Isolation and purification of TDEVs
There is no perfect method to isolate EVs from tea leaves. We had made some improvements to isolate tea EVs. Fresh tea leaves were purchased from tea growing base (Zhaoqing, Guangdong, China) and washed with deionized water three times at room temperature. Tea juice was obtained from tea leaves in a slow grinder. Then, the juice was filtered with 200 mesh nylon membrane, and sequentially centrifuged at 2000×g for 20 min and 10,000×g for 30 min to remove large particles and fibers. The supernatant was collected and mixed with 6% PEG 6000 solution in equal volume and placed at 4℃ for 10 min, then centrifuged at 10,000×g for 10 min. And finally centrifuged at 100,000×g for 90 min (Beckman Optima XE-100, Beckman, USA). The pellets were resuspended in PBS, and ultra centrifuged at 100,000×g for 60 min. Finally, the TDEVs were diluted in sterile PBS. The resuspension was filtered (0.22um) and used freshly or stored at -80。C for further use.
Characterization of TDEVs
Size distribution and particle numbers of the isolated TDEVswere determined by Nanosight (Malvern Instruments Ltd, Malvern, Worcestershire, UK). TDEVs samples were diluted in 1 ml distilled water and the characterization and component of all samples were analyzed at room temperature at least 3 replicates. TDEVs were resuspended in 2% paraformaldehyde (PFA) and loaded onto formvar carbon-coated grids, fixed in 1% glutaraldehyde, washed, and contrasted with a solution of uranyl oxalate (pH7), then embedded in a mixture of 4% uranyl acetate and 2% methylcellulose subsequently, and finally, the morphology of TDEVs was evaluated by transmission electron microscopy (Hitachi electron microscope H-600, Japan).
Assessment of TDEVs components by High performance liquid chromatography
EGCG is the most extensively studied tea catechin. A number of studies in vitro and in vivo demonstrated potential benefits of EGCG in animal models and in patients with NAFLD by clinical trials18. Therefore, we used HPLC (Elite EClassical 3100, Dalian, China) to measure the EGCG in TDEVs.The polyphenol composition in TDEVs was identified by the gradient of binary elution phase (A: acetonitrile, B: 0.1% acetic acid in water) as follows: 0-15 min (90% A), 15-20 min (40% A), 20-20.5 min (40% A), 20.5-25 min (90% A) at a flow rate of 0.5 mL/min. The detection wavelength was 278 nm and C18 Column (Elite, SinoPak SP C18, Dalian, China). The external standard method was used to detect the polyphenol compounds in TDEVs. The data were expressed as milligram per 100 g fresh weight of tea (mg/100 g FW), and reported as mean ± SD in triplicate.
Establishment of NAFLD cell model
Human hepatoblastoma HepG2 cells were purchased from the China Center for Type Culture Collection (Wuhan, China) and routinely stored in a humidified incubator with atmosphere of 5% CO2. The medium was α-minimum essential medium (α-MEM) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL. To establish a cell model, oleic acid (A502071, Sangon biotech, China) was added into the culture medium at a final concentration of 0.7 mM, and cells were cultured for 24 h. Oil red O (G1015, Servicebio, China) staining method was used to observe fat droplets in HepG2 cells. The red droplets in HepG2 cells indicated the successful establishment of non-alcoholic fatty liver cell model.
TDEVs treatment of NAFLD cell models
TDEVs were added to NALD HepG2 cell models with the final concentration of 50ug/ml, 100ug/ml, 200ug/ml and 300ug/ml respectively. The HepG2 cells were added in equal volume culture medium as blank control group. NALD HepG2 cell models were treated with equal volume culture medium as positive control group. After 24h, Oil red O staining was used to assess fat droplets in HepG2 cells. TDEVs of different concentrations were labeled with PKH67 dye (Solebaol, Beijing, china) and co-cultured with HepG2 cells for 24h. The absorptivity of TDEVs by HepG2 cells was observed by fluorescence microscopy.
Real-Time Quantitative PCR (RT-PCR) analysis
Total RNA was extracted from HepG2 cell using the TRIzol® reagent (DP424, Tiangen Biotech, China) and quantified using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). Reverse Transcription Kit (AE341, TransGen Biotech, China) was used for the conversion of isolated RNA into cDNA. SYBR Green Two-Step RT-PCR SuperMix (AQ301,TransGen Biotech, China) was used to analyze the mRNA expression level of MTTP，PNPLA3，PI3K genes. Reaction mixture consisted of SYBR Green PCR mix, universal primer, RNase-DNase free water and 0.5ug cDNA for each sample, and reactions were conducted according to the manufacturer’s protocol with 7500 Fast Real-Time PCR system (Applied biosystems; Thermo Fisher Scientific, USA). Gene expression levels were determined using the 2−∆∆ Cq method, and all data were normalized based on GAPDH expression. Each measurement was performed in three biological replicates.
Western blotting analysis
HepG2 Cells (2x106) were lysed in RIPA buffer (G2002, Servicebio, China) containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich, USA.) for 30 min at 4℃, followed by centrifugation at 3000x g for 30 min. Protein concentrations were determined using a BCA Protein Assay kit (Beyotime, China). Subsequently, immunological analyses were performed using Anti-MTTP antibody (GTX80580, GeneTex, USA), Anti-PNPLA3 antibody (GTX32794, GeneTex, USA), and Anti-PI3K antibody (GTX100462, GeneTex, USA). Densitometry analysis of each band was performed using Image J software.
Enzyme‑linked immunosorbent assay
Cells were lysed in RIPA buffer containing 1% protease inhibitor cocktail for 5 min on ice. Lysates were centrifuged at 2000×g for 10 min at 4 °C. TG ELISA kits was used to analyze TG levels in HepG2 cellular lysates.Detailed procedure was under manufacturer’s guide.
Bisulfite genomic sequencing analysis
All reagents were purchased from Sigma (St Louis, MO). Genomic DNA was isolated from HepG2 cells using the sodium dodecylsulfate (SDS) and proteinase K, and DNA bisulfite modified cation was performed as previously described. Bisulfite modified DNA was amplified with primers (Table 1) designed using the Meth Primer program (http://www.urogene.org/-methprimer/index.html). Direct sequencing and classic cloning/sequencing were used to detect DNA methylation. Briefly, promoters of target genes were amplified with Taq polymerase (Tiangen Biotech, Beijing, China). Then the PCR products were purified by shrimp alkaline phosphatase-exonuclease I (SAP-ExonI) and subjected to sequencing using a DNA sequencer (ABI PRISM 3730, Applied Biosystems) with Big dye terminator v3.1 cycle sequencing kit (Applied Biosystems). If there was a single “C” at a cytosine phosphodiester bond guanine (CpG) site, it was defined as complete methylation; if there was a single “T” at the CpG site, it was considered as no methylation. Overlapping of “C” and “T” was ranked as partial methylation. Classic cloning/sequencing was also used to accurately measure the levels of DNA methylation. The proportion of methylation for each CpG site was calculated as the number of methylated cytosine divided by total number of samples in each group.
Statistical analysis
All data were analyzed by one-way ANOVA using SPSS software package (version 22.0, USA). Data were expresses as the mean ± standard deviation. P < 0.05 was considered statistically significant.