Introduction
Atherosclerosis is one of the most common chronic progressive inflammatory processes affecting large and medium-sized arteries [1]. Epidemiological studies have revealed that cardiovascular diseases (CVDs) caused by atherosclerosis account for ca.50% of deaths worldwide. Despite significant advances in pharmacological treatments of atherosclerosis in recent decades, CVDs continue to pose a worldwide health threat due to their ever-increasing morbidity and mortality [2]. As a result, finding efficacious molecular targets may provide a new research direction in order to understand the pathogenesis of atherosclerosis and help predict, prevent, and treat atherosclerosis.
A variety of theories have been proposed to explain the pathogenesis of atherosclerosis, including the hyperlipidemia theory [3], the lipid peroxidation theory [4], the thrombosis theory [5], the senescence-related theory [6], and the inflammatory response theory [7]. Of these, the inflammatory response theory has been the most widely researched and is internationally recognised as being closely related to the development of atherosclerosis [8, 9]. However, the mechanism for regulating inflammatory responses, and preventing or reversing atherosclerosis, remains unknown.
Pyroptosis is a type of pro-inflammatory programmed cell death characterised by an intense inflammatory response and rapid plasma membrane rupture [10]. Pyroptosis has received much attention in recent years because of its role in several biological processes, including atherosclerosis [11]. Recent studies have indicated that pyroptosis in endothelial cells (ECs) [12], vascular smooth muscle cells (VSMCs), and macrophages [13] could promote the development of atherosclerosis and lead to instability in the atherosclerotic lesion. However, the molecular mechanisms of how pyroptosis is involved in atherosclerosis are still not fully understood.
Long non-coding RNAs (lncRNAs), a class of RNAs with transcripts longer than 200 nucleotides but no protein-coding capacities [14], have been shown to play critical roles in the regulation of gene expression through gene imprinting [15], chromatin remodelling [16], cell cycle regulation [17], regulation of pre-mRNA degradation [18], and transcriptional control [19]. Recently, a growing number of lncRNAs have been identified as being involved in the development of numerous diseases, such as CVDs and several cancers [20, 21]. lncRNAs ANRIL, CDKN2B-AS1, RNCR3, APPAT, H19, and FA2H-2, in particular, have been shown to influence the occurrence and progression of atherosclerosis by regulating vascular function, cholesterol efflux, cell proliferation, survival and migration, apoptosis, autophagy, and many more signal pathways [22–27].
Clarifying the biological function of lncRNAs would provide fresh insight into exploiting new targets for atherosclerosis treatment and prevention. The lncRNA NEXN-AS1 is a newly discovered non-coding RNA with low expression in atherosclerotic plaques [28]. Our previous study showed that the lncRNA NEXN-AS1 exerted anti-atherosclerotic effects by downregulating the expression of adhesion molecules and inflammatory cytokines and inhibiting monocyte adhesion to endothelial cells [28].
Atherosclerosis, pyroptosis, and lncRNA NEXN-AS1 are all closely related to inflammation in different ways. However, the precise inter-relationships between atherosclerosis, pyroptosis, and lncRNA NEXN-AS1 in the inflammatory response process are still unclear. We recently discovered that lncRNA NEXN-AS1 could mediate the inhibition effects of atorvastatin on pyroptosis [29]. These findings suggested potential links between pyroptosis and lncRNA NEXN-AS1 that needed to be further clarified.
NEXN is a cognate gene of NEXN-AS1 that encodes the F-actin protein NEXN (also known as nexilin or nelin) found at cell junctions. NEXN has been reported to have an anti-atherosclerotic effect [28]. Furthermore, studies have also revealed that mutations in the NEXN gene can cause cardiomyopathy [30], and it is a susceptible gene for cardiovascular disease (CVD) in the Chinese Han population [31]. Although the above data established NEXN as a protective gene in the development of CVD, the mechanisms by which NEXN influences atherosclerosis require further investigation.
In this study, we found that lncRNA NEXN-AS1 could inhibit key molecules of the pyroptosis canonical inflammasome pathway in human umbilical vein endothelial cells (HUVECs) and VSMCs by regulating its adjacent gene NEXN to exert its anti-inflammatory and anti-atherosclerotic effects.
MATERIALS AND METHODS
Materials
We purchased a reverse transcription kit (PrimeScript RT Reagent Kit, DRR037A) and a real-time polymerase chain reaction kit (SYBR® Premix Ex Taq™ II, DRR820A) from TaKaRa Bio, Inc. (Shiga, Japan). We also obtained a transfection reagent (Lipofectamine 3000) from Invitrogen (Carlsbad, CA, USA) and Optimised medium (Opti-MEM) from Gibco BRL (Paisley, UK). All other chemicals used in this study were of pharmaceutical grade and purchased from commercial suppliers.
Cells and culture parameters
Human umbilical vein endothelial cells (HUVECs) and human vascular smooth muscle cells (VSMCs) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% foetal bovine serum (NLRP3, Gaithersburg, MD, USA) and antibiotics (penicillin, 100 U/mL; streptomycin, 100 mg/mL) at 37°C in a humidified incubator containing 5% CO2.
Construction of lentiviral vectors
The lentivirus-mediated overexpression vector of lncRNA NEXN-AS1 was constructed using previously reported methods [28]. In summary, the total RNA was extracted from the cultured cells using a TRIzol reagent (Invitrogen). First- -strand complementary DNA (cDNA) of lncRNA NEXN-AS1 was synthesised using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and a PrimeScript RT reagent kit (TaKaRa). NEXN-AS1 cDNA was amplified using PCR and inserted into the pLKD-CMV-EGFP-2A-Puro-U6-shRNA vector as described earlier [28]. The proper sequence of the NEXN-AS1 gene in this construct was then verified by sequencing. This construct (henceforth referred to as LV-NEXN-AS1) and the control vector (Obio Technology Corp. Ltd., Shanghai, China) were used to transfect both cultured HUVECs (CRL-1730, ATCC) and VSMCs (CRL-1999, ATCC) at an infection multiplicity of 20 transfection units per cell in the presence of 8 mg/mL of polybrene. After 24 hours, the cells were rinsed with fresh complete medium. We used quantitative RT-PCR to confirm that the NEXN-AS1 overexpression was effective [28].
RNA interference using small interfering RNA (siRNA) mimics in transfection
The siRNA against NEXN (siNEXN) and a non-relevant 21-nucleotide control siRNA (negative control, siNC) were procured from Ribo Biotechnology (Guangzhou, China). The sequences of the siRNAs are set out in Table 1. HUVECs (2×106 cells/well) were seeded into the wells of 6-well plates (2 × 106 cells/well). After achieving 80% confluence, the cells were cultured at room temperature for 20 min in Opti-MEM medium (Gibco BRL, Big Island, NY, USA) containing 5 µL of Lipofectamine 3000 reagent (Invitrogen) and 10 µL of 50 nM siRNA (Ribo) or siNC. Subsequently, the siRNA combination (siNC, control/single LV-NEXN-AS1 group; siNEXN, single siNEXN/LV-NEXN-AS1 and siNEXN group) was carefully and gradually added to each well. qRT-PCR and Western blot analyses were conducted after 24 h of treatment.
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Table 1. Oligonucleotide sequences used in this study |
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|
Gene |
Sequence |
Gene |
Sequence |
|
NEXN-AS-RT |
5'-GGGAGCAATAGACAGATGAAG-3' |
Caspase-1-F |
5'-AGTGCAGGACAACCCAGCTATG-3' |
|
NEXN-AS1-F |
5'-AAGGAATGAGGCTGAAATGG-3' |
Caspase-1R |
5'-CAAGACGTGTGCGGCTTGA-3' |
|
NEXN-AS1-R |
5'-AGGAAAACTTGGCCAAAGGT-3' |
GSDMD-F |
5'-GGGGACTGCTGGGATGAAAA-3' |
|
U6-RT |
5'-AACGCTTCACGAATTTGCGT-3v |
GSDMD-R |
5'-GGAACAGGGAGGCATAGAGC-3' |
|
U6-F |
5'-CTCGCTTCGGCAGCACA-3v |
IL-1β-F |
5'-TCGCCAGTGAAATGATGGCTTA-3' |
|
U6-R |
5'-AACGCTTCACGAATTTGCGT-3v |
IL-1β-R |
5'-GTCCATGGCCACAACAACTGA-3' |
|
NEXN-F |
5'-ACTGTGAAGGGTAGATTTGCTG-3' |
IL-18-F |
5'-GACCTTCCAGATCGCTTCCTC-3' |
|
NEXN-R |
5'-TCTGCGTTTTCGTTCCTCCT-3' |
IL-18-R |
5'-GATGCAATTGTCTTCTACTGGTTC-3' |
|
NLRP3-F |
5'-AAAGCCAAGAATCCACAGTGTAAC-3' |
GADPH-F |
5'-GCACCGTCAAGGCTGAGAAC-3' |
|
NLRP3-R |
5'-TTGCCTCGCAGGTAAAGGT-3' |
GADPH-R |
5'-TGGTGAAGACGCCAGTGGA-3' |
|
si-h-NEXN |
5'-GGAGATGATTCACTACTTA-3' |
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RNA isolation and qRT-PCR analysis
The total RNA was extracted from the cultured cells using the TRIzol reagent (Invitrogen). The first-strand complementary DNA (cDNA) of lncRNA NEXN-AS1 and mRNAs of NEXN, NLRP3, caspase-1, IL-1β and IL-18 were synthesised using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and the PrimeScript RT reagent kit (TakaRa), respectively. Both reactions contained 2 µg of total RNA as template strands in a reaction volume of 20 µL. qRT-PCR was performed to measure the expression levels of lncRNA NEXN-AS1, with U6 as an endogenous control and mRNAs with GAPDH as an endogenous control, using a SYBR Green Master Mix (TaKaRa) and a LightCycler 480 Fast Real-Time PCR system (Roche Molecular Systems, Inc., Pleasanton, CA, USA). The fold-differences in expression levels were calculated using the 2-ΔΔCt method. All experimental procedures were carried out following the manufacturer’s instructions. Each sample was measured in triplicate, and the mean values were used for comparative analysis. The primer sequences are listed in Table 1, and all primers for qRT-PCR were synthesised by Sangon Biotech (Shanghai, China). The qRT-PCR cycling protocol consisted of an initial preincubation step at 95°C for 20 seconds, followed by 40 cycles at 95°C for 10 seconds, 60°C for 20 seconds, and a final extension step at 70°C for 1 second.
Western blot analysis
The method we used for harvesting the cells and preparing protein extracts in this study was as described in [32]. Protein extraction from the cultured cells was performed using a radio immunoprecipitation assay buffer (Biocolor Ltd., Belfast, UK), quantified with a BCA protein assay kit (KeyGen Biotechnologies, Nanjing, China), and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a concentration of 12%. The separated proteins were then transferred to cellulose acetate membranes and incubated overnight at 4°C with the following antibodies (all at 1:1,000 dilution): mouse polyclonal anti-nexilin antibodies (ab213628, Abcam, Cambridge, UK); rabbit recombinant monoclonal anti-GSDMD antibody (ab210070, Abcam); rabbit polyclonal anti-NLRP3 (A5652, ABclonal, Wuhan, China); caspase-1 (2225, Cell Signaling Technology, Beverly, MA, USA), -IL-1β (sc-12742, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and -IL-18 (A16288, ABclonal), as well as mouse polyclonal anti-β-tubulin-specific antibodies (diluted 1:2,000; ab6046, Abcam) as an internal control. After being conjugated with primary antibodies, the membranes were incubated with goat anti-rabbit or goat anti-mouse secondary antibodies (diluted 1:5,000; Bioss Antibodies, Beijing, China). The proteins were visualised using the chemiluminescence detection method (ECL Plus Western Blot Detection System, GE Healthcare, Piscataway, NJ, USA) and analysed using Image J software (free web version, https://www.imagej.net/).
IL-18 concentration in cell supernatants
The concentration of IL-18 in HUVECs and VSMCs supernatants after treatment with LV-NEXN-AS1 or siNEXN was measured using a commercially available specific enzyme-linked immunosorbent assay (ELISA) kit (RK00001, ABclonal), as per the manufacturer’s instructions. The optical density (OD) value was measured using an ELISA plate reader (R&D Systems, Minneapolis, MN, USA) at a wavelength of 450 nm. The standard substances and sample dilutions were added to the ELISA plate in duplicate, and the average concentration was calculated as the protein level (pg/mL) in the cell culture supernatant. The average OD values of the standards were used to create a standard curve. The results were calculated with Excel 2010 (Microsoft, Redmond, WA, USA).
Statistical analysis
Data was analysed using IBM SPSS Statistics for Windows, version 23.0 (IBM Corporation, Armonk, NY, USA) and GraphPad software (GraphPad Software, Inc., La Jolla, CA, USA). Unless otherwise indicated, data was presented as mean ± standard deviation or median (interquartile range). The results of the RT-PCR and Western blot analyses were assessed with a Student’s t-test for two-group comparisons and one-way analysis of variance followed by a Student-Newman-Keuls test for comparison of more than two groups. A two-tailed probability (P) value of < 0.05 was considered statistically significant.
RESULTS
Overexpression of lncRNA NEXN-AS1 increased expression of NEXN in HUVECs and VSMCs
Our bioinformatics analysis showed that lncRNA NEXN-AS1 and NEXN were situated nearby on chromosome 1 and had opposite transcription directions. Previously, we found that lncRNA NEXN-AS1 could regulate the expression of its cognate gene NEXN in HUVECs and murine animal models [28]. In this study, we further explored the effects of lncRNA NEXN-AS1 on NEXN in both HUVECs and VSMCs. The NEXN-AS1 gene was transferred into HUVECs and VSMCs by lentiviral vectors (LV-NEXN-AS1), with empty lentiviral vectors (LV-Mock) as negative controls. We used qRT-PCR and Western blot to measure the expression level of NEXN-AS1.
The lncRNA levels of NEXN-AS1 were enhanced 52-fold in HUVECs and 80-fold in VSMCs by LV-NEXN-AS1 transfection, and the mRNA levels of NEXN were very significantly increased, by 1,060% in HUVECs and 833% in VSMCs (Figs. 1A, B). Western blot analysis showed that NEXN protein levels were also significantly upregulated, by 2.99-fold in HUVECs and 4.32-fold in VSMCs, compared to the control group (Figs. 1C, D). These results suggest that overexpression of NEXN-AS1 could increase the expression of NEXN in HUVECs and VSMCs at both mRNA and protein levels.
Overexpression of lncRNA NEXN-AS1 inhibited pyroptosis via canonical inflammasome pathway
Pyroptosis was recently identified as a new type of cell death accompanied by the activation of many cytokines, including inflammatory cytokines.
We hypothesised that because inflammatory cytokines were found to be regulated by lncRNA NEXN-AS1 in our previous research [28], there might be some relationship between lncRNA NEXN-AS1 and pyroptosis with the same inflammatory response features. Therefore, in this study we investigated the effects of lncRNA NEXN-AS1 on pyroptosis.
Firstly, we transfected HUVECs and VSMCs with LV-NEXN-AS1 to overexpress lncRNA NEXN-AS1. Next, we used qRT-PCR and Western blot analyses to assess the expression levels of the canonical inflammasome pathway molecules of pyroptosis (NLRP3, caspase-1, GSDMD, IL-1β, and IL-18). The supernatant of both cell types was collected to determine IL-18 concentration using an ELISA kit. The qRT-PCR results showed that the overexpression of lncRNA NEXN-AS1 significantly downregulated mRNA levels of canonical inflammasome pathway molecules (NLRP3, caspase-1, GSDMD, IL-1β and IL-18) in two different cell types (Figs. 2A, B). Western blot analysis showed that the overexpression of lncRNA NEXN-AS1 decreased protein levels of the canonical inflammasome pathway proteins [NLRP3, caspase-1p45 full length, cleavage p20 fragment, cleavage IL-1β p17, IL-18 and C-terminal GSDMD (GSDMD-C)], while there were no significant differences in the expression levels of the inactive precursors of IL-1β and GSDMD full length (Figs. 2B, D, F). Notably, the expression levels of IL-18 detected by ELISA were also similarly decreased in the supernatant of the abovementioned two cell types (Figs. 2C, G).
The results of this study demonstrate that the overexpression of lncRNA NEXN-AS1 in HUVECs and VSMCs inhibits the expression levels of pyroptosis-associated canonical inflammasome pathway molecules.
Knock-down of NEXN did not affect expression of lncRNA NEXN-AS1
Because NEXN gene expression was upregulated by the overexpression of lncRNA NEXN-AS1, we investigated the effects of NEXN on lncRNA NEXN-AS1. We transfected HUVECs and VSMCs with siNEXN to explore whether NEXN affected the expression level of lncRNA NEXN-AS1. As shown in Figs. 3A and B, HUVECs and VSMCs were transfected with small interference vectors carrying NEXN (siNEXN) to downregulate the expression of NEXN with small interference control vectors (siNC) as a negative control, and then total RNA and proteins were extracted for qRT-PCR and Western blot, respectively.
The findings suggested that siRNA-induced knockdown of NEXN had no influence on NEXN-AS1 expression, but did markedly reduce the expression of NEXN at both the mRNA and protein levels compared to the siNC group (Figs. 3A–D).
Knockdown of NEXN upregulated expression of biomarkers in canonical inflammasome pathway of pyroptosis
Based on the above data, we have confirmed that the overexpression of lncRNA NEXN-AS1 could suppress the expression level of pyroptosis-related molecules. Subsequently, in order to investigate whether the function of NEXN was consistent with lncRNA NEXN-AS1 in regulating pyroptosis, we measured the expression levels of the proteins of pyroptosis canonical inflammasome pathway by knockdown of NEXN in HUVECs and VSMCs.
As shown in Figs. 4A and 4E, the mRNA levels of the inflammasome pathway molecules (NLRP3, caspase-1, GSDMD, IL-1β, and IL-18) significantly increased in the siNEXN group. Meanwhile, siRNA-induced knockdown of NEXN increased the protein expression level of NLRP3, cleavage caspase-1 p45 and p20 fragments, GSDMD-C, cleavage IL-1β p17, and IL-18, but did not change the expression level of precursors of IL-1β (p31 form) and GSDMD full length (Fig. 4B, D, and F). Similarly, the level of IL-18 was significantly increased in the supernatant of the above two cell types (Fig. 4C, G).
These results indicated that in both studied cell lines, NEXN exerted the regulatory effects of pyroptosis through the canonical inflammasome pathway, which was consistent with the effects of lncRNA NEXN-AS1.
LncRNA NEXN-AS1 regulated NEXN expression, but NEXN did not affect lncRNA NEXN-AS1 expression
We further examined lncRNA levels of NEXN-AS1 in VSMCs and HUVECs treated with both LV-NEXN-AS1 and siNEXN to discover whether lncRNA NEXN-AS1 could be regulated by NEXN. Compared to the negative control, our results showed that the experimental group treated with both LV-NEXN-AS1 and siNEXN showed significant upregulation of NEXN-AS1 at RNA levels similar to the LV-NEXN-AS1 group (Fig. 5A). However, both the mRNA (Figure 5B) and protein levels (Figs. 5C, D) of NEXN recovered to normal.
These findings showed that the upregulation effects of LV-NEXN-AS1 on NEXN expression could be neutralised by knockdown of NEXN. They also indicated that lncRNA NEXN-AS1 could regulate the expression levels of NEXN, but NEXN could not influence the expression level of lncRNA NEXN-AS1.
LncRNA NEXN-AS1 inhibited pyroptosis via NEXN
We found that lncRNA NEXN-AS1 and NEXN regulated the expression levels of the canonical inflammasome pathway molecules of pyroptosis, but whether lncRNA NEXN-AS1 inhibited pyroptosis via NEXN remains unclear. In this study, we transfected HUVECs and VSMCs with an LV-mock or an LV-NEXN-AS1 and then incubated them with siNC or siNEXN. We found that the expression of genes encoding the molecules of pyroptosis (NLRP3, caspase-1, GSDMD, IL-1β, and IL-18) was significantly suppressed by LV-NEXN-AS1, and they could be reversed at the levels of mRNA by treatment with siNEXN (Figs. 6A, E).
Consistent with the mRNA expression, Western blot analysis showed that the protein levels of the molecules of pyroptosis (NLPR3, cleavage caspase-1 p20, cleavage IL-1β p17, IL-18, and GSDMD-C) were downregulated by LV-NEXN-AS1, upregulated by siNEXN, and recovered to normal levels by the synergistic effects of LV-NEXN-AS1 and siNEXN. There were no significant differences in the mRNA (Figs. 6B, E, F) and protein (Figs. 6C, G) levels of the precursors of caspase-1, IL-1β, and GSDMD in two cell types. In addition, our results showed that after transfection with LV-NEXN-AS1, the level of IL-18 produced by HUVECs and VSMCs and secreted into the supernatant decreased, but after transfection with siNEXN, the level of IL-18 increased. Nevertheless, after co-transfection with LV-NEXN-AS1 and siNEXN, the level of IL-18 returned to the normal level (Figs. 6C, G).
All these results indicated that lncRNA NEXN-AS1 could suppress the protein levels of pyroptosis-associated molecules via the regulation of its downstream gene, NEXN.
DISCUSSION
In this study, we have found that in HUVECs and VSMCs, overexpression of lncRNA NEXN-AS1 upregulated the expression of its cognate gene NEXN and downregulated both mRNA and protein levels of pyroptosis-associated molecules in the canonical inflammation pathway (NLRP3, caspase-1, GSDMD, IL-1β, and IL-18). Knockdown of NEXN upregulated the expression of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 genes. Furthermore, the downregulation effects on NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 induced by the overexpression of lncRNA NEXN-AS1 could be reversed by knocking down NEXN.
Atherosclerosis is a chronic, progressive vascular pathology in which cell death plays a critical role. Pyroptosis is a relatively novel form of programmed cell death characterised by flattening-like rupture, the release of inflammatory factors (such as IL-1β and IL-18), and severe inflammatory reaction [34]. Pyroptosis has been implicated in the atherosclerotic process in several cell type models, including vascular wall cells such as endothelial and smooth muscle cells, and immune cells (such as T-lymphocytes and macrophages) [32–34].
During pyroptosis, a chain reaction occurs in which many molecules and cytokines, including NLRP3, inflammatory caspases (caspase-1 and -11), gasdermin D, and proinflammatory interleukins (IL-1β, IL-18) are involved in a sequence. There are two signals leading to pyroptosis, each with a different initiation mechanism, i.e. the canonical inflammasome pathway and the non-canonical inflammasome pathway. The former is usually stimulated by danger-associated molecular patterns (DAMPs), while the latter is usually stimulated by pathogen-associated molecular patterns (PAMPs) [35]. Cholesterol crystals and hyperlipidemia serve as DAMPs to induce cell pyroptosis via the canonical inflammasome pathway to promote atherosclerosis [36].
NLRP3 organising inflammasomes are recruited, oligomerised, and activated by DAMPs. NLRP3’s N-terminal pyrin segment (PYD) recruits pro-caspase 1 to the inflammasome [37]. A caspase-1 p20 or p10 fragment is then produced following caspase-1 oligomerisation and autoproteolytic cleavage. Caspase-1 that has been activated performs a catalytic function acting as cysteine-dependent protease, causing the cleavage of pro-IL-1β and pro-IL-18 into the biologically active cytokines IL-1β and IL-18, respectively [38, 39]. Caspases also cleave gasdermin D into C-terminal and N-terminal fragments (GSDMD-N), the latter of which initiates pyroptosis by forming non-selective membranes. The GSDMD N-terminal fragment is released from membrane pores when active caspase-1 cleaves GSDMD [38].
Endothelial cells are neatly arranged on the inner wall of blood vessels, forming a physical barrier to maintain blood vessel integrity. Endothelial injury has been shown to be involved in many vascular pathological processes, including thrombosis and atherosclerosis, resulting from the physical barrier destruction and altered secretory profile of ECs [40]. Activated or injured HUVECs secrete large amounts of inflammatory molecules (caspase-1, IL-1β, and IL-18) and adhesion molecules [P-selectin, intercellular adhesion molecule-1 (ICAM-1), as well as vascular cell adhesion molecule-1 (VCAM-1)] [41].
Inflammatory molecules promote the expression of NLRP3, caspase-1, and GSDMD-C by activating the inflammatory reaction, while adhesion molecules recruit monocytes and other inflammatory cells to infiltrate the endothelium and expose it to increased levels of lipids, the main characteristics of hyperlipidemia. NLRP3 and GSDMD expression levels are promoted by caspase-1 in the endothelial injury, which are all the main components of the canonical inflammasome pathway of pyroptosis [42]. They are related to the pyroptosis of HUVECs and could greatly affect the progression of atherosclerosis. Furthermore, the pyroptosis of HUVECs promotes the production of adhesion molecules, the accumulation of inflammatory factors, and the infiltration of multiple leukocytes into the intima. Consequently, these factors lead to the further deterioration of atherosclerosis. Therefore, control of pyroptosis in HUVECs may be a potential strategy to mitigate atherosclerosis, and in this respect the findings from our current study are consistent with previous research.
Our results showed that lncRNAs NEXN-AS1 and NEXN could regulate the expression of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18, and particularly activate the caspase-1 p20 fragment, GSDMD-N, and cleavage of IL-1β p17. These findings suggest that lncRNA NEXN-AS1 and NEXN could regulate cell pyroptosis through the canonical inflammasome pathway because key molecules of this pathway were all affected. As a result, lncRNA NEXN-AS1 and NEXN have for the first time been reported in this current study as molecular targets for regulating pyroptosis in HUVECs.
VSMCs are important components of the medium layer of the arterial wall that play a vital role in the progression of atherosclerosis. On the one hand, migration of VSMCs from the media to the intima of the arteries promotes atherosclerosis [43]. On the other hand, VSMCs keep atherosclerotic plaques stable by producing extracellular matrix molecules such as collagen and elastic fibres [44]. However, VSMC pyroptosis can lead to the loss of collagen fibres and trigger a severe inflammatory response, causing the fibres and plaques to become thinner and unstable. Many pro-atherosclerotic factors, such as a high concentration of oxidized low density lipoprotein cholesterin, can induce VSMC pyroptosis. Pyroptosis could further increase the levels of inflammatory factors by activating in the inflammasomes GSDMD-C, NLRP3, and caspase-1. As a result, the plaques become prone to rupture promoting thrombus formation, which can result in cardiovascular events [11].
Therefore, it is particularly important to identify targets to inhibit pyroptosis of VSMC. In our study, we are the first to find that lncRNA NEXN-AS1 inhibits the expression level of pyroptosis-related molecules in VSMCs via the regulation of its downstream gene, NEXN.
In addition, emerging research is confirming that an increasing number of lncRNAs are involved in pyroptosis. For example, lncRNA MALAT1 has been found to affect the occurrence and development of experimental diabetic nephropathy by interacting with miR-23c to inhibit the expression of closely related pyroptosis molecules such as ELAVL1, NLRP3, caspase-1, and IL-1β in HK-2 cells [45]. LncRNA LINC00339 promotes renal tubular epithelial pyroptosis by regulating the miR-22-3p/NLRP3 pathway [46], and lncRNA MEG3 mediates EC pyroptosis via the miR-223/NLRP3 pathway [47]. These results demonstrate the vital role played by some lncRNAs in regulating pyroptosis.
Consistent with the above research, we found that a previously unknown lncRNA, NEXN-AS1, involved in inflammation and atherosclerosis, could regulate pyroptosis in both HUVECs and VSMCs. Interestingly, both inflammation and pyroptosis were found to be closely related to atherosclerosis [48–50]. Therefore, precise regulation of cell pyroptosis and the release of inflammatory molecules could serve as a new direction for the treatment and prevention of atherosclerosis. Our findings have demonstrated that lncRNA NEXN-AS1 could regulate the expression levels of pyroptosis-related molecules, which should offer new insights into the therapeutic and preventive potential of lncRNA in atherosclerosis. However, we have only demonstrated the expression of pyroptosis molecule alterations. The specific processes of activation of such molecules remain to be identified.
In summary, we have found that lncRNA NEXN-AS1 inhibited pyroptosis via the canonical inflammasome pathway in HUVECs and VSMCs by regulating the expression of its adjacent gene NEXN, suggesting that lncRNAs NEXN-AS1 and NEXN could have anti-inflammatory and anti-atherosclerotic effects by regulating pyroptosis.
These findings have provided a new direction for determining the mechanisms of lncRNAs action in the regulation of atherosclerotic process, as well as new insights into the potential of lncRNAs in the prevention and treatment of atherosclerosis.
Article information and declarations
Data availability statement
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.
Ethics statement
Not applicable.
Authors’ contributions
FC: Acquisition of data, writing of manuscript, obtaining financing; YYL: acquisition, analysis and interpretation ofdata; RC: analysis and interpretation of data, statistical analysis; WJL: acquisition of data, critical revision of manuscript for intellectual content; YWW: statistical analysis, critical revision of manuscript for intellectual content; SQ: conceptualisation and design of research, critical revision of manuscript for intellectual content; LW: conceptualisation and design of research, acquisition of data, obtaining financing.
All authors read and approved the final draft.
Funding
This study was supported by: (1) the Science and Technology Programme of Guangzhou city (grant numbers: 2023A03J0492 and 202102010049); and (2) Research project of Traditional Chinese Medicine Bureau of Guangdong Province (grant number: 20231235).
Acknowledgements
We would like to acknowledge the hard and dedicated work of all the staff who implemented the intervention and evaluation components of this study.
Conflicts of interest
The authors declare that they have no competing interests.
