Objective: Hepatic encephalopathy (HE) characterized by neuropsychiatric abnormalities is a major complication of cirrhosis with high mortality. However, the pathogenesis of HE has not been fully elucidated. This study aimed to determine endogenous hydrogen sulfide (H₂S) in the blood of HE patients and investigate the role of H₂S in an astrocytic model of HE.

Methods: Patients with and without HE were recruited to determine plasma H₂S levels and blood microbial 16S rRNA gene. Rat astrocytes were employed as a model of HE by treatment of NH₄Cl. Exogenous H₂S was preadded. Cell viability was measured by Cell Counting Kit-8 (CCK-8) assay, and cell death was evaluated by lactate dehydrogenase (LDH) release. Apoptosis was determined by Hoechst 33342/Propidium Iodide (PI) Double Staining and Western blot analysis of apoptosis-related protein expression. Intracellular reactive oxygen species (ROS) levels were assessed by flow cytometer. Expressions of Nrf2 and its downstream regulated genes were examined by immunofluorescence staining and Western blot, respectively. Nrf2 gene knockdown was performed by antisense shRNA of Nrf2 gene.

Results: There was a significant decrease in H₂S levels in cirrhotic patients with HE compared with without HE. Blood microbiota analyses revealed that certain strains associated with H₂S production were negatively correlated with HE. In vitro, H₂S markedly attenuated NH₄Cl-induced cytotoxicity, oxidative stress, and apoptosis. This effect was mediated by Nrf2/ARE signaling, and knockdown of Nrf2 expression abolished the antagonistic effect of H₂S on NH₄Cl-induced neurotoxicity in astrocytes.

Conclusion: Levels of H₂S and bacteria associated with H₂S production are decreased in HE, and H₂S functions as the neuroprotector against NH₄Cl-induced HE by activating Nrf2/ARE signaling of astrocytes.

目的：以神经精神异常为特征的肝性脑病（HE）是高死亡率的肝硬化的主要并发症。然而，HE的发病机理尚未完全阐明。本研究旨在确定HE患者血液中的内源性硫化氢（H ₂ S），并研究H ₂ S在HE星形细胞模型中的作用。

方法：招募有和没有HE的患者，以确定血浆H ₂ S水平和血液微生物16S rRNA基因。通过处理NH ₄ Cl ，将大鼠星形胶质细胞用作HE的模型。外源H ₂S已预先添加。通过细胞计数试剂盒8（CCK-8）测定来测量细胞活力，并通过乳酸脱氢酶（LDH）释放评估细胞死亡。通过Hoechst 33342 /碘化丙啶（PI）双重染色和凋亡相关蛋白表达的蛋白质印迹分析来确定细胞凋亡。通过流式细胞仪评估细胞内活性氧（ROS）水平。Nrf2及其下游调控基因的表达分别通过免疫荧光染色和蛋白质印迹法进行检测。Nrf2基因敲低是通过Nrf2基因的反义shRNA进行的。

结果：与没有HE的肝硬化患者相比，肝硬化患者的H ₂ S水平显着降低。血液微生物群分析显示，某些与H ₂ S产生相关的菌株与HE呈负相关。体外，H ₂ S显着减弱了NH ₄ Cl诱导的细胞毒性，氧化应激和细胞凋亡。这种作用是由Nrf2 / ARE信号介导的，而敲低Nrf2表达消除了H ₂ S对星形胶质细胞中NH ₄ Cl诱导的神经毒性的拮抗作用。

结论：HE中H ₂ S和与H ₂ S产生有关的细菌的水平降低，并且H ₂ S通过激活星形胶质细胞的Nrf2 / ARE信号传导而成为针对NH ₄ Cl诱导的HE的神经保护剂。