背景:心肌中的炎症在心脏重塑和心力衰竭(HF)的病理生理学中起关键作用。先前的研究表明,线粒体DNA(mtDNA)可以以不同的拓扑形式存在。然而,超螺旋/松弛mtDNA比例对心肌细胞炎症反应的具体影响尚不清楚.这项研究的目的是阐明不同mtDNA类型通过调节ZBP1对心肌细胞炎症的不同影响。
方法:通过横向主动脉缩窄(TAC)或阿霉素(Doxo)诱导建立HF小鼠模型。通过HE染色评估组织病理学变化。ELISA用于测量细胞因子水平(IL-1β和IL-6)。进行Southern印迹分析以检查mtDNA的不同拓扑结构。使用Pearson相关性分析来确定超螺旋/松弛mtDNA的比率与炎性细胞因子之间的相关性。逆转录定量PCR(RT-qPCR)检测细胞因子(IL-1β,IL-6)和Dloop,作为mtDNA标记。
结果:在Doxo诱导的小鼠心肌中,超螺旋与松弛的mtDNA的比率显着增加,而在TAC诱导的小鼠中没有观察到显著的变化。IL-1β和IL-6水平与胞质mtDNA超螺旋/松弛环比率呈正相关。不同的mtDNA拓扑结构对炎症通路有不同的影响。低超螺旋mtDNA主要通过ZBP1激活NF-κB(Ser536)途径,而高超螺旋mtDNA显着影响STAT1和STAT2途径。RIPK3-NF-κB通路,作为ZBP1的下游靶标,介导低超螺旋mtDNA诱导的炎症反应。敲除TLR9增强ZBP1、p-NF-κB的表达,和RIPK3在用低超螺旋mtDNA处理的心肌细胞中,表明TLR9参与ZBP1在低超螺旋mtDNA诱导的炎症中的抗炎作用。
结论:超螺旋与松弛mtDNA的不同比例影响心肌细胞的炎症反应,并通过ZBP1参与导致HF。ZBP1及其下游炎症机制,介导低比例超螺旋mtDNA诱导的炎症反应。
BACKGROUND: Inflammation in the myocardium plays a critical role in cardiac remodeling and the pathophysiology of heart failure (HF). Previous studies have shown that mitochondrial DNA (mtDNA) can exist in different topological forms. However, the specific influence of the ratio of supercoiled/relaxed mtDNA on the inflammatory response in cardiomyocytes remains poorly understood. The aim of this study was to elucidate the differential effects of different mtDNA types on cardiomyocyte inflammation through regulation of ZBP1.
METHODS: A mouse model of HF was established by transverse aortic constriction (TAC) or doxorubicin (Doxo) induction. Histopathological changes were assessed by HE staining. ELISA was used to measure cytokine levels (IL-1β and IL-6). Southern blot analysis was performed to examine the different topology of mtDNA. Pearson correlation analysis was used to determine the correlation between the ratio of supercoiled/relaxed mtDNA and inflammatory cytokines. Reverse transcription quantitative PCR (RT-qPCR) was used to measure the mRNA expression levels of cytokines (IL-1β, IL-6) and Dloop, as an mtDNA marker.
RESULTS: The ratio of supercoiled to relaxed mtDNA was significantly increased in the myocardium of Doxo-induced mice, whereas no significant changes were observed in TAC-induced mice. The levels of IL-1β and IL-6 were positively correlated with the cytoplasmic mtDNA supercoiled/relaxed circle ratio. Different mtDNA topology has different effects on inflammatory pathways. Low supercoiled mtDNA primarily activates the NF-κB (Ser536) pathway via ZBP1, whereas high supercoiled mtDNA significantly affects the STAT1 and STAT2 pathways. The RIPK3-NF-κB pathway, as a downstream target of ZBP1, mediates the inflammatory response induced by low supercoiled mtDNA. Knockdown of
TLR9 enhances the expression of ZBP1, p-NF-κB, and RIPK3 in cardiomyocytes treated with low supercoiled mtDNA, indicating the involvement of
TLR9 in the anti-inflammatory role of ZBP1 in low supercoiled mtDNA-induced inflammation.
CONCLUSIONS: Different ratios of supercoiled to relaxed mtDNA influence the inflammatory response of cardiomyocytes and contribute to HF through the involvement of ZBP1. ZBP1, together with its downstream inflammatory mechanisms, mediates the inflammatory response induced by a low ratio of supercoiled mtDNA.