Kaempferol Attenuates Gouty Arthritis by Regulating the Balance of Th17/Treg Cells and Secretion of IL-17

Kaempferol is a common flavonoid aglycone widely found in plants. It exhibits beneficial therapeutic effects in the treatment of arthritis. However, the effects of kaempferol on gouty arthritis (GA) have not been verified. This study aimed to explore the potential mechanisms by which kaempferol regulates GA by network pharmacology and experimental validation. Potential drug targets for GA were identified with a protein–protein interaction network. Then, we performed a KEGG pathway analysis to elucidate the major pathway involved in the kaempferol-mediated treatment of GA. In addition, the molecular docking was performed. A rat model of GA was constructed to verify the results of network pharmacology analysis and investigate the mechanism of kaempferol against GA. The network pharmacology study indicated that there were 275 common targets of kaempferol and GA treatment. Kaempferol exerted therapeutic effects on GA, in part, by regulating the IL-17, AGE-RAGE, p53, TNF, and FoxO signaling pathways. Molecular docking results showed that kaempferol stably docked with the core MMP9, ALB, CASP3, TNF, VEGFA, CCL2, CXCL8, AKT1, JUN, and INS. Experimental validation suggested that kaempferol eased MSU-induced mechanical allodynia, ankle edema, and inflammation. It significantly suppressed the expression of IL-1β, IL-6, TNF-α, and TGF-β1 and restored Th17/Treg imbalance in MSU-induced rats and IL-6-induced PBMCs. Kaempferol also affected RORγt and Foxp3 through IL-17 pathway. The present study clarifies the mechanism of kaempferol against GA and provides evidence to support its clinical use.


INTRODUCTION
Gouty arthritis (GA) is the most common inflammatory arthritis worldwide.It is a painful inflammatory disease induced by the deposition of monosodium urate (MSU) crystals in the joints and periarticular tissues [1].Currently, benzbromarone, colchicine, glucocorticoids, indomethacin, and probenecid are commonly used in the treatment of GA.These drugs have a beneficial anti-inflammatory and analgesic effect, promoting uric acid excretion and inhibiting uric acid production.However, they have many side effects, such as gastrointestinal reactions, allergies, and kidney toxicity [2,3].Therefore, the exploration of safe and novel compounds for GA treatment has become an important issue.
Kaempferol is a major flavonoid aglycone found in many natural products, such as beans, cabbage, fennel, and Kaempferia rotunda L. [4].Kaempferol and its glycosylated derivatives have anti-inflammatory, antitumour, antidiabetic, antioxidant, cardioprotective, neuroprotective, and antimicrobial activities [5][6][7].Our previous research showed that kaempferol inhibited the migration and invasion of fibroblast-like synoviocytes and joint destruction in rheumatoid arthritis [8].However, to our knowledge, the molecular mechanism of kaempferol in GA has not yet been reported.
Network pharmacology is a practical tool that integrates multiple subjects, including bioinformatics, chemoinformatics, traditional pharmacology, and network biology [9].It aims to study the complex, varied relationships among targets, drugs, diseases, and pathways, which presents a new approach for clarifying a drug's possible pharmacological mechanisms [10].Therefore, the present work combined network pharmacology and experimental validation to clarify the mechanism of action of kaempferol against GA to provide a novel candidate for GA treatment.
In this study, firstly, a protein-protein interaction network for GA was used to identify the potential drug targets.KEGG pathway analysis was then performed to elucidate the signaling pathway in the kaempferol-mediated treatment of GA.In addition, experimental evidence showed positive effects of kaempferol in treating a rat model of MSU-induced GA, which revealed the underlying mechanisms of the role it plays in treating GA.This research provides evidence that kaempferol has potential as a therapeutic agent for GA.

Construction of the Kaempferol-GA PPI Network
The targets of kaempferol and GA treatment were imported into Jvenn (http:// jvenn.toulo use.inra.fr/ app/ examp le.html).A Venn diagram with the common targets of kaempferol and GA treatment was obtained.
The common targets were then imported into the STRING database (https:// www.string-db.org/) to obtain the kaempferol-GA PPI network, together with the organism set to Homo sapiens, and the minimum required interaction score was set to medium confidence (0.400).Text mining, experiments, and databases were selected as the interaction sources.
The network was imported into the Cytoscape 3.9 software to screen out the core kaempferol-GA PPI network through the maximal clique centrality (MCC) algorithm in the CytoHubba plug-in.

Enrichment Analysis
The R package "ClusterProfiler" (http:// www.bioco nduct or.org/) was used to carry out Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of common targets of kaempferol and GA treatment.The GO enrichment analysis items were biological process (BP), cellular component (CC), and molecular function (MF).The organism was set to Homo sapiens, and the P value was set to ≤ 0.05.

Kaempferol-Target-KEGG Pathway Network Construction
The affiliation between kaempferol, common targets, and the top 30 enriched KEGG pathways was further imported into the Cytoscape 3.9 software to construct the kaempferol-target-KEGG pathway network.The network was analyzed using the network analysis function.

Molecular Docking
Kaempferol and the top 10 core targets of the kaempferol-GA PPI network were used for molecular docking to understand the docking mode and the reliability of the docking.The structure of kaempferol was retrieved from TCMSP database.The structures of target proteins ALB (PDB ID: 6YG9), TNF (PBD ID: 7IRA), AKT1 (PBD ID: 1UNR), MMP9 (PBD ID: 4XCT), JUN (PBD ID: 5FV8), CASP3 (PBD ID: 1NME), CXCL8 (PBD ID: 1QE6), VEGFA (PBD ID: 4GLS), INS (PBD ID: 3W7Y), and CCL2 (PBD ID: 1DOK) were obtained from RCSB PDB database (https:// www.rcsb.org/).The target proteins and the ligand were imported into Auto-DockTools 1.5.7 to perform molecular docking.Before docking, water molecules, co-crystallized ligands and ions in the protein were removed, and hydrogens and Kollman partial charges were added.The docking results were visualized using the PyMOL 2.4.0 and Ligplus 2.25 software.

Animals
Forty-two male Sprague − Dawley (SD) rats (180 − 220 g) were housed in a room with a 12/12 h light/ dark cycle at 22 ± 1 °C and 65 ± 5% humidity.Ethical clearance was approved by the Research Ethics Committee of the First Affiliated Hospital, Guangzhou University of Chinese Medicine.All experiments of animals were conducted in conformity with the Animal Care and Use Committee of Institute of Materia Medica, PR China.

Induction of GA in Rats with MSU
The rats were randomly divided into eight groups (n = 6): normal, model, indomethacin (3.0 mg/kg), colchicine (1.5 mg/kg), low-dose (50 mg/kg), middle-dose (100 mg/kg), and high-dose (200 mg/kg) kaempferol.The dosages of kaempferol were based on our previous report [8].Sterile water was used to make an MSU suspension (25 mg/mL), which was injected (0.2 mL) into the medial side of the right ankle joint of the hind limbs in the model, indomethacin, colchicine, and kaempferol groups based on previous studies tested in rodents [3].The normal group was injected with 0.2 mL saline.The successful establishment of the gouty arthritis model was estimated by obvious swelling and mechanical hyperalgesia 2 h after MSU injection [11].Indomethacin, colchicine, and kaempferol were injected intraperitoneally 3 days before the day of MSU injection for 7 consecutive days, and the model group and the normal group were given normal saline.

Evaluation of Ankle Joint Hypersensitivity and Edema
We applied a plantar test apparatus (Ugo Basile, Italy) to assess hypersensitivity to heat stimulation (heat hyperalgesia) in rats.Experimental rats were habituated for 30 min prior to this test, after which the right hind paw was irradiated with a radiation beam from a light bulb to determine the paw withdrawal latency (the time spent removing the paw from the stimulus).To avoid excessive heating that could lead to injury, a 20 s cut-off threshold was set.Significant decreases in paw withdrawal latency were interpreted as heat hyperalgesia according to the previous report [12].

N. Li et al
The degree of joint swelling was quantified by measuring the ankle circumference at 4 h, 8 h, 24 h, 48 h, and 72 h after the MSU injections.The ankle swelling ratio (%) = (ankle circumference after MSU injection/ankle circumference before MSU injection − 1) × 100%.
All of the tests above were conducted by an experimenter blinded to the experimental conditions.

Histopathological Assessment of the Ankle Joints
The ankles of the experimental rats were harvested and fixed in 4% paraformaldehyde for 24 h, and then decalcified in 10% decalcification solution for 1 month, embedded in paraffin and sectioned.Hematoxylin and eosin (H&E) staining was performed on the knee sections.The number of infiltrating inflammatory cells in each observation field of diverse groups was calculated in a completely blinded manner using × 40 and × 100 objectives and normalized with different groups.

Real-time Quantitative PCR (RT-qPCR)
Total RNA of the ankle joint samples were extracted using TRIzol reagent (Invitrogen).The RNA was reverse transcribed into cDNA using TaKaRa Pri-meScript RT reagent kit in accordance with the manufacturer's instructions (TaKaRa Bio, Shiga, Japan).RT-qPCR was performed using the Bio-Rad CFX96 system with the SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) according to the manufacturer's protocol.The instrument was programmed as described below: 40 cycles of 5 s denaturation at 95 ℃ and 34 s amplification at 60 ℃.Gene corresponding to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal standard.The relative levels of mRNAs were assessed by 2 −ΔΔCT method.

Cell Viability Assays
The isolated PBMCs were obtained as reported previously [13].Cells were incubated with kaempferol (0, 5, 10, 20, and 40 μM) for 24 h and then treated with MTT.After that, the medium was then removed, DMSO was added, and optical density (OD) was measured using a microplate reader.

Flow Cytometry
The PBMCs were isolated from the blood of rats.Antibodies against CD4 and IL-17A were selected for Th17 cell staining; meanwhile, CD4, CD25, and Foxp3 were used for Treg staining.In vitro, the isolated PBMCs were stimulated with IL-6 (20 ng/ml) and treated with or without kaempferol.Finally, flow cytometry was used to analyze the stained cells.

Statistical Analysis
All experiments were performed at least three times.All data were first subjected to normality test and expressed as mean ± standard deviation.One-way ANOVA was performed with GraphPad Prism 9.0 for comparisons and analysis.A p-value < 0.05 was considered statistically significant.

Kaempferol-GA PPI Network Analysis
A schematic flow chart of the study was shown in Fig. 1.
The 2D structure of kaempferol was obtained (Fig. 2a).Two hundred and eighty-nine targets of kaempferol were retrieved from CTD, SEA, SymMap, Target-Net, TCMSP, and Swiss Target Prediction databases.Eleven thousand nine hundred and twenty seven targets of GA treatment were retrieved from DisGeNET, Phenopedia, OMIM, TTD, GeneCards, CTD, and DRUGBANK databases.Two hundred and seventy-five common targets of kaempferol and GA treatment were obtained and are shown in a Venn diagram (Fig. 2b).The kaempferol-GA PPI network (Fig. 2c) shows the interactions among the common targets.

Enrichment Analysis
GO enrichment analysis and KEGG pathway enrichment analysis of the common targets of kaempferol and GA treatment were performed using the R package "ClusterProfiler." The analysis results of the GO functions, including the top 10 enriched biological processes, cellular components, and molecular functions, are shown in a bar chart (Fig. 3a-c).For the biological processes, the common targets were mainly enriched in rhythmic process, cellular response to chemical stress, response to oxidative stress, cellular response to oxidative stress, peptidyl-serine phosphorylation, response to xenobiotic stimulus, response to reactive oxygen species, peptidyl-serine modification, response to peptide, and positive regulation of MAPK cascade.For cellular components, the targets were enriched in protein kinase complex, serine/threonine protein kinase complex, transcription regulator complex, cyclin-dependent protein kinase holoenzyme complex, transferase complex, transferring phosphorus-containing groups, membrane raft, membrane microdomain, plasma membrane raft, endoplasmic reticulum lumen, and cytoplasmic side of membrane.For molecular functions, the targets were enriched in protein serine/threonine/tyrosine kinase activity, protein serine/threonine kinase activity, protein serine kinase activity, nuclear receptor activity, ligand-activated transcription factor activity, RNA polymerase II-specific DNA-binding transcription factor binding, DNA-binding transcription factor binding, alditol: NADP + 1 − oxidoreductase activity, nuclear receptor binding, and cytokine receptor binding.
These GO functions and pathways may be the potential mechanisms of kaempferol in GA treatment.

Kaempferol-Target-KEGG Pathway Network Analysis
The relationships between kaempferol, common targets of kaempferol and GA treatment, and the top 30 enriched KEGG pathways are shown in Fig. 4. The network analysis results showed that AKT1, PIK3R1, RELA, MAPK3, and IKBKB had relatively more connected edges than other targets.Among the pathways, IL-17 signaling pathway, p53 signaling pathway, TNF signaling pathway, apoptosis, FoxO signaling pathway, MAPK signaling pathway, HIF-1 signaling pathway, NFkappa B signaling pathway, osteoclast differentiation, EGFR tyrosine kinase inhibitor resistance, and PI3K-Akt signaling pathway are closely related to the treatment of GA with kaempferol.These pathways are mainly related to T cell function, inflammation, cell proliferation, cell differentiation, and apoptosis.The core network of the  The target with the lowest binding energy is MMP9 (PBD ID: 4XCT) with binding energy of − 9.87 kcal/mol.

Kaempferol Alleviated MSU-Induced Mechanical Allodynia, Ankle Edema, and Inflammation
We first investigated the effect of kaempferol on the degree of joint swelling in a rat model induced by MSU.As expected, injection of MSU significantly induced ankle swelling, which usually appeared 2 h after injection and persisted up to 72 h.We found that indomethacin, colchicine, and kaempferol significantly reduced ankle swelling of rats with MSU treatment (Fig. 6a).We then assessed the effect of kaempferol on pain induced by MSU using a plantar test apparatus.We found that MSU injection resulted in significant mechanical allodynia, represented by a reduction in the 50% paw withdrawal threshold.The results also showed that kaempferol, indomethacin, and colchicine had significant anti-allodynia effects (Fig. 6a).
These results show that kaempferol can inhibit MSU-induced ankle edema and mechanical allodynia.
We then used histopathological assessment to examine the effects of kaempferol on inflammatory cell infiltration and joint destruction induced by MSU in the ankle joint.Kaempferol, indomethacin, and colchicine all reduced MSU-induced inflammatory cell infiltration in the ankle joint (Fig. 6b-c).Bone destruction was not observed because of the short duration of acute GA.

Kaempferol Affected the Core Target Expression and Th17/Treg Imbalance in Rats
We verified the results of network pharmacology.As shown in Fig. 7a, Kaempferol significantly downregulated the expression of MMP9, CASP3, TNF-β, VEGFA, CCL2, CXCL8, AKT1, and JUN in GA rats.We also found that kaempferol upregulated ALB and INS expression.
The proportion of Th17 cells in the blood of MSUinduced rats were significantly increased, and this effect was alleviated by kaempferol.In addition, the proportion of Treg cells were significantly decreased, and this effect was enhanced by kaempferol.Kaempferol effectively restored Th17/Treg imbalance in MSU-induced rats (Fig. 7b).

Kaempferol Affected the Cytokine Expression
In the rats with MSU treatment, in comparison with the normal group, the levels of IL-1β, IL-6, and TNF-α increased, while the levels of TGF-β1 decreased in the model group.Kaempferol significantly downregulated the expression of IL-1β, IL-6, and TNF-α and upregulated TGF-β1 expression (Fig. 8a).We isolated PBMCs in MSU-induced rats for in vitro culture and treated with kaempferol.As depicted in Fig. S1, kaempferol (0, 5, 10, and 20 μM) did not inhibit cell viability after 24 h of intervention.We then used IL-6 and kaempferol (0, 5, 10, and 20 μM) to intervene the cells, and found that kaempferol could reduce IL-6-induced IL-1β, IL-6, and TNF-α levels and increase IL-6-induced TGF-β1 level (Fig. 8b).

Kaempferol Restored IL-6-Induced Th17/Treg Imbalance and Affected Transcription Factor Expression of PBMCs Through IL-17 Pathway
In order to investigate whether kaempferol reduces the disease severity of GA via regulation of Th17 and Treg balance, we extracted PBMCs from MSU-induced rats, stimulated cell activation with IL-6, and intervened with different concentrations of kaempferol.As shown in Fig. 9a, kaempferol treatment inhibited the proportion of Th17 cells and increased the proportion of Treg cells.Kaempferol effectively restored IL-6-induced Th17/Treg imbalance.Kaempferol significantly downregulated the expression of RORγt and upregulated Foxp3 in IL-6-induced PBMCs and in MSU-induced rats (Fig. 9b).Th17 secretes IL-17, which has six family members, we measured the expression of common IL-17A, IL-17E, and IL-17F.We found that kaempferol treatment decreased IL-17A, IL-17E, and IL-17F levels in IL-6-induced PBMCs and in MSU-induced rats (Fig. 9c-d), revealing that the treatment of GA by kaempferol is related to the IL-17 signaling pathway.

DISCUSSION
A certain number of natural products derived from plants have been found to have anti-GA properties.Compared to conventional synthetic chemical drugs, they might have fewer side effects.Kaempferol, one of the active components extracted from various plants [14], has been demonstrated to exhibit a broad spectrum of biological activities [15], including antiarthritis [8], antioxidative stress [16], antitumor [17], immunomodulatory [18], and bone protective activities [19].However, whether kaempferol has the potential to treat GA is currently unknown.
In the present study, the potential components and targets of kaempferol were analyzed through a network pharmacology approach, and common targets, enriched biological processes and pathways were discovered.In addition, the molecular docking was performed.Furthermore, we estimated the anti-inflammatory and analgesic effects of kaempferol in a rat model induced by MSU.We also observed the effect on IL-17 signaling pathway and related cytokine expression of kaempferol, providing a basis for the development of more effective and less toxic natural medicines for GA, which has not been reported in previous articles.
The core of network pharmacology is constructing a "network target."It is a potentially useful tool can help to illustrate the function of complex biological systems, and it also has theoretical and methodological implications for drug design [20].For biological processes, the common targets of kaempferol and GA treatment are mainly enriched in metabolic processes, response to oxidative stress and inflammatory processes.Kaempferol has been found to have potent antidiabetic and antiobesity properties, which suggest therapeutic effects of kaempferol  for metabolic disorders [21,22].It is also reported that kaempferol achieves the bone-protective effects by inhibiting adipogenesis, inflammation, oxidative stress, osteoclastic autophagy, and osteoblastic apoptosis while activating osteoblastic autophagy [23].Our previous report has shown that kaempferol inhibited the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes by blocking activation of the MAPK pathway [8].These observations suggest therapeutic effects of kaempferol for the treatment of joint diseases.
GA is a chronic disease caused by the deposition of MSU crystallites in joints and tissues, and its pathogenesis is similar to obesity and metabolic syndrome, both of which are closely related to metabolic disorders [24,25].In this study, an experimental model of GA was created via the injection of MSU.Unlike humans, rats express the enzyme uricase.Thus, the acute inflammation in the GA model would self-heal in approximately 3 days [26].Based on previous reports, we treated each group before and after MSU injection [3].We showed that kaempferol remarkably reduced ankle edema, mechanical allodynia, and macroscopic inflammation scores in ankle joint induced by MSU and was as effective as indomethacin and colchicine.
We further investigated the mechanisms of kaempferol intervention for GA.The results of KEGG pathway analysis indicated that kaempferol mainly regulated the IL-17, AGE-RAGE, p53, TNF, and FoxO signaling pathways, etc. IL-17 is a proinflammatory cytokine produced by activated T cells, and its major transcription factor is RORγt [27].The IL-17 cytokine has 6 subtypes, including IL-17A (IL-17), IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F [28].IL-17 can promote the activation of T cells, if Th17/Treg balance is deviated in favor of Th17 cells and against Treg cells, the symptoms of GA were exacerbated [29,30].IL-17 also stimulate endothelial cells, fibroblasts, and other cells to produce cytokines such as IL-1β, IL-6, and TNF-α, resulting in the production of inflammation [31].In contrast, Foxp3 and TGF-β1 negatively regulate IL-17 production [32].IL-17 blocking agents have been widely used to treat various arthritic conditions [33,34].Recent studies have proven that the application of a neutralizing antibody against IL-17 effectively eases joint symptoms, swelling and leukocyte infiltration into the inflamed tissue of GA [30,35].Investigators have accrued compelling evidence that the IL-17 pathway is central to the pathogenesis of arthritis [29].In the present study, we demonstrated that kaempferol significantly suppressed the expression of MMP9, CASP3, TNF, VEGFA, CCL2, CXCL8, AKT1, JUN, IL-1β, IL-6, TNF-α, and RORγt and upregulated ALB, INS, TGF-β1, and Foxp3 expression.Kaempferol also restored IL-6-induced Th17/Treg imbalance and inhibited inflammatory factor release of PBMCs through IL-17 pathway.These experimental results are consistent with those of the network pharmacology.

Fig. 1
Fig.1The graphical abstract of this study.

Fig. 2 a
Fig. 2 a The 2D structure of kaempferol.b Venn diagram of kaempferol and GA treatment.c Kaempferol-GA PPI network.

Fig. 4 a
Fig. 4 a Kaempferol-target-KEGG pathway network of kaempferol, common targets of kaempferol and GA treatment, and top 30 enriched KEGG pathways.b Core kaempferol-GA PPI network.c The IL-17 signaling pathway.

Fig. 6
Fig. 6 Therapeutic effect of kaempferol on the MSU-induced GA rat model.a Kaempferol alleviated MSU-induced mechanical allodynia and ankle edema.b H&E staining of ankle joints.Effects of kaempferol on inflammatory cell infiltration in ankle joint tissues on histopathological examination.c The inflammation scores of H&E staining.n = 6 mice per group.L-Kae: Low dose kaempferol, M-Kae: Middle dose kaempferol, H-Kae: High dose kaempferol.* P < 0.05, ** P < 0.01 vs. model group.

Fig. 9
Fig. 9 Kaempferol restored IL-6-induced Th17/Treg imbalance and inhibited inflammatory factor release of PBMCs through IL-17 pathway.The isolated PBMCs were isolated from the blood of rats.a Effects of kaempferol on the percentage of Th17 (CD4 + IL-17A +) and Treg (CD4 + CD25 + Foxp3 +) and the ratio of Th17/Treg in the IL-6-induced PBMCs.b Effects of kaempferol on RORγt and Foxp3 expression in the IL-6-induced PBMCs and in MSU-induced rats.c Effects of kaempferol on IL-17A, IL-17E, and IL-17F levels in the IL-6-induced PBMCs and in MSU-induced rats (d).* P < 0.05, vs. model group or IL-6 model.

Table 1
The Top 10 Core Targets of the Kaempferol-GA PPI Network