Maternal sleep deprivation induces gut microbial dysbiosis and neuroinflammation in offspring rats
摘要: 孕期睡眠剥夺(Maternal sleep deprivation, MSD)已成为影响孕产妇身心健康及新生儿早期发育的全球公共卫生问题。最新的研究进展表明，睡眠剥夺(Sleep deprivation, SD)会破坏肠道微生物群，导致宿主神经炎症和精神异常。然而，尚不清楚MSD是否会影响新生儿肠道菌群的建立和神经炎症。该研究对妊娠晚期(妊娠15～21天)的Sprague-Dawley大鼠进行MSD处理，然后在出生后不同时间点(P1、P7、P14、P56)收集子代的肠道内容物和脑组织。通过菌群测序发现，受MSD影响的孕鼠粪便微生物多样性和丰富度增加，主要表现为厚壁菌门显著增加；同时我们在MSD子代中也观察到以厚壁菌门类细菌增多为标志的肠道菌群失调。进一步的qRT-PCR和ELISA检测发现，MSD成年后代脑内促炎细胞因子IL-1β和TNF-α的表达显著高于对照组(P56)。Spearman相关性分析显示，IL-1β和TNF-α与Ruminococcus_1和Ruminococcaceae_UCG-005这两类厚壁菌门的丰度呈正相关，从而提示肠道菌群与宿主神经发育密切相关。综上所述，MSD改变了母体肠道菌群，并影响了子代早期肠道菌群的建立，最后导致MSD后代出现特定菌群相关的神经炎症。因此，揭示肠道菌群在个体生理发育过程中的作用，将可能为治疗MSD后代的认知功能障碍提供潜在的干预措施。Abstract: Maternal sleep deprivation (MSD) is a global public health problem that affects the physical and mental development of pregnant women and their newborns. The latest research suggests that sleep deprivation (SD) disrupts the gut microbiota, leading to neuroinflammation and psychological disturbances. However, it is unclear whether MSD affects the establishment of gut microbiota and neuroinflammation in the newborns. In the present study, MSD was performed on pregnant Sprague-Dawley rats in the third trimester of pregnancy (gestational days 15–21), after which intestinal contents and brain tissues were collected from offspring at different postnatal days (P1, P7, P14, and P56). Based on microbial profiling, microbial diversity and richness increased in pregnant rats subjected to MSD, as reflected by the significant increase in the phylum Firmicutes. In addition, microbial dysbiosis marked by abundant Firmicutes bacteria was observed in the MSD offspring. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that the expression levels of proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were significantly higher in the MSD offspring at adulthood (P56) than in the control group. Through Spearman correlation analysis, IL-1β and TNF-α were also shown to be positively correlated with Ruminococcus_1 and Ruminococcaceae_UCG-005 at P56, which may determine the microbiota-host interactions in MSD-related neuroinflammation. Collectively, these results indicate that MSD changes maternal gut microbiota and affects the establishment of neonatal gut microbiota, leading to neuroinflammation in MSD offspring. Therefore, understanding the role of gut microbiota during physiological development may provide potential interventions for cognitive dysfunction in MSD-impacted offspring.
Figure 1. Experimental design
Pregnant rats were subjected to MSD for 6 h per day by acclimation to gentle handling in third trimester of pregnancy (gestational days 15–21). Fecal samples of mother rats (M) and intestinal contents and brain tissues of offspring were collected at different postnatal days (P1, P7, P14, and P56).
Figure 2. Microbial richness and diversity increased in sleep-deprived pregnant rats
A: Alpha diversity indices increased in both richness and diversity of gut microbiota in MSD group (MSD-M). B: Weighted UniFrac PCoA showed no significant difference in microbial composition between MSD-M and control (C-M) groups. C, D: Relative abundance of top 20 microbial phyla (C) and genera (D) were analyzed to show subtle differences between MSD-M and C-M groups. All OTUs under 1% were classified as “others”. Data are expressed as means±SD, *: P<0.05.
Figure 3. Microbial diversity and richness increased in both control and MSD offspring
A: Alpha diversity indices steadily increased in diversity and richness of gut microbiota in control offspring. B: Weighted UniFrac PCoA analysis showed significant differences in microbial composition at different postnatal time points in control offspring. C, D: Relative abundances of top 20 microbial phyla (C) and genera (D) were clustered to show changes from mothers to offspring in control group. E: Alpha diversity indices showed a steady increase in diversity and richness of gut microbiota in MSD offspring. F: Weighted UniFrac PCoA showed significant differences in microbial composition at different postnatal time points in MSD offspring. G, H: Relative abundances of top 20 microbial phyla (G) and genera (H) were clustered to show changes from mother rats to offspring in MSD group. Data are expressed as means±SD, *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001.
Figure 4. Changes in gut microbiota of MSD offspring
Alpha diversity indices showed changes in richness and diversity of microbiota at P1 (A), P7 (E), P14 (I), and P56 (M) in both control and MSD offspring. Weighted UniFrac PCoA showed microbial composition at P1 (B), P7 (F), P14 (J), and P56 (N) in both control and MSD offspring. Relative abundances of top 20 microbial phyla (C, G, K, O) and genera (D, H, L, P) were clustered to show differences between control and MSD offspring. Data are expressed as means±SD, *: P<0.05.
Figure 5. Dominant bacteria in MSD offspring at different postnatal time points
Linear discriminant analysis (LDA) effect size (LEfSe) showed different abundant bacterial taxa in mother rats (A) and offspring at P1 (C), P7 (D), P14 (F), and P56 (H). Different bacteria were defined by LDA scores with a cut-off of 3.5. Pie diagram summarized phylogenetic changes in mother rats (B) and their offspring at P7 (E) and P14 (G).
Figure 6. MSD increased proinflammatory cytokines in brains of MSD offspring
A–C: mRNA expression of proinflammatory cytokines IL-1β (A), IL-6 (B), and TNF-α (C) in brains of MSD offspring measured by qRT-PCR. D–F: Proinflammatory cytokines IL-1β (D), IL-6 (E), and TNF-α (F) in brains measured by ELISA. G–I: Spearman heat-map showed correlations among top 30 microbial genera and proinflammatory cytokines IL-1β, IL-6, and TNF-α at P1 (G), P7 (I), and P56 (H). Data are expressed as means±SD, *: P<0.05; **: P<0.01.
 Alvarenga TA, Aguiar MFP, Mazaro-Costa R, Tufik S, Andersen ML. 2013. Effects of sleep deprivation during pregnancy on the reproductive capability of the offspring. Fertility and Sterility, 100(6): 1752−1757. doi: 10.1016/j.fertnstert.2013.08.014  Argeri R, Nishi EE, Volpini RA, Palma BD, Tufik S, Gomes GN. 2016. Sleep restriction during pregnancy and its effects on blood pressure and renal function among female offspring. Physiological Reports, 4(16): e12888. doi: 10.14814/phy2.12888  Aswathy BS, Kumar VM, Gulia KK. 2018. The effects of rapid eye movement sleep deprivation during late pregnancy on newborns' sleep. Journal of Sleep Research, 27(2): 197−205. doi: 10.1111/jsr.12564  Baratta AM, Kanyuch NR, Cole CA, Valafar H, Deslauriers J, Pocivavsek A. 2020. Acute sleep deprivation during pregnancy in rats: rapid elevation of placental and fetal inflammation and kynurenic acid. Neurobiology of Stress, 12: 100204. doi: 10.1016/j.ynstr.2019.100204  Benedict C, Vogel H, Jonas W, Woting A, Blaut M, Schürmann A, et al. 2016. Gut microbiota and glucometabolic alterations in response to recurrent partial sleep deprivation in normal-weight young individuals. Molecular Metabolism, 5(12): 1175−1186. doi: 10.1016/j.molmet.2016.10.003  Chassaing B, Gewirtz AT. 2014. Gut microbiota, low-grade inflammation, and metabolic syndrome. Toxicologic Pathology, 42(1): 49−53. doi: 10.1177/0192623313508481  Chien MY, Chen HC. 2015. Poor sleep quality is independently associated with physical disability in older adults. Journal of Clinical Sleep Medicine, 11(3): 225−232. doi: 10.5664/jcsm.4532  Crusell MKW, Hansen TH, Nielsen T, Allin KH, Rühlemann MC, Damm P, et al. 2018. Gestational diabetes is associated with change in the gut microbiota composition in third trimester of pregnancy and postpartum. Microbiome, 6(1): 89. doi: 10.1186/s40168-018-0472-x  Du M, Liu J, Han N, Zhao ZL, Yang J, Xu XR, et al. 2021. Maternal sleep quality during early pregnancy, risk factors and its impact on pregnancy outcomes: a prospective cohort study. Sleep Medicine, 79: 11−18. doi: 10.1016/j.sleep.2020.12.040  El Aidy S, Bolsius YG, Raven F, Havekes R. 2020. A brief period of sleep deprivation leads to subtle changes in mouse gut microbiota. Journal of Sleep Research, 29(6): e12920.  Francis AP, Dominguez-Bello MG. 2019. Early-life microbiota perturbations and behavioral effects. Trends in Microbiology, 27(7): 567−569. doi: 10.1016/j.tim.2019.04.004  Fricke EM, Elgin TG, Gong HY, Reese J, Gibson-Corley KN, Weiss RM, et al. 2018. Lipopolysaccharide-induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood. American Journal of Reproductive Immunology, 79(5): e12816. doi: 10.1111/aji.12816  Gao T, Wang ZX, Dong YL, Cao J, Lin RT, Wang XT, et al. 2019. Role of melatonin in sleep deprivation-induced intestinal barrier dysfunction in mice. Journal of Pineal Research, 67(1): e12574.  García-Gómez E, González-Pedrajo B, Camacho-Arroyo I. 2013. Role of sex steroid hormones in bacterial-host interactions. BioMed Research International, 2013: 928290.  Goyal D, Ali SA, Singh RK. 2021. Emerging role of gut microbiota in modulation of neuroinflammation and neurodegeneration with emphasis on Alzheimer's disease. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 106: 110112. doi: 10.1016/j.pnpbp.2020.110112  Grech A, Collins CE, Holmes A, Lal R, Duncanson K, Taylor R, et al. 2021. Maternal exposures and the infant gut microbiome: a systematic review with meta-analysis. Gut Microbes, 13(1): 1897210. doi: 10.1080/19490976.2021.1897210  Hajela N, Ramakrishna BS, Nair GB, Abraham P, Gopalan S, Ganguly NK. 2015. Gut microbiome, gut function, and probiotics: implications for health. Indian Journal of Gastroenterology, 34(2): 93−107. doi: 10.1007/s12664-015-0547-6  Harskamp-van Ginkel MW, Ierodiakonou D, Margetaki K, Vafeiadi M, Karachaliou M, Kogevinas M, et al. 2020. Gestational sleep deprivation is associated with higher offspring body mass index and blood pressure. Sleep, 43(12): zsaa110. doi: 10.1093/sleep/zsaa110  Herlemann DP, Labrenz M, Jürgens K, Bertilsson S, Waniek JJ, Andersson AF. 2011. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. The ISME Journal, 5(10): 1571−1579. doi: 10.1038/ismej.2011.41  Hutchison BL, Stone PR, Mccowan LM, Stewart AW, Thompson JM, Mitchell EA. 2012. A postal survey of maternal sleep in late pregnancy. BMC Pregnancy and Childbirth, 12: 144. doi: 10.1186/1471-2393-12-144  Jiang Y, Li PX, Zhong L, Liu BY, Gao XY, Ning L, et al. 2021. The influence of changes in work stressors and coping resources on sleep disturbances: evidence from the OHSPIW cohort study. Sleep, 44(8): zsab039. doi: 10.1093/sleep/zsab039  John GK, Mullin GE. 2016. The gut microbiome and obesity. Current Oncology Reports, 18(7): 45. doi: 10.1007/s11912-016-0528-7  Khalyfa A, Mutskov V, Carreras A, Khalyfa AA, Hakim F, Gozal D. 2014. Sleep fragmentation during late gestation induces metabolic perturbations and epigenetic changes in adiponectin gene expression in male adult offspring mice. Diabetes, 63(10): 3230−3241. doi: 10.2337/db14-0202  Koliada A, Syzenko G, Moseiko V, Budovska L, Puchkov K, Perederiy V, et al. 2017. Association between body mass index and Firmicutes/Bacteroidetes ratio in an adult Ukrainian population. BMC Microbiology, 17(1): 120. doi: 10.1186/s12866-017-1027-1  Koren O, Goodrich JK, Cullender TC, Spor A, Laitinen K, Bäckhed HK, et al. 2012. Host remodeling of the gut microbiome and metabolic changes during pregnancy. Cell, 150(3): 470−480. doi: 10.1016/j.cell.2012.07.008  Li DT, Wang P, Wang PP, Hu XS, Chen F. 2016. The gut microbiota: a treasure for human health. Biotechnology Advances, 34(7): 1210−1224. doi: 10.1016/j.biotechadv.2016.08.003  Li YY, Zhang B, Zhou Y, Wang DM, Liu XC, Li L, et al. 2020. Gut microbiota changes and their relationship with inflammation in patients with acute and chronic insomnia. Nature and Science of Sleep, 12: 895−905. doi: 10.2147/NSS.S271927  Lima ILB, Rodrigues AFAC, Bergamaschi CT, Campos RR, Hirata AE, Tufik S, et al. 2014. Chronic sleep restriction during pregnancy-repercussion on cardiovascular and renal functioning of male offspring. PLoS One, 9(11): e113075. doi: 10.1371/journal.pone.0113075  Luczynski P, McVey Neufeld KA, Oriach CS, Clarke G, Dinan TG, Cryan JF. 2016. Growing up in a bubble: using germ-free animals to assess the influence of the gut microbiota on brain and behavior. International Journal of Neuropsychopharmacology, 19(8): pyw020. doi: 10.1093/ijnp/pyw020  Maki KA, Burke LA, Calik MW, Watanabe-Chailland M, Sweeney D, Romick-Rosendale LE, et al. 2020. Sleep fragmentation increases blood pressure and is associated with alterations in the gut microbiome and fecal metabolome in rats. Physiological Genomics, 52(7): 280−292. doi: 10.1152/physiolgenomics.00039.2020  Matenchuk BA, Mandhane PJ, Kozyrskyj AL. 2020. Sleep, circadian rhythm, and gut microbiota. Sleep Medicine Reviews, 53: 101340. doi: 10.1016/j.smrv.2020.101340  Mindell JA, Cook RA, Nikolovski J. 2015. Sleep patterns and sleep disturbances across pregnancy. Sleep Medicine, 16(4): 483−488. doi: 10.1016/j.sleep.2014.12.006  Nam S, Whittemore R, Jung S, Latkin C, Kershaw T, Redeker NS. 2018. Physical neighborhood and social environment, beliefs about sleep, sleep hygiene behaviors, and sleep quality among African Americans. Sleep Health, 4(3): 258−264. doi: 10.1016/j.sleh.2018.03.002  Nuriel-Ohayon M, Neuman H, Ziv O, Belogolovski A, Barsheshet Y, Bloch N, et al. 2019. Progesterone increases Bifidobacterium relative abundance during late pregnancy. Cell Reports, 27(3): 730−736.e3. doi: 10.1016/j.celrep.2019.03.075  Okun ML, Coussons-Read ME. 2007. Sleep disruption during pregnancy: how does it influence serum cytokines?. Journal of Reproductive Immunology, 73(2): 158−165. doi: 10.1016/j.jri.2006.06.006  Peng Y, Wang W, Tan T, He WT, Dong ZF, Wang YT, et al. 2016. Maternal sleep deprivation at different stages of pregnancy impairs the emotional and cognitive functions, and suppresses hippocampal long-term potentiation in the offspring rats. Molecular Brain, 9: 17. doi: 10.1186/s13041-016-0197-3  Pien GW, Schwab RJ. 2004. Sleep disorders during pregnancy. Sleep, 27(7): 1405−1417. doi: 10.1093/sleep/27.7.1405  Pires GN, Benedetto L, Cortese R, Gozal D, Gulia KK, Kumar VM, et al. 2021. Effects of sleep modulation during pregnancy in the mother and offspring: evidences from preclinical research. Journal of Sleep Research, 30(3): e13135.  Poroyko VA, Carreras A, Khalyfa A, Khalyfa AA, Leone V, Peris E, et al. 2016. Chronic sleep disruption alters gut microbiota, induces systemic and adipose tissue inflammation and insulin resistance in Mice. Scientific Reports, 6: 35405. doi: 10.1038/srep35405  Qi XY, Yun CY, Pang YL, Qiao J. 2021. The impact of the gut microbiota on the reproductive and metabolic endocrine system. Gut Microbes, 13(1): 1894070. doi: 10.1080/19490976.2021.1894070  Radhakrishnan A, Aswathy BS, Kumar VM, Gulia KK. 2015. Sleep deprivation during late pregnancy produces hyperactivity and increased risk-taking behavior in offspring. Brain Research, 1596: 88−98. doi: 10.1016/j.brainres.2014.11.021  Rao JJ, Qiao Y, Xie RN, Lin L, Jiang J, Wang CM, et al. 2021. Fecal microbiota transplantation ameliorates stress-induced depression-like behaviors associated with the inhibition of glial and NLRP3 inflammasome in rat brain. Journal of Psychiatric Research, 137: 147−157. doi: 10.1016/j.jpsychires.2021.02.057  Roque A, Ochoa-Zarzosa A, Torner L. 2016. Maternal separation activates microglial cells and induces an inflammatory response in the hippocampus of male rat pups, independently of hypothalamic and peripheral cytokine levels. Brain, Behavior, and Immunity, 55: 39−48. doi: 10.1016/j.bbi.2015.09.017  Thomal JT, Palma BD, Ponzio BF, do Carmo Pinho Franco M, Zaladek-Gil F, Fortes ZB, et al. 2010. Sleep restriction during pregnancy: hypertension and renal abnormalities in young offspring rats. Sleep, 33(10): 1357−1362. doi: 10.1093/sleep/33.10.1357  Tochitani S. 2021. Vertical transmission of gut microbiota: points of action of environmental factors influencing brain development. Neuroscience Research, 168: 83−94. doi: 10.1016/j.neures.2020.11.006  Vecsey CG, Baillie GS, Jaganath D, Havekes R, Daniels A, Wimmer M, et al. 2009. Sleep deprivation impairs cAMP signalling in the hippocampus. Nature, 461(7267): 1122−1125. doi: 10.1038/nature08488  Vecsey CG, Wimmer MEJ, Havekes R, Park AJ, Perron IJ, Meerlo P, et al. 2013. Daily acclimation handling does not affect hippocampal long-term potentiation or cause chronic sleep deprivation in Mice. Sleep, 36(4): 601−607. doi: 10.5665/sleep.2556  Wang HX, Wang YP. 2016. Gut microbiota-brain axis. Chinese Medical Journal, 129(19): 2373−2380. doi: 10.4103/0366-6999.190667  Wang Z, Chen WH, Li SX, He ZM, Zhu WL, Ji YB, et al. 2021. Gut microbiota modulates the inflammatory response and cognitive impairment induced by sleep deprivation. Molecular Psychiatry, 26(11): 6277−6292. doi: 10.1038/s41380-021-01113-1  Yoo JY, Groer M, Dutra SVO, Sarkar A, Mcskimming DI. 2020. Gut microbiota and immune system interactions. Microorganisms, 8(10): 1587. doi: 10.3390/microorganisms8101587  Yu YZ, Huang ZL, Dai CF, Du YH, Han HL, Wang YT, et al. 2018. Facilitated AMPAR endocytosis causally contributes to the maternal sleep deprivation-induced impairments of synaptic plasticity and cognition in the offspring rats. Neuropharmacology, 133: 155−162. doi: 10.1016/j.neuropharm.2018.01.030  Zhao Q, Elson CO. 2018. Adaptive immune education by gut microbiota antigens. Immunology, 154(1): 28−37. doi: 10.1111/imm.12896  Zhao QY, Peng C, Wu XH, Chen YB, Wang C, You ZL. 2014. Maternal sleep deprivation inhibits hippocampal neurogenesis associated with inflammatory response in young offspring rats. Neurobiology of Disease, 68: 57−65. doi: 10.1016/j.nbd.2014.04.008  Zhao QY, Xie XF, Fan YH, Zhang JQ, Jiang W, Wu XH, et al. 2015. Phenotypic dysregulation of microglial activation in young offspring rats with maternal sleep deprivation-induced cognitive impairment. Scientific Reports, 5: 9513. doi: 10.1038/srep09513