2.1 Cell culture and hydrogen treatment
A549 cells were resuscitated according to the routine. Cell culture was carried out using RPMI-1640 medium containing 10% fetal bovine serum. The cells were passed 1 times every 1-2 days, according to cell growth. After reaching the expected number of cells, the cells were intervened in hydrogen incubator for 2 hours with different concentrations of hydrogen. The control group was placed in the common cell culture box. After 3 days of intervention, the cells were collected and the total protein was extracted to carry out the experiment.
2.2 Detection of apoptosis by flow cytometry
Trypsin was used to digest and collect cells, then centrifugation and discarding supernatant. The cells were added with ice PBS 250 μl and then 750 μl ice ethanol was added by drop. The final ethanol concentration is up to 75%. The suspension is placed at -20 C for at least 2 hours in the refrigerator. Take the fixed cells from the fridge, centrifuge for 3 minutes with 2000 turns, discard the supernatant, and add PI/RNase Staining Buffer.
2.3 Subcutaneous tumor formation in nude mice
Acquisition of 5-6 week male nude mice from card Vince experimental Animals Inc, Changzhou, Jiangsu. The nude mice were anaesthetized with ether. First, the skin was sterilized with alcohol, and the A549 cell suspension was inoculated into the axilla of the forelimb of nude mice. The cell concentration was 250х106 /ml, each was inoculated with 0.1 ml. After subcutaneous tumor formation, the control group was still fed normally, and the hydrogen group was given 60% concentration of hydrogen inhalation once a day for four hours each time. Cisplatin group was given intraperitoneal injection of cisplatin 4 mg/kg for one times a week. After 3 weeks, the nude mice were sacrificed, then the tumor tissue was removed and fixed in 4% neutral formaldehyde.
The fresh tumor tissue was immobilized in 4% neutral formaldehyde for 48-72 hours. The tissue was first dehydrated in gradient alcohol, then transparent in turpentine, and finally immersed in a wax cylinder and embedded into wax blocks. The wax blocks were cut into pathological sections with a thickness of 4 μm. The tissue section was followed by roasting, dewaxing, hydration, antigenic heat repair, 3% hydrogen peroxide blocking endogenous peroxidase activity, 5% goat serum blocked for 60 minutes at room temperature. The sections were subsequently incubated overnight at 4˚C with primary antibody. The negative control group was incubated with PBS instead of the primary antibody. The slides were incubated with a biotinylated goat anti-mouse horseradish peroxidase conjugated secondary antibody (proteintech, Wuhan, China) for 30minutes at 37℃ centigrade bath. The slices were stained with DAB reagent and distilled water to stop dyeing. After DAB staining, first use of hematoxylin to stain the slices, followed by 1% hydrochloric acid alcohol for color separation, and then use ammonia water to return blue, using anhydrous ethanol soaked for 5 minutes. After the tissue slices were dried, the neutral gum was used to seal them.
2.5 Transcript Analysis
After cultured A549 cells reached the experimental number, the control group was routinely cultured, and the experimental group was treated with 60% hydrogen. Two groups of specimens were sent to Source of NOA in Beijing to extract total RNA by TRIzol method. Samples were tested by agarose gel electrophoresis, NanoPhotometer® spectrophotometer， Qubit® 2.0 Fluorometer，Agilent 2100 bioanalyzer and so on. After the establishment of the library, the quality of the library was checked to ensure the quality of the library, followed by Illumina HiSeq/MiSeq sequencing. In comparison between the two groups, the differential genes were screened from two levels of significant and multiple differences ( Pvalue(pval)<0.005, │log2FoldChange│>1). KEGG Pathway enrichment was used to analyze the major signaling pathways involved in differential genes.
2.6 Western blot
First, SDS-PAGE gel was prepared, then samples and marker were added to the lane. A constant voltage 110 V was used to separate the different molecular weight proteins. We used 200mA steady current to conduct transmembrane at 4 ℃. 5% skimmed milk was used to block the Polyvinylidene Fluoride(PVDF) membrane for 1 hours at room temperature. PVDF membrane was incubated overnight at 4℃ with the following primary antibodies: XIAP(1:1000 proteintech, Wuhan, China), BIRC3(1:750 proteintech, Wuhan, China), BAX(1:1000 Cell Signaling Technology, Beverly, MA, USA) or β-actin(1:1000 proteintech, Wuhan China). PVDF membrane immersed in secondary antibody (1:8000 proteintech, Wuhan, China) solution and incubated at room temperature for one and a half hours. Use the ECL developer to develop. All the antibody reagents were brand Proteintech.
2.7 Statistical Analysis
SPSS21.0 statistical software was used to analyze the data and the experimental data were expressed in x±s. The average number between the two groups was compared with the t test of two independent samples. The multiple groups of data were normal distribution and Fang Chaqi test, and a single factor analysis of variance was used for statistical test. The test level was set at α=0.05, and all results were statistically significant as compared with P < 0.05.