The Study Of TRAIL Signal Iter Mediate Apoptosis In Rat Renal IRI Model And Intervention Of Minocycline

Objective : To construct the model of rat renal ischemia reperfusion injury(IRI) in order to observe the IRI,s influence of the rat renal function and morphology changes ;to approach the IRI model , s regularity of apoptosis and diversify;to detect IRI model,s the express and variation of TNF-related apoptosis Inducing ligand(TRAIL) and caspase- 3 ,and to approach the apoptotic mechanism;to study Minocycline,s protective function and role of restraining apoptosis, and to understand the mechanism.Methods: 72 male Sprague-Dawley rats which weight 220g²60g are randomly divided into three groups: sham-operated(Control group),ischemia-reperfusion(IR) group,minocycline (M) group, there are 24 rats in each group and 6 rats for each of the four phases (6h,12h,24h,72h).On 45 minutes after the left renal artery occlusion on the basis of the right renal resection, we restored perfusion under modeling of ischemia reperfusion injury; we only cut right kidney of rats in control group but not to clamp left renal artery. To give the rats in M group intragastric administration in 36 hours before operation, the initial dose is 45mgï¹’ Kg^-1 , then to intragastric 22.5mgï¹’ Kg^-1 every 12 hours, the all of the dosage frequency is four times, whose method of operation is identical with the IR group. To reflect the extent of renal function lesion by determining the level of serum creatinine; To observe renal histological and ultrastructural changes through light microscope and transmission electron microscope; To detect the express of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) and then to compute Apoptotic index; To detect the express level of TRAIL and Caspase-3 by immunohistochemistry PV6001.Results:1.there are not obvious abnormity of nephridial histopathology ultrastructure in control group; we observe conspicuous nephridial tissue,s injury of histopathology and ultramicrostructure in IR group, whose main appearance is necrosis,amotic of renal tubular epithelial cell, destruction of tubal wall,s Integrity and apoptosis cell; All of the damage in M group is gentle than which in IR group.2. The level of Ser in IR group start to step up on postinjury 6H, arrive the peak in 24 hours , then descend after 72H, which is obviously more high than control group and M group. The level of Ser in M group is more high than Control group.3. In substantia corticalis renis, there are almost no apoptosis in Control group, whose apoptotic index is about 0.5%; the apoptotic index in IR group start to step up in 6H after reperfusion (1 7.76%±0.9%), arrive the peak in 24 hours( 2 3.39%±1.8%), then descend after 72H ( 6.84%±0.9%),all of which are more high than control group; The apoptotic index in M group is lower than IR group. In kidney medulla, there almost is not the express of apoptosis, whose apoptotic index is only 0.29%±0.27%～0.65%±0.63%.The apoptotic index in IR group 6H is 44.29%±2.45%;arrive the peak in 24 hours (4 9.46%±2.86%, we still can see the express of apoptosis in 72H, whose apoptotic index is 32.91%±3.78%,which is obviously more high than control group. The express of apoptosis in M group is obviously attenuated. Apoptotic index in kidney medulla is more high than which in cortex of kidney.4.In cortex of kidney, there almost are not express of TRAIL in control group, IOD is only about 0.00015μ㎡. In IR group, the express of TRAIL in 6H after reperfusion is 0.00574±0.000477μ㎡; which in 24H is 0.0215±0.000130μ㎡;which in 72H obviously down regulation to 0.000636±0.0000439μ㎡, that is obviously more high than control group. In M group, the express of TRAIL in 6H,12H,24H are obviously lower than which in IR group, but there is not difference between M group and IR group in 72H. IN kidney medulla, there almost are not express of TRAIL in control group, IOD is only about 0.000136～0.000152μ㎡. IOD in IR group and M group are only 0.0001～0.0002, which is more high than Control group, there are not obviously difference between the IOD of IR group and M group.5.The express of CASPASE-3 is concord with apoptotic disposition. In cortex of kidney, IOD of Caspase-3 is 0.0001～0.0002μ㎡; the express of which in 6H after reperfusion is enhanced to 0.0197±0.00107μ㎡,in 24H which is 0.0268±0.00076μ㎡,in 72H which is 0.0145±0.00214μ㎡, both of which are more high than control group; the express of CASPASE-3 in M group is attenuated than IR group, the result have statistical significance. In kidney medulla, there are not express of Caspase-3, the IOD is only 0.000096～0.000176μ㎡; which in 6H is 0.0238±0.00073μ㎡,which in 24H is 0.0307±0.00078μ㎡,which in 72H is obviously enhanced than Control group ,the IOD is 0.0168±0.00070μ㎡; which is more attenuated than IR group.6.In IR group, in cortex of kidney, there are positive correlation between apoptosis,TRAIL,Caspase-3; in kidney medulla ,the apoptosis and Caspase-3 are abviously positive correlation, but there are not dependability between apoptosis and TRAIL; there are not dependability between Caspase-3 and TRAIL.Conclusion:1.Ischemia reperfusion for more than 45 minutes, which may result in damage of rats nephridial tissue and kidney hypofunction,however the damage is reverse.2.In the model of IRI , apoptosis is obviously increasing, which illustrate that apoptosis participate ischemical reperfusion injury, and mainly in juncture of cortex and medulla.3.TRAIL and Caspase-3 both high express in proximal convoluted tubule, which manifest that TRAIL angd Caspase-3 participate apoptosis of proximal convoluted tubule; in kidney medulla, the express of TRAIL have not been enhanced, and Caspase-3 is high expressing, which indicate that the iter of TRAIL do not participate apoptosis of kidney medulla,it is other iters activate Caspase-3 to apoptosis.4.Minocycline can lessen the damage of renal function and nephridial tissue after IRI, and can protect the renal function and histomorphology.The intervention of Minocycline can down regulation the express of TRAIL and Caspase-3 to decrease apoptosis.