目的：阻塞性睡眠呼吸暂停（obstructive sleep apnea，OSA）是最常见的睡眠呼吸障碍疾病。OSA 引起的间歇性低氧（Intermittent hypoxia，IH）会导致严重的神经认知功能障碍。既往研究观察到 OSA 患者存在肠道微生物群及衍生代谢物水平异常，但尚未发现 OSA 与肠道菌群及衍生代谢物的因果关联证据，并且 OSA 介导的菌群及代谢环境紊乱对相关认知障碍的影响尚不明确。研究目的包括：1.基于孟德尔随机化（Mendelian randomization，MR）分析全面评估 196 种肠道微生物群丰度及 83 种衍生代谢物水平与 OSA 的双向因果关系，获得肠道菌群与 OSA 及相关认知障碍的关联证据。2.明确 IH 对小鼠学习记忆功能的影响。3.探究 IH 对小鼠肠道菌群、代谢物、海马基因调控和神经功能水平的多维度动态影响，确定菌群及代谢紊乱在 IH 所致认知障碍中的作用。4.明确外源性短链脂肪酸（Short-chain fatty acids, SCFAs，丁酸钠 [sodium butyrate，SB]）补充通过调节肠脑轴改善 IH 相关认知功能障碍的潜在机制。

方法：1. OSA表型相关全基因组关联分析（genome-wide association study，GWAS）汇总数据来自FinnGen项目，肠道菌群和衍生代谢物GWAS汇总数据来自MiBioGen项目和独立数据集。本研究设计了多重 MR 分析策略，采用双向两样本 MR 分析筛选与 OSA 相关的潜在暴露或结局菌群和代谢物集合，通过多变量 MR（Multivariate Mendelian randomization，MVMR）分析针对肥胖、男性和吸烟进行调整，获得菌群及衍生代谢物与 OSA 风险的独立因果证据。2. 通过 IH 干预建立 OSA 小鼠模型，利用行为学实验检测 IH 小鼠认知功能，通过静息态功能核磁检测 IH 对小鼠神经功能水平的影响。3. 通过粪便宏基因组和代谢组、海马转录组分析，明确 IH 导致的肠道菌群结构、代谢物水平和海马基因调控变化；通过多组学关联分析构建 IH 相关肠脑轴调控网络，利用荧光定量PCR（quantitative polymerase chain reaction，qPCR）验证肠脑轴调控网络关键通路基因在小鼠海马的表达；使用 Luminex 定量分析检测IH 小鼠海马细胞因子水平。4. 探究 SB 补充对 IH 认知功能障碍的改善情况及相关肠脑轴调控机制。

结果：1.多重 MR 分析确定 3 种肠道菌群衍生代谢物血清含量增加（亮氨酸、硫酸表雄酮和 3-脱氢肉碱）与 OSA 患病风险增加独立相关，瘤胃球菌科为代表的多种产 SCFAs 细菌在矫正混杂偏倚后仍对 OSA 风险表现出独立的保护作用。2. 行为学实验发现 IH 会导致小鼠出现学习记忆功能障碍，功能核磁显示 IH 小鼠出现全脑神经活动强度不一致，认知相关脑区神经功能水平下降；多组学联合分析发现 IH小鼠肠脑轴出现多维度病理性改变，包括 SCFAs 产生菌丰度降低、代谢途径水平改变、海马小胶质细胞过度激活、炎症因子累积和炎性通路激活。3. 外源性 SB 补充显著改善 IH 小鼠学习记忆功能障碍，上调认知功能相关脑区神经活性水平；菌群重构、调节代谢环境以及抑制小胶质细胞激活和炎症因子表达可能是 SB 改善 IH 认知障碍的潜在机制。

结论：多种肠道微生物菌群和衍生的代谢物与 OSA 具有独立因果关系；外源性SCFAs（丁酸）补充可以显著改善 IH 相关认知功能障碍。本研究为肠道菌群与 OSA 及相关认知功能障碍的关联提供了证据支持，基于肠道菌群代谢物的SCFAs 补充治疗可作为 OSA 相关认知功能障碍的潜在药物治疗手段。

Objective: Obstructive sleep apnea (OSA) is the most common sleep disordered breathing disorder. Intermittent hypoxia (IH) caused by OSA can lead to severe neurocognitive impairment. Previous studies have observed abnormal levels of gut microbiota and derived metabolites in OSA patients. However, there is no evidence of causal association between OSA and gut microbiota and derived metabolites. The effect of OSA mediated microbiota and metabolic environment disturbance on related cognitive impairment is still unclear. The objectives of the study were as follows: 1. Based on Mendelian randomization (MR) analysis, we comprehensively evaluated the bidirectional causal relationship between the abundance of 196 gut microbiota and the levels of 83 derived metabolites and OSA, and obtained evidence of the association between gut microbiota and OSA and related cognitive impairment. 2. To determine the effects of IH on learning and memory function in mice. 3. To explore the multi-dimensional dynamic effects of IH on gut microbiota, metabolites, hippocampal gene regulation and neural function in mice, and determine the role of flora and metabolic disorders in IH-induced cognitive impairment. 4. Identify potential mechanisms of exogenous Short-chain fatty acids (SCFAs, eg. sodium butyrate, SB) supplementation to improve IH-related cognitive dysfunction by regulating the gut-brain axis.

Methods: 1. OSA genome-wide association study (GWAS) summary data were obtained from the FinnGen project, and gut microbiota and derived metabolites GWAS summary data were obtained from the MiBioGen project and independent data sets. In this study, multiple MR Analysis strategies were designed, using two-way two-sample MR Analysis to screen for potential exposure or outcome microbiota and metabolite collections associated with OSA. Independent causal evidence was obtained from Multivariate Mendelian randomization (MVMR) analyses adjusted for obesity, masculinity, and smoking to establish the relationship between the microbiota and derived metabolites and OSA risk. 2. The mouse model of OSA was established by IH intervention, the cognitive function of IH mice was detected by behavioral experiment, and the effect of IH on the neural function of mice was detected by resting state functional MRI. 3. Through fecal metagenome, metabolome and hippocampal transcriptome analysis, the changes in gut microbiota structure, metabolite level and hippocampal gene regulation caused by IH were identified; The IH-related gut-brain axis regulatory network was constructed by multi-omics association analysis, and quantitative polymerase chain reaction (qPCR) was used to verify the expression of key pathway genes of the gut-brain axis regulatory network in the hippocampus of mice. Luminex quantitative assay was used to detect the level of cytokines in IH mice. 4. To explore the improvement of SB supplementation on IH cognitive dysfunction and the related gut-brain axis regulation mechanism.

Results: 1. Multiple MR Analysis determined that increased serum levels of three gut microbiota derived metabolites (leucine, epimandrosterone sulfate and 3-dehydrocarnitine) were independently associated with increased risk of OSA, and multiple SCFAs producing bacteria represented by Rumenococcaceae showed independent protective effects on OSA risk even after correction of confounding bias. 2. Behavioral experiments found that IH would lead to learning and memory dysfunction in mice. Functional MRI showed that IH mice showed inconsistent intensity of nerve activity in the whole brain and decreased nerve function in cognitive-related brain areas. Multi-omics analysis revealed multi-dimensional pathological changes in the gut-brain axis of IH mice, including reduced abundance of SCFAs producing bacteria, altered metabolic pathway levels, over-activation of hippocampal microglia, accumulation of inflammatory factors and activation of inflammatory pathways. 3. Exogenous SB supplementation significantly improved the learning and memory dysfunction of IH mice, and up-regulated the level of neural activity in brain regions related to cognitive function; Microbiota remodeling, regulation of metabolic environment, inhibition of microglia activation and expression of inflammatory factorsmay be the potential mechanisms by which SB improves IH cognitive impairment.

Conclusion: Multiple gut microbiota and derived metabolites have independent causal relationship with OSA. Exogenous SCFAs (butyric acid) supplementation can significantly improve IH-related cognitive dysfunction. This study provides evidence for the association between gut microbiota and OSA and related cognitive dysfunction, and SCFAs supplementation based on gut microbiota metabolites can be used as a potential drug therapy for OSA related cognitive dysfunction.