Preventing necroptosis by scavenging ROS production alleviates heat stress-induced intestinal injury
Preventing necroptosis by scavenging ROS production alleviates heat stress-induced intestinal injury
复制标题
通过清除ROS产生来预防坏死性凋亡可减轻热应激引起的肠道损伤
DOI：
10.1080/02656736.2020.1763483
复制
发表时间：
2020-01-01
影响因子：
3.1
通讯作者：
Gu, Zhengtao
中科院分区：
文献类型：
--
作者：
Li, Li;Tan, Hongping;Gu, Zhengtao
摘要背景近年来，全球范围内中暑发病率呈上升趋势，并伴有高发病率和死亡率。因此，确定介导中暑的机制至关重要。先前的研究表明，小肠损伤可能是热中风相关发病率和死亡率的主要因素。然而，中暑相关的小肠损伤的机制仍不清楚。方法采用小鼠和IEC-6细胞热应激（HS）模型，在体内和体外模拟中暑，探讨中暑对肠道损伤的影响。通过WST-1和LDH释放测定来评估存活率和细胞死亡的百分比。通过流式细胞术用Annexin V-FITC/PI染色分析HS诱导的细胞死亡的诱导。流式细胞仪分析HS诱导的线粒体超氧化物歧化酶（MitoSOX）染色。酶联免疫吸附法检测丙二醛（MDA）和超氧化物歧化酶（SOD）的含量。流式细胞术分析HS诱导的线粒体去极化（低Δ Δ Km），JC-1染色。采用H&E染色法观察回肠组织学变化，透射电镜观察回肠超微结构变化。通过Western blot检测RIPK 1、RIPK 3、磷酸化MLKL和MLKL水平。通过免疫沉淀法测定RIPK 1-RIPK 3复合物。结果HS可增加IEC-6细胞的坏死率和RIPK 1、RIPK 3及磷酸化MLKL的表达水平。这些增加的表达水平促进更高的RIPK 1-RIPK 3复合物形成，导致体内和体外的坏死体形成。此外，HS引起稳态失调、氧化应激反应和线粒体损伤，沿着小肠组织损伤和细胞死亡。然而，用RIPK 1活性化学抑制剂Nec-1或RIPK 3活性化学抑制剂GSK'872预处理的IEC-6细胞或小鼠显着逆转了这些现象，并促进了氧化应激反应稳态的平衡。更重要的是，活性氧（ROS）清除剂N-乙酰-L-半胱氨酸（NAC）预处理显着抑制HS诱导的RIPK 1/RIPK 3依赖性坏死性凋亡的形成在体内和体外，表明通过清除ROS的产生防止坏死性凋亡可能会减轻HS诱导的小肠组织损伤和细胞死亡。结论HS对小肠和肠上皮细胞均有损伤作用，清除ROS可明显减轻RIPK 1/RIPK 3依赖的坏死性凋亡，介导HS诱导的肠损伤。这些发现为未来诊断为中暑患者的基于机制的治疗策略提供了明确的目标。
Abstract Background Worldwide heat stroke incidence has increased in recent years and is associated with high morbidity and mortality. Therefore, it is critical to identify mechanisms that mediate heat stroke. Previous studies suggested that damage to the small intestine may be a major factor in heat stroke-related morbidity and mortality. However, the mechanism underlying heat stroke related small intestine injury remains unclear. Methods To explore how heat stroke promotes intestinal damage, we applied two well established models: mouse and IEC-6 cells heat stress (HS) to mimic heat stroke both in vivo and in vitro. The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Induction of HS-induced cell death was analyzed by flow cytometry with Annexin V-FITC/PI staining. Flow cytometry was used to analyze HS-induced mitochondrial superoxide with MitoSOX staining. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) levels were detected by ELISA. Flow cytometry was used to analyze HS-induced mitochondrial depolarization (low ΔΨm) with JC-1 staining. Histopathology changes in the ileum were detected by H&E staining.The ileum ultrastructure was observed by transmission electron microscopy (TEM). RIPK1, RIPK3, phosphorylated MLKL, and MLKL levels were detected by Western blot. RIPK1-RIPK3 complexes were measured by immunoprecipitation assay. Results HS increased both necrotic cell rate and RIPK1, RIPK3, and phosphorylated MLKL expression levels in IEC-6 cells. These increased expression levels promoted higher RIPK1-RIPK3 complex formation, leading to necrosome formation both in vivo and in vitro. Moreover, HS caused dyshomeostasis, an oxidative stress response, and mitochondrial damage, along with small intestinal tissue injury and cell death. However, IEC-6 cells or mice pretreated with the RIPK1 activity chemical inhibitor Nec-1 or RIPK3 activity chemical inhibitor GSK'872 significantly reversed these phenomena and promoted balance in oxidative stress response homeostasis. More importantly, the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) pretreatment significantly inhibited HS-induced RIPK1/RIPK3-dependent necroptosis formation both in vivo and in vitro, suggesting that preventing necroptosis via scavenging ROS production might alleviate HS-induced small intestinal tissue injury and cell death. Conclusion This study provides strong evidence that HS causes damage to both the small intestine and intestinal epithelial cells, scavenging ROS production can significantly alleviate such RIPK1/RIPK3-dependent necroptosis, mediating HS-induced intestinal damage both in vitro and in vivo. These findings provide a clear target for future mechanism-based therapeutic strategies for patients diagnosed with heat stroke.