| PurPose：This experiment was designed to observe the vitality of the PC12cells by differentconcentrations of KCl on different time points, in order to select the concentration and thetime points of KCl inducing PC12cell injury, and simulate nerve cells’ injury from calciumoverload; Through observation of PC12cell morphology and survival rate, the protectiveeffect of liu wei di huang pill contained serum was discussed; Through observation of thechange of the flow intensity of extracellular calcium, the intracellular calcium ionconcentration was measured in order to evaluate the influence of liu wei di huang pillcontained serum; By measuring the expressions of CaMKII, CREB and BDNF in both proteinand gene levels, the effects of liuwei dihuang pill contained serum on CaMKII-CREB-BDNF signaling pathway were discussed. At last, the experiment was also used to providescientific experimental data and theoretical basis for the prevention and treatment ofAlzheimer’s disease with liuwei dihuang pill, and partly illustrate the molecular mechanism ofthe protective effect of liu wei di huang pills on nerve cells.Material and method：Male SD rats were randomly divided into2groups as the normal group and liuweidihuang pill group. Rats in liuwei dihuang pill group were gavaged twice with10mL/kgaqueous solution in0.75kg/L crude drug concentration, and the rats in normal group weregavaged with double volume evaporate water for4days, and on the5th day, abdominal aortablood was collected1.5h after the first dose, and the blood was centrifuged for10min at2000r/min to separate serum. After56℃,30min inactivating,0.22mu m filter membranefiltration sterilizating, the serum was saved at–20℃for later use. PC12cells in thelogarithmic phase taken from the culture flask were digested by0.25%trypsin at37℃, andadded DMEM containing10%fetal bovine serum, which were continually cultured for12hin the conditions of cell density of1x105/mL,37℃and5%CO2, and then the cells beganto grow adherent to the wall, after switching to serum-free DMEM, the cells were cultured for another12h in order to be used in the experiment. Preparing KCl with differentconcentrations of50,100,150,200mmol/L to injure the cells for10,20,30min respectively,the activity of PC12cells were determined by MTT method. The cells were divided into sixgroups：（1） the control group：10%normal serum;（2） model group:10%normal serum plus150mmol/L KCl;（3） traditional Chinese medicine（TCM） intervention group:10%LiuWeiserum+150mmol/L KCl;（4） western medicine intervention group:10%normal serum+10umol/L nimo+150mmol/L KCl;（5） pre-intervention model group：10%normal serum （pre-intervention48h）+150mmol/L KCl;（6） pre-intervention in Chinese traditionalmedicine（TCM） group:10%LiuWei serum （pre-intervention48h）+150mmol/L KCl.（2）,（3） and （4） group were cultured in DMEM contained fetal bovine serum after48hours, andwashed by the PBS twice, and then the drug serum and KCl were added at the same time.20min later, with PBS washing twice, the cells were added corresponding serum and drugs toculture for another6h;（5） and （6） groups were added KCl after48h early intervention, and20min later, with PBS washing twice, the cells were added serum-free DMEM to culture foranother6h. The cell morphology of each groups was observed under inverted microscope，and the cells’ survival rate and LDH were determined by MTT method. After48h culturing,Fluo-3load and laser scanning confocal microscopy detected the changes of meanfluorescence intensity in intracellular calcium ion. The total RNA was extracted from eachgroup of cells, and mRNA expression of CaMK II, CREB, BDNF were measured by RT-PCR.After fixing of the cells climbing in each group, the protein expressions of CaMK II, CREB,BDNF were measured by SABC. The cells were collected, total proteins of cells in eachgroup were extracted, and the protein expression of CaMK II, CREB, and BDNF weremeasured by western blot.Results：1. Successful establishment of KCl induced cell injury modelStatistical results show：compared with the control group, there were significantly decreasedin cell vitality with150mmol/L and200mmol/L KCl in20min （P<0.05）， and there weresignificantly decreased in cell vitality with100mmol/L,150mmol/L and200mmol/L KCl in30min （P<0.05）. 2. The influence of liu wei di huang pill contained serum on KCl-induced cell injury2.1Morphological observation: From an inverted microscope, the cells of the control groupappear fusiform, close intercellular contacts, and extend staggered pseudopodia, strongproliferation and diopter; model group, pre-intervention model group show that pseudopodsof cell disappear, decreased diopter, some of the cell rounded shrinkage and float off in themedium; some cells swell with decreased diopter, and some float off in TCM interventiongroup and western medicine intervention group. Cells in pre-intervention groups show strongproliferation and diopter.2.2The influence of KCl induced cells injury on cell vitality of every groups （MTT）.Compared to control group， cell survival rate of model groups was decreased significantly（P<0.01）, and cell survival rate of pre-intervention model group, TCM intervention group andwestern medicine intervention group were also significantly decreased （P<0.05）; Compared tothe model group, cell survival rate of TCM intervention group and the western medicineintervention group was significantly increased （P<0.05）; Compared to pre-intervention modelgroup, the cell survival rate of pre-Chinese Medicines intervention was significantly increased（P<0.05）.2.3The influence of KCl injured cells on LDH activity. Compared to blank control group,LDH activity in supernatant of model group of cells was significantly increased （P<0.01）, inpre-intervention model group, TCM intervention group and the western medicine interventiongroup cell LDH activity in supernatant was significantly increased （P<0.05）; Compared to themodel group, the TCM intervention group and the western medicine intervention group, LDHactivity in supernatant was decreased significantly （P<0.05）; Compared to thepre-intervention model group, pre-intervention in TCM group of cells LDH activity insupernatant was decreased significantly （P<0.05）.3. The influence of liuwei dihuang pill contained serum on Intracellular free calciumconcentration of PC12cellsThe changes of extracellular calcium influx fluorescence intensity were observed by the laserconfocal microscope, and the statistical results showed that under quiescent condition, themean intracellular calciumion fluorescence intensity pre-intervention of TCM wassignificantly lowered （P<0.05） than the control group, model group, TCM intervention group, western medicine intervention group and pre-intervention model group. There was nosignificant difference among the other groups. Under dynamic condition, compared to thecontrol group, there were significantly increased in model group， TCM intervention group，western medicine intervention group，pre-intervention model group（P<0.01）; Compared to themodel group, there was significantly decreased in TCM intervention group（P<0.05）, thewestern medicine intervention group（P<0.01）; Compared to the pre-intervention model group,pre-intervention in TCM group of the mean intracellular calciumion fluorescence intensitywas decreased significantly（P<0.01）;Compared with TCM intervention group and the westernmedicine intervention group, pre-intervention TCM group was decreased significantly（P<0.01）; Compared with quiescent condition, there was a significant increased （P<0.01） inmodel group, intervention group，western medicine intervention group，and pre-interventionmodel group, while pre-intervention TCM group has significant increased （P<0.05） underdynamic condition.4. The influence of liuwei dihuang pill contained serum on the signaling pathway of CaMK II,CREB, BDNF in each group4.1Variations in the cells gene expression of CaMK II CREB, the BDNF in each groupStatistical results showed：Compared with the control group, model group, TCM interventiongroup, western medicine intervention group, pre-intervention model group, pre-interventionTCM group expression of CaMK II mRNA had significantly decreased （P<0.01）,comparedwith the model group, the expression of CaMK II mRNA in TCM intervention group andwestern medicine intervention group, had significantly increased （P<0.01）, compared with thepre-intervention model group, the expression of CaMK II mRNA in pre-intervention TCMgroup had significantly increased （P<0.01）; Compared with the control group, model group,TCM intervention group, the expression of CREB mRNA had significantly decreased（P<0.01）, and expression of CREB mRNA in western medicine intervention group hadsignificantly decreased （P<0.05）, compared with the model group, expression of CREBmRNA in TCM intervention group had significantly increased （P<0.05）, and expression ofCREB mRNA in western medicine intervention group, pre-intervention TCM group hadsignificantly increased （P<0.01）, compared with the pre-intervention model group, expressionof CREB mRNA in pre-intervention TCM group had significantly increased （P<0.01）, compared with the TCM intervention group, expression of CREB mRNA in pre-interventionTCM group had significantly increased （P<0.05）; Compared with the control group,expression of BDNF mRNA in model group and pre-intervention model group hadsignificantly decreased （P<0.01）, and expression of BDNF mRNA in TCM intervention group,western medicine intervention group had significant decreased （P<0.05）, compared with themodel group, the expression of BDNF mRNA in TCM intervention group had significantlyincreased （P<0.05）, and expression of BDNF mRNA in western medicine intervention group,pre-intervention in TCM had significant increased （P<0.01）, compared to pre-interventionmodel group， expression of BDNF mRNA in pre-intervention TCM group had significantlyincreased （P<0.01）, and expression of BDNF mRNA in pre-intervention TCM group hadsignificantly increased compared with the TCM intervention group （P<0.05）, and the westernmedicine intervention group （P<0.01）.4.2Variation of protein expression of CaMK II， CREB and of BDNF in each group cells4.2.1statistical results of SABC immunohistochemical assayThe statistical results showed：Compared with the control group, protein expression of CaMKII in model group, intervention group, western medicine intervention group, pre-interventionmodel group had significantly decreased （P<0.01）, compared with the model group, proteinexpression of CaMK II in TCM intervention group, western medicine intervention group hadsignificantly increased （P<0.01）, compared with the pre-intervention model group, proteinexpression of CaMK II in pre-intervention TCM group had significantly increased （P<0.01）,compared with the TCM intervention group, western medicine intervention group, proteinexpression of CaMKII in pre-intervention TCM group had significantly increased （P<0.01）;Compared with the control group, protein expression of CREB in the model group, TCMintervention group, western medicine intervention group, pre-intervention model group,pre-intervention TCM group had significantly decreased （P<0.01）, compared with the modelgroup, protein expression of CREB in the TCM intervention group, western medicineintervention group had significant increased （P<0.01）, compared with the pre-interventionmodel group, protein expression of CREB in pre-intervention TCM group had significantlyincreased （P<0.01）, compared to TCM intervention group, protein expression of CREB in thepre-intervention TCM group had significantly increased （P<0.01）; Compared with the control group， protein expression of BDNF in model group， TCM intervention group， westernmedicine intervention group, and pre-intervention group had significant decreased （P<0.01）,compared with the model group, protein expression of BDNF in TCM intervention group,western medicine intervention group had significantly increased（P<0.01）, compared withpre-intervention model group, protein expression of BDNF in pre-intervention TCM grouphad significantly increased （P<0.01）,compared with the TCM intervention group and westernmedicine intervention group, protein expression of CREB in pre-intervention TCM group hadsignificant increased （P<0.05）.4.2.2Statistical results of western blot assayThe statistical results showed: compared with the control group, protein expression of CaMKII in model group, intervention group, western medicine intervention group, pre-interventionmodel group had significantly decreased （P<0.01）, compared with the model group, proteinexpression of CaMKII in TCM intervention group, western medicine intervention group hadsignificantly increased（P<0.01）, compared with the pre-intervention model group，proteinexpression of CaMKII in pre-intervention TCM group had significantly increased （P<0.01）,compared with the TCM intervention group, the western medicine intervention group, proteinexpression of CaMKII had significantly increased in pre-intervention TCM group （P<0.01）;compared with the control group, protein expression of CREB in model group andpre-intervention model group had significantly decreased （P<0.01）, and TCM interventiongroup， western medicine intervention group （P<0.05）, compared with the model group，protein expression of CREB in TCM intervention group, western medicine intervention group,pre-intervention TCM group had significantly increased （P<0.01）, compared with thepre-intervention model group, protein expression of CREB in pre-intervention TCM grouphad significantly increased （P<0.01）, compared with the TCM intervention group and westernmedicine intervention group, protein expression of CREB in pre-intervention TCM group hadsignificantly increased （P<0.05）; Compared with the control group, protein expression ofBDNF in the model group, TCM intervention group, western medicine intervention group,pre-intervention model group had significantly decreased （P<0.01）, compared with the modelgroup, protein expression of BDNF in TCM intervention group, western medicineintervention group had significantly increased, compared with the pre-intervention model group， protein expression of BDNF in pre-intervention TCM group had significantlyincreased （P<0.01）, compared with the TCM intervention group and western medicineintervention group, protein expression of BDNF in pre-intervention TCM group had asignificantly increased （P<0.01）.Conclusion：1. By different KCl concentrations and time points on PC12cells, with the increase of KClconcentration and lasting times, cell viability was gradually weakened. The KCl injured cellsshowed the dose–timeliness dependence. The final selection of150mmol/L KCl as theconcentration of cell injuries was used to simulate neural cell injury.2. Liu Wei Di Huang pill-contained serum could improve the vitality of PC12cells.3. Liu Wei Di Huang pill-contained serum could significantly reduce the influx ofextracellular calcium, maintain and regulate intracellular homeostasis of Ca2+4. Liu Wei Di Huang pill contained serum could significantly improve gene and proteinexpression of CaMKII, CREB, BDNF. |
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