Materials
Weak anion exchange gel (DEAE-650M) was provided by TOSOH Corporation (Tokyo, Japan). Dialysis tubes with molecular weight cutoff of 3.5 kDa, 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH·), lactate detection kit and phosphatase inhibitor cocktail were purchased from Solarbio Life Sciences (Beijing, China). All monosaccharide standards, 1-phenyl-3-methyl-5-pyrazolone (PMP), lipppolysaccharide (LPS; from Escherichia coli O111:B4), 1,2-naphthoquinone-4-sulfonic acid sodium salt (NQS) were purchased from Sigma-Aldrich (Shanghai, China). Dextran standards (Mw: 73300, 95800, 206800, 544800, 1194000, 2667000) were purchased from American Polymer Standards Corporation (Mentor, Ohio, USA). Mycoplasma detection and prevention reagents were purchased from Shanghai Yise Medical Technology Co., Ltd. (Shanghai, China) and TransGen Biotech Co., Ltd. (Beijing, China), respectively. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Gibco Life Technologies (Shanghai, China). Cell counting kit-8 (CCK-8) was purchased from MCE-China (Shanghai, China). ELISA kits (tumor necrosis factor (TNF)-α, IL (interleukin)-6), nitric oxide (NO), neutral red uptake detection kits, BCA protein detection kit, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS·+), ferric ion reducing antioxidant power (FRAP) detection kit, Western blocking buffer and glucose detection kit were acquired from Beyotime Biotechnology (Beijing, China). Total RNA extraction kit was purchased from Promega Corporation (Shanghai, China). Polyvinylidene fluoride (PVDF) transfer membrane (0.22 µm) was purchased from Millipore (MA, USA). Pattern recognition receptor (PRR) antibodies including anti-mannose receptor antibody (anti-MR, ab300621), anti-Scavenger receptor antibody (anti-SR, ab36970), anti-complement receptor type 3 antibody (anti-CR3, ab8878), anti-Toll-like receptor 2 antibody (anti-TLR2, ab16894), anti-Toll-like receptor 4 antibody (anti-TLR4, ab22048), anti-CD14 antibody (anti-CD14, ab221678) and anti-dendritic cell-associated C-type lectin-1 antibody (anti-Dectin-1, ab287961) were bought from Abcam, Inc. (Cambridge, England). Antibodies against β-actin (sc-47778), phospho-nuclear factor kappa-B p65 (p-NF-κB p65, sc-136548), NF-κB p65 (sc-8008), inhibitor of NF-κB (IκBα, sc-1643), protein kinase B (Akt, sc-81434), phospho-Akt (sc-514032), myeloid differentiation factor88 (MyD88, sc-74532) and phosphatidylinositol 3 kinase (PI3K, sc-374534) were purchased from Santa Cruz, Inc. (California, USA). Antibodies against phospho-PI3K (4228T), phospho-IκBα (2859T), glucose transporter1 (GLUT1, 12939S), lactate dehydrogenase A (LDHA, 2012S) and anti-rabbit IgG (H + L) (#5366) and anti-mouse IgG (H + L) (#5470) secondary antibodies were purchased from Cell Signaling Technology (MA, USA). The other chemicals used were of analytical grade or the best available.
SAV with an aging time of 3 years from the same bach (physicochemical indexes of the vinegar sample were listed in Supplementary Table 6) was supplied by a vinegar factory (Taiyuan City, Shanxi Province). SAV melanoidins (3.5VM) were immediately collected by dialyzing vinegar with dialysis tubes (3.5 kDa) against distilled water and then lyophilized using an ALPHA 2–4 LD plus lyophilizer (Christ, Germany). Model melanoidins (3.5MM) synthesized by D-glucose and L-alanine were prepared according to our previous method64.
Isolation and fractionation of SBMSAV
SAV was firstly centrifuged (8000 g, 30 min) and then evaporated to remove volatiles. SBMSAV were precipitated with ethanol at a final concentraction of 70% (v/v). The precipitation was redissolved in deionized water and centrifuged (8000 g, 30 min) to remove denatured impurities. The supernatant was lyophilized, giving the crude portion (SAV-CP70). The proteins were removed from SAV-CP70 utilizing Sevag reagent. Then the as-obtained SBMSAV (2.0 mg/mL) were decolored using activated charcoal (200 mesh) under an optimized condition (1% m/v, pH4.0 with acetic acid, 70℃, 40 min). The decolored SBM solution was concentrated and dialyzed against distilled water using 3.5 kDa dialysis tubes. The final SBM solution was lyophilized to yield the refined portion (SAV-RP70).
DEAE-650M column chromatography was used for further fractionation. SAV-RP70 (~ 300 mg) was dissolved in 10 mL water and passed through the column at a flow rate of 15.8 mL/min. The sub-fractions were eluted with distilled water, 0.1 M and 0.3 M NaCl solutions and monitored by phenol-sulfuric method19. Each of these sub-fractions was pooled together, concentrated, dialysed and then lyophilized to obtain the purified sections, which were named as SAV-RP70-W, SAV-RP70-N1 and SAV-RP70-N2, respectively. Besides, the fraction which was eluted by 1.0 M NaCl, namely in the column regeneration procedure, was also collected and performed the same handling as above and given a name as SAV-RP70-X.
Characterization and comparative analysis of SBMSAV
Basic chemical composition analysis
Total neutral sugar and uronic acid contents were determined by phenol-sulfuric acid method and m-hydroxybiphenyl assay, respectively19. The total phenolic contents were measured according to the Folin-Ciocalteu method using gallic acid as standard8.
Molecular weight (Mw) distribution
The Mw distribution of the analytes was determined by a HPLC (Agilent 1260, Santa Clara, USA) equipped with TSKgel G5000PWXL column (10 µm × 7.8 mm × 300 mm, TOSOH, Tokyo, Japan) and refractive index detector. Milli-Q water was used as mobile phase at a flow rate of 0.6 mL/min. The oven temperature was kept at 40℃, the injection volume was 20 µL. Serial dextrans were used as standards.
Organic element analysis
Organic elements of C, H, O and N were quantified using a Vario EL Ⅲ elemental analyser (Elementar, Hanau, Germany) with thermal conductivity detector65.
Monosaccharide composition analysis
Monosaccharide composition of all analytes was assayed by HPLC of precolumn derivatization with PMP. Ten milligrams of each analyte were hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120℃ for 4 h. After cooling, methanol was added into the hydrolysate allowing TFA to be evaporated under nitrogen flow. This procedure was repeated thrice. Then 3.0 mL water was added to the dried hydrolysate and the solution was used for subsequent derivatization. In detail, 250 µL hydrolysate solution was mixed with 250 µL 0.6 M sodium hydroxide and 500 µL 0.4 M methanolic solution of PMP. The mixed solution was heated to 70℃ and kept for 1 h. After cooling to ambient temperature, the reaction mixture was neutralized with 0.3 M hydrochloric acid (500 µL), and the redundant PMP was removed through extracting by chloroform for several times. The monosaccharide standards were treated simultaneously under the same condition. The final aqueous solution was filtered through a 0.22 µm membrane. A LC-20AD HPLC (Shimadzu, Tokyo, Japan) equipped with an Xtimate C18 column (5 µm, 200 × 4.6 mm; Welch Materials Inc., Shanghai, China) was used for HPLC analysis. The HPLC was operated using following parameters: column oven 30℃, flow rate 1.0 mL/min, UV wavelength 250 nm, injection volume 20 µL. The elution phase was a mixture of 0.05 M potassium dihydrogen phosphate solution (pH6.70) and acetonitrile in a ratio of 83:17 (v/v).
Spectral characterization
Ultraviolet-visible (UV-Vis) spectroscopy
All analytes were dissolved in distilled water to proper concentration for the UV-Vis spectral analysis using an UV-2550 spectrometer (Shimazu, Tokyo, Japan). Specific extinction coefficients (Kmix) at 284, 336 and 420 nm were calculated according to the law of Lambert-Beer: Abs = Kmix (L/g/cm) × concentration (g/L) × length of light path (cm)27. Similarities between two UV-Vis spectra were calculated as reported by Cheng et al66.
Fourier transform infrared (FT-IR) spectroscopy
All FT-IR spectra of the analytes were performed with a Nicolet IS50 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, each 0.2–0.5 mg analyte was ground with KBr powder and then pressed into a pellet and recorded on the spectrometer in the frequency range from 4000 to 400 cm− 1 at a resolution of 4 cm− 1.
Excitation-emission matrix (EEM) spectroscopy
The EEM spectra of all analytes (~ 0.1 mg/mL of each sample in distilled water) were acquired by a F-7100 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) at an excitation wavelength (λEx) of 200–450 nm and emission wavelength (λEm) of 250–550 nm. Excitation and emission sampling intervals were 5 nm, the slits were 10 nm, and the photomultiplier tube voltage was 400 V.
Nuclear magnetic resonance (NMR) spectroscopy
For nuclear magnetic resonance (NMR) assay, 50–80 mg of each sample was dissolved in 0.8 mL D2O and transferred to separate NMR tube for testing. The 1H and 13C NMR spectra were collected using a Bruker 600 MHz NMR spectrometer (Avance NEO 600, Bruker, Germany). All NMR experiments were conducted at 298 K.
Pyrolysis characteristic analysis
The pyrolysis assay was conducted under fixed temperature or linear temperature. For pyrolytic characterization and comparative analysis of SAV-RP70 and 3.5VM, pyrolysis- gas chromatography/mass spectrometry (Py-GC/MS) was conducted using an EGA/PY-3030D pyrolyzer (Frontier lab, Fukushima, Japan) coupled to a QP2010 Ultra GC/MS (Shimadzu, Tokyo, Japan) with a Rtx-5MS column (30 m × 0.25 mm × 0.25 µm, Shimadzu, Tokyo, Japan). In detail, 0.40 mg of each sample was pyrolyzed at 900℃ for 12 s, and the GC was programmed from 40 to 280℃ holding the initial temperature for 1 min, then 5℃/min to 80℃, then 15℃/min to the final temperature holding for 15 min. The carrier gas was helium (He) with a flow rate of 1.0 mL/min. The MS adopted an electron ionization source being set an electron energy of 70 eV, transmission line and ion source temperature at 280 and 230℃, respectively. The mass spectra were obtained from m/z 40 to 550. The qualitative analysis of the pyrolyzates was based on the NIST library. Peak area percentages were used to perform a relative quantification analysis.
For the pyrolysis analysis of other analytes, thermogravimetry-mass spectrometry (TG-MS) via a thermogravimetric mass spectrometer (Thermo Mass Photo, Rigaku, Tokyo, Japan) was performed. Detailedly, a certain amount (~ 2.5 mg) of each sample was pyrolyzed programmatically from ambient temperature to 800℃ with a heating rate of 10℃/min under He atmosphere, and the evolved gas was carried by He with a flow rate of 300 mL/min and monitored simultaneously by mass spectrometer under electron ionization mode from m/z 1-400.
Micromorphology imaging
The micromorphology of all samples was observed through field-emission scanning electron microscope (SEM; TESCAN MIRA4, Brno-Kohoutovice, Czech Republic) and atomic force microscope (AFM; Bruker Dimension Icon, Billerica, MA, USA). In SEM assay, all samples were sputter-coated with platinum and a voltage of 10.0 kV was chosen. For AFM assay, all analytes were prepared in solution with a concentration of 10 µg/mL, and a 5 µL of each solution was pipetted onto a freshly cleaved mica plate. After air-dried, all samples were observed following operation instructions in a non-contact mode.
Particle size distribution and dynamic viscosity analysis
Particle size distribution of each sample was measured utilizing a Zeta potential analyzer (ZetaPALS, Brookhaven, Holtsville, NY, USA). Briefly, each sample was dissolved in distilled water to 4.0 mg/mL and dispersed ultrasonically for 5 s. The measurement temperature was 25℃ and scattering angle was 90.00°.
Dynamic viscosity data of all analytes in aqueous solution (5.0 mg/mL) were collected via a rolling-ball viscometer (Lovis 2000 M, Anton Paar, Oosterhout, Netherlands) under 25℃.
Chromaticity characterization
Color values of all test samples were measured resorting to a Lico 690 professional spectral colorimeter (Hach, Loveland, Colorado, USA). Firstly, the colorimeter was calibrated with distilled water in a 11-mm round glass cuvette. Then, 2.0 mg/mL of each sample was performed the same operation. The chromaticity coordinates of all analytes were expressed by adopting the Commission Internationale de l'Eclairage (CIE)LAB color space (http://www.colorbasics.com/ColorSpace/) and the CIExyY color space. The chromaticity coordinates derived from CIExyY color space were depicted in the CIE1931 chromaticity diagram.
Antioxidant activity assay
The antioxidant activities of all samples were evaluated using three methods, including DPPH, ABTS and FRAP assays. The DPPH assay was performed according to guidelines reported67, ABTS and FRAP assays were conducted following the instructions in commercial kits.
Cellular experiment
The murine macrophage RAW264.7 cells were acquired from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and performed mycoplasma detection. Cells without contamination were then incubated in a humidified chamber at 37℃ under 5% CO2 in DMEM medium containing 10% FBS and 0.1% mycoplasma prevention reagent.
Cell viability assay
Cell viability assay was conducted referring to the CCK-8 method. In detail, RAW264.7 cells were incubated in 96-well plates at a density of 5×104 cells/cm2 and allowed to adhere for 4 h. Then, the cells were treated with different concentrations (25–800 µg/mL) of SBMSAV and melanoidins (3.5VM and 3.5MM) for 24 h. Following that, the culture medium was discarded and 110 µL fresh medium with 10% CCK-8 was added to each well. After incubated for another 3 h, the absorbance at 450 nm was measured using a microplate reader (Infinite 200 pro, Tecan, Männedorf, Switzerland). In parallel, cells cultured only in medium and with LPS (1 µg/mL) were adoptted as normal control (NC) and positive control group, respectively.
Cellular morphology and neutral red uptake assay
The 96-well plates tested after cell viability were conveniently transferred under a FV1000 confocal laser scanning microscopy (Olympus, Tokyo, Japan) for cellular morphology observation in differential intereference constrast mode.
Neutral red uptake assay was used to evaluate the phagocytic activity of each sample on RAW264.7 cells. Macrophages with a density of 1×105 cells/cm2 were incubated in 96-well plate and treated with each sample as described above. After treatment for 24 h, medium in each well was superseded by neutral red in fresh DMEM. After lysed by lysis buffer, the phagocytic activity was reflected by measuring absorbance at 540 nm as instructed in commercial kit.
Determination of NO and cytokines
Macrophages were placed in 24-well plates at a dense of 1×105 cells/cm2 and preincubated for 12 h. After that, LPS and different samples (25–200 µg/mL) were added with DMEM medium for 24 h-incubation. NO along with TNF-α and IL-6 were determined by corresponding NO detection and ELISA kits, respectively.
Investigation of membrane receptors
The membrane receptor assay was conducted referring to literature51. In detail, RAW264.7 cells were seeded in 96-well plate with a density of 1×105 cells/cm2 and precultured for 12 h. Then the cells were treated with antibodies (5 µg/mL) of PRR (anti-MR, anti-CR3, anti-LTR2, anti-TLR4, anti-CD14, anti-Dectin-1, anti-SR) or the mixed antibodies (anti-Mix) for 2 h. After that, the cells were treated with/without SAV-RP70-X (200 µg/mL) or LPS (1 µg/mL) for 24 h. The group cultured only with SAV-RP70-X was used as control, the untreated cells were normal control (NC), and LPS treated cells were positive control group. Finally, the levels of NO and TNF-α in supernatants were measured as stated above.
Determination of glucose uptake and lactate production
The glucose uptake and lactate production levels were quantified by detecting glucose and lactate concentrations in cell culture supernatant retrieved in RT-qPCR assay (see below). The consumption of glucose was calculated as the difference of glucose levels between the final and the initial medium. All operations were performed in accordance with the instructions in detection kits.
Reverse transcription quantitative PCR (RT-qPCR) and Western blotting (WB)
Macrophages with a density of 1×105 cells/cm2 were incubated in 6-well plates and treated with/without SAV-RP70-X or LPS for 24 h. Then the cells were washed with PBS and collected under 4℃. Total RNA in cells was extracted complying with the manufacturer’s specification. Then the total RNA was reverse-transcribed into cDNA with a PCR amplifier (VeritiPro, ThermoFisher Scientific, USA). Transcription levels of Nos2, Il6, Tnf, Glut1, Ldha and Actb (internal reference) were quantitated by RT-qPCR amplifier (SteponePlus, Applied Biosystems, Carlsbad, CA, USA) with AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, China). All primer sequences for RT-qPCR were listed in Supplementary Table 7.
Macrophages at a dense of 5×104 cells/cm2 were cultured in 10-cm plates and allowed for adhere for 12 h. Then the cells were treated with LPS or SAV-RP70-X for 1 h (for phosphorylated protein detection) or 24 h (for MyD88, GLUT1 and LDHA detection) and then washed with cold PBS and lysed with RIPA buffer containg phosphatase inhibitor. Following protein quantification, the proteins were denatured at 95℃ for 5 min. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separated proteins. After proteins were transferred to PVDF membrane and blocked with Western blocking buffer for 13 min at ambient temperature, the blots were incubated with target primary antibodies (anti-p-NF-κB p65, anti-NF-κB p65, anti-p-IκBα, anti-IκBα, anti-p-PI3K, anti-PI3K, anti-p-Akt, anti-Akt, anti-MyD88, anti-GLUT1, anti-LDHA) under 4℃ overnight. Subsequently, the PVDF membrane was rinsed with TBST and incubated with corresponding secondary antibodies (DyLight 680 conjugated anti-rabbit or anti-mouse IgG) for 2h. After washing the membrane with TBST, protein bands were visualized by an Odyssey infrared imaging system (Li-Cor, Licoln, NE, USA). The protein quantification was performed using ImageJ software (version 1.53e, National Institutes of Health, Bethesda, USA).
Statistical analysis
All biochemical analyses were conducted in triplicate. Data were presented as mean ± standard deviation. Differences were deemed as significant and highly significant with the p-values less than 0.05 and 0.01, respectively, followed by one-way ANOVA or Student’s t-test.
Data Availability
The authors declare that the data supporting the findings of this study are presented in the article. The raw data are available on request.