慢性间歇性缺氧（Chronic intermittent hypoxia, CIH）是阻塞性睡眠呼吸暂停（Obstructive sleep apnea, OSA）的典型病理生理改变，与心血管、肾脏、神经等多器官系统疾病相关。据统计，OSA患病率约9%~38%^[1]，并且伴随生活水平提高以及人口增加，发病率逐年上升，已成为全球热点关注的健康问题。CIH作为OSA的典型病理生理改变，可造成认知损伤，包括注意力和警觉性损害、执行及记忆功能障碍等。动物研究表明，睡眠期间暴露于CIH环境会产生与OSA类似的夜间缺氧/再氧合循环，以及同OSA患者相似的认知功能损伤。目前CIH造成认知损伤的分子学机制尚未阐明，仍有待深入探索。蛋白质翻译后修饰（Post-translational modifications, PTMs）是CIH调节蛋白质功能的机制之一。常见的蛋白质翻译后修饰，如磷酸化修饰和乙酰化修饰，在突触可塑性和认知过程中起关键作用。目前，PTMs在CIH调节海马功能中发挥的作用有待进一步研究。

目的：

研究CIH小鼠模型海马组织中蛋白质磷酸化修饰的昼夜节律改变及认知损伤的分子机制。

研究CIH小鼠模型海马组织中蛋白质乙酰化修饰图谱变化及认知功能相关的重要分子通路的改变。

方法：

1、构建CIH组小鼠和常氧对照组（Control, CON）小鼠模型。统计分析小鼠生理代谢指标，包括饮食饮水量、运动量和体重。通过行为学实验（新物体识别实验、Y迷宫及巴恩斯迷宫）评估CIH对小鼠新物体记忆和空间工作记忆能力的影响。通过血氧水平依赖功能磁共振成像（Blood oxygenation level dependent-functional magnetic resonance imaging, Bold-fMRI）评估CIH对脑功能的影响。通过苏木精-伊红（Hematoxylin and eosin, H&E）染色和尼氏染色来观察小鼠海马区神经元病理学变化。通过免疫荧光染色技术，对小鼠全脑区域进行NEUN、GFAP、IBA-1和DCX指标染色，观察海马组织上述因子表达变化。为评估海马整体炎症状态，通过Luminex多因子液相芯片技术检测小鼠海马组织中IFN-γ、IL-1β、IL-2、1L-4、1L-6、1L-10、1L-16等31种细胞因子/趋化因子水平。

2、利用液相色谱-串联质谱(LC-MS/MS)、RNA-Seq技术获得一天之中（24小时）6个时间点（ZT 0、ZT 4、ZT 8、ZT 12、ZT 16、ZT 20）的CON组小鼠海马组织蛋白质组、磷酸化修饰组数据，以及2个时间点（ZT 4、ZT 16）CON组和CIH组小鼠海马组织转录组、蛋白质组、磷酸化修饰组数据。通过多组学联合分析（包括GO注释、KEGG通路富集、亚细胞定位、蛋白-蛋白相互作用、激酶预测分析等）确定关键致病因子和通路。基于R语言加载Metacycle软件包计算不同蛋白质磷酸化修饰位点的昼夜节律性。ARSER分析方法计算同一参数在6个时间点的昼夜节律显著性，非配对t检验计算两组间同一参数在某一时间点表达的差异显著性。免疫荧光实验检测CON组和CIH组小鼠海马组织神经胶质细胞标志物（GFAP、IBA-1）昼夜节律动态变化。腹腔注射雷帕霉素（mTOR通路抑制剂）对CIH组小鼠药物干预，通过行为学实验、脑组织病理染色、Bold-fMRI等方法观察雷帕霉素干预后小鼠海马组织功能和结构改变，同时免疫印迹分析验证mTOR信号通路关键因子磷酸化修饰（p-mTOR、p-RPS6）的节律变化。

3、利用LC-MS/MS技术首次研究CIH小鼠海马组织乙酰化修饰组图谱的变化。通过生物信息学技术（GO注释、KEGG通路富集、基序分析、二级结构预测、亚细胞定位、蛋白质结构域分析、蛋白-蛋白相互作用分析）对组学数据分析，筛选差异乙酰化修饰蛋白、差异KEGG通路、差异结构域等。通过免疫印迹分析验证部分乙酰化蛋白质（VDAC、Ywhaz、Camk2a）水平。腹腔注射丁酸钠（Sodium butyrate，NaB）（组蛋白去乙酰化酶抑制剂）对CIH组小鼠进行药物干预（命名为CIH+NaB组），通过行为学实验、脑组织病理染色方法观察CIH+NaB组记忆能力和海马组织病理结构改变，同时免疫组化实验验证抗乙酰化组蛋白3赖氨酸9位点（anti-acetyl-Histone H3 (Lys9)，H3K9ac）、抗乙酰化组蛋白3赖氨酸27位点（anti-acetyl-Histone H3 (Lys27)，H3K27ac）水平。

结果：

1、行为学实验（新物体识别、Y-迷宫、巴恩斯迷宫）显示CIH显著降低小鼠记忆能力。颅脑Bold-fMRI显示CIH会显著抑制小鼠脑功能，尤其是右侧海马区功能。病理学染色（H&E、尼氏染色）结果显示CIH组海马DG区经元受损、出现细胞溶解和胞质空泡化。免疫荧光染色结果显示CIH降低小鼠海马组织NEUN、DCX表达水平，增加GFAP、IBA-1表达水平。炎症因子芯片结果显示CIH诱导海马组织CXCL1、IL-10、CCL24、CXCL5、IL-6、TNF-α、CXCL16、CCL 1、IL-4、CXCL12共10种细胞因子和趋化因子水平显著升高。

2、基于CON组小鼠海马组织磷酸化修饰蛋白质组6个时间点的数据分析，我们发现60%（1980个）磷酸化修饰蛋白呈现节律表达，GO功能注释结果显示，1980个节律表达的蛋白中，87个和学习或记忆相关，134个和认知相关。时序分析显示蛋白和磷酸化修饰位点均倾向于在ZT 4（白天）和ZT 16（夜晚）两个时间点出现显著波动。KEGG结果显示，上述节律波动的蛋白主要富集在mTOR信号（mTOR signaling pathway）、轴突导向（Axon guidance）、丙酮酸代谢（Pyruvate metabolism）等通路。GO注释显示节律波动的磷酸化修饰位点主要富集在学习（Learning）、神经发生（Neurogenesis）、神经肌肉接头发育（Neuromuscular junction development）等与神经、突触功能相关的生物学过程。免疫荧光显示神经胶质细胞的特征性标志物GFAP、IBA-1存在昼夜节律性，并且节律性受CIH干扰，以ZT 4和Z16两个时间点受干扰最显著。选择ZT 4和ZT 16两个差异显著时间点，进行CON组和CIH组小鼠海马组织转录、蛋白组、磷酸化修饰组的数据分析，结果发现CIH对磷酸化修饰组学的节律性干扰显著，对转录组影响很小。腹腔注射雷帕霉素(mTOR抑制剂)后，行为学、颅脑Bold-fMRI、病理染色显示CIH组记忆能力、海马区脑功能显著改善、受损神经元修复。免疫印迹实验显示，CIH组干扰的p-mTOR、p-RPS6节律表达模式可以通过雷帕霉素得到挽救。

3、乙酰化修饰蛋白组学显示，CIH调控213个蛋白质上的280个乙酰化位点，其中35.75%乙酰化蛋白位于线粒体。KEGG通路富集分析显示，CIH显著扰乱氧化磷酸化（Oxidative phosphorylation）、三羧酸循环(Citrate cycle)和糖酵解（Glycolysis/Gluconeogenesis）等通路。基序分析结果显示CIH组上调的乙酰化修饰位点更偏好于亮氨酸，而下调的乙酰化修饰位点更倾向于异亮氨酸。通过与PLMD数据库比对分析，我们成功鉴定了292个蛋白质和977个赖氨酸乙酰化位点。值得注意的是，在已鉴定的赖氨酸位点中，我们还发现了其它多种类型的PTMs，包括泛素化（920）、琥珀酰化（776）、丙酰化（604）以及戊二酰化（209）。最后，丁酸钠（组蛋白去乙酰化酶抑制剂）对CIH组小鼠干预后，新物体识别实验发现CIH+NaB组记忆能力改善，免疫组化结果显示NaB可以显著降低CIH诱导的H3K27ac和H3K9ac表达上调。

结论：

1、慢性间歇性缺氧可能通过蛋白质翻译后修饰（乙酰化修饰、磷酸化修饰）调节海马功能。

2、生物钟对海马组织认知功能的相关分子机制同样具有重要影响。

Chronic intermittent hypoxia (CIH) is a typical pathophysiological change of obstructive sleep apnea (OSA), which is related to cardiovascular, renal, neurological, and other multiple organ system diseases. According to statistics, the prevalence rate of OSA is about 9% to 38%^[1], and with the improvement of living standards and the increase of population, the incidence rate is increasing year by year, which has become a global focus of health issues. As a typical pathological change of OSA, CIH can lead to cognitive impairment, including attention, vigilance impairment, executive and memory impairment and so on. Animal studies have shown that exposure to CIH during sleep produces nocturnal hypoxia/reoxygenation cycles like OSA and cognitive impairment like those in OSA patients. At present, the molecular mechanism of cognitive impairment caused by CIH remains to be further explored. Protein post-translational modification (PTMs) is one of the mechanisms by which CIH regulates protein function. Common PTMs, such as phosphorylation and acetylation, play a key role in synaptic plasticity and cognitive processes. At present, the role of PTMs in the regulation of hippocampal function by CIH needs further study.

Objective

1. To study the circadian rhythm changes of protein phosphorylation in hippocampal tissue of CIH mouse model and the molecular mechanism of cognitive impairment.

2. To study changes in protein acetylation modification patterns and changes in important molecular pathways related to cognitive function in the CIH mouse model.

Methods

1. To establish the mouse model of CIH group and normal oxygen control group (Control, CON). The physiological metabolic indexes of diet and drinking water, exercise and body weight of mice were statistically analyzed. The effects of CIH on new object memory and spatial working memory in mice were evaluated by behavioral experiments (new object recognition test, Y-maze, and Barnes maze). The effect of CIH on brain function was evaluated by Blood oxygenation level dependant-functional magnetic resonance imaging (Bold-fMRI). The pathological changes of hippocampal neurons in mice were observed by hematoxylin and eosin staining(H&E) and Nissl staining. The whole brain tissue of mice was stained with NEUN, GFAP, IBA-1 and DCX by immunofluorescence staining, and the expression of these factors in hippocampus was observed. To evaluate the inflammatory state of hippocampus, the levels of IFN-γ, IL-1β, IL-2, 1L-4, 1L-6, 1L-10, 1L-16 and other 31 kinds of cytokines /chemokines in mouse hippocampal tissue were detected by Luminex multifactor liquid chip technique.

2. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA-Seq techniques were used to obtain proteome and phosphorylation modification group data of hippocampal tissue of CON group at 6 time points (ZT 0, ZT 4, ZT 8, ZT 12, ZT 16, ZT 20), and hippocampal tissue transcriptome, proteome and phosphorylation modification group data of CON group and CIH group at 2 time points (ZT 4, ZT 16). The key pathogenic factors and pathways were identified by multiplex analysis (including GO annotation, KEGG pathway enrichment, subcellular localization, protein-protein interaction, kinase prediction analysis, etc.). The circadian rhythms of different protein phosphorylation modification sites were calculated based on R language loading Metacycle software package. The circadian rhythm of the same parameter was calculated by ARSER algorithm at 6 time points, and the difference of the expression of the same parameter at a certain time point between the two groups was calculated by unpaired t-test. Immunofluorescence assay was used to detect the circadian rhythm of glial cell markers (GFAP and IBA-1) in hippocampus of mice in CON group and CIH group. CIH mice were intervened by intraperitoneal injection of rapamycin (inhibitor of mTOR pathway). The functional and structural changes of hippocampus were observed by behavioral experiment, brain pathological staining and Bold-fMRI. At the same time, the rhythm changes of phosphorylation modification (p-mTOR, p-RPS6) of mTOR signal pathway were verified by Western blotting.

3. The map changes of acetylated hippocampal tissue of CIH mice were studied by LC-MS/MS technique for the first time. Bioinformatics techniques (GO annotation, KEGG pathway enrichment, motif analysis, secondary structure prediction, subcellular localization, protein domain analysis, protein-protein interaction analysis) were used to screen differential acetylation modified proteins, differential KEGG pathway, differential domain and so on. Western blot analysis was used to verify the levels of partially acetylated proteins (VDAC, Ywhaz, Camk2a). Intraperitoneal injection of sodium butyrate (Sodium butyrate, NaB) (histone deacetylase inhibitor) was used to intervene the mice in CIH group (named CIH+NaB group). The memory ability and pathological changes of hippocampus in CIH+NaB group were observed by behavioral experiment and brain pathological staining, and the anti-acetylated histone 3 lysine 9 site (anti-acetyl-Histone H3 (Lys9)) was verified by immunohistochemistry. H3K9ac) and anti-acetyl-Histone H3 (Lys27) (H3K27ac) level of antiacetylated histone 3 lysine..

Results:

1. Behavioral experiments (new object recognition, Y-maze, Barnes maze) showed that CIH significantly decreased the memory ability of mice. Brain Bold-fMRI showed that CIH could significantly inhibit the brain function of mice, especially in the right hippocampus. The results of pathological staining (H&E and Nissl staining) showed that the hippocampal DG region in CIH group was damaged, cell lysis and cytoplasmic vacuolization were found. The results of immunofluorescence staining showed that CIH decreased the expression of NEUN and DCX and increased the expression of GFAP and IBA-1 in hippocampus of mice. The results of inflammatory factor microarray showed that CIH significantly increased the levels of CXCL1, IL-10, CCL24, CXCL5, IL-6, TNF-α, CXCL16, CCL-1, IL-4 and CXCL12 in hippocampus.

2. Based on the data analysis of phosphorylated proteomics at 6 time points in hippocampal tissue of mice in CON group, we found that 1980 phosphorylated proteins expressed rhythmically. GO functional annotations showed that of the 1980 rhythmic proteins expressed, 87 were related to learning or memory and 134 were related to cognition. Time sequence analysis showed that both protein and phosphorylation modification sites tended to fluctuate significantly at ZT 4 (day) and ZT 16 (night). KEGG results showed that the above rhythmic proteins were mainly concentrated in mTOR signaling, axon guidance, pyruvate metabolism and other pathways. GO annotations showed that the phosphorylation modification sites of rhythm fluctuation were mainly concentrated in biological processes related to neural and synaptic functions, such as learning, neurogenesis, neuromuscular junction development and so on. Immunofluorescence showed that the characteristic markers GFAP and IBA-1 of glial cells had circadian rhythm, and the rhythm was interfered by CIH, especially at ZT 4 and Z16 time points. Two significant time points of ZT 4 and ZT 16 were selected to analyze the data of hippocampal transcription, proteome and phosphorylation modification group in CON group and CIH group. The results showed that CIH had significant interference on the rhythm of phosphorylation modification histology but had little effect on transcriptome. After intraperitoneal injection of rapamycin (mTOR inhibitor), behavior, brain Bold-fMRI and pathological staining showed that memory ability, hippocampal brain function and damaged neurons were significantly improved and repaired in CIH group. Western blotting showed that the rhythmic expression patterns of p-mTOR and p-RPS6 interfered by CIH could be saved by rapamycin.

3. Acetylome showed that CIH regulates 280 acetylation sites on 213 proteins, of which 35.75% are in mitochondria. KEGG pathway enrichment analysis showed that CIH significantly disrupted pathways such as oxidative phosphorylation, Citrate cycle, and glycolysis/Gluconeogenesis. Motif analysis results showed that the up-regulated acetylation modification sites in the CIH group preferred leucine, while the down-regulated acetylation modification sites preferred isoleucine. By comparison with the PLMD database, we successfully identified 292 proteins and 977 lysine acetylation sites. It is worth noting that among the identified lysine sites, we have also found multiple other types of PTMs, including ubiquitination (920), succinylation (776), propionylation (604), and glutarylation (209). Finally, after sodium butyrate (histone deacetylase inhibitor) intervened in CIH group mice, new object recognition experiments found that memory ability in the CIH+NaB group was improved. Immunohistochemical results showed that NaB could significantly reduce the CIH-induced upregulation of H3K27ac and H3K9ac expression.

Conclusions:

1. Chronic intermittent hypoxia may regulate hippocampal function through protein post-translational modifications (acetylation, phosphorylation).

2. The biological clock also has an important impact on the molecular mechanisms related to cognitive function in the hippocampus.