The Study Of Buyang Huanwu Decoction On GLT1and GS In Ischemic Astrocyte

ObjectiveCerebrovascular disease is a common and frequently-occurring disease that seriously endangers human health. It is one of the leading causes of death and disability worldwide, especially ischemic cerebrovascular disease, which is the most frequently prevalent. The molecular mechanism of ischemic injury including energy decreasing, free radicals, NO&NOS, intracellular calcium increasing, inflammatory factor, apoptosis factor and exciatory amino acid. And the exciatory amino acid is the major pathogenesis in stroke. Glutamate (GLU) is the main exciatory neurotransmitter in the mammalian brain, however, it can also aggravate the injury of brain when it is released too much. Recent studies indicated that Buyang Huanwu Decoction (BYHWD) can reduce glutamate concentration, in addition, the metabolism of Glu mainly depended on GLT1and GS which were located in astrocyte. Based on the above, we used immunofluorescent staining and western-blot to investigate whether BYHWD can increase the function of GLTl and GS in astrocyte after middle cerebral artery occlusion(MCAO). Meanwhile, this study would elucidate the mechanism about how BYHWD reduce glutamate concentration, which may provide novel evidence for the treatment on ischemia injury by traditional Chinses medicine.Methods1. To investigate whether Buyang Huanwu Decoction can incraese the expression of GLT1and GSAdult male SD rats were used in these studies, and after middle cerebral artery occlusion, rats were randomly divided into ischemic group (1d,3d,5d,7d) and BYHWD (Id,3d,5d,7d). Rats in BYHWD were administrated by BYHWD (16g/kg, twice a day). The vehicle treated rats were administrated with equal volumes of normal saline (NS). At last, the expression of GLT1, GS and GFAP protein in brain tissues was determined by immunofluorescent staining.2. To investigate the mechanism of how Buyang Huanwu Decoction treatment increase GLT1and GS expressionAdult male SD rats were randomly divided into control, ischemia, BYHWD and BYHWD+PACAP(6-38), each group contained18rats. And then BYHWD+PACAP(6-38) were first given PACAP(6-38)(1.49mmol/ml, the others were given lml NS which contained15%acetonitrile/water) by intravenous injection at15min before ischemia reperfusion, and then the second time at48h after ischemic reperfusion; meanwhile rats in both BYHWD and BYHWD+PACAP(6-38) were intragastricaly administrated by BYHWD(16g/kg, others were given2ml NS) twice a day for3days. Finally, the expression of the PACAP, GLT1, GS, GFAP protein in brain tissues was determined by immunofluorescent staining and western-blot (except PACAP), and glutamate conentration was assayed by UV.ResultsThe expression of GLT1and GS protein were increased on day1and peaked on day3after ischemic reperfusion, and compared with ischemia (Id、3d、5d、7d), GLT1and GS expression was elevated in ischemia+BYHWD (Id,3d,5d,7d)(P<0.01).On day3, BYHWD significantly increased the expression of GLT1and GS (P<0.01).2. The expression of PACAP was significantly increased on group ischem-ia+BYHWD, and after i. v PACAP (6-38), GLT1and GS expression was significantly decreased, meanwhile the glutamate was elevated, and the expression of GFAP was also increased.ConclusionBuyang Huanwu Decoction treatment increased the expression of GLT1and GS protein in astrocyte by PACAP, which can delay neuronal death after ischemia by reducing extracellular glutamate.