Department of Anesthesiology, The Fourth Central Clinical School, Tianjin Medical University, Tianjin 300140, China; Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300052, China; Tianjin Research Institute of Anesthesiology, Tianjin 300052, China.
Department of Anesthesiology, The Fourth Central Clinical School, Tianjin Medical University, Tianjin 300140, China.
Int Immunopharmacol. 2023 May;118:110009. doi: 10.1016/j.intimp.2023.110009. Epub 2023 Mar 22.
Sepsis-associated encephalopathy (SAE) is a complication of the central nervous system in patients with sepsis. Currently, no effective treatment for sepsis is available. Hydrogen plays a protective role in different diseases; however, the detailed mechanism of hydrogen-treated disease remains unclear. The purpose of this study was to investigate the effect of hydrogen on SAE in vitro and in vivo and the mechanism of hydrogen in mitochondrial dynamics and its function in astrocytes and microglia stimulated by lipopolysaccharides (LPSs).
Animal models of SAE were generated by cecal ligation and puncture, and the SAE model was established by in vitro LPS stimulation. MTT, lactate dehydrogenase (LDH), reactive oxygen species (ROS), heme oxygenase-1 (HO-1) activity, mitochondrial membrane potential (MMP), and cell apoptosis assays were used to determine the effect of hydrogen on astrocytes and microglia stimulated by LPSs. The relationships between nuclear factor erythroid 2-related factor 2 (Nrf2), YY1, and HO-1 were examined by chromatin immunoprecipitation and co-immunoprecipitation. Mitochondrial homeostasis-related proteins in LPS-stimulated glial cells and brain tissues of SAE mice were detected by western blotting. The effects of hydrogen treatment in the SAE mouse model were investigated using Morris water maze and Y-maze analyses.
After performing experiments with different concentrations of LPSs in vitro, we selected 1000 ng/ml for subsequent experiments. Hydrogen attenuated the increase in ROS, LDH, and apoptosis and promoted decreases in cell activity and MMP, further promoting an increase in HO-1 expression induced by LPSs in astrocytes and microglia. Moreover, hydrogen further promoted the expression of Nrf2, HO-1, PGC-1α, TFAM, PARKIN, and PINK1, inhibited LPS-induced OPA1 and MFN2 expression in astrocytes and microglia, and downregulated the expression of DRP1 after LPS induction. Intriguingly, hydrogen treatment enhanced the binding between Nrf2 and YY1. However, silencing Nrf2 or YY1 abolished the protective effects of hydrogen on cell activity, LDH, ROS, and MMP; apoptosis; and regulation of Nrf2, HO-1, PGC-1α, TFAM, OPA1, DRP1, MFN2, PARKIN, and PINK1 in microglia. Finally, hydrogen treatment improved the results of behavioral detection, apoptosis, Nrf2, HO-1, PGC-1α, TFAM, OPA1, DRP1, MFN2, PARKIN, PINK1, and cytokines in SAE in vivo.
Hydrogen improved cell injury and mitochondrial quality, which were associated with HO-1 expression promoted by the Nrf2/YY1 complex in vitro. Thus, hydrogen treatment may represent a novel therapeutic method for treating SAE.
脓毒症相关性脑病（SAE）是脓毒症患者中枢神经系统的并发症。目前，尚无有效的脓毒症治疗方法。氢气在不同疾病中发挥保护作用；然而，氢气治疗疾病的详细机制尚不清楚。本研究旨在探讨氢气对体内和体外 SAE 的影响，以及氢气在脂多糖（LPSs）刺激的星形胶质细胞和小胶质细胞中线粒体动力学及其功能中的作用。
通过盲肠结扎和穿刺建立 SAE 动物模型，通过体外 LPS 刺激建立 SAE 模型。MTT、乳酸脱氢酶（LDH）、活性氧（ROS）、血红素加氧酶-1（HO-1）活性、线粒体膜电位（MMP）和细胞凋亡检测用于确定氢气对 LPS 刺激的星形胶质细胞和小胶质细胞的影响。通过染色质免疫沉淀和共免疫沉淀检测核因子红细胞 2 相关因子 2（Nrf2）、YY1 和 HO-1 之间的关系。通过蛋白质印迹检测 LPS 刺激的神经胶质细胞和 SAE 小鼠脑组织中线粒体稳态相关蛋白。通过 Morris 水迷宫和 Y 迷宫分析研究氢气处理对 SAE 小鼠模型的影响。
在体外进行不同浓度 LPSs 的实验后，我们选择 1000ng/ml 进行后续实验。氢气减轻了 ROS、LDH 和细胞凋亡的增加，并促进了 LPSs 诱导的星形胶质细胞和小胶质细胞中细胞活性和 MMP 的降低，进一步促进了 HO-1 的表达。此外，氢气进一步促进了 Nrf2、HO-1、PGC-1α、TFAM、PARKIN 和 PINK1 的表达，抑制了 LPS 诱导的星形胶质细胞和小胶质细胞中 OPA1 和 MFN2 的表达，并下调了 LPS 诱导后的 DRP1 表达。有趣的是，氢气处理增强了 Nrf2 和 YY1 之间的结合。然而，沉默 Nrf2 或 YY1 消除了氢气对细胞活性、LDH、ROS 和 MMP、细胞凋亡以及 LPS 诱导的 Nrf2、HO-1、PGC-1α、TFAM、OPA1、DRP1、MFN2、PARKIN 和 PINK1 调节的保护作用。最后，氢气处理改善了体内 SAE 的行为检测结果、细胞凋亡、Nrf2、HO-1、PGC-1α、TFAM、OPA1、DRP1、MFN2、PARKIN、PINK1 和细胞因子的表达。
氢气改善了细胞损伤和线粒体质量，这与体外 Nrf2/YY1 复合物促进的 HO-1 表达有关。因此，氢气治疗可能代表治疗 SAE 的一种新的治疗方法。
