Pathological damage of medullary visceral zone induced by early sepsis and intervention effect of the cholinergic anti-inflammatory pathway

Li Chujuan; Li Hongbing; Shi Lan; Liu Xian; Wang Ying; Zheng Quan

doi:10.13406/j.cnki.cyxb.003575

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Pathological damage of medullary visceral zone induced by early sepsis and intervention effect of the cholinergic anti-inflammatory pathway

Basic research | 更新时间：2025-10-21

- Pathological damage of medullary visceral zone induced by early sepsis and intervention effect of the cholinergic anti-inflammatory pathway
- Journal of Chongqing Medical University Vol. 49, Issue 9, Pages: 1095-1104(2024)
- 作者机构：
  
  1.湖北省肿瘤医院妇科肿瘤科，武汉 430060
  2.贵阳市第一人民医院全科医学科，贵阳　550002
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.003575
  CLC： R114
- Received：31 July 2023，
  
  Revised：2024-01-09，
  
  Accepted：12 March 2024，
  
  Published：28 September 2024
- 稿件说明：
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李楚娟,李红兵,史兰,等.早期脓毒症对延髓内脏带的病理损害及胆碱能抗炎通路的干预效应[J].重庆医科大学学报,2024,49(9):1095-1104.

Li Chujuan,Li Hongbing,Shi Lan,et al.Pathological damage of medullary visceral zone induced by early sepsis and intervention effect of the cholinergic anti-inflammatory pathway[J].Journal of Chongqing Medical University,2024,49(9):1095-1104.
李楚娟,李红兵,史兰,等.早期脓毒症对延髓内脏带的病理损害及胆碱能抗炎通路的干预效应[J].重庆医科大学学报,2024,49(9):1095-1104. DOI： 10.13406/j.cnki.cyxb.003575.

Li Chujuan,Li Hongbing,Shi Lan,et al.Pathological damage of medullary visceral zone induced by early sepsis and intervention effect of the cholinergic anti-inflammatory pathway[J].Journal of Chongqing Medical University,2024,49(9):1095-1104. DOI： 10.13406/j.cnki.cyxb.003575.

摘要

目的

探索脓毒症对延髓内脏带（medullary visceral zone，MVZ）的影响及胆碱能抗炎通路（cholinergic anti-inflammatory pathway，CAP）的干预效应。

方法

64只成年SPF级雄性Sprague-Dawley大鼠纳入本研究，随机分为对照组8只：正常饲养；假手术组8只：大鼠剖腹，游离盲肠后缝合腹腔，并予哌拉西林（50 mg/kg，腹腔注射，1次/d，连续3 d）预防感染及生理盐水（1 mL/100 g，腹腔注射，1次/d，连续3 d）；脓毒症组共48只，采用盲肠结扎穿刺（cecal ligation and puncture，CLP）制备脓毒症模型后随机分为：模型组16只：CLP后，余处理同假手术组；GTS-21［α7-烟碱样乙酰胆碱受体（nicotinic acetylcholine receptors，nAChRs）激动剂，用于激活CAP］组16只，哌拉西林使用同脓毒症组，并以GTS-21（4 mg/kg，腹腔注射，1次/d，连续3 d）进行干预；甲基牛扁亭（methyllycaconitine，MLA）强效选择性α7nAChRs拮抗剂，用于阻断CAP组16只：哌拉西林使用同脓毒症组。干预3 d后，收集每组大鼠延髓样本，用于延髓内脏带的病理观察、TdT介导的dUTP缺口末端标记（TdT mediated dUTP Nick End Labeling，TUNEL）检测凋亡、酪氨酸羟化酶（tyrosine hydroxylase，TH）/Caspase 3或胆碱乙酰转移酶（choline acetyltransferase，CHAT）/Caspase 3的双重免疫荧光标记检测两种神经元的凋亡情况，PCR检测关键基因如生长相关蛋白43（grouth associated protein-43，GAP-43）mRNA，少突胶质细胞转录因子2（oligodendrocyte transcription factor 2，Olig-2） mRNA，血管内皮生长因子（vascular endothelial growth factor，VEGF） mRNA，胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP） mRNA，和基质金属蛋白酶（matrix metalloprotein，MMP）-9 mRNA的表达。

结果

模型组MVZ凋亡指数明显高于对照组（

0.05），GTS-21具有降低脓毒病导致的凋亡的趋势（

0.05），MLA较GTS-21明显增加凋亡（

0.05）；脓毒症诱导MVZ儿茶酚能神经元和胆碱能神经元较对照组Caspase 3表达明显升高

0.05），TH明显降低（

0.05），但CHAT仅有下降的趋势（

0.05）；GTS-21具有降低脓毒症儿茶酚胺能和胆碱能神经元Caspase 3表达的趋势（

0.05），并上调脓毒症TH与CHAT表达的趋势（

0.05）；MLA则促进脓毒症导致的儿茶酚胺能和胆碱能神经元Caspase 3表达（

0.05），明显降低TH与CHAT表达（

0.05）。此外，与对照组相比，脓毒症明显上调了关键基因GAP-43 mRNA、GFAP mRNA、VEGF mRNA和MMP-9 mRNA的表达（

0.05），下调了Olig-2 mRNA的表达（

0.05）。GTS-21的干预明显下调了脓毒症GAP-43 mRNA、GFAP mRNA、VEGF mRNA和MMP-9 mRNA的表达（

0.05），具有上调Olig-2 mRNA的表达趋势（

0.05）；反之，与GTS-21相比，MLA则明显上调了GAP-43 mRNA、GFAP mRNA、VEGF mRNA和MMP-9 mRNA的表达（

0.05），明显下调了Olig-2 mRNA的表达（

0.05）。

结论

激活CAP可以有效地恢复早期脓毒症引起MVZ的病理变化和功能抑制，可能是激活CAP发挥对系统性炎症和神经炎症的抑制的另外一个重要机制，该研究揭示了MVZ和CAP可能是遏制早期脓毒症炎症风暴的潜在关键靶点。

Abstract

Objective

To investigate the influence of sepsis on medullary visceral zone（MVZ） and the intervention effect of the cholinergic anti-inflammatory pathway（CAP）.

Methods

A total of 64 specific pathogen-free adult male Sprague-Dawley rats were randomly divided into control group with 8 rats（normal feeding）；sham-operation group with 8 rats undergoing laparotomy and suture of the abdominal cavity after isolation of the cecum，followed by piperacillin（50 mg/kg，intraperitoneal injection，once a day for 3 consecutive days） to prevent infection and normal saline（1 mL/100 g，intraperitoneal injection，once a day for 3 consecutive days）；sepsis group with 48 rats undergoing cecal ligation and puncture（CLP） to establish a models of sepsis，then they were randomly divided into model group（given the same treatment as the sham-operation group），GTS-21（a selective α7 nicotinic acetylcholine receptor agonist used to activate the cholinergic anti-inflammatory pathway[CAP]） group with 16 rats treated with piperacillin（the same treatment as the sepsis group） and intervened with GTS-21（4 mg/kg，intraperitoneal injection，once a day for 3 consecutive days），and the methyllycaconitine（MLA，a potent and selective nicotine acetylcholine receptor antagonist used to block CAP） group with 16 rats given the same piperacillin treatment as the sepsis group and intervened with MLA（4.8 mg/kg，intraperitoneal injection，once a day for 3 consecutive days）. After 3 days of intervention，medulla oblongata samples were collected from each group for pathological observation；TdT-mediated dUTP nick end labeling（TUNEL） was used to measure apoptosis；double immunofluorescence labeling for tyrosine hydroxylase（TH）/Caspase 3 or choline acetyltransferase（CHAT）/Caspase 3 was used to observe the apoptosis of neurons；PCR was used to measure the mRNA expression levels of the key genes such as growth-associated protein-43（GAP-43），oligodendrocyte transcription factor 2（Olig-2），vascular endothelial growth factor（VEGF），glial fibrillary acidic protein（GFAP），and matrix metalloprotein-9（MMP-9）.

Results

The model group had a significantly higher apoptosis index of MVZ than the control group（

0.05），and GTS-21 tended to reduce apoptosis induced by sepsis（

0.05），while MLA significantly increased apoptosis compared with GTS-21（

0.05）. Compared with the control group，sepsis induced a significant increase in the expression of Caspase 3 in MVZ catecholaminergic and cholinergic neurons（

0.05），as well as a significant reduction in the expression of TH（

0.05），while there was only a tendency of reduction in the expression of CHAT（

0.05）；GTS-21 tended to reduce the expression of Caspase 3 in catecholaminergic and cholinergic neurons in sepsis（

0.05） and upregulate the expression of TH and CHAT in sepsis（

0.05）；MLA promoted the expression of Caspase 3 in catecholaminergic and cholinergic neurons caused by sepsis（

0.05） and significantly reduced the expression of TH and CHAT（

0.05）. In addition，compared with the control group，sepsis significantly upregulated the mRNA expression levels of the key genes such as GAP-43，GFAP，VEGF，and MMP-9（

0.05） and significantly downregulated the mRNA expression level of Olig-2（

0.05）. GTS-21 significantly downregulated the mRNA expression levels of GAP-43，GFAP，VEGF，and MMP-9 in sepsis（

0.05） and tended to upregulate the mRNA expression level of Olig-2（

0.05）；on the contrary，compared with GTS-21，MLA significantly upregulated the mRNA expression levels of GAP-43，GFAP，VEGF，and MMP-9（

0.05） and significantly downregulated the mRNA expression level of Olig-2（

0.05）.

Conclusion

Activating CAP can effectively restore the pathological changes and functional inhibition of MVZ caused by early sepsis，which may be another important mechanism to inhibit systemic inflammation and neuroinflammation by activating CAP，and this study shows that MVZ and CAP may be potential key targets for curbing inflammatory storm in early sepsis.

关键词

Keywords

references

Bréchot N ， Hajage D ， Kimmoun A ， et al . Venoarterial extracorporeal membrane oxygenation to rescue sepsis-induced cardiogenic shock：a retrospective，multicentre，international cohort study [J ] . Lancet ， 2020 ， 396 （ 10250 ）： 545 - 552 ．

Carlet J ， Payen D ， Opal SM . Steroids for sepsis and ARDS：this eternal controversy remains with COVID-19 [J ] . Lancet ， 2020 ， 396 （ 10259 ）： e61 - e62 ．

Pavlov VA ， Tracey KJ . Neural regulation of immunity：molecular mechanisms and clinical translation [J ] . Nat Neurosci ， 2017 ， 20 （ 2 ）： 156 - 166 ．

Hu S ， Wang YD ， Li HB . The regulation effect of α 7nAChRs and M1AChRs on inflammation and immunity in sepsis [J ] . Mediators Inflamm ， 2021 ， 2021 ： 9059601 ．

Ye ZP ， Zhu YJ ， Tang NN ， et al . α7 nicotinic acetylcholine receptor agonist GTS-21 attenuates DSS-induced intestinal colitis by improving intestinal mucosal barrier function [J ] . Mol Med ， 2022 ， 28 （ 1 ）： 59 ．

Pastor V ， Castillo Díaz F ， Sanabria VC ， et al . Prefrontal cortex nicotinic receptor inhibition by methyllycaconitine impaired cocaine-associated memory acquisition and retrieval [J ] . Behav Brain Res ， 2021 ， 406 ： 113212 ．

Li HB ， Zhou Y ， Zhao AH ， et al . Exploring the mechanism on the medullary visceral zone inhibiting the cholinergic anti-inflammatory pathway induced by sepsis [J ] . Mediators Inflamm ， 2020 ， 2020 ： 1320278 ．

Stevens KE ， Cornejo B ， Adams CE ， et al . Continuous administration of a selective alpha7 nicotinic partial agonist，DMXBA，improves sensory inhibition without causing tachyphylaxis or receptor upregulation in DBA/2 mice [J ] . Brain Res ， 2010 ， 1352 ： 140 - 146 ．

Liu Y ， Hu J ， Wu J ， et al . α7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation [J ] . J Neuroinflammation ， 2012 ， 9 ： 98 ．

Takata K ， Amamiya T ， Mizoguchi H ， et al . Alpha7 nicotinic acetylcholine receptor-specific agonist DMXBA（GTS-21） attenuates Aβ accumulation through suppression of neuronal γ-secretase activity and promotion of microglial amyloid-β phagocytosis and ameliorates cognitive impairment in a mouse model of Alzheimer’s disease [J ] . Neurobiol Aging ， 2018 ， 62 ： 197 - 209 ．

Ren C ， Li XH ， Wang SB ， et al . Activation of central alpha 7 nicotinic acetylcholine receptor reverses suppressed immune function of T lymphocytes and protects against sepsis lethality [J ] . Int J Biol Sci ， 2018 ， 14 （ 7 ）： 748 - 759 ．

Wu KLH ， Chan SHH ， Chan JYH . Neuroinflammation and oxidative stress in rostral ventrolateral medulla contribute to neurogenic hypertension induced by systemic inflammation [J ] . J Neuroinflammation ， 2012 ， 9 ： 212 ．

Amorim MR ， de Deus JL ， Cazuza RA ， et al . Neuroinflammation in the NTS is associated with changes in cardiovascular reflexes during systemic inflammation [J ] . J Neuroinflammation ， 2019 ， 16 （ 1 ）： 125 ．

Witlox J ， Eurelings LS ， de Jonghe JF ， et al . Delirium in elderly patients and the risk of postdischarge mortality，institutionalization，and dementia：a meta-analysis [J ] . JAMA ， 2010 ， 304 （ 4 ）： 443 - 451 ．

郝军舰，冯智明，才　新，等 . 血必净对盲肠结扎穿孔手术诱导的脓毒症模型大鼠肺损伤的治疗作用 [J ] . 中华实验外科杂志， 2019 ， 36 （ 6 ）： 1141 ．

Hao JJ ， Feng ZM ， Cai X ， et al . Therapeutic effect of Xuebijing on sepsis model rats induced by cecal ligation and perforation [J ] . Chin J Exp Surg ， 2019 ， 36 （ 6 ）： 1141 ．

周文海，李建国 . 脓毒症和胆碱能抗炎通路模型的建立 [J ] . 武汉大学学报（医学版）， 2016 ， 37 （ 5 ）： 748 - 751，771 ．

Zhou WH ， Li JG . Establishment of sepsis and cholinergic anti-inflammatory pathway model [J ] . Med J Wuhan Univ ， 2016 ， 37 （ 5 ）： 748 - 751，771 ．

苗珍花，王　银，田建英，等 . α7nACh受体竞争性拮抗剂甲基牛扁亭对昆明小鼠认知功能的损伤 [J ] . 解剖学杂志， 2010 ， 33 （ 6 ）： 764 - 767 ．

Miao ZH ， Wang Y ， Tian JY ， et al . Mechanism of cognitive impairment by α7nAChR competitive antagonist methyllycaconitine in mouse [J ] . Chin J Clin Anat ， 2010 ， 33 （ 6 ）： 764 - 767 ．

江秀慧，张子莘，张信鸽 . α7nAchR烟碱型乙酰胆碱受体拮抗剂对高氧保护脓毒症小鼠效应的影响 [J ] . 中外健康文摘， 2014 （ 3 ）： 44 - 46 ．

Jiang XH ， Zhang ZX ， Zhang XG . Effect of α7nAchR nicotinic acetylcholine receptor antagonist on the protective effect of high oxygen on septic mice [J ] . Chinese and foreign health abstracts ， 2014 （ 3 ）： 44 - 46

Shrum B ， Anantha RV ， Xu SX ， et al . A robust scoring system to evaluate sepsis severity in an animal model [J ] . BMC Res Notes ， 2014 ， 7 ： 233 ．

Sulzbacher MM ， Sulzbacher LM ， Passos FR ， et al . Adapted murine sepsis score：improving the research in experimental sepsis mouse model [J ] . Biomed Res Int ， 2022 ， 2022 ： 5700853 ．

李红兵，李　媛，王武石，等 . 胆碱能抗炎通路对脓毒症大鼠早期炎症与免疫反应具有负向调控作用 [J ] . 南方医科大学学报， 2020 ， 40 （ 5 ）： 647 - 653 ．

Li HB ， Li Y ， Wang WS ， et al . Cholinergic anti-inflammatory pathway plays negative regulatory role in early inflammatory and immune responses in septic rats [J ] . J South Med Univ ， 2020 ， 40 （ 5 ）： 647 - 653 ．

Mrak R E ， Griffin W . Glia and their cytokines in progression of neurodegeneration .[J ] . Neurobiology of Aging ， 2005 ， 26 （ 3 ）: 349 - 354 .

Wang Z ， Wu LY ， You WC ， et al . Melatonin alleviates secondary brain damage and neurobehavioral dysfunction after experimental subarachnoid hemorrhage：possible involvement of TLR4-mediated inflammatory pathway [J ] . J Pineal Res ， 2013 ， 55 （ 4 ）： 399 - 408 ．

Brites D ， Fernandes A . Neuroinflammation and depression：microglia activation，extracellular microvesicles and microRNA dysregulation [J ] . Front Cell Neurosci ， 2015 ， 9 ： 476 ．

Zhang F ， Zhang JG ， Yang W ， et al . 6-Gingerol attenuates LPS-induced neuroinflammation and cognitive impairment partially via suppressing astrocyte overactivation [J ] . Biomedecine Pharmacother ， 2018 ， 107 ： 1523 - 1529 ．

Wang C ， Zhang L ， Ndong JC ， et al . Progranulin deficiency exacerbates spinal cord injury by promoting neuroinflammation and cell apoptosis in mice [J ] . J Neuroinflammation ， 2019 ， 16 （ 1 ）： 238 ．

Yu HY ， Cleveland DW . Tuning apoptosis and neuroinflammation：TBK1 restrains RIPK1 [J ] . Cell ， 2018 ， 174 （ 6 ）： 1339 - 1341 ．

Su Y ， Zhang W ， Zhang RX ， et al . Activation of cholinergic anti-inflammatory pathway ameliorates cerebral and cardiac dysfunction after intracerebral hemorrhage through autophagy [J ] . Front Immunol ， 2022 ， 13 ： 870174 ．

Giebelen IA ， van Westerloo DJ ， LaRosa GJ ， et al . Stimulation of alpha 7 cholinergic receptors inhibits lipopolysaccharide-induced neutrophil recruitment by a tumor necrosis factor alpha-independent mechanism [J ] . Shock ， 2007 ， 27 （ 4 ）： 443 - 447 ．

Wang KF ， Chen QJ ， Wu NH ， et al . Berberine ameliorates spatial learning memory impairment and modulates cholinergic anti-inflammatory pathway in diabetic rats [J ] . Front Pharmacol ， 2019 ， 10 ： 1003 ．

Matteoli G ， Gomez-Pinilla PJ ， Nemethova A ， et al . A distinct vagal anti-inflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen [J ] . Gut ， 2014 ， 63 （ 6 ）： 938 - 948 ．

Pavlov VA ， Parrish WR ， Rosas-Ballina M ， et al . Brain acetylcholinesterase activity controls systemic cytokine levels through the cholinergic anti-inflammatory pathway [J ] . Brain Behav Immun ， 2009 ， 23 （ 1 ）： 41 - 45 ．

Rinaman L . Ascending projections from the caudal visceral nucleus of the solitary tract to brain regions involved in food intake and energy expenditure [J ] . Brain Res ， 2010 ， 1350 ： 18 - 34 ．

Duris K ， Lipkova J ， Jurajda M . Cholinergic anti-inflammatory pathway and stroke [J ] . Curr Drug Deliv ， 2017 ， 14 （ 4 ）： 449 - 457 ．

Krafft PR ， McBride D ， Rolland WB ， et al . α 7 nicotinic acetylcholine receptor stimulation attenuates neuroinflammation through JAK2-STAT3 activation in murine models of intracerebral hemorrhage [J ] . Biomed Res Int ， 2017 ， 2017 ： 8134653 ．

Ellis JM . Cholinesterase inhibitors in the treatment of dementia [J ] . J Am Osteopath Assoc ， 2005 ， 105 （ 3 ）： 145 - 158 ．

Waldburger JM ， Boyle DL ， Edgar M ， et al . Spinal p38 MAP kinase regulates peripheral cholinergic outflow [J ] . Arthritis Rheum ， 2008 ， 58 （ 9 ）： 2919 - 2921 ．

Holahan MR . A shift from a pivotal to supporting role for the growth-associated protein（GAP-43） in the coordination of axonal structural and functional plasticity [J ] . Front Cell Neurosci ， 2017 ， 11 ： 266 ．

Castrén E ， Antila H . Neuronal plasticity and neurotrophic factors in drug responses [J ] . Mol Psychiatry ， 2017 ， 22 （ 8 ）： 1085 - 1095 ．

Zhu XK ， Wang P ， Liu HJ ， et al . Changes and significance of SYP and GAP-43 expression in the hippocampus of CIH rats [J ] . Int J Med Sci ， 2019 ， 16 （ 3 ）： 394 - 402 ．

Heuser K ， Hoddevik EH ， Taubøll E ， et al . Temporal lobe epilepsy and matrix metalloproteinase 9：a tempting relation but negative genetic association [J ] . Seizure ， 2010 ， 19 （ 6 ）： 335 - 338 ．

Li HM ， Yue PJ ， Su YY ， et al . Plasma levels of matrix metalloproteinase-9：a possible marker for cold-induced stroke risk in hypertensive rats [J ] . Neurosci Lett ， 2019 ， 709 ： 134399 ．

Stawarski M ， Stefaniuk M ， Wlodarczyk J . Matrix metalloproteinase-9 involvement in the structural plasticity of dendritic spines [J ] . Front Neuroanat ， 2014 ， 8 ： 68 ．

Lepeta K ， Kaczmarek L . Matrix metalloproteinase-9 as a novel player in synaptic plasticity and schizophrenia [J ] . Schizophr Bull ， 2015 ， 41 （ 5 ）： 1003 - 1009 ．

Oeckl P ， Weydt P ， Steinacker P ， et al . Different neuroinflammatory profile in amyotrophic lateral sclerosis and frontotemporal dementia is linked to the clinical phase [J ] . J Neurol Neurosurg Psychiatry ， 2019 ， 90 （ 1 ）： 4 - 10 ．

Abu-Rumeileh S ， Steinacker P ， Polischi B ， et al . CSF biomarkers of neuroinflammation in distinct forms and subtypes of neurodegenerative dementia [J ] . Alzheimers Res Ther ， 2019 ， 12 （ 1 ）： 2 ．

Touyz RM ， Lang NN . Hypertension and antiangiogenesis：the Janus face of VEGF inhibitors [J ] . JACC CardioOncol ， 2019 ， 1 （ 1 ）： 37 - 40 ．

Ureña-Guerrero ME ， Castañeda-Cabral JL ， Rivera-Cervantes MC ， et al . Neuroprotective and neurorestorative effects of epo and VEGF：perspectives for new therapeutic approaches to neurological diseases [J ] . Curr Pharm Des ， 2020 ， 26 （ 12 ）： 1263 - 1276 ．

Beigi Boroujeni F ， Pasbakhsh P ， Mortezaee K ， et al . Intranasal delivery of SDF-1α-preconditioned bone marrow mesenchymal cells improves remyelination in the cuprizone-induced mouse model of multiple sclerosis [J ] . Cell Biol Int ， 2020 ， 44 （ 2 ）： 499 - 511 ．

Ferensztajn-Rochowiak E ， Tarnowski M ， Samochowiec J ， et al . Increased mRNA expression of peripheral glial cell markers in bipolar disorder：the effect of long-term lithium treatment [J ] . Eur Neuropsychopharmacol ， 2016 ， 26 （ 9 ）： 1516 - 1521 ．

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