Simiao San were purchased from Jiling Zixin Pharmaceutical Industrial Company, LTD. (Jiling,China) and authenticated by Guang’anmen Hospital, China Academy of Chinese Medical Sciences (Beijing, China). SMS comprises a mixture of 66.7 g of Phellodendron chinese SCHNEID, 33.3 g of Atractylodes lancea, 33.3 g of Coix lacryma-jobi L., and 66.7 g of Achyranthes bidentata BL. SMS medicinal juice was prepared using a well established protocol (18). The four herbs were mixed with distilled water for 2 h, added to a volatile oil extractor, refluxed, and extracted twice for 1.5 h each. A 7-fold excess of distilled water was added for the first extraction, whereas a 6-fold excess of distilled water was added for the second extraction. The mixture was then filtered, sealed for storage, concentrated, and mixed with the volatile oil. Finally, we dispensed the mixture into a 1 g/mL medicinal juice and sterilized it for packaging.

Mass spectrometry as performed using an Agilent 1290 UHPLC and 6530 QTOF-MS/MS LC-MS system (Agilent Technologies, Santa Clara, CA, USA), and an Agilent Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) column (Agilent Technologies) was used for the acquisition of metabolic data. Gradient elution was performed using 0.1% formic acid and methanol.

For sample preparation, 1 mL of SMS concentrate was added to 4 mL of methanol solution, and the mixture was sonicated for 30 min, centrifuged, and filtered through a 0.1-µm microporous filter.

Liquid chromatography–mass spectrometry was performed on an Agilent 1290 UHPLC and 6530 QTOF-MS/MS system with a 2.1 m × 100 mm × 1.8 μm Agilent Eclipse Plus C18 column. The atomizing temperature was 350°C, the nebulizer flow rate was 10 L/min, the atomization gas pressure was 30 psi, the capillary voltage was 3500 V, the Skimmer voltage was 65 V, the octopole RF voltage was 750 V, and the papillary outlet voltage was 150 V. The data matrix comprised the retention times, m/z 50–1500 values, and the corresponding peak areas for subsequent statistical analysis.

MSU crystals were prepared as we previously reported (23) and then assessed using compensated polarized light microscopy. Endotoxin was present at <0.015 EU/mL in the MSU crystal preparations, as determined using the Limulus amebocyte lysate assay (Sigma-Aldrich, St Louis, MO, USA). Before each experiment, the MSU crystals were milled and then sterilized for 2 h.

THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in complete medium comprising RPMI supplemented with 2 mM l-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA). THP-1 cells were used after treatment with 100 ng/μL PMA for 48 h. Cells were seeded in 24-well culture plates (2×10⁵ cells/mL/well), pretreated with 1 or 2 μg/mL SMS for 12 h, and then incubated with 100 μg/mL MSU for 48 h. To block the PI3K/Akt pathway, cells were pretreated with 50 μM LY294002 (Selleckchem) for 1 h before MSU treatment. SMS was added for 30min after LY294002 treatment.

Specific pathogen-free male C57BL/6 mice (6-8 weeks old) were purchased from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, China) and maintained at the Animal Center of Shanghai Medical School of Fudan University. This study was approved by the Ethics Committee of the Department of Laboratory Animal Science, Fudan University (No. 20160981A302).

To establish mice with gout, 50 μL of an MSU suspension (1 mg/50 μL Normal saline) was intra-articularly injected in the right footpad of each animal, whereas the left footpad was injected with 50 μL of Normal saline (N.S). The joint index evaluation was performed as we previously reported (23–27). In the SMS treatment group, mice were intra-gastrically injected with a low dose (1 mg/kg·day) or high dose (10 mg/kg·day) of SMS for 14 days before MSU administration, samples were collected at 8h after MSU injection. The foot joint tissues were immediately isolated, snap-frozen in liquid nitrogen, and stored at -80°C.

Hyperuricemic mice were established via treatment with yeast polysaccharide (YP) and potassium oxonate (OP) for 3 weeks as previously described (28). Mice were orally feed containing 1/4 yeast polysaccharide (Sigma, USA) or intraperitoneally injected with potassium oxonate (250 mg/kg; OP, Sigma, USA) at 8:00 a.m. every day. In the SMS treatment group, different concentrations (1 or 10 mg/kg·day) of SMS were intra-gastrically administered to mice simultaneously with OP for 3 weeks. Samples were collected after the last drug administration. Blood samples were obtained from the eye socket vein of each mouse and centrifuged at 2500rpm for 10 min at 4°C. The kidney tissues were immediately isolated, snap-frozen in liquid nitrogen, and stored at -80°C.

After the joint index evaluation, mice were sacrificed, and joint tissue and kidneys sections were prepared for hematoxylin and eosin (H&E) staining and immunostaining. The paraffin-embedded sections were cut at a thickness of 4 μm and placed on positively charged slides for staining. In H&E staining, inflammatory cells were counted using ImageJ software (version 1.51p, National Institutes of Health, Bethesda, MD, USA).

For immunofluorescence, slides were placed in target retrieval solution, and staining was performed manually at room temperature with anti–Arg-1 antibody (Clone #CI:A3-1, 1:500 dilution, Abcam, Cambridge, MA, USA) or anti-F4/80 antibody (Clone #SP156, 1:400 dilution, Abcam, Cambridge, MA, USA). Arg-1 expression was visualized using Alexa Fluor 647 secondary antibody (ab150115, 1:400 dilution, Abcam, Cambridge, MA, USA). F4/80 expression was visualized using Alexa Fluor 488 secondary antibody (ab150077, 1:400 dilution, Abcam, Cambridge, MA, USA). Slides were counterstained with Vectashield hard-set mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and kept in the dark at 4°C until visualization using fluorescence microscopy. Images were obtained via optical sectioning using a Nikon epifluorescence microscope for analysis. F4/80⁺/Arg-1⁺ cells (M2 macrophage) analysis was performed using ImageJ software from randomly chosen fields [the average of 5 to 10 fields (× 200) in the section]. F4/80⁺/Arg-1⁺ cells were determined by using a standardized custom histogram-based colored thresholding technique and then subjected to “particle analysis” using ImageJ.

For immunohistochemistry, slides were placed in target retrieval solution, and staining was performed manually at 4°C for 18 hours with Akt (Clone #11E7, 1:1000 dilution, Cell Signaling Technology, Boston, USA). Then goat anti-Rabbit IgG H&L (HRP) (ab6721, 1:500 dilution, Abcam, Cambridge, MA, USA) was added and incubation continued for 20 minutes. Staining was visualized using DAB and the sections were then observed. Finally, sections were further dyed and sealed with hematoxylin. Images were obtained via optical sectioning using a Nikon epifluorescence microscope for analysis. To display the results more intuitively, the average optical density of positive areas (40×,100X) was calculated using ImageJ software.

SUA concentrations and creatinine levels were separately determined using colorimetric method (#MAK077, #MAK080, Sigma, USA).

Total RNA was extracted from joint tissue or cells with Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and reverse translation was conducted using an iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The PCR primers (BioTNT, Shanghai, China) used for qPCR were as shows in Supplementary Table 1 .

The RNA expression in samples was normalized to that of control housekeeping genes (murine Gapdh), and the relative mRNA levels of target genes were calculated using the 2^−ΔΔCt method.

The joint samples were homogenized by tissue tearor, and added with 10 equivalent volumes of RIPA lysis buffer supplemented with 1 mM PMSF (protease inhibitor) in an ice bath for 30 min and then centrifuged (12,000g, 10 min) to extract total proteins. The protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Mouse joint tissues or cell lysates from different groups were prepared, and equal aliquots of protein extract were electrophoresed via SDS-PAGE. Protein levels were expressed as a ratio of the protein level to that of β-actin.

The following antibodies were obtained from commercial sources as indicated: NLRP3(Clone #Ala306, 1:1000 dilution, Cell Signaling Technology, Boston, USA), p-Akt (Ser473) (Clone #587F11, 1:1000 dilution, Cell Signaling Technology, Boston, USA), Akt (Clone #11E7, 1:1000 dilution, Cell Signaling Technology, Boston, USA), β-actin(Clone #8H10D10, 1:1000 dilution, Cell Signaling Technology, Boston, USA), anti-rabbit IgG HRP-conjugated antibody(#7074, 1:5000 dilution, Cell Signaling Technology, Boston, USA), anti-moude IgG HRP-conjugated antibody(#7076, 1:5000 dilution, Cell Signaling Technology, Boston, USA). The immunoblots were visualized via ECL (Pierce, Rockford, IL, USA). The results were normalized to β-actin. Gel quantification was performed using the ImageJ software.

Data were analyzed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) and presented as the mean ± SEM or median (range). Repeated-measures analysis of variance followed by the Student-Newman-Keuls test was used for post hoc analyses of differences among groups. P < 0.05 indicated statistical significance.

Mice were pretreated with different doses of SMS (1 or 10 mg/kg.day) for 2 weeks, and then acute gouty arthritis was induced by MSU. Eight hours after MSU injection, joint thickness and the mRNA expression of Nlrp3, IL-6, IL-1β, IL-4, Tgf-β in the joints were significantly elevated. The joint swelling was significantly reduced in the mice treated with SMS compared with that in the MSU mice ( Figure 1A ). Moreover, the over-expression of Nlrp3, IL-6, and IL-1β mRNA in MSU-injured joints was inhibited by SMS, especially the high-dose SMS (10mg/kg.day). Notably, the anti-inflammatory factors, IL-4 and Tgf-β, were further increased by SMS treatment ( Figure 1B ). H&E pathological staining further revealed that inflammatory cell infiltration in gouty joints was notably inhibited by SMS pretreatment, especially in the SMS high-dose group ( Figure 1C ).

Nlrp3 protein expression was significantly increased in the injured joints of MSU mice, whereas p-Akt (S473) expression was decreased. SMS treatment suppressed the upregulation of Nlrp3, but further increased the expression of p-Akt (S473), especially in the SMS high-dose group (10mg/kg) ( Figure 2 Aa ). No significant difference of the total Akt expression was detected in mice joint of all the groups.

In vitro, inflammatory factors NLRP3, IL-6, and IL-1β were also reduced by SMS treatment in the MSU stimulated THP-1 cells, while p-AKT (S473) was upregulated ( Figures 2 Ab , 2Ba ). However, the inhibitory effects of SMS on NLRP3 were notably suppressed by pre-treatment with the PI3K/Akt inhibitor LY294002 ( Figure 2 Bb ).

To identify the macrophage polarization, joint tissue from mice was subjected to double immunofluorescence staining for the marker F4/80 and Arg-1. F4/80⁺/Arg-1⁺ cells were ubiquitously observed in mouse joints ( Figure 3 Aa ), and were significantly increased by SMS treatment ( Figure 3 Ab ). In MSU-treated THP-1 cells, iNOS expression was induced, whereas ARG-1 remained stable. Upon SMS administration, iNOS expression was inhibited but ARG-1 was elevated. After blocking Akt activation with LY294002, Arg-1 upregulation and the anti-inflammatory effects of SMS were simultaneously suppressed in macrophages ( Figure 3B ).

In this study, we further evaluated the effects of SMS on SUA levels. As presented in Figure 4A , hyperuricemia was induced in mice treated with PO and YP for 3 weeks. However, hyperuricemia was inhibited when the mice were simultaneously treated with SMS. Compared with the control group, the hyperuricemia and the SMS-treated mice did not show histological changes in the kidney ( Figure 4B ).

Because of the anti-inflammatory and uric acid lowering effects of SMS, mass spectrometry was used to screen the ingredients responsible for the observed benefits. Total Ion Chromatography of SMS was presented in Figure 5A with mass spectrometry. SMS did not exhibit any ion currents of the common ingredients of steroid (the spectra of the ingredients screened were listed in the Supplement Table 2 ) or non-steroidal anti-inflammatory drugs (the spectra of the ingredients screened were listed in the Supplement Table 3 ). Then phytohormones, estrogen and its analogs, and other potential hormones in plants were further examined by mass spectrometry ( Figure 5B ), ecdysone and estrone were finally detected in SMS ( Figure 5C ).