细胞外RNA和PKR-NLRP3炎性体系对小鼠心肌缺血再灌注损伤的影响

Double-stranded RNA-dependent protein kinase (PKR) can regulate inflammatory responses via mediate NLRP3 inflammasome. Our previous studies suggest that injection of RNaseA can extenuate I/R injury through reducing the expression of myocardial PKR protein and inflammatory cytokinesin (such as IL-1β、HMGB-1) in ischemic region. Myocardial I/R injury and inflammatory responses were also relieved in mice with PKR gene knockdown (PKR-/-). Thus, we hypothesized that extracellular RNA (exRNA) released by myocardial ischemia-reperfusion (I/R) injury might activate PKR, mediated inflammasome to promote the increase of inflammatory cytokines, thereby aggravating myocardial I/R injury. In this study, PKR, NLRP3 and down-stream inflammatory factors, myocardial infarction zone, area at risk and cardiac function were observed in myocardium I/R injury mice after injection of RNaseA. Similar parameters were detected in PKR or NLRP3 knockdown mice. Accordingly, In vitro experiments, inflammatory response of inhibited or over-expressed PKR and NLRP3 in cardiomyocytes and macrophages which induced by necrotic cells were also needed to observed to further clarify the molecular mechanisms of extracellular RNA and PKR- NLRP3 inflammasome in myocardial I/R injury, and provide new targets for intervention.

双链RNA 依赖性蛋白激酶(PKR)能介导炎性小体NLRP3，调控炎症反应。我们的前期研究表明，注射核糖核酸酶干预心肌缺血再灌注(I/R)小鼠，缺血区PKR及IL-1β、HMGB-1等表达下降，I/R损伤减轻；PKR基因敲除小鼠的心肌缺血区上述炎症因子亦减少、I/R损伤减轻。我们推测，细胞损伤释放的细胞外RNA(exRNA) 激活PKR，介导NLRP3，促进IL-1β、HMGB-1等炎症因子释放，加重I/R损伤。本研究拟对心肌I/R小鼠注射核糖核酸酶，比较PKR、NLRP3及下游炎症因子、梗死和损伤面积、心功能；比PKRˉ/ˉ及NLRP3基因敲除小鼠心肌I/R的缺血区炎症反应、梗死和损伤面积、心功能；观察抑制或过表达PKR、NLRP3的心肌细胞、巨噬细胞对坏死细胞介导的炎症反应。阐明exRNA和PKR-NLRP3炎性体系在心肌I/R损伤中的作用，为防治心肌I/R损伤提供新的干预靶点。

双链RNA依赖性蛋白激酶（PKR）能介导炎性小体NLRP3，调控炎症反应。课题组前期研究表明，注射核糖核酸酶干预心肌缺血再灌注（I/R）小鼠，缺血区PKR及IL-1β、HMGB-1等表达下降，I/R损伤减轻；PKR基因敲除小鼠的心肌缺血区上述炎症因子亦减少、I/R损伤减轻。因此，该课题组推测，细胞损伤释放的细胞外RNA（exRNA） 激活PKR，介导NLRP3，促进IL-1β、HMGB-1等炎症因子释放，加重I/R损伤。项目主要研究内容：(1)动物实验：明确exRNA通过调节PKR，激活NLRP3炎性小体，促进炎症反应，介导心肌I/R损伤；(2)体外细胞实验：证实exRNA通过调节巨噬细胞PKR，进而激活NLRP3炎性小体，促进炎症反应，从而参与心肌细胞缺氧-复氧损伤。经开展研究，本课题组在动物实验中证实了exRNA能加重小鼠心肌I/R损伤，抑制exRNA可减轻心肌I/R损伤；exRNA主要通过上调PKR，激活NLRP3炎性小体，介导炎症反应加重心肌I/R损伤，而PKR是NLRP3炎性小体的上游调节子；细胞实验证实了exRNA介导巨噬细胞炎症反应参与心肌细胞缺氧-复氧损伤，其主要通过上调巨噬细胞PKR，进而激活NLRP3炎性小体，促进炎症反应，损伤心肌细胞。项目结论：exRNA主要通过上调巨噬细胞PKR，激活NLRP3炎性小体，促进炎症反应，参与心肌I/R损伤；使用RNase1抑制exRNA可有效减轻小鼠心肌I/R损伤。