前列腺素E1对大鼠小肠缺血再灌注损伤保护作用的实验研究
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摘要
目的：通过应用前列腺素E1对大鼠小肠缺血／再灌注损伤影响的研究,从肠粘膜的损伤程度,促炎因子和抗炎因子的释放,以及细菌移位等不同方面探讨前列腺素E1对大鼠小肠缺血再灌注损伤的是否具有保护作用,从而为临床防治小肠缺血再灌注损伤提供一个新的策略。
方法：SPF级健康成年雄性SD大鼠80只,体重280g-320g,随机分为假手术(Sham group, S)组、缺血／再灌注损伤(ischemia/reperfusion injury, IRI)组、缺血预处理组(ischemia preconditioning, IPC)和前列腺E1(Prostaglandin E1,PGE1)组(n=20),每组根据再灌注后采集标本的时间分为两个亚组(n=10),实验前12h使大鼠禁食,自由饮水,并于实验前2h行含绿色荧光素蛋白大肠杆菌(Ecdi DH5α)2ml灌胃。
①S组：麻醉后取腹正中切口,长约3cm,分离肠系膜上动脉(SMA)后关腹；
②IRI组：同S组分离SMA后,用无创微动脉夹夹闭SMA后关腹,45min后取出动脉夹,分别于再灌注2h、4h时采集标本；
③IPC组：同S组分离SMA后,给予SMA 3个循环的预处理(缺血2min后再灌注2min为一个循环)后关腹,余同IRI组；
④PGE1组：同S组分离SMA后,用无创微动脉夹夹闭SMA后关腹,缺血45min后,于再灌注前5min经尾静脉按20ug／kg注入PGE1,余同IRI组。
ELISA法检测血清中IL-1β,IL-6,IL-10和TNF-α的含量,同时取回肠壁组织长1cin行肠粘膜病理学检测并行HE染色,取肝脏组织、脾脏组织及门静脉血行细菌移位检测。
结果：
1、小肠病理变化：S组大鼠肠绒毛完整,排列整齐,镜下无中性粒细胞浸润。IRI组大鼠在再灌注2h和4h后小肠绒毛上皮顶端与固有层分离并见固有层毛细血管暴露、出血和溃疡,粘膜绒毛坏死,脱落,镜下见大量中性粒细胞浸润。与IRI组相比,IPC组和PGE1组在各时间点肠粘膜绒毛损伤减轻,坏死,脱落少见,镜下中性粒细胞浸润轻。IPC组和PGE1组损伤评分明显低于IRI组(P<0.05),IPC组和PGE1组相比,差异无统计学意义(P>0.05)。
2、细胞因子改变：IRI组、IPC组和PGE1组再灌注2h、4h,大鼠动脉血清中炎性细胞因子IL-1β,IL-6,TNF-α含量明显高于S组(P<0.05)；IPC组和PGE1组IL-1p,IL-6,TNF-a则低于IRI组(P<0.05)；IPC组和PGE1组相比,差异无统计学意义(P>0.05)。大鼠动脉血清中抗炎因子IL-10含量在IRI组、IPC组和PGE1组各时间点明显低于S组(氏0.05)；IPC组和PGE1组各时间点高于IRI组(P<0.05)；IPC组和PGE1相比,差异无统计学意义(P>0.05)。
3、细菌移位改变：再灌注2h、4h,S组大鼠脾脏、肝脏及门静脉血未检出移位细菌生长；IPC组和PGE1组明显低于IRI组(P<0.05),IPC组和PGE1组相比,差异无统计学意义(P>0.05)。
结论：
PGE1和IPC能减轻大鼠小肠缺血再灌注损伤后小肠粘膜绒毛的坏死程度：PGE1和IPC能抑制大鼠小肠缺血再灌注损伤后促炎细胞因子(IL-1p,IL-6,TNF-α)的释放和促进抗炎细胞因子IL-10的表达：PGE1和IPC能减少大鼠小肠缺血再灌注损伤后的细菌移位。
Objective:
Through used prostaglandin E1 in the rat model of ischemia/reperfusion injury, we investigate the protective effects of PGE1 on small intestinal ischemia/reperfusion injury by analyzing the small intestinal mucous membrane's extent of damage、the inflammatory cell factor's release and bacterial translocation, to provide a new strategy for clinical to prevent from small intestine ischemia/reperfusion injury.
Methods:
Eighty male SD rats were weighed about 280-320g, then they were divided randomly into four groups:Sham group(S group), ischemia/reperfusion injury group(IRI group), ischemia preconditioning group(IPC group), Prostaglandin El group(PGE1 group)(n=20).Each group was divided into two subgroups according to time of specimen collection after reperfusion(n=10).Before 12 hours of testing, the testing rats were gaven fasting and free drinking; and before 2 hour of testing, the testing rats were irrigated colon bacillus (E.cdi DH5a)which contained green fluorescent protein into stomach.
①The S group:the rats underwent a 3cm medium length-wise laparotomy after anesthesia, identification and dissection of the superior mesenteric artery(SMA)and then, closed abdominal cavity.
②The IRI group:identification and dissection of the SMA as S group, the SMA was clipped by a un-wound micro artery clip, and then, closed abdominal cavity; after 45min,taken out the un-wound micro artery clip; then collected specimen at the time of 2h and 4h.
③The IPC group:identification and dissection of the SMA as S group,the SMA was subjected to 3 circles of 2min ischemia and 2min reperfusion before ischemia, and then, same as IRI group.
④The PGE1:group identification and dissection of the SMA as S group, t the SMA was clipped for 45 min,5 min of reperfusion,inject PGE1 from tail vein(20ug/kg), and then, same as IRI group.
At the end of the before operation, we dissected 1cm segment of ileum to determination of intestines mucous membrane's injury. Then we measured the serum of TNF-a, IL-1β, IL-6, IL-10 by ELISA. The last,the rats were sacrificed, we cut tissues of liver、spleen at the same weight(2g) and portal vein blood to train bacterial translocation。
Results:
①The changes of intestinal mucosa:In Sham group, under microscope, the intestinal villus were completely and linedly;In IRI group, the intestinal mucosa presented dilated and exposed capillaries and denuded villi, some of villi hemorrhage were ulceration;the lamina propria was edema and infiltrated with inflammatory cells. The intestinal mucosal structure maintain intact with lifting of the epithelial layer from the lamina propria and moderate extension of the subepithelial space in PGE1 group and in IPC group. In PGE1 group and IPC group, histopathologic scores were significantly lower than that of IPI group(P<0.05). There were no significant difference between PGE1 group and IPC group (P>0.05).
②Compared with S group, the serum contents of TNF-a,IL-1β,IL-6 in IRI group, IPC group and PGE1 group were obviously higher, but the PGE1 group and IPC were lower than that of IRI group. The contents of IL-10 in other three groups were lower than that of S group, and The PGE1 group and IPC group were higher than that of IRI group. There were no significant differences between PGE1 group and IPC group (P>0.05).
③acterial translocation:In S group, the bacteria wasn't checked out from portal vein blood、liver tissues and spleen tissues at 2h and 4h. But PGE1 group and IPC were lower than that of IRI group (P>0.05).There were no significant differences between PGE1 group and IPC group (P>0.05).
Conclusion:
The PGE1 and IPC make rats small intestinal mucosa's extent of damage alleviated after reperfusion, can protect small intestinal mucosal barrier; can obviously inhibit the release of TNF-a,IL-1β,IL-6 and promote anti-inflammatory medium of IL-10 expression after reperfusion;can obviously inhibit the bacteria transposition after reperfusion。
方法：SPF级健康成年雄性SD大鼠80只,体重280g-320g,随机分为假手术(Sham group, S)组、缺血／再灌注损伤(ischemia/reperfusion injury, IRI)组、缺血预处理组(ischemia preconditioning, IPC)和前列腺E1(Prostaglandin E1,PGE1)组(n=20),每组根据再灌注后采集标本的时间分为两个亚组(n=10),实验前12h使大鼠禁食,自由饮水,并于实验前2h行含绿色荧光素蛋白大肠杆菌(Ecdi DH5α)2ml灌胃。
①S组：麻醉后取腹正中切口,长约3cm,分离肠系膜上动脉(SMA)后关腹；
②IRI组：同S组分离SMA后,用无创微动脉夹夹闭SMA后关腹,45min后取出动脉夹,分别于再灌注2h、4h时采集标本；
③IPC组：同S组分离SMA后,给予SMA 3个循环的预处理(缺血2min后再灌注2min为一个循环)后关腹,余同IRI组；
④PGE1组：同S组分离SMA后,用无创微动脉夹夹闭SMA后关腹,缺血45min后,于再灌注前5min经尾静脉按20ug／kg注入PGE1,余同IRI组。
ELISA法检测血清中IL-1β,IL-6,IL-10和TNF-α的含量,同时取回肠壁组织长1cin行肠粘膜病理学检测并行HE染色,取肝脏组织、脾脏组织及门静脉血行细菌移位检测。
结果：
1、小肠病理变化：S组大鼠肠绒毛完整,排列整齐,镜下无中性粒细胞浸润。IRI组大鼠在再灌注2h和4h后小肠绒毛上皮顶端与固有层分离并见固有层毛细血管暴露、出血和溃疡,粘膜绒毛坏死,脱落,镜下见大量中性粒细胞浸润。与IRI组相比,IPC组和PGE1组在各时间点肠粘膜绒毛损伤减轻,坏死,脱落少见,镜下中性粒细胞浸润轻。IPC组和PGE1组损伤评分明显低于IRI组(P<0.05),IPC组和PGE1组相比,差异无统计学意义(P>0.05)。
2、细胞因子改变：IRI组、IPC组和PGE1组再灌注2h、4h,大鼠动脉血清中炎性细胞因子IL-1β,IL-6,TNF-α含量明显高于S组(P<0.05)；IPC组和PGE1组IL-1p,IL-6,TNF-a则低于IRI组(P<0.05)；IPC组和PGE1组相比,差异无统计学意义(P>0.05)。大鼠动脉血清中抗炎因子IL-10含量在IRI组、IPC组和PGE1组各时间点明显低于S组(氏0.05)；IPC组和PGE1组各时间点高于IRI组(P<0.05)；IPC组和PGE1相比,差异无统计学意义(P>0.05)。
3、细菌移位改变：再灌注2h、4h,S组大鼠脾脏、肝脏及门静脉血未检出移位细菌生长；IPC组和PGE1组明显低于IRI组(P<0.05),IPC组和PGE1组相比,差异无统计学意义(P>0.05)。
结论：
PGE1和IPC能减轻大鼠小肠缺血再灌注损伤后小肠粘膜绒毛的坏死程度：PGE1和IPC能抑制大鼠小肠缺血再灌注损伤后促炎细胞因子(IL-1p,IL-6,TNF-α)的释放和促进抗炎细胞因子IL-10的表达：PGE1和IPC能减少大鼠小肠缺血再灌注损伤后的细菌移位。
Objective:
Through used prostaglandin E1 in the rat model of ischemia/reperfusion injury, we investigate the protective effects of PGE1 on small intestinal ischemia/reperfusion injury by analyzing the small intestinal mucous membrane's extent of damage、the inflammatory cell factor's release and bacterial translocation, to provide a new strategy for clinical to prevent from small intestine ischemia/reperfusion injury.
Methods:
Eighty male SD rats were weighed about 280-320g, then they were divided randomly into four groups:Sham group(S group), ischemia/reperfusion injury group(IRI group), ischemia preconditioning group(IPC group), Prostaglandin El group(PGE1 group)(n=20).Each group was divided into two subgroups according to time of specimen collection after reperfusion(n=10).Before 12 hours of testing, the testing rats were gaven fasting and free drinking; and before 2 hour of testing, the testing rats were irrigated colon bacillus (E.cdi DH5a)which contained green fluorescent protein into stomach.
①The S group:the rats underwent a 3cm medium length-wise laparotomy after anesthesia, identification and dissection of the superior mesenteric artery(SMA)and then, closed abdominal cavity.
②The IRI group:identification and dissection of the SMA as S group, the SMA was clipped by a un-wound micro artery clip, and then, closed abdominal cavity; after 45min,taken out the un-wound micro artery clip; then collected specimen at the time of 2h and 4h.
③The IPC group:identification and dissection of the SMA as S group,the SMA was subjected to 3 circles of 2min ischemia and 2min reperfusion before ischemia, and then, same as IRI group.
④The PGE1:group identification and dissection of the SMA as S group, t the SMA was clipped for 45 min,5 min of reperfusion,inject PGE1 from tail vein(20ug/kg), and then, same as IRI group.
At the end of the before operation, we dissected 1cm segment of ileum to determination of intestines mucous membrane's injury. Then we measured the serum of TNF-a, IL-1β, IL-6, IL-10 by ELISA. The last,the rats were sacrificed, we cut tissues of liver、spleen at the same weight(2g) and portal vein blood to train bacterial translocation。
Results:
①The changes of intestinal mucosa:In Sham group, under microscope, the intestinal villus were completely and linedly;In IRI group, the intestinal mucosa presented dilated and exposed capillaries and denuded villi, some of villi hemorrhage were ulceration;the lamina propria was edema and infiltrated with inflammatory cells. The intestinal mucosal structure maintain intact with lifting of the epithelial layer from the lamina propria and moderate extension of the subepithelial space in PGE1 group and in IPC group. In PGE1 group and IPC group, histopathologic scores were significantly lower than that of IPI group(P<0.05). There were no significant difference between PGE1 group and IPC group (P>0.05).
②Compared with S group, the serum contents of TNF-a,IL-1β,IL-6 in IRI group, IPC group and PGE1 group were obviously higher, but the PGE1 group and IPC were lower than that of IRI group. The contents of IL-10 in other three groups were lower than that of S group, and The PGE1 group and IPC group were higher than that of IRI group. There were no significant differences between PGE1 group and IPC group (P>0.05).
③acterial translocation:In S group, the bacteria wasn't checked out from portal vein blood、liver tissues and spleen tissues at 2h and 4h. But PGE1 group and IPC were lower than that of IRI group (P>0.05).There were no significant differences between PGE1 group and IPC group (P>0.05).
Conclusion:
The PGE1 and IPC make rats small intestinal mucosa's extent of damage alleviated after reperfusion, can protect small intestinal mucosal barrier; can obviously inhibit the release of TNF-a,IL-1β,IL-6 and promote anti-inflammatory medium of IL-10 expression after reperfusion;can obviously inhibit the bacteria transposition after reperfusion。
引文
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38、 Matlin KS, Haus B, Zuk A. In tegrins in epithelial cell polaritv:using antibody to analyze adhesive function and morphogenesis [J]. Methods 2003; 30:235-245
39、刘卉,刘延香.细胞凋亡与活性氧.现代肿瘤学.2008.10(16)：1830-1832
40、 Tsurg A,Kaizu T, Nakao A,et al. Ethyl pyruvate ameliorates liver ischemia-reperfusion injury by decreasing hepatic necrosis and apoptosis.[J].Transpiantation,2005,79:196-204.