein structure interactions, with hydrogen
   bonds marked by dark blue dashed lines. D. The levels of MDA
   (Malonaldehyde) in kidney tissues of mice.

3.5. Inhibition of EMT in HK2 cells by myricetin through NOX4 expression

   The production of ROS is closely linked to the activity of NOX4, a
   pivotal player that is upregulated in DN and contributes to increased
   oxidative stress. This oxidative stress, in turn, activates NF-κB, a
   key transcription factor known to regulate the expression of Snail.
   Snail is a crucial transcription factor in the EMT process, exerting
   its influence by suppressing the expression of E-cadherin and promoting
   the expression of mesenchymal markers such as vimentin, thereby driving
   the transition of epithelial cells into mesenchymal cells.

   Complementing the in vivo findings, in vitro experiments conducted on
   high glucose-induced HK2 cells, a human renal proximal tubule
   epithelial cell line. We observed that high glucose conditions led to
   an increase in NOX4 fluorescence and nuclear translocation of NF-κB,
   indicative of elevated oxidative stress and NF-κB activation. However,
   the introduction of Myricetin treatment effectively attenuated these
   changes, providing compelling evidence of its ability to reduce
   oxidative stress ([94]Fig. 5, [95]Fig. 6 A). Western blot analysis
   further corroborated these findings by revealing dose-dependent
   alterations in the expression levels of NOX4, phosphorylated NF-κB, and
   key EMT markers, including Snail, E-cadherin and vimentin ([96]Fig. 5B
   and C). These changes were consistent with the reversal of EMT
   processes observed in our in vivo experiments, highlighting the ability
   of Myricetin to modulate NOX4 and the NF-κB signaling pathway,
   ultimately inhibiting EMT.

Fig. 5.

   [97]Fig. 5
   [98]Open in a new tab

   Myricetin attenuates EMT in HK2 cells by downregulating NOX4
   expression.

   A. HK-2 cells cultured and treated with glucose ± Myricetin for 24 h,
   followed by immunocytochemical analysis using NOX4-specific primary
   antibody and Alexa Fluor 488-conjugated secondary antibody. Nuclei
   stained with DAPI (blue). (Scale bar = 20 μM) B–C. Protein expression
   analysis of Snail, E-cadherin, Vimentin, NOX4, and P–NF-κB in
   HK-2 cells post-treatment, evaluated via Western blot (n = 3).
   Quantification conducted with ImageJ, normalized to GAPDH. Data
   represented as mean ± standard deviation (n = 3/group), indicating
   significance with ##p < 0.01, ###p < 0.001, and ####p < 0.0001, ns = no
   significant, versus control (Ctrl); *p < 0.05, **p < 0.01,
   ***p < 0.001, ****p < 0.0001, ns = no significant, versus 35 mM glucose
   (HG).

Fig. 6.

   [99]Fig. 6
   [100]Open in a new tab

   Reduction of NF-κB activation and NOX4 expression in DN mice via
   myricetin.

   A. NF-κB staining in HK-2 cells treated with glucose ± Myricetin for
   24 h, using NF-κB-specific primary antibody and Alexa Fluor
   488-conjugated secondary antibody. Nuclei counterstained with DAPI
   (blue). (Scale bar = 20 μm) B–C. Validation of NF-κB and NOX4 protein
   levels in renal tissues by Western blot (n = 3), with β-actin as
   internal reference. D-E. Immunohistochemical assessment of P–NF-κB and
   NOX4 expression in the renal cortex of DN mice, accompanied by
   statistical analysis. (×200 magnification, Scale bar = 60 μm) G.
   Elucidation of Myricetin's mechanism in mitigating EMT in diabetic
   nephropathy via inhibition of the NOX4-NF-κB-Snail signaling pathway.
   Data depicted as mean ± standard deviation (n = 3/group), with
   significance denoted by ##p < 0.01, ###p < 0.001, and ####p < 0.0001,
   ns = no significant, versus control (Ctrl); *p < 0.05, **p < 0.01,
   ***p < 0.001, ****p < 0.0001, ns = no significant, versus diabetic
   nephropathy (DN).

3.6. Myricetin modulates NF-κB activation and NOX4 expression in DN contexts

   To confirm the mechanisms underlying the therapeutic effects of
   Myricetin in diabetic nephropathy (DN), we conducted Western blot
   analysis on kidney tissues from DN mice. This analysis revealed
   elevated levels of NOX4 and phosphorylated NF-κB, indicative of
   increased oxidative stress and NF-κB activation in the DN context.
   However, the introduction of Myricetin treatment resulted in a
   dose-dependent reversal of these effects ([101]Fig. 6B and C).

   Furthermore, we employed immunohistochemistry to assess the expressions
   of Nox4 and phosphorylated NF-κB in the renal cortex of DN mice.
   Consistently, we observed increased expressions of both Nox4 and
   phosphorylated NF-κB in DN mice compared to the control group
   ([102]Fig. 6D and E). Notably, Myricetin treatment markedly attenuated
   these expressions, highlighting its potential in mitigating
   NF-κB-mediated inflammatory pathways in the context of diabetic
   nephropathy ([103]Fig. 6 G).

4. Discussion

   Diabetic nephropathy (DN), a formidable complication of diabetes
   mellitus, is a principal contributor to the global prevalence of
   end-stage renal disease [[104]25,[105]26] While primarily affecting the
   kidneys, diabetes can also exacerbate risks of hepatotoxicity,
   complicating the overall management of the disease [[106]27]. The
   pathophysiological progression of DN, characterized by the
   epithelial-mesenchymal transition (EMT) of renal tubular cells
   [[107]28,[108]29], presents a complex therapeutic challenge,
   underscoring the urgent need for innovative treatment strategies. In
   this context, our comprehensive study on Myricetin, a flavonoid with
   well-documented pharmacological properties [[109]30], marks a
   significant advancement in understanding potential therapeutic
   interventions for DN, particularly in modulating the pivotal EMT
   process.

   The role of flavonoids in influencing cellular processes is widely
   recognized within the scientific community [[110]31]. However, the
   specific mechanisms by which Myricetin affects the EMT process in DN
   have remained largely unexplored. Our investigation aimed to fill this
   critical gap by elucidating the effects of Myricetin on DN's
   pathogenesis. We observed that Myricetin administration significantly
   ameliorates renal dysfunction and fibrosis in an HFD/STZ-induced model
   of DN [[111]32]. This effect is manifested through Myricetin's
   modulation of key EMT biomarkers, including a marked downregulation of
   Snail, a central transcription factor in EMT induction, and Vimentin, a
   mesenchymal marker [[112]33], coupled with an up-regulation of
   E-cadherin, an epithelial hallmark [[113]34]. These molecular
   alterations underscore Myricetin's capacity to directly counteract the
   EMT process, providing a plausible therapeutic mechanism for its action
   against DN.

   Delving deeper into the molecular underpinnings of Myricetin's action,
   our utilization of network pharmacology and molecular docking
   approaches revealed a notable interaction between Myricetin and NADPH
   oxidase 4 (Nox4). This interaction suggests Myricetin's potential role
   in modulating Nox4 expression and ROS generation, which plays a
   critical role in exacerbating DN. Furthermore, our findings extend to
   the identification of Myricetin's inhibitory effects on the NF-κB
   signaling pathway, a key regulator of inflammatory and fibrotic
   responses. The suppression of NF-κB activity by Myricetin likely
   mediates the observed downregulation of Snail, further impeding the EMT
   process and its contribution to renal fibrosis in DN.

   Our research identifies Myricetin as a potent candidate for treating DN
   [[114]35], primarily by inhibiting Nox4 expression, reducing ROS
   levels, and suppressing the NF-κB signaling pathway. These mechanisms
   work together to weaken Snail expression and reverse the
   epithelial-mesenchymal transition (EMT) process, presenting a
   comprehensive strategy against the challenges of DN. Additionally, as a
   natural compound, Myricetin offers a cost-effective alternative to
   synthetic drugs, potentially improving patient accessibility.

   However, the clinical application of Myricetin is limited by its low
   bioavailability in the body, a challenge that can be mitigated through
   advanced delivery systems such as nanomaterials. For instance, a recent
   study demonstrated that Myricetin-loaded solid lipid nanoparticles
   (SLNs) significantly alleviate DN pathology more effectively than
   Myricetin in its natural form by reducing oxidative stress and
   enhancing antioxidant enzyme levels [[115]36]. This highlights that SLN
   encapsulation successfully addresses the bioavailability issues of
   Myricetin, underscoring its potential as a valuable natural compound
   for future clinical needs.

5. Conclusions

   Through an integrative approach that combines network pharmacology with
   advanced bioinformatics, our study provides detailed insights into the
   therapeutic mechanisms and key molecular targets of Myricetin in the
   modulation of EMT in diabetic nephropathy. Our findings illuminate
   Myricetin's action through the modulation of the NOX4/NF-κB/Snail
   signaling pathways, alongside a significant reduction in ROS
   production, delineating a multi-targeted, safe therapeutic strategy for
   DN. This investigation not only highlights Myricetin as a potent
   therapeutic agent with the potential to ameliorate DN but also sets a
   foundation for further clinical research to exploit its therapeutic
   benefits fully. The insights gained here advocate for Myricetin's
   inclusion in future treatment paradigms for diabetic nephropathy,
   emphasizing the critical role of targeted molecular interventions in
   overcoming this complex disease.

Funding

   This work was supported by the National Natural Scientific Foundation
   of China (Grant numbers 82174322 and 82074257), the Natural Science
   Foundation of Guangdong Province (Grant numbers 2023A1515011200,
   2023A1515011483), the Innovation Team Project of Guangdong Province
   (Grant number 2023KCXTD019)

Institutional review board statement

   This study was reviewed and approved by the Animal Ethics Committee of
   Southern Medical University with the approval number: SMUL2022007. The
   date of resolution: March 10, 2022.

Data availability statement

   Data are contained within the article.

CRediT authorship contribution statement

   Ningning Yuan: Writing – original draft, Visualization, Validation,
   Project administration, Methodology, Data curation. Yuchi Chen: Writing
   – original draft, Visualization, Software, Data curation,
   Conceptualization. Yangtian Yan: Software, Resources, Investigation,
   Formal analysis, Data curation. Fujing Wang: Methodology. Xinyao Xu:
   Data curation. Mingqing Wang: Validation, Project administration,
   Funding acquisition. Jianxin Diao: Validation, Resources, Project
   administration. Wei Xiao: Writing – review & editing, Supervision,
   Resources, Project administration, Funding acquisition,
   Conceptualization.

Declaration of competing interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Footnotes

   ^Appendix A

   Supplementary data to this article can be found online at
   [116]https://doi.org/10.1016/j.heliyon.2024.e35234.

Contributor Information

   Mingqing Wang, Email: mingqing@smu.edu.cn.

   Jianxin Diao, Email: jianxindiao@163.com.

   Wei Xiao, Email: xw7688@smu.edu.cn.

Appendix A. Supplementary data

   The following are the Supplementary data to this article:
   Multimedia component 1
   [117]mmc1.txt^ (2KB, txt)
   Multimedia component 2
   [118]mmc2.xlsx^ (11.5KB, xlsx)
   Multimedia component 3
   [119]mmc3.xlsx^ (10.9KB, xlsx)
   Multimedia component 4
   [120]mmc4.csv^ (723B, csv)

References