Studies On Protective Effects Of Cerebrospinal Fluid Contain Tongqiaohuoxue Decotion On The PC12 Cells Injury Induced By Glutamate

Object:Through the investigation of the protective effect of rat cerebrospinal fluid containing Tongqiaohuoxue Decoction on the survival rate in PC12 cells,We can get rid of the disturb of BBB to the study on pharmacodynamics.It can also Provide data support in clinical application.Methods:Every male rat had surgery to cause cerebral ischemia model.Then,They were fed by gavage with TQHXT.After that,we collect the rat cerebrospinal fluid and comed together.We should have blank control at the same time.We cultured PC12induced by NGF in blank cell culture media contain GLU with debita spiss-itudo and choice the debita spissitudo by the methods of MTT to investigate the PC12 cells’proli-feration and activity.In the study of TQHXT Decoction containing rat cerebrospinal fluid,we establish control group,model group,TQHXT group.After the culture,inverted microscope,MTT method,trypan blue staining,LDH methods are used to inspect the protection of PC12.In the next study,we inspect the changes of intracellular NO,MDA,ATPase,SOD levels with kit,intracel-lular ROS levels with a fluorescence spectrophotometer after DCFH-DA dyeinged,intracellular free calcium concentration levels after Fura-2/AM dyeinged,cells mitochondrial trans-membrane potential levels after Rhodamine 123 dyeinged,Bax/bcl-2 detected by Westen-blotting method.Results:Results of MTT assay showed that,inhibition rate of PC12 cells is about50%when the concentration of GLU is 20mmol/L;When the concentration of rat cerebrospinal fluid is 5%、10%、20%,the influence of the GLU injured PC12 cells is indifference.Compared with model group,control group and TQHX group is closer to normal cell.After trypan blue staining,the ratio of living cells of control group and TQHX group is higher than model group.After AO/EB staining,control group and TQHX group has less number of EB staining.Compare with model group,LDH in cell culture supernatant,intracellular NO,MDA,ROS dropped.SOD,ATPase activity increased.Concentration of intracellular free calcium became lower.Bax/bcl-2 shows a lower ratio.Conclusion:Chose 20mmol/L as glutamate concentration to establish a PC12 cell injury model.Cell culture fluid contain 5%,10%,20%drug containing rat cerebrospinal fluid is used in the study,and cell culture fluid contain 20%containing rat cerebrospinal fluid is also used as the blank group.TQHXD containing rat cerebrospinal fluid shows protective effect on GLU injuryed PC12 cells in proliferation activity and membrane permeability.TQHXD drug containing rat cerebrospinal fluid can reduce the generation of NO,ROS and MDA to prevent the occurrence of oxidative damage;rise activities of SOD and ATPase to keep the normal function of cells;restrain Ca²⁺overload to reduce the toxic effects.