糖原合成酶激酶-3抑制剂与1，6-二磷酸果糖联合应用对大鼠肝脏创伤救治的影响
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摘要
目的:
在战、创伤所致肝脏破裂伤急救手术后仍然有相当高的死亡率及术后并发症发生率。咎其原因,在复杂的肝脏破裂伤手术操作时,为了及时控制肝脏破裂创面的大出血或便于破碎创面的清创(切除)止血,手术过程中常采用的阻断入肝血流是一个重要原因。入肝血流阻断后无疑将会进一步给肝脏带来不可低估的热缺血损伤,在手术成功止血清创后,恢复肝脏血流供应,又会产生对肝脏的再灌注损伤,这种损伤在某种程度上往往比前期阻断肝脏血流所致热缺血引起的损伤更为严重。并且,在战、创伤所致肝脏破裂伤救治过程中,机体正处于急性应激状态,此时肝脏的一系列病理生理改变就是肝脏在应激条件下的缺血再灌注损伤!
目前普遍认为,肝细胞生物能量缺乏是引发肝脏缺血再灌注损伤一系列病理改变的始动因素。业已证实,如果在阻断肝门前有效地提高肝脏糖原储备,则能有效降低后续的缺血再灌注损伤。但是在抢救肝脏破裂伤的过程中,为避免失血引起的休克,抢救人员必须尽快手术阻断肝脏供血以止血清创,这就必须在短时间内提高肝脏的糖原储备。如何在短时间内提高肝脏的糖原储备?一个是有效地增加合成糖原的底物,另一个就是加快糖原的合成。
已知1,6-二磷酸果糖(fructose-1,6-diphosphate,FDP)作为能量代谢调节剂,能减轻各种组织器官包括肝脏的缺血再灌注损伤,同时FDP作为细胞内糖代谢的中间产物,还是糖酵解和糖异生的共同通路;另外,糖原合成酶激酶-3(glycogen synthase kinase-3,GSK-3)可以磷酸化糖原合成酶的丝氨酸位点使其失活,进而可抑制糖原的合成;相反GSK-3抑制剂氯化锂能有效抑制GSK-3活性,从而激活糖原合成酶,促进糖原的合成。
基于以上认识,本课题通过建立大鼠肝脏撞击破裂伤模型,探讨在战、创伤致肝脏破裂伤的急救或手术过程中,通过联合使用GSK-3抑制剂氯化锂和FDP,能否在短时间内(如1h)增加肝细胞内糖原含量,从而减轻手术过程中阻断入肝血流带来的肝脏热缺血再灌注损伤。
方法:
采用SD大鼠为实验对象,进行下列三方面的研究。
1选取健康SD大鼠42只,随机分为3组(n=14)。通过对第三军医大学大坪医院野战外科研究所全军战创伤中心研制的BIM-Ⅳ型生物撞击机增加T型二次打击头进行改良,用轻、中、重三种质量的钢球分别撞击一组大鼠,复制出3组损伤程度不同的大鼠肝脏钝性撞击伤模型,即轻度致伤组、中度致伤组、重度致伤组。各组再按观察指标不同,随机平分为A组和B两组。测定A组实验动物致伤前后生命体征变化及自然存活数,B组致伤前后血清中AST、ALT、TNF-α含量及致伤后腹腔内出血质量,所有大鼠死亡后均进行肝损伤分级并进行评分。
2选取健康SD大鼠49只,采用前部分建立的大鼠肝脏撞击破裂伤模型。先取42只建模后大鼠随机分为对照组、葡萄糖组、FDP组(n=14),分别于致伤10min后静脉注射0.9%氯化钠、5%葡萄糖、15%FDP注射液2ml(均在10min内静注完)。各组再按处死取材时相点随机均分为缺血前和再灌注4h两个亚组(n=7)。剩余7只建模后大鼠为再灌注4h时相点假手术组。测定各组动物血清AST、ALT含量,肝组织糖原含量、SOD活性、MDA及ATP含量,并行肝组织HE染色、荧光实时定量PCR检测肝组织Bcl-2 mRNA表达、肝组织细胞凋亡检测(TUNEL法)。
3选取健康SD大鼠49只,采用前部分建立的大鼠肝脏撞击破裂伤模型。先取42只建模后大鼠随机分为对照组、FDP组、FGI组(FDP+GSK-3抑制剂联合应用组)(n=14)。对照组于致伤10min后静脉注射0.9%氯化钠注射液2ml,FDP组于致伤10min后静脉注射15% FDP注射液2ml,FGI组致伤后除在10min静注完15% FDP注射液2ml外,尚通过腹腔注射一次性给予0.5% GSK-3抑制剂氯化锂(3mmol/kg)(均在10min内静注完)。3组再按处死取材时相点随机平分为缺血前和再灌注4h两个亚组(n=7)。剩余7只建模后大鼠为再灌注4h时相点假手术组。测定各组血清中AST、ALT含量、肝组织糖原含量、SOD活力、MDA含量,并行肝组织糖原合成酶活性测定和肝组织免疫组化GSK-3表达测定。
结果:
1各组平均股动脉压致伤后均低于致伤前(P<0.01),且致伤后平均股动脉压随损伤程度的增加而降低(P<0.01或P<0.05)。各组血清AST、ALT含量致伤后均明显高于致伤前(P<0.01或P<0.05);致伤后各组AST、ALT含量均随损伤程度的增加而增高(P<0.01或P<0.05)。各组腹腔出血量随损伤程度的增加而增多(P<0.01或P<0.05)。各组肝脏损伤评分随损伤程度的增加而增高(P<0.01)。致伤后血清TNF-α含量均明显高于致伤前(P<0.01);致伤后各组间血清TNF-α含量随损伤程度的增加而增高(P<0.01)。
2在缺血前时相点,肝糖原含量,对照组<葡萄糖组葡萄糖组>FDP组>假手术组(P<0.01或P<0.05),AST水平,对照组>葡萄糖组/FDP组>假手术组(P<0.01或P<0.05),FDP组与葡萄糖组比较差异无统计学意义;肝组织SOD活性对照组<葡萄糖组葡萄糖组>FDP组>假手术组(P<0.01或P<0.05)。各组再灌注4h时相点的肝脏组织ATP含量均明显低于缺血前时相点(P<0.01);ATP含量在缺血前时相点,对照组分别低于葡萄糖组(P<0.05)和FDP组(P<0.01),葡萄糖组和FDP组之间差异无统计学意义;在再灌注4h时相点,肝脏组织ATP含量,对照组<葡萄糖组葡萄糖组>FDP组>假手术组(P<0.01或P<0.05)。
3在缺血前时相点,肝脏糖原含量,对照组FDP组>FGI组>假手术组(P<0.01),肝脏组织SOD活力,对照组FDP组>FGI组>假手术组(P<0.01)。将缺血前后各组进行比较,对照组和FDP组再灌注4h时相点的肝组织糖原合成酶活性均明显低于缺血前时相点(P<0.01),余组无差异;在缺血前时相点,肝组织糖原合成酶活性,FGI组分别大于FDP组和对照组(P<0.01),对照组和FDP组之间差异无统计学意义;在再灌注4h时相点,肝组织糖原合成酶活性,对照组FDP组>FGI组>假手术组(P<0.01)。
结论:
1各组大鼠肝脏损伤程度及应激反应随钢球质量的增加而加重,分级明显。该大鼠肝脏创伤模型建模过程简单,具有稳定的肝脏损伤的伤情特点,可重复性好,损伤程度可分级调节,费效比低,其中度致伤组可用于本课题的进一步病理机制和伤后救治的研究。
2 FDP不仅作为代谢调节剂,还是细胞内糖代谢的中间产物,是糖原合成的又一种有效底物,可以独立地在缺血再灌注损伤中发挥重要作用,而且在创伤应激条件下,相对于同摩尔浓度的葡萄糖,能更有效地在短时间内促进肝细胞内的糖原合成,提高肝糖原含量,从而减轻了手术过程中阻断入肝血流带来的术后肝脏的热缺血再灌注损伤。
3在输注FDP的同时,应用GSK-3抑制剂氯化锂,间接地增强糖原合成酶的活性,在短时间内促进肝细胞内糖原合成,比单纯应用FDP更加有效地提高了肝细胞内的糖原含量,从而减轻了手术过程中阻断入肝血流带来的术后肝脏的热缺血再灌注损伤。
Objective:
There is a high mortality rate and incidence of postoperative complications after emergency operation of liver trauma caused by war or external injury. It is an important factor that surgical procedures to block hepatic blood flow are often used to control bleeding or facilitate debridement (removal) of trauma liver. Hepatic inflow occlusion, no doubt, will bring about the liver warm ischemia injury that can not be underestimated. After the successful operation to stop bleeding or debride, recovery of liver blood supply will bring about the liver injury again, which to some extent can be more serious than damage of the liver warm ischemia caused by pre-blocking blood flow. Moreover, during treatment process of liver trauma, the body is in a state of acute stress, and this time a series of pathophysiological changes in the liver is ischemia reperfusion injury of the liver under acute stress!
Now It is widely recognized that energy deficiency of liver cells is the initial factor inducing a series of pathological changes of hepatic ischemia reperfusion injury. It has been proved that if effectively increasing liver glycogen reserves before blocking hepatic blood flow would effectively reduce subsequent ischemic reperfusion injury. However, in order to avoid the shock caused by loss of blood, rescue personnel must stop bleeding as soon as possible in the treatment process. So it is imperative to increase the liver glycogen reserves within a short time. How to increase the liver glycogen reserves within a short time? One is to increase the effective substrate for glycogen synthesis, and the other is to speed up the synthesis of glycogen.
It is known that fructose-1, 6-diphosphate (FDP) as a regulator of energy metabolism can reduce ischemia reperfusion injury of a variety of tissues and organs including the liver, meanwhile FDP as the middle product in intracellular glucose metabolism is the common pathway of glycolysis and gluconeogenesis; in addition, glycogen synthase kinase-3 (GSK-3) can phosphorylate serine sites of glycogen synthase to inhibit the synthesis of glycogen, oppositely GSK-3 inhibitor lithium chloride can inhibit GSK-3 activity to activate glycogen synthase and promote glycogen synthesis.
Based on the above understanding, this study investigated effects of associated application of GSK-3 inhibitor lithium chloride and FDP on warm ischemia reperfusion injury of liver in rats caused by surgery during the emergency through the establishment of liver trauma model.
Methods:
1. 42 healthy Sprague-Dawley (SD) rats were randomly divided into 3 groups (mild injury group, moderate injury group and severe injury group, n=14). By improving the biological impact machine-Ⅳdesigned by Trauma Center of PLA, Institute of Surgery Research, Daping Hospital, Third Military Medical University with a T-type second-hit head, 3 groups of liver blunt impact injury model were established respectively with three different-quality steel ball. Then each group was randomly divided into A group and B group according to the difference of measured items. The diversity in vital signs before and after injury and the number of natural survival animals after injury in A group were measured. AST and ALT content in serum before and after injury, TNF-αcontent, the quality of intra-abdominal hemorrhage of animals after injury in B group were measured. The liver injury of all rats was classified and given a score after the death.
2. After the establishment of a liver trauma model on 49 SD rats, 42 out of the animals were randomly divided into control group, glucose group and FDP group (n=14), which were respectively injected 2ml of 0.9% sodium chloride, 5% glucose and 15% FDP injection at 10min after injury. Then each group was randomly divided into pre-ischemia group and 4h reperfusion group (n=7) according to time points when animals were executed before and after ischemia. The remaining seven for sham operation group (SH group) were executed in 4h reperfusion time point. The AST and ALT content in serum and glycogen content, SOD vitality, MDA content and ATP content in liver tissue were determined. It was also carried out that HE staining, fluorescence real-time quantitative PCR detection of liver Bcl-2 mRNA expression, cell apoptosis detection (TUNEL method).
3. After the establishment of a liver trauma model in 49 SD rats, 42 out of the animals were randomly divided into control group, FDP group, FGI group (FDP and GSK-3 inhibitor in combination group) (n=14). control group was injected 2ml of 0.9% sodium chloride injection. FDP group was injected 2ml of 15% FDP injection. FGI group was injected 2ml of 15% FDP injection, in addition, 0.5% GSK-3 inhibitor lithium chloride (3mmol/kg) by intraperitoneal injection (all at 10min after injury). Then each group was randomly divided into pre-ischemia group and 4h reperfusion group (n=7) according to time point when animals were executed before and after ischemia. The remaining seven for sham operation group (SH group) were executed in 4h reperfusion time point. The AST and ALT content in serum and glycogen content, SOD vitality and MDA content, liver glycogen synthase activity and immunohistochemical determination of GSK-3 expression in liver tissue were determined.
Results:
1. The average femoral artery pressure in each group after injury was significantly lower than that before injury (P<0.01), and decreased with the increased degree of injury (P<0.01 or P<0.05). AST and ALT content in each group after injury were significantly higher than those before injury (P<0.01 or P<0.05), and increased with the increased degree of injury (P<0.01 or P<0.05). The quality of intra-abdominal hemorrhage in each group significantly increased with the increased degree of injury (P<0.01 or P<0.05). The score for the hepatic injury significantly increased with the increased degree of injury (P<0.01). TNF-αcontents in serum were significantly higher than those before injury (P<0.01), and increased with the increased degree of injury (P<0.01).
2. At pre-ischemia time point, liver glycogen content, control groupglucose group>FDP group>SH group (P<0.01 or P<0.05); except that differences between glucose group and FDP group, AST content, control group>glucose group/FDP group>SH group (P<0.01 or P<0.05); SOD vitality, control groupglucose group>FDP group>SH group, (P<0.01 or P<0.05); ATP content, the control groupglucose group>FDP group>SH group (P<0.01 or P<0.05).
3. At pre-ischemia time point, liver glycogen content, the control groupFDP group>FGI group>SH group (P<0.01); SOD vitality, control groupFDP group>FGI group>SH group (P<0.01). As compared with pre-ischemia time point, liver glycogen synthase activity of the control group and the FDP group at 4h reperfusion time point were significantly lower (P<0.01); at pre-ischemia time point, liver glycogen synthase activity, FGI group>FDP group (P<0.01); at 4h reperfusion time point, liver glycogen synthase activity, control groupFDP group>FGI Group>SH group (P<0.01).
Conclusion:
1. The degree of liver damage and stress in each group obviously increases with the increase of quality of the steel ball. As an ideal experimental animal model, this liver trauma model in rat has simple, stable features of liver injury, good repeatability, adjustability and high cost-effective rate. The moderate injury group can be used in this study for further pathological mechanisms and treatment of injury.
2. As compared with glucose, the application of FDP has better protective role on rat liver trauma. The mechanism may be related to that it is more effective that the glycogen synthesis of FDP as substrate in the liver in stress conditions, which more increase reserves of liver glycogen before ischemia, thus reducing warm ischemia reperfusion injury caused by surgery during the emergency after rat liver trauma.
3. The associated application of FDP and GSK-3 inhibitor lithium chloride can enhance the effect of FDP on the protective role of rat liver trauma. The mechanism may be related to that GSK-3 inhibitor can effectively enhanced the role of glycogen synthesis of FDP as substrate before liver ischemia, which increases the liver glycogen reserves more effectively than the single application of FDP in a short period of time, thus more reducing warm ischemia reperfusion injury caused by surgery during the emergency after rat liver trauma.
在战、创伤所致肝脏破裂伤急救手术后仍然有相当高的死亡率及术后并发症发生率。咎其原因,在复杂的肝脏破裂伤手术操作时,为了及时控制肝脏破裂创面的大出血或便于破碎创面的清创(切除)止血,手术过程中常采用的阻断入肝血流是一个重要原因。入肝血流阻断后无疑将会进一步给肝脏带来不可低估的热缺血损伤,在手术成功止血清创后,恢复肝脏血流供应,又会产生对肝脏的再灌注损伤,这种损伤在某种程度上往往比前期阻断肝脏血流所致热缺血引起的损伤更为严重。并且,在战、创伤所致肝脏破裂伤救治过程中,机体正处于急性应激状态,此时肝脏的一系列病理生理改变就是肝脏在应激条件下的缺血再灌注损伤!
目前普遍认为,肝细胞生物能量缺乏是引发肝脏缺血再灌注损伤一系列病理改变的始动因素。业已证实,如果在阻断肝门前有效地提高肝脏糖原储备,则能有效降低后续的缺血再灌注损伤。但是在抢救肝脏破裂伤的过程中,为避免失血引起的休克,抢救人员必须尽快手术阻断肝脏供血以止血清创,这就必须在短时间内提高肝脏的糖原储备。如何在短时间内提高肝脏的糖原储备?一个是有效地增加合成糖原的底物,另一个就是加快糖原的合成。
已知1,6-二磷酸果糖(fructose-1,6-diphosphate,FDP)作为能量代谢调节剂,能减轻各种组织器官包括肝脏的缺血再灌注损伤,同时FDP作为细胞内糖代谢的中间产物,还是糖酵解和糖异生的共同通路;另外,糖原合成酶激酶-3(glycogen synthase kinase-3,GSK-3)可以磷酸化糖原合成酶的丝氨酸位点使其失活,进而可抑制糖原的合成;相反GSK-3抑制剂氯化锂能有效抑制GSK-3活性,从而激活糖原合成酶,促进糖原的合成。
基于以上认识,本课题通过建立大鼠肝脏撞击破裂伤模型,探讨在战、创伤致肝脏破裂伤的急救或手术过程中,通过联合使用GSK-3抑制剂氯化锂和FDP,能否在短时间内(如1h)增加肝细胞内糖原含量,从而减轻手术过程中阻断入肝血流带来的肝脏热缺血再灌注损伤。
方法:
采用SD大鼠为实验对象,进行下列三方面的研究。
1选取健康SD大鼠42只,随机分为3组(n=14)。通过对第三军医大学大坪医院野战外科研究所全军战创伤中心研制的BIM-Ⅳ型生物撞击机增加T型二次打击头进行改良,用轻、中、重三种质量的钢球分别撞击一组大鼠,复制出3组损伤程度不同的大鼠肝脏钝性撞击伤模型,即轻度致伤组、中度致伤组、重度致伤组。各组再按观察指标不同,随机平分为A组和B两组。测定A组实验动物致伤前后生命体征变化及自然存活数,B组致伤前后血清中AST、ALT、TNF-α含量及致伤后腹腔内出血质量,所有大鼠死亡后均进行肝损伤分级并进行评分。
2选取健康SD大鼠49只,采用前部分建立的大鼠肝脏撞击破裂伤模型。先取42只建模后大鼠随机分为对照组、葡萄糖组、FDP组(n=14),分别于致伤10min后静脉注射0.9%氯化钠、5%葡萄糖、15%FDP注射液2ml(均在10min内静注完)。各组再按处死取材时相点随机均分为缺血前和再灌注4h两个亚组(n=7)。剩余7只建模后大鼠为再灌注4h时相点假手术组。测定各组动物血清AST、ALT含量,肝组织糖原含量、SOD活性、MDA及ATP含量,并行肝组织HE染色、荧光实时定量PCR检测肝组织Bcl-2 mRNA表达、肝组织细胞凋亡检测(TUNEL法)。
3选取健康SD大鼠49只,采用前部分建立的大鼠肝脏撞击破裂伤模型。先取42只建模后大鼠随机分为对照组、FDP组、FGI组(FDP+GSK-3抑制剂联合应用组)(n=14)。对照组于致伤10min后静脉注射0.9%氯化钠注射液2ml,FDP组于致伤10min后静脉注射15% FDP注射液2ml,FGI组致伤后除在10min静注完15% FDP注射液2ml外,尚通过腹腔注射一次性给予0.5% GSK-3抑制剂氯化锂(3mmol/kg)(均在10min内静注完)。3组再按处死取材时相点随机平分为缺血前和再灌注4h两个亚组(n=7)。剩余7只建模后大鼠为再灌注4h时相点假手术组。测定各组血清中AST、ALT含量、肝组织糖原含量、SOD活力、MDA含量,并行肝组织糖原合成酶活性测定和肝组织免疫组化GSK-3表达测定。
结果:
1各组平均股动脉压致伤后均低于致伤前(P<0.01),且致伤后平均股动脉压随损伤程度的增加而降低(P<0.01或P<0.05)。各组血清AST、ALT含量致伤后均明显高于致伤前(P<0.01或P<0.05);致伤后各组AST、ALT含量均随损伤程度的增加而增高(P<0.01或P<0.05)。各组腹腔出血量随损伤程度的增加而增多(P<0.01或P<0.05)。各组肝脏损伤评分随损伤程度的增加而增高(P<0.01)。致伤后血清TNF-α含量均明显高于致伤前(P<0.01);致伤后各组间血清TNF-α含量随损伤程度的增加而增高(P<0.01)。
2在缺血前时相点,肝糖原含量,对照组<葡萄糖组
3在缺血前时相点,肝脏糖原含量,对照组
结论:
1各组大鼠肝脏损伤程度及应激反应随钢球质量的增加而加重,分级明显。该大鼠肝脏创伤模型建模过程简单,具有稳定的肝脏损伤的伤情特点,可重复性好,损伤程度可分级调节,费效比低,其中度致伤组可用于本课题的进一步病理机制和伤后救治的研究。
2 FDP不仅作为代谢调节剂,还是细胞内糖代谢的中间产物,是糖原合成的又一种有效底物,可以独立地在缺血再灌注损伤中发挥重要作用,而且在创伤应激条件下,相对于同摩尔浓度的葡萄糖,能更有效地在短时间内促进肝细胞内的糖原合成,提高肝糖原含量,从而减轻了手术过程中阻断入肝血流带来的术后肝脏的热缺血再灌注损伤。
3在输注FDP的同时,应用GSK-3抑制剂氯化锂,间接地增强糖原合成酶的活性,在短时间内促进肝细胞内糖原合成,比单纯应用FDP更加有效地提高了肝细胞内的糖原含量,从而减轻了手术过程中阻断入肝血流带来的术后肝脏的热缺血再灌注损伤。
Objective:
There is a high mortality rate and incidence of postoperative complications after emergency operation of liver trauma caused by war or external injury. It is an important factor that surgical procedures to block hepatic blood flow are often used to control bleeding or facilitate debridement (removal) of trauma liver. Hepatic inflow occlusion, no doubt, will bring about the liver warm ischemia injury that can not be underestimated. After the successful operation to stop bleeding or debride, recovery of liver blood supply will bring about the liver injury again, which to some extent can be more serious than damage of the liver warm ischemia caused by pre-blocking blood flow. Moreover, during treatment process of liver trauma, the body is in a state of acute stress, and this time a series of pathophysiological changes in the liver is ischemia reperfusion injury of the liver under acute stress!
Now It is widely recognized that energy deficiency of liver cells is the initial factor inducing a series of pathological changes of hepatic ischemia reperfusion injury. It has been proved that if effectively increasing liver glycogen reserves before blocking hepatic blood flow would effectively reduce subsequent ischemic reperfusion injury. However, in order to avoid the shock caused by loss of blood, rescue personnel must stop bleeding as soon as possible in the treatment process. So it is imperative to increase the liver glycogen reserves within a short time. How to increase the liver glycogen reserves within a short time? One is to increase the effective substrate for glycogen synthesis, and the other is to speed up the synthesis of glycogen.
It is known that fructose-1, 6-diphosphate (FDP) as a regulator of energy metabolism can reduce ischemia reperfusion injury of a variety of tissues and organs including the liver, meanwhile FDP as the middle product in intracellular glucose metabolism is the common pathway of glycolysis and gluconeogenesis; in addition, glycogen synthase kinase-3 (GSK-3) can phosphorylate serine sites of glycogen synthase to inhibit the synthesis of glycogen, oppositely GSK-3 inhibitor lithium chloride can inhibit GSK-3 activity to activate glycogen synthase and promote glycogen synthesis.
Based on the above understanding, this study investigated effects of associated application of GSK-3 inhibitor lithium chloride and FDP on warm ischemia reperfusion injury of liver in rats caused by surgery during the emergency through the establishment of liver trauma model.
Methods:
1. 42 healthy Sprague-Dawley (SD) rats were randomly divided into 3 groups (mild injury group, moderate injury group and severe injury group, n=14). By improving the biological impact machine-Ⅳdesigned by Trauma Center of PLA, Institute of Surgery Research, Daping Hospital, Third Military Medical University with a T-type second-hit head, 3 groups of liver blunt impact injury model were established respectively with three different-quality steel ball. Then each group was randomly divided into A group and B group according to the difference of measured items. The diversity in vital signs before and after injury and the number of natural survival animals after injury in A group were measured. AST and ALT content in serum before and after injury, TNF-αcontent, the quality of intra-abdominal hemorrhage of animals after injury in B group were measured. The liver injury of all rats was classified and given a score after the death.
2. After the establishment of a liver trauma model on 49 SD rats, 42 out of the animals were randomly divided into control group, glucose group and FDP group (n=14), which were respectively injected 2ml of 0.9% sodium chloride, 5% glucose and 15% FDP injection at 10min after injury. Then each group was randomly divided into pre-ischemia group and 4h reperfusion group (n=7) according to time points when animals were executed before and after ischemia. The remaining seven for sham operation group (SH group) were executed in 4h reperfusion time point. The AST and ALT content in serum and glycogen content, SOD vitality, MDA content and ATP content in liver tissue were determined. It was also carried out that HE staining, fluorescence real-time quantitative PCR detection of liver Bcl-2 mRNA expression, cell apoptosis detection (TUNEL method).
3. After the establishment of a liver trauma model in 49 SD rats, 42 out of the animals were randomly divided into control group, FDP group, FGI group (FDP and GSK-3 inhibitor in combination group) (n=14). control group was injected 2ml of 0.9% sodium chloride injection. FDP group was injected 2ml of 15% FDP injection. FGI group was injected 2ml of 15% FDP injection, in addition, 0.5% GSK-3 inhibitor lithium chloride (3mmol/kg) by intraperitoneal injection (all at 10min after injury). Then each group was randomly divided into pre-ischemia group and 4h reperfusion group (n=7) according to time point when animals were executed before and after ischemia. The remaining seven for sham operation group (SH group) were executed in 4h reperfusion time point. The AST and ALT content in serum and glycogen content, SOD vitality and MDA content, liver glycogen synthase activity and immunohistochemical determination of GSK-3 expression in liver tissue were determined.
Results:
1. The average femoral artery pressure in each group after injury was significantly lower than that before injury (P<0.01), and decreased with the increased degree of injury (P<0.01 or P<0.05). AST and ALT content in each group after injury were significantly higher than those before injury (P<0.01 or P<0.05), and increased with the increased degree of injury (P<0.01 or P<0.05). The quality of intra-abdominal hemorrhage in each group significantly increased with the increased degree of injury (P<0.01 or P<0.05). The score for the hepatic injury significantly increased with the increased degree of injury (P<0.01). TNF-αcontents in serum were significantly higher than those before injury (P<0.01), and increased with the increased degree of injury (P<0.01).
2. At pre-ischemia time point, liver glycogen content, control group
3. At pre-ischemia time point, liver glycogen content, the control group
Conclusion:
1. The degree of liver damage and stress in each group obviously increases with the increase of quality of the steel ball. As an ideal experimental animal model, this liver trauma model in rat has simple, stable features of liver injury, good repeatability, adjustability and high cost-effective rate. The moderate injury group can be used in this study for further pathological mechanisms and treatment of injury.
2. As compared with glucose, the application of FDP has better protective role on rat liver trauma. The mechanism may be related to that it is more effective that the glycogen synthesis of FDP as substrate in the liver in stress conditions, which more increase reserves of liver glycogen before ischemia, thus reducing warm ischemia reperfusion injury caused by surgery during the emergency after rat liver trauma.
3. The associated application of FDP and GSK-3 inhibitor lithium chloride can enhance the effect of FDP on the protective role of rat liver trauma. The mechanism may be related to that GSK-3 inhibitor can effectively enhanced the role of glycogen synthesis of FDP as substrate before liver ischemia, which increases the liver glycogen reserves more effectively than the single application of FDP in a short period of time, thus more reducing warm ischemia reperfusion injury caused by surgery during the emergency after rat liver trauma.
引文
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[4]吴红涛,麻晓林,蒋耀光.实验性胸腹撞击伤动物模型的建立及其伤情特点研究[J].创伤外科杂志, 2004, 6(4): 279-281.
[5]麻晓林,杨志焕,王正国,等.实验性肝脏撞击伤的病理形态学改变[J].中华创伤杂志, 2001, 17(12): 745-746.
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[7]陈卫华(综述),郭松超(审校).肝脏损伤动物模型及观察指标研究现状[J].广西医科大学学报, 2007, 24(1): 156-158.
[1] Cywes R, Greig PD, Morgan GR, et al. Rapid donor liver nutritional enhancement in a large animal model. [J]. Hepatology, 1992, 16(5): 1271.
[2]汤礼军,田伏洲,王雨,等.兔肝冷保存后复温缺血-再灌注损伤及其相关机制的研究[J].中国病理生理杂志, 2001, 17(2): 161-163.
[3] Tang L, Tian F, Tao W. Hepatocellular glycogen in alleviation of liver ischemia-reperfusion injury during partial hepatectomy.[J]. World J Surg, 2007, 31 (10): 2039-2043.
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[11]卢卫新,载瑞鸿. 1, 6-二磷酸果糖对组织缺血与缺氧的保护作用[J].中国临床医学, 2002, 9(1): 91.
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[1] Patel S, Doble B, Woodgett J R. Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword? [J]. Biochem Soc Trans, 2004, 32(Pt5): 803-808.
[2] Lochhead PA, Coghlan M, Rice SQ, et al. Inhibition of GSK-3 seletively reduces glucose-6-phosphatase and phosphatese and phosphoenolpyruvate carboxykinase gene expression. [J]. Diabetes, 2001, 50(5): 937-946.
[3]罗皓,汤礼军,谭积善,等.糖原合成酶激酶-3β抑制剂对肝热缺血再灌注损伤的保护[J].西南国防医药, 2009, 19(5): 468-470.
[4]陆海一,刘亚林.锂盐药物浓度与临床应用进展[J].南京军医学院学报, 2001, 23(1): 33-35.
[5] Kockefitz L, Doble B, Patel S, et a1. Glycogen synthase kinase-3, an overview of an over-achieving protein kinase. [J]. Curr Drug Targets, 2006, 7: 1377-1388.
[6] Woodgett JR . cDNA cloning and properties of glycogen synthase kinase-3. [J]. Methods Enzymol, 1991, 200: 564-577.
[7] Frame S, Cohen P. GSK-3 takes centre stage more than 20 years after its discovery. [J]. Biochem J, 2001, 359: l-l6.
[8] Takada Y, Fang X, Jamaluddin MS, et al.Genetic deletion of glycogen synmase kinase-3beta abrogates activation of Ikappa-Balpha kinase, JNK, Akt and p44/p42 MAPK but potentiates apoptosis induced by tumor necrosis factor. [J]. J Biol Chem, 2004, 279: 39541-39554.
[9]刘率男,申竹芳.糖尿病治疗新靶点糖原合成酶激酶-3抑制剂的研究进展[J].药学学报, 2007, 42(12): 1227-1231.
[10]柯文波,郑启昌.应激状态对肝脏代谢的影响[J].实用肝脏病杂志, 2006, 9(2): 107-110.
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[2] Moore EE, Cogbill TH, Jurkovich GJ, et al. Organ injury scaling: spleen and liver (1994 revision). [J]. J Trauma, 1995, 38(3): 323-324.
[3]田华,张喜平,陈力,等.重症急性胰腺炎大鼠模型制备的技术改良[J].中华急诊医学杂志, 2005, 14(3): 211-214.
[4]吴红涛,麻晓林,蒋耀光.实验性胸腹撞击伤动物模型的建立及其伤情特点研究[J].创伤外科杂志, 2004, 6(4): 279-281.
[5]麻晓林,杨志焕,王正国,等.实验性肝脏撞击伤的病理形态学改变[J].中华创伤杂志, 2001, 17(12): 745-746.
[6]尹志勇,王正国.多功能小型生物撞击机的研制与应用[J].生物医学工程学杂志, 2000, 17(3): 309-312.
[7]陈卫华(综述),郭松超(审校).肝脏损伤动物模型及观察指标研究现状[J].广西医科大学学报, 2007, 24(1): 156-158.
[1] Cywes R, Greig PD, Morgan GR, et al. Rapid donor liver nutritional enhancement in a large animal model. [J]. Hepatology, 1992, 16(5): 1271.
[2]汤礼军,田伏洲,王雨,等.兔肝冷保存后复温缺血-再灌注损伤及其相关机制的研究[J].中国病理生理杂志, 2001, 17(2): 161-163.
[3] Tang L, Tian F, Tao W. Hepatocellular glycogen in alleviation of liver ischemia-reperfusion injury during partial hepatectomy.[J]. World J Surg, 2007, 31 (10): 2039-2043.
[4] Moore EE, Cogbill TH, Jurkovich GJ, et al. Organ injury scaling: spleen and liver (1994 revision). [J]. J Trauma, 1995, 38(3): 323.
[5] Kenneth J, Livak K, Thomas D, et al. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta Ct) method. [J]. Methods, 2001, 25(4): 402-408.
[6]汤礼军,田伏洲.肝细胞内糖原含量与肝脏缺血再灌注损伤关系的实验研究[J].中国普通外科杂志, 2000, 9(1): 35.
[7]柯文波,郑启昌.应激状态对肝脏代谢的影响[J].实用肝脏病杂志, 2006, 9(2): 107.
[8]孙宏斌.以糖代谢干预为基础的新药研究与开发[J].中国药科大学学报, 2006, 37(1): 1.
[9]周爱儒,查锡良.生物化学,第5版,人民卫生出版社, 2002. 101.
[10]方家驹.葡萄糖激酶是己糖激酶的同功酶D[J].生命的化学, 1992, 12(5): 35.
[11]卢卫新,载瑞鸿. 1, 6-二磷酸果糖对组织缺血与缺氧的保护作用[J].中国临床医学, 2002, 9(1): 91.
[12]蔡广. 1, 6-二磷酸果糖的临床应用进展[J].心脏杂志, 2005, 17(1): 87.
[1] Patel S, Doble B, Woodgett J R. Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword? [J]. Biochem Soc Trans, 2004, 32(Pt5): 803-808.
[2] Lochhead PA, Coghlan M, Rice SQ, et al. Inhibition of GSK-3 seletively reduces glucose-6-phosphatase and phosphatese and phosphoenolpyruvate carboxykinase gene expression. [J]. Diabetes, 2001, 50(5): 937-946.
[3]罗皓,汤礼军,谭积善,等.糖原合成酶激酶-3β抑制剂对肝热缺血再灌注损伤的保护[J].西南国防医药, 2009, 19(5): 468-470.
[4]陆海一,刘亚林.锂盐药物浓度与临床应用进展[J].南京军医学院学报, 2001, 23(1): 33-35.
[5] Kockefitz L, Doble B, Patel S, et a1. Glycogen synthase kinase-3, an overview of an over-achieving protein kinase. [J]. Curr Drug Targets, 2006, 7: 1377-1388.
[6] Woodgett JR . cDNA cloning and properties of glycogen synthase kinase-3. [J]. Methods Enzymol, 1991, 200: 564-577.
[7] Frame S, Cohen P. GSK-3 takes centre stage more than 20 years after its discovery. [J]. Biochem J, 2001, 359: l-l6.
[8] Takada Y, Fang X, Jamaluddin MS, et al.Genetic deletion of glycogen synmase kinase-3beta abrogates activation of Ikappa-Balpha kinase, JNK, Akt and p44/p42 MAPK but potentiates apoptosis induced by tumor necrosis factor. [J]. J Biol Chem, 2004, 279: 39541-39554.
[9]刘率男,申竹芳.糖尿病治疗新靶点糖原合成酶激酶-3抑制剂的研究进展[J].药学学报, 2007, 42(12): 1227-1231.
[10]柯文波,郑启昌.应激状态对肝脏代谢的影响[J].实用肝脏病杂志, 2006, 9(2): 107-110.
[1] Cooper GJ, Taylor DEM. Biophysics of impact injury to the chest and abdomen. [J]. Army Med Corp, 1989, 135(5): 56-58.
[2]安波.腹部撞击伤时肝脏损伤生物力学效应的初步探讨[J].生物医学工程学杂志, 1994, 11(8): 75-79.
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