Search
Close this search box.

Sargassum horneri extract fermented by Lactiplantibacillus pentosus SH803 mediates adipocyte metabolism in 3T3-L1 preadipocytes by regulating oxidative damage and inflammation – Scientific Reports

Materials

S. horneri samples were collected along the shores of Jeju Island, South Korea. LAB strains, Lactiplantibacillus pentosus SH803 (100% identity, accession no. NR_029133.1), Lactobacillus plantarum HJ617 (99.8% identity, accession no. NR_115605.1), Lactobacillus acidophilus SJ422 (100% identity, accession no. NR_113638.1), and Lactobacillus acidophilus SH123 (99.86% identity, accession no. NR_117062.1), were obtained from the Food Microbiology Laboratory at Korea University (Seoul, South Korea). Additionally, 16S rRNA sequencing was performed by Macrogen (Seoul, South Korea) for strain identification. Sequence datasets generated during and/or analyzed during the current study are available in the GeneBank (NCBI-Nucleotide Database) repository and summarized in Supplementary Table ST. 1. All strains were grown in De Man, Rogosa, and Sharpe (MRS) broth (Kisan Bio, Seoul, South Korea) at 37 °C for 18 h and subcultured thrice before use.

Preparation of LAB-fermented S. horneri

S. horneri water extracts and fermented products of those were prepared44. S. horneri was washed, dried at 50 °C for 24 h, and ground. The resulting powder was mixed with distilled water (1:50 w/v) and autoclaved (121 °C, 15 min) for sterilization. After cooling, cellulase (1:50 v/v, Sigma-Aldrich, MO, USA) was added, incubated (50 °C, pH 4.5) for 48 h, and autoclaved (121 °C, 15 min) for cellulase denaturation and sample sterilization44. The resulting mixture was termed S. horneri water extract (SHWE). Subsequently, the minimal broth (peptone [1:100 w/v], sodium acetate 3H2O [1:200 w/v], magnesium sulfate 7H2O [1:10,000 w/v], manganese sulfate 4H2O [1:20,0000 w/v], 5 mL tween 80 [1:100 v/v], diammonium citrate [1:500 w/v], dipotassium phosphate [1:500 w/v], and distilled water adjusted to a total volume of 500 mL) was mixed with SHWE and used as bacterial strain culture medium. Thereafter, SH803 culture was centrifuged at 10,800 × g for 3 min (VS-180Cfi, Vision Scientific Co., Daejeon, Korea) and washed twice with phosphate-buffered saline (PBS). Next, the optical density of the harvested bacterial pellets at 600 nm was adjusted to 0.3; the pellets were added to S. horneri culture medium (1:100 v/v) and incubated for 48 h. Sample preparations were done at 0 h and 48 h fermentation and spread on MRS agar plates (Kisan Bio) to assess bacterial growth. SHWE fermented with SH803 was termed F-SHWE. Subsequently, F-SHWE was filtered using 0.45 μm Stericup filters (Merck, Darmstadt, Germany), freeze dried, and kept at − 80 °C until use.

Antioxidative activity evaluation using DPPH assay

Investigation of antioxidant activity was measured by the DPPH method, with slight modifications45. DPPH (2,2-diphenyl-1-picrylhydrazyl) solutions were prepared by dissolving 0.4 mM DPPH (Sigma-Aldrich) in ethanol (Duksan Chemicals, Incheon, South Korea) until the absorbance at 517 nm was 0.94–0.97. In addition, ascorbic acid solutions were prepared dose-dependently (0.6, 1.2, and 2.4 mM) and used as positive controls. Next, the samples (60 μL) were mixed with 1000 μL DPPH solution and kept in complete darkness for 30 min at room temperature (25 °C). After incubation, absorbance was determined at 517 nm using the Epoch microplate spectrophotometer (BioTek, VT, USA). DPPH radical scavenging activity was calculated using the following equation.

$$ {text{DPPH radical scavenging activity }}left( % right) , = , left( {{1} – left[ {{text{Abs}}_{{{text{sample}}}} } right]/left[ {{text{Abs}}_{{{text{blank}}}} } right]} right) , times {1}00 $$

where Abssample is the absorbance of the sample mixed with DPPH solution, and Absblank is the absorbance of the sample solvent mixed with DPPH solution.

Cell culture

HT-29 human colorectal adenocarcinoma cells were obtained from the Korean Cell Line Bank (KCTC, Seoul, South Korea). The cells were maintained in culture dishes containing Roswell Park Institute 1640 Medium (RPMI, Gibco, Dublin, Ireland) with 10% fetal bovine serum (FBS, Hyclone, MA, USA) and 1% penicillin/streptomycin (P/S, GE Healthcare, Chicago, IL, USA). Furthermore, 3T3-L1 murine preadipocytes were also obtained from KCTC. The cells were maintained in cell culture dishes containing Dulbecco’s Modified Eagle Medium (DMEM), low glucose (Gibco, Dublin, Ireland) with 10% bovine calf serum (Hyclone, MA, USA), and 1% P/S (GE Healthcare). Both the cell types were incubated in a humid atmosphere (37 °C, 5% CO2).

Cell cytotoxicity assessment using the MTT assay

Cell cytotoxicity was measured by the MTT assay with slight modifications45. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay was performed to assess the toxicity of F-SHWE on HT-29 and 3T3-L1 cells. The cells were seeded at a density of 5 × 105 cells/well in 96-well plates. After 24 h incubation, the cells were pre-treated with F-SHWE dose-dependently (0.2, 1, 2, and 10%) and incubated for 24 h (37 °C, 5% CO2). Subsequently, the cells were treated with MTT solution (5 mg/mL, Sigma-Aldrich) and incubated for 1 h. After media removal, the plates were washed with PBS and treated with dimethyl sulfoxide (Sigma-Aldrich). Eventually, absorbance was measured at 540 nm using the Epoch microplate spectrophotometer (BioTek, VT, USA), and the relative percentage of cell proliferation was calculated.

Gene expression measurement using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)

Gene expressions of biomarkers were measured using RT-qPCR46. For antioxidative activity evaluation, cells were seeded at a density of 5 × 105 cells/well in 12-well plates. After 24 h incubation, the cells were pre-treated with F-SHWE (1:2 v/v, diluted with RPMI) and incubated for 24 h (37 °C, 5% CO2). To induce oxidative stress, the cells were treated with 100 μM hydrogen peroxide (H2O2, Duksan, Ansan, South Korea) diluted with RPMI and incubated for 24 h (37 °C, 5% CO2). Next, total mRNA was extracted using TRIzol reagent (Life Technologies, CA, USA) following the manufacturer’s instructions. The concentration and purity of the extracted RNA were assessed using the NanoDrop spectrophotometer (BioTek, VT, USA) and standardized to a final concentration of 0.1 μg/μL. Following this, cDNA was synthesized using the reverse transcription kit (Thermo- Fisher). The polymerase chain reaction (PCR) cycling conditions were 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. Subsequently, reverse transcription quantitative real-time PCR (RT-qPCR) was performed using the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-rad, Hercules, CA, USA). Targeted genes were quantified using MG2 x qPCR MasterMix (SYBR green) (MGmed, South Korea). The RT-qPCR cycling conditions were: an initial denaturation cycle at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s, annealing at 55–65 °C for 30 s, and extension at 70 °C for 5 s. Lastly, the mRNA expression levels of each targeted gene were analyzed and normalized to the internal standard gene, GAPDH, using Bio-Rad CFX Maestro (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences used in this study are listed in Supplementary Table ST. 2.

For anti-inflammatory activity evaluation, cells were seeded at a density of 5 × 105 cells/well in 12-well plates. After 24 h incubation, the cells were pre-treated with F-SHWE (1:2 v/v, diluted with RPMI) and incubated for 24 h (37 °C, 5% CO2). To stimulate inflammatory responses, 1 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich) diluted with RPMI was added and incubated for 24 h (37 °C, 5% CO2). Subsequent procedures were similar to those performed for evaluating antioxidative activity using RT-qPCR.

For evaluation of adipocyte differentiation-related gene expression levels, 3T3-L1 cells were treated with post-differentiation media as described in Section “Gas Chromatography/Mass Spectrometry (GC/MS) instrumentation and chromatographic condition” and incubated for 6 days. The media was replaced every 2 days. Following this, the total mRNA was extracted using TRIzol reagent (Life) according to the manufacturer’s instructions. The RNA, PCR, and RT-qPCR analyses were performed as described in section “Gene expression measurement using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)”. The primer sequences used in this study are listed in Supplementary Table ST. 3.

Lipid accumulation assessment by Oil Red O Staining

Lipid accumulation was evaluated with Oil Red O staining47. Initially, 3T3-L1 cells were seeded at a density of 0.8 × 105 cells/well in six-well plates and incubated for 48 h. After incubation, the cell culture media was replaced and incubated for 72 h. Further, cells were co-treated with differentiation medium (DMI; 0.5 mM 3-isobutyl-1-methylanthine [IBMX], 1 μM dexamethasone, 5 μg/mL insulin, 10% FBS, and 1% P/S mixed in DMEM, low glucose) and F-SHWE (1:2 v/v, diluted with differentiation medium) and incubated for 48 h. Subsequently, the cells were treated with post-differentiation media (5 μg/mL insulin, 10% FBS, and 1% P/S mixed in DMEM, low glucose) and incubated for 6 days; the media was replaced every 2 days. After the 6-day incubation, cells were fixed with formaldehyde (10% v/v) for 10 min and washed with isopropanol (60% v/v). Subsequently, 1 mL Oil Red O staining solution (0.5% v/v in isopropanol, Sigma-Aldrich) was added to the cells and incubated at room temperature for 20 min. Finally, the cells were washed with PBS and observed using the Olympus CKX41 inverted phase contrast microscope (Olympus, Tokyo, Japan). Oil Red O-stained area ratio (%) was measured using Image J software (National Institutes of Health, MA, USA).

Gas chromatography/mass spectrometry (GC/MS) instrumentation and chromatographic condition

Short chain fatty acid (SCFA) contents of SHWE and F-SHWE were determined using GC/MS, per the method described by Eor et al. with slight modifications48. Briefly, 50μL of SHWE or F-SHWE samples were combined with 100 µL crotonic acid, 50 µL HCl, and 200 µL ether, and then homogenized and centrifuged at 1000 ×g for 10 min. Supernatants were transferred to vials and 16 µL N-tert-butyldimethylsilyl-N-methylttrifluoroacetamide (Sigma‒Aldrich, USA) was added. After mixing, vials were sealed and heated at 80 ℃ for 20 min, and then kept in room temperature for 48 h. Samples were placed in a 6890N Network GC System (Agilent Technologies, California, USA) with a HP-5MS column (30 m, 0.25 mm, 0.25 µm) and 5973 Network Mass Selective Detector (Agilent Technologies, USA). Helium (99.9999% purity) was used as a delivery gas at a flow rate of 1.2 mL/min. The head pressure was 97 kPa and the split was 20:1. The inlet and transfer line temperatures were 250 and 260 ℃, respectively. The following temperature program was used: 60 ℃ (3 min), 60–120 ℃ (5 ℃ min), 120–300 ℃ (20 ℃ min). One microliter of sample was injected with 30 min of run time. SCFA concentrations were qualified by comparing their peak areas with those of the standards.

Statistical analyses

Statistical analyses were performed using IBM SPSS Statistics software version 25.0 (IBM, Armonk, NY, USA). One-way analysis of variance with Tukey’s test was used to analyze the statistical difference between the mean values of samples. Statistical significance was set at p < 0.05. All figures were drawn using GraphPad Prism 9.0 (GraphPad Software, Boston, MA, USA). Correlation based analyses and visualization were performed using R studio (RStudio, United States) and related packages.