Effect Of Huwentoxin-Ⅰ On The TNF Apoptosis Pathway In Hippocampus Of Rat With Global Cerebral Ischemia Reperfusion

Objective Observing the effects of Huwentoxin-Ⅰ(HWTX-Ⅰ) on protein and gene expressions of related factors in the TNF apoptosis pathway of the hippocampal tissue in rats with global cerebral ischemia reperfusion injury, and investigate the possible molecular mechanism for the neuroprotective effect of HWTX-Ⅰon the rat models of global cerebral ischemia reperfusion in jury, so as to provide experimental evidence for the new method of the clinical treatment of cranio-cerebral injury.Methods 48 grown-up male SD rats were randomly divided into sham-operation group, non-administered group and administered group, adopting themodel of improved Pulsinelli 4-vessel occluded global cerebral ischemia damage combined with the method of chronic intrathecal catheterization, observing the morphological change of pyramidal neurons through Nissl staining, the protein and gene expressions of TNFα(tumor necrosis factor),TNFRI(TNF receptorⅠ),TRADD (TNFR-associated death domain),FADD (Fas-associated death domain) and Caspase8 in TNF death receptor apoptosis pathway in the hippocampal CA1 region of the rats with global cerebral ischemia reperfusion injury with microscope, electron microscope, immunohistochemistry and RT-PCR.ResultsResults(1) By Nissl staining, in the sham-operated group, multilayer pyramidal neurons ranged orderly and densely were clearly observed; In the HWTX-Ⅰadministered group, pyramidal neurons of several layers in orderly arrangement and sparsely distributed were observed, the characters of the concentration and deep staining of a few cell body and volume constriction of cells appeared, however those in the non-administered group, ranged disorderly and distributed sparsely with incomplete layers, the characters of the concentration and deep staining of a lot cell body and volume constrictionof cells appeared.Results(2) By electron microscope, in the sham-operated group, mitochondrial cristae were clear, and nobreakage of mitochondrial cristae was observed; In the non-administered group, mitochondrial cristae were unclear, vacuolization occurred; In the HWTX-Ⅰadministered group, mitochondrial Cristae were clear, and no obvious breakage of mitochondrial cristae was observed.(3) By immuno-histochemistry staining, there were almost no TNFα,TNFRI,TRADD,FADD,Caspase8 protein expressions in the hippocampal CA1 region of rats in the sham-operated group (yellow represented the positive expression of proteins); In the non-administered group, stronger expressions of each factor protein occurred in the hippocampal CA1 region of rats, and there were many positive cells; In the HWTX-Ⅰadministered group, strong expressions of each factor protein could be observed, but there were fewer and sparser positive cells than the non-administered group.Results(4) By RT-PCR detection, the expressions of TNFα,TNFRI, TRADD,FADD,Caspase 8 mRNA were the highest in the non-administered group, followed by the HWTX-Ⅰadministered group, and the lowest in the sham-operated group.Conclusions(1) The administration of HWTX-Ⅰin intrathecal space could maintain the morphological stability of pyramidal cells structure in hippocampus CA1 region of global cerebral ischemic reperfusion rats.(2) The administration of HWTX-Ⅰin intrathecal space decreased the protein and gene expressions of related factors of TNFα, TNFRI, TRADD, FADD and Caspase8 in TNF death receptor apoptosis pathway, which has obviously neuroprotective effects on rats hippocampus neurons damage induced by global cerebral ischemic reperfusion.(3) Signal transduction channel mediated by death receptor is closely correlated with cerebral ischemia reperfusion injury. It is of important theoretical significance to explain the mechanism of TNF death receptor channel in the global cerebral schemia reperfusion injury.