158 patients from the oncology department of Jingzhou hospital who received radiation after neck cancers participated and their arterial function was monitored by B ultrasound. Similarly, HDL and other blood lipid indexes were also tested. All patients provided written informed consent.
C57BL/6 J mice were used for MAECs isolation. DMEM medium was obtained from Gibco Company. The Institutional Animal Care and Use Committee approved the experiments involved the use of animals. The Ethics Committee of Soochow University approved the present study protocol (clearance No: 2019854268).
2.3. Cell culture
C57BL/6 J mice were employed for isolating MAECs by applying an outgrowth technique which has been described earlier [14, 15]. DMEM medium was employed which contained 10% calf serum for maintaining MAECs by providing a temperature of 37 °C within a humidified incubator provided with 5 percent CO2. Serum-free DMEM medium was used for performing overnight (12–16 hr) serum starvation of MAECs cells after they attained their growth to that of near-confluence for all the experiments.
2.4. Preparation of HDL
Following the procedure, HDL was prepared . Briefly, Human serum obtained from Jingzhou central hospital (Jingzhou, Hubei) was taken to overlay with potassium bromide (KBr) gradient solution that possessed a density of 1.063 g/mL. After that, these samples were passed through ultracentrifugation for removing low as well as very low-density lipoproteins at 35,000 rpm for a period of 18hr. The adjustment of infranatant was made to 1.21 g/mL along with solid KBr and blended with that of 1.21 g/ml buffered KBr solution, which was then passed through the process of centrifugation for a span of 48 hr at 48,000 rpm. After collecting HDL, it was dialyzed three times for 48 hr at a temperature of 4 ℃ against phosphate buffer saline (PBS) containing 1 mM EDTA. Finally, it was passed through PBS for dialysis with no EDTA for a period of 8 hr and then filtered with the help of a 0.22-mm filter.
2.5. MTT assay
MTT assay was performed for measuring cell growth. A 96-well plate was employed for sowing cells at a density of 5 × 103 cells/well which were cultured for specific time intervals. Besides that, a fresh cell culture medium that contained 0.5 mg/mL MTT for 4 hr replaced the medium at each time interval. Then we added 150 µL DMSO to each well and shook on the low-speed rotation for 10 min until the crystal was fully dissolved, then measurement at 490 nm was performed by employing a Multiskan MS ELSA reader (xMark Microplate Absorbance Spectrophotometer, BioRad, CA, USA). For normalizing the relative cell number, the absorbance from control cells was used.
2.6. SDS-PAGE and Western blotting
Western blot assay was performed over total protein by adopting the standard western blotting protocol (Molecular Clone, Edition II). The concentrations that were employed for primary antibodies: PI3K (1:1500, sc-374534,Santa Cruz,USA), p-ERK(1:500, sc-7383,Santa Cruz, USA), ERK(1:500, sc-94,Santa Cruz, USA), p-AKT(1:500, sc-7985R, Santa Cruz,USA), AKT(1:500, sc-8312,Santa Cruz,USA), cleaved caspase 3(1:500, CST 9579, Boston, USA), ATF-4(1:500, abcam,ab-25331, London ,England)and GAPDH (1:1000, Santa sc-575,Cruz,USA). Furthermore, we had to apply secondary antibodies that were already labeled with respective horseradish peroxidase (HRP) which was followed by performing enhanced chemiluminescence (ECL) detection while adopting the company’s instructions (Pierce, Rockford, IL, USA). For the analysis of the integrated density mean grey value of the band, the ImageJ software was employed, and calculation was performed for the corresponding relative expression ratio.
2.7. Statistical analysis
Dates were expressed as means ± SE. The two-tailed Student’s t-test (for 2 groups) was used to assess the differences of means among multiple groups in addition to the analysis of variance (ANOVA, for > 2 groups). P ≤ 0.05 was taken as statistically significant for all analyses.