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.

摘要

背景：近年来全球范围内的中暑发病率上升，并且与高发病率和高死亡率有关。因此，确定介导中暑的机制至关重要。先前的研究表明，对小肠的损害可能是中暑相关发病率和死亡率的主要因素。但是，中暑相关的小肠损伤的机制尚不清楚。

方法：为了探讨中暑如何促进肠道损伤，我们应用了两个公认的模型：小鼠和IEC-6细胞热应激（HS）在体内和体外模拟中暑。通过WST-1和LDH释放测定法评估生存力和细胞死亡的百分比。通过膜联蛋白V-FITC / PI染色的流式细胞仪分析HS诱导的细胞死亡的诱导。流式细胞仪用于通过MitoSOX染色分析HS诱导的线粒体超氧化物。通过ELISA检测丙二醛（MDA）水平和超氧化物歧化酶（SOD）水平。流式细胞仪用于分析HS诱导的线粒体去极化（低ΔΨm）和JC-1染色。通过H＆E染色检测回肠的组织病理学变化，通过透射电镜（TEM）观察回肠的超微结构。Western blot检测RIPK1，RIPK3，磷酸化MLKL和MLKL水平。通过免疫沉淀测定法测量RIPK1-RIPK3复合物。

结果： HS增加了坏死细胞率和IEC-6细胞中的RIPK1，RIPK3和磷酸化的MLKL表达水平。这些增加的表达水平促进了更高的RIPK1-RIPK3复合物形成，导致体内和体外的坏死体形成。。此外，HS引起动态不平衡，氧化应激反应和线粒体损害，以及小肠组织损伤和细胞死亡。但是，用RIPK1活性化学抑制剂Nec-1或RIPK3活性化学抑制剂GSK'872预处理的IEC-6细胞或小鼠可显着逆转这些现象，并促进氧化应激反应稳态的平衡。更重要的是，活性氧（ROS）清除剂N-乙酰基-L-半胱氨酸（NAC）预处理在体内和体外均显着抑制HS诱导的RIPK1 / RIPK3依赖性坏死病的形成，这表明通过清除ROS产生来预防坏死病可能减轻HS引起的小肠组织损伤和细胞死亡。

结论：本研究提供了有力的证据表明HS造成损害的小肠和肠上皮细胞二者，清除活性氧的产生能显著减轻这种RIPK1 / RIPK3依赖性坏死的，介导两个HS-诱导肠损伤的体外和体内。这些发现为诊断为中暑的患者提供了基于未来机制的治疗策略的明确目标。