The Effect Of Tetrahydroxystilbene Glucoside On The Protein Expression Of NGF,PKAc And GAP-43 In Cerebral Cortex Of Rats After Cerebral Ischemia-reperfusion

Background and objective The cell survival and proliferation following ischemic brain damage was modulated by many signal transduction pathways. The expression of cAMP increased when treated with exogenous NGF,which activated the PKA pathyway and increased the expression of GAP-43 that made the neurite regenerate. Presently, it was reported that Chinese medicine polygonum multiflorum thunb and its active pharmaceutical ingredients tetrahydroxystilbene glucoside (TSG) had protective effects on cerebral ischemia,but the mechanism and Dose-effect relationship was not fully understood. Studies have shown the expression of NGF increased when treated with NGF in AD rats. In this study, we observed the protein expression of NGF,PKAc and GAP-43 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 the experimental bases for the new drug development and therapeutic strategy on cerebral ischemia.Methods The healthy male sprague-dawley rats weighting 200-300g were randomly divided into four groups: control group (n=24), I/R(ischemia/reperfusin)group (n=24), low dose (60mg/kg/d, n=24) and high dose(120mg/kg/d, n=24) TSG treatment group. After 6 days intragastric (ig) administration of TSG (treatment groups) or natural saline (I/R 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 protein expression of NGF,PKAc and GAP- 43 were measured with methods of immunohistochemistry.Results1. Compared with I/R group, treated 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 control group. Apoptotic cells' numbers increased significantly at 6 h after reperfusion in I/R group and both TSG groups compared with in control group. The peak of apoptotic cells' numbers appeared at 24 h after reperfusion, and lasted to 48 h and 7 d after reperfusion. Treated 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 protein expression of NGF,PKAc and GAP-43 were detected few in control group. The protein expression of NGF and PKAc increased at 6h after cerebral reperfusion, reached maximum at 24h, reduced at 48h and maintained few at 7d in I/R group and both TSG groups. The protein expression of GAP-43 increased at 24h after cerebral reperfsion, reached maximum at 48h, and maintained positive at 7d in I/R group and both TSG groups. Compared with I/R group, treated with TSG could significantly increase the protein expression of NGF,PKAc and GAP-43 after reperfusion. There was no significant difference between low and high dose TSG treatment group.Conclusions1. The protein expression of NGF increased when treated with TSG after cerebral ischemia-reperfusion ,which activated the PKA pathyway and increased the protein expression of GAP-43 that made the neurite regenerate.2. There was no significant difference between low and high dose TSG treatment group on the protein expression of NGF,PKAc and GAP-43.