肠缺血/再灌注损伤及高渗盐溶液干预作用的实验研究
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摘要
目的:研究高渗盐溶液对兔肠缺血再灌注所致肠与下游远隔器官肝脏和肺脏损伤保护作用及可能的机制,同时探讨高渗盐溶液对脏器功能保护作用的规律。
方法:新西兰白兔108只,按随机原则,分6组,每组18只:正常对照组,缺血对照组,NS组,4%HS组,7.5%HS组,7.5%HS延迟组。正常对照组只游离SMA,但不夹闭SMA。其他组均游离并夹闭SMA1小时。NS组,4%HS组和7.5%HS组在SMA开放前5分钟由颈内静脉输注相应浓度的氯化钠溶液。而7.5%HS延迟组在开放再灌注2小时时才输注高渗盐溶液。选择缺血前与再灌注后2、4、6小时4个时间点抽血行动脉血气分析测Na~+,CL~-,PH,取血清测定TNF-α、MCP-Ⅰ,IL-10,ICAM-1,LPS,D-1ac,ALT,AST水平。每组动物随机抽取8只用于再灌注6小时时处死,取小肠、肝脏及肺组织标本,测定组织中的MPO、MDA、SOD浓度,光镜下对病理形态进行观察,TUNEL法检测肠上皮细胞凋亡。其余10只用于观察存活时间。
结果:(1)肠缺血再灌注后血液中LPS、D-lac、TNF-α、MCP-Ⅰ,ICAM-1显著增加,均于2小时达高峰;肝酶ALT、AST明显升高,分别于开放灌注后4小时、6小时达高峰;小肠、肝脏及肺组织中MDA、MPO含量显著升高,SOD活性降低;小肠病理显示肠绒毛缺损、肿胀明显,有溃疡形成,血管周围出血明显,有中性粒细胞浸润,基底层断裂;肠上皮细胞凋亡增加;肺组织病理显示肺组织充血、肺泡间质水肿、炎性细胞渗出,部分肺泡壁破坏,肺泡不张。(2)NS治疗组肠道局部损伤、全身炎症介质以及肝脏和肺脏损伤和缺血对照组对照无显著差异。(3)4%HS组肠道局部损伤、全身炎症介质以及肝脏和肺脏损伤较肠缺血再灌注对照组减轻,动物牛存时间延长,但肠上皮细胞凋亡增加,两组之间有显著差异。(4)7.5%HS组肠道局部损伤、全身炎症介质以及肝脏和肺脏损伤较4%HS组减轻,动物生存时间进一步延长,肠上皮细胞凋亡进一步增加,两者之间有显著差异。(5)7.5%HS延迟组肠道局部损伤、全身炎症介质以及肝脏和肺脏损伤和缺血对照组无显著差异,且动物生存时间较缺血对照组下降。
结论:(1)肠缺血再灌注损伤可以导致肠缺血局部以及位于下游的其他脏器功能损伤;(2)适时应用高渗盐溶液对肠缺血再灌注引起的局部及远隔的脏器功能有保护作用;(3)高渗盐溶液能调控肠缺血再灌注损伤动物模型的体内炎症反应,一方面降低促炎介质的释放,一方面增加抗炎介质的释放;(4)0.9%-7.5%的范围内随着浓度的增加,高渗盐溶液对肠缺血再灌注引起的局部和远隔器官的损伤保护作用逐渐增强;(5)高渗环境可以诱导肠缺血再灌注肠上皮细胞凋亡,避免缺血后肠上皮坏死;(6)高渗盐溶液作为限制全身炎症反应的工具应尽早使用,否则甚至会加重全身炎症反应。
Objective: To study the protective effects and possible mechanisms of the hypertonic saline solution on intestine and remote organs (liver and lung ) injuries induced by intestinal ischemia reperfusion; To investigate the rule of organ protection by hypertonic saline.
Methods: Rabbits were randomly assigned to control group (no treatment, no superior mesenteric artery occlusion[SMAO] , n=18), ischemia reperfusion group (no treatment,no SMA0,n=18), NS group (6mL/kg 0.9% NS, SMAO,n=18), 4%HS group (6mL/kg 4%HS, SMAO,n=18), 7.5%HS group (6mL/kg 7.5%HS, SMAO,n=18), 7.5%HS delayed group (6mL/kg 7.5%HS delayed, SMAO,n=18). The SMAO was clamped for 60 minutes and boluses given 5 minutes before clamp removal. But in 7.5%HS delayed group hypertonic saline was infused two hours after reperfusion. Serum lipopolysaccharide (LPS ) and D-lac as markers of gut barrier, serum TNF-α, MCP-I, IL-10 and ICAM-1 as markers of systemic inflammation response , serum ALT and AST as markers of liver injury were detected before ischemia and 2,4,6 hours after reperfusion .At the same time arterial blood gas analysis was performed for Na~+,CL~- and PH. After 6 hours of reperfusion, ileum ,liver and lungs of 8 rabbits each group were harvested for myeloperoxidase (MPO) as an index of neutrophil mediated injury, superoxide dismutase (SOD )and malondialdehyde (MDA) as an index of oxygen free radical mediated injury, analysis of histologic injury of gut and lung,and analysis of apoptosis of enteric epithelium. The survival time of Other 10 rabbits each group was observed.
Results: (1) The markers of gut barrier: LPS, D-lac and inflammation factors:TNF-α, MCP-I, ICAM-1 significantly increased after intestinal reperfusion ,reaching peak after 2 hours of reperfusion ; markers of liver injury:ALT and AST increased significantly, and they reached peak respectively at the time 4 and 6 hours after reperfusion; intestinal pathology showed intestinal villi defect, swelling obviously,ulcer,perivascular hemorrhage, neutrophil infiltration and basal fracture; Apoptosis of intestinal epithelial cells increased ;lung pathology showed congestive, interstitial pulmonary edema, inflammatory exudate, alveolar wall destruction. (2) There are no significant difference of intestinal injury, systemic inflammatory mediators, as well as liver and lung injury between NS group (isotonic liquid treatment group) and ischemia reperfusion group. (3) In 4%HS group ,intestinal injury, systemic inflammatory mediators, as well as liver and lung injury reduced ; survival time of rabbits extended, but apoptosis of intestinal epithelial cells increased. (4) In 7.5%HS group,intestinal injury, systemic inflammatory mediators, as well as liver and lung injury improved more than 4%HS group; survival time of rabbits extended longer than 4%HS group, but apoptosis of intestinal epithelial cells increased more than 4%HS group. (5) In 7.5%HS delayed group, there are no significant difference of intestinal injury, systemic inflammatory mediators, as well as liver and lung injury as ischemia reperfusion group. But the survival time of rabbits decreased significantly.
Conclusion: (1) Intestinal ischemia-reperfusion injury leaded to the injury of gut, as well as the remote organs. (2) Hypertonic saline had the protective effect on intestine and remote organs injuries induced by intestinal ischemia reperfusion. (3)Hypertonic saline could modulate systemic inflammatory response, regulate the balance between pro-inflammatory and anti-inflammatory response. (4) With the increasing concentration of hypertonic saline from 0.9% to 7.5%, the protective effects on organs function were gradually strengthened. (5) Hypertonic environment could promote apoptosis of intestinal epithelial cells by ischemic reperfusion, avoiding necrosis . (6) Hypertonic saline should be used early as tool of systemic inflammatory response limitation, otherwise could even worse systemic inflammatory response.
方法:新西兰白兔108只,按随机原则,分6组,每组18只:正常对照组,缺血对照组,NS组,4%HS组,7.5%HS组,7.5%HS延迟组。正常对照组只游离SMA,但不夹闭SMA。其他组均游离并夹闭SMA1小时。NS组,4%HS组和7.5%HS组在SMA开放前5分钟由颈内静脉输注相应浓度的氯化钠溶液。而7.5%HS延迟组在开放再灌注2小时时才输注高渗盐溶液。选择缺血前与再灌注后2、4、6小时4个时间点抽血行动脉血气分析测Na~+,CL~-,PH,取血清测定TNF-α、MCP-Ⅰ,IL-10,ICAM-1,LPS,D-1ac,ALT,AST水平。每组动物随机抽取8只用于再灌注6小时时处死,取小肠、肝脏及肺组织标本,测定组织中的MPO、MDA、SOD浓度,光镜下对病理形态进行观察,TUNEL法检测肠上皮细胞凋亡。其余10只用于观察存活时间。
结果:(1)肠缺血再灌注后血液中LPS、D-lac、TNF-α、MCP-Ⅰ,ICAM-1显著增加,均于2小时达高峰;肝酶ALT、AST明显升高,分别于开放灌注后4小时、6小时达高峰;小肠、肝脏及肺组织中MDA、MPO含量显著升高,SOD活性降低;小肠病理显示肠绒毛缺损、肿胀明显,有溃疡形成,血管周围出血明显,有中性粒细胞浸润,基底层断裂;肠上皮细胞凋亡增加;肺组织病理显示肺组织充血、肺泡间质水肿、炎性细胞渗出,部分肺泡壁破坏,肺泡不张。(2)NS治疗组肠道局部损伤、全身炎症介质以及肝脏和肺脏损伤和缺血对照组对照无显著差异。(3)4%HS组肠道局部损伤、全身炎症介质以及肝脏和肺脏损伤较肠缺血再灌注对照组减轻,动物牛存时间延长,但肠上皮细胞凋亡增加,两组之间有显著差异。(4)7.5%HS组肠道局部损伤、全身炎症介质以及肝脏和肺脏损伤较4%HS组减轻,动物生存时间进一步延长,肠上皮细胞凋亡进一步增加,两者之间有显著差异。(5)7.5%HS延迟组肠道局部损伤、全身炎症介质以及肝脏和肺脏损伤和缺血对照组无显著差异,且动物生存时间较缺血对照组下降。
结论:(1)肠缺血再灌注损伤可以导致肠缺血局部以及位于下游的其他脏器功能损伤;(2)适时应用高渗盐溶液对肠缺血再灌注引起的局部及远隔的脏器功能有保护作用;(3)高渗盐溶液能调控肠缺血再灌注损伤动物模型的体内炎症反应,一方面降低促炎介质的释放,一方面增加抗炎介质的释放;(4)0.9%-7.5%的范围内随着浓度的增加,高渗盐溶液对肠缺血再灌注引起的局部和远隔器官的损伤保护作用逐渐增强;(5)高渗环境可以诱导肠缺血再灌注肠上皮细胞凋亡,避免缺血后肠上皮坏死;(6)高渗盐溶液作为限制全身炎症反应的工具应尽早使用,否则甚至会加重全身炎症反应。
Objective: To study the protective effects and possible mechanisms of the hypertonic saline solution on intestine and remote organs (liver and lung ) injuries induced by intestinal ischemia reperfusion; To investigate the rule of organ protection by hypertonic saline.
Methods: Rabbits were randomly assigned to control group (no treatment, no superior mesenteric artery occlusion[SMAO] , n=18), ischemia reperfusion group (no treatment,no SMA0,n=18), NS group (6mL/kg 0.9% NS, SMAO,n=18), 4%HS group (6mL/kg 4%HS, SMAO,n=18), 7.5%HS group (6mL/kg 7.5%HS, SMAO,n=18), 7.5%HS delayed group (6mL/kg 7.5%HS delayed, SMAO,n=18). The SMAO was clamped for 60 minutes and boluses given 5 minutes before clamp removal. But in 7.5%HS delayed group hypertonic saline was infused two hours after reperfusion. Serum lipopolysaccharide (LPS ) and D-lac as markers of gut barrier, serum TNF-α, MCP-I, IL-10 and ICAM-1 as markers of systemic inflammation response , serum ALT and AST as markers of liver injury were detected before ischemia and 2,4,6 hours after reperfusion .At the same time arterial blood gas analysis was performed for Na~+,CL~- and PH. After 6 hours of reperfusion, ileum ,liver and lungs of 8 rabbits each group were harvested for myeloperoxidase (MPO) as an index of neutrophil mediated injury, superoxide dismutase (SOD )and malondialdehyde (MDA) as an index of oxygen free radical mediated injury, analysis of histologic injury of gut and lung,and analysis of apoptosis of enteric epithelium. The survival time of Other 10 rabbits each group was observed.
Results: (1) The markers of gut barrier: LPS, D-lac and inflammation factors:TNF-α, MCP-I, ICAM-1 significantly increased after intestinal reperfusion ,reaching peak after 2 hours of reperfusion ; markers of liver injury:ALT and AST increased significantly, and they reached peak respectively at the time 4 and 6 hours after reperfusion; intestinal pathology showed intestinal villi defect, swelling obviously,ulcer,perivascular hemorrhage, neutrophil infiltration and basal fracture; Apoptosis of intestinal epithelial cells increased ;lung pathology showed congestive, interstitial pulmonary edema, inflammatory exudate, alveolar wall destruction. (2) There are no significant difference of intestinal injury, systemic inflammatory mediators, as well as liver and lung injury between NS group (isotonic liquid treatment group) and ischemia reperfusion group. (3) In 4%HS group ,intestinal injury, systemic inflammatory mediators, as well as liver and lung injury reduced ; survival time of rabbits extended, but apoptosis of intestinal epithelial cells increased. (4) In 7.5%HS group,intestinal injury, systemic inflammatory mediators, as well as liver and lung injury improved more than 4%HS group; survival time of rabbits extended longer than 4%HS group, but apoptosis of intestinal epithelial cells increased more than 4%HS group. (5) In 7.5%HS delayed group, there are no significant difference of intestinal injury, systemic inflammatory mediators, as well as liver and lung injury as ischemia reperfusion group. But the survival time of rabbits decreased significantly.
Conclusion: (1) Intestinal ischemia-reperfusion injury leaded to the injury of gut, as well as the remote organs. (2) Hypertonic saline had the protective effect on intestine and remote organs injuries induced by intestinal ischemia reperfusion. (3)Hypertonic saline could modulate systemic inflammatory response, regulate the balance between pro-inflammatory and anti-inflammatory response. (4) With the increasing concentration of hypertonic saline from 0.9% to 7.5%, the protective effects on organs function were gradually strengthened. (5) Hypertonic environment could promote apoptosis of intestinal epithelial cells by ischemic reperfusion, avoiding necrosis . (6) Hypertonic saline should be used early as tool of systemic inflammatory response limitation, otherwise could even worse systemic inflammatory response.
引文
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1. Ding LA, Li JS.Intestinal failure: pathophysiological elements and clinical diseases. World J Gastroenterol.2004 Aprl; 10(7):930-3.
2. Hassoun HT, Kone BC, Mercer DW, et al. Post-injury multiple organ failure:the role of the gut.Shock,2001,5:1210.
3. Deitch EA.Bacterial translocation or lymphatic drainage of toxic products from the gut: what is important in human beings? Surgery, 2002, 131 (3):241-244.
4. Fang WH , Yao YM, Shi GZ , et al. The mRNA expression patterns of tumor necrosis factor alpha and TNFR-I in some vital organs after thermal injury. World J Gastroenterol, 2003 , 9 :103 8-1044.
5. Jackson GD , Dai Y, Sewell WA. Bile mediates intestinal pathology inendotoxemia in rats. Infect Immunol, 2000 , 68 :4714-4719 .
6. Deitch EA. Role of the gut lymphatic system in multiple organ failure.Curr Opin Crit Care, 2001,7:92-98
7. Dayal SD , Hauser CJ , Feketeova E , et al. Shock mesenteric lymph induced rat polymorphonuclear neutrophil activation and endothelial cell injury is mediated by aqueous factors. J Trauma, 2002 , 52 :1048-1055.
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