| PrefaceHepatic ischemia reperfusion injury ( HIRI) is a common pathological progress in hepatic surgery and traumatic surgery. Many diseases are related to this procedure, such as severe hepatic injury, hepatic tumor, extensive hepatic ecto-my, hepatic transplantation, shock and infection. There are many facts that leads to HIRI, it is generally accepted that the mechanism is related to free oxygen radical, calcium overload, dysfunction of mitochondria. Oxygen radical, endothelin, prostaglandin and nitrogen monoxidum can be involved in the HIRI, and it has been confirmed in many experiments.As a relatively stable free radical, NO has two kinds of biological functions to human. It can regulate the normal physiologic function such as blood circulation, information transferation, energy metabolism and immunological regulation. On the other hand, the quality of NO in human is harm to organism more or less, for example, hypotensive shock, cell damage or hypertension, arthero-sclerosis and ischemic.The NOS in liver have two types: endothelial NOS ( eNOS) and induced NOS (iNOS). eNOS is mainly in the endothelial cell, smooth muscle cells and platelets and can react under physiological circumstances. iNOS mainly in hepatic cell, macrophages, Kupffers cells, can transcript, express, synthesis under the effects of stimulating factor.The mechanism of hepatic reperfusion injury still needs further researches, but it plays important parts in the prevention and treatment of many diseases. The research on the reperfusion injury have been changed from the pathogensisto exploration of preventative drugs, and many experiments demonstrate that Vit E, Vit C, coenzyme Q, biologic agent SOD have intensive preventional function on reperfusion injury of tissues and organs. There are many reports on the prevention function of Shen Fu injection on reperfusion injury of cardiac muscles, kidney, medulla spinalis, brain and intestinal mucosa. But fewer researches on function of Shen Fu injection in HIRI are reported. This experiment investigated the protection function of Shen Fu injection on the HIRI of rats by made the HIRI model of rats. By investigating the influence on NO and NOS in HIRI, we try to approach its mechanism and supply the experimental evidence of its clinical use.Materials and MethodsMale Wister rats weighing 200 - 250g and feeding food and water conventionally and giving light in 12 hour were fed on food in 3 days in order to adapt environment. Rats were randomly divided into experimental ( SF) group and control ( NS) group. Rats in SF group were administred Shen Fu Injection (10ml/kg) through intraperitoneal injection once per day and continued 5 days, on the sixth day, rats also were received Shen Fu Injection at the same method 30 minutes before operation. Rats in NS group were received 0.9% saline solution at the same method. At the sixth day, the rats were anesthetized before all operative procedures, and the abdomen was opened through a median laparotomy and the liver was detached from there ligaments. According to Pringle's maneuver, total hepatic ischemia with portal congestion was produced by clamping the hepatoduodenal ligament for 15 minutes with a micro vascular clamp, declamping allowed reperfusion until sampling after specified periods. After reperfusion, 24 hours respectively, blood samples were taken from the abdominal aorta. Serum concentrations of alanine aminotransferase ( ALT), lactic dehydrogenase (LDH) and aspartate aminotransferase (AST) were measured by auto -biochemical assay instrument. Hepatic tissue was removed after 1 hour and 24 hours respectively and determined the level of NO according to the introduction of the agent. Finally hepatic tissue was immersed in 10% formalin solution forfixation and determination of liver iNOS and eNOS expression and was conducted by sp immunohistochemistry. The data of serous enzymes, the level of NO were expressed as meant ± standard deviation (x ± s) and analyzed by independent -sample t test. The express of NOS of hepatic tissues is examined by line x array exact probabilistic method. All of the analyses were performed by SPSS11. 0 software. P value <0.05 was considered to be significant.Results1. The concentrations of serum biochemical enzymes: After ischemia and reperfusion 1 hour, the serum ALT, LDH and AST concentrations of the group SF were 125.38 ±40.32 U/L,2433.88 ± 1346.44U/L ,330. 38 ± 142.38 U/L respectively , while the group NS were 381. 63 ± 202. 88U/L,3652. 63 ± 2687. 24U/L.940.25 ±762. 83 U/L respectively, and the ALT and AST level in SF group was significantly lower than NS group ( P < 0. 05 ) , the LDH level in SF group were lower than NS group, but no significantly( P > 0.05 ) . After ischemi-a and reperfusion 3 hour, the serum ALT, LDH and AST concentrations of the group SF were 142.00 ± 110. 24U/L,2026. 56 ± 1031.72U/L ,342. 11 ± 132. 80U/L respectively,while the group NS were 334. 56 ±214. 16U/L,2722.44 ± 1185.45U/L,720.44 ±428.32 U/L respectively,and the ALT and AST level in SF group was significantly lower than NS group ( P < 0.05 ) , the LDH level in SF group were lower than NS group, but no significantly ( P > 0. 05 ). Then after reperfusion 24 hours,those of the group SF were 109.43 ±45.11U/L,900.43 ± 527.18U/L,344. 86 ± 108.60U/L respectively,while the group NS were 116.43±23. 14U/L,657. 71 ± 174. 76 U/L,337. 71 ± 129. 81U/L respectively, and the levels of those between were no significantly difference both group ( P >0. 05).2. The level of NO in liver tissue: After ischemia and reperfusion 1 hour,in the group SF the level of NO in the liver tissue (31. 88 ±9.78) was significantly lower than that in the group NS (51.75 ±22.30) ( P <0. 05). After ischemia and reperfusion 3 hour,in the group SF the level of NO in the liver tissue (38. 78 ± 10.67) was significantly lower than that in the group NS (73.22 ±46. 89)( P <0.05). After reperfusion 24 hours that of the group SF (66.29 ±57. 55 ) was no significantly difference with that of trie group NS{47.14 ±21. 37 )( P> 0.05).3. eNOS and iNOS expression of liver tissue: After ischemia and reperfusion 1 hour and 3 hour,the iNOS expression in the group SF was significantly lower than that in the group NS( P <0. 05). After ischemia and reperfusion 24 hours, that was no significantly difference between the group SF and the NS . After ischemia and reperfusion 1 hours ,3 hours and 24 hours,t eNOS was no significantly difference between the group SF and the NS.DiscussionHepatic ischemia reperfusion injury ( HIRI) is a common pathological progress in hepatic surgery and traumatic surgeiy. Many diseases are related to this procedure, such as severe hepatic injury, hepatic tumor, extensive hepatic ecto-my, hepatic transplantation, shock and infection. There are many facts leads to HIRI, it is generally accepted that the mechanism is related to free oxygen radical, calcium overload, dysfunction of mitochondria. Oxygen radical, endothe-lin, prostaglandin and nitrogen monoxidum can be involved in the HIRI, and it has been confirmed in many experiments. The research on the reperfusion injury has been changed from the pathogenety to exploration of the preventative drugs.The NO can be made from L - arginine ( L - Arg) sloughing the guanidino nitrogen by the NOS. L - Arg→NO pathway exists extensuvely in the tissue and cells. NOS can be divided into architectonic type and induced type, the former can synthesis low level NO in the normal physiological, which can diastole the vescular smooth muscle, inhibit the aggregation of platelets, resist lipid peroxi-dation. The latter can produce a great quantity of NO induced by the LPS, IL -1 and TNF, which take part in some pathological procedures. NO is a new type of messenger gases between the cells and intra - cells. It is also a kind of high activity gas molecular free radical, more animation than active oxygen. NO made by inducible nitric oxide synthase can regulate cGMP and influence the hepatocyte function by inhibit or activate some enzyme, then display the damageand prevention to the hepatic tissues. When the hepatic cell was oxidized, NO can prevent the cell by cleaning up the 02 ? But excess NO can not be cleaned up by the electron transfer, it even can form the peroxynitral ion, peroxynitral ion can form the hydroxyl free radical which has more toxicity thanO-2 , then lipid peroxidize injury was occurred which can induce the cell dysfunction even death. The role of NO in HIRI is still conversional. The two contradistinction result may be related to the production, distribution and enzymatic activity of two NO synases (iNOS and eNOS). The NO'S in liver have two types: endothelial NOS (eNOS) and induced NOS (iNOS). eNOS is mainly in the endothelial cell, smooth muscle cell and blood disc and can react under physiological circumstances. iNOS mainly in hepatic cell, macrophage, Kupffer's cells, can transcript, express, synthesis under the stimulating factor's effect. Under the normal circumstance, there is only trace express of NOS, which is difficult to be detected. In reperfusion of liver after ischemic, the eNOS of hepatic and inflammatory cell are decreased, iNOS over expressed, so it plays a important role in HIRI.Overdose NO has cytotoxicity: ( 1) damage the mitochondria of hepatic cell, influence the energy metabolism of hepatic cell; ( 2 ) react with the activity free radical and form the more noxious free radical, induce the injury of liver; (3) NO can mediate the apoptosis and directly induce the cell death.In our research, ALT, AST and LDH demonstrate the hepatic function after HIRI, the result demonstrate after 1h and 3hs reperfusion of liver, the hepatic function in SF group is better than NS group obviously, the NO in SF group is less than NS group, they are both have statistical significance, illustrate NO can aggravate the HIRI. After 1h and 3hs reperfusion of liver, the express of iNOS in liver of SF group is lower than NS group, there is no significant difference between the two groups of iNOS. This demonstrate that Shen Fu injection can degrade the NO content by inhibit the express of iNOS, then prevent the liver in HIRI. 24hs after reperfusion, there is no significant difference in two groups of ALT, AST, LDH, NO and iNOS, it may be related to the already repaired hepatic function.Shen Fu injection can improve the symptom of surgery patients, decrease... |
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