Study of TrkA、Bcl-2 Expression and Exffect of Tetrahydroxystilbene Glucoside on Them Following Cerebral Ischemic/Reperfusion Injury in Rats

Background and objective Many signal transduction pathways modulate cell survival and proliferation following ischemia brain damage. Nerve growth factor receptor A(TrkA)broadly participates in regulating cell proliferation,differentiation,apoptosis and other inflammatory process.It plays an important role in cerebral ischemia/reperfusion injury. Bcl-2,downstream effector of TrkA,is the firstly found anti-apoptotic protein of Bcl-2 family.Bcl-2 triggered by activated TrkA by several ways is involved in preventing the apoptosis pathway.Previous studies suggested TrkA/Bcl-2 could prevent cell apoptosis and promote nerve survival.However,there isn't study about important role of TrkA/Bcl-2 pathway during cerebral ischemia.Presently,it was reported that Chinese medicine polygonum multiflorum thunb and its active pharmaceutical ingredients tetrahydroxystilbene glucoside(TSG)have protective effects on cerebral ischemia.However,the mechanism and Dose-effect relationship is not fully understood.In this study,we observed the the mRNA and/or protein expression of TrkA and Bcl-2 and the effect of TSG.Furthermore,the impairment of brain tissue and apoptotic cells were detected to investigate the mechanism of neuroprotective and dose effect of TSG.The results may provide new drug development and therapeutic strategy for cerebral ischemia.Methods The healthy male sprague-dawley rats weighting 250～350g were randomly divided into five groups:normal control group(n=6), sham operation group(n=6),cerebral ischemia/reperfusion(I/R)group (n=48),low dose(60g/kg/d,n=48)and high dose TSG treatment group (120g/kg/d,n=48).After 6 days intragastric(ig)administration of TSG (treatment groups)or natural saline(sham-operation group and model group).Transient focal cerebral ischemia was induced by middle cerebral artery occlusion(MCAO).Then,neurological behavior evaluation was performed by the method of Longa's scoring.The pathologic changes were observed by hematoxylin and eosin(HE)staining at 6 h,12 h,24 h and 7 d after reperfusion in cerebral cortex of rats.Meanwhile,apoptotic cells were detected by terminal deoxynucleotide transferase-mediated nick end labeling(TUNEL)technique.The mRNA and/or protein expression of TrkA and Bcl-2 were measured with methods of immunohistochemistry and in situ hybridization respectively.Results1.Compared with I/R group,treatment with both dose of TSG could decrease the grade of the rat neurological defects except at 6 h after reperfusion.2.Typical neural necrosis could be observed in I/R group and both TSG groups by HE staining at 24 h after reperfusion.3.Few apoptotic cells were detected in normal group and sham operation group.Apoptotic cells' numbers was increased significantly at 6 h after reperfusion in I/R group and both TSG groups compared with in normal group and sham operation group.The peak of apoptotic cells' numbers was appeared at 24 h after reperfusion,and lasted to 48 h and 7 d after reperfusion.Treatment with both doses of TSG could reduce apoptotic cells' numbers compared with I/R group except at 7 d after reperfusion.There was no significant difference between low and high dose TSG treatment group.4.The mRNA and/or protein expression of TrkA and Bcl-2 were not detected normal group and sham operation group.The mRNA and/or protein expression of TrkA and Bcl-2 elevated at 6 h after cerebral reperfusion,reached maximum at 24 h,reduced at 48 h and maintained few at 7 d in I/R group and both TSG groups.Compared with I/R group,treatment with TSG could significantly increase TrkA and Bcl-2 expression except at 7 d after reperfusion observed.There was no significant difference between low and high dose TSG treatment group.Conclusions1.The TrkA/Bcl-2 pathway may be activated after cerebral ischemia.2.The mechanism of neuroprotective effect of TSG may be related to up-regulating TrkA/Bcl-2 pathway,thereby reducing the impairment of brain tissue,neurological defects and apoptotic cells.3.The cerebral protection of two doses of TSG is not significant difference.Maybe the dose-related differences are too small.