5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Abstract Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.

anti-oxidant and cytoprotective properties, 14 which may be related to its participation in the regulation of apoptosis and inflammation. 13 In addition, pharmacological studies have demonstrated that TDD can ameliorate myocardial ischaemia-reperfusion injury and prevent arteriosclerosis. 10,11 Taken together, these findings indicate that TDD could improve endothelial function, reduce oxidative stress and suppress inflammatory reactions. Thus, studies aiming towards understanding the molecular mechanisms underlying TDD activities are imperative.
HMBOX1, considered to be a transcription repressor, was first identified and synthesized from a human pancreatic cDNA library. 16 HMBOX1 is expressed in many human tissues, including the cytoplasm of the human umbilical vein endothelial cells (HUVECs). The lack of HMBOX1 in HUVECs can induce cell apoptosis and inhibit cell autophagy, which are indispensable for HUVEC survival. 17 In addition, HMBOX1 is essential for the maintenance of endothelial function and has the potential to be a novel therapeutic target for atherosclerosis (AS). 18 However, the precise role of HMBOX1 in the context of anti-inflammatory and anti-oxidative effects of TDD in EA.hy926 cells remains unknown.
Endothelial dysfunction is a critical factor in the pathogenesis of AS. [19][20][21] An increasing number of studies have demonstrated the close association of vascular inflammatory response and oxidative stress with endothelial dysfunction. [22][23][24] Thus, the inhibition of vascular inflammatory response and oxidative stress may be a promising therapeutic approach in the treatment of AS and other cardiovascular diseases. To investigate the key molecules and pathways of TDD, gene expression profiles of control and TDD-treated EA.hy926 cells were studied using microarray analysis. Our results indicated that HMBOX1 was dramatically upregulated in TDD-treated EA.hy926 cells. One of the biological processes most significantly influenced by TDD was the response to lipopolysaccharide (LPS); this result is in agreement with the results of previous investigations. 13 Reportedly, LPS can initiate vascular inflammatory response and oxidative stress. 25,26 Therefore, we sought to understand the correlation between HMBOX1 activation and the antiinflammatory and antioxidant effects of TDD in LPS-treated EA.hy926 cells and further explored the possible underlying mechanisms.

| Materials
TDD was synthesized in our laboratory as previously reported (Supplementary Material online, Figure S1, 99% purity). 14

| Microarray analysis
Total RNA was extracted from EA.hy926 cells, which were either untreated or exposed 10 μmol/L TDD for 1 hour. An Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) was used to evaluate RNA purity and concentration. The EA.hy926 cell gene expression profiles of different groups were analysed using the Agilent Human 8 × 60 K.

| Cell adhesion assay
Monocytes adhering to EA.hy926 cells were evaluated by using THP-1 cells as previously described. 28

| Cytokine assays
The concentrations of the cytokines IL-6 and TNF-α in the supernatants were measured using ELISA kits according to the manufacturer's recommendations.

| Measurement of ROS generation
The production of intracellular reactive oxygen species (ROS) was evaluated using the DCFH-DA probe (Sigma-Aldrich) as previously described. 27 Then, DCF fluorescence was detected by flow cytometric analysis at 488 and 525 nm using a BD Biosciences flow cytometer.

| Western blot analysis
Extracts from cell cytoplasm and nuclear proteins were prepared as previously described. 29 Sample proteins (30 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After the membranes were blocked with 5% non-fat milk, they were probed overnight at 4°C with the indicated antibodies. Then, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies as previously described. 27,29 Immunoreactivity was detected by ECL reagents and data were analysed using Adobe Photoshop CC software.

| Confocal microscopy
EA.hy926 cells were cultured in four-chambered coverglass and treated with TDD (20 μmol/L) for 4 hours before the addition of LPS (1 μg/mL) for a further 2 hours. An NF-κB nuclear translocation assay was performed using an NF-κB activation-nuclear translocation assay kit. Briefly, after the cells were fixed and blocked, they were incubated with NF-κB p65 antibody at 4°C overnight. Next, the cells were incubated with a Cy3-conjugated secondary antibody at room temperature for 1 hour and stained with DAPI for 5 minutes.

| HMBOX1 siRNA transfection
The HMBOX1 gene silencer was designed and synthesized by Gene-Pharma (Shanghai, China). Transfection experiments were performed using 50 nmol/L siRNA and siRNA-mate Reagent (GenePharma) according to the manufacturer's protocol.

| Statistical analysis
All experiments were conducted in triplicates. All results are expressed as the mean ± SD. The data were analysed using the Student's t-test and differences were considered statistically significant at P < 0.05.  Figure S3). Therefore, in the subsequent experiments, TDD was used at a concentration between 5 and 20 μmol/L.

T A B L E 1 List of primer sequences
Primer name Primer sequence  Figure S4). Among them the most prominently regulated gene induced by TDD was HMBOX1. Therefore, we focused study on HMBOX1. As shown in Figure 1, treatment with TDD in EA.hy926 cells significantly increased the expression level of HMBOX1 mRNA and protein in a dosedependent manner. Biological processes related to genes with differential expression were done using GO analysis. Among them the response to the LPS pathway was shown to be significantly impacted by TDD treatment (Figure 2).

| TDD inhibited LPS-induced THP-1 adhesion
Inflammation-induced mononuclear cell adhesion to the endothelium is believed to be one of the earliest events in the development of AS. 30

| TDD inhibited LPS-induced NF-κB activation by activating HMBOX1
Previous studies have shown that NF-κB is a key regulator of pro-inflammatory cytokines. 32 Therefore, the effect of TDD on LPS-induced NF-κB activation in EA.hy926 cells was examined. Our results indicate that TDD pre-treatment inhibited LPS-increased p65 NF-κB translocation to the nuclear fraction in EA.hy926 cells (Figure 5A). The inhibitory effect of TDD on LPS-induced NF-kB p65 nuclear translocation was consistent with protein expression levels and further confirmed using confocal microscopy ( Figure 5B).
Next, HMBOX1 knockdown analysis was used to investigate whether HMBOX1 influenced the effect of TDD on LPS-induced NF-κB activation. As shown in Figure 5C Figure 5D).

| DISCUSSION
Inflammation and oxidative stress play key roles in the progression of cardiovascular diseases, including AS. [33][34][35] Therefore, the inhibition of inflammatory response and ROS production may be beneficial in preventing the development of AS. 33 Endothelial cell dysfunction is  [42][43][44] Epidemiological studies also indicate that an elevated serum LPS level constitutes a critical risk factor for the development of AS in humans. [45][46][47] A pivotal mechanism of AS aggravation is proposed to be a part of an endothelial injury pathway induced by LPS. 48,49 Pharmacological studies have demonstrated that TDD can ameliorate myocardial ischaemia-reperfusion injury and prevent arteriosclerosis, 10,11 indicating that TDD could improve endothelial function, suppress the inflammatory reaction and reduce oxidative stress. To investigate TDD target genes, the microarray analysis of differences between TDD-treated and control EA.hy926 cells was conducted. In this study, approximately 42 545 mRNAs with differential changes induced by TDD were identified in EA.hy926 cells.
Among these, the highest fold change in upregulation induced by TDD was in HMBOX1.
On the basis of the results of the GO analysis of microarray data, biological processes influenced by TDD were determined to be involved in the response to LPS, which is in agreement with previous results. 13 Exposing vascular endothelial cells to LPS generated inflammatory cytokines and ROS. 25,26,50 Here, we investi- In addition to NF-kB, this secretion of CAMs and pro-inflammatory cytokines is always dependent on the activation of MAPKs. 58,60 Our results indicated that TDD treatment inhibits the LPS-stimulated activation of p38 and ERK1/2. In addition, when LPS-stimulated EA.hy926 cells were exposed to PDTC (a selective NF-κB inhibitor), In conclusion, TDD may be a potential novel anti-inflammatory and anti-oxidative drug for treating endothelial cell dysfunction in AS.

CONFLI CT OF INTEREST
All authors declare no competing interests.