Male Sprague Dawley (SD) rats (n=50; body weight, 125–155 g; age, 7 weeks) were purchased from the Experimental Animal Center of Southern Medical University. Rats were housed at a constant temperature of 22–25°C and 50–60% relative humidity under a controlled 16-h light/8-h dark cycle for at ≥5 days; they had free access to rat chow and water. Experimental protocols involving animals followed the guidelines approved by the Chinese Association of Laboratory Animal Care and approved by the Institutional Animal Care and Use Committee of Nanfang Hospital (approval no. NFYY-2019-176; Guangzhou, China).

Primary PMVECs were isolated from SD rats as previously described (19). Briefly, after anesthesia with pentobarbital sodium (50 mg/kg, i.p.), the distal lung tissues (diameter, 3–5 mm) were resected from rat lungs, cut into small pieces and digested with type I collagenase (2 mg/ml) and neutral protease (0.6 U/ml) for 1 h. The mixture was centrifuged at 800 × g for 10 min at 37°C. Subsequently, cells were resuspended and plated with Dynabeads (Invitrogen; Thermo Fisher Scientific, Inc.) and rat anti-mouse platelet endothelial adhesion molecule-1 antibody (Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h. The cell-bead mixture was separated using a magnetic particle concentrator (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated in EGM-2 culture media containing 10% FBS with growth factor bullet kit (Lonza Group, Ltd.). For HS treatment, the bottom of each culture dish was placed into a circulating water bath maintained at 43±0.5°C for 2 h and PMVECs were further incubated at 37°C for periods as indicated.

Cells were seeded in a 96-well plate at a density of 1×10⁵/ml and incubated at 37°C overnight. Subsequently, cells were cultured at 43°C for 2 h or treated with p65 small interfering (si)RNA or IκBα mutant construct, mixed with 100 µl CCK-8 solution (Dojindo Molecular Technologies, Inc.) and incubated for 1 h. Cell viability was assessed following the manufacturer's protocol.

Cell apoptosis was detected using an Annexin V-FITC apoptosis detection kit and flow cytometry according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc). A total of 1×10⁶ cells were collected and washed three times in PBS. Subsequently, cells were incubated in binding buffer containing Annexin V-FITC for at 4°C 15 min, the buffer was removed by centrifugation at 1,000 × g for 5 min at 4°C and the cells were resuspended in buffer containing PI solution. After 5 min, apoptotic cells were detected using a FACSCalibur cytometer (Becton, Dickinson and Company). The data were analyzed using Flowing version 2.5.3 software (Turku Bioscience) and Origin version 7 software (OriginLab). The results of a previous study have suggested that Annexin V-positive cells in the right quadrant are apoptotic, including the early and late apoptotic cells (20).

Cells were treated with 2′-7′-dichlorofluorescin diacetate (Beyotime Institute of Biotechnology) at 37°C for 20 min in the dark as previously described (21). For live imaging, DNA was stained with 0.2 mg/ml Hoechst 33342 (Thermo Fisher Scientific, Inc.) at 37°C for 10 min and PMVECs were washed with DMEM Fluoro Brite (Gibco; Thermo Fisher Scientific, Inc.). Cells were analyzed using a confocal fluorescent microscope (magnification, ×400; LI-COR Biosciences, Inc.).

Cells were exposed to HS treatment and a nuclear extraction kit (Active Motif, Inc.) was used to obtain cytoplasmic and nuclear protein extracts, according to the manufacturer's protocol. Total protein was quantified using an Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). Western blotting was performed as previously described (22), the proteins (40 µg per lane) were separated via 12% SDS-PAGE and further transferred onto PVDF membranes. Then, the membranes were blocked with 5% skimmed milk for 1 h at room temperature, and cultured with the following primary antibodies: p65 (cat. no. ab16502; 1:1,000; Abcam), IκBα (cat. no. 4812; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p)-IκBα (cat. no. 2859; 1:1,000; Cell Signaling Technology, Inc.), p-p65 (cat. no. 3303; 1:1,000; Cell Signaling Technology, Inc.), Lamin B (cat. no. 13435; 1:1,000; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.) overnight at 4°C. Following primary incubation, membranes were incubated with HRP-conjugated anti-rabbit IgG antibody (cat. no. ZB-2301; 1:5,000; OriGene Technologies, Inc.) at room temperature for 1 h. Protein bands were visualized using ECL reagents (Pierce; Thermo Fisher Scientific, Inc.) with GAPDH and Lamin B as the internal control. ImageJ (version 6.0; National Institutes of Health) was used for the semi-quantification of protein expression.

The luciferase activity was determined using the Dual-Glo luciferase assay system (cat. no. E2980; Promega Corporation) following the manufacturer's protocol. Briefly, plasmids were purified from bacterial cultures using the QIAGENP plasmid Midi kit (Qiagen GmbH). Rat PMVECs were transfected with the vector constructs using Lipofectamine^® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the pGL3-Basic plasmid without promoter as the negative control. The pGl4.74 vector (Promega Corporation) was used as an internal control to determine the efficiency of transfection. After 24 h of transfection, cells were washed with PBS and resuspended in Passive Lysis Buffer (Promega Corporation). Cells were treated with HS at 43°C for 2 h, followed by a recovery period at 37°C. Activity was measured using a Centro LB 960 Luminometer (Titertek-Berthold).

The pcDNA3.1-IκBα-mutant (M) and pcDNA3.1-IκBα-wild-type (wt) were synthesized by Cyagen Biosciences, Inc. Cells were transfected with the corresponding vectors according to the Lipofectamine^® 2000 reagent procedure (Thermo Fisher Scientific, Inc.) and transfected cells were incubated for 48–72 h at 37°C before further experiments.

siRNA targeting p65 was designed and synthesized by Shanghai GenePharma Co., Ltd. The siRNA sequences for each gene and their corresponding negative controls are demonstrated in Table I. PMVECs were seeded into 6-well plates (Wuxi NEST Biotechnology, Co., Ltd.) at 30–50% confluence for transfection. After 24 h, PMVECs were incubated with TurboFect siRNA Transfection reagent (Shanghai GenePharma Co., Ltd.) and 10 nM siRNAs for 6 h at 37°C according to the manufacturer's protocol. Transfected cells were incubated for 24 h at 37°C before further experiments.

Cells were subjected to HS treatment at 43°C for 2 h, followed by an additional incubation period at 37°C, or exposed to 1 µg/ml lipopolysaccharide (LPS; Sigma-Aldrich; Merck KGaA) for the times indicated. Co-IP assays were subsequently performed using the Co-IP kit (Pierce; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Cells were lysed with immunol precipitation lysis buffer (Beyotime Institute of Biotechnology) for 30 min on ice, and the buffer was removed via centrifugation at 16,000 × g for 10 min at 4°C. The cell lysates were then immunoprecipitated at 4°C overnight using anti-IκBα (cat. no. 4812; 1:1,000; Cell Signaling Technology, Inc.) and normal IgG complexes. Then, 40 µl protein A+G agarose beads were used and spun at 4°C for 3 h. After centrifugation at 250 × g for 5 min at 4°C, the supernatant was discarded. Finally, 1X loading buffer was added and boiled for 5 min for western blot analysis. Protein expression levels were semi-quantified using ImageJ (version 6.0; National Institutes of Health).

All data were analyzed for statistical significance using SPSS V.23.0 software (IBM Corp.). Data are presented as the mean ± standard deviation from at least three independent experiments, each performed in duplicate. The comparisons of multiple groups were analyzed using the Kruskal-Wallis test followed by a Dunn's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

As demonstrated in Fig. 1A, nuclear and cytosolic fractions were isolated from rat PMVECs and PMVECs were exposed to HS for 2 h followed by further incubation for 0, 6 and 12 h. The results of the present study indicated a significant amount of NF-κB p65 and IκBα nuclear translocation in a time-dependent manner. As shown in Fig. S1A, the infection efficiency of p65siRNA was observed with p65 expression. Knockdown of p65 significantly decreased the viability of PMVECs, as demonstrated by CCK-8 assays (Fig. 1B). Furthermore, fluorescence confocal assays revealed that siRNA knockdown of NF-κB p65 significantly aggravated ROS accumulation in HS-induced rat PMVECs compared with the control (Fig. 1C). Collectively, the results of the present study demonstrated that NF-κB p65 served a key role in HS-induced cytotoxicity and ROS production.

IκB is an inhibitor of NF-κB, which interacts with the nuclear localization sequences of the Rel homology domain. IκBα was the first protein described in the IκB protein family (23). To investigate the interaction between NF-κB p65 and IκBα, Co-IP experiments of NF-κB p65 and IκBα were performed. As demonstrated in Fig. 2A, Co-IP assays revealed that NF-κB p65 co-immunoprecipitated with IκBα following HS treatment. Furthermore, treatment with LPS lead to a decrease in the co-immunoprecipitation of IκBα and NF-κB p65. The results of the present study demonstrated that IκBα had the ability to bind to NF-κB and IκBα was not degraded or dissociated from the NF-κB complex during the recovery period following HS. To further investigate the direct interaction between NF-κB p65 and IκBα in HS-induced PMVECs, western blot analysis was used. Western blotting revealed that protein expression of IκBα was significantly reduced following knockdown of NF-κB p65 during the recovery period following HS (Fig. 2B and C). Collectively, the findings of the present study suggested that upstream NF-κB p65 mediated the HS-induced expression of IκBα in HS-induced rat PMVECs.

The results of our previous study confirmed that HS induced the phosphorylation and subsequent activation of IκBα on Ser32 and Ser36 in rat PMVECs (9). To determine whether phosphorylation of Ser32 and Ser36 was responsible for NF-κB activity and nucleus translocation induced by HS, IκBα mutants with substitutions of Ser/Thr with Ala residues at Ser32 and Ser36 were used (pcDNA3.1-IκBα-M; Fig. 3A). The infection efficiency of pcDNA3.1-IκBα-M was observed with levels of phosphorylated IκBα (Fig. S1B). A significant decrease in the phosphorylation of IκBα was observed in the pcDNA3.1-IκBα-M group compared with the pcDNA3.1-IκBα-wt and control groups (Fig. 3B). By contrast, no significant changes in the phosphorylation and nucleus translocation of p65 were observed following transfection with pcDNA3.1-IκBα-M (Fig. 3C). Furthermore, measurement of NF-κB activity indicated that pcDNA3.1-IκBα-M failed to attenuate activation of NF-κB in a time-dependent manner following HS (Fig. 3D). Thus, the results of the present study revealed that HS-mediated NF-κB activity and nucleus translocation are independent of IκBα phosphorylation.

The role of IκBα phosphorylation on Ser32 and Ser36 in ROS accumulation and cell apoptosis induced by HS was investigated, following transfection of PMVECs with pcDNA3.1-IκBα-M. Compared with the control group, pcDNA3.1-IκBα-M significantly increased the level of apoptosis and significantly decreased the proliferation of PMVECs, as demonstrated by flow cytometry and CCK-8 assays, respectively (Fig. 4A and C). Additionally, the results of the fluorescence confocal assay demonstrated that pcDNA3.1-IκBα-M significantly aggravated ROS generation in HS-induced rat PMVECs (Fig. 4B). Collectively, these findings revealed that IκBα may serve an essential role in HS-induced rat PMVEC apoptosis and ROS generation.