Quercetin Ameliorates Renal Injury and Pyroptosis in Lupus Nephritis through Inhibiting IL-33/ST2 Pathway In Vitro and In Vivo

Lupus nephritis (LN) is a common and serious symptom in patients with systemic lupus erythematosus (SLE). Tubular interstitial fibrosis is a common underlying mechanism in the development of lupus nephritis to end-stage renal failure (ESRD). Quercetin is widely proven to prevent tissue fibrosis. Therefore, the purpose of this study is to investigate the beneficial effects of quercetin on the inhibition of fibrosis and inflammation pathways in in vitro and in vivo lupus nephritis models. In the current study, MRL/lpr mice as animal models, and HK-2 human renal tubular epithelial cells were stimulated by interleukin-33 (IL-33) to mimic the cellular model of lupus nephritis. Immunohistochemical staining, immunoblotting assay, immunofluorescence staining, and quantitative real-time polymerase chain reaction assay were used. The in vivo results showed that quercetin improved the renal function and inhibited both fibrosis- and inflammation-related markers in MRL/lpr mice animal models. The in vitro results indicated that quercetin ameliorated the accumulation of fibrosis- and inflammation-related proteins in IL-33-induced HK-2 cells and improved renal cell pyroptosis via the IL33/ST2 pathway. Overall, quercetin can improve LN-related renal fibrosis and inflammation, which may offer an effective potential therapeutic strategy for lupus nephritis.


Introduction
Systemic lupus erythematosus (SLE) is the prototype systemic autoimmune disease, characterized by chronic and aberrant immune activation against nuclear self-antigens and drives the production of pathogenic autoantibodies and activation of immune complexes (ICs) that mediate organ and tissue damage, especially in the skin and kidneys [1]. Lupus nephritis (LN) is the most serious manifestation in patients with SLE, and about 35-60% to investigate whether quercetin has anti-fibrosis and anti-inflammation effects on both MRL/prl mice in vivo and IL-33-induced lupus nephritis in vitro cell models, and we also aim to elucidate the underlying mechanism, which might contribute to better utilization of quercetin for lupus nephritis therapy.

Reagent Preparation
The yellow powder quercetin was purchased from Sigma-Aldrich (St. Louis, MI, USA) (catalog# 6151-25-3) and stored as a stock solution with a concentration of 100 mM in dimethyl sulfoxide (DMSO). Recombinant human IL-33 (carrier-free) was purchased from Biolegend (San Diego, CA, USA) (catalog# 581802) and stored as a stock solution at a concentration of 200 µg/mL in 0.1% bovine serum albumin (BSA). During the in vitro experiment, each stock solution was diluted to the desired final concentration in the medium, while DMSO and/or 0.1% BSA served as a vehicle control.

MRL/lpr Mice
MRL/lpr mice (characterized by lpr mutations associated with the Fas gene) are a model of spontaneous SLE that develop lupus with human-like symptoms, such as erythema and nephritis with proteinuria [29][30][31]. The 8-week-old MRL/lpr female mice were a generous gift from Dr. Yong-Han, Hong (Department of Nutrition, I-SHOU University, Kaohsiung City, Taiwan). All mice were divided into two groups: (1) SLE group: MRL/lpr mice (n = 5); (2) Quercetin group: MRL/lpr mice were given 40 mg/kg quercetin by gavage daily (n = 5). Mice were gavaged with quercetin dissolved in sterile water in a suspension form after shaking while the control group was also gavaged with sterile water every day. The body weight was recorded, and the state of the mice was observed every week. The whole experimental period was three months. At the end of the experiment, the mice were euthanized with mixture solution (1 mL Zoletil and 0.1 mL Rompun in 3.9 mL normal saline), and brain, spleen, kidneys, and uterine weights were recorded, as well as blood being collected. Harvested samples were used for analyzing the expression of specific proteins mainly in the kidneys. All animal studies were conducted according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University (LAC-2016-0215).

Renal Function Assessment
Renal function was assessed through the measurement of the kidney index, blood urea nitrogen (BUN), and plasma creatinine (Cr) concentration [32]. The kidney index (mg/g) was calculated as the ratio of the weight of the two kidneys to the body weight of the mice. BUN and Cr were determined by the Department of Medical Laboratory (Taipei Medical University, Taipei, Taiwan).

Immunohistochemical Staining
Kidney tissue specimens were fixed using 4% paraformaldehyde and embedded in paraffin, and the subsequent staining was entrusted to BIO-CHECK LABORATORIES LTD (New Taipei City, Taiwan) for processing. Kidney sections were stained with Hematoxylin and Eosin (H&E) stain, Sirius Red stain, and Toluidine Blue stain, as well as different antibodies IL-33 (1:400, Affinity Biosciences, Cincinnati, OH, USA), NLRP3 (1:400, Novus Biologicals, Centennial, CO, USA), and IL-1β (1:400, Abcam, Cambridge, UK), and the expression changes were recorded using an EVOS ® FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). The determination of tubulointerstitial injury, degree of interstitial collagen deposition, and tubulointerstitial infiltration was based on previous literature [28,33,34].

Cell Culture and Cell Viability
HK-2 (human kidney 2, human renal tubule epithelial cell line) was purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and grown in Dulbecco's Modified Eagle Medium/Ham's F-12 Medium in a 1:1 ratio (CAISSON Labs, Smithfield, UT, USA), which was supplemented with 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA), 100 U/mL penicillin, and streptomycin in a humidified incubator at 37 • C with 5% CO 2 .

Immunofluorescence Staining
HK-2 cells (5 × 10 4 ) were seeded in a 6-well microplate with glass slides (18 × 18 mm). After the cells were adhered stably, IL-33 and quercetin were added into the cells, as explained in figure legends. HK-2 cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min, then permeabilized with 0.25% Triton X-100 for 10 min and blocked by 5% bovine serum albumin (BSA) for 30 min. Adherent cells were washed twice with PBS at room temperature (RT) between each step on a shaker. HK-2 cells were then incubated

Cytoplasmic and Nucleus Extraction Preparation
Nuclear and cytoplasmic proteins were isolated with nuclear and cytoplasmic extraction reagents, according to the manufacturer's protocol (Nuclear/Cytosol Fractionation Kit, BioVision, Waltham, MA, USA), and the expression levels of anti-NF-κB (Cell Signaling Technology, Beverley, MA, USA) in the cytoplasmic or nuclear fractions were determined, respectively. Anti-α-tubulin (Abcam, Cambridge, UK) and anti-histone H3 (Abcam) were used as housekeeping genes to determine cytoplasmic and nuclear protein extraction purity.

Western Blotting Analysis
The extraction procedure for total protein was the same as in our previous study [35]. Briefly, HK-2 cells treated with a combination of IL-33 and quercetin were extracted with ice-cold lysis buffer containing protease (Roche, Basel, Switzerland) and/or phosphatase inhibitor cocktail (Roche). Cell lysates were quantified at 562 nm using an ELISA reader (BioTek, Winooski, VT, USA) after reaction with T-Pro BCA Protein Assay Kit (OMICS BIOTECHNOLOGY CO., LTD., New Taipei City, Taiwan). The proteins were mixed with SDS-PAGE loading buffer and boiled for 5 min before being subjected to SDS-PAGE gels, followed by protein transfer to a PVDF membrane. After blocking non-binding sites with 5% BSA (BioShop, Burlington, ON, Canada) for 1 h at RT, the membrane was sequentially incubated with primary antibodies of interest (at 4 • C overnight) and the corresponding goat anti-rabbit/mouse secondary antibody IgG (Abcam, Cambridge, UK) (for 1 h at RT). Membranes were washed three times with Tris-buffered saline buffer with Tween-20 (TBST, OMICS BIOTECHNOLOGY CO., LTD.) for 30 min (60 rpm) before changing each buffer. After reacting with electrochemiluminescence (ECL; Thermo Fisher Scientific) in the dark, imaging was performed with an eBlot Touch ImagerTM (eBlot Photoelectric Technology, Shanghai, China). Densitometric estimations were quantified using the Image J software  Supplementary Table S1.

RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)
Procedures of total RNA isolation, reverse transcription, and qPCR were modified from our previous study [35]. In brief, total RNA from HK-2 cells treated with a combination of IL-33 and quercetin-treated was extracted using TRIzol™ Reagent (Invitrogen, Carlsbad, CA, USA) followed by purification with Direct-zol RNA MiniPrep (Thermo Fisher Scientific, Waltham, MA, USA), and samples were quantified at 260 and 280 nm using an ELISA reader (BioTek, Winooski, VT, USA). The cDNA was reverse transcribed from 2 µg of total RNA using the PrimeScript ® RT Kit (Takara, Kusatsu, Shiga, Japan), followed by mixing with gene-specific primers and Power SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The mRNA level of the gene of interest was analyzed using the Applied Biosystems StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), and the amplification conditions were: denaturation at 95 • C for 10 min followed by 40 cycles for 15 s at 95 • C and 30 s at 60 • C. Synthesis of DNA product of the expected size was confirmed by DNA electrophoresis and melting curve analysis. All reactions were performed in triplicate, and relative gene expression was calculated by normalization to GAPDH and calculated by 2 −∆∆Ct method. The primer sequences used are detailed in Supplementary Table S2.

Statistical Analysis
All data were presented as the mean ± standard deviation (SD). For comparison of two groups, statistical differences between the means were analyzed by Student's t-test using the SigmaPlot, version 12.5 (SoftHome, Taipei, Taiwan). The difference between two means was considered statistically significant when p < 0.05 and highly significant when p < 0.001.

Quercetin Relieves Symptoms of Lupus Nephritis in MRL/lpr Mice Model
MRL/lpr mice are often used as an animal model to study lupus erythematosus nephritis [31]; the experimental process is shown in Figure 1A. The results showed that the body weight of MRL/lpr mice did not change significantly during the whole experimental period ( Figure 1B), while it was found that daily treatment with 40 mg/kg quercetin induced hyperactivity, but there was no significant difference in the weight and appearance of the brains among the groups (results not shown). The spleen is a crucial organ in the occurrence of immune inflammation. Our results found that the spleen weight of MRL/lpr mice in the quercetin group was significantly lower than that in the untreated group ( Figure 1C), indicating that quercetin reduced the enlargement of the spleen and improved the abnormal immune function or the inflammatory status of MRL/lpr mice. In addition, our results showed that the kidneys of treated MRL/lpr mice were heavier than the untreated group, which may be related to the improvement of renal atrophy ( Figure 1C). Furthermore, quercetin significantly reduced the BUN and creatinine concentration of treated MRL/lpr mice, compared with the untreated group ( Figure 1D).
( Figure 1C), indicating that quercetin reduced the enlargement of the spleen and improved the abnormal immune function or the inflammatory status of MRL/lpr mice. In addition, our results showed that the kidneys of treated MRL/lpr mice were heavier than the untreated group, which may be related to the improvement of renal atrophy ( Figure  1C). Furthermore, quercetin significantly reduced the BUN and creatinine concentration of treated MRL/lpr mice, compared with the untreated group ( Figure 1D).

Quercetin Attenuates the Pathological Changes in MRL/lpr Mice Model
To confirm that quercetin has a protective effect on the kidney tissue of MRL/lpr mice, immunohistostaining was used. The morphological alterations were evaluated by H&E staining, collagen deposition in the tubulointerstitium was assessed by Sirius Red staining, as well as mast cell infiltration into the tubulointerstitium being measured by Toluidine Blue staining. As shown in Figure 1E, the structure of the kidney tissue was scattered in the untreated MRL/lpr group, while daily administration of 40 mg/kg quercetin improved renal integrity and the level of collagen deposition ( Figure 1E). However, the differences in the degree of staining after quantification did not confirm the improvement of tubulointerstitial infiltration by quercetin due to the excessively dispersed renal tissue architecture in the untreated MRL/lpr mice group.
In addition, we measured inflammation-related indicators in kidney tissue of MRL/lpr mice using immunohistochemical staining and found that quercetin showed a tendency to reduce the distribution of positive staining indicators IL-33, NLRP3, and IL-1β ( Figure 1F). Summarizing the results of animal experiments, we speculate that quercetin has potential benefits in ameliorating the symptoms and progression of lupus nephritis.

Quercetin Reduces IL-33-Induced Fibrosis in Human Renal Tubular Epithelial Cell Line
Based on the abundant expression of IL-33 cytokines in the kidney tissue of MLR/lpr mice, we speculated that IL-33 may be involved in the development of LN. To verify the ameliorating effect of quercetin on lupus nephritis phenotype, we first used recombinant human IL-33 to induce fibrosis and inflammation in HK-2 renal tubular epithelial cells. The results of immunofluorescence staining, Western blotting, and qPCR analysis showed that 50 and 100 ng/mL IL-33 significantly upregulated the expression level of fibrosisrelated indicators vimentin (

Quercetin Suppresses IL-33-Stimulated Inflammasome and Pyroptosis-Associated Protein in Human Renal Tubular Epithelial Cell Line
Furthermore, we presumed that IL-33 can induce systemic inflammatory responses and damage by triggering pyroptosis in HK-2 cells, as canonical inflammasome and GS-DMD activation are considered key factors in LN pathogenesis [36]. The results of immunofluorescence staining, Western blotting, and qPCR showed that IL-33 markedly increased the activation of NLRP3 ( Figure 4A,C,D), ASC ( Figure 4B,C,E,G), and caspase-1 ( Figure 4C,F); IL-33 also enhanced the level of N-GSDMD ( Figure 5B,C,D) and IL-1β secretion ( Figure 5A,C,E), especially at a dose of 100 ng/mL. However, quercetin reversed the expression of NLRP3, ASC, caspase-1, N-GSDMD, and IL-1β induced by IL-33, indicating that quercetin can relieve inflammasome-and GSDMD-mediated pyroptosis in the cellular model of lupus nephritis.

Quercetin Regulates IL-33-Stimulated Inflammatory Pathways in Human Renal Tubular Epithelial Cell Line
To further clarify the mechanism of quercetin effects in HK-2 cells' differing IL-33 we first verified the expression of various receptors, including ST2 ( Figure 6A,B), IL1RAP ( Figure 6A,C), TLR4 ( Figure 6A,D), TLR7 ( Figure 6A,E), and TLR9 ( Figure 6A,F) using immunoblotting. IL-33 significantly increased the activation of ST2, IL1RAP, TLR4, TLR7 and TLR9, whereas quercetin was more effective in reducing the expression of ST2 and

Quercetin Regulates IL-33-Stimulated Inflammatory Pathways in Human Renal Tubular Epithelial Cell Line
To further clarify the mechanism of quercetin effects in HK-2 cells' differing IL-33, we first verified the expression of various receptors, including ST2 ( Figure 6A,B), IL1RAP ( Figure 6A,C), TLR4 ( Figure 6A,D), TLR7 ( Figure 6A,E), and TLR9 ( Figure 6A,F) using immunoblotting. IL-33 significantly increased the activation of ST2, IL1RAP, TLR4, TLR7, and TLR9, whereas quercetin was more effective in reducing the expression of ST2 and TLR4 ( Figure 6A,B,D). The expression of NF-κB, the most important transcription factor in the inflammatory pathway, was stimulated in response to IL-33, and it decreased following quercetin combination. However, its expression of downregulation was after quercetin intervention did not reach statistical significance due to a large standard deviation ( Figure 7A,B). Likewise, the protein expression levels of MyD88 ( Figure 7C,D) and IκBα ( Figure 7C,E), as downstream targets, were not statistically different between groups. On the other hand, concerning AMPK, which is an important molecule involved in several antiinflammatory pathways, our results showed that quercetin significantly increased p-AMPK activation, even though exposure to IL-33 did not significantly reduce p-AMPK expression ( Figure 7C,F). To sum up, these results show that quercetin ameliorates inflammasome-and GSDMD-mediated pyroptosis, primarily through the regulation of IL-33/ST2 and possibly partly through the IL-33/TLR4 pathway. the other hand, concerning AMPK, which is an important molecule involved in several anti-inflammatory pathways, our results showed that quercetin significantly increased p-AMPK activation, even though exposure to IL-33 did not significantly reduce p-AMPK expression ( Figure 7C,F). To sum up, these results show that quercetin ameliorates inflammasome-and GSDMD-mediated pyroptosis, primarily through the regulation of IL-33/ST2 and possibly partly through the IL-33/TLR4 pathway.

Discussion
Lupus nephritis is one of the most common complications in patients with systemic lupus erythematosus; this study investigated whether quercetin has the potential to improve lupus nephritis in vivo and in vitro. Notably, quercetin decreased the plasma levels of BUN and creatinine, as well as ameliorating the degree of pathological progression in the kidney tissue sections of MRL/lpr mice as in in vivo models of SLE. In addition, quercetin reversed IL-33-induced fibrosis, inflammasome, and pyroptosis-related proteins expression while improving the molecular expression of inflammation-related pathways in HK-2 cells in vitro (Figure 8). The results of this study confirmed that quercetin has the potential to improve lupus nephritis.

Discussion
Lupus nephritis is one of the most common complications in patients with systemic lupus erythematosus; this study investigated whether quercetin has the potential to improve lupus nephritis in vivo and in vitro. Notably, quercetin decreased the plasma levels of BUN and creatinine, as well as ameliorating the degree of pathological progression in the kidney tissue sections of MRL/lpr mice as in in vivo models of SLE. In addition, quercetin reversed IL-33-induced fibrosis, inflammasome, and pyroptosis-related proteins expression while improving the molecular expression of inflammation-related pathways in HK-2 cells in vitro (Figure 8). The results of this study confirmed that quercetin has the potential to improve lupus nephritis. Antioxidants 2022, 11, x FOR PEER REVIEW 14 of 20 Mice have the advantages of a shortened lifespan, ease of reproduction, ease of genetic manipulation, and the fact that standardized conditions can be maintained [37]. Spontaneous and induced models of lupus are useful tools for the study of the disease etiology. The classic models of spontaneous lupus include the NZB/W F1, MRL/lpr, and BXSB/Yaa strains, whereas induced models include the pristane-induced model and the chronic graft-versus-host-disease models (cGVHD) [38].
Studies have pointed out that several cytokines, chemokines, or growth factors have been utilized to determine the clinical pathogenesis of SLE patients [8], including interleukin subfamily, CC chemokine ligand (CCL) subfamily, transforming growth factor-β (TGFβ), etc. [39]. It is worth noting that serum IL-33 levels were found to be upregulated in SLE patients, suggesting that IL-33 may serve as a biomarker and therapeutic target for SLE [17]. A recent piece of research indicated that neutrophils from SLE patients release neutrophil extracellular traps (NETs) complexed with IL-33 in the blood and other inflamed tissues, such as kidneys and skin, and contribute to the production of type I IFNs through interactions with the ST2L receptor on plasmacytoid dendritic cells (pDCs) [40], implying that IL-33 and its receptor ST2 may contribute to the pathogenesis of autoimmune diseases, especially in patients with SLE. Moreover, Li et al. (2014) indicated that MRL/lpr mice receiving the anti-IL-33 antibody showed significantly less severe proteinuria than untreated control; moreover, serum creatinine was significantly reduced by the anti-IL-33 Ab treatment [19]. Taken together, these studies highlight the significant role of IL-33 in the development of SLE.
Mice have the advantages of a shortened lifespan, ease of reproduction, ease of genetic manipulation, and the fact that standardized conditions can be maintained [37]. Spontaneous and induced models of lupus are useful tools for the study of the disease etiology. The classic models of spontaneous lupus include the NZB/W F1, MRL/lpr, and BXSB/Yaa strains, whereas induced models include the pristane-induced model and the chronic graft-versus-host-disease models (cGVHD) [38].
Studies have pointed out that several cytokines, chemokines, or growth factors have been utilized to determine the clinical pathogenesis of SLE patients [8], including interleukin subfamily, CC chemokine ligand (CCL) subfamily, transforming growth factor-β (TGFβ), etc. [39]. It is worth noting that serum IL-33 levels were found to be upregulated in SLE patients, suggesting that IL-33 may serve as a biomarker and therapeutic target for SLE [17]. A recent piece of research indicated that neutrophils from SLE patients release neutrophil extracellular traps (NETs) complexed with IL-33 in the blood and other inflamed tissues, such as kidneys and skin, and contribute to the production of type I IFNs through interactions with the ST2L receptor on plasmacytoid dendritic cells (pDCs) [40], implying that IL-33 and its receptor ST2 may contribute to the pathogenesis of autoimmune diseases, especially in patients with SLE. Moreover, Li et al. (2014) indicated that MRL/lpr mice receiving the anti-IL-33 antibody showed significantly less severe proteinuria than untreated control; moreover, serum creatinine was significantly reduced by the anti-IL-33 Ab treatment [19]. Taken together, these studies highlight the significant role of IL-33 in the development of SLE. Tubulointerstitial fibrosis is a common defect in the progression of lupus nephritis to end-stage renal disease (ESRD) [7]. Proinflammatory cytokines or chemokines stimulate the epithelial-mesenchymal transition (EMT) pathway, which converts epithelial cells into fibroblasts, which, in turn, causes renal fibrosis [41]. Recent studies provide evidence that tubular epithelial cells may contribute to renal fibrogenesis through partial epithelialmesenchymal transition [42]. IL-33 is not only a fundamental cytokine in inflammatory diseases but also a profibrotic protein [43]. In a previous study, IL-33 could induce EMT in HK-2 cells by promoting fibronectin expression [44]. This was consistent with our results where IL-33 significantly increased the expression of vimentin and fibronectin and stimulated the production of proinflammatory cytokines IL-6, IL-8, and HMGB1 proteins.
A previous study showed that 100 mg/kg quercetin attenuated renal interstitial fibrosis in renal tissues of UUO rats, while 40 µM quercetin inhibited the EMT and extracellular matrix (ECM) accumulation in TGF-β1-treated NRK-52E cells [45]. Our study results were consistent as collagen deposition in kidney tissue was reduced after quercetin administration, and HK-2 renal tubular epithelial cells had lower expression of vimentin and fibronectin after quercetin intervention, indicating renal fibrotic alteration in MRL/lrp mice and IL-33-induced renal tubular epithelial cell fibrosis were both improved after quercetin treatment.
Many studies have shown that quercetin and its metabolites can inhibit the production of inflammatory cytokines IL-6, IL-8, and tumor necrosis factor (TNF-α) [20]. In addition, a recent study showed that quercetin was able to reduce the inflammatory response of cells in the contrast-induced acute kidney injury (CI-AKI) model via downregulation of HMGB1 protein expression [46]. Similarly, we observed consistent results that IL-33induced proinflammatory cytokines IL-6, IL-8, and HMGB1 in HK-2 cells were ameliorated following quercetin treatment. Thus, the secretion of pro-fibrotic proteins and cytokines, triggered in response to IL-33, were significantly reduced when quercetin was used as a treatment. These findings highlight the benefits of quercetin as a potential treatment for LN patients.
Accumulating evidence supports the involvement of the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome in driving renal fibrosis and inflammatory responses [47,48]. The activation of the NLRP3 inflammasome promotes the activation of inflammatory caspases (caspase-1)-dependent pro-inflammatory hormones IL-1β and IL-18, which, in turn, promotes renal fibrosis [49]. Our study showed that the positive staining ratio of NLRP3 in renal tissue was decreased after quercetin administration, and the expression of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 in renal epithelial cells was induced by IL-33 and decreased after quercetin intervention, suggesting that renal inflammation in MRL/lrp mice and IL-33-induced inflammation-related molecules in HK-2 cells were both ameliorated after quercetin treatment.
Pyroptosis is a type of lytic cell death triggered by inflammatory caspases that are normally activated in multiprotein inflammasome complexes assembled in response to pathogens and endogenous danger signals [50]. Notably, gasdermin D (GSDMD) was identified as a caspase substrate and considered an important mediator of pyroptosis in processes involving cleavage by active caspase-1 within the junction of the N-and Cterminal domains of GSDMD. Afterward, N-GSDMD promotes the formation of the plasma membrane pore [51], as well as stimulating pyroptosis with a subsequent production of cytokines IL-1β and IL-18 [52]. Our study showed that the expression of N-GSDMD and IL-1β in renal epithelial cells was induced by IL-33 and decreased after quercetin intervention, concluding that IL-33-induced pyroptosis-related molecules in HK-2 cells were ameliorated after quercetin treatment.
The canonical pathway elicited by IL-33 results in stimulation of a receptor complex composed of ST2L and interleukin 1 receptor accessory protein (IL-1RAP) [43], which triggers the recruitment of MyD88, IRAK1, IRAK4, and TRAF6, followed by activation of downstream signaling pathways, such as NF-κB, c-Jun N-terminal kinase (JNK), p38, and ERK [53]. A previous study in STZ-induced diabetic rats found that quercetin intervention downregulated the expression of NF-κB in the kidney [54]. Similarly, our current data showed that IL-33 significantly induced ST2, IL-1RAP, and NF-κB expression, whereas quercetin only reduced ST2 expression, indicating that quercetin can affect the inflammatory status of renal tubular epithelial cells by inhibiting ST2 receptors. However, the changes of downstream mechanisms still need to be further explored.
Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that play an important role in innate immunity and are typically expressed on macrophages, dendritic cells, and B lymphocytes, and/or epithelial and mesangial cells of the kidney [55]. In the pathogenesis of SLE, TLRs are activated by exogenous ligands of infection and/or endogenous ligands of apoptotic debris that are not cleared, and downstream signaling cascades are initiated, leading to the production of inflammatory cytokines [55]. A recent study by Upadhyay et al. found that the knockdown of TLR9 and reactive oxygen species (ROS) inhibition ameliorated EMT, mitochondrial dysfunction, and inflammatory response in proximal renal tubular cells induced by aristolochic acids [56]. In the present study, our findings showed that IL-33 significantly induced the expression of TLR4, TLR7, and TLR9 proteins, whereas quercetin only reduced IL-33-induced TLR4 expression, suggesting that quercetin may alter the release of inflammation-related proteins through TLR4, located on the cell surface, rather than TLR7 and TLR9 on the membranes of endosomes.
AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine protein kinase, is an important regulator of whole-body energy metabolism [57]. It is well known that activation of AMPK contributes to the prevention of inflammation and NF-κB pathway transactivation [58]. A concurrent study showed that quercetin attenuated oxidative stress-mediated endoplasmic reticulum (ER) stress and mitochondrial dysfunction through activating SIRT1/AMPK signal pathways in lipopolysaccharide (LPS)-stimulated murine lung epithelial (MLE-12) cells [59]. Consistent with our observations, quercetin significantly induced p-AMPKα expression in HK-2 cells even under IL-33 induction, indicating an anti-inflammatory effect of quercetin. On the other hand, previous literature showed that IL-33 induced an increase in p-AMPK expression in group 2 innate lymphoid cells (ILC2) in a short period of time, but the negative feedback would decrease p-AMPK expression due to the prolonged induction of IL-33 [60]. In addition, IL-33 induced p-AMPK expression in monocytes to promote mitophagy [61]. However, in our experiment, the intervention of IL-33 did not significantly change the expression of p-AMPK. We speculate that it may be closely related to the time of intervention, which needs more experiments to confirm.
There are some limitations to be aware of in this experiment. First, quercetin is considered a potent and well-known antioxidant [21], however, our present results found that IL-33-induced renal epithelial cell injury does not appear to occur through the promotion of oxidative stress (shown in Supplementary Figure S2). Furthermore, after searching the database with the same criteria, it was found that IL-33 was less explored in renal epithelial cells and oxidative stress studies. Therefore, the antioxidant capacity of quercetin does not apply to this conclusion after being challenged with IL-33. Second, the results indicated that changes in AMPKα were not the main factor of IL-33-induced inflammation and fibrosis, although quercetin induced a significant increase in AMPKα expression. Therefore, we speculate that other mechanisms may be involved in the anti-inflammatory or anti-fibrotic effects of quercetin. However, the exact pathway requires more experimental confirmation. Finally, many studies remain skeptical about the role of IL-33 in SLE patients, and two opposing views have emerged. Fortunately, this study proposes novel findings. We used IL-33 to induce renal proximal tubule epithelial cells to mimic the fibrosis and inflammatory responses that LN may encounter, which was then treated with quercetin. This positive impact indicates that quercetin can indeed improve the renal fibrosis by retarding the inflammatory response in LN development.

Conclusions
In summary, our findings showed that quercetin was able to decrease the plasma levels of BUN and creatinine and ameliorate the degree of pathological deterioration in kidney tissue sections in MRL/lpr mice in an in vivo SLE animal model. Furthermore, we reported for the first time that quercetin reversed IL-33-induced fibrosis, inflammasome, and pyroptosis-related protein expression in HK-2 cells via IL-33/ST2 in vitro. Collectively, these findings propose quercetin as a potential therapeutic option in the management of lupus nephritis.