Dataset Information

MicroRNA-146a-5p alleviates lipopolysaccharide-induced NLRP3 inflammasome injury and pro-inflammatory cytokine production via the regulation of TRAF6 and IRAK1 in human umbilical vein endothelial cells (HUVECs).

ABSTRACT:

Background

Microribonucleic acids (miRNAs) have an evident role in regulating endothelial inflammation and dysfunction, which characterizes the early stages of atherosclerosis. The NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome has been reported to contribute to the endothelial inflammatory response that promotes atherosclerosis development and progression. This study sought to investigate the effects of miR-146a-5p on lipopolysaccharide (LPS)-induced NLRP3 inflammasome injury and pro-inflammatory cytokine production in human umbilical vein endothelial cells (HUVECs).

Methods

HUVECs were transfected with a miR-146a-5p mimic, small-interfering RNA (siRNA) (si-TRAF6, and si-IRAK1), and were then stimulated with LPS for 24 h. The messenger (mRNA) and the protein levels of p-NF-κB/NF-κB, NLRP3, Caspase-1, pro-inflammatory cytokine [interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α)] in the HUVECs were analyzed by quantitative real-time polymerase chain reactions (PCRs) and western blot assays, respectively. The secretion of IL-6 from the cells was detected by enzyme-linked immunoassay (ELISA). Bioinformatic and dual-luciferase reporter assays were performed to identify the targets of miR-146a-5p.

Results

LPS promoted pro-inflammatory cytokine expression in a dose-dependent manner and significantly increased the expression levels of p-NF-κB/NF-κB p65, NLRP3, and Caspase-1. After transfection with a miR-146a-5p mimic, or si-TRAF6 or si-IRAK1, we observed that the mRNA and protein levels of NF-κB/p-NF-κB, NLRP3, Caspase-1, and pro-inflammatory cytokine in the HUVECs were all down-regulated, and the secretion of IL-6 from cells declined significantly. After transfection with a miR-146-5p mimic, the expression of TRAF6 and IRAK1 in HUVECs were both down-regulated. Dual-luciferase reporter assays confirmed that miR-146-5p directly targets the 3'-untranslated region (3'-UTR) of TRAF6 and IRAK1 to regulate their expression.

Conclusions

As a modulator of TRAF6 and IRAK1, miR-146a-5p negatively regulated LPS-induced NF-κB activation and the NLRP3 inflammasome signaling pathway in HUVECs. Thus, miRNA-146a-5p may serve as a potential therapeutic target for atherosclerosis.

SUBMITTER: Hou J

PROVIDER: S-EPMC8506750 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Publications

MicroRNA-146a-5p alleviates lipopolysaccharide-induced NLRP3 inflammasome injury and pro-inflammatory cytokine production via the regulation of TRAF6 and IRAK1 in human umbilical vein endothelial cells (HUVECs).

Hou Jingyuan J Deng Qiaoting Q Deng Xunwei X Zhong Wei W Liu Sudong S Zhong Zhixiong Z

Annals of translational medicine 20210901 18

<h4>Background</h4>Microribonucleic acids (miRNAs) have an evident role in regulating endothelial inflammation and dysfunction, which characterizes the early stages of atherosclerosis. The NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome has been reported to contribute to the endothelial inflammatory response that promotes atherosclerosis development and progression. This study sought to investigate the effects of miR-146a-5p on lipopolysaccharide (LPS)-induced NLR ...[more]

PMID: 34733985

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Project description:BackgroundmicroRNA-146a-5p (miRNA-146a-5p) is a key molecule in the negative regulation pathway of TLRs and IL-1 receptor (TIR) signaling. Our recent study demonstrated that MyD88-dependent signaling pathway of TIR in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) plays a role in peripheral nerve injury-induced neuropathic pain. However, it was not clear whether and how miRNA-146a-5p regulates the TIR pathway of DRG and SDH in the development of neuropathic pain.MethodsThe sciatic nerve chronic constriction injury (CCI) model of rat was used to induce chronic neuropathic pain. The levels and cellular distribution of miRNA-146a-5p were detected with quantitative real-time PCR (qPCR) and fluorescent in situ hybridization (FISH). The RNA level, protein level, and cellular distribution of IRAK1 and TRAF6 that is targeted by miRNA-146a-5p were detected with qPCR, western blot, and immunofluorescent. The pain-related behavioral effect of miRNA-146a-5p was accessed after intrathecal administration. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia.ResultsWe found that the level of miRNA-146a-5p significantly increased in L4-L6 DRGs and SDH after CCI surgery; meanwhile, the protein level of IRAK1 and TRAF6 in DRGs was significantly increased after CCI. Intrathecal injection of miR146a-5p agomir or miRNA-146a-5p antagomir regulates miRNA-146a-5p level of L4-L6 DRGs and SDH. We found that intrathecal injection of miR146a-5p agomir can alleviate mechanical and thermal hyperalgesia in CCI rats and reverse the upregulation of IRAK1 and TRAF6 of L4-L6 DRGs and SDH induced by CCI. We furthermore found that intrathecal injection of miRNA-146a-5p antagomir can exacerbate the mechanical and thermal pain-related behavior of CCI rats and meanwhile increase IRAK1 and TRAF6 of L4-L6 DRGs and SDH expression even further.ConclusionsmiRNA-146a-5p of DRG and SDH can modulate the development of CCI-induced neuropathic pain through inhibition of IRAK1 and TRAF6 in the TIR signaling pathway. Hence, miRNA-146a-5p may serve as a potential therapeutic target for neuropathic pain.

Project description:Bovine endometritis is characterized by reduced milk production and high rates of infertility. Prior research has indicated that melatonin may possess anti-inflammatory and antioxidant properties that can counteract the progression of inflammatory diseases. In this research, we attempted to elucidate the protective effects of melatonin on LPS-induced endometritis. The results obtained from enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR) revealed that melatonin effectively reduced the production and release of pro-inflammatory cytokines in an LPS-induced bovine endometrial epithelial cell line (BEND cells). Furthermore, western blotting demonstrated that melatonin treatment reduced the expression levels of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-related proteins, including NLRP3, activated caspase-1, and cleaved IL-1β. Importantly, we further demonstrated that the anti-inflammatory effect of melatonin on BEND cells was related to autophagy by western blotting. Moreover, we used western blotting to detect autophagy-related proteins, MitoSOX to detect mitochondrial reactive oxygen species production (mtROS), and mitochondrial membrane potential (MMP) assay to detect mitochondrial membrane potential. The administration of melatonin demonstrated a significant enhancement in autophagy within BEND cells, leading to the effective elimination of impaired mitochondria. This process resulted in a reduction in the generation of reactive oxygen species within the mitochondria, restoration of mitochondrial membrane potential, and inhibition of the NLRP3 inflammasome activation. Moreover, in a mouse model of LPS-induced endometritis, melatonin treatment repressed the expression of pro-inflammatory cytokines by ELISA and qRT-PCR, alleviated pathological changes by hematoxylin-eosin staining (H&E), and inhibited myeloperoxidase (MPO) activity. In conclusion, our study showed that melatonin inhibited the activation of the NLRP3 inflammasome in BEND cells through autophagy, which may provide a novel therapeutic strategy for bovine endometritis.

Dataset Information

MicroRNA-146a-5p alleviates lipopolysaccharide-induced NLRP3 inflammasome injury and pro-inflammatory cytokine production via the regulation of TRAF6 and IRAK1 in human umbilical vein endothelial cells (HUVECs).

Background

Methods

Results

Conclusions

Publications

MicroRNA-146a-5p alleviates lipopolysaccharide-induced NLRP3 inflammasome injury and pro-inflammatory cytokine production via the regulation of TRAF6 and IRAK1 in human umbilical vein endothelial cells (HUVECs).

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