The miR-223-3p Regulates Pyroptosis Through NLRP3-Caspase 1-GSDMD Signal Axis in Periodontitis.

Salivary exosomes contain various components and may play important roles in oral diseases. The purpose of this study was to verify the possible function of miR-223-3p from salivary exosomes in periodontitis. We isolated the salivary exosomes and found that the miR-223-3p content of salivary exosomes from periodontitis was less than the healthy control. Furthermore, we performed dual-luciferase reporter assay and real-time PCR to verify that (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) was the target of miR-223-3p. When we knocked down the miR-223-3p expression in THP-1-derived macrophages, the expression of NLRP3 and the downstream inflammatory mediators interleukin-1β (IL-1β) and IL-6 were upregulated. By using integrated bioinformatics analysis, we found that pyroptosis and cytokine secretion participated in inflammatory gingival tissues. In addition, NLRP3, and the pyroptosis executioner, gasdermin D (GSDMD) was highly active in inflammatory gingival tissues compared with healthy controls by western blotting and immunohistochemistry. In summary, we speculated that miR-223-3p in salivary exosomes might regulate GSDMD-mediated pyroptosis by targeting NLRP3 in periodontitis. Detection of miR-223-3p expression in salivary exosomes could be used as an important non-invasive method to diagnose and evaluate the severity of periodontitis.

Xia Y ，Zhou K ，Sun M ，Shu R ，Qian J ，Xie Y ... -  《-》

MiR-139 protects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced nerve injury through targeting c-Jun to inhibit NLRP3 inflammasome activation.

Cerebral ischemia/reperfusion (I/R) injury after ischemic stroke is usually accompanied with the activation of inflammasome which seriously impairs neurological function. MiR-139 has been reported to be associated with inflammatory regulation in multiple diseases. However, its effect and mechanism on inflammation regulation after cerebral I/R injury are still poorly understood. An in vitro model of cerebral I/R injury was constructed with oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. TargetScan bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the targeted relationship between miR-139 and c-Jun. Cell pyroptosis was verified by flow cytometry and Caspase-1 Detection Kit. qRT-PCR assay was performed to detect the expression levels of miR-139, c-Jun, NLRP3 and ASC. Western blotting was applied to measure the protein levels of c-Jun and pyroptosis-related markers NLRP3, ASC, caspase-1, GSDMDNterm. The ELISA assay was applied to measure the release of IL-1β, IL-18 and LDH. MiR-139 was significantly downregulated whereas c-Jun was obviously upregulated after OGD/R treatment. TargetScan analysis predicted that c-Jun was a potential target of miR-139, which was verified by the dual-luciferase reporter assay. Also, overexpression of miR-139 repressed c-Jun expression. Furthermore, miR-139 inhibited OGD/R-induced cell pyroptosis and the upregulation of NLRP3, caspase-1, ASC, GSDMDNterm, and the release of IL-1β, IL-18 and LDH, while miR-139 inhibition exerted the opposite effects. However, overexpression of c-Jun aggravated OGD/R-induced nerve injury and partly abolished the neuroprotective effect of miR-139. Upregulation of miR-139 exerted neuroprotection against OGD/R-induced nerve injury by negatively regulating c-Jun/NLRP3 inflammasome signaling. This study offered insights for providing potential therapeutic targets for treating cerebral I/R injury.

Wang QS ，Luo XY ，Fu H ，Luo Q ，Wang MQ ，Zou DY ... -  《-》

Suppression of the caspase-1/GSDMD-mediated pyroptotic signaling pathway through dexamethasone alleviates corneal alkali injuries.

The pathological mechanism of corneal injuries mediated by alkali burns are associated with Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 protein (NLRP3)-related corneal sterile inflammation. Whether the executive protein gasdermin D (GSDMD) of pyroptosis mediated by the NLRP3 inflammasome is present in alkali-induced corneal lesions remains unclear. Dexamethasone (Dex) is a commonly used drug for ocular surface diseases that can maintain corneal transparency and anti-inflammatory effects by topical administration. Here, we presented evidence that the effect of Dex on the pyroptosis-related caspase-1/GSDMD pathway in corneal alkali burns (CABs). We assessed the clinical manifestations and histological characteristics of the placebo group, 0.05% Dex group, 0.1% Dex group on day 3 or day 7 postburn and the control group (healthy corneas). The expression of factors (including NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N termini, pro-interleukin(IL)-1β, IL-1β, pro-IL-18 and IL-18) involved in the pyroptosis related caspase-1/GSDMD signaling pathway was demonstrated by molecular experiments in CAB. Alkali burns can upregulate the originally relatively dim expression of NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, pro-IL-1β, pro-IL-18, IL-1β and IL-18 in the healthy corneal epithelium and stroma. However, Dex can reverse the enhanced expression at the two timepoints. Corneal sterile inflammation can activate the NLRP3 inflammasome through the innate immune response mechanism and then activate the pyroptosis-related caspase-1/GSDMD signaling pathway. In addition, Dex can inhibit pyroptosis through this pathway.

Tan Y ，Zhang M ，Pan Y ，Feng H ，Xie L ... -  《-》

MiR-223-3p inhibits inflammation and pyroptosis in monosodium urate-induced rats and fibroblast-like synoviocytes by targeting NLRP3.

Down-regulated miR-223-3p was found in rheumatoid arthritis. This study aimed to further explore the level and role of miR-223-3p in gout arthritis (GA). After monosodium urate (MSU)-induced GA rat and fibroblast-like synoviocytes (FLSs) models were established, the rat paw volume and gait score were documented and the FLSs were transfected with miR-223-3p mimic/inhibitor or NLR family pyrin domain containing 3 (NLRP3) over-expression plasmids. The MiR-223-3p target was found through bioinformatics and the dual-luciferase reporter. The rat joint pathological damage was observed by hematoxylin and eosin staining. The levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and articular elastase in rats were detected by enzyme-linked immunosorbent assay (ELISA). The viability and pyroptosis of FLSs were detected by methyl thiazolyl tetrazolium (MTT) and flow cytometry. The expressions of miR-223-3p, NLRP3, cleaved caspase-1, IL-1β, apoptosis-associated speck-like protein (AS) and cleaved N-terminal gasdermin D (GSDMD) in FLSs or rat synovial tissues were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, Western blot or immunohistochemistry analysis. MSU increased the paw volume, gait score, inflammation in synovial tissues and increased the levels of IL-1β, TNF-α and articular elastase in rats. MSU decreased the viability and increased the pyroptosis of FLSs, up-regulated the expression of NLRP3, ASC, cleaved caspase-1, cleaved N-terminal GSDM, and IL-1β, and down-regulated miR-223-3p expression in synovial tissues of rat joints and FLSs. MiR-223-3p mimic reversed the effect of MSU on lowering cell viability, increasing pyroptosis in FLSs, while miR-223-3p inhibitor further enhanced the effect of MSU on FLSs. NLRP3 was a target of miR-223-3p. Also, NLRP3 over-expression reversed the effects of miR-223-3p on MSU-induced FLSs. MiR-223-3p inhibited pyroptosis in MSU-induced rats and FLSs by targeting NLRP3.

Tian J ，Zhou D ，Xiang L ，Liu X ，Zhang H ，Wang B ，Xie B ... -  《-》

MiR-223-3p attenuates radiation-induced inflammatory response and inhibits the activation of NLRP3 inflammasome in macrophages.

Macrophage pyroptosis plays an important role in the development of radiation-induced cell and tissue damage, leading to acute lung injury. However, the underlying mechanisms of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3)-mediated macrophage pyroptosis and the regulatory factors involved in radiation-induced pyroptosis are unclear. In this study, the expression of the NLRP3 inflammasome and pyroptosis-associated factors in murine macrophage cell lines was investigated after ionizing radiation. High-throughput RNA sequencing was performed to identify and characterize miRNAs and mRNA transcripts associated with NLRP3-mediated cell death. Our results demonstrated that cleaved-caspase-1 (p10) and N-terminal domain of gasdermin-D (GSDMD-N) were upregulated, and the number of NLRP3 inflammasomes and pyroptotic cells increased in murine macrophage cell lines after irradiation (8 Gy). Comparativeprofiling of 300miRNAs revealed that 41 miRNAsexhibited significantly different expression after 8 Gy of irradiation. Granulocyte-specific microRNA-223-3p (miR-223-3p) is a negative regulator of NLRP3. In vitro experiments revealed that the expression of miR-223-3p was significantly altered by irradiation. Moreover, miR-223-3p decreased the expression of NLRP3 and proinflammatory factors, resulting in reduced pyroptosis in irradiated murine macrophages. Subsequently, in vivo experiments revealed the efficacy of miR-223-3p supplementation in ameliorating alveolar macrophage (AM) pyroptosis, attenuating the infiltration of inflammatory monocytes, and significantly alleviating the severity of acute radiation-induced lung injury (ARILI). Our findings suggest that the miR-223-3p/NLRP3/caspase-1 axis is involved in radiation-induced AM pyroptosis and ARILI.

Zhang M ，Lan H ，Peng S ，Zhou W ，Wang X ，Jiang M ，Hong J ，Zhang Q ... -  《-》