Effect Of Substance P On Acute Pancreatitis With Hepatic Injury In Rats

Acute pancreatitis(AP) is a frequent acute abdomen in clinic, causes damages not only to pancreas, but also to distant organs, such as liver, kidney, lung, intestines etc. The mechanisms of hepatic injury during AP are not clear yet. The existing studys shows that, hepatic injury is a complicatide pathology process with many facters such as inflammatory cytokines, hepatic apoptosis, oxidative stress, microcirculation disturbance etc.Substance P is an important member of tachykinin family which belong to neuropeptide. It is generally distributed in central nervous system and peripheral nerveous system. Substance P has many biological activities, such as algesia conduction, neurogenic inflammation, visceral smooth muscle contraction and angiectasis etc. It has important effect in regulating inflammatory cells transmigration. It is a medium between nerve fiber and inflammatory cell, becaues it can promote inflammatory reaction happening by neutrophil aggregation, interleukin 8 synthesis and release, and mastocyte release histamine. Substance P has the strongest avidity with neurokinin receptor-1(NK-1R). So NK-1R also called substance P receptor. NK-1R express at cell envelope of nerve cell, vascular endothelial cell, fibroblast and inflammatory cell (such as lymphocyte, mononuclear macrophage , neutrophil). As substance P combine with NK-1R, it can inhibit exocrine pancreas, lighten pancreas and peripancreatic tissue damage caused by various digestive enzymes. But it also causes angiectasis, blood plasma exosmose, white blood cell adhesion and emigration out of blood vessel. The reaction caused by substance P is a defensive reaction of organsim to external destructive stimulus virtually, but it can leads to harmful result if it over servere. Patients with AP have a high rate of hepatic disfunction, and hepatic injury level is direct correlation with AP condition. It means unfavourable prognosis, when hepatic failure happens. Therefor, it is important to study the effect of substance P in AP hepatic injury. And it may helps to reveal the mechanism of AP hepatic injury and provide an idea for treatment.Objective:To investigate expressions of substance P, NK-1R and hepatic apoptosis by establishing AP model and administrating L-703,606 which is an antagon of NK-1R, to explore the effect of substance P in hepatic injury in AP and the therapeutical effect of NK-1R antagon.Method:1 Animal mode: The experiment was performed in SD rats under anesthesia injected intra-peritoneally with 10% chloral hydrate. Then, the abdomen median incision was performed. Use #4 ligature to ligate bile duct near hepatic portal before bile duct was injected antidromicly by microinjector with 5% Sodium Cholate (0.1ml/100g) at the speed of 0.2ml/min. Then press bile duct entering duodenum by thumb of left hand. 3 minutes later, when pancrea appears obviously hyperemia, cut off ligature and suture abdomen.2 Animal groups: All SD rats (n=54) were divided into 3 groups at random: sham-operation group (n=18), AP group (n=18) and AP+L-703,606 group (n=18). Then every group was divided into 3 subgroups (n=6) at different time points: 3h,6h,12h. The rats in AP+L-703,606 group were injected intra-peritoneally with L-703,606(250nmol/kg) 15 minutes before injected Sodium Cholate.3 Mensuration of biochemical parameters: The levels of serum amylase, lipase, alanine aminotransferase were determined respectively by automatic biochemical analyzer.4 Pancreatic gland and liver histological analysis: Paraffin section was stained by hematoxylin-eosin (HE).5 Immunohistochemical staining of substance P and NK-1R: After dehydration and antigen heat plerosis, the sections were incubated with 3% hydrogen peroxide for 15min. The sections were dripped with goat serum for 30min, then incubated with first antibody at 4℃overnight. In negative control group, PBS replaced first antibody. Dripping successively the second antibody and the third antibody, which were kept 15min respectively. The results were visualized by using 3,3-diaminobenzidine (DAB) as chromogen. At last, the sections were redyed with hematoxylin.Hepatic apoptosis: Using TUNEL detect and measurement kit to detect apoptotic hepatocyte.Results:1 Histological findings of pancrea: It was seen by naked eyes and light microscope that the pancreatic gland and its surrounding tissue of sham-operation group was approximatly normal; areas and degrees of dropsy, hemorrhage and necrosis of pancreatic gland and its surrounding tissue were being more and more severe, and the seroperitoneum and and neutrophil were growing in AP group and AP+L-703,606 group.2 Histological findings of hepatic: It was seen by naked eyes and light microscope that the liver tissue of sham-operation group was approximatly normal; areas and degrees of cellular swelling, denaturation, necrosis and hepatic cord structure decollement were being more and more severe, and inflammatory cell infiltrate were growing in in AP group and AP+L-703,606 group. Compared with AP group the hepatic injury was lighter in AP+L-703,606 group.3 Serum amylase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum amylas were increased in AP group and AP+L-703,606 group (P<0.05). But there was no significant difference between AP group and AP+L-703,606 group. 4 Serum lipase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum lipase were increased in AP group and AP+L-703,606 group (P<0.05). But there was no significant difference between AP group and AP+L-703,606 group.5 Serum alanine aminotransferase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum alanine aminotransferase were increased in AP group and AP+L-703,606 group (P<0.05). There was no significant difference between AP group and AP+L-703,606 group, but AP+L-703,606 group had lower level than AP group at each time point.6 Expression of substance P: In sham-operation group, substance P was a little expressed in intracytoplasm of hepatocyte cells. We found an overexpression of substance P in AP group and AP+L-703,606 group. Immunohistochemistry score showed that substance P expressed in AP+L-703,606 group was lower than in AP group.7 Expression of NK-1R: In sham-operation group, NK-1R was expressed a little in intracytoplasm of hepatocyte cells. We found an overexpression of NK-1R in AP group and AP+L-703,606 group. Immunohistochemistry score showed that substance P expressed in AP+L-703,606 group was lower than in AP group. 8 Hepatic apoptosis: There were hardly any apoptotic cells in sham-operation group. In AP group and AP+L-703,606 group, we can see many apoptotic cells tincted buffy and increased by the time. But AP+L-703,606 group had less amount than AP group.Conclusions:1 On the basis of histological findings and increase of serum amylase and lipase from this study, we concluded that AP model group has been successfully manufactured by bile duct inject antidromic with 5% Sodium Cholate.2 On the basis of histological findings ,increase of serum alanine aminotransferase and hepatic apoptosis from this study, we concluded that AP rats have apparente hepatic injury.3 The overexpression of substance P and NK-1R in AP rats shows substance P play role in hepatic injury.4 L-703,606 is an specific antagon of NK-1R. It play biological effect by block NK-1R to combine with substance P. It can decrease serum alanine aminotransferase, hepatocyte apoptosis, expression of substance P and NK-1R, ameliorate liver tissue damage when give to acute pancreatitic rats. We suppose that substance P play an important role in hepatic injury by combined to NK-1R during acute pancreatitis.