| Hepatic ischemia reperfusion injury ( HIRI) is a common pathological progress in hepatic surgery and traumatic surgery. HIRI can both cause hepatic injury and lead to multiple organs dysfunction even failure in the whole body. It can also lead to death if it is serious. Generally, its exact mechanism is mainly thought to be related to free oxygen radicals, calcium overload, etal. It is also related to cellular signal transduction, activation of cytokines, cellular apoptosis.Recently, lots of methods and drugs have been used to protect or reduce liver against HIRI. Both ischemia preconditioning and drugs preconditioning are main methods . But it is difficult to carry out those and the effects are not satisfactory, the drugs which testified by some researches are expensive prices and hardly used in clinic. So it is essential to find a drug with moderate price and satisfactory treatment and be used to protect or reduce liver against HIRI.Shen Fu Injection is made of red ginseng and prepared aconite root. Gin-senoside and aconitine are its effective ingredients. Recently, it is be widely used to heart failure, bronchus asthma, shock, myocardial ischemia, arrhythmia and multiple organs dysfunction in clinic. Studies have demonstrated that it can protect tissues from the ischemia reperfusion injuries such as myocardium, kidney, spinal cord,cerebrum and intestine. However,the related reports were rare about its protective effects on HIRI or effects on superoxide dismutase (SOD) level, glutathione ( GSH) level and nuclear factor - kB ( NF - kB ) expression inhepatic tissues.In our study, we primarily investigated whether Shen Fu Injection has a protective effects on HIRI through observing the effect of Shen Fu Injection on concentrations of the hepatic biochemical enzymes, and furtherly discussed the possible mechanisms through observing the effect on tissue SOD and GSH level and the expression of NF - kB in the HIRI model of rats.Materials and MethodsMale Wister rats weighing 200 - 250g and feeding food and water conventionally and giving light in 12 hour was 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 received Shen Fu Injection (10ml/kg)through intraperitoneal injection each day only once and continued 5 days . At 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 by 10% chlorali hydras ( 3 ml/kg) through intraperitoneal injection before all operative procedures, and the abdomen was opened through a median laparotomy and the liver was detached from there ligaments. According to Pringles maneuver, total hepatic ischemia with portal congestion was produced by clamping the hepatoduodenal ligament for 15 minutes with a micro vascular clamp, and declamping allowed reperfusion until sampling after specified periods. After reperfusion, 1, 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 1,24 hours respectively and determined the level of SOD and GSH according to the introduction of the agent. Finally hepatic tissue was immersed in 10% formalin solution for fixation and determination of liver NF - kB expression which was conducted by sp immunohisto-chemistry. The data of serous enzymes,the level of SOD and GSH,the percent of NF - kB positive cell were expressed as mean ± standard deviation (x ± s) andanalyzed by independent - sample t test. All of the analysis 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 136.17 ±72.34 U/L,2190. 50 ± 854.70 U/L and 308. 33 ± 131. 28 U/L respectively, while the group NS were 298. 67 ± 158.21 U/L,3990.00 ±3076. 61 U/L and 977.17 ±868. 29 U/L respectively, and the ALT level in SF group was significantly lower than NS group ( P <0. 05) , the LDH and AST levels 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 116. 83 ±44. 51 U/L,884. 83±575.72 U/L and 369.17 ±95. 85 U/L respectively,while the group NS were 121.16 ±21.30 U/L.619.33 ± 155. 81 U/L and 350.17 ± 137.54 U/L respectively , and the levels of those between were no significantly difference both group( P>0.05).2. The level of SOD and GSH in liver tissue: After ischemia and reperfusion 1 hour,in the group SF the level of SOD in the liver tissue (109.40 ± 13.95 U/ mgprot) was significantly higher than that in the group NS (88. 69 ± 17. 83 U/ mgprot) ( P <0.05). After reperfusion 24 hours that of the group SF (96.33 ± 18.48 U/mgprot) was no significantly difference with that of the group NS(98. 93 ±9.96 U/mgprot) ( P >0.05). After ischemia and reperfusion 1 hour and 24 hours ,the level of GSH in liver tissue was no significantly differences between the group SF and the group NS (47. 59 ± 19.07 mg/gprot vs 37.32 ±4.71 mg/ gprot,33.56 ±6.59mg/gprot vs 38.69 ±6.35mg/gprot; P >0.05).3. NF - KBp65 expression of liver tissue: After HIRI there was the expression of NF - KBp65 in the hepatocytes and the Kuffer cells. After ischemia and reperfusion 1 hour,the percent of NF - kBp65 positive cell in the group SF (59. 33 ± 11. 06% ) was significantly lower than that in the group NS(75. 83 ±11. 46% ) ( P <0.05). After ischemia and reperfusion 24 hours,that was no significantly difference between the group SF and the group NS (58. 33 ± 12.91% vs 66.00 ±22.27% ;P>0.05).DiscussionIn our study,the results show that after ischemia and reperfusion 1 hour,the level of ALT and the percent of NF - KBp65 positive cell in the group SF were significantly lower than those in the group NS, the level of SOD in the group SF was significantly higher than that in the group NS ;while the levels of LDH.AST in the group SF were lower than those in the group NS, but no significantly and the level of GSH in the group SF was higher than that in the group NS, but no significantly. After ischemia and reperfusion 24 hours, those were no significantly differences between both group. Our study results demonstrated Shen Fu Injection had a protective effects on early hepatic ischemia reperfusion injury in rats, but show a unsignificant protective effects on late hepatic ischemia reperfusion injury.Free oxygen radicals and reactive oxygen species are important injury factors , that have an important effects on HIRI. SOD is one of natural antioxidant protecting tissue against oxygen - derived free oxygen radicals. There are lot of SOD in normal body which can eliminate effectually the free oxygen radicals producing by the metabolism of the body. During HIRI,the level of malondialdehyde which was the metabolism of lipid peroxidation was markedly increased, the level of SOD was significantly decreased, the morphological changes of heptocytes were obvious. Some drugs can alleviate the injury of HIRI by eliminating free oxygen radicals and increasing the level of SOD in tissue. The studies indicated that ginsenoside Rb1, Rb2, Rb3 and Rc resisted the level of free oxygen radicals producing by xanthine dehydro genase - xanthine oxidase , inhibited lipid peroxidation , eliminated free oxygen radicals and increased the level of SOD in tissue. Our study demonstrated Shen Fu Injection could protect the early hepatic ischemia reperfusion injury in rats through increasing the level of SOD in tissue.GSH is another of important antioxidant protecting tissue against oxygen -derived free oxygen radicals injury. GSH resisted the injury causing by free oxygen radicals and alleviated the tissue injury. During HIRI the studies indicated the level of GSH in hepatocyte was markedly decreased and the degree of inju-ries was inverse ratio to the level of GSH in the cells proischemia and the level and consumption of GSH was used as one of the sensitive signs indicating the severe degrees of tissue injury. Our study didn' t show Shen Fu Injection could protect the hepatic ischemia reperfusion injury in rats through increasing the level of GSH in tissue.NF - kB is a new member of the family of transduction factors and is a important nuclear transduction factor in cell. In motionless state, NF - kB which is integrated with inhibitive protein IkB through the dimmer of p50 and p65 is un-active . External stimulation of a series of signal transduction such as lipopo ly-saccharide, ischemia and reperfusion, tumor necrosis factor - α (TNF - α) , etal can lead to the decomposition of IkB and the composition of NF - kB - IkB , lead to the activation of NF - kB and enter to cellular nucleus through nucleus location sign and integrate with the special sequence of target gene and start to transduction and control. The activation of NF - kB can control with lots of the expression of series genes such as interleukin - 1 (IL - 1) , interleukin - 6 (IL -6) , TNF - α, inducible nitric oxide synthase ( iNOS) , intercellular adhesion molecule -1 (ICAM - 1) , etal. During HIRI the activation of NF - kB which was integrated with the special sequence and increased the expression of TNF -a and ICAM - 1 mRNA lead to HIRI. Inhibiting the activation of NF - kB can alleviate the HIRI. Shen Fu Injection could protect the ischemia reperfusion small intestinal mucosa through inhibiting the activation of NF - kB and the expression of ICAM - 1, iNOS and TNF - a. In our study, the results indicated Shen Fu Injection could protect the early hepatic ischemia reperfusion injury in rats through inhibiting the activation of NF - kB.The possible mechanisms of Shen Fu Injection protecting the early HIRI through increasing the level of SOD in tissue and inhibiting the activation of NF - kB are involved: (1) Ginsenoside Rb1, Rb2, Rb3 and Rc resisted the level of free oxygen radicals producing by xanthine dehydrogenase - xanthine oxidase, inhibited lipid peroxidation , eliminated free oxygen radicals and increased the level of SOD in tissue and inhibited the activation of NF - kB , protected HIRI directively; (2) Shen Fu Injection could protect the ischemia reperfusion small intestinal mucosa, reduce the translocation of bacterial endotoxin to portal vein...
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