Elsevier

Immunobiology

Volume 226, Issue 5, September 2021, 152133
Immunobiology

Long non-coding RNA RP11-490M8.1 inhibits lipopolysaccharide-induced pyroptosis of human umbilical vein endothelial cells via the TLR4/NF-κB pathway

https://doi.org/10.1016/j.imbio.2021.152133Get rights and content

Abstract

Background and aims

Pyroptosis is a relatively newly discovered form of programmed cell death that plays an important role in the development of atherosclerosis. Many studies have reported that lncRNAs participated in the regulation of atherosclerosis development. However, the regulatory mechanism of lncRNAs in pyroptosis must be studied further.

Methods

In a previous study, microarray analysis was used to detect the lncRNA expression profile in three human advanced atherosclerotic plaques and three normal arterial intimae. In the present research, in vitro assays were performed to investigate the role of lncRNA RP11-490M8.1 on pyroptosis. The relative gene mRNA and lncRNA expression levels were tested by quantitative real-time PCR, and protein levels were evaluated by western blot analysis. The RNA hybrid structure was analyzed using the DINAMelt server.

Results

The lncRNA RP11-490M8.1 was significantly downregulated in atherosclerotic plaques and serum. Lipopolysaccharide (LPS) markedly reduced the expression of lncRNA RP11-490M8.1 and induced pyroptosis by increasing the mRNA and protein levels of NLRP3, caspase-1, ASC, IL-1β, and IL-18 in HUVECs. The promotion effects of LPS on pyroptosis were markedly suppressed by overexpression of lncRNA RP11-490M8.1. In addition, LPS increased the mRNA and protein levels of TLR4 and NF-κB, which was also markedly offset by overexpression of lncRNA RP11-490M8.1.

Conclusions

These findings indicated that lncRNA RP11-490M8.1 inhibited LPS-induced pyroptosis via the TLR4/NF-κB pathway. Thus, lncRNA RP11-490M8.1 may provide a therapeutic target to ameliorate atherosclerosis.

Introduction

Atherosclerosis is a chronic low grade inflammatory disease affecting the large- and medium-sized arteries, and is considered to be the main cause of cardiovascular disease (Ruiz-León et al., 2019). Research has shown that inflammation plays a pivotal role in atherosclerosis (Li et al., 2017, Cheng et al., 2019). Notably, the inflammatory disorder is initiated via vascular injury induced by infection, and damage to the vascular endothelium may predispose to further injury, including the formation of atherosclerotic plaques. Many factors can stimulate damage to vascular endothelial cells. For example, dead endothelial cells can release pro-inflammatory cytokines, amplifying the inflammatory response. In turn, the release of inflammatory factors can result in the aggregation and necrosis of other cells, where the accumulated cells gradually form atherosclerotic plaques with a necrotic core.

Pyroptosis, also known as inflammatory cell necrosis, is a type of programmed cell death that relies on the enzymatic activities of inflammatory proteases belonging to the cysteine-dependent aspartate-specific protease (caspase) family (Vande Walle and Lamkanfi, 2016). Activated caspase-1 recognizes and converts inactive interleukin (IL)-1β and IL-18 precursors to mature forms (Yu et al., 2019). A large number of vesicles or pyroptotic corpuscles can be seen in pyroptotic cells under the electron microscope. Subsequently, pores form on the cell membrane and the cells swell and rupture the membrane, releasing inflammatory factors and resulting in inflammation (Ge et al., 2018). Pyroptosis promotes the formation of unstable atherosclerotic plaques through important functional molecules, such as the NOD-like receptor family, pyrin domain-containing 3 (NLRP3), and caspase-1 (Yu et al., 2020). Hence, reducing pyroptosis may play a significant part in slowing down the atherosclerosis process (Nguyen et al., 2019). Although pyroptosis participates in in the development of atherosclerosis (Xu et al., 2018), its underlying mechanism remains unclear.

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs longer than 200 nucleotides. Emerging evidence suggests that lncRNAs play a critical role in many diseases, especially atherosclerosis (Wang et al., 2019, Sha et al., 2019, Li et al., 2019). For example, lncRNA NEXN-antisense RNA 1 (NEXN-AS1) mitigates atherosclerosis by regulating the actin-binding protein nexilin (Hu et al., 2019). In addition, lncRNA CDKN2B-AS1 reduces the inflammatory response and accelerates cholesterol efflux in atherosclerosis by suppressing ADAM10 (Li et al., 2019). Although thousands of lncRNAs have been described, many gaps remain in the understanding of the lncRNA mechanism in atherosclerosis. LncRNA RP11-490M8.1 is a 366-kb lncRNA located on chromosome 2p22.3. To date, there have been no reports examining the role of lncRNA RP11-490M8.1 in pyroptosis and atherosclerosis.

Human umbilical vein endothelial cells (HUVECs) are used to study a variety of diseases, including atherosclerosis and the inflammatory process (Shi et al., 2004). The HUVEC-related inflammatory response is led by various mediators, including lipopolysaccharide (LPS) (Kim et al., 2014). LPS, also known as endotoxin, is an outer membrane component of Gram-negative bacteria. LPS increases reactive oxygen species (ROS) production and upregulates cytokines in HUVECs (Liu et al., 2010), which contributes to endothelial dysfunction and may evoke atherosclerosis. Notably, it has been confirmed that LPS induced NLRP3 inflammasome-mediated cell pyroptosis and promoted pro-inflammatory cytokine (IL-1β and IL-18) generation and secretion (Zhang et al., 2021). However, it is still unclear how LPS contributes to pyroptosis.

In a previous study, microarray analysis revealed that lncRNA RP11-490M8.1 expression was downregulated in atherosclerotic plaques (Bai et al., 2019). Furthermore, it was found that LPS inhibited lncRNA RP11-490M8.1 and upregulated mRNA and protein levels of biological markers for pyroptosis (NLRP3, apoptosis-associated speck-like protein containing a CARD [ASC], caspase-1, IL-1β, and IL-18) in HUVECs. In addition, lncRNA RP11-490M8.1 inhibited LPS-induced pyroptosis via the Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway.

Section snippets

Reagents

Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Shanghai, China). Polyclonal anti-rabbit antibodies against TLR4 (catalog no. AF7017) and NF-κB (AF5006-50) were purchased from Affinity Biologicals, Inc. (Ancaster, ON, Canada); those against NLRP3 (A12694), IL-1β (A1112), caspase-1 (A16792), ASC (A11433), and IL-18 (A16737) were purchased from ABclonal Technology (Woburn, MA, USA). SYBR green detection reagents were

LPS increases pyroptosis of HUVECs

Recent studies have confirmed that LPS induced NLRP3/caspase-1 activity and pyroptosis in mouse astrocytes (Li et al., 2019). Therefore, we were interested in exploring the effects of LPS on pyroptosis in HUVECs. In this research, HUVECs with 0 ng/mL of LPS were regarded as the negative control. Test samples of HUVECs were treated with LPS at a concentration of 0, 250, 500, or 1000 ng/mL, for 0, 12, 24, or 48 h. Western blot and qRT-PCR analyses were used to measured expression of related

Discussion

Pyroptosis, a form of necrotic and inflammatory procedural cell death, depends on caspase-1 activation (Jiang et al., 2018, Li et al., 2018, Jia et al., 2019). The NLRP3 inflammasome plays an important role in pyroptosis by responding to the upstream signaling molecules via the NOD-like receptor protein and transmitting the pyroptosis signal to the downstream executive proteins via activated caspase-1. Previous studies have reported that the NLRP3 inflammasomes were triggered in cells by

Conclusions

The results of the present research demonstrated that lncRNA RP11-490M8.1 inhibited LPS-induced pyroptosis via the TLR4/NF-κB signaling axis in HUVECs, suggesting that lncRNA RP11-490M8.1 might serve as a therapeutic target for pyroptosis. However, there were several deficiencies in this study that should be pointed out. First, further studies are required to better understand the underlying mechanism of LPS-induced inhibition of lncRNA RP11-490M8.1. Second, the potential mechanism of lncRNA

Financial support

This work was supported by the National Natural Sciences Foundation of China (grant number: 81871701), the Natural Science Foundation of Guangdong (grant numbers: 2020B1515020013 and 2018A030313533), the Science and Technology Program of Guangzhou (grant number: 201707010156 and 202102080398), and Medical Science and Technology Research Fundation of Guangdong (grant numbers: A2020140)

Author contributions

Yan-Wei Hu, Shao-Guo Wu, and Xue-Hui Liu designed the study; Li-Mei Wu, Jia-Li Wang, Xian-hui Dong, Shun-Chi Zhang, Xue-Hen Li, Hui Xu, Zhi-hai Li, Zhe-Ming Liu, and Da-Bin Liu acquired the data and conducted the experiments; Shao-Guo Wu, Xue-Hui Liu, and Li-Mei Wu wrote the manuscript; Jia-Li Wang, Xue-Hen Li, and Zhi-hai Li analyzed the data and revised the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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