| Objective:(1)to build stable model of orthotopic liver transplantation in rat in order to further experiment study for ischemia-reperfusion injury and others. (2)to investigate the protective role of shenfu (SF) ischemia-reperfusion injury of rat liver tissue through comparing serum transaminases and liver histology. (3)to observe the changes of glucocorticoid receptor (glucocorticoid receptor, GR), nuclear factorκB (NF-κB) in Kupffer cells from rat liver graft which pretreat with SF and supernatant fluid tumor necrosis factor-α(TNF-α) level. Data was analyzed to elucidate the protective mechanism of SF pretreatment against ischemia-reperfusion injury.Methods:(1)The animal model of orthotopic liver transplantation was conducted with two-cuff technique, in which suprahepatic vena cava (SVC) was anastomosed with continuous suture,portal vein (PV) and infrahepatic vena cava(IVC) were anastomosed with cuff method, and the bile duct was reconstructed by interior stent. (2)Male Sprague-Dawley rats, weighing 180-220g, were divided into three groups in a randomized, blinded fashion. the control group (n=6) did not perform any surgery, donor animals of the the SF group (n=6) were treated with SF(2ml/100g, iv)1h before surgery, and the ischemia-reperfusion group (IR) (n=6) were treated with physiological saline. At 1h, 3h, 6h and 24h after reperfusion, serum transaminases were investigated and compared between SF and IR group. At the period of reperfusion 1 week, samples of hepatic tissue were taken for liver histopathological examination. (3) KCs were isolated from normal rat liver and liver grafts at 6h after transplantation. The expression of GR, NF-κB in KCs and TNF-αsecretion were measured by RT-PCR, Western blot ,laser confocal scanning microscope and ELISA.Results: (1) The pre-experiment of orthotopic liver transplantation were conducted in 40 cases of 80 rats by"two-cuff technique"methods. The operative successful rate and one-month survival rate were 25% and 80%. 30 cases of orthotopic liver transplantation were operated in formal experiments when the operative successful rate and one-month survival rate were 90% and 90%. The donor and the receptor surgery respectively cost 35±2.5min and 46±2.8min.The liver repairing cost 16±4.1min. Ischemia period≤19min, the cold storage time≤60min. (2) The serum transaminases (ALT) of the control group is 50.8±23.6.Between IR group and SF group, the rate of ALT have a significant difference in the experiment at every observation point (P>0.05), especially in 3 hours and 6 hours after transplantation. They were at a high rate in both of the two groups. The ALT of IR group: 1075.01±134.02 U/L(3h) and 958.15±35.70 U/L(6h); The ALT of SF group: 808.79±44.74 U/L(3h) and 655.55±69.18 U/L(6h ).(3)The mRNA and protein expression of GR in the IR group were significantly lower (P<0.01), and the NF-κB expression and TNF-αlevel (830.19±71.34) were obviously higher as compared with the control group ( TNF-αlevel 65.41±37.63,P<0.01). SF treatment (TNF-αlevel 543.59±41.27) significantly reversed the changes in IR group (P<0.01).Conclusion: SF can protect IR injury after liver transplantation and improve liver graft structure and function. IR injury can down-regulate the expression of GR and up-regulate NF-κB and TNF-αlevel in Kupffer cells, SF pretreatment can meliorate activation of KCs after IRI.
|
PDF Full Text Request