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

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

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（P<0.05），GTS-21具有降低脓毒病导致的凋亡的趋势（P>0.05），MLA较GTS-21明显增加凋亡（P<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P<0.05），TH明显降低（P<0.05），但CHAT仅有下降的趋势（P>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（P>0.05），并上调脓毒症TH与CHAT表达的趋势（P>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（P>0.05），明显降低TH与CHAT表达（P<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（P<0.05），下调了Olig-2 mRNA的表达（P<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（P<0.05），具有上调Olig-2 mRNA的表达趋势（P>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（P<0.05），明显下调了Olig-2 mRNA的表达（P<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（P<0.05），and GTS-21 tended to reduce apoptosis induced by sepsis（P>0.05），while MLA significantly increased apoptosis compared with GTS-21（P<0.05）. Compared with the control group，sepsis induced a significant increase in the expression of Caspase 3 in MVZ catecholaminergic and cholinergic neurons（P<0.05），as well as a significant reduction in the expression of TH（P<0.05），while there was only a tendency of reduction in the expression of CHAT（P>0.05）；GTS-21 tended to reduce the expression of Caspase 3 in catecholaminergic and cholinergic neurons in sepsis（P>0.05） and upregulate the expression of TH and CHAT in sepsis（P>0.05）；MLA promoted the expression of Caspase 3 in catecholaminergic and cholinergic neurons caused by sepsis（P>0.05） and significantly reduced the expression of TH and CHAT（P<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（P<0.05） and significantly downregulated the mRNA expression level of Olig-2（P<0.05）. GTS-21 significantly downregulated the mRNA expression levels of GAP-43，GFAP，VEGF，and MMP-9 in sepsis（P<0.05） and tended to upregulate the mRNA expression level of Olig-2（P>0.05）；on the contrary，compared with GTS-21，MLA significantly upregulated the mRNA expression levels of GAP-43，GFAP，VEGF，and MMP-9（P<0.05） and significantly downregulated the mRNA expression level of Olig-2（P<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

脓毒症 / 延髓内脏带 / 胆碱能抗炎通路 / 神经炎症

Key words

sepsis / medullary visceral zone / cholinergic anti-inflammatory pathway / neuroinflammation

基金资助

贵州省自然科学基金重点课题资助项目(黔科合基础-ZK[2023]重点001)

贵州省科技厅科研课题资助项目(黔科合基础[2019]1005)

贵州省卫健委科学技术基金资助项目(gzwkj2021-001)