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论文题名(中文):

 睡眠障碍对消化道肿瘤患者术后胃肠道功能恢复的影响:菌群机制及营养干预初探    

姓名:

 陈沫汐    

论文语种:

 chi    

学位:

 博士    

学位类型:

 学术学位    

学校:

 北京协和医学院    

院系:

 北京协和医学院北京协和医院    

专业:

 临床医学-外科学    

指导教师姓名:

 于健春    

校内导师组成员姓名(逗号分隔):

 陈伟    

论文完成日期:

 2025-05-25    

论文题名(外文):

 The Impact of Sleep Disturbances on Postoperative Gastrointestinal Recovery in Patients with Gastrointestinal Tumors: Gut Microbiota Mechanisms and Nutritional Interventions    

关键词(中文):

 睡眠障碍 术后胃肠道功能障碍 肠道菌群 氧化应激 营养干预    

关键词(外文):

 Sleep Wake Disorders Postoperative Gastrointestinal Motility Disorders Gastrointestinal Microbiome Oxidative Stress Nutritional Support    

论文文摘(中文):

第一部分 术前睡眠障碍对消化道肿瘤患者术后胃肠道功能及肠屏障的影响

 

研究背景:围手术期睡眠障碍(Sleep Disturbances, SD)是指患者在手术前后出现的入睡困难、睡眠中断或睡眠碎片化等睡眠问题,其通过激活下丘脑-垂体-肾上腺(Hypothalamic-Pituitary- Adrenal,HPA)轴、诱导全身炎症及肠道菌群失调等机制,与术后恢复不良密切相关。肠内营养不耐受(Enteral Nutrition Intolerance,ENI)是消化道肿瘤患者术后常见并发症,但其与术前SD的关系尚不明确。本研究旨在探讨术前SD对术后胃肠道功能及肠屏障的影响,并揭示其潜在机制。

研究方法:本研究采用前瞻性队列研究设计,研究对象为2022年8月至2023年12月期间在北京协和医院进行消化道肿瘤手术并接受规范化营养治疗的患者,共67例(SD组26例,非SD组41例),采用匹兹堡睡眠质量指数及焦虑/抑郁自评量表评估术前睡眠及情绪状态,肠内营养耐受性评分表评估术后ENI发生情况。检测围术期血清肠屏障标志物(D-乳酸、二胺氧化酶、人脂多糖结合蛋白)、激素(皮质醇、5-羟色胺)、炎症指标(C反应蛋白、全身免疫炎症指数、预后营养指数;IL-6、IL-10、TNF-α等)、铁死亡标志物(Fe2+、还原型谷胱甘肽、脂质过氧化物丙二醛)及肠道菌群特征,分析其在SD与非SD组间的差异。

研究结果:SD组ENI发生率为53.8%,显著高于非SD组(26.8%,P=0.038)。多因素Logistic回归显示,入睡困难(OR=2.755,P=0.033)为ENI独立危险因素。SD组术前皮质醇水平显著升高(P<0.05),且与肠屏障标志物D-乳酸(r=0.4874,P=0.0012)及人脂多糖结合蛋白(r=0.3233,P=0.0367)水平的升高呈正相关。SD组D-乳酸、二胺氧化酶及人脂多糖结合蛋白(P<0.05)均高于非SD组,且与C反应蛋白(r=0.3538,P=0.0215)水平的升高呈正相关。SD组术前还原型谷胱甘肽显著降低(P<0.01),脂质过氧化物丙二醛显著升高(P<0.001),且丙二醛水平与皮质醇(r=0.3660,P=0.0171)及人脂多糖结合蛋白(r=0.3621,P=0.0170)的水平呈正相关。SD组机会致病菌(Enterobacteriaceae、Burkholderiaceae等)相对丰度升高,益生菌(Bifidobacteriaceae、Akkermansiaceae等)相对丰度降低。代谢通路预测显示,SD组“查加斯病”及“蛋白质N-糖基化”通路上调,提示致病风险增加。

研究结论:术前SD通过HPA轴激活、氧化应激及菌群失调等机制加重消化道肿瘤患者的肠屏障损伤,导致术后ENI风险升高。临床需重视围术期睡眠管理,联合抗氧化应激治疗与肠道微生态干预,以改善术后胃肠道功能恢复。

 

第二部分 术前睡眠剥夺对小鼠腹部手术后胃肠道功能及肠屏障的影响

 

研究背景:术前睡眠剥夺(Sleep Deprivation)对术后胃肠道功能恢复的影响尚未明确,且急慢性睡眠剥夺的作用效果存在差异。急性睡眠剥夺可能通过激活副交感神经和5-羟色胺分泌短暂增强肠道动力,而慢性睡眠剥夺则因下丘脑-垂体-肾上腺(Hypothalamic-Pituitary- Adrenal,HPA)轴失衡、氧化应激及菌群失调导致肠屏障损伤,可能延缓胃肠道蠕动。本研究旨在探讨不同时长术前睡眠剥夺对小鼠腹部手术后肠道动力、屏障功能及菌群结构的影响,并揭示其潜在机制,包括铁死亡、炎症反应和菌群代谢通路的调控作用。

研究方法:24只C57BL/6J雄性小鼠随机分为短睡眠剥夺组(1周,SD组)、长睡眠剥夺组(4周,LSD组)及对应仅手术对照组(POI组,POI_L组)。通过干扰棒睡眠剥夺仪构建睡眠剥夺模型,随后实施标准化腹部手术。术后评估胃肠道运输功能(异硫氰酸荧光素-右旋糖酐分布)、肠道通透性(血清异硫氰酸荧光素-右旋糖酐浓度)、血清激素(5-羟色胺、促肾上腺皮质激素释放激素)及炎症因子(TNF-α、IL-6等)水平,检测肠道铁死亡标志物(Fe2+、还原型谷胱甘肽、脂质过氧化物丙二醛等)及紧密连接蛋白(Occludin和E-cadherin)表达。采用16SrRNA测序技术解析肠道微生物群落组成,并基于KEGG与MetaCyc两大生物信息学数据库对菌群功能进行系统评估,揭示样本间潜在的代谢通路差异。

研究结果:长睡眠剥夺组小鼠术后胃肠道运输功能显著延缓(荧光分布几何中心:2.64 vs. 4.15,P=0.003),肠黏膜损伤加重及肠道通透性升高。睡眠剥夺组小鼠血清促炎因子TNF-α(SD组62.53±3.33 vs POI组48.81±1.84 pg/ml, P<0.01;LSD组68.66±4.45 vs POI_L组53.28±3.76 pg/ml, P<0.01)、IL-6水平显著上升(SD组 131.74±7.03 vs POI组112.88±10.48 pg/ml, P<0.01;LSD组144.94±7.00 vs POI_L组127.74±4.34 pg/ml, P<0.01),抗炎因子IL-10降低(SD组13.58±1.33 vs POI组 16.25±0.82 pg/ml, P<0.01;LSD组12.32±1.03 vs POI_L组15.00±1.38 pg/ml, P<0.01),5-羟色胺水平下降(P<0.05),促肾上腺皮质激素释放激素升高(P<0.05)。肠道铁死亡相关指标显示,还原型谷胱甘肽水平显著降低(SD组5.32±0.16 vs POI组6.33±0.24μmol/L, P<0.01;LSD组5.01±0.19 vs POI_L组5.83±0.42μmol/L, P<0.01),脂质过氧化物丙二醛升高(SD组5.65±0.64 vs POI组4.92±0.46 nmol/ml, P<0.01;LSD组6.61±0.35 vs POI_L组5.02±0.52 nmol/ml, P<0.01),长链脂酰辅酶A合成酶家族成员4表达上调(P<0.05)而血红素加氧酶-1下调(P<0.05)。菌群分析发现,睡眠剥夺组机会致病菌(Enterobacteriaceae、Sphingobacteriaceae等)丰度升高,有益菌(Bifidobacteriaceae、Akkermansiaceae等)减少,代谢通路中四环素合成、Wnt信号通路及甲基乙二醛降解途径发生显著改变。

研究结论:术前睡眠剥夺通过HPA轴紊乱、氧化应激及炎症反应加剧术后肠道功能障碍,且影响呈时间依赖性。慢性睡眠剥夺导致肠道菌群失调,机会致病菌富集及代谢通路异常,进一步破坏肠屏障完整性。研究提示,改善围术期睡眠质量或针对性调节菌群和抗氧化通路可能有助于减轻术后胃肠功能障碍。

 

第三部分 营养干预改善睡眠剥夺小鼠腹部手术后肠道菌群紊乱及肠屏障功能

 

研究背景:睡眠剥夺引发的肠道炎症与氧化应激已成为影响术后康复的重要风险因素。近年研究表明,营养干预与肠道微生态疗法可能通过调节肠屏障功能、免疫应答及氧化还原平衡改善术后认知及胃肠道功能。本研究尝试应用色氨酸、谷氨酰胺、鱼油等免疫营养素,以及三联益生菌、盐酸小檗碱(Berberine Hydrochloride,BBH)、尿石素A(Urolithin A,UA)、吲哚-3-丙酸(Indole-3-propionic acid,IPA)等微生态干预措施,探讨其对术前睡眠剥夺小鼠术后肠道炎症、氧化应激及菌群稳态的调控作用,并探索其潜在机制。

研究方法:采用48只SPF级C57BL/6J雄性小鼠构建术前睡眠剥夺模型(20小时/天,持续1周),随机分为对照组及7个干预组(每组6只)。干预措施包括色氨酸(200 mg/kg)、鱼油(2.5 mL/kg)、谷氨酰胺(450 mg/kg)、三联益生菌(345 mg/kg)、BBH(150 mg/kg)、UA(20 mg/kg)及IPA(60 mg/kg),每日睡眠剥夺前灌胃干预,持续1周后实施标准化腹部手术。检测血清激素(5-羟色胺、促肾上腺皮质激素释放激素)、炎症因子(TNF-α、IL-6等)、肠道形态学(HE染色)、紧密连接蛋白(Occludin和E-cadherin)表达,以及铁死亡标志物(Fe²⁺、还原型谷胱甘肽、脂质过氧化物丙二醛等)水平。通过16S rRNA测序技术,系统评估肠道微生物群落的组成特征,并通过生物信息学方法解析不同样本间代谢通路的差异性。

研究结果:免疫营养素干预及肠道微生态疗法均能显著改善肠道细胞形态,上调紧密连接蛋白Occludin和E-cadherin表达(P<0.05),并降低血清5-羟色胺、促肾上腺皮质激素释放激素及促炎因子(TNF-α、IL-6)水平,同时升高抗炎因子IL-10水平(P<0.05)。此外,营养干预还降低了铁死亡标志物Fe²⁺和脂质过氧化物丙二醛水平(对照组7.35±0.46 vs 色氨酸组5.23±1.08 vs 鱼油组4.22±1.40 vs 谷氨酰胺组3.86±0.51 vs 三联益生菌组3.39±0.80 vs BBH组2.88±0.57 vs IPA组4.06±0.71 vs UA组2.69±0.49 nmol/ml)、提高了抗氧化物还原型谷胱甘肽水平(对照组5.74±0.38 vs 色氨酸组6.94±1.58 vs 鱼油组9.15±1.38 vs 谷氨酰胺组7.98±0.32 vs 三联益生菌组7.84±0.76 vs BBH组10.37±1.66 vs IPA组8.07±0.77 vs UA组10.77±0.55 μmol/L);铁死亡相关蛋白长链脂酰辅酶A合成酶家族成员4的表达被显著抑制,而抗氧化酶血红素加氧酶-1的表达增强(P<0.05)。肠道微生态疗法提高了术前睡眠剥夺小鼠肠道菌群中Akkermansiaceae、Lactobacillaceae和Bifidobacteriaceae等有益菌的相对丰度,降低了Burkholderiaceae和Enterococcaceae等机会致病菌的丰度,色氨酸相关代谢通路的激活可能是其作用的主要机制。

研究结论:术前短期免疫营养素与肠道微生态干预可有效缓解睡眠剥夺诱导的肠屏障损伤、全身炎症及氧化应激。肠道菌群分析表明,干预措施通过调节益生菌丰度、激活色氨酸代谢通路及抑制机会致病菌增殖等机制改善菌群稳态。未来需结合长期干预和临床转化研究,优化围术期睡眠及肠屏障功能管理策略。

论文文摘(外文):

Part I: Impact of Preoperative Sleep Disturbances on Postoperative Gastrointestinal Function and Intestinal Barrier in Gastrointestinal Cancer Patients

 

Background:Perioperative sleep disturbances (SD) are characterized by difficulties in falling asleep, sleep interruptions, or fragmented sleep before and after surgery, and have been closely linked to poor postoperative recovery. These effects are mediated through mechanisms such as hypothalamic-pituitary-adrenal (HPA) axis activation, systemic inflammation induction, and gut microbiota dysbiosis. Enteral nutrition intolerance (ENI) is a common postoperative complication in gastrointestinal cancer patients, but its relationship with preoperative SD remains unclear. This study aimed to investigate the effects of preoperative SD on postoperative gastrointestinal function and intestinal barrier integrity and to elucidate the underlying mechanisms.

Methods:A prospective cohort study was conducted on 67 gastrointestinal cancer patients undergoing surgery at Peking Union Medical College Hospital from August 2022 to December 2023 (SD group: 26 cases; non-SD group: 41 cases). Preoperative sleep quality and emotional status were assessed using the Pittsburgh Sleep Quality Index and anxiety/depression self-rating scales. Postoperative ENI was evaluated using an enteral nutrition tolerance assessment scale. Perioperative serum markers of intestinal barrier integrity (D-lactate, diamine oxidase, lipopolysaccharide-binding protein), hormones (cortisol, 5-hydroxytryptamine), inflammatory indicators (C-reactive protein, systemic immune-inflammatory index, prognostic nutritional index; IL-6, IL-10, TNF-α), ferroptosis markers (Fe²⁺, reduced glutathione, malondialdehyde), and gut microbiota profiles were analyzed to compare differences between SD and non-SD groups.

Results:The incidence of ENI in the SD group (53.8%) was significantly higher than in the non-SD group (26.8%, P=0.038). Multivariate logistic regression identified difficulty falling asleep (OR=3.168, P=0.037) and preoperative hypertension (OR=25.717, P=0.010) as independent risk factors for ENI. Preoperative cortisol levels were significantly elevated in the SD group (P<0.05) and positively correlated with intestinal barrier markers D-lactate (r=0.4874, P=0.0012) and lipopolysaccharide-binding protein (r=0.3233, P=0.0367). The SD group exhibited higher D-lactate, diamine oxidase, and lipopolysaccharide-binding protein levels (P<0.05) and a positive correlation with C-reactive protein (r=0.3538, P=0.0215). Reduced glutathione (P<0.01) and increased malondialdehyde (P<0.001) were observed in the SD group, with malondialdehyde levels positively correlated with cortisol (r=0.3660, P=0.0171) and lipopolysaccharide-binding protein (r=0.3621, P=0.0170). Gut microbiota analysis revealed increased relative abundance of opportunistic pathogens (Enterobacteriaceae, Burkholderiaceae, Streptococcaceae) and decreased probiotics (Bifidobacteriaceae, Akkermansiaceae) in the SD group. Metabolic pathway predictions indicated upregulation of "Chagas disease" and "protein N-glycosylation" pathways, suggesting elevated pathogenic risks.

Conclusion:Preoperative SD exacerbates intestinal barrier damage and increases postoperative ENI risk in gastrointestinal cancer patients via HPA axis activation, oxidative stress, and microbiota dysbiosis. Clinical emphasis on perioperative sleep management, combined with antioxidant therapy and gut microbiota interventions, is critical to improving postoperative gastrointestinal recovery.

 

Part II: Effects of Preoperative Sleep Deprivation on Gastrointestinal Function and Intestinal Barrier in Mice After Abdominal Surgery

 

Background:The impact of preoperative sleep deprivation (SD) on postoperative gastrointestinal recovery remains unclear, with differential effects between acute and chronic SD. Acute SD may transiently enhance intestinal motility via parasympathetic activation and 5-hydroxytryptamine secretion, while chronic SD disrupts intestinal barrier integrity due to HPA axis imbalance, oxidative stress, and microbiota dysbiosis. This study aimed to explore the effects of varying SD durations on postoperative intestinal motility, barrier function, and microbiota structure in mice, focusing on ferroptosis, inflammation, and microbial metabolic pathways.

Methods:Twenty-four C57BL/6J male mice were randomized into short-term SD (1 week), long-term SD (4 weeks), and surgery-only control groups. A standardized abdominal surgery was performed after establishing SD models using a sleep deprivation apparatus. Postoperative assessments included gastrointestinal transit (fluorescein isothiocyanate-dextran distribution), intestinal permeability (serum FITC-dextran), serum hormones (5-hydroxytryptamine, corticotropin-releasing hormone), inflammatory cytokines (TNF-α, IL-6), ferroptosis markers (Fe²⁺, glutathione, malondialdehyde), and tight junction proteins (occludin, E-cadherin). Gut microbiota composition was analyzed via 16S rRNA sequencing, with KEGG and MetaCyc databases predicting metabolic pathways.

Results:Long-term SD mice exhibited delayed gastrointestinal transit (geometric center of fluorescence: 2.64 vs. 4.15, P=0.003), increased intestinal permeability (P<0.01), elevated pro-inflammatory cytokines (TNF-αSD 62.53±3.33 vs POI 48.81±1.84 pg/ml, P<0.01;LSD 68.66±4.45 vs POI_L 53.28±3.76 pg/ml, P<0.01;IL-6 SD  131.74±7.03 vs POI 112.88±10.48 pg/ml, P<0.01;LSD 144.94±7.00 vs POI_L 127.74±4.34 pg/ml, P<0.01), reduced anti-inflammatory IL-10 (SD 13.58±1.33 vs POI  16.25±0.82 pg/ml, P<0.01;LSD 12.32±1.03 vs POI_L 15.00±1.38 pg/ml, P<0.01), decreased 5-hydroxytryptamine (P<0.05), and increased corticotropin-releasing hormone (P<0.05). Ferroptosis markers showed reduced glutathione (SD 5.32±0.16 vs POI 6.33±0.24μmol/L, P<0.01;LSD 5.01±0.19 vs POI_L 5.83±0.42μmol/L, P<0.01), elevated malondialdehyde (SD 5.65±0.64 vs POI 4.92±0.46 nmol/ml, P<0.01;LSD 6.61±0.35 vs POI_L 5.02±0.52 nmol/ml, P<0.01), upregulated acyl-CoA synthetase long-chain family member 4, and downregulated heme oxygenase-1 (P<0.05). Microbiota analysis revealed increased opportunistic pathogens (Enterobacteriaceae, Sphingobacteriaceae) and decreased probiotics (Bifidobacteriaceae, Akkermansiaceae), with altered pathways in tetracycline synthesis, Wnt signaling, and methylglyoxal degradation.

Conclusion:Preoperative SD exacerbates postoperative intestinal dysfunction via HPA axis dysregulation, oxidative stress, and inflammation, with time-dependent severity. Chronic SD promotes microbiota dysbiosis and pathogenic pathway activation, impairing barrier integrity. Improving perioperative sleep quality and targeting microbiota or antioxidant pathways may mitigate gastrointestinal complications.

 

Part III: Nutritional Interventions Alleviate Gut Microbiota Dysbiosis and Intestinal Barrier Dysfunction in Sleep-Deprived Mice After Abdominal Surgery

 

Background:Sleep deprivation-induced intestinal inflammation and oxidative stress are critical risk factors for postoperative recovery. Emerging evidence suggests that immunonutrients and microbiota-targeted therapies may improve cognitive and gastrointestinal function by modulating barrier integrity, immune responses, and redox balance. This study evaluated the efficacy of tryptophan, glutamine, fish oil, probiotics, berberine hydrochloride (BBH), urolithin A (UA), and indole-3-propionic acid (IPA) in mitigating SD-induced intestinal damage.

Methods:Forty-eight SPF C57BL/6J male mice underwent 1-week SD (20 hours/day) and were randomized into control and seven intervention groups (n=6/group). Interventions included tryptophan (200 mg/kg), fish oil (2.5 mL/kg), glutamine (450 mg/kg), probiotics (345 mg/kg), BBH (150 mg/kg), UA (20 mg/kg), and IPA (60 mg/kg), administered daily before SD. Postoperative assessments included serum hormones, inflammatory cytokines, intestinal histology, tight junction proteins, ferroptosis markers, and 16S rRNA-based microbiota analysis.

Results:Interventions improved intestinal morphology, upregulated occludin and E-cadherin (P<0.05), reduced pro-inflammatory cytokines (TNF-α, IL-6) and corticotropin-releasing hormone, and increased IL-10 (P<0.05). Ferroptosis markers showed reduced Fe²⁺ and malondialdehyde (Control 7.35±0.46 vs Trp 5.23±1.08 vs Fish Oil 4.22±1.40 vs Gln 3.86±0.51 vs Bifico 3.39±0.80 vs BBH 2.88±0.57 vs IPA 4.06±0.71 vs UA 2.69±0.49 nmol/ml) and increased glutathione (Control 5.74±0.38 vs Trp 6.94±1.58 vs Fish Oil 9.15±1.38 vs Gln 7.98±0.32 vs Bifico 7.84±0.76 vs BBH 10.37±1.66 vs IPA 8.07±0.77 vs UA 10.77±0.55μmol/L) , with suppressed acyl-CoA synthetase long-chain family member 4 and enhanced heme oxygenase-1 expression (P<0.05). Microbiota analysis demonstrated increased probiotics (Akkermansiaceae, Lactobacillaceae, Bifidobacteriaceae) and reduced opportunistic pathogens (Burkholderiaceae, Enterococcaceae), linked to activated tryptophan metabolism.

Conclusion:Short-term immunonutrient and microbiota interventions effectively alleviate SD-induced barrier damage, inflammation, and oxidative stress. Modulation of probiotics, tryptophan pathways, and suppression of opportunistic pathogens underlie these benefits. Future studies should optimize perioperative strategies integrating sleep management, anti-inflammatory therapies, and microbiota modulation to enhance clinical outcomes.

开放日期:

 2025-05-26    

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