胆碱能抗炎通路对家兔肺缺血再灌注损伤的影响
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摘要
目的分别采用电刺激迷走神经和电针足三里穴的方法激活胆碱能抗炎通路,观察胆碱能抗炎通路对家兔肺缺血再灌注损伤家兔的影响,并探讨其可能的作用机制。
方法电刺激迷走神经部分:健康成年雄性新西兰大白兔36只(由北京市结核病胸部肿瘤研究所动物实验室提供),体重(2.5±0.5)kg,随机分为4组:①手术对照组(SHAM组):游离左肺门假手术;②肺缺血再灌注组(I/R组):左肺缺血再灌注手术;③迷走神经切断组(VGX组):左肺缺血再灌注手术+双侧颈迷走神经干离断术;④迷走神经刺激组(STM组):左肺缺血再灌注手术+双侧颈迷走神经干离断术+左侧颈迷走神经干电刺激。各组均需先行双侧颈部迷走神经干分离术。除SHAM组外,各组夹闭左肺门60min后再行肺灌注120min。再灌注即刻,VGX组剪断双侧颈迷走神经干;STM组以5V、2ms、1Hz强度持续刺激左颈迷走神经干20min。电针足三里穴部分:A组(假手术组)、B组(缺血再灌注组)和E组(缺血再灌注+迷走神经切断组)同电刺激迷走神经部分。另取健康成年雄性新西兰大白兔21只,体重(2.5±0.5)kg,随机分为3组。C组:缺血再灌注+非经非穴组,阻断左肺门60min后,再灌注即刻电针足三里穴旁5mm处30min,继续双肺再灌注90min。D组:缺血再灌注+电针足三里穴组,阻断左肺门60min后,再灌注即刻电针足三里穴30min,继续双肺再灌注90min。F组:缺血再灌注+迷走神经切断+电针足三里穴组,钝性分离双侧颈迷走神经,阻断左肺门60min后,再灌注即刻剪断迷走神经,并电针足三里穴30min,继续双肺再灌注90min。各组动物均于再灌注120min后,从颈总动脉取血行动脉血气分析,阻断上下腔静脉处死动物,采集标本。ELISA法检测左肺组织和血浆中肿瘤坏死因子α(TNFα)含量;Western Blot法检测左肺组织核转录因子κB (NFκB)P65含量;比色法测定左肺组织髓过氧化物酶(MPO)和丙二醛(MDA)含量和肺组织湿-干重比(W/D);HE染色观察左肺组织病理学改变。
结果电刺激迷走神经部分:再灌注50min后VGX组MAP变化率值显著低于IR组(P<0.05或0.01)。动脉血气分析显示VGX组pH、PaO2和SaO2值均明显低于IR组(P<0.01);STM组明显升高(P<0.01)。VGX组与IR组比较,血浆中TNFα水平显著增高(P<0.05);STM组左肺和肝中TNFα表达明显弱于IR组(P<0.05或0.01)。STM组与IR组比较,NFκB P65表达减弱(P<0.05),VGX组显著增强(P<0.05)。VGX组MPO活性和W/D值均显著高于IR组(P<0.01);STM组则显著降低(P<0.05或0.01)。肺脏病理组织学观察发现,VGX组的组织破坏较IR组更加严重,STM组相对减轻。电针足三里穴部分:与A组比较,B组血浆中TNFα含量和肺组织中MPO、TNFα含量及NFκB活性显著升高(P<0.05或0.01),pH和PaO2值显著降低(P<0.05或0.01),PaCO2值升高(P<0.05),肺组织炎症反应程度最重。C组各项检测指标与B组比较无统计学意义(P>0.05)。D组血浆中TNFα含量和肺组织中MPO、TNFα含量及NFκB活性与B组比较明显降低(P<0.05或0.01),血气指标好转,病理改变减轻。E组血浆中TNFα含量和肺组织中MPO、TNFα含量及NFκB活性与B组比较明显升高(P<0.05或0.01),肺功能损害和肺组织炎症反应程度加重。与D组比较,F组血浆中TNFα含量和肺组织中MPO、TNFα含量及NFκB活性明显升高(P<0.05或0.01),肺功能损害和肺组织炎症反应程度加重。
结论切断迷走神经阻断胆碱能抗炎通路可以加重肺缺血再灌注损伤;电刺激迷走神经和电针足三里穴兴奋胆碱能抗炎通路可以减轻肺再灌注损伤。胆碱能抗炎通路的保护作用与削弱NFκB活性,抑制白细胞聚集活化,减少TNFα产生有关。
Objective To investigate the effect and mechanism of vagotomy, vagus nerve stimulation and electro-acupuncture at Zusanli points against the lung ischemia-reperfusion injury of rabbits.
Methods The part of vagus nerve stimulation: 36 male healthy rabbits were randomly divided into four equal groups:①sham operation (SHAM) group,②ischemia reperfusion (IR) group,③bilateral cervical vagotomy (VGX) group,④vagus nerve stimulation (STM) group. The bilateral cervical vagus nerve trunks and hilum of left lung were exposed and isolated from surrounding tissue. The animals were blocked hilum of left lung for 60 minutes except the SHAM group. VGX group was subjected to bilateral cervical vagotomy, and constant voltage stimuli (5V, 2ms, 1Hz) were applied to the left cervical vagus nerve trunk of STM group for 20 min immediately after loosing the clamp. The part of electro-acupuncture at Zusanli points: A, B and E group were as same as the first part. Twenty-one healthy male New Zealand white rabbits weighting (2.5±0.5)kg were randomly divided into other three groups: Non-acupoints+ischemia -reperfusion (group C): left hilum was isolated and clamped for 60min, after declamping the lungs electro-acupuncture the non-acupoints for 30min and the lungs were ventilated for another 90min; Electro-acupuncture to“ZuSanLi”Points+ischemia-reperfusion (group D): left hilum was isolated and clamped for 60min, after declamping the lungs electro-acupuncture the“ZuSanLi”Points for 30min and the lungs were ventilated for another 90min; Bilateral cervical vagotomy+ electro-acupuncture to“ZuSanLi”Points+ischemia-reperfusion (group F): left hilum was isolated and clamped for 60min, before declamping the lungs, the animals were subjected to bilateral cervical vagotomy and electro-acupuncture the“ZuSanLi”Points for 30min and the lungs were ventilated for another 90min. The common carotid artery were implanted into a blood pressure transducer for continuous registration of mean arterial blood pressure (MBP).Left lung was excised for measurement of MDA﹑MPO﹑TNFαcontent and NFκB activity and wet-dry ration(W/D) and histopathologic changes. Blood samples of these rabbits were collected after the operation complete for measurement of TNFαcontent and blood gas analysis.
Results The part of vagus nerve stimulation: The MAP change Ratios of VGX group were lower than those of IR group subsequent to 50min reperfusion. pH, PaO2 and SaO2 values of VGX group decreased substantially as compared with those of IR group; STM group produced opposite result. Vagotomy caused a farther increase of serous TNF in VGX group as compared with IR group. Whereas STM group observably inhibited pulmonary and hepatic TNF. VGX group had a higher NFκB P65 level than IR group, STM group can markedly modify the rise. STM group prevented the increases of activity of MPO and W/D value caused by IR, but VGX group aggravated those rises. As compared with IR group, STM group suppressed inflammatory responses, and pathological depravation was induced by vagotomy. The part of electro-acupuncture at Zusanli points: As compared with A groups, B groups produced a remarkable increase of lung MPO、MDA﹑TNFαcontent and NFκB activity, and lead to acidosis and hypoxia and pathological depravation. C groups showed no difference as compared with B groups in statistical manner. On the contrary, D groups attenuated significantly lung MPO、MDA﹑TNFαcontent and effectively inhibited NFκB activity and reverted the value of blood gas and pathological changes. E groups offered a significantly increase in lung MPO、MDA﹑TNFαcontent and NFκB activity and W/D ration, and lead to acidosis and hypoxia and pathological depravation as compared with B groups. F groups offered same results as E groups as compared with D groups, and showed no difference as compared with E groups in statistical manner.
Conclusions Vagotomy can interdict cholinergic anti-inflammatory pathway and aggravate lung ischemia-reperfusion injury. Vagus nerve stimulation and electro-acupuncture at Zusanli points can protect the lung against ischemia-reperfusion injury via activating cholinergic anti-inflammatory pathway. The mechanisms that CAP alleviated lung ischemia-reperfusion injury were likely to inhibit the leukocyte aggregation and activity, suppress NFκB and TNFαexpression.
方法电刺激迷走神经部分:健康成年雄性新西兰大白兔36只(由北京市结核病胸部肿瘤研究所动物实验室提供),体重(2.5±0.5)kg,随机分为4组:①手术对照组(SHAM组):游离左肺门假手术;②肺缺血再灌注组(I/R组):左肺缺血再灌注手术;③迷走神经切断组(VGX组):左肺缺血再灌注手术+双侧颈迷走神经干离断术;④迷走神经刺激组(STM组):左肺缺血再灌注手术+双侧颈迷走神经干离断术+左侧颈迷走神经干电刺激。各组均需先行双侧颈部迷走神经干分离术。除SHAM组外,各组夹闭左肺门60min后再行肺灌注120min。再灌注即刻,VGX组剪断双侧颈迷走神经干;STM组以5V、2ms、1Hz强度持续刺激左颈迷走神经干20min。电针足三里穴部分:A组(假手术组)、B组(缺血再灌注组)和E组(缺血再灌注+迷走神经切断组)同电刺激迷走神经部分。另取健康成年雄性新西兰大白兔21只,体重(2.5±0.5)kg,随机分为3组。C组:缺血再灌注+非经非穴组,阻断左肺门60min后,再灌注即刻电针足三里穴旁5mm处30min,继续双肺再灌注90min。D组:缺血再灌注+电针足三里穴组,阻断左肺门60min后,再灌注即刻电针足三里穴30min,继续双肺再灌注90min。F组:缺血再灌注+迷走神经切断+电针足三里穴组,钝性分离双侧颈迷走神经,阻断左肺门60min后,再灌注即刻剪断迷走神经,并电针足三里穴30min,继续双肺再灌注90min。各组动物均于再灌注120min后,从颈总动脉取血行动脉血气分析,阻断上下腔静脉处死动物,采集标本。ELISA法检测左肺组织和血浆中肿瘤坏死因子α(TNFα)含量;Western Blot法检测左肺组织核转录因子κB (NFκB)P65含量;比色法测定左肺组织髓过氧化物酶(MPO)和丙二醛(MDA)含量和肺组织湿-干重比(W/D);HE染色观察左肺组织病理学改变。
结果电刺激迷走神经部分:再灌注50min后VGX组MAP变化率值显著低于IR组(P<0.05或0.01)。动脉血气分析显示VGX组pH、PaO2和SaO2值均明显低于IR组(P<0.01);STM组明显升高(P<0.01)。VGX组与IR组比较,血浆中TNFα水平显著增高(P<0.05);STM组左肺和肝中TNFα表达明显弱于IR组(P<0.05或0.01)。STM组与IR组比较,NFκB P65表达减弱(P<0.05),VGX组显著增强(P<0.05)。VGX组MPO活性和W/D值均显著高于IR组(P<0.01);STM组则显著降低(P<0.05或0.01)。肺脏病理组织学观察发现,VGX组的组织破坏较IR组更加严重,STM组相对减轻。电针足三里穴部分:与A组比较,B组血浆中TNFα含量和肺组织中MPO、TNFα含量及NFκB活性显著升高(P<0.05或0.01),pH和PaO2值显著降低(P<0.05或0.01),PaCO2值升高(P<0.05),肺组织炎症反应程度最重。C组各项检测指标与B组比较无统计学意义(P>0.05)。D组血浆中TNFα含量和肺组织中MPO、TNFα含量及NFκB活性与B组比较明显降低(P<0.05或0.01),血气指标好转,病理改变减轻。E组血浆中TNFα含量和肺组织中MPO、TNFα含量及NFκB活性与B组比较明显升高(P<0.05或0.01),肺功能损害和肺组织炎症反应程度加重。与D组比较,F组血浆中TNFα含量和肺组织中MPO、TNFα含量及NFκB活性明显升高(P<0.05或0.01),肺功能损害和肺组织炎症反应程度加重。
结论切断迷走神经阻断胆碱能抗炎通路可以加重肺缺血再灌注损伤;电刺激迷走神经和电针足三里穴兴奋胆碱能抗炎通路可以减轻肺再灌注损伤。胆碱能抗炎通路的保护作用与削弱NFκB活性,抑制白细胞聚集活化,减少TNFα产生有关。
Objective To investigate the effect and mechanism of vagotomy, vagus nerve stimulation and electro-acupuncture at Zusanli points against the lung ischemia-reperfusion injury of rabbits.
Methods The part of vagus nerve stimulation: 36 male healthy rabbits were randomly divided into four equal groups:①sham operation (SHAM) group,②ischemia reperfusion (IR) group,③bilateral cervical vagotomy (VGX) group,④vagus nerve stimulation (STM) group. The bilateral cervical vagus nerve trunks and hilum of left lung were exposed and isolated from surrounding tissue. The animals were blocked hilum of left lung for 60 minutes except the SHAM group. VGX group was subjected to bilateral cervical vagotomy, and constant voltage stimuli (5V, 2ms, 1Hz) were applied to the left cervical vagus nerve trunk of STM group for 20 min immediately after loosing the clamp. The part of electro-acupuncture at Zusanli points: A, B and E group were as same as the first part. Twenty-one healthy male New Zealand white rabbits weighting (2.5±0.5)kg were randomly divided into other three groups: Non-acupoints+ischemia -reperfusion (group C): left hilum was isolated and clamped for 60min, after declamping the lungs electro-acupuncture the non-acupoints for 30min and the lungs were ventilated for another 90min; Electro-acupuncture to“ZuSanLi”Points+ischemia-reperfusion (group D): left hilum was isolated and clamped for 60min, after declamping the lungs electro-acupuncture the“ZuSanLi”Points for 30min and the lungs were ventilated for another 90min; Bilateral cervical vagotomy+ electro-acupuncture to“ZuSanLi”Points+ischemia-reperfusion (group F): left hilum was isolated and clamped for 60min, before declamping the lungs, the animals were subjected to bilateral cervical vagotomy and electro-acupuncture the“ZuSanLi”Points for 30min and the lungs were ventilated for another 90min. The common carotid artery were implanted into a blood pressure transducer for continuous registration of mean arterial blood pressure (MBP).Left lung was excised for measurement of MDA﹑MPO﹑TNFαcontent and NFκB activity and wet-dry ration(W/D) and histopathologic changes. Blood samples of these rabbits were collected after the operation complete for measurement of TNFαcontent and blood gas analysis.
Results The part of vagus nerve stimulation: The MAP change Ratios of VGX group were lower than those of IR group subsequent to 50min reperfusion. pH, PaO2 and SaO2 values of VGX group decreased substantially as compared with those of IR group; STM group produced opposite result. Vagotomy caused a farther increase of serous TNF in VGX group as compared with IR group. Whereas STM group observably inhibited pulmonary and hepatic TNF. VGX group had a higher NFκB P65 level than IR group, STM group can markedly modify the rise. STM group prevented the increases of activity of MPO and W/D value caused by IR, but VGX group aggravated those rises. As compared with IR group, STM group suppressed inflammatory responses, and pathological depravation was induced by vagotomy. The part of electro-acupuncture at Zusanli points: As compared with A groups, B groups produced a remarkable increase of lung MPO、MDA﹑TNFαcontent and NFκB activity, and lead to acidosis and hypoxia and pathological depravation. C groups showed no difference as compared with B groups in statistical manner. On the contrary, D groups attenuated significantly lung MPO、MDA﹑TNFαcontent and effectively inhibited NFκB activity and reverted the value of blood gas and pathological changes. E groups offered a significantly increase in lung MPO、MDA﹑TNFαcontent and NFκB activity and W/D ration, and lead to acidosis and hypoxia and pathological depravation as compared with B groups. F groups offered same results as E groups as compared with D groups, and showed no difference as compared with E groups in statistical manner.
Conclusions Vagotomy can interdict cholinergic anti-inflammatory pathway and aggravate lung ischemia-reperfusion injury. Vagus nerve stimulation and electro-acupuncture at Zusanli points can protect the lung against ischemia-reperfusion injury via activating cholinergic anti-inflammatory pathway. The mechanisms that CAP alleviated lung ischemia-reperfusion injury were likely to inhibit the leukocyte aggregation and activity, suppress NFκB and TNFαexpression.
引文
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2. Michael J Eppinger, Michael L Jones, Micheal Deeb, et al. Pattern of injury and the role of neutrophils in reperfusion injury of lung. Surg Res, 1995, 58: 713-718.
3. Koyama I, Toung TJK, Rogers MC, et al. O2 radicals mediate reperfusion lung injury in ischemic O2-ventilated canine pulmonary lobe. Appl Physiol, 1987, 63(1)111.
4. Bryan CL, Cohen DI, Gibbons WJ, et al. Lung transplantation:the reimplantation response. Crit Care Rep, 1991,2(3):217.
5. Bergers M, Thorne F, Reempts JV, et al. The role of calciumin cellular dysfunction. Am J Emerg Med, 1983, 2(2)154.
6. Eppinger MJ, Ward PA, Jones ML, et al. Disparate effects of nitric oxide on lung ischemia-reperfusion injury. Ann Thorac Surg, 1995, 60(5):1169.
7. 林文注. 实验针灸学. 上海: 上海科学技术出版社, 1999, 283.
8. Lu YT, Hellewell PG, Evans TW. Ischemia reperfusion lung injury:contribution of ischemia,neutrophils and hydrostatic pressure. Am J Physiol, 1997, 273(17):L46-54.
9. Al-Mehdi AB, Fisher AB. Invited editorial on“tumor necrosis factor-alpha in ischemia and reperfusion injury in rat lungs”. Appl Physiol, 1998, 85(6):2003-2004.
10. Abolhoda A, Brooks A, Choudhry M, et al. Characterization of local inflammatory response in an isolated lung perfusion model. Ann Surg Oncol, 1998, 5(1):87-92.
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20. Borovikova L V, Ivanova S, Zhang M, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin.Nature,2000,405:438 462.
21. Bluthe RM, et al. Lipolysaccharide induce sickness behaviour in rats by a vagal mediated mechanism. C. R. Acad. Sci. Paris. 1994,317:499-503.
22.. Maier SF, Goehler LE, Fleshner M, Watkins LR. The role of the vagus nerve in cytokine-to-brain communication. Ann.NYA.cad. Sci. 1998,840,289-300
23. 王景杰,黄裕新,王键,等.c fos 在电针调控大鼠胃运动中的表达及其意义.针刺研究,2001,26:274 278.
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