Bakkenolide‑IIIa ameliorates lipopolysaccharide‑induced inflammatory injury in human umbilical vein endothelial cells by upregulating LINC00294

Abstract

Inflammation, which causes injury to vascular endothelial cells, is one of the major factors associated with atherosclerosis (AS); therefore, inhibition of endothelial inflammation is a key step toward preventing AS. The present study aimed to investigate the effects of bakkenolide‑IIIa (Bak‑IIIa), an important active component of bakkenolides, on endothelial inflammation, as well as the mechanisms underlying such effects. Lipopolysaccharide (LPS)‑damaged human umbilical vein endothelial cells (HUVECs) were treated with Bak‑IIIa. The results of the MTT assay and enzyme‑linked immunosorbent assay indicated that Bak‑IIIa significantly alleviated survival inhibition, and decreased the levels of LPS‑induced TNF‑α, interleukin (IL)‑1β, IL‑8, and IL‑6. Furthermore, long noncoding RNA (lncRNA) microarray analyses revealed 70 differentially expressed lncRNAs (DELs) in LPS‑damaged HUVECs treated with Bak‑IIIa. lncRNA target prediction results revealed that 44 DELs had 52 cis‑targets, whereas 12 DELs covered 386 trans‑targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses of the trans‑targets indicated that three GO terms were associated with inflammation. Therefore, 17 targets involved in these GO terms and six relevant DELs were screened out. Validation via reverse transcription‑quantitative PCR indicated that the fold change of NR_015451 (LINC00294) was the highest among the six candidates and that overexpression of LINC00294 significantly alleviated LPS‑induced survival inhibition and inflammatory damage in HUVECs. In conclusion, Bak‑IIIa ameliorated LPS‑induced inflammatory damage in HUVECs by upregulating LINC00294. Thus, Bak‑IIIa exhibited potential for preventing vascular inflammation.

Keywords: endothelial inflammation; bakkenolide‑IIIa; LINC00294.

Figure 1.

Bak-IIIa alleviates LPS-induced survival inhibition in HUVECs. (A) Molecular structure of Bak-IIIa. (B) Effect of 0, 10, 20, 50, 100 and 200 µM Bak-IIIa on the viability of HUVECs. *P<0.05 vs. 0 group. (C) Effect of 0, 10, 20 and 50 µM Bak-IIIa on the viability of LPS-injured HUVECs. ^#P<0.05 vs. control group; *P<0.05 vs. LPS group. Bak-IIIa, bakkenolide-IIIa; HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide.

Figure 2.

Bak-IIIa ameliorates LPS-induced inflammatory damage in HUVECs. Effect of 0, 10, 20 and 50 µM Bak-IIIa on the levels of (A) TNF-α, (B) IL-1β, (C) IL-8 and (D) IL-6 in LPS-injured HUVECs. ^#P<0.05 vs. control group; *P<0.05 vs. LPS group. Bak-IIIa, bakkenolide-IIIa; HUVECs, human umbilical vein endothelial cells; IL, interleukin; LPS, lipopolysaccharide.

Figure 3.

Volcano plot of differentially expressed long non-coding RNAs in LPS-damaged human umbilical vein endothelial cells treated with Bak-IIIa. Bak-IIIa, bakkenolide-IIIa; FC, fold change; LPS, lipopolysaccharide.

Figure 4.

Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the cis-targets of differentially expressed long non-coding RNAs.

Figure 5.

Reverse transcription-quantitative PCR validation of six candidate differentially expressed lncRNAs. NR_015451 is also known as LINC00294. *P<0.05 vs. LPS group. Bak-IIIa, bakkenolide-IIIa; lncRNA, long non-coding RNA; LPS, lipopolysaccharide.

Figure 6.

Overexpression of LINC00294 alleviates LPS-induced inflammatory damage in HUVECs. (A) Reverse transcription-quantitative PCR analysis of LINC00294 expression levels in HUVECs overexpressing LINC00294. *P<0.05 vs. NC group. (B) Effect of overexpressed LINC00294 on the viability of LPS-injured HUVECs. *P<0.05. Effect of overexpressed LINC00294 on the levels of (C) TNF-α, (D) IL-1β, (E) IL-8 and (F) IL-6 in LPS-injured HUVECs. *P<0.05. Bak-IIIa, bakkenolide-IIIa; HUVECs, human umbilical vein endothelial cells; IL, interleukin; LPS, lipopolysaccharide; NC, negative control.