Caco-2 cell line was from cell bank of Wuhan University (Wuhan, China). The MA104 cell line was from the cell bank of Sun Yat-Sen University (Guangzhou, China). The RV Wa strain (G1P) was was from the immunology institute of the Third Military Medical University (Chongqing, China). The glucose assay kit was obtained from Shanghai Rong Sheng Biotechnology Co., LTD (Shanghai, China). The glucose 6-phosphatase and phosphoenolpyruvate carboxylase assay kits were from Solarbio (Beijing, China). Anti-JNK, anti-AKT, anti-SIK2 and anti-PDK1 were obtained from Proteintech (Wuhan, China). Anti-beta-actin, anti-rabbit IgG were obtained from Cell Signaling Technology (Boston, USA).
2.2. Cell culture
Caco-2 and MA104 cells were cultured with high-sugar DMEM medium containing 1% anti-penicillin, anti-streptomycin and 10% FBS, which were placed in the cell incubator at constant 37℃ and 5% CO2 for conventional culture. When the cells grew to about 85%~90%, original medium was disposed. And an appropriate amount of trypsin containing 0.25% EDTA was added and incubated for 2 min~7min. Then fresh culture medium was added to stop digestion. Next, the mixed suspension of cells was centrifuged at 900 rpm/min for 3 min, and the supernatant was removed. Then 4ml DMEM culture medium was added for cell suspension, which was divided into new culture bottles according to a certain proportion. Finally, 7 ml fresh culture medium was added to continue the routine culture.
2.3. Cultivation and amplification of RV on MA104 cells
Virus were removed from -80℃ and dissolve at 4℃. 10 μg/ml trypsin without EDTA was added and incubated at 37℃ for 30min. MA104 cells, which had grown to a monolayer, were added with incubated RV venom. MA104 cells with RV were routinely cultured at 37℃ and 5% CO2. After the cytopathic effect of MA104 cells were observed under an inverted microscope, the cell culture bottle was taken out and put at -20℃ for 12 h. Next, the cell was placed at 4℃ for natural melting, and the above operations were repeated for 3 times. The cell freeze-thaw solution was collected in the centrifuge tube, and centrifuged at 12000 g for 30 min. Finally, the supernatant RV venom was collected and stored at -80℃. For the titer determination of RV, the incubated RV venom was first diluted into a series of concentrations, including 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6, in DMEM culture solution without fetal bovine serum, and then applied to MA104 cells at 100 μl/well. The pathological changes of MA104 cells after RV infection were observed at different times. When a CPE was no longer present in the 96-well plates of RV venom with the lowest dilution, the number of wells with a CPE was recorded for each dilution. The median tissue culture infective dose (TCID50) of the virus was calculated using the Reed and Muench method.
2.4. Preparation and grouping of Caco-2 cells infected with RV
The RV venom at a virus titer of 105 TCID50/ml (the virus reacted with 10 μg/ml trypsin for 30min) was added to the 100mm × 100mm culture dish where Caco-2 cells grew to a monolayer. At the same time, the uninfected Caco-2 cells was set and the equal volume DMEM culture medium was added. After incubated at 37℃ with 5% CO2 for 2 h, the cells’ RV venom was replaced with DMEM culture solution. After 48 hours of continuous culture, the RV-infected Caco-2 cells model was successfully constructed.
2.5. GC-MS analysis of cellular metabolites
Preparation of samples: the RV-infected and uninfected Caco-2 cells were scraped off by adding 4℃ pre-cooled methanol and transferred into the centrifuge tube. The mixture was centrifuged at 4℃ and 1.4 x 104 g for 15 min and take 500µl supernatant with 10 µl internal standard (50 µg/ml L-norvaline). After dried under nitrogen, 40µl methylamine hydrochloride pyridine was added and the supernatant was incubated at 37℃ for 90 min. Finally, added with 40 LBSTFA (including 1% trimethyl chlorosilane), it vortexed for 30 seconds and derived at 70℃ for 60 minutes. Samples derived from trimethylsilane were obtained for GC-MS analysis.
GC-MS conditions: Agilent gas chromatogram mass spectrometer (7890A/5975C) and MACHEREY-NAGEL OPTIMA®5 MSAccent fused silicon capillary column (30 m x 0.25 mm x 0.25 μm) were used to conduct metabolomics tests on derived samples. The obtained data files were imported into SIMCA software (version 14.1) of UmetricsAB for multidimensional statistical analysis such as principal component analysis (PCA), partial least-square-discriminant analysis (PLS-DA) and orthogonal filtering partial least-square-discriminant analysis (OPLS-DA). The model quality is described by the R2X or R2Y and Q2 values. R2X (PCA) or R2Y (PLS-DA and OPLS-DA) is defined as the proportion of variance in the data explained by the models and indicates the goodness of fit. Q2 is defined as the proportion of variance in the data predictable by the model and indicates the predictability of current model, calculated by cross-validation procedure. Generally, their value is greater than 0.5, which means that the model quality is better.
Metabolite structure identification method: The AMDIS software was used for GC-MS deconvolution analysis of original data automatically, which was matched self-built standard database (including retention time and mass spectrum), Golm metabolome database and Agilent Fiehn GC/MS metabonomics RTL database.
2.6. Analysis of gluconeogenesismetabolic enzymes
The supernatant of cell culture with RV infection was disposed, and then cells were scraped and collected in the centrifuge tube. The cells were centrifuged at 10000 g for 10min, and the precipitation was preserved. 1ml of working solution was added into the precipitate, which crushed by ultrasonic. According to the enzyme assay kit instructions, G-6-Pase and PEPCK activity were detected.
2.7. Analysis of glucose consumption
The glucose assay kit (glucose oxidase-peroxidase method) was used to detect the glucose content in the supernatant of cell culture in Caco-2 cells infected by RV, in order to determine the glucose consumption of Caco-2 cells after RV treatment.
2.8. Western blot
After Caco-2 cells were grown in a 6 cm petri dish and the experimental interventions were conducted, Radio immunoprecipitation assay lysis buffer (containing 1% PMSF) was added for cleavage. The protein samples were then added to the 5x SDS loading buffer and denatured at 95℃ for 5min. The denatured protein samples were separated by SDS-PAGE. It was transferred to PVDF membrane through wet transfer and incubated with corresponding antibodies. The dilution multiples of antibodies were as follows: JNK (1:500), PDK1 (1:500), AkT (1:2000), SIK2 (1:1000), and beta-actin (1:1000). Finally, protein expression levels were analyzed by Image J software, with beta-actin as internal reference.
2.9. Statistical approach
SPSS 13.0 statistical software was used for data analysis. T test was used to compare the two groups of control experiments, and One-Way ANOVA method was used to compare the mean of multiple samples. The experimental results were expressed as mean ± standard deviation (x ± s), and P < 0.05 was statistically significant.