Escherichia coli LPS and CA were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were resolved in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum (FBS). EA.hy926 human endothelial cells were obtained from the American Type Culture Collection (Rockville, MD, USA). EA.hy926 cells were propagated in RPMI-1640 medium supplemented with 10% FBS (Gibco Life Technologies, Rockville, MD, USA) at 37°C under 5% CO₂, or maintained in RPMI-1640 medium supplemented with 2% FBS. EA.hy926 cells at ~85% confluence were treated with 0, 10, 100 or 1,000 ng/ml LPS and/or with 0, 10 or 50 µM CA for 0, 3, 6 or 12 h. Cells were subsequently lysed for mRNA and protein expression analysis. For the TNF-α and IL-1β assays, the supernatant of the LPS- and/or CA-treated EA.hy926 cells was collected and centrifuged at for 15 min at 4°C and 13,200 × g. The supernatant was then transferred to new tubes and stored at −20°C until required for assays.

For the determination of the expression levels of IL-1β and TNF-α, the supernatants of the LPS- and/or CA-treated EA.hy926 cells were analyzed using an human IL-1β or TNF-α ELISA kit (Shanghai ExCell Biology Inc., Shanghai, China) according to the manufacturer's instructions. In brief, standards and samples were diluted with phosphate-buffered saline (PBS), loaded onto a 96-well plate and incubated for 90 min at 37°C. Next, biotin-labeled antibodies against IL-1β or TNF-α were utilized for specific binding. Finally, an avidin-labeled enzyme and substrate were used to quantitatively examine the levels of IL-1β and TNF-α using a spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

EA.hy926 endothelial cells post treatment were treated with ice-cold cell lysis reagent (Sigma-Aldrich) according to the manufacturer's instructions, and each protein sample was supplemented with protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), then quantified by bicinchoninic acid assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein samples were separated using SDS-PAGE (10–12%) and detected by western blot analysis using polyclonal rabbit antibodies against p38, phosphorylated p38 (#sc-535, 1:500), c-Jun N-terminal kinase (JNK; #sc-571, 1:400), phosphorylated JNK (#sc-135642, 1:200), extracellular signal-regulated kinase (ERK), phosphorylated ERK (#sc-23759-R, 1:200), TLR4 (#sc-10741, 1:200) and β-actin (#sc-130656, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (Pierce) and an electrochemiluminescence detection system (Amersham Pharmacia Biotech, Amersham, UK) were used for detection.

Total mRNA was extracted from cell samples using an RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions, and supplemented with RNase inhibitor (Takara Bio, Inc., Tokyo, Japan). RT-qPCR analysis of the TLR4 mRNA level was performed using a QuantiTect SYBR Green PCR kit (Qiagen, Inc.). All mRNA expression levels were normalized against β-actin. The 2^−∆∆Ct method was used for the relative quantification of TLR4 mRNA expression (28).

Data are presented as the mean ± standard error, and were analyzed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference. All tests were performed using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA).

In order to determine whether CA inhibits the LPS-induced secretion of IL-1β and TNF-α, EA.hy926 endothelial cells were treated with LPS (10, 100 or 1,000 ng/ml) and/or CA (0, 10 or 50 µM) for 12 h. Subsequently, the IL-1β and TNF-α content in the cell supernatant was determined using an ELISA assay. Firstly, it was demonstrated that the LPS treatment significantly promoted the expression of IL-1β and TNF-α in the cell supernatant. Treatment with 10 ng/ml LPS induced a 5.8-fold elevation in the levels of IL-1β and a 19-fold elevation of TNF-α levels in the EA.hy926 cells (P<0.01 and P<0.001, respectively; Fig. 1A and B). The promotion of IL-1β and TNF-α secretion was dose-dependent; significant difference in the levels of IL-1β and TNF-α were observed between the 10 and 100 or 1,000 ng/ml groups, as follows: 10 vs. 100 ng/ml (P<0.01) and 100 vs. 1,000 ng/ml (P<0.05) for IL-1β; 10 vs. 100 ng/ml and 100 vs. 1,000 ng/ml (P<0.01) for TNF-α. Secondly, the induction of IL-1β and TNF-α in EA.hy926 cells was reexamined following combined treatment with LPS and CA. CA was observed to significantly attenuate the LPS-induced increase in the expression of IL-1β and TNF-α (Fig. 1C and D). Reduced levels of IL-1β or TNF-α were observed in EA.hy926 cells treated with a combination of 1 µg/ml LPS and 10 or 50 µM CA compared with the levels in EA.hy926 cells treated with 1 µg/ml LPS only (P<0.01 and P<0.001, respectively, for IL-1β; P<0.05 and P<0.01, respectively, for TNF-α). This attenuation was dose-dependent, as the LPS-induced levels of IL-1β and TNF-α in the cells treated with 50 µM CA were observed to be lower than those in the cells treated with 10 µM CA. Notably, the inhibition of IL-1β or TNF-α expression by CA was not significant in the EA.hy926 cells that were not treated with LPS, possibly as the background secretion of cytokines was insufficient to discriminate the inhibitory effect of CA.

In order to further investigate the CA-mediated inhibition of proinflammatory cytokines in endothelial cells, the levels of IL-1β and TNF-α were determined in the supernatants of EA.hy926 cells at various periods following treatment with 1 µg/ml LPS and/or with 50 µM CA. As presented in Fig. 2A, the inhibitory effect of CA on IL-1β secretion was significant at 3 h post-treatment. The levels of IL-1β were found to be reduced in cells treated with a combination of 50 µM CA and 1 µg/ml LPS compared with those in the cells treated with LPS alone (P<0.05), and the inhibitory effect was greater at 6 or 12 h post-treatment (P<0.01). Furthermore, the inhibitory effect of TNF-α secretion was initially observed at 6 h post-treatment (P<0.01), then reconfirmed at 12 h post-treatment (P<0.01). Therefore, the results indicate that the CA-mediated inhibition of proinflammatory cytokines in the endothelial cells was time-dependent.

In order to investigate how CA affected the secretion or expression of IL-1β or TNF-α, the degree of activation of MAPK signaling in the LPS- and/or CA-treated EA.hy926 cells was analyzed, as MAPK signaling is known to be promoted by LPS and to upregulate proinflammatory cytokines (29,30). Western blot analysis was performed to examine the expression and phosphorylation of p38, JNK and ERK in EA.hy926 cells following various treatments. Fig. 3 indicates that levels of phosphorylated p38, JNK and ERK were significantly elevated as a result of treatment with 1 µg/ml LPS for 12 h (P<0.01). However, this promoted phosphorylation of p38, JNK and ERK was significantly attenuated by treatment with 50 µM CA (P<0.01), with JNK phosphorylation detectably attenuated by 10 µM CA (P<0.05). Furthermore, the attenuation was dose-dependent, as a significant difference was detected between the 10 µM- and 50 µM-treated cells (P<0.01 for p38 or JNK phosphorylation; P<0.05 for ERK phosphorylation). Therefore, the results indicate that CA treatment attenuated MAPK activation in the LPS-induced inflammatory cells.

TLRs, such as TLR4, are type-I transmembrane receptors expressed on the cell membrane following LPS stimulation (31). Activation of TLR4 signaling by LPS is associated with the release of LPS-induced inflammatory cytokines (32). In order to determine whether TLR4 signaling is targeted by CA to attenuate the LPS-induced release of inflammatory cytokines, the expression of TLR4 mRNA and protein was determined in EA.hy926 cells treated with LPS and/or CA. As shown in Fig. 4A, the mRNA expression levels of TLR4 were significantly upregulated by LPS treatment (P=0.003); however, treatment with 10 or 50 µM CA alone exerted no upregulatory effect on TLR4 mRNA (P=0.177 and 0.140, respectively). Furthermore, experiments in which combined treatments were applied indicated that treatment with 10 or 50 µM CA did not exert a significant effect on the LPS-induced promotion of TLR4 mRNA levels (P=0.511 or 0.366; Fig. 4B). Western blot analysis indicated that no CA-mediated regulation of TLR4 expression occurred at the protein level in EA.hy926 cells, as no significant difference in TLR4 expression levels was observed between the cells treated with LPS alone and those treated with a combination of LPS and CA (Fig. 4C and D).