迷走神经对内毒素血症大鼠心、肺炎症反应影响的初步研究
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摘要
目的：采用静注内毒素(主要成分脂多糖Lipopolysaccharide，LPS)造成大鼠LPS血症和脏器炎症损害的模型，研究迷走神经对LPS引起的心、肺组织炎症反应及脏器损伤的影响。探讨胆碱能神经在调节机体抗炎反应中的作用，为临床上脓毒症、多脏器功能障碍综合征(MODS)以及过度炎症反应性疾病的防治提供实验依据。
方法：雄性Wistar大鼠70只，采用乌拉坦腹腔注射麻醉(25％，1.2g·kg~(-1))，颈动脉插管，切断双侧颈部迷走神经或做假手术对照，将左侧迷走神经远端连接刺激与记录电极，监测神经动作电位及心率和血压(Pclab生物信号电脑采集系统，北京威信斯达公司)。鼠阴茎背静脉静注LPS(O111：B4，Sigma，10mg·kg~(-1))即刻持续电刺激(5V，2ms，1Hz)迷走神经远端20min，每间隔10min后重复刺激，每次20min，共刺激3次。随机分组如下，每组10只：
实验Ⅰ组：手术+静注LPS(以下简称：LPS组)；
实验Ⅱ组：手术+双侧颈部迷走神经切断+静注LPS(以下简称：V-LPS组)：
实验Ⅲ组：手术+双侧颈部迷走神经切断+静注LPS+左迷走神经远端电刺激(以下简称：VS-LPS组)；
对照Ⅰ组：手术对照，只行麻醉、颈部切口神经分离、动脉插管测压和监测(以下简称：C组)；
对照Ⅱ组：手术+双侧颈部迷走神经切断(以下简称：V组)；
对照Ⅲ组：手术+左颈部迷走神经远端电刺激(以下简称：S组)：
对照Ⅳ组：手术+双侧颈部迷走神经切断+左迷走神经远端电刺激(以
下简称:VS组)。
静注LPS后1 .sh处死动物。分别检测各组动物心肌和肺组织中肿瘤坏
死因子一a(T切叮or necrosis factor一Q，TNF一a)、丙二醛(Malondialdehyde，
MDA)量、髓过氧化物酶(Myel叩eroxidase，MpO)活性变化以及血肌酸
激酶同工酶(CK-MB)、TNF一Q、乙酞胆碱(Acetyicholine，ACh)和皮质
醇水平。
结果:1、动物双侧迷走神经被切断后，心率、血压明显上升，呼吸减
慢变深。电刺激后心率、血压平均下降12%，呼吸转快，停刺激后心率、
血压迅速上升。2、C组和未静注LPS组动物，心肌和肺组织中TNF一Q、
MPO、MDA值以及血CK.MB、TNF一Q和皮质醇水平较低，组间两两比较，
数值无统计学意义，心肌及肺组织病理检查无异常。血ACh水平随迷走神
经的切断或刺激而明显降低或升高(P<0.01)。3、LPS组动物心肌和肺组
织中TNF一Q、MDA量、MPO活性以及血CK一MB、TNF一a和皮质醇水平
显著高于C组(P<0.01)，血ACh也升高(P<0.05)。心肌及肺组织出现炎
性病理改变，可见间质血管扩张充血，组织内炎性细胞浸润等。4、V一LPS
组动物较LPS组动物心肌和肺组织中TNF一Q、MDA量及MPO活性和血
CK一MB、TNF一Q水平显著升高(P<0.05)，血ACh和皮质醇水平却明显下
降(P<0.01)。心肌和肺组织炎性病理改变加重，可见血管充血和炎性细胞
浸润明显增加，出现组织点状坏死。5、VS一LPS组心肌和肺组织中TNF-
Q、MDA量和MPO活性和血CK一MB、TN下一Q、皮质醇水平较LPS组显
著降低(P<0.01)，血中ACh水平明显上升(P<0.05)。心肌和肺组织炎性
病理改变减轻，可见血管扩张充血和炎性细胞浸润明显减轻，组织的点状
坏死消失。
结论:1、电刺激切断的迷走神经能使LPS血症大鼠血CK一MB、TNF
水平及心、肺组织中TNF、MDA量以及MPO活性明显下降，组织炎症病
理改变减轻，揭示迷走神经通过抑制TNF的合成参与LPS血症时组织炎
症反应的调节;2、切断迷走神经对正常大鼠血CK一MB、TNF水平及心、
肺组织中TNF、MDA量和MPO活性并无显著影响，但能使LPS血症大
Vl
鼠血CK－MB、WF水平及心、肺组织中IN’－－、MDA量和WO活性明显
升高，组织炎症反应加重，表明切断迷走神经不影响正常机体炎症介质的
水平，但加重LPS血症时组织的炎症反应。3、切断LPS血症大鼠的迷走
神经可显著降低血皮质醇水平，提示迷走神经的完整性对于维持体内皮质
醇的水平，调节机体全身炎症反应，保护心、肺组织免受过度炎症损害具
有重要意义。
Aim: To expound the effects of vagus nerve on the inflammatory response in heart and lung tissues in rats with endotoximia and investigate the role of the cholinergic nervous system in modulating systemic inflammatory response to endotoxin.
Methods: Seventy male Wistar rats, weighting 220 20g, were anaesthetized with urethane(25 %, 1.2g-kg-1). The common carotid artery were cannulated and connected to a blood pressure transducer.An acquisition system(Pclab System, Wisinstar CO.BeiJing) was used to monitor mean arterial blood pressure and heart rate continuously. Animals were subjected to bilateral cervical vagtotomy. A bipolar electrodes was placed at the distal end of one side vagus nerve trunk and connected to a stimulation module. The electrical stimulation was controlled by an acquisition system for continuous registration of nerve action potential. Stimuli with constant voltage (5V,2ms,1Hz) were administered at the nerve for 20 min immediately after administration of lipopolysaccharide(LPS, 10mg kg-1,E coli O111:B4, Sigma), and then were repeated 2 times after each of 10 min interval.
Rats were randomly divided into 7 groups as follow:
(1)sham operation(C, n=10);
(2)vagotomy alone(V, n=10);
(3)vagus nerve stimulation alone(S, n=10);
(4)vagotomy plus vagus nerve stimulation(VS, n=10);
(5)LPS injection alone(LPS, n=10);
(6)vagotomy plus LPS injection(V-LPS, n=10);
(7)vagotomy plus LPS injection and vagus nerve stimulation(VS-LPS, n=10).
Blood and tissue pieces from rats heart and lung were collected at 1.5h after LPS administration in all groups. Tumor necrosis factor- a (TNF- ), malondialdehyde (MDA) and myeloperoxidase (MPO) in heart and lung tissues and serum TNF-a , acetylcholine (ACh), CK-MB and corticosterone were determined.
Results: (1)Bilateral cervical vagotomy significantly elevated blood pressure , speeded up heart rate and slowed down respiratory rate, but vagus never stimulation reversed changes above. (2)The TNF- a and MDA contents and MPO activity in serum and tissues were very lower in the operation control group and other groups without LPS injection.There were no significant differences in the content of TNF- a and MDA and MPO activity in tissue and CK-MB and corticosterone in serum between each groups without LPS injection. Pathologic lesion was not found in these groups either. (3)LPS administration significantly elevated the levels of TNF- a and ACh and CK-MB and corticosterone in serum and the contents of TNF- a and MDA and MPO in heart and lung tissues. Inflammatory changes including interstitial vascular dilatation and hyperemia and inflammatory cells infiltration were found in heart and lung tissues. (4)Vagotomy without stimulation resulted in significant increase in TNF and MDA contents and MPO activity in tissues as well as TNF- a and CK-MB in serum,but significant decrease in corticosterone and ACh in serum. A more severe inflammatory response than LPS
administration alone in heart and lung tissues was observed. (5)Vagotomy compounded stimulation significantly decreased in TNF-α and MDA and MPO in heart and lung tissues and TNF-α and corticosterone and CK-MB in serum, but increased in ACh in serum. Vagotomy compounded stimulation also atteuated inflammatory response in heart and lung tissues compared with LPS administration alone.
Conclusion: (1) Vagus nerve excitation can significantly reduce the levels of proinflammatory mediator and lipid peroxidation product in tissues and serum in endotoxin challenged rats, alleviate the inflammatory response in heart and lung tissues. It might provide a potential protective effect on heart and lung from endotoximia. (2)Vagotomy resulted in significant increase in TNF and MDA contents, and MPO activity in tissues as well as TNF- α and CK-MB in serum and aggravating the inflammatory response in heart and lung tissues. (3)The results suggested that vagus nerve play a very important role in modulating the anti-inflammatory response against endotoxin and preventing tissue from over-inflammatory injury.
方法：雄性Wistar大鼠70只，采用乌拉坦腹腔注射麻醉(25％，1.2g·kg~(-1))，颈动脉插管，切断双侧颈部迷走神经或做假手术对照，将左侧迷走神经远端连接刺激与记录电极，监测神经动作电位及心率和血压(Pclab生物信号电脑采集系统，北京威信斯达公司)。鼠阴茎背静脉静注LPS(O111：B4，Sigma，10mg·kg~(-1))即刻持续电刺激(5V，2ms，1Hz)迷走神经远端20min，每间隔10min后重复刺激，每次20min，共刺激3次。随机分组如下，每组10只：
实验Ⅰ组：手术+静注LPS(以下简称：LPS组)；
实验Ⅱ组：手术+双侧颈部迷走神经切断+静注LPS(以下简称：V-LPS组)：
实验Ⅲ组：手术+双侧颈部迷走神经切断+静注LPS+左迷走神经远端电刺激(以下简称：VS-LPS组)；
对照Ⅰ组：手术对照，只行麻醉、颈部切口神经分离、动脉插管测压和监测(以下简称：C组)；
对照Ⅱ组：手术+双侧颈部迷走神经切断(以下简称：V组)；
对照Ⅲ组：手术+左颈部迷走神经远端电刺激(以下简称：S组)：
对照Ⅳ组：手术+双侧颈部迷走神经切断+左迷走神经远端电刺激(以
下简称:VS组)。
静注LPS后1 .sh处死动物。分别检测各组动物心肌和肺组织中肿瘤坏
死因子一a(T切叮or necrosis factor一Q，TNF一a)、丙二醛(Malondialdehyde，
MDA)量、髓过氧化物酶(Myel叩eroxidase，MpO)活性变化以及血肌酸
激酶同工酶(CK-MB)、TNF一Q、乙酞胆碱(Acetyicholine，ACh)和皮质
醇水平。
结果:1、动物双侧迷走神经被切断后，心率、血压明显上升，呼吸减
慢变深。电刺激后心率、血压平均下降12%，呼吸转快，停刺激后心率、
血压迅速上升。2、C组和未静注LPS组动物，心肌和肺组织中TNF一Q、
MPO、MDA值以及血CK.MB、TNF一Q和皮质醇水平较低，组间两两比较，
数值无统计学意义，心肌及肺组织病理检查无异常。血ACh水平随迷走神
经的切断或刺激而明显降低或升高(P<0.01)。3、LPS组动物心肌和肺组
织中TNF一Q、MDA量、MPO活性以及血CK一MB、TNF一a和皮质醇水平
显著高于C组(P<0.01)，血ACh也升高(P<0.05)。心肌及肺组织出现炎
性病理改变，可见间质血管扩张充血，组织内炎性细胞浸润等。4、V一LPS
组动物较LPS组动物心肌和肺组织中TNF一Q、MDA量及MPO活性和血
CK一MB、TNF一Q水平显著升高(P<0.05)，血ACh和皮质醇水平却明显下
降(P<0.01)。心肌和肺组织炎性病理改变加重，可见血管充血和炎性细胞
浸润明显增加，出现组织点状坏死。5、VS一LPS组心肌和肺组织中TNF-
Q、MDA量和MPO活性和血CK一MB、TN下一Q、皮质醇水平较LPS组显
著降低(P<0.01)，血中ACh水平明显上升(P<0.05)。心肌和肺组织炎性
病理改变减轻，可见血管扩张充血和炎性细胞浸润明显减轻，组织的点状
坏死消失。
结论:1、电刺激切断的迷走神经能使LPS血症大鼠血CK一MB、TNF
水平及心、肺组织中TNF、MDA量以及MPO活性明显下降，组织炎症病
理改变减轻，揭示迷走神经通过抑制TNF的合成参与LPS血症时组织炎
症反应的调节;2、切断迷走神经对正常大鼠血CK一MB、TNF水平及心、
肺组织中TNF、MDA量和MPO活性并无显著影响，但能使LPS血症大
Vl
鼠血CK－MB、WF水平及心、肺组织中IN’－－、MDA量和WO活性明显
升高，组织炎症反应加重，表明切断迷走神经不影响正常机体炎症介质的
水平，但加重LPS血症时组织的炎症反应。3、切断LPS血症大鼠的迷走
神经可显著降低血皮质醇水平，提示迷走神经的完整性对于维持体内皮质
醇的水平，调节机体全身炎症反应，保护心、肺组织免受过度炎症损害具
有重要意义。
Aim: To expound the effects of vagus nerve on the inflammatory response in heart and lung tissues in rats with endotoximia and investigate the role of the cholinergic nervous system in modulating systemic inflammatory response to endotoxin.
Methods: Seventy male Wistar rats, weighting 220 20g, were anaesthetized with urethane(25 %, 1.2g-kg-1). The common carotid artery were cannulated and connected to a blood pressure transducer.An acquisition system(Pclab System, Wisinstar CO.BeiJing) was used to monitor mean arterial blood pressure and heart rate continuously. Animals were subjected to bilateral cervical vagtotomy. A bipolar electrodes was placed at the distal end of one side vagus nerve trunk and connected to a stimulation module. The electrical stimulation was controlled by an acquisition system for continuous registration of nerve action potential. Stimuli with constant voltage (5V,2ms,1Hz) were administered at the nerve for 20 min immediately after administration of lipopolysaccharide(LPS, 10mg kg-1,E coli O111:B4, Sigma), and then were repeated 2 times after each of 10 min interval.
Rats were randomly divided into 7 groups as follow:
(1)sham operation(C, n=10);
(2)vagotomy alone(V, n=10);
(3)vagus nerve stimulation alone(S, n=10);
(4)vagotomy plus vagus nerve stimulation(VS, n=10);
(5)LPS injection alone(LPS, n=10);
(6)vagotomy plus LPS injection(V-LPS, n=10);
(7)vagotomy plus LPS injection and vagus nerve stimulation(VS-LPS, n=10).
Blood and tissue pieces from rats heart and lung were collected at 1.5h after LPS administration in all groups. Tumor necrosis factor- a (TNF- ), malondialdehyde (MDA) and myeloperoxidase (MPO) in heart and lung tissues and serum TNF-a , acetylcholine (ACh), CK-MB and corticosterone were determined.
Results: (1)Bilateral cervical vagotomy significantly elevated blood pressure , speeded up heart rate and slowed down respiratory rate, but vagus never stimulation reversed changes above. (2)The TNF- a and MDA contents and MPO activity in serum and tissues were very lower in the operation control group and other groups without LPS injection.There were no significant differences in the content of TNF- a and MDA and MPO activity in tissue and CK-MB and corticosterone in serum between each groups without LPS injection. Pathologic lesion was not found in these groups either. (3)LPS administration significantly elevated the levels of TNF- a and ACh and CK-MB and corticosterone in serum and the contents of TNF- a and MDA and MPO in heart and lung tissues. Inflammatory changes including interstitial vascular dilatation and hyperemia and inflammatory cells infiltration were found in heart and lung tissues. (4)Vagotomy without stimulation resulted in significant increase in TNF and MDA contents and MPO activity in tissues as well as TNF- a and CK-MB in serum,but significant decrease in corticosterone and ACh in serum. A more severe inflammatory response than LPS
administration alone in heart and lung tissues was observed. (5)Vagotomy compounded stimulation significantly decreased in TNF-α and MDA and MPO in heart and lung tissues and TNF-α and corticosterone and CK-MB in serum, but increased in ACh in serum. Vagotomy compounded stimulation also atteuated inflammatory response in heart and lung tissues compared with LPS administration alone.
Conclusion: (1) Vagus nerve excitation can significantly reduce the levels of proinflammatory mediator and lipid peroxidation product in tissues and serum in endotoxin challenged rats, alleviate the inflammatory response in heart and lung tissues. It might provide a potential protective effect on heart and lung from endotoximia. (2)Vagotomy resulted in significant increase in TNF and MDA contents, and MPO activity in tissues as well as TNF- α and CK-MB in serum and aggravating the inflammatory response in heart and lung tissues. (3)The results suggested that vagus nerve play a very important role in modulating the anti-inflammatory response against endotoxin and preventing tissue from over-inflammatory injury.
引文
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