The Experimental Study In Vitro Of Buyang Huanwu Decoction On GLT-1 And GS In Ischemic Astrocyte

ObjectiveCerebrovascular disease is one of the main diseases endangering human health after malignant tumor and heart disease, which has been described as the "health public enemy". Ischemic cerebrovascular disease is the most common type of cerebrovascular diseases,the prophylaxis and treatment of ischemic cerebrvascular disease has become a hot area in medicine today. The pathogenesis of cerebral ischemia is involved in various-links, the neurotoxicity of excitatory amino acid is a hub to the pathogenesis among these links. Glutamic acid is the most important excitatory neurotransmitter in the central nervous system, too high concentrations of amino acids in the extracellular can cause cell apoptosis, then cause brain damage. It is commonly believed that there is no glutamic acid inactivation of hydrolytic enzyme system in the extracellular fluid, the removal and inactivation of the glutamic acid released from the presynaptic terminals is implemented through the uptaking from the glutamate transporter and the synergetic transforming from the glutamine synthetase, which are both located on the cell membrane of presynaptic membrane and glial.Some studies have reported that Buyang Huanwu Decoction(BYHWD) could reduce the concentration of glutamic in brain tissue and the cerebrospinal fluid, and it could also reduce the GFAP expression in astrocytes. Our previous experiment in vivo has confirmed that, BYHWD could effect the expression of GLT-1 and GS protein in astrocyte by PACAP, so that it could reduce the extracellular fluid glutamate concentration after cerebral ischemia. The potential function chennel and the mechanism of it should be discussed. Therefore, this experiments researched the influence of BYHWD for GLT-1 and GS in vitro culture of rat astrocytes by using Western Blot, CCK-8, glutamate kits and other technique, and selected the MAPK pathway as breakthrough point, further elucidated the mechanism of it, so that it could lay the foundation of the further research of the resistance mechanism of cerebral ischemia injury by glutamate metabolic pathways about BYHWD, which may provide novel evidence for the treatment on ischemia injury by BYHWD.Methods1. Effect of serum containing drug of BYHWD on astrocytic GLT-1 and GSThe newborn SD rat brain astrocytes was cultured, and detected the purity of astrocytes by immunofluorescence. The groups of the experiments were:the control group, the OGD group, the OGD with SD rats’control serum group and the OGD with SD rats’drug-containing serum group. After oxygen glucose deprivation (OGD) treatment for 2 h, Western Blot technique was used to examine the expression of GLT-1, GS and GFAP protein in astrocytes at some indicated time points (12,24 and 48 h), chose the most obvious temporal points about the difference in expression between the OGD with SD rats’control serum group and the OGD with SD rats’drug-containing serum group, then the concentration of glutamate in the extracellular fluid was measured by glutamic acid measurement kit.2. the functional mechanism of influence in GLT-1 and GS in astrocyte by BYHWDChose the most obvious temporal points about the difference in expression between the OGD with SD rats’control serum group and the OGD with SD rats’ drug-containing serum group as the experimental time point. Western Blot technique was used to examine the expression of ERK, JNK and p38MAPK protein in astrocytes. Selected the pathway of which expression changed significantly, to add a inhibitors group on the basis of the original group, Western Blot technique was used to examine the expression of GLT-1, GS and GFAP protein in astrocytes, and application method of CCK-8 was used for testing energetic differences of each groups.ResuIts1. Compared with control group, the expression of GLT-1 and GS protein was down-regulated by OGD/reoxygenation (0/R). However, drug-serum of BYHWD up-regulated the decreased expression of GLT-1 and GS, also down-regulated the raised expression of GFAP, among which the level of GLT-1 and GS protein at 24 h after 0/R was the highest and the level of GFAP protein at 24 h after 0/R was the lowest, and the differences were significant. Correspondingly, the concentration of glutamate in the extracellular fluid was also decreased in BYHWD drug-serum group2. Compared with control group, the expression of p-ERK, p-JNK and p-p38MAPK protein was up-regulated by OGD/reoxygenation (0/R). However, drug-serum of BYHWD up-regulated the expression of p-p38MAPK, while the expressions of p-ERK and p-JNK had no obvious change. And the inhibitor group that by application of SB203580 blocking p38MAPK pathway activation compared to the OGD with SD rats’drug-containing serum group, the expression of GLT-1 and GS significantly decreased, and the expression of GFAP significantly raised. Correspondingly, the concentration of glutamate in the extracellular fluid was also decreased in BYHWD drug-serum group. Correspondingly, the energetic of the inhibitor group was also lower than that of the OGD with SD rats’drug-containing serum group.ConclusionIn vitro cultured astrocytes after lack of oxygen and glucose processing, the expression of GLT-1 and GS protein in cells decreased significantly. And the glutamic acid and glutamine circulation anomalied, which was participated by GLT-1 and GS, then a dramatic increase in the extracellular glutamate concentration happened and caused excitatory neurotoxicity, so did the damage of astrocyte. BYHWD increased the expression of GLT-1 and GS protein in astrocyte by p38MAPK pathway, which could reduce the extracellul-ar fluid glutamate concentration, and then achieved the purpose of protection of astrocytes.