Puerarin inactivates NLRP3-mediated pyroptotic cell death to alleviate cerebral ischemia/reperfusion (I/R) injury through modulating the LncRNA DUXAP8/miR-223-3p axis

Zhenguo Shi,Songtie Ying,Haiyan Shi,Liang Tao,Qiaoyun Wu

doi:10.32604/biocell.2022.015345

Abstract

NLRP3 inflammasome-mediated cell pyroptosis aggravates the development of cerebral ischemia/reperfusion (I/R) injury, and the aim of this study is to investigate the potential utilization of the Chinese medicine, Puerarin, in treating this disease. Through conducting in vitro and in vivo experiments, the present study illustrated that Puerarin regulated LncRNA double homeobox A pseudogene 8 (DUXAP8)/miR-223-3p axis to inactivate NLRP3-mediated pyroptotic cell death, resulting in the attenuation of I/R injury. Specifically, the cerebral I/R injury in rat models and hypoxia/reoxygenation (H/R) in primary hippocampus neuron (PHN) cells were inducted, which were subsequently exposed to Puerarin treatment. As expected, we validated that Puerarin suppressed cell pyroptosis and rescued cell viability in I/R rat hippocampus tissues and H/R PHN cells. Next, through bioinformatics analysis, we noticed that miR-223-3p targeted both LncRNA DUXAP8 and NLRP3 mRNA, and both LncRNA DUXAP8 ablation and miR-223-3p overexpression inactivate NLRP3-mediated cell pyroptosis to rescue cell viability in H/R PHN cells. Interestingly, we evidenced that Puerarin restrained LncRNA DUXAP8 expressions, but upregulated miR-223-3p in I/R rat tissues and H/R PHN cells, and the protective effects of Puerarin on H/R PHN cells were abrogated by overexpressing LncRNA DUXAP8 and silencing miR-223-3p. Collectively, we concluded that Puerarin regulated LncRNA DUXAP8/miR-223-3p/NLRP3 signaling cascade to attenuate I/R injury.

Highlights

The main treatment therapies for cerebral ischemia includes pharmacological and mechanical reperfusion strategies (Zhang et al, 2016; Zhao et al, 2017), and researchers and clinicians agreed that timely ischemia-reperfusion strategy is effective to prevent neurological death and dysfunction (Zhang et al, 2016; Zhao et al, 2017), the adverse effects caused by this strategy, including intracerebral hemorrhage (Kimiwada et al, 2019; Zhang et al, 2017a), brain edema (King et al, 2018; Nakano et al, 2018), etc., still remains a serious challenge in ischemic stroke treatment
Involvement of NLRP3-mediated cell pyroptosis in I/R rat models and H/R cellular models Initially, according to the previous publications (An et al, 2019; He et al, 2019; Ma et al, 2019; Qu et al, 2019; Ye et al, 2019), we established the in vivo I/R rat models
The overexpression and silencing vectors for LncRNA double homeobox A pseudogene 8 (DUXAP8) were delivered into the H/R primary hippocampus neuron (PHN) cells (Fig. 4E), by performing Real-Time qPCR and Western Blot, we found that knock-down of LncRNA DUXAP8 decreased the mRNA and protein levels of NLRP3 in H/R-treated PHN cells, which were reversed by silencing miR-223-3p (P < 0.05, Figs. 4F and 4G)

Summary

Introduction

Ischemic stroke accounts for approximately 87% of all stroke patients worldwide, and about 15 million people suffer from stroke each year (Benjamin et al, 2018). Puerarin exerted its neuroprotective effects to alleviate cerebral I/R injury in rat models (Ling et al, 2018; Liu et al, 2013; Wang et al, 2018; Zhou et al, 2014), and the available clinical data. Hinted that Puerarin was effective for the treatment of ischemic stroke in China (Liu et al, 2016). JinFeng Wang et al (2018) found that Puerarin protected rat brain against I/R injury by inactivating the AMPK-mTORULK1 pathway mediated autophagy, and Liu et al (2013) evidenced that Puerarin activated the cholinergic antiinflammatory pathway to alleviate cerebral I/R injury. The data provided by Zi-Kuang Wang indicated that Puerarin regulated the SIRT1/NF-kB pathway to suppress NLRP3 inflammasome in myocardial I/R injury (Wang et al, 2020), and Guan et al (2020) proved that Puerarin ameliorated I/R-induced retinal ganglion cell damage through regulating the TLR4/NLRP3 pathway, which enlightened us that Puerarin might protect neurons from cerebral I/R-induced death through inhibiting NLRP3mediated pyroptotic cell death

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