Neuroprotective Effect Of Polysaccharides From Gastrodia Elata And Its Sulfated Derivative And Their Mechanism

Objective:The purpose of this study was to examine the neuroprotective effect and relative mechanisms of polysaccharides from Gastrodia elata(GEP)and sulfated polysaccharides from Gastrodia elata(GEPs)against corticosterone-induced apoptosis in PC 12 cells.Simultaneously,the antiproliferative activity of GEPs against the C6 glioma cell(C6 cell)in vitro was also evaluated.Here,cell model of PC 12 cell induced by CORT and cell model of C6 cell were established.In terms of providing theoretical basis for explotiation of GEP into a new,safe and effective neuroprotective natural medicine and seeking of novel original bioactive of GEPs,cells survived rate,lactate dehydrogenase(LDH)release,intracellular ROS level,WB were measured,and the cell morphology and wound healing were observed.Methods:To study the neuroprotective effect of GEP and GEPs,cell model of CORT-induced apoptosis in PC 12 cell was established.The experiment was divided into control groups,CORT-induced groups(200?M CORT),low concentration groups(200 ?M CORT+250?g/ml GEP/GEPs),medium concentration groups(200?M CORT+500?g/ml GEP/GEPs),high concentration groups(200 ?W CORT+1000?g/ml GEP/GEPs).PC12 cells were treated with 200 ?M CORT in the absence or presence of different concentrations of GEP/GEPs's pretreatment for 48h.In order to detect the mechanism of GEP's neuroprotective effect,cell survival rate was assessed by CCK-8 assay,LDH release rate and intracellular ROS level were measured,cell apoptosis rate was assessed by Hoechst33258/PI double staining,endoplasmic reticulum(ER)stress in PC 12 cells was assessed by 3,3-Dihexyloxacarbocyanine iodide fluorescent staining and the expression level of GRP78,XBP-1,GADD153,caspase9 and caspase12 was measured by WB.In order to detect the neuroprotetive effect of GEPs,cell survival rate was assessed by CCK-8 assay,LDH release rate was measured,the morphological changes of PC 12 cell was observed by microscope,DAPI staining was observed and the expression level of BAX and Bcl-2were measured by WB.On the purpose of researching the antiproliferative activity of GEPs against C6 cell,the experiment was divided into control groups,0.1 mg/ml GEPs group,0.25 mg/ml GEPs group,0.5 mg/ml GEPs group,1 mg/ml GEPs group,1.5 mg/ml GEPs group.Cell growth inhibition in vitro was assessed by CCK-8 assay.The morphorlogical changes of C6 cell pre-and post-treatment were observed by microscope.The cell apoptosis was observed through DAPI fluorescent staining by microsope.Cellular migration ability was assessed by scratch test.Results:The results showed that pretreatment with GEP prior to 200 ?M CORT exposure significantly protected PC 12 cell from CORT-induced cell death,reduced LDH leakage and intracellular reactive oxygen species(ROS)level,attenuated the ER stress.In addition,pretreatment with GEP can also restrain the activation of GRP78,XBP-1,GADD153,caspase9 and caspasel2.Similarly,the pretreatment with GEPs prior to 200?M CORT exposure protected PC 12 cell from CORT-induced cell death,reduced LDH leakage.Moreover,GEPs can also restrain the activation of Bcl-2/BAX.Last but not the least,GEPs restrained the proliferation of C6 cell,induced C6 cell apoptsis and significantly restrain C6 cell migration with a dose-dependent manner.Conclusion:The mechanism of neuroprotective effect of GEP was likely connected with the attenuation of intracellular ROS,down-regulation of GRP78,XBP-1,GADD153,caspasel2 and caspase9 protein expressions,which means protective effect is probably associated with the inhibition of ER stress-mediated pathway.In addition,GEPs also showed the similar neuroprotective effect.However,compared to GEP,this protective effect of GEPs did not measurably increase.Finally,GEPs can effectively restrain proliferation activity of C6 cell.