A total of 60 female C57BL/6 mice (weight, 18-20 g; age, 10 weeks), were purchased from Nanjing Medical University Animal Experiment Center (Nanjing, China). All experimental mice were housed under specific pathogen-free conditions in static microisolator cages with tap water ad libitum in a temperature-controlled room (25±2°C, 50±5% humidity) with a 12-h light/dark cycle and were fed a standard laboratory rodent diet with water ad libitum. The mice experiments were performed in a way to minimize animal suffering according to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health from 1996. The Institutional Animal Care and Use Committee at Huai'an First People's Hospital, Nanjing Medical University (Huai'an, China) approved the animal study protocols. The animals were divided into 5 groups (n=10/group): Control (Con; without any treatment); PRI group; PRI+25 mg/kg fisetin; PRI+50 mg/kg fisetin and PRI+100 mg/kg fisetin (20,21). Fisetin (purity, >98%) was from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and was administered orally by gavage once a day. In order to induce lupus in mice, the animals received an intraperitoneal (i.p.) injection of 0.5 ml PRI (Sigma-Aldrich; Merck KGaA), as previously described (22). PBS was administered to the control group. At 30 weeks following treatment with PRI or PBS, blood samples were collected through retroorbital bleeding with capillary tubes to obtain the serum. Next, the mice were sacrificed, and renal and spleen tissue samples were immediately isolated and stored at −80°C for the further experiments described below. Proteinuria was measured through urine albumin-to-creatinine ratio determined using the Albumin assay kit (A028-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and Creatinine Assay kit (C011-2; Nanjing Jiancheng Bioengineering Institute).

The RAW264.7 murine macrophage cell line and the 293FT cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% (v/v) penicillin/streptomycin in an incubator at 37°C in a humidified atmosphere containing 5% CO₂. For the experiments, cells were pre-treated with 100 ng/ml lipopolysaccharide (LPS; Sigma-Aldrich; Merck KGaA) for 2 h, followed by incubation with fisetin (15 and 30 µM) for another 24 h. Subsequently, all cells were harvested for reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot and flow cytometric analysis.

In order to measure the cytotoxicity of fisetin on normal cells, 293FT and RAW264.7 cells were seeded into 96-well plates at 1×10³ cells/well in complete growth medium. On the following day, the cells were treated with different concentrations of fisetin ranging from 0-160 µM at 37°C for 24, 48 or 72 h. The cell viability was determined by an MTT assay with the absorbance read at 570 nm (23).

The serum concentrations of anti-dsDNA antibodies (5110; Alpha Diagnostic, San Antonio, TX, USA), anti-small nuclear ribonucleoprotein antibody (AB-23240-A; anti-snRNP; Alpha Diagnostic), IL-1β (MLB00C), IL-6 (M6000B), TNF-α (MTA00B), interferon-γ (IFN-γ) (DY485), CXCL-1 (DY453), MCP-1/CCL-2 (MJE00), chemokine (C-C motif) ligand 3 (CCL-3) (MMA00) and CXCL-2 (MM200; all from R&D Systems, Minneapolis, MN, USA) were measured with corresponding ELISA kits following the manufacturers' protocols.

To histologically evaluate the renal tissue samples after various treatments, kidney tissue specimens were fixed in 10% formalin at room temperature for 48 h and embedded in paraffin. A series of 4-µm sections containing the hilum of the renal tissue samples were stained with a periodic acid-Schiff (PAS) Stain Kit (Abcam, Cambridge, MA, USA) following the manufacturer's protocols. In brief, a semi-quantitative scoring system was applied to calculate 10 various parameters, including mesangial deposits, mesangial hypercellularity, mesangial sclerosis, endocapillary sclerosis, endocapillary cellular infiltrate, endocapillary organized crescents, endocapillary cellular crescents, tubular atrophy, interstitial inflammation and interstitial fibrosis: 0, no involvement; 0.5, minimal involvement (<10%); 1, mild involvement (10-30%); 2, moderate involvement (31-60%); 3, severe involvement (60%). An activity and chronicity index was determined through compiling scores from groups of associated parameters (as for the activity: Mesangial deposits, mesangial hypercellularity and endocapillary cell infiltration; and as for the chronicity: Tubular atrophy, endocapillary organized crescents, endocapillary sclerosis and interstitial fibrosis). Spleen tissue samples were soaked in 4% formalin overnight at room temperature, embedded in paraffin and sectioned at 4 µm thickness. Sections were stained with hematoxylin and eosin (H&E) and visualized by light microscopy at a magnification of x400.

The spleen tissue was disaggregated into single cells as described previously (24) and stained for 30 min in dark at room temperature for surface markers with the following antibodies: CD3 (554832; 1:100), CD4 (553651; 1:100) and CD8 (557085; 1:100) purchased from BD Biosciences (Franklin Lakes, NJ, USA). Spleen cells were stimulated and then stained for intracellular cytokines. RAW264.7 cells were treated as indicated above and then harvested for CD3 conjugation. The following fluorochrome-conjugated antibodies were used: CD4 (563933; 1:100 dilution, GK1.5), CD3 (561042; 1:100 dilution, 145-2C11), IL-17 (eBio17B7; 1:100 dilution) and RAR-related orphan receptor (ROR)γt (12-6988-82; 1:100 dilution, AFKJ-9), and all were purchased from BD Biosciences or eBioscience (Thermo Fisher Scientific, Inc.). The antibodies were stained in dark for 30 min Finally, the labeled cells were quantified with a FACSCanto instrument (BD Biosciences).

CD4⁺CD25⁻ T cells were isolated from spleens of 6-month-old mice as described previously (25). Cells were purified using a MACS magnetic column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with a CD4⁺ T cell enrichment kit (11-0041-82; eBioscience; Thermo Fisher Scientific, Inc.). The cells were then stimulated with a plate-bound anti-CD3e antibody (5 µg/ml; 11-0038-42, eBioscience; Thermo Fisher Scientific, Inc.) and anti-CD28 antibody (2 µg/ml; 16-0281-82, eBioscience; Thermo Fisher Scientific, Inc.). For Th17-cell polarization, the following exogenous cytokines and antibodies were added: Transforming growth factor-β (5 ng/ml, R&D Systems); anti-IL-4 antibody (46-7041-82; 10 µg/ml; eBioscience; Thermo Fisher Scientific, Inc.); IL-6 (MAB406; 30 ng/ml; R&D Systems); anti-IFN-γ antibody (MAB485; 10 µg/ml; eBioscience; Thermo Fisher Scientific, Inc.) and IL-1β (AF-401-NA; 20 ng/ml; R&D Systems). After 5 days of culture, the proportion of Th17 cells among all cells was determined by flow cytometry.

Total RNA extraction of cells and spleen tissue samples was performed using TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was reverse transcribed using the Moloney murine leukemiavirus RT system (Promega Corp., Madison, WI, USA). A mixture of 2 µl RT product with 7.3 µl nuclease-free H₂O, 0.4 µl ROX I (50X), 10 µl TaqMan PCR Mixture (2X) and 0.3 µl TaqMan MiRNA Assay (20X) primer was prepared. The reaction was performed at 42°C for 1 h and terminated by deactivation of the enzyme at 70°C for 10 min. PCR was performed using SYBR-Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in an ABI PRISM 7900HT detection systems (Applied Biosystems; Thermo Fisher Scientific, Inc.). Equal amounts of cDNA were diluted and amplified through real-time PCR using All-in-One qPCR Mix (GeneCopoeia, Maryland, USA) in a 20-µl reaction volume containing 10 µl of 2X All-in-One qPCR Mix (GeneCopoeia, Maryland, USA), 1 µl of 2 µM forward primer, 1 µl of 2 µM reverse primer, 1 µl of cDNA and 6 µl of nuclease-free water. All of the primers were from Invitrogen (Thermo Fisher Scientific, Inc.). Amplification of pre-denatured products was performed at 94°C for 60 sec followed by 45 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec; this was followed by 95°C for 10 sec, 65°C for 45 sec and 40°C for 60 sec. Fold induction values were calculated according to the 2^−ΔΔCq method, where ΔCt represents the differences in cycle threshold number between the target gene and GAPDH, and ΔΔCt represents the relative change in the differences between control and treatment groups (26). The following primers were used: IL-6 forward, 5'-CAA GCA ACA ATG GAC TTT AGG-3' and reverse, 5'-GAG CCA TTA ACT ATG GAA CCA-3'; IL-1β forward, 5'-AGA CTC CAC ATT ACA GGC AAT GCC-3' and reverse, 5'-AAT CGC TAC GGA TCT CCA GA-3'; MCP-1 forward, 5'-AGA TCA GGC GAT GCA TGT AGA CG-3' and reverse, 5'-CAA TAC CAG TGG ACA AAC GAC-3'; IFN-γ forward, 5'-ACA AGC TCA AGT ACC CAA GGT ACA-3' and reverse, 5'-GTG CTA CTG GTG TGA TCA-3'; TNF-α forward, 5'-ATG TCC GTC CTC ATC GGT AGC-3' and reverse, 5'-CCG GAG GAC ACA GTC CAC C-3'; IL-17 forward, 5'-CCC ATG AGA TAC ACG GGT A-3' and reverse, 5'-CCG ATA CTG CAA GGT GAA C-3'; GAPDH forward, 5'-TCA CAT GAA CCG GAC AGA GG-3' and reverse, 5'-CCA ATC CTA CGA CAC CGA CTA AC-3'.

The cells and spleen tissue samples were homogenized with hypotonic buffer [25 mM Tris-HCl (pH 8.0), 1 mM EDTA, 5 µg/ml leupeptin, 1 mM Pefabloc SC, 50 µg/ml aprotinin, 5 µg/ml soybean trypsin inhibitor and 4 mM benzamidine] at 10% (wt/vol) to yield a homogenate. The final supernatants were the obtained by centrifugation at 12,000 x g for 20 min at 4°C. The protein concentration was determined with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) with bovine serum albumin as a standard. Subsequently, equal amounts of total protein (20-40 µg) were subjected to 10% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked with 5% skimmed milk Tris buffered saline with 0.1% Tween 20 (TBST), washed, and then incubated with the following primary antibodies at 1:1,000 dilution overnight at 4°C: Rabbit anti-IL-17 (13838), rabbit anti-CXCL-1 (PA1-29220), rabbit anti-CD3 (4443), rabbit anti-CXCL-2 (701126), rabbit anti-CCL-3 (MA5-24364), rabbit anti-CXCR-2 (PA5-38620) and rabbit anti-GAPDH (2118; Cell Signaling Technology, Inc., Danvers, MA, USA), followed by incubation with HRP-conjugated goat anti rabbit secondary antibody (1:5,000; ab6721; Abcam) for 2 h at room temperature. Western blot bands were observed using the GE Healthcare ECL Western Blotting Analysis System (GE Healthcare, Little Chalfont, UK) and exposed to X-ray film (Eastman Kodak, Rochester, NY, USA). Protein expression levels were quantified by determining the grey value, standardized to the housekeeping gene (GAPDH) and expressed as a fold of the control.

Values are expressed as the mean ± standard error of the mean. Statistical analyses were performed using GraphPad PRISM (version 6.0; GraphPad Inc., La Jolla, CA, USA). Analysis of variance with Dunnet's least-significant difference post-hoc test was performed for comparison between groups. P<0.05 was considered to indicate a statistically significant difference.

Compared with the Con group, anti-dsDNA antibodies were significantly increased in PRI-induced mice (Fig. 1A). Of note, fisetin administration significantly reduced PRI-induced anti-dsDNA antibody levels in a dose-dependent manner. In addition, the levels of anti-snRNPs were markedly increased in PRI-treated mice, but were reduced by fisetin in a dose-dependent manner (Fig. 1B). Finally, the ratio of urine albumin to creatinine was evaluated to assess lupus nephritis after PRI administration. As presented in Fig. 1C, PRI treatment significantly enhanced the ratio of albumin to creatinine, suggesting that it caused injury in mice, which was ameliorated by fisetin treatment. These results indicated that fisetin may improve various autoimmune features of lupus in mice induced by PRI.

In order to determine the effect of fisetin on PRI-induced nephritis in mice, the renal sections were histologically examined by PAS staining, which was evaluated using indexes of activity and chronicity. As presented in Fig. 2A and B, the activity index was markedly enhanced by PRI induction, which was comparable to that in the control group. Fisetin administration significantly reduced the activity index, indicating that lupus nephritis was attenuated by fisetin. Consistently, the PRI-induced elevation in the chronicity index was also reduced by fisetin treatment (Fig. 2A and C). In addition, serum BUN and creatinine levels were highly induced by PRI, which was inhibited by fisetin, indicating renal injury induced by PRI and that fisetin had the capacity to attenuate nephritis (Fig. 2D and E). Furthermore, infiltration of CD3⁺ cells was assessed to calculate the effects of fisetin on SLE. The immunohistochemical analysis demonstrated that PRI treatment led to increases of CD3⁺ cells throughout the kidney tissue compared with the control group. Of note, fisetin-treated mice exhibited significantly reduced infiltration of CD3⁺ cells compared with that in the PRI group (Fig. 3A). Western blot analysis further indicated that PRI treatment increased CD3 levels, which was inhibited by fisetin (Fig. 3B). Taken together, the above results demonstrated similar results to those of other PRI-induced animal models of lupus, and various features associated with lupus nephritis induced by PRI administration were dose-dependently reduced by fisetin treatment.

In order to assess the role of fisetin in circulating inflammatory biomarkers associated with leukocyte recruitment, the serum levels of CXCL-1, CXCL-2, CCL-3, CXCR-2, IL-6, IFN-γ, TNF-α, IL-1β and MCP-1 were evaluated in PRI-induced mice treated with or without fisetin. In the pathogenesis of lupus, leukocyte extravasation has an important role (27). The results of the present study indicated that serum CXCL-1 (Fig. 4A), CXCL-2 (Fig. 4B), CCL-3 (Fig. 4C) and CXCR-2 (Fig. 4D) were highly increased in PRI-induced mice compared with those in the Con group. However, after fisetin treatment, reduced levels of these signals were observed. Next, the inflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β, and the chemokines MCP-1 were assessed in the plasma of mice after various treatments (Fig. 4E-I). The results indicated that PRI treatment enhanced the levels of these factors, which were reduced by administration of fisetin in a concentration-dependent manner. Thus, fisetin may attenuate SLE due to reducing chemokines and cytokines in PRI-induced mice.

According to previous studies, splenomegaly is a characteristic of mice with SLE (27). As presented in Fig. 5A, the spleen size of mice induced with PRI was larger than that of mice without any treatment. By contrast, fisetin administration reduced the spleen size in PRI-induced mice. In addition, the total number of lymphocytes in the spleen tissue samples was markedly increased in PRI-treated mice, which was reduced by fisetin treatment (Fig. 5B). Subsequently, the composition of splenic lymphocytes was explored by flow cytometric analysis. The results presented in Fig. 5C indicated that the proportion of CD3⁺CD4⁺ T cells was highly augmented in PRI-induced mice, which was inhibited by fisetin administration. The ratio of CD4⁺ vs. CD8⁺ T cells among the total splenic lymphocytes was significantly increased in the PRI-treated mice, which was attenuated by fisetin administration (Fig. 5D). Of note, a reduced CD4⁺/CD8⁺ ratio was observed after fisetin administration compared with that in mice treated with PRI only. Furthermore, the B-cell number was also increased in the PRI-induced mice compared with that in the control group (Fig. 5E). Finally, the H&E staining indicated that PRI induced histological changes compared with control group, which was attenuated by fisetin administration (Fig. 5F). These results demonstrated that PRI treatment caused an abundance of different lymphocyte subsets in mice, which was reduced by fisetin administration, indicating its role in improving SLE induced by PRI in mice.

The present study attempted to evaluate the proportion of Th17 cells among the splenic CD3⁺CD4⁺ T cells through flow cytometric analysis. As presented in Fig. 6A-C, the percentage of RORγ⁺ cells among CD3⁺CD4⁺ T cells was significantly increased in PRI-induced mice, as well as the percentage of IL-17-generating CD3⁺CD4⁺ T cells, which were reduced by fisetin treatment in a dose-dependent manner. In addition, the pro-inflammatory cytokines in splenic cells were determined by RT-qPCR analysis. The mRNA expression of pro-inflammatory cytokines and MCP-1 was highly induced by PRI, which was in line with the circulating levels. Of note, fisetin administration markedly reduced the mRNA levels of these cytokines and MCP-1, indicating its role in suppressing the inflammatory response in mice with SLE (Fig. 6D). Finally, in order to further confirm the capacity of fisetin to ameliorate SLE induced by PRI in mice, the mRNA and protein levels of IL-17 were assessed in the splenic cells through RT-qPCR, western blotting and ELISA. As presented in Fig. 6E, PRI induced IL-17, which was inhibited by fisetin.

The present study further investigated how CXCLs signaling was altered in PRI-induced mice with or without fisetin treatment. Western blot analysis indicated that the CXCL signaling pathway was activated by PRI in mice with SLE, supported by an elevation in CXCL-1, CXCL-2, CCL-3 and CXCR-2 protein expression levels. Of note, fisetin administration significantly reduced the abundance of CXCL-1, CXCL-2, CCL-3 and CXCR-2 protein, which may be involved in the mechanisms by which fisetin attenuates SLE induced by PRI treatment (Fig. 7A and B).

According to the in vivo experiment, induction of inflammation was a key molecular mechanism by which SLE progressed. Hence, RAW264.7 cells were pre-treated with 100 ng/ml LPS for 2 h, followed by fisetin administration for another 24 h. As presented in Fig. 8A and B, the LPS-induced increases in CD3 expression levels were significantly reduced by fisetin. Furthermore, the mRNA levels of the cytokines IL-6, IFN-γ, TNF-α and IL-1β, and the chemokine MCP-1 were all increased by LPS, which was inhibited by fisetin treatment (Fig. 8C). Consistently, the protein expression of CXCL-1, CXCL-2, CCL-3 and CXCR-2 was also enhanced by LPS, which was markedly reduced by fisetin treatment (Fig. 8D). In conclusion, the above results indicated that in vitro, fisetin reduced inflammation and CXCL pathway activation triggered by LPS as a possible molecular mechanism by which it also attenuates SLE in vivo. Finally, to assess whether fisetin had any possible cytotoxic effects, the cell line 293FT and the RAW264.7 mouse macrophage cell line of were treated with fisetin at 0, 5, 10, 20, 40, 80 or 160 µM for 24, 48 or 72 h. Subsequently an MTT assay was used to measure the cell viability. The results presented in Fig. 9A and B indicated no significant difference in the number of viable 293FT and RAW264.7 cells after fisetin treatment for 24, 48 and even 72 h, which suggested that fisetin was safe for SLE treatment under the above conditions.