高炎症反应在心肌细胞衰老的病理生理学中至关重要,与IL33作为一个关键的促炎介质。我们之前的发现强调了RND3对IL33表达的抑制作用。本研究旨在探讨RND3在IL33/ST2信号激活和心肌细胞衰老中的作用。心肌内注射外源性IL33可降低大鼠射血分数和缩短,诱导心肌组织中衰老相关分泌表型(SASP)的出现。重组IL33处理AC16心肌细胞显着上调SASP因子如IL1α的表达,IL6和MCP1,并增加了p-p65/p65比率和SA-β-gal和γH2AX阳性细胞的比例。NF-κB抑制剂吡咯烷二硫代氨基甲酸酯铵(PDTC)和ST2抗体astegolimab处理减轻了上述作用。使用CRISPR/Cas9技术敲除RND3基因H9C2心肌细胞上调IL33,ST2L,IL1α,IL6和MCP1水平,sST2水平降低,和增加SA-β-gal和γH2AX阳性细胞。通过分子对接和免疫共沉淀显示RND3和IL33蛋白之间结合的可能性很高,和RND3的损失减弱泛素化介导的IL33降解;更重要的是,使用qPCR阵列筛选了一组与RND3密切相关的泛素化调节基因。相比之下,通过注射AAV9-CMV-RND3颗粒在大鼠中的RND3过表达抑制IL33,ST2L,IL1α,IL6和MCP1在心脏组织中的表达,降低血清IL33水平,并增加sST2水平。这些结果表明,RND3在心肌细胞中的表达通过抑制IL33/ST2/NF-κB信号通路调节细胞衰老,强调其作为心血管衰老治疗靶点的潜力。
Hyperinflammatory responses are pivotal in the cardiomyocyte senescence pathophysiology, with IL33 serving as a crucial pro-inflammatory mediator. Our previous findings highlighted
RND3\'s suppressive effect on IL33 expression. This study aims to explore the role of
RND3 in IL33/ST2 signaling activation and in cardiomyocyte senescence. Intramyocardial injection of exogenous IL33 reduces the ejection fraction and fractional shortening of rats, inducing the appearance of senescence-associated secretory phenotype (SASP) in myocardial tissues. Recombinant IL33 treatment of AC16 cardiomyocytes significantly upregulated expression of SASP factors like IL1α, IL6, and MCP1, and increased the p-p65/p65 ratio and proportions of SA-β-gal and γH2AX-positive cells. NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) and ST2 antibody astegolimab treatments mitigated above effects.
RND3 gene knockout H9C2 cardiomyocytes using CRISPR/Cas9 technology upregulated IL33, ST2L, IL1α, IL6, and MCP1 levels, decreased sST2 levels, and increased SA-β-gal and γH2AX-positive cells. A highly possibility of binding between RND3 and IL33 proteins was showed by molecular docking and co-immunoprecipitation, and loss of RND3 attenuated ubiquitination mediated degradation of IL33; what\'s more, a panel of ubiquitination regulatory genes closely related to
RND3 were screened using qPCR array. In contrast,
RND3 overexpression in rats by injection of AAV9-CMV-
RND3 particles inhibited IL33, ST2L, IL1α, IL6, and MCP1 expression in cardiac tissues, decreased serum IL33 levels, and increased sST2 levels. These results suggest that RND3 expression in cardiomyocytes modulates cell senescence by inhibiting the IL33/ST2/NF-κB signaling pathway, underscoring its potential as a therapeutic target in cardiovascular senescence.