2.1 Experimental Materials
2.1.1 Experimental Animals
SPF-grade female BALB/c mice aged 5-6 weeks (17-19g) were purchased from Qinglongshan Animal Breeding Center in Jiangning District, NanJing City, China. Permit Number: SCXK (su) 2017-0001.
2.1.2 Cells and Viruses
Human epidermoid larynx carcinoma cells contaminated with Hela cells were purchased from ATCC (CCL-23). Cells in passages 5-30 used for experiments. Human respiratory syncytial virus (RSV) strain A2 Long was purchased from China Center for Type Culture Collection (Wuhan, China). Cells and viruses were preserved in Jiangsu Provincial Key Laboratory of Childhood Respiratory Diseases (Traditional Chinese Medicine).
2.1.3 Drugs and Reagents
LUT-7G purchased from Nanjing Liangwei Biotechnology Co., Ltd. (Nanjing, China)
Carboxymethyl cellulose sodium salt (CMC-Na, C4888), hydrochloride (98%), pyridine (99.8%), 1, 2-13C2-glucose (SH2326V, 99%), N,O-Bis(trimethylsilyl)-trifluoroacetamide (BSTFA), pyruvate (Batch no: SLBP4879V), glucose (Batch no: WXBC1347V), lactate (Batch no: BCBS2643V) , 3-phosphoglycerate (Batch no: SLBW5317), malate (Batch no: WXBB0572) , succinate (Batch no: SLBR5477V) , citrate (Batch no: MKBS5294V) , fumarate (Batch no: WXBB1420V) and α-ketoglutarate (Batch no: BCBT6944) were purchased from Sigma-Aldrich Company (St Louis, MO, USA). Phosphoenolpyruvate (PEP, Batch no: D1712023) and cis-aconitate (Batch no: C1806046) were purchased from Shanghai Aladin Biochemical Technology Co. LTD. Isocitrate (Batch no: H06S7B19343) was purchased from Source Leaf Creature (Shanghai, China).
TRIzol reagents was purchased from Ambion (Texas, USA). Reverse transcription reagents, Taq DNA polymerase, quantitative real time PCR reagents were purchased from Vazyme Biotech Co., Ltd (Nanjing, China).
2.1.4 Primers for Real-Time PCR
PCR primers (Table 1) were synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).
2.2 Experimental Methods
2.2.1 Animal Modeling, Drug Administration and Material Collection
RSV was amplified on Hep-2 cells and cultured in DMED [DMEM containing 2% FBS (v/v), 100 U/mL penicillin, and 0.1 mg/mL streptomycin] at 37°C, 5% CO2. When cell fusion lesions caused 50%-80% of Hep-2 cells to shed (typically occurs between 48h and 72h after infection), both supernatant and cells were collected, vortexed vigorously for 5-10 s, centrifuged at 3,000 rpm for 10 min, aspirated the supernatant, and then stored at -80°C for use. The titer of RSV was quantified by a plaque assay.
The experimental protocol was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine. After 1 week of adaptive feeding, the mice were anesthetized by ether inhalation. The control group was given 80 μL of saline through the nose, and the other groups were given 80 μL of RSV virus solution containing 5 × 105 PFU. LUT-7G was given daily (60mg/kg·d-1) intragastrically for at least 12 hours after the mice were infected with RSV. The control group and the model group were given saline containing 0.5% CMC-Na, once a day. Both groups were intervened for 4 days.
The mice were anesthetized within 24 hours after the last administration, and blood was collected from the eyeballs. Then the mice were sacrificed by dislocation, lung tissues were obtained for HE staining, PCR, GC-MS and other experiments.
2.2.2 Lung Histopathological Evaluation
Left-lung tissues of mice were fixed with 4% paraformaldehyde overnight, dehydrated by gradient ethanol, then embedded in paraffin, sectioned to a thickness of about 4 μm and dried for later use. HE staining was performed, microscope images were acquired and analyzed. HE staining was mainly used to evaluate the infiltration of airway inflammatory cells, with a score of 0 being no inflammation, 1 being slight or very little, 2 being moderate or more, 3 being severe or much, and 4 being very severe or heavy. 0 is normal, 1, 2, 3, and 4 are graded from mild to severe. The pathological scores of 5 mice in each group were counted.
2.2.3 Detection of Viral Surface Proteins and Inflammatory Factors by RT-PCR
RNA was extracted from mouse lung tissues with TRIzol reagents and reverse transcribed. Quantitative PCR was performed on a Quant Studio 7 Flex real-time PCR system using Eva Green Master Mix reagents. The data were calculated and analyzed by the 2-△△CT method, and normalized with GAPDH as the internal reference.
2.2.4 Detection of Glycolysis and TCA Intermediates by GC-MS
184.108.40.206 Preparation of Reference Solution
Accurately weighed an appropriate amount of glucose, 3-phosphoglycerate, PEP, pyruvate, citrate, cis-aconitate, isocitrate, and succiniate, placed the above reference substance in a 10mL volumetric flask, and diluted with water. Proper amounts of fumarate, malate, lactate and α-ketoglutarate were placed in a 10mL volumetric flask, diluted with methanol to prepare a mother solution, and then diluted with methanol solution to a predetermined gradient concentration (Supplementary Table 1).
220.127.116.11 GC-MS-based Samples Preparation for Metabolomics
Taked 50 μL of serum samples, lung tissue lysates or 50 μL mixed reference solution, added 200 μL of ice methanol (containing 10 ug/mL internal standard 1,2-13C-myristic acid), vortexed for 3 min, and incubated at 4°C , 18,000 rpm, centrifugation for 10 min. Taked 100 μL of the supernatant, put it in a centrifugal concentrator and evaporate to dryness for 2 h, added 30 μL of 10 mg/mL methoxyamine pyridine solution, mixed for 3 min, and shaked in a constant temperature shaker at 30 °C , 450rpm for 1.5 h. Added 30 μL of BSTFA, mixed well, shaked at 37°C , 450rpm for 0.5 h, then centrifuged at 18,000 rpm for 10 min, taked the supernatant, transfered the mixture to a sampling bottle with a glass insert and conducted GC-MS analysis .
18.104.22.168 Gas Phase Conditions and Mass Spectrometry Conditions
The gas chromatograph equipped with the Trace 1310 automatic sampling system, the chromatographic column was a TG-5MS capillary chromatographic column, and the temperature program was adopted. The initial temperature was kept at 60 °C for 1 min, then increased to 100 °C at 30 °C/min, and finally increased to 300 °C at 20 °C/min and held for 2 min. The inlet temperature was 300 °C; the split mode was 20:1; the carrier gas was high-purity helium, and the flow rate was 1.2 mL/min; the injection volume was 1 μL.
Electron impact (EI) was used as an ionization source for GC-MS analysis. The ion source temperature and transfer line temperature were both 300 °C; the ionization energy was 70 eV; the solvent peak delay time was 3.8 min. The retention time of each target metabolite was obtained by full scan mode, each metabolite was quantitatively determined by selective reaction monitoring (SRM) mode, and the collision gas was high-purity argon. The specific parameters of each metabolite are shown in Supplementary Table 2, and the extracted ion chromatogram of each reference substance is shown in Supplementary Figure 1-2.
2.2.5 Statistical Analysis
GraphPad Prism 7.0 was used for data processing and graphing, and measurement data were expressed as Mean ± SD. One-way ANOVA was used for comparisons among multiple groups, Dunnett's post-hoc was used for multiple comparisons, P<0.05 was considered as statistically significant difference. MetaboAnalyst 3.0 (http://www.metaboanalyst.ca) was used to perform principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA) and metabolic pathway analysis on metabolomic data (Supplementary Table 3).