Experimental Study On Protective Effects Of RhKD/APP On Liver Injury Of Rats

There are various kinds of causative agents that will cause liver injury in different degree. These years the study about the mechanism of liver injury and its prevention have become the hot spot in biology and medical study in domestic and oversea. Protease inhibitors are a group of enzymes that exist in biological body and keep a dynamic equilibrium between proteases and their specific inhibitors. The protease inhibitors play an important role in regulating physiological process, such as coagulation, fibrinolysis, inflammatory response, cell migration, cell differentiation, complement activation and release of hormone of peptide and protein. BPTI( Bovine pancreatictrypsin inhibitor),or aprotinin, is a representative serine protease inhibitor of Kunitz family. It is a nonspecific and broad-spectrum serine protease inhibitor which affects known serine proteases. It can defend inflammatory reaction, formation of thrombus, improve microcirculation, and decrease the effect of oxygen free radicals, which have been used in clinic already.But infusion of high doses of these nonmammalian inhibitor could result in immunologic reactions in patients undergoing by frequent use. So we need to find a substitute for BPTI which is preferable, small, potent and human-derived.The amyloidβ-protein precursor contains a domain homologous to Kunitz-type serine protease inhibitors. The sequence of hKD/APP which is consisted of 57 amino acids has 43% sequence identity with that of BPTI. The spatial conformation, reactive center of hKD/APP and its mechanism of function is much similar to that of BPTI. The isoforms of APP containing KD and hKD/APP show great ability to inhibit a variety of serine proteases. They are involed in coagulation, fibrinolysis, inflammatory response, complement activation and cell apoptosis, etc.The pathological and physiological role of this protease inhibitor domains which concerns with abnormal metabolism of rhKD/APP are not well understood. So we need to produce large quantity of rhKD/APP to discover and study the possible mechanism on pharmacodynamics. The aim of this study is to investigate the protective effect of rhKD/APP on liver injury induced by D-GalN and hepatic ischemia/reperfusion of rats. And probable mechanism of this effect was discussed in the aspects of free radical, expression of cytokines mRNA and protein by immunohistochemistry and in situ hybridization technology, and cell apoptosis.Part 1 The experimential study of the protective effect of rhKD/APP on liver injury induced by D-GalN of ratsPathological model of hepatic injury of rats caused by D-GalN was set up . All rats were divided into 6 groups randomly, 10 in every group. rhKD/APP with different dosage were administrated (ip) to rats at daily on 7 day.Rats were injected with D-GalN at a dose of 600 mg?kg-1 body weight as a 10% saline solution and control ones with the same dose of saline ,4 h after the last injection of rhKD/APP.1. The influence of rhKD/APP on levels of serum ALT,AST,ALPThe levels of serum ALT,AST,ALP were measured. The rats treated with rhKD/APP with different dosage exhibited that the amount of ALT,AST,ALP in blood serum were decreased.in different degree(P<0.01).2. The influence of rhKD/APP on liver weight coefficentCompared with the model control ,the liver weight coefficent decreased,but it has no statistical meaning in the groups of rats treated with rhKD/APP.3. The influence of rhKD/APP on changes of liver hisopathologyUnder light microscopy , the extent of hepatic tissue damage caused by D-GalN , including necrosis and hydropic swelling of hepatocytes and inflammatory cell infiltration, were decreased in the groups treated with rhKD/APP with different dosage .4. The influence of rhKD/APP on histological changes of liver under electronic microscopyUnder electronic microscopy , the extent of swelling of cell body and endotheliocyte were decreased in the groups treated with rhKD/APP with different dosage.The swelling of chondrosome and cavitation in the intracytoplasm were also decreased. The nuclear became normal.5. The influence of rhKD/APP on levels of MDA,SOD,GSH-PX in tissueThe activities of SOD, GSH-PX and the content of MDA in liver tissue were measured. In model rats, the activities of SOD and GSH-PX were all decreased and the content of MDA was significantly increased (P<0.01) compared with the controls. After treated with the rhKD/APP, the activities of SOD, GSH-PX were significantly increased, while the content of MDA were obviously decreased (P<0.01). These results showed that pretreatment with rhKD/APP could reduce oxygen free radicals and reinforce elimination.6. The influence of rhKD/APP on expression of TNF-α,IFN-γmRNA and protein in tissueThe expression of TNF-α,IFN-γmRNA and protein in tissue was determined by immunohistochemistry and in situ hybridization technology. The results showed that after treated with rhKD/APP, expression of TNF-α,IFN-γmRNA and protein in tissue was significantly reduced compared with model group(P<0.01).The results have shown that rhKD/APP has the protective effect through inhibiting the inflammatory process.7. The influence of rhKD/APP on apoptosis of D-GalN induced liver injury of ratsHematoxylin-Eosin (HE) staining, in situ end-labeling of nuclear DNA fragmentation (TUNEL) were employed to determine the level of apoptosis. The increase numbers of TUNEL-positive staining cells were significantly observed in model. The immunoreactivity was inhibited by rhKD/APP(P<0.01). It indicated that rhKD/APP could protect liver cells through inhibting the cell apoptosis.Part 2 The experimential study of the protective effect of rhKD/APP on liver ischemia-reperfusion injury of ratsPathological model of liver ischemia-reperfusion injury of rats was set up . All rats were divided into 6 groups randomly, 10 in every group. Rats were pretreated with rhKD/APP 30min before operation and then subjected to liver ischemia/reperfusion injury induced by a left and middle hepatic artery oCClusion. Rats were killed after 40min ischemia and 3h reperfusion.1. The influence of rhKD/APP on levels of serum ALT,AST,ALP The levels of serum ALT,AST,ALP were measured. The rats treated with rhKD/APP with different dosage exhibited that the amount of ALT,AST,ALP in blood serum were decreased in different degree(P<0.01).2. The influence of rhKD/APP on liver weight coefficientCompared with the model control ,the liver weight coefficient decreased, but it has no statistical meaning in the groups of rats treated with rhKD/APP.3. The influence of rhKD/APP on histological changes of liver under light microscopyUnder light microscopy , the extent of hepatic tissue damaged , including necrosis and hydropic swelling of hepatocytes and inflammatory cell infiltration, were decreased in the groups treated with rhKD/APP with different dosage .4. The influence of rhKD/APP on histological changes of liver under electronic microscopyUnder electronic microscopy , the extent of swelling of cell body and endotheliocyte were decreased in the groups treated with rhKD/APP with different dosage.The swelling of chondrosome and cavitation in the intracytoplasm were also decreased. The nuclear became normal.5. The influence of rhKD/APP on levels of MDA,SOD,GSH-PX in tissueThe activities of SOD, GSH-PX and the content of MDA in liver tissue were measured. After treated with the rhKD/APP, the activities of SOD, GSH-PX were significantly increased, while the content of MDA were obviously decreased (P<0.01). These results showed that pretreatment with rhKD/APP could reduce oxygen free radicals and reinforce elimination.6. The influence of rhKD/APP on levels of TNF-α,IFN-γmRNA and protein in tissueThe expression of TNF-α,IFN-γmRNA and proteinin tissue was determined by immunohistochemistry and in situ hybridization. The results showed that after treated with rhKD/APP, expression of TNF-α,IFN-γmRNA and protein in tissue wassignificantly reduced compared with model group(P<0.01).The results have shown that rhKD/APP has the protective effect through inhibiting the inflammatory process.7. The influence of rhKD/APP on apoptosis of liver ischemia-reperfusion injury of ratsTUNEL were employed to determine the level of apoptosis. The numbers of TUNEL-positive staining cells were significantly decreased in the rats pretreated with rhKD/APP(P<0.01). It indicated that rhKD/APP could protect liver cells through inhibting the cell apoptosis.Conclusions:The results showed that rhKD/APP has protective effect on liver injury induced by D-GalN and hepatic ischemia/reperfusion of rats. rhKD/APP can improve the liver fuction, reduce liver weight ,relieve the extent of hepatic tissue damage , including necrosis and hydropic swelling of hepatocytes and inflammatory cell infiltration.And there is a dose–dependent on its protective effect. rhKD/APP can obviously reduce oxygen free radicals and reinforce oxygen free radicals elimination. rhKD/APP can obviously inhibit the inflammatory process . rhKD/APP can inhibit the cell apoptosis . Through those mechanisms, rhKD/APP may have the protective effect of liver injury.