2.1. Cell culture protocol
To evaluate the treatment protocol, we selected human umbilical vein endothelial cells (HUVECs). The cells were expanded in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F‐12; Gibco) under reasonable condition consisted of 37°C temperature, 5% CO2 and 95-98% humidity. To induce cell proliferation, culture media is supplemented with 10% 10% fetal bovine serum (FBS) and 1% Pen-Strep solution. Cells were detached using 0.25% Trypsin‐EDTA solution (Gibco). We enrolled the cells to different analyses between passages 3-6.
2.2. Treatment protocol
HUVECs were allocated into seven different groups as follows; the non-treated Control cells; Pal; DHA, DHA+ Pal; Insulin, Insulin +Pal, and Insulin + DHA + Pal. To induce atherosclerotic changes, cells were treated with culture medium containing 1 mM Pal (Cat no. P0500‐10G, Sigma) and 2% FBS for 48 hours. We also incubated HUVECs with 50μM insulin for the induction of hyperinsulinemia conditions for 48 hours. After completion of hyperinsulinemia condition and atherosclerotic changes, we added the 50μMDHA (Cat no. D253425MG, Sigma) to the respective groups and cells allowed to expose to DHA for 48 hours.
2.3. Preparation of Albumin (Alb)-Pal conjugate
To increase the solubility of Pal, we prepared conjugated Alb-Pal composition according to our previously published protocols[2]. In brief, 2.267g bovine serum albumin (Sigma-Aldrich) was dissolved in 150mM NaCl solution and maintained at 37°C. Then, Pal (30.6 mg) was added to the solution and heated up to 70°C. The mixture was gently stirred at 40°C for 1 hour. The pH was adjusted to 7.4. The final solution was passed through the 0.22‐μm pore size micro-filter (Membrane Solution).
2.4. Confirmation of intracellular Pal accumulation by Oil red O staining
The oil red O staining method was used to evaluate the intracellular deposition of Pal inside the HUVECs. To this end, 3× 105 HUVECs were placed in each well of 24-well plates. After the completion of experiments, the supernatant medium was discarded, and treated cells from different groups were fixed with pre-cooled 4% paraformaldehyde (PFA) solution at 4°C for 20 minutes. After that, 200 µl Oil red O solution (0.5% w/v) was added to each well, and plates were kept at room temperature for 20-30 minutes. Then, cells were washed twice with PBS and imaged by using an inverted microscope. The number of red-colored cells in the group was counted in 10 random serial fields and compared to each other.
2.5. MTT assay
200 µl medium containing 10% FBS and 104 HUVECs were poured in each well of96‐well plates (SPL) and kept for 24 days. Next, the exhausted medium was replaced with 200 fresh culture medium supplemented with 2% FBS. After completion of the experimental period, 50 μl MTT (dilution: 5 mg/ml, 3‐ (4, 5‐dimethylthiazol‐2‐yl) ‐2, 5‐diphenyltetrazolium bromide; Cat no: M5655.100MG; Sigma-Aldrich) was added to each well and kept in an incubator for next 4 hours. After the formation of formazan crystal, 100 μl diethyl sulfoxide (Merck; Darmstadt; Germany) was added to dissolve the crystals and generate blue-to-purple appearance. Cell viability was measured by reading optical density at 570nm wavelength by using BioTek microplate reader and expressed as % of the control group. This assay was performed octuplicate.
2.6. Measuring the levels of inflammatory cytokines
In this research, the content of paracrine inflammatory factors such as NF-ƙB, TNF-α, and IL-6 was measured. For this purpose, supernatants from different groups were collected and subjected to the ELISA assay (IL-6; Cat no: E0090Hu, TNF-α; Cat no: E0082Hu, NFƙ-B; Cat no: E0690Hu). Supernatants were centrifuged at 400g for 5 min to exclude the cell debris. After that, the contents of the cytokines were measured according to the manufacturer's instruction. This assay was performed in triplicate.
2.7. Measuring pro-inflammatory status by using immunofluorescence staining
We also performed immunofluorescence staining (IF) to detect the intracellular level of TNF-α after exposure to the DHA, Pal, and insulin. In brief, 1 × 104 cells were placed in each well of 8 well slide chambers (SPL). After reaching 70-80% confluence, cells were subjected to the experimental protocol at the respective time point. Cells were fixed with 4% PFA and permeablized using permeabilizing buffer (Ref no: 00-0055-56; eBioscience; USA) for 20 minutes. Cells were then incubated with anti-TNF-α antibody (dilution: 1:100; Cat no: ab34674; Abcam) for 1 hour followed by PBS wash (3 × 10 minutes). After that, we added FITC-conjugated secondary antibody (1: 1000; eBioscience; USA) for 1 hour at RT. After PBS wash, the nuclei were stained with 1 µg/ml for 30 seconds.
2.8. Evaluation of NO content by Griess assay
The intracellular level of NO was measured using the Griess assay, as previously described (Ref). HUVECs (5 × 104cells/well) were plated in each well of 96-well plates and enrolled in the experimental procedure as those mentioned above. After completion of treatment, 200 μl supernatant was mixed with 15 μl of Griess A solution and maintained at 37̊C for 20 minutes. Then, we added 15 μl of Griess B reagent to samples, and the optical density (OD) was determined at 450 nm using a microplate reader. NO content was expressed in nM. The final NO contents were calculated after comparison with standard control (Sodium nitrite).
2.9. Flow cytometry analysis of apoptosis
To measure the occurrence of apoptosis in cells from different groups, the percent of HUVECs entering apoptosis was determined by using flow cytometry analysis. For this purpose, 2× 105 HUVECs were seeded in each well of 24-well plates and allowed to reach 70-80% confluence. The cells were subjected to the treatment protocol. After 48 hours, cells were detached using 0.25% Trypsin-EDTA solution and washed twice with PBS. After that, cells were permeabilized using a permeabilizing buffer (eBioscience, USA) at room temperature for 30 minutes. In the current experiment, we incubated cells from different groups with 2.5μl FITC-tagged Annexin-V antibody at 4̊C for 30 minutes. After twice washing with PBS, we added 2.5 μl propidium iodide and incubated cells for b5 minutes. The cells were analyzed by using BD FACSCalibur® system and FlowJo software ver.7.6.1
2.10. PCR array
To investigate the effect of DHA on atherosclerotic changes in HUVECs under treatment with hyper insulin concentration, we performed PCR array analysis. After completion of the experimental procedure, RNAs were extracted from each group by using an RNA extraction kit (Cat no.11828665001, Roche) followed by cDNA synthesis (Cat no. YT4500, Yekta Tajhiz Azma). Using the RT2 Profiler PCR Array, we monitored the expression of 86 genes participating in the atherosclerosis signaling pathway. Raw data were processed using the 2−ΔΔCT method to determine statistically significant between groups.
2.11. Statistical analysis
Data are presented as mean ± SD. One-way ANOVA and Tukey postdoc analysis was used to find statistically significant differences between the groups. P<0.05 was considered statistically significant. All experiments were repeated three times unless mentioned.