Cell culture

The HUVECs cells (BeNa Culture Collection, Suzhou, China) were cultured in specialized culture medium (Procell Life Technology Co., Ltd, Wuhan, China) in a humidified atmosphere at 37℃ containing 5% CO2 and were sub-cultured every two to three days to maintain an exponential growth phase during experiments.

Preparation for RSO

The RSO used in this study was provided by Huakun Biotechnology Co., Ltd. In Xishuangbanna, Yunnan, China. The seed oil was made from Brazilian rubber tree seeds and processed through raw material cleaning, drying, shelling, pressing, and refining. RSO was sterilized by filtration though a 0.2 µm membrane (Jiete Biofiltration Co., Ltd, Guangzhou, China) and dissolved in acetone (Wansheng Chuandong Chemical Co., Ltd, Chongqing).

Cell viability testing

In the cell viability testing, cells were inoculated into the 96-well plate (Beyotime Biotechnology, Shanghai, China with a density of 1 × 105 cells/well and cultured for 3 h until the cells adhered to the plate Then, the cells were treated with RSO in different concentration (25, 50, 75, 100, 125, 150, 200 µg/mL), as the previous study described (Liu et al. 2022), and ox-LDL (Solaybao Technology Co., Ltd, Beijing, China) at 12.5, 25, 50, 100, 150, 200, 250, 300 µg/mL, respectively, for 24 h.

After 24 h treatment, the original culture medium from the 96 well plate was extracted and 10μL CCK-8 reagent (Proteintech, Chicago, USA) was added to each well. After 1 h incubation, Optical Density (OD) was measured at 450 nm in the culture plate using a plate reader (BioTek, Vermont, USA).

Determination of inflammatory cytokines

Supernatant from each treatment group described above was collected and stored at -20℃ until analyses. Levels of IL-1β, IL-6, IL-10, GSH and TNF-α were determined by an ELISA kit (Huamei Bioengineering Co., Ltd, Wuhan China) at 450 nm in triplicates (Kaval et al. 2022; Chen et al. 2021). Concentrations of inflammatory cytokines were calculated by standard curves and expressed as microgram or nanogram per milliliter per manufacturer’s instructions.

Detection of intracellular ROS levels

The levels of intracellular ROS were analyzed using a ROS Assay Kit (Fu et al. 2023), a fluorescence enzyme-linked immunosorbent assay (Solaybao Technology Co., Ltd, Beijing, China). The DCFH-DA was diluted to 10 µmol/L and added to each well so that it can cover all cells. Next, the cells were incubated at 37℃ for 20 min. After discarding the culture medium, serum-free culture medium was used to wash the cells three times with gentle shaking. ROS levels were detected and plate reader, with parameter settings of 488 nm excitation wavelength and 525 nm emission wavelength (BioTek, Vermont, USA).

Determination of NO

NO levels were measured with a NO detection kit, an enzyme-linked immunosorbent assay (Beyotime Biotechnology Co., Ltd, Shanghai, China). 60 μl of cell supernatant was added to each well in a 96 well plate with 5 μl NADPH, FAD, and Nitrate Reductase reagents incubated at 37 º C for 30 min. Then, the cells were incubated with LDH Buffer and LDH at 37 ℃ for 30 min. Next, 50 µl Griess reagent I and 50 µl Griess reagent II were added to each well at room temperature (20–30 ℃) for 10 min, and the OD value at 540 nm was measured with a plate reader (BioTek, Vermont, USA).

RNA extraction and PCR

Cells were washed with PBS for three times after treatment. Total RNA was extracted from cells with Trizol (Solaybao Technology Co., Ltd, Beijing, China) and chloroform (Shandian Pharmaceutical Co., Ltd, Yunnan, China). The total RNA was collected in the aqueous phase after centrifugation ands precipitated by isopropanol (Jingrui Technology Co., Ltd, Yunnan, China). The pellet was washed with 75% ethanol, and redissolved in RNAase-free water. The concentration and purity of the total RNA were determined using a spectrophotometer. Quantitive RT-PCR (Sangon Biotech, Shanghai, China) was used measure the expression levels of genes are below (Table 1) in triplicate assays. Based on the threshold cycle (Ct) values of the target genes and internal control gene, the relative expression level of target genes in each group was calculated by the 2−ΔΔct method.

Table 1 The primers of genesWestern blot

After the cells were washed twice with PBS, 200 µl of lysing buffer was added to each well, and placed it on ice for 30 min; 4 ℃, 12,000 rpm, centrifugation for 30 min; Then the protein concentration was detected using the BCA kit (Biyuntian Biotechnology Co., Ltd, Shanghai, China), followed by heating in a metal bath at 95 ℃ for 15 min and cooling at room temperature for denaturation.

SDS-PAGE separated the total protein, and the protein was transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore, Massachusetts, USA). The PVDF membrane was sealed at room temperature for 1 h on a shaking table using a sealing solution prepared with TBST and skimmed milk powder (Wandashan Dairy Industry Co., Ltd, Hulin China). Then washed it with TBST three times, and immersed it in the prepared primary antibodies at 4 ℃ overnight. Then, washed it with TBST three times. Next, put the membrane into the secondary antibodies, which were incubating for 1 h. Finally, protein imprinting on the PVDF film can be displayed with a visualizer. Meanwhile, data was analyzed by Image J.

Antibodies: eNOS (1:2000, ABclonal, Wuhan, China), NF-κB p65 (1:1000, ABclonal, Wuhan, China), MCP-1 (1:2000, Proteintech, Chicago, USA), TLR4 (1:1000, Proteintech, Chicago, USA), VCAM-1 (1:2000, Hua'an Biotechnology Co., Ltd, Hangzhou, China), p-NF-κB p65 (1:500, Affinity Biosciences, Cincinnati, USA).

Statistical analysis

All experimental data were statistically analyzed using GraphPad Prism. Comparison between groups were conducted using conventional one-way ANOVA and the t-test. The statistical results were expressed as mean ± standard deviation (χ ± s), and P < 0.05 was considered statistically significant.