Cell culture, treatment, lentiviral SIRT1 shRNA preparation and transduction

HRECs [21] were obtained from Shanghai Xuanke Biotechnology co. LTD (Shanghai, China) and cultured in endothelial cell medium (Sciencell, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA). Cells were incubated at 37 ℃ under 5% CO2. Unless specially explained, the cells were used after they reached 85% confluence at passages 3–6.

The chemically synthesized sense (5′-TGCGGGAATCCAAAGGATAATTTTCAAGAGAAATTATCCTTTGGATTCCCGCTTTTTC-3′) and antisense (5′-TCGAGAAAAAGCGGGAATCCAAAGGAGAAGGTCTCTTGAAAATTATCCTTTGGATTCCCGA-3′) chains were annealed to form SIRT1 shRNA expression templates, which were then connected to the vector pLentilox3.7 containing green fluorescent protein (GFP). The obtained plasmids were cotransfected with lentiviral packaging plasmids pLP1, pLP2, and pLP/VSVG into 293FT cells. After 24 h of transfection, the culture medium was changed. After 48 h of cultivation, the cell supernatant rich in lentivirus particles was collected and concentrated 20 times to obtain a high titer lentivirus concentrate.

To knockdown the expression of SIRT1 in HRECs, cells were transduced with lentiviral SIRT1 shRNA for 48 h, followed by evaluating the transduction efficacy using the Western blotting assay.

Lactate dehydrogenase (LDH) release

In a 96-well plate, cell supernatant was collected and implanted, followed by adding LDH solution to be incubated for 90 min in the dark. Subsequently, the absorbance at 490 nm was detected utilizing the microplate reader (PerkinElmer, USA). The release of LDH was calculated based on the absorbance value of the testing sample and standards.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

HRECs were implanted in a 96-well plate and incubated at 37 ℃ for 1 day. Different concentrations of Liraglutide (5, 10, 50, 100, 500, 1000 nM) were introduced, incubated for 1 day, and then 10 μL of MTT (Sigma-Aldrich, USA) solution was added. Lastly, the microplate reader (PerkinElmer, USA) was utilized to detect the OD value at 490 nm [22].

Real-time polymerase chain reaction (PCR)

RNAs were isolated from treated HRECs with the TRIzol solution and centrifuged at 16,000 g for 10 min, followed by being dissolved in ddH2O. The transcription to cDNA was conducted using a reverse transcription kit (QIAGEN, Germany). The PCR was then carried out using the ABI 7500 Real-time PCR system (Applied Biosystems, USA) and the SYBR green (Sigma, USA). After normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the level of genes was calculated with the 2−ΔΔCt method. The following primers were used in this study: GLP-1R (F, 5′-CTACGCACTCTCCTTCTCTGCT-3′; R, 5′- CGGACAATGCTCGCAGGATGAA-3′), p53 (F, 5′-CCTCAGCATCTTATCCGAGTGG-3′; R, 5′-TGGATGGTGGTACAGTCAGAGC-3′), p21 (F, 5′'-AGGTGGACCTGGAGACTCTCAG-3′; R, 5′-TCCTCTTGGAGAAGATCAGCCG-3′), SIRT-1 (F, 5′-TAGACACGCTGGAACAGGTTGC-3′; R, 5′-CTCCTCGTACAGCTTCACAGTC-3′), GAPDH (F, 5′-GTCTCCTCTGACTTCAACAGCG-3′; R, 5′-ACCACCCTGTTGCTGTAGCCAA-3′).

Western blotting assay

The bicinchoninic acid (BCA) method was used to measure the concentration of proteins after they had been isolated from HRECs using various treatment methods before being further separated using 12% SDS-PAGE. Then, proteins were transferred onto the PVDF membrane, which was blocked with skim milk for 2 h. Subsequently, the membrane was incubated with primary antibodies against GLP-1R (1:800; cat. no. sc-390774, Santa Cruz Biotechnology, USA), p53 (1:2000; cat. no. sc-126, Santa Cruz Biotechnology, USA), p21 (1:2000; cat. no. sc-6246, Santa Cruz Biotechnology, USA), SIRT1 (1:3000, cat. no. sc-74465, Santa Cruz Biotechnology, USA), or GAPDH (1:5000, cat. no. sc-365062, Santa Cruz Biotechnology, USA) for 12 h at 4 ℃, followed by adding the secondary antibody (1:2000, cat. no. 7074 or cat. no.7076, Cell Signaling Technology, USA) for 1.5 h. Lastly, Bio-Rad Quantity One software was used for the quantitative analysis of bands.

Enzyme-linked immunosorbent assay (ELISA)

The precipitates were discarded after centrifugation at 1000 × g for 10 min at 4 °C, and then the standard dilution and test samples were added to the 96-well plate. After introducing the biotin-labeled antibody, samples were incubated at 37 ℃ for 1 h and the liquid was then discarded, followed by adding the HRP solution. After incubation at 37 ℃ for half an hour, substrate A and substrate B were introduced into each well and incubated at 37 ℃ for 15 min, and the stop solution was added subsequently. Finally, the microplate reader (PerkinElmer, USA) was utilized to detect the optical density (OD) value at 450 nm.

Senescence-associated β-galactosidase (SA-β-gal) staining

HRECs were washed with the phosphate-buffered saline (PBS) buffer and treated with 1 mL fixing solution for 15 min, followed by being rinsed with 2 mL PBS buffer. Then, cells were incubated with 1 mL SA-β-gal staining solution and the culture plate was cultured at 37 ℃ without CO2 for 15 h. After sealing, the number of positive cells was counted using a microscope (Leica, Germany) [23].

The telomerase activity

HRECs were lysed with the CHAPS buffer and the sample was centrifuged at 15,000 × g for one and a half hours, followed by being quantified with the BCA method. The activity of telomerase was then determined utilizing the TeloTAGGG Telomerase PCR ELISA Plus kit (Roche, Switzerland) based on the instruction of the product. The TRAP-PCR system, the samples, and the primers were introduced together, followed by conducting the quantification utilizing the RT-PCR assay.

Statistical analysis

Achieved data were expressed as mean ± standard deviation (S.D.) and were analyzed with the GraphPad software. The analysis of variance (ANOVA) method with Tukey’s post hoc test was applied for comparison. P < 0.05 was regarded as a significant difference.