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. 2021 Mar 17;23(5):377. doi: 10.3892/mmr.2021.12016

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

Jichong Xu 1,*, Hao Feng 1,*, Lin Ma 1, Huaqiao Tan 1, Shuo Yan 1,✉, Chun Fang 1,✉
PMCID: PMC7986008  PMID: 33760129

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

Introduction

Atherosclerosis (AS) is the main cause of cardiovascular diseases and is a chronic progressive disease initiated by vascular inflammation (1,2). The endothelium is the innermost layer of blood vessels and changes in blood environment serve an important role in vascular homeostasis. Several factors, such as hyperlipidemia, tobacco use, hypercysteinemia, diabetes and hypertension (3,4), may induce vascular injury by activating endothelial cells, which then release increased amounts of proinflammatory cytokines, chemokines and adhesion molecules (5,6). Subsequently, circulating monocytes are recruited to the lesion site, further triggering a series of pathogenic processes, including excessive lipid accumulation, tunica smooth muscle cell migration and plaque formation (7). Therefore, inhibition of endothelial inflammatory processes may constitute a key step toward preventing AS.

Bakkenolides are a relatively rare class of natural products, which can be isolated from plants of the genus Petasites, including Petasites japonicus (Sieb. et Zucc.) F. Schmidt and Petasites tricholobus Franch (8), which have been used in Chinese folk medicine to treat palsy, hypertension, cough and snakebite for hundreds of years (9). Compared with three other bakkenolides, bakkenolide-IIIa (Bak-IIIa), which is an important active component of bakkenolides, has been reported to display the best neuroprotective effect against oxygen-glucose deprivation-induced neuronal injuries (9). Another study involving animal cerebral damage models indicated that Bak-IIIa could exert neuroprotective effects by inhibiting nuclear factor (NF)-κB activation (10). Inflammation is a fundamental, yet complex, biological response initiated by the body to counter injuries, as well as infections, and to eliminate the initial causes of cell injury (11). Although the inflammatory levels of injured neurons were not evaluated in the two aforementioned studies, it is reasonable to surmise that inflammation may have been decreased, considering the significant neuroprotective effect of Bak-IIIa.

Long noncoding RNAs (lncRNAs) belong to an important class of ncRNAs that are involved in the regulation of various physiological and pathological processes, including AS (12). Recently, some lncRNAs have been identified as novel regulators of vascular inflammation, which constitutes an initial step toward AS (12,13). For example, the lncRNA HOXA-AS3 has been revealed to be a critical activator that promotes NF-κB-mediated endothelial inflammation (14). In addition, the lncRNA H19 was upregulated in the serum of patients with AS and in oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs), whereas knockdown of its expression significantly ameliorated inflammation, apoptosis and oxidative stress in ox-LDL-injured HUVECs (15). In addition, the lncRNAs HOXA-AS2 and MALAT1 have been shown to participate in the regulation of endothelial inflammation (16,17).

The present study investigated the effect of Bak-IIIa on lipopolysaccharide (LPS)-induced inflammatory injury in HUVECs and identified potential lncRNAs involved in this process. The present study may provide a candidate molecule for the treatment of AS.

Materials and methods

Cells and cell culture

HUVECs were purchased from American Type Culture Collection, and were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO2.

Cell viability assay

HUVECs were seeded in a 96-well plate at 8,000 cells/well. The next day, the cells were exposed to different concentrations (0, 10, 20, 50, 100 and 200 µM) of Bak-IIIa (CSNpharm, Inc.) for 72 h at 37°C. The supernatant was then removed, and 100 µl medium containing 50 µg MTT was added to the wells. After incubation at 37°C for 3 h, accumulated formazan was dissolved in 100 µl DMSO (Sangon Biotech Co., Ltd.), and absorbance was determined at 490 nm using a microplate reader (ELx800; BioTek Instruments, Inc.).

To observe the effect of Bak-IIIa on LPS-induced cellular injury, HUVECs were first exposed to 10 ng/ml LPS (Sigma-Aldrich; Merck KGaA) for 4 h at 37°C, and then treated with 0, 10, 20 and 50 µM Bak-IIIa for 24 h at 37°C. Cell survival was assessed using the aforementioned MTT assay. Cells treated without LPS and Bak-IIIa were used as the control.

Enzyme-linked immunosorbent assay (ELISA)

HUVECs were seeded in a 12-well plate at 3×104 cells/well, and were treated with LPS for 4 h, and with 0, 10, 20 and 50 µM Bak-IIIa for 24 h, respectively. Thereafter, cell culture supernatants were collected and filtered through a 0.45-µm cell Nalgene syringe filter (Thermo Fisher Scientific, Inc.). TNF-α (cat. no. PT518), interleukin (IL)-1β (cat. no. PI305), IL-8 (cat. no. PI640) and IL-6 (cat. no. PI330) levels in the supernatants were analyzed using corresponding ELISA kits (Beyotime Institute of Biotechnology), according to the manufacturer's instructions.

Microarray analysis

After LPS-treated HUVECs were treated with 0 or 50 µM Bak-IIIa for 24 h (named as LPS or Bak-IIIa group respectively), total RNA from these two groups was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequent microarray hybridization and data analysis were performed by Bhbio Ltd. Co (http://www.shbio.com/). Briefly, purified RNA from each sample was amplified and labeled using a Low Input Quick Amp Labeling kit (Agilent Technologies, Inc.). Thereafter, labeled cRNA was hybridized in a hybridization oven (Agilent Technologies, Inc.), and the hybridized slides were washed and scanned using a Microarray Scanner (Agilent Technologies, Inc.). Raw data were extracted using Feature Extraction software (v10.7.1.1; Agilent Technologies, Inc.) and normalized via a quantile algorithm. Differentially expressed lncRNAs (DELs) were filtered through fold change absolute values (FC abs ≥2) and the significance of P-values (P<0.5).

Prediction of lncRNA targets

For predicting cis-regulation, genes transcribed within 10 kb upstream or downstream of DELs were considered as cis-targets. DELs and potential cis-targets were paired using the UCSC genome browser (18). For trans-target prediction, complementary or similar sequences were first selected via BLAST software (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/). Thereafter, complementary energy between the two sequences was calculated using RNAplex software (v2.1.8) (19) and energy ≤-30 was selected as the trans-target of DELs.

Bioinformatics analyses

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses (https://david.ncifcrf.gov/) were conducted to determine enrichment of the predicted targets of DELs. Both cis- and trans-targets were first classified into corresponding GO terms or KEGG pathways according to each database, and the top 30 GO terms or KEGG pathways showing a higher enrichment factor and a significant P-value (<0.5) were selected.

Reverse transcription-quantitative PCR (RT-qPCR) analysis

Total RNA was isolated from cells in the LPS and Bak-IIIa groups using TRIzol® reagent (Thermo Fisher Scientific, Inc.) and cDNA was synthesized using PrimeScript™ One Step RT-PCR kit (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. RT-qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. Thermocycling was: 94°C for 5 min, then 40 cycles at 94°C for 5 sec and 60°C for 1 min. Expression levels were calculated using the 2−ΔΔCq method (20), and relative levels of lncRNA were normalized to GAPDH. Primer sequences for lncRNAs and GAPDH are listed in Table I.

Table I.

Primer sequences for reverse transcription-quantitative PCR.

LncRNA/Gene Forward (5′→3′) Reverse (5′→3′)
NONHSAT089168 TGTCATTCAGCCCAAACTCA CGCCAGGTTAGTTCAACACA
NR_015451 TCTCGATCTCCTGACCTCGT ACAGAGCAGCCTCCACCTTA
NR_038275 CCCTCCTATGTGCTCGTTTC GAGAAGTCCTCGTCGGTCAG
lnc-MUC20-9:1 GCCTGTCTCTGCCTCTGAAC CCAGGCCAGACCAATCTAAA
NR_034072 TCCCAAAGTGCTGGGATTAC CAGTTGGGAACTTGGGAAAA
lnc-HSP90B1-3:1 ACTTGAGGCCAGGAGTTTGA AACCTCTCAGGCTCAAGCAA
GAPDH CGACCACTTTGTCAAGCTCA AGGGGAGATTCAGTGTGGTG
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lncRNA, long non-coding RNA.

Transfection assay

HUVECs were seeded in 6-well plates (2×105 cells/well) and transfected with 2 µg pCDH-CMV-MCS-EF1-GFP-T2A-Puro plasmid encoding full length LINC00294 (Hanbio Biotechnology Co., Ltd.) or the empty vector, using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. After transfection at 37°C for 48 h, cells were collected for RT-qPCR validation. HUVECs transfected with the plasmid or empty vector were categorized as LINC00294 or negative control (NC) groups, respectively and were then used for LPS treatment, followed by cell viability and ELISA assay.

Statistical analysis

SPSS v16.0 (SPSS, Inc.) was used for data processing. Data are presented as the mean ± standard deviation and each experiment was performed three times. Differences between two groups were determined using Student's t-test, whereas one-way analysis of variance and Tukey's post hoc test were used to compare three or more groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Bak-IIIa ameliorates the LPS-induced inflammatory damage in HUVECs

Initially, the cytotoxic effects exerted by Bak-IIIa on HUVECs were assessed (Fig. 1A). MTT assays indicated that the viability of HUVECs was not significantly changed by treatment with lower concentrations (10–50 µM) of Bak-IIIa, whereas viability was reduced by 12–35% by high doses (100 and 200 µM) (Fig. 1B). Hence, low doses of Bak-IIIa (10–50 µM) were selected for use in subsequent studies. HUVECs were then exposed to LPS, and the results of the MTT assay indicated that the survival of HUVECs was decreased to ~63% compared with the control group (Fig. 1C). Following further treatment with 20 and 50 µM Bak-IIIa, the viability of damaged HUVECs was increased by ~11 and ~16%, respectively (Fig. 1C).

Figure 1.

Figure 1.

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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.

ELISA revealed that the levels of TNF-α, IL-1β, IL-8 and IL-6 were significantly increased following LPS-induced stimulation compared with in the control group, indicating that inflammatory damage was inflicted on the HUVECs (Fig. 2A-D). The LPS-injured HUVECs were then treated with 10–50 µM Bak-IIIa, and the results indicated that the levels of the four proinflammatory factors were gradually decreased in response to increasing Bak-IIIa concentrations (Fig. 2A-D). Of the three dose groups, the 20 and 50 µM groups induced a significant reduction in TNF-α, IL-1β, IL-8 and IL-6 levels compared with in the LPS group (Fig. 2A-D), thus suggesting that Bak-IIIa treatment was effective in ameliorating LPS-induced inflammatory damage in HUVECs.

Figure 2.

Figure 2.

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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.

Microarray analysis

In order to investigate the mechanisms underlying the effect of Bak-IIIa on HUVECs damaged by inflammation, cells from the LPS and Bak-IIIa groups were subjected to lncRNA microarray analyses, which identified 70 DELs in these two groups. A volcano plot showed the presence of 29 significantly upregulated (red dots) and 41 significantly downregulated (blue dots) DELs in HUVECs that had undergone LPS-induced inflammatory damage and subsequent Bak-IIIa treatment (Fig. 3). These 70 DELs were then ranked according to FC abs values in descending order; the top 10 are listed in Table II. The FC abs of most DELs was ~4, whereas the lncRNA ENST00000441971 had the highest FC abs of ~7.

Figure 3.

Figure 3.

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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.

Table II.

Top 10 differentially expressed lncRNAs after lipopolysaccharide-damaged human umbilical vein endothelial cells were treated with bakkenolide-IIIa.

Number lncRNA P-value FC FC (abs) Regulation Source
1 ENST00000441971 0.026 6.807 6.807 Up ENSEMBL_GENCODE
2 lnc-NR2F1-8:1 0.048 4.810 4.810 Up lncipedia
3 ENST00000609511 0.015 4.343 4.343 Up ENSEMBL_GENCODE
4 NONHSAT089168 0.045 4.250 4.250 Up NONCODE
5 ENST00000451766 0.039 0.239 4.190 Down ENSEMBL_GENCODE
6 NONHSAT113888 0.005 3.920 3.920 Up NONCODE
7 ENST00000618397 0.037 3.788 3.788 Up ENSEMBL_GENCODE
8 lnc-COMTD1-1:1 0.006 0.281 3.560 Down lncipedia
9 lnc-MOK-1:2 0.030 0.284 3.523 Down lncipedia
10 NR_015451 0.034 3.320 3.320 Up RefSeq
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abs, absolute; FC, fold change; lncRNA, long non-coding RNA.

lncRNA target prediction and bioinformatics analyses

To obtain a better understanding of the DELs, the cis- and trans-targets were predicted. As a result, 52 cis-regulated targets were obtained from 44 DELs (Table SI), whereas 386 trans-regulated targets (111 overlaps among all 497 rows) were predicted from only 12 DELs (Table SII); nine of the 12 DELs, listed in Table III, covered a total of 382 targets (some overlapped). In addition, the trans-target number for NONHSAT089168, NR_015451 and lnc-MUC20-9:1 reached 100 (Table III).

Table III.

Nine differentially expressed lncRNAs covering 382 trans-targets.

Number lncRNA Trans-target number FC FC (abs) Regulation Sources
1 NONHSAT089168 113 4.250 4.250 Up NONCODE
2 NR_015451 154 3.320 3.320 Up RefSeq
3 NR_027411 10 2.650 2.650 Up RefSeq
4 NR_033350 10 2.578 2.578 Up RefSeq
5 NR_033353 10 2.435 2.435 Up RefSeq
6 NR_038275 13 2.395 2.395 Up RefSeq
7 lnc-MUC20-9:1 100 2.366 2.366 Up lncipedia
8 NR_034072 29 2.188 2.188 Up RefSeq
9 lnc-HSP90B1-3:1 54 2.037 2.037 Up lncipedia
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abs, absolute; FC, fold change; lncRNA, long non-coding RNA.

Among the top 30 targets found to be enriched according to GO analysis, some cis-targets were involved in ion homeostasis and ‘autophagy’ (Fig. S1), whereas trans-targets were enriched in ‘cis-Golgi network’ and ‘IL-12 production’ (Fig. S2). KEGG pathway analysis showed that cis-targets were mainly enriched in the ‘AMPK signaling pathway’, whereas several targets were involved in the ‘p53 signaling pathway’ (Fig. 4). trans-targets were enriched in ‘purine metabolism’ and ‘glycosylphosphatidylinositol-anchor biosynthesis’ (Fig. S3).

Figure 4.

Figure 4.

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

RT-qPCR validation of the DELs corresponding to inflammation-related trans-targets

The key words ‘inflammation’ and ‘inflammatory’ were searched during GO and KEGG analyses of trans-targets (Tables SIII and SIV), and three inflammation-related GO terms, ‘inflammatory response’, ‘positive regulation of inflammatory response’, and ‘regulation of inflammatory response’, involving a total of 17 targets (some overlapped) were screened (Table IV). The corresponding DELs are listed in Table V, and nine (XIAP, CCL5, ACER3, OPRM1, IDO1, LYZ, HMGB1, LDLR and F2R) of the 17 targets were those of LINC00294. RT-qPCR indicated that the expression levels of most of the six candidate DELs were consistent with those revealed by the results of microarray analyses. Subsequently, NR_015451 (also termed LINC00294), which displayed the highest verified expression level, was selected for further evaluation (Fig. 5).

Table IV.

Inflammation-related GO terms and trans-targets.

GO ID Description Count Trans-targets
GO:0006954 Inflammatory response 17 XIAP, CCR6, CCL5, MEFV, ACER3, OPRM1, IDO1, AXL, TLR10, REL, LYZ, CCL22, NLRP12, HMGB1, LDLR, CAMK1D, F2R
GO:0050729 Positive regulation of inflammatory response 4 IDO1, NLRP12, TLR10, LDLR
GO:0050727 Regulation of inflammatory response 7 NLRP12, CCL5, MEFV, LDLR, XIAP, IDO1, TLR10
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GO, Gene Ontology.

Table V.

The 17 trans-targets and corresponding lncRNAs.

Trans-targets lncRNAs
XIAP NONHSAT089168, NR_015451
CCR6 lnc-MUC20-9:1
CCL5 lnc-MUC20-9:1, NR_015451, NR_034072
MEFV lnc-HSP90B1-3:1
ACER3 lnc-MUC20-9:1, NR_015451
OPRM1 NR_015451
IDO1 NONHSAT089168, NR_015451
AXL NR_034072
TLR10 NONHSAT089168
REL NR_038275
LYZ NR_015451
CCL22 lnc-HSP90B1-3:1
NLRP12 lnc-MUC20-9:1, NONHSAT089168
HMGB1 NONHSAT089168, NR_015451
LDLR NR_015451
CAMK1D lnc-MUC20-9:1
F2R NR_015451
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lncRNA, long non-coding RNA.

Figure 5.

Figure 5.

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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.

Overexpression of LINC00294 alleviates the LPS-induced inflammatory damage in HUVECs

The RT-qPCR results indicated that LINC00294 was successfully overexpressed in HUVECs post-transfection with the LINC00294 plasmid (Fig. 6A). Subsequently, cells were treated with LPS and subjected to an MTT assay, which indicated that overexpression of LINC00294 significantly reversed the survival inhibition induced by LPS, compared with cells in the NC group (Fig. 6B). Furthermore, ELISA indicated that the levels of the four proinflammatory factors TNF-α, IL-1β, IL-8 and IL-6 were all significantly decreased in HUVECs overexpressing LINC00294 (Fig. 6C-F), suggesting that upregulation of LINC00294 significantly alleviated the inflammatory damage in HUVECs induced by LPS.

Figure 6.

Figure 6.

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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.

Discussion

To the best of our knowledge, the present study was the first to assess the effects of Bak-IIIa treatment on LPS-induced inflammatory damage in HUVECs and the lncRNAs involved. The present results indicated that Bak-IIIa ameliorated inflammatory damage in HUVECs.

Vascular inflammation is usually caused by endothelial injury, and thus, prevention of endothelial injury may be considered a key step toward limiting the development of vascular inflammatory processes. LPS acts as a common exogenous factor that stimulates endothelial injury (21). Firstly, LPS treatment generally increases cell permeability and impairs the endothelial barrier, which constitutes an essential step toward the initiation of inflammation (22,23). Secondly, exposure of endothelial cells to LPS leads to inflammation via the production of proinflammatory cytokines/chemokines, cell adhesion molecules and nitric oxide (NO) (24). Hence, LPS-induced inflammatory damage in HUVECs has been widely used as a model for developing potentially effective compounds, as well as for exploring the underlying mechanisms (25). In the present study, HUVECs were exposed to LPS, following which the levels of TNF-α, IL-1β, IL-8 and IL-6 were all significantly enhanced, thereby substantiating the results of previous studies (26,27). TNF-α and IL-1β are two predominant proinflammatory factors involved in the pathogenesis of vascular inflammation. Firstly, increased levels of TNF-α may promote the expression of the inducible form of NO synthase, which in turn can lead to excessive synthesis of NO, resulting in endothelial dysfunction, apoptosis and vascular damage (28). Secondly, IL-1β can stimulate the synthesis of monocyte chemoattractant protein 1, resulting in the adhesion of monocytes and the subsequent induction of proliferative, proinflammatory vascular smooth muscle cell phenotypes, thereby initiating the pathological progression of vascular inflammation (29). In the present study, the levels of TNF-α and IL-1β, as well as two other proinflammatory factors, IL-8 and IL-6, were all significantly decreased following the treatment of HUVECs with Bak-IIIa, which effectively inhibited vascular inflammation.

In order to clarify the potential mechanism underlying the effect of Bak-IIIa on damaged HUVECs, the DELs present in the two groups were analyzed and 70 were identified. lncRNAs may regulate gene expression in a cis- or trans-acting manner (30). The difference between these two mechanisms is that cis-acting lncRNAs generally influence the expression of nearby genes, whereas trans-acting lncRNAs usually leave the transcription site to execute an array of cellular functions by interacting with promoters, enhancers or other proteins (30). Therefore, as trans-targets of lncRNAs are always abundant, potential ways of interacting with these are diverse. The lncRNA target predictions in the present study indicated that 12 DELs covered a total of 386 trans-targets, suggesting a predominant role for these 12 DELs in the amelioration of inflammatory damage in HUVECs by Bak-IIIa. Furthermore, a bioinformatics-based analysis of trans-targets was performed to determine the DELs that may be potentially involved. However, the GO and KEGG pathway enrichment analysis results only indicated those that were significant (P<0.5); thus, it was difficult to establish certain associations between these and inflammation. Therefore, ‘inflammation’ and ‘inflammatory’ were searched in the bioinformatics analyses of trans-targets; this resulted in the screening out of 17 involved targets and six candidate DELs.

Further validation via RT-qPCR and ELISA indicated that upregulation of LINC00294 in the Bak-IIIa group was an important mechanism underlying the ameliorative effect of Bak-IIIa on LPS-induced survival inhibition and inflammatory damage in HUVECs. Among the 17 inflammation-related targets, nine were targets of LINC00294. C-C motif chemokine ligand 5 (CCL5) is a key proinflammatory chemokine expressed in endothelial cells, smooth muscle cells and macrophages, which has been reported to activate various inflammation-related pathological processes, including those of vascular inflammation (31,32). CCL5 may be involved in emodin-alleviated cytotoxicity and high glucose-induced inflammatory events in HUVECs (33), indicating that CCL5 may potentially regulate viability and inflammatory damage in HUVECs. Furthermore, high mobility group box 1 (HMGB1), a ubiquitously expressed nuclear protein, has been reported to serve a predominantly proinflammatory role in the progression of inflammation (34). Several studies confirmed that HMGB1 may have an important regulatory role in vascular endothelial inflammation (35,36). Therefore, it may reasonably be surmised that CCL5 and HMGB1 serve a role in Bak-IIIa-alleviated survival inhibition and inflammatory damage in HUVECs; however, this requires further study.

Although this study focused on the trans-targets of DELs, KEGG pathway-based enrichment analyses of cis-targets provided further leads regarding potential mechanisms that may be involved. AMPK acts as a pivotal cellular energy sensor in the regulation of bioenergy metabolism (37), and exerts anti-inflammatory and antioxidative effects (38). AMPK is widely expressed in the endothelium, and modulates vascular functions via both endothelium-dependent and -independent mechanisms (39). Previous reports have indicated that activation of the AMPK signaling pathway could serve an important role in the amelioration of endothelial inflammation and barrier dysfunction (40,41). Hence, the AMPK signaling pathway and the cis-targets adrenoceptor alpha 1A, tuberous sclerosis 2 and stearoyl-CoA desaturase (SCD) that are enriched in it, as well as the corresponding DELs, lnc-PNMA2-5:1, ENST00000570072 and lnc-SCD-1:8, may serve a role in the alleviation of inflammatory damage in HUVECs via Bak-IIIa.

To date, studies focusing on Bak-IIIa are very limited, and one previous study revealed that the protective effect of Bak-IIIa against cerebral damage were induced through the inactivation of NF-κB signaling (10), which is not only involved in the pathogenesis of several diseases but also has an important role in transcriptional regulation. Increasing evidence has uncovered that NF-κB could be the downstream effector of lncRNAs, but in some instances, NF-κB may also be able to regulate lncRNA expression (42). For example, cardiac and apoptosis-related lncRNA was revealed to be translocated to the cytoplasm after NF-κB activation in macrophages, and its expression levels were increased in an NF-κB dependent manner (43). NF-κB has also been shown to bind to the HOX antisense intergenic RNA promoter and induce its expression by 16-fold in ovarian cancer cells leading to the maintenance of DNA damage response (44). Moreover, other transcription factors, including p53 and FOXC1, have been reported to regulate lncRNA expression in a direct or indirect manner (45,46). Hence, the increased level of LINC00294 in injured-HUVECs treated with Bak-IIIa may be attributed to the potential regulatory role of NF-κB, p53 or other transcription factors.

There are some limitations in the present study. First, apoptosis often occurs alongside inflammation in HUVECs stimulated by LPS (47); however, the role of Bak-IIIa in apoptosis of injured HUVECs was not investigated. Secondly, the underlying mechanisms by which Bak-IIIa induced the expression of LINC00294 and how LINC00294 protected HUVECs remain unknown; therefore, further studies are required.

In conclusion, the present findings indicated that Bak-IIIa ameliorated LPS-induced inflammatory damage in HUVECs by upregulating LINC00294, and that Bak-IIIa may have potential as a therapeutic agent against vascular inflammation.

Supplementary Material

Supporting Data
Supporting Data
Supplementary_Data2.xlsx (12.3KB, xlsx)
Supporting Data
Supplementary_Data3.xlsx (28.6KB, xlsx)
Supporting Data
Supplementary_Data4.xlsx (73.3KB, xlsx)
Supporting Data

Acknowledgements

Not applicable.

Funding Statement

This work was supported by the Project from Shanghai Natural Science Fund Project (grant no. 19ZR1449000), the Training Program of Shanghai Tongji Hospital (grant no. GJPY1812), the National Natural Science Foundation of China (grant no. 81702872) and the Program of Outstanding Young Scientists of Tongji Hospital of Tongji University (grant no. HBRC1808).

Funding

This work was supported by the Project from Shanghai Natural Science Fund Project (grant no. 19ZR1449000), the Training Program of Shanghai Tongji Hospital (grant no. GJPY1812), the National Natural Science Foundation of China (grant no. 81702872) and the Program of Outstanding Young Scientists of Tongji Hospital of Tongji University (grant no. HBRC1808).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

CF and SY worked on the conception and design of the study. JX and HF performed the experiments. JX, LM and HT collected and analyzed data. CF, SY and JX confirmed the authenticity of all the raw data. All authors contributed to the preparation of the manuscript, and read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

All authors declare that they have no competing interests.

References

  • 1.Roth GA, Johnson C, Abajobir A, Abd-Allah F, Abera SF, Abyu G, Ahmed M, Aksut B, Alam T, Alam K, et al. Global, regional, and national burden of cardiovascular diseases for 10 causes, 1990 to 2015. J Am Coll Cardiol. 2017;70:1–25. doi: 10.1016/j.jacc.2017.04.052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Wolf MP, Hunziker P. Atherosclerosis: Insights into vascular pathobiology and outlook to novel treatments. J Cardiovasc Transl Res. 2020;13:744–757. doi: 10.1007/s12265-020-09961-y. [DOI] [PubMed] [Google Scholar]
  • 3.Tabas I, Garcia-Cardeña G, Owens GK. Recent insights into the cellular biology of atherosclerosis. J Cell Biol. 2015;209:13–22. doi: 10.1083/jcb.201412052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Libby P, Buring JE, Badimon L, Hansson GK, Deanfield J, Bittencourt MS, Tokgözoğlu L, Lewis EF. Atherosclerosis. Nat Rev Dis Primers. 2019;5:56. doi: 10.1038/s41572-019-0106-z. [DOI] [PubMed] [Google Scholar]
  • 5.Meng G, Liu Y, Lou C, Yang H. Emodin suppresses lipopolysaccharide-induced pro-inflammatory responses and NF-κB activation by disrupting lipid rafts in CD14-negative endothelial cells. Br J Pharmacol. 2010;161:1628–1644. doi: 10.1111/j.1476-5381.2010.00993.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Zeng XK, Guan YF, Remick DG, Wang X. Signal pathways underlying homocysteine-induced production of MCP-1 and IL-8 in cultured human whole blood. Acta Pharmacol Sin. 2005;26:85–91. doi: 10.1111/j.1745-7254.2005.00005.x. [DOI] [PubMed] [Google Scholar]
  • 7.Moroni F, Ammirati E, Norata GD, Magnoni M, Camici PG. The role of monocytes and macrophages in human atherosclerosis, plaque neoangiogenesis, and atherothrombosis. Mediators Inflamm. 2019;2019:7434376. doi: 10.1155/2019/7434376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Pei L, Dai D, Bao Y, Chen F, Zheng J, Li J, Liu S, Chen X. Determination of bakkenolide A in rat plasma using liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study. J Mass Spectrom. 2012;47:962–968. doi: 10.1002/jms.3042. [DOI] [PubMed] [Google Scholar]
  • 9.Wang YL, Li RP, Guo ML, Zhang G, Zhang N, Ma YL. Bakkenolides from Petasites tricholobus and their neuroprotective effects related to antioxidant activities. Planta Med. 2009;75:230–235. doi: 10.1055/s-0028-1088377. [DOI] [PubMed] [Google Scholar]
  • 10.Jiang Q, Li RP, Tang Y, Wang YQ, Liu C, Guo ML. Bakkenolide-IIIa protects against cerebral damage via inhibiting NF-κB activation. CNS Neurosci Ther. 2015;21:943–952. doi: 10.1111/cns.12470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Skaper SD, Facci L, Zusso M, Giusti P. An inflammation-centric view of neurological disease: Beyond the neuron. Front Cell Neurosci. 2018;12:72. doi: 10.3389/fncel.2018.00072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Aryal B, Suárez Y. Non-coding RNA regulation of endothelial and macrophage functions during atherosclerosis. Vascul Pharmacol. 2019;114:64–75. doi: 10.1016/j.vph.2018.03.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhang HN, Xu QQ, Thakur A, Alfred MO, Chakraborty M, Ghosh A, Yu XB. Endothelial dysfunction in diabetes and hypertension: Role of microRNAs and long non-coding RNAs. Life Sci. 2018;213:258–268. doi: 10.1016/j.lfs.2018.10.028. [DOI] [PubMed] [Google Scholar]
  • 14.Zhu X, Chen D, Liu Y, Yu J, Qiao L, Lin S, Chen D, Zhong G, Lu X, Wang Y, et al. Long noncoding RNA HOXA-AS3 integrates NF-κB signaling to regulate endothelium inflammation. Mol Cell Biol. 2019;39:e00139–19. doi: 10.1128/MCB.00139-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cao L, Zhang Z, Li Y, Zhao P, Chen Y. LncRNA H19/miR-let-7 axis participates in the regulation of ox-LDL-induced endothelial cell injury via targeting periostin. Int Immunopharmacol. 2019;72:496–503. doi: 10.1016/j.intimp.2019.04.042. [DOI] [PubMed] [Google Scholar]
  • 16.Zhu X, Liu Y, Yu J, Du J, Guo R, Feng Y, Zhong G, Jiang Y, Lin J. LncRNA HOXA-AS2 represses endothelium inflammation by regulating the activity of NF-κB signaling. Atherosclerosis. 2019;281:38–46. doi: 10.1016/j.atherosclerosis.2018.12.012. [DOI] [PubMed] [Google Scholar]
  • 17.Li S, Sun Y, Zhong L, Xiao Z, Yang M, Chen M, Wang C, Xie X, Chen X. The suppression of ox-LDL-induced inflammatory cytokine release and apoptosis of HCAECs by long non-coding RNA-MALAT1 via regulating microRNA-155/SOCS1 pathway. Nutr Metab Cardiovasc Dis. 2018;28:1175–1187. doi: 10.1016/j.numecd.2018.06.017. [DOI] [PubMed] [Google Scholar]
  • 18.Casper J, Zweig AS, Villarreal C, Tyner C, Speir ML, Rosenbloom KR, Raney BJ, Lee CM, Lee BT, Karolchik D, et al. The UCSC genome browser database: 2018 update. Nucleic Acids Res. 2018;46D:D762–D769. doi: 10.1093/nar/gkx1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Tafer H, Hofacker IL. RNAplex: A fast tool for RNA-RNA interaction search. Bioinformatics. 2008;24:2657–2663. doi: 10.1093/bioinformatics/btn193. [DOI] [PubMed] [Google Scholar]
  • 20.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  • 21.Song L, Kang C, Sun Y, Huang W, Liu W, Qian Z. Crocetin inhibits lipopolysaccharide-induced inflammatory response in human umbilical vein endothelial cells. Cell Physiol Biochem. 2016;40:443–452. doi: 10.1159/000452559. [DOI] [PubMed] [Google Scholar]
  • 22.Yin X, Liang Z, Yun Y, Pei L. Intravenous transplantation of BMP2-transduced endothelial progenitor cells attenuates lipopolysaccharide-induced acute lung injury in rats. Cell Physiol Biochem. 2015;35:2149–2158. doi: 10.1159/000374020. [DOI] [PubMed] [Google Scholar]
  • 23.Uhlig S, Yang Y, Waade J, Wittenberg C, Babendreyer A, Kuebler WM. Differential regulation of lung endothelial permeability in vitro and in situ. Cell Physiol Biochem. 2014;34:1–19. doi: 10.1159/000362980. [DOI] [PubMed] [Google Scholar]
  • 24.Kim JH, Jeong JH, Jeon ST, Kim H, Ock J, Suk K, Kim SI, Song KS, Lee WH. Decursin inhibits induction of inflammatory mediators by blocking nuclear factor-kappaB activation in macrophages. Mol Pharmacol. 2006;69:1783–1790. doi: 10.1124/mol.105.021048. [DOI] [PubMed] [Google Scholar]
  • 25.Zhang M, Pan H, Xu Y, Wang X, Qiu Z, Jiang L. Allicin decreases lipopolysaccharide-induced oxidative stress and inflammation in human umbilical vein endothelial cells through suppression of mitochondrial dysfunction and activation of Nrf2. Cell Physiol Biochem. 2017;41:2255–2267. doi: 10.1159/000475640. [DOI] [PubMed] [Google Scholar]
  • 26.Hou X, Yang S, Yin J. Blocking the REDD1/TXNIP axis ameliorates LPS-induced vascular endothelial cell injury through repressing oxidative stress and apoptosis. Am J Physiol Cell Physiol. 2019;316:C104–C110. doi: 10.1152/ajpcell.00313.2018. [DOI] [PubMed] [Google Scholar]
  • 27.Makó V, Czúcz J, Weiszhár Z, Herczenik E, Matkó J, Prohászka Z, Cervenak L. Proinflammatory activation pattern of human umbilical vein endothelial cells induced by IL-1β, TNF-α, and LPS. Cytometry A. 2010;77:962–970. doi: 10.1002/cyto.a.20952. [DOI] [PubMed] [Google Scholar]
  • 28.Mangoni AA, Zinellu A, Sotgia S, Carru C, Piga M, Erre GL. Protective effects of methotrexate against proatherosclerotic cytokines: A review of the evidence. Mediators Inflamm. 2017;2017:9632846. doi: 10.1155/2017/9632846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Khan R, Rheaume E, Tardif JC. Examining the role of and treatment directed at IL-1β in atherosclerosis. Curr Atheroscler Rep. 2018;20:53. doi: 10.1007/s11883-018-0754-6. [DOI] [PubMed] [Google Scholar]
  • 30.Kopp F, Mendell JT. Functional classification and experimental dissection of long noncoding RNAs. Cell. 2018;172:393–407. doi: 10.1016/j.cell.2018.01.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Marques RE, Guabiraba R, Russo RC, Teixeira MM. Targeting CCL5 in inflammation. Expert Opin Ther Targets. 2013;17:1439–1460. doi: 10.1517/14728222.2013.837886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Nosalski R, Guzik TJ. Perivascular adipose tissue inflammation in vascular disease. Br J Pharmacol. 2017;174:3496–3513. doi: 10.1111/bph.13705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Gao Y, Zhang J, Li G, Xu H, Yi Y, Wu Q, Song M, Bee YM, Huang L, Tan M, et al. Protection of vascular endothelial cells from high glucose-induced cytotoxicity by emodin. Biochem Pharmacol. 2015;94:39–45. doi: 10.1016/j.bcp.2015.01.006. [DOI] [PubMed] [Google Scholar]
  • 34.Sachdev U, Lotze MT. Perpetual change: Autophagy, the endothelium, and response to vascular injury. J Leukoc Biol. 2017;102:221–235. doi: 10.1189/jlb.3RU1116-484RR. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Cai W, Duan XM, Liu Y, Yu J, Tang YL, Liu ZL, Jiang S, Zhang CP, Liu JY, Xu JX. Uric acid induces endothelial dysfunction by activating the HMGB1/RAGE signaling pathway. Biomed Res Int. 2017;2017:4391920. doi: 10.1155/2017/4391920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Tang ST, Wang F, Shao M, Wang Y, Zhu HQ. MicroRNA-126 suppresses inflammation in endothelial cells under hyperglycemic condition by targeting HMGB1. Vascul Pharmacol. 2017;88:48–55. doi: 10.1016/j.vph.2016.12.002. [DOI] [PubMed] [Google Scholar]
  • 37.Kim WH, Lee JW, Suh YH, Lee HJ, Lee SH, Oh YK, Gao B, Jung MH. AICAR potentiates ROS production induced by chronic high glucose: Roles of AMPK in pancreatic beta-cell apoptosis. Cell Signal. 2007;19:791–805. doi: 10.1016/j.cellsig.2006.10.004. [DOI] [PubMed] [Google Scholar]
  • 38.Bijland S, Mancini SJ, Salt IP. Role of AMP-activated protein kinase in adipose tissue metabolism and inflammation. Clin Sci (Lond) 2013;124:491–507. doi: 10.1042/CS20120536. [DOI] [PubMed] [Google Scholar]
  • 39.Ewart MA, Kennedy S. AMPK and vasculoprotection. Pharmacol Ther. 2011;131:242–253. doi: 10.1016/j.pharmthera.2010.11.002. [DOI] [PubMed] [Google Scholar]
  • 40.Hu R, Wang MQ, Ni SH, Wang M, Liu LY, You HY, Wu XH, Wang YJ, Lu L, Wei LB. Salidroside ameliorates endothelial inflammation and oxidative stress by regulating the AMPK/NF-κB/NLRP3 signaling pathway in AGEs-induced HUVECs. Eur J Pharmacol. 2020;867:172797. doi: 10.1016/j.ejphar.2019.172797. [DOI] [PubMed] [Google Scholar]
  • 41.Dennhardt S, Finke KR, Huwiler A, Coldewey SM. Sphingosine-1-phosphate promotes barrier-stabilizing effects in human microvascular endothelial cells via AMPK-dependent mechanisms. Biochim Biophys Acta Mol Basis Dis. 2019;1865:774–781. doi: 10.1016/j.bbadis.2018.12.022. [DOI] [PubMed] [Google Scholar]
  • 42.Gupta SC, Awasthee N, Rai V, Chava S, Gunda V, Challagundla KB. Long non-coding RNAs and nuclear factor-κB crosstalk in cancer and other human diseases. Biochim Biophys Acta Rev Cancer. 2020;1873:188316. doi: 10.1016/j.bbcan.2019.188316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Castellanos-Rubio A, Kratchmarov R, Sebastian M, Garcia-Etxebarria K, Garcia L, Irastorza I, Ghosh S. Cytoplasmic form of carlr lncRNA facilitates inflammatory gene expression upon NF-κB activation. J Immunol. 2017;199:581–588. doi: 10.4049/jimmunol.1700023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Özeş AR, Miller DF, Özeş ON, Fang F, Liu Y, Matei D, Huang T, Nephew KP. NF-κB-HOTAIR axis links DNA damage response, chemoresistance and cellular senescence in ovarian cancer. Oncogene. 2016;35:5350–5361. doi: 10.1038/onc.2016.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Chen S, Thorne RF, Zhang XD, Wu M, Liu L. Non-coding RNAs, guardians of the p53 galaxy. Semin Cancer Biol. 2020 Sep 11; doi: 10.1016/j.semcancer.2020.09.002. (Epub ahead of print). doi: 10.1016/j.semcancer.2020.09.002. [DOI] [PubMed] [Google Scholar]
  • 46.Sun CC, Zhu W, Li SJ, Hu W, Zhang J, Zhuo Y, Zhang H, Wang J, Zhang Y, Huang SX, et al. FOXC1-mediated LINC00301 facilitates tumor progression and triggers an immune-suppressing microenvironment in non-small cell lung cancer by regulating the HIF1α pathway. Genome Med. 2020;12:77. doi: 10.1186/s13073-020-00773-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Jung TW, Park HS, Choi GH, Kim D, Ahn SH, Kim DS, Lee T, Jeong JH. Maresin 1 attenuates pro-inflammatory reactions and ER stress in HUVECs via PPARα-mediated pathway. Mol Cell Biochem. 2018;448:335–347. doi: 10.1007/s11010-018-3392-y. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Data
Supplementary_Data1.pdf (1.1MB, pdf)
Supporting Data
Supplementary_Data2.xlsx (12.3KB, xlsx)
Supporting Data
Supplementary_Data3.xlsx (28.6KB, xlsx)
Supporting Data
Supplementary_Data4.xlsx (73.3KB, xlsx)
Supporting Data
Supplementary_Data5.xlsx (20KB, xlsx)

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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