NAC and Escherichia coli LPS were obtained from Sigma-Aldrich; Merck KGaA. BAY11-7082 was obtained from Biomol GmbH. Cell Counting Kit-8 (CCK-8) reagent was purchased from Dojindo Molecular Technologies. IL-8 and TNF-α ELISA kits were manufactured by BioLegend, Inc. Antibodies against ICAM-1 (cat. no. ab53013), NF-κB (p65; cat. no. ab32536) and phosphorylated NF-κB (p-p65; cat. no. ab76302) were obtained from Cell Signaling Technology, Inc. NO and inducible nitric oxide synthase (iNOS) assay kits were purchased from the Nanjing Institute of Jiancheng Bioengineering. Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone; GE Healthcare Life Sciences. All other supplies used in the experiments were reagent grade.

HUVECs were purchased from the American Type Culture Collection. These were cultured in high glucose DMEM with the addition of 10% FBS, 100 mg/ml streptomycin, and 100 units/ml penicillin in a 5% CO₂ environment at 37°C. The entire medium was replaced once every 48 h. HUVECs were used in cell culture at passages 2–5 as previously described (1). NAC and LPS were dissolved in pure medium and BAY11-7082 was dissolved in dimethyl sulfoxide (DMSO, 200 µl).

The experiment consisted of seven groups: The control group, the LPS group (only LPS), the NAC group (only NAC), the LPS+NAC group (cells pre-treated with NAC for 1 h before the addition of LPS, L+N), the BAY11-7082 group (only BAY11-7082; 5 µmol/l) (24), the LPS+BAY11-7082 group (cells pretreated with BAY11-7082 for 1 h before the addition of LPS, L+B), the DMSO group (the final concentration was 0.05%). The groups are presented in Table I.

The effect of NAC on cell viability in the HUVECs model was first investigated by CCK-8 assay. In this process, HUVECs were treated with different concentrations of NAC (0.1, 0.25, 0.5, 1, 5 and 10 mM) for 24 h. Subsequently, the effect of NAC was evaluated on LPS-induced inflammation of HUVECs by pre-treatment of cells with different concentrations of NAC (0.1, 0.25, 0.5, 1, 5 mM) for 1 h before LPS (100 ng/ml) treatment of cells. HUVECs (density 1×10⁵/ml) were cultivated in 96-well plates at 37°C in 5% CO₂ for 24 h followed by their incubation with CCK-8 reagent for 2 h. An MCC 340 microplate reader (Thermo Fisher Scientific, Inc.), wavelength set at 450 nm, was used to detect the absorbance value representing cell proliferation. In the seven treatment groups, the morphology of HUVECs was observed using a phase contrast microscope. The experiments were repeated three times.

HUVECs were seeded in 6-well plates, treated with drugs corresponding to their group for 24 h. Total RNA was extracted from HUVECs using TRIzol reagent (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's recommendations. The cDNA was then reverse transcribed from total RNA using the PrimeScript™ RT reagent kit (Takara Bio, Inc.) and maintained at 42.8°C for 1 h under the use of oligo (dT) primers. The prepared cDNA was subjected to PCR amplification according to the manufacturer's instructions. The ABI Prism 7300 Sequence Detection PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to monitor the fluorescence signal intensity during PCR reaction in real time. The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec, with a final extension at 75°C for 10 min. The following primer sequences were used: IL-8 forward, 5′-AACTGAGAGTGATTGAGAGT-3′ and reverse, 5′-ATGAATTCTCAGCCCTCTTC-3′; TNF-α forward, 5′-AGCTGGTGGTGCCATCAGAG-3′ and reverse, 5′-TGGTAGGAGACGGCGATGCG-3′; iNOS forward, 5′-AGGCCACCTCGGATATCTCT-3′ and reverse, 5′-GCTTGTCTCTGGGTCCTCTG-3′; ICAM-1 forward, 5′-CGAAGGTTCTTCTGAGC-3′ and reverse, 5′-TCTGCTGAGACCCCTCTTG-3′. The housekeeping gene glyceraldehyde-phosphate dehydrogenase (GAPDH) served as the control.

HUVECs (1.0×10⁵ cells/well) were seeded into a 24-well plate, randomly divided into seven groups and treated with the corresponding agents (Control group, LPS group, NAC group, LPS+NAC group, BAY group, LPS+NAY group, DMSO group) and cultured for 24 h. The supernatant was collected to detect TNF-α and IL-8 protein concentrations using ELISA kits (BioLegend, Inc.) according to the manufacturer's recommended protocol. All experiments were conducted in triplicate.

The activity of iNOS in HUVECs was detected using the nitric reductase method. After treatment of cells for 24 h (as described in the section Real time semi-quantitative polymerase chain reaction (RT-qPCR) for mRNA expression of TNF-α, IL-8, iNOS, and ICAM-1), these were digested with 0.25% trypsin and stopped with 10% FBS high glucose DMEM medium. Cell pellets were obtained after centrifugation for 5 min at 200 × g. The cells were rinsed with an appropriate amount of Hank's solution; subsequently, these were centrifuged again for 5 min at 200 × g, and the supernatant was removed to obtain cell pellets. The washing steps were repeated twice. Then 500 µl Hank's solution was added to each group of cells, mixed well, and pulverized ultrasonically. The supernatant was obtained for protein concentration measurement and iNOS detection. After cell treatment for 24 h, the media supernatant was collected from each group, followed by the addition of reagents 1 and 2, according to the manufacturer's recommended protocol for the NO assay kit. Finally, the microplate reader, wavelength set at 550 nm, was used to detect the absorbance values after mixing. All experiments were repeated three times.

After seven groups of cells were treated with the corresponding drugs [detection of NF-κB protein expression after drug treatment for 2 h (25); detection of ICAM-1 protein expression after drug treatment for 24 h], the cells were rinsed with an appropriate amount of phosphate-buffered saline (PBS). Then 200 µl lysate was added to each group of cells, mixed well, and pulverized ultrasonically for 5 min. Then the samples were lysed on ice at 4°C for 20 min, followed by centrifugation for 15 min at 12,000 × g. Then, the protein expression in the supernatant was determined using the Bicinchoninic Acid (BCA) method. Depending on the sample loading, 5X loading buffer was added and boiled for 10 min. Denatured protein sample (80 µg) was electrophoresed at 100 V [100 mg/l sodium dodecyl sulfate-polyacrylamide (SDS-PAGE)] and transferred to a polyvinylidene fluoride (PVDF) film (350 mA, 2 h). Next 50 g/l bovine serum albumin (BSA) was added at normal temperature for 2 h and incubated overnight with p65 (cat. no. ab32536; 1:1,000 dilution; Abcam), phosphorylated p65 (cat. no. ab76302; 1:1,000 dilution; Abcam), ICAM-1 (cat. no. ab53013; 1:2,000 dilution; Abcam), and β-actin (cat. no. bs-0061R; 1:1,000 dilution; Beijing Boaosen Biological Co., Ltd.) at 4°C. On the subsequent day, the solution was thoroughly washed with TBST. Next, the horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat. no. bs-0295G-HRP; 1:3,000 dilution; Beijing Boaosen Biological Co., Ltd.) was added with the solution and incubated together for 2 h at room temperature, which was added with TBST to wash again. Then the developer was added for development, and the exposed negative was developed, fixed, baked and photographed in a dark room. The gray values of the NF-κB pathway protein, the ICAM-1 protein, and the internal reference protein β-actin expression band were analyzed using the Quantity One gel imaging scanning system. Representation of the NF-κB signaling pathway protein expression level was the gray scale ratio of phosphorylated p65 and p65; the protein expression level of ICAM-1 is represented by the gray scale ratio of ICAM-1 and the internal reference β-actin.

Experimental data are expressed as the mean ± standard deviation (SD) values from three experiments. One-way ANOVA followed by LSD post-hoc tests were used to assess between-group differences; P-values <0.05 were considered indicative of statistical significance.

When the cells were treated with different concentrations of NAC (0.1, 0.25, 0.5, 1, 5 and 10 mM), there was no significant change in cell viability up to 5 mM relative to the control. However, cytoxicity was observed at NAC concentration of 10 mM (Fig. 1A). In the experiments to assess the effects of NAC on LPS-mediated inflammation, the LPS group (1 µg/ml) exhibited significantly reduced cell viability compared with the control group. However, pretreatment with NAC (0.1, 0.25, 0.5, 1, 5 mM) significantly alleviated the LPS-induced inhibition of cell viability, especially at NAC concentration of 1 mM (Fig. 1B). Therefore, this was selected as the optimal concentration of NAC. When HUVECs were treated with the appropriate concentration of the corresponding drug, the morphology of the seven groups of HUVECs was not significantly altered under phase contrast microscope (Fig. 1C).

The mRNA and protein expression of IL-8 and TNF-α were examined in LPS-stimulated HUVECs using RT-qPCR and ELISA. It was revealed that 100 ng/ml of LPS enhanced the mRNA expression of IL-8 (Fig. 2A) and TNF-α (Fig. 2C). However, pretreatment of cells with 1 mM NAC inhibited the mRNA expression of IL-8 and TNF-α. The effect of NAC was similar to that observed after pretreatment of cells with BAY11-7082 (a specific NF-κB inhibitor). Moreover, a similar phenomenon was observed with respect to the protein expression of IL-8 and TNF-α (Fig. 2B and D). This result indicated that 1 mM of NAC could significantly reduce the production of inflammatory mediators (IL-8 and TNF-α) in LPS-stimulated HUVECs.

iNOS mRNA was detected by RT-qPCR and the NO content and the viability of iNOS were determined in LPS-stimulated HUVECs using the nitric reductase method. As revealed in Fig. 3A, it was revealed that LPS induced an increase in the iNOS mRNA expression (P<0.01), while pretreatment with NAC and BAY11-7082 significantly reduced the iNOS mRNA expression. Fig. 3B and C revealed that NAC significantly reduced the activity of iNOS and the NO content (P<0.05). This indicated that NAC can decrease the viability of iNOS and the production of NO in HUVECs stimulated by LPS.

ICAM-1 is a major proinflammatory chemokine involved in vascular response and migration of neutrophils to inflammatory foci (10). RT-qPCR and western blotting were performed to assess the effects of NAC on LPS-induced expression of ICAM-1. LPS stimulation significantly increased the mRNA expression of ICAM-1. However, pretreatment with NAC and BAY11-7082 significantly attenuated the increase (P<0.05; Fig. 4A). Consistent with the results of RT-qPCR, western blot results revealed that LPS stimulation significantly increased ICAM-1 secretion compared with the control group (P<0.01; Fig. 4B and D). However, pre-treatment of cells with NAC and BAY11-7082 significantly reduced the expression of ICAM-1 protein. These results revealed that NAC significantly attenuated the LPS-induced increased expression of ICAM-1 in HUVECs, which supported the protective effect of NAC on HUVECs.

The protein expression levels of p65 and phosphorylated p65, which represent excitation of the NF-κB pathway, were examined by western blot analysis. The protein expression of p-p65/p65 in the LPS group was significantly higher than that in the control group (P<0.01). However, the expression of p-p65/p65 appeared to decrease in the NAC and BAY11-7082 groups, compared with the control group. Similarly, upon comparison of the LPS group with the LPS+NAC and LPS+BAY11-7082 groups, the p-p65/p65 expression in the latter two groups was significantly lower (P<0.05; Fig. 4C and D). These findings indicated that LPS can activate the NF-κB signaling pathway in HUVECs, whereas NAC restrained the LPS-mediated activation of NF-κB. The effect of NAC was similar to that of BAY-117082.