| Hepatic ischemia-reperfusion injury(HIRI) is commonly found in liver transplantation, hemorrhagic shock, trauma, etc. HIRI have complex mechanism. The early period of HIRI is often light ,the liver cells is mild swelling , liver function was no significant change. With the increase of the reperfusion time, vascular constriction, interleukin incarcerated. microcirculatory disturbance caused by platelet aggregation. swelling of liver cells, endothelial cells, kupffer cells are caused by lacking of energy, the liver microcirculation suffere furthe damage,and then it activate kupffer cells and neutrophil that release inflammatory cytokines and oxygen free radicals. The external stimuli role in the cells to produce biological effects,this process is very complicated, intracellular signal transduction pathway play a intermediary role in the process.studies have show that Janus kinase signal transduc-ers and activators of transcription(JAK-STAT)singal transduction pathway and HIRI-related. and it have relationship with apoptosis.ObjectiveTo build the rat model of hepatic ischemia and reperfusion injury and to investigate the expression changes of STAT1 and Caspase-3 protein during the periods of hepatic ischemia reperfusion in rats. The protective effect of salviae miltiorrhiae (SM) pretreatment was also invastigated. discussion the significance of JAK-STAT singal transduction pathway during the HIRI. Further Learn the HIRI s Signal transduction mechanism, provided new ideas to treat the HIRI.Method1 Eighty SD rats were randomly divided into four groups: normal group, sham operated group, ischemia and reperfusion group, SM pretreatment group. Then the groups were divided into five subgroups by different reperfusion time (Oh, 3h, 12h, 24h, 72h). Every group has five rats.2 Normal group: Inject 40ml/kg saline from tail vein 30 min before operation . 3% pentobarbital sodium 40mg/kg inject in aperitoneal, incision that on abdominal is about 3 cm,and then cut the liver tissue. ischemia and reperfusion group: infusion before operation like the former, find hepatic pedicle in peritoneal,and block it with artery forceps,remove artery forceps after 45min. cut the liver tissue at Oh ,3h ,12h ,24h and 72h after reperfusion. sham operated group: infusion before operation like the former, and also find hepatic pedicle in peritoneal but not block it. cut the liver tissue like ischemia and reperfusion group. SM-pretreated group: inject 40ml/kg salvia miltiorrhiza injection from tail vein 30 min before operation , narcotic , operation and get the liver tissue like before.3 The liver preparation was put into 10% formalin, dehydration with alcohol, embedded with paraffin,make slice that is 4μm.The preparation for elentron microscope was put into 3 % glutaric dialdehyde for twenty minutes. Then repair the block and draw material.4 Hematoxylin and eosin stain and mmunohistochemical SABC method were operated according to the procedure of the kit. rabbit anti HIF-1αand Caspase-3 (both working concentration was 1 : 100 ) were purchased from Wuhan Bosto biotechnological company.5 The stat1 positive criteria is brown granules that is found in nucleus or cytoplasmic, the caspase-3 positive criteria is brown granules that is found in cytoplasmic . The results were quantitated by HPIAS—1000 pathological image analytical system, ten 200-fold visions are found in each slice to record PU, and get average.the size of PU represent the number of positive production6 Date express by (x|-)±s . and was disposed in statistics by SPSS13.0. Factorial design ANOVE was used to analyze the main effects and interaction of all groups. The difference of every groups was tested by SNK method .Between two variables correlation analysis use Bivariate.P<0.05 means that there was statistical difference of experimental groups.Results1 The expressions of STAT1 were in a low levels in normal group and sham operated group. There was no statistical difference between them (P>0.05). The expression of STAT1 was increased after hepatic ischemia, it was increasing with the reperfusion time,and the converge district also appeared to express .The peak of STAT1’s expression appeared at 12～24 hours reperfusion, and the expression decreased but also have some expression at 72 hours. In SM-pretreated group , the expression level of STAT1 reduced as compared with ischemia and reperfusion group at the same periods(P < 0. 05).2 The expressions of Caspase-3 were in a low levels in normal group and sham operated group. There was no statistical difference between them (P>0.05). The expression of Caspase-3 was increased after hepatic ischemia, it was increasing with the reperfusion time. The expression peak appeared at 24 hours and it decreased to basic level at 72 hours. The expression of Caspase-3 in SM pretreatment group was lower than that in ischemia reperfusion group excepet 72h (P<0.05).3 Ischemia and reperfusion group:STAT1 and caspase3 was a positive correlation( P< 0. 01), SM-pretreated group: STAT1 and caspase3 was also a positive correlation( P< 0. 01).4 HE dyeing section was observed in light microscope: the liver structure in normal group and sham operated group is basically entire, hepatocyte continues ready-made a rope, cell nucleus is big but round. At 45min after hepatic ischemia and Oh after reperfusion, liver cells was mild edema, crowded with disorder,the gap of sinus hepaticus is narrow; At 3h after reperfusion, liver cells was edema, the loose memorial of cytoplasm is netlike, semi-transparent; At 12h after reperfusion, liver cells was exceeding edema and like a balloon.there were some shape necrosis and eosinophilic body in lobuli hepatis. damage wais the heaviest time 24 h,some lobuli hepatis were deformated. some inflammatory cells were found in health district. At 72h after reperfusion, the narrow gap of sinus hepaticus was relieved, lobuli hepatis was reconstructed. In SM-pretreated group , the damage of liver reduced as compared with ischemia and reperfusion group at the same periods5 The pathohistolgical changes were observed in electron microscope: the liver structure in normal group and sham operated group is basically entire; At 45min after hepatic ischemia and Oh after reperfusion, mitochondria was mild edema,other had no obvious abnormalities; At 3h after reperfusion, mitochondria and endoplasmic reticulum were edema, mitochondrial cristae became shorter, chromatin concentrated; At 12h after reperfusion, chromatin concentrated, the cell implements were region-like distribution, glycogen disappeared; At 24h after reperfusion,some liver cells died and some nowborn liver cells; At 24h after reperfusion ,the Ultrastructure of liver cell become normal. The injury of SM pretreatment groups was lower than that of ischemia and reperfusion groups.ConclusionThe expressions of STAT1 were in a low levels in normal group and sham operated group. The expression of STAT1 was increased after hepatic ischemia, it was increasing with the reperfusion time, and the district also appeared to express,the peak of STAT1’s expression appeared at 12～24 hours reperfusion, and the expression decreased but also have some expression at 72 hours. The expression of Caspase-3 was increased after hepatic ischemia, it was increasing with the reperfusion time,the expression peak appeared at 24 hours and it decreased to basic level at 72 hours. Correlation analysis between STAT1 and caspase3 show positive correlation, the dynamic changes of expression between the two are basically the same. The activate mechanism of STAT1 is not clear, possible mechanism that some inflammatory cytokines were released when liver suffere ischemia-reperfusion injury(for exampleIL-1、IL-6、TNF-α、IFN-γ).this cytokines can activate the JAK-STAT singal transduction pathway. In particular, the expression of STAT1 induced by IFN-γ,at the some time, endotoxin that come from intestinal can indirect activate the JAK-STAT singal transduction pathway. Other study indicate that oxidative stress can also activate the JAK-STAT singal transduction pathway . ROS that was producted when liver suffere ischemia-reperfusion injury can activate the JAK-STAT singal transduction pathway,and STAT1 was producted. At 12～24 hours reperfusion, inflammatory cytokines and ROS were still produced ,but at this time STAT1 had no increase,this may have connected with SOCS, the expression of SOCS can induced by cytokines,and then SOCS inhibite the JAK-STAT singal transduction pathway that be activated by cytokines. SOCS have negative feedback effect on the expresstion of STAT1, and make it in a balanced status. At the early of reperfusion, STAT1 was found in the liver cell that around the central vein area,the expression of STAT1 increased with the increase of the reperfusion time,and was found aroud converge district. Analyzes its reason: the ventilation of the cell that aroud the central vein area is bad,this cells are most sensitive to the oxygen deficit damage,and implicated first when block the liver blood stream, inflammatory cytokines were producted, this cytokines can stimulates STAT1 to express, with the increase of the reperfusion time, endothelial cells that in sinus hepaticus swelling, blood can not inflow successfully in central venous,a lot of inflammatory cytokines and endotoxin that come from intestinal accumulated at the converge district,and this stimulates STAT1 to express. Observation the spatial distribution of STAT1 and caspase3,we found that the expression region between the two are basically the same ,and primarily located in the central vein and around the converge district .Combination of the spatial distribution and dynamic changes we speculate : external stimuli signal may promoted the expression of caspase3 through the activation of STAT1, and induced apoptosis.Many research shows that SM have protective effects when some important organ suffere ischemia-reperfusion injury,because the SM can relief platelet aggregation, improve microcirculation,antioxidant free radical. In SM pretreatment group, the expressions of STAT1 and Caspase-3 were lower than that in ischemia reperfusion group. SM improve the microcirculation to enable reduction of inflammatory cytokine production, and it can inhibite the oxidative stress response,so it inhibit the high expression of STAT1 in some extent. This study hint SM can influence JAK-STAT signaling pathway that is intermediate material ,but this impaction which is through reduce the indirect role of triggering factors or direct role in the JAK-STAT signaling pathway depende further study... |
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