Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, China.
Department of Cardiology, General Hospital, Tianjin Medical University, Tianjin, China.
Free Radic Res. 2022 Jan;56(1):115-126. doi: 10.1080/10715762.2022.2052057. Epub 2022 Mar 16.
While the zinc transporter ZIP2 (Slc39a2) is upregulated STAT3 as an adaptive response to protect the heart from ischemia/reperfusion (I/R) injury, the precise mechanism underlying its upregulation remains unclear. The purpose of this study was to investigate the role of NADPH oxidase (NOX) isoform NOX2-derived ROS in the regulation of ZIP2 expression, focusing on the role of the NOX2 cytosolic factor p67. Mouse hearts or H9c2 cells were subjected to I/R. Protein expression was detected with Western blotting. Infarct size was measured with TTC staining. The cardiac-specific p67 conditional knockout mice (p67 cKO) were generated by adopting the CRISPR/Cas9 system. I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression was reversed by the ROS scavenger N-acetylcysteine (NAC) and the NOX inhibitor diphenyleneiodonium (DPI). p67 but not NOX2 expression was increased 30 min after the onset of reperfusion, and downregulation of p67 by siRNA or cKO invalidated I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression. Both NAC and DPI prevented upregulation of STAT3 phosphorylation and ZIP2 expression induced by overexpression of p67, whereas the STAT3 inhibitor stattic abrogated upregulation ZIP2 expression, indicating that the increase of p67 at reperfusion is an upstream signaling event responsible for ZIP2 upregulation STAT3. Experiments also showed that chelation of Zn markedly enhanced p67 and ZIP2 expression as well as STAT3 phosphorylation, whereas supplementation of Zn had the opposite effects, indicating that cardiac Zn loss upon reperfusion triggers p67 upregulation. Furthermore, ischemic preconditioning (IPC) upregulated ZIP2 p67, and cKO of p67 aggravated cardiac injury after I/R, indicating that p67 upregulation is cardioprotective against I/R injury. In conclusion, an increase of p67 expression in response to Zn is an intrinsic adaptive response to I/R and leads to cardioprotection against I/R by upregulating ZIP2 STAT3.
虽然锌转运蛋白 ZIP2（Slc39a2）作为一种适应性反应而上调 STAT3，以保护心脏免受缺血/再灌注（I/R）损伤，但它上调的确切机制尚不清楚。本研究旨在探讨 NADPH 氧化酶（NOX）同工型 NOX2 衍生的 ROS 在调节 ZIP2 表达中的作用，重点研究 NOX2 细胞溶质因子 p67 的作用。将小鼠心脏或 H9c2 细胞进行 I/R 处理。通过 Western blot 检测蛋白表达。用 TTC 染色测量梗塞面积。采用 CRISPR/Cas9 系统生成心脏特异性 p67 条件性敲除小鼠（p67 cKO）。ROS 清除剂 N-乙酰半胱氨酸（NAC）和 NOX 抑制剂二苯基碘（DPI）逆转了 I/R 诱导的 STAT3 磷酸化和 ZIP2 表达的上调。再灌注 30 分钟后 p67 表达增加，而 siRNA 或 cKO 下调 p67 则使 I/R 诱导的 STAT3 磷酸化和 ZIP2 表达上调无效。NAC 和 DPI 均可阻止 p67 过表达诱导的 STAT3 磷酸化和 ZIP2 表达的上调，而 STAT3 抑制剂 stattic 则阻断了 ZIP2 表达的上调，表明再灌注时 p67 的增加是导致 STAT3 上调 ZIP2 的上游信号事件。实验还表明，Zn 的螯合显著增强了 p67 和 ZIP2 的表达以及 STAT3 的磷酸化，而 Zn 的补充则产生相反的效果，表明再灌注时心脏 Zn 的丢失触发了 p67 的上调。此外，缺血预处理（IPC）上调了 ZIP2 和 p67，而 p67 的 cKO 加重了 I/R 后的心脏损伤，表明 p67 的上调对 I/R 损伤具有心脏保护作用。总之，Zn 再灌注后 p67 表达的增加是 I/R 的一种内在适应性反应，通过上调 ZIP2 STAT3 来保护心脏免受 I/R 损伤。
