六味地黄丸对KCl诱导PC12细胞钙超载损伤的保护作用

【作者】 王俊；

【导师】 柳春；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2013， 博士

【摘要】 目的：本实验通过不同浓度的KCl不同时间点作用于PC12细胞，观察细胞活力的变化，选取KCl诱导PC12细胞损伤模拟神经细胞钙超载的损伤；通过对PC12细胞形态及细胞存活率的观察，探讨六味地黄丸含药血清对PC12细胞的保护作用；通过对细胞内钙荧光强度变化的观察，观察六味地黄丸含药血清对细胞内钙离子浓度的影响；通过对CaMKII、CREB、BDNF mRNA和蛋白水平表达的测定，探讨六味地黄丸含药血清对Ca²⁺-CaMKII-CREB-BDNF信号通路的影响。为六味地黄丸用于防治神经细胞内钙超载提供科学的实验数据与理论基础，初步探讨六味地黄丸对神经细胞保护作用的分子机制。材料与方法：按照随机数字法将雄性SD大鼠分为2组，即正常组、六味地黄丸组。六味地黄丸组，将生药浓度为0.75kg/L药液，按10mL/kg灌胃，每天2次，正常组给予同体积双蒸水灌胃，连续给药4天，第5天首次给药1.5h后腹主动脉取血，静置2h，2000r/min离心10min后分离血清。经56℃，30min灭活，0.22μm滤膜过滤灭菌，-20℃保存备用。取培养瓶中处于对数生长期的PC12细胞，经0.25%胰蛋白酶于37℃消化，加入含10%胎牛血清的DMEM完全培养液，细胞密度1×105/mL，37℃，5%CO2下继续培养12h后，细胞贴壁生长，换用无血清的DMEM培养液继续培养12h后用于实验。配置浓度为50、100、150、200mmol/L的KCl，分别损伤10、20、30min后采用MTT法检测PC12细胞的活性，依据时间短、损伤小但钙超载模型成功的原则选取KCl的浓度。将PC12细胞分为六组：（1）空白对照组：10%空白血清；（2）模型组：10%空白血清+150mmol/L KCl；（3）中药干预组：10%六味血清+150mmol/L KCl；（4）西药干预组：10%空白血清+尼莫地平10umol/L+150mmol/L KCl；（5）预干预模型组：10%空白血清（预干预48h）+150mmol/L KCl；（6）预干预中药组：10%六味血清（预干预48h）+150mmol/L KCl。（2）、（3）和（4）组是含有胎牛血清的DMEM中培养48小时后，PBS洗2遍后药物血清与KCl同时加入，作用20min后用PBS洗2遍后加入对应血清及药物继续培养6h；（5）和（6）组是预干预48h后加KCl，作用20min后用PBS洗2遍后加入无血清DMEM继续培养6h。倒置显微镜下观察各组细胞形态，MTT法测定细胞存活率，细胞上清液中LDH活性测定。各组细胞经Fluo-3的负载后激光共聚焦显微镜检测细胞内钙离子平均荧光强度。各组细胞内总RNA的提取和RT-PCR法检测CaMKII、CREB、BDNF的mRNA表达。各组细胞爬片经固定后，SABC法检测细胞CaMKII、CREB、BDNF的蛋白表达。收集细胞，提取各组细胞总蛋白，western blot法检测CaMKII、CREB、BDNF的蛋白表达。结果：1KCl诱导细胞损伤模型建立统计结果显示：与对照组比较，150mmol/L、200mmol/L的KCl作用20min后细胞活力明显性降低（P<0.05），100mmol/L、150mmol/L、200mmol/L的KCl作用30min细胞活力明显性降低（P<0.05）。2六味地黄丸含药血清对KCl诱导PC12细胞损伤的影响2.1形态学观察倒置显微镜下，空白对照组细胞呈梭形，胞间接触紧密，并伸出伪足，伪足有交错，增殖旺盛，折光度强；模型组、预干预模型组细胞伪足大部分消失，折光度减弱，部分细胞皱缩、变圆、脱落，漂浮于培养基中；中药干预组和西药干预组，细胞肿胀，少数折光度减弱，少数细胞脱落；预干预中药组细胞折光度强，细胞增殖旺盛。2.2KCl损伤后对各组细胞活力（MTT）的影响与空白对照组比较，模型组细胞存活率显著降低（P<0.01），预干预模型组、中药干预组和西药干预组细胞存活率明显降低（P<0.05）；与模型组相比，中药干预组和西药干预组细胞存活率明显提高（P<0.05）；与预干预模型组相比，预干预中药组细胞存活率显著提高（P<0.01）。2.3KCl损伤对各组细胞上清液中LDH活力的影响与空白对照组比较，模型组细胞上清液中LDH活力显著提高（P<0.01），预干预模型组、中药干预组和西药干预组细胞上清液中LDH活力明显提高（P<0.05）；与模型组相比，中药干预组和西药干预组细上清液中LDH活力明显降低（P<0.05）；与预干预模型组相比，预干预中药组细胞上清液中LDH活力明显降低（P<0.05）。3六味地黄丸含药血清对细胞内游离钙浓度的影响激光共聚焦观察细胞内钙荧光强度的变化，统计结果显示：静息时，预干预中药组细胞内钙离子平均荧光强度明显低于空白对照组、模型组、中药干预组、西药干预组、预干预模型组（P<0.05），其余各组间无显著性差异；动态时，与空白对照组相比，模型组、中药干预组、西药干预组、预干预模型组细胞内钙荧光强度显著性增高（P<0.01），与模型组相比，中药干预组细胞内钙荧光强度明显降低（P<0.05），西药干预组细胞内钙荧光强度显著性降低（P<0.01），与预干预模型组相比，预干预中药组细胞内钙荧光强度显著性降低（P<0.01），与中药干预组相比，预干预中药组细胞内钙荧光强度显著性降低（P<0.01）；与静息时比较，动态时模型组、中药干预组、西药干预组、预干预模型组细胞内钙荧光强度显著性增高（P<0.01），动态时预干预中药组胞内钙荧光强度明显增高（P<0.05）。4六味地黄丸含药血清对信号通路中CaMKII、CREB、BDNF的影响4.1各组细胞CaMKII、CREB、BDNF mRNA表达的变化统计结果显示：与空白对照组相比，模型组、中药干预组、西药干预组、预干预模型组、预干预中药组的CaMKII mRNA表达显著性降低（P<0.01），与模型组相比，中药干预组、西药干预组的CaMKII mRNA表达显著性增高（P<0.01），预干预模型组相比，预干预中药组的CaMKII mRNA表达显著性增高（P<0.01）；与空白对照组相比，模型组、中药干预组CREB mRNA表达显著性降低（P<0.01），西药干预组CREB mRNA表达明显降低（P<0.05），与模型组相比，中药干预组CREB mRNA表达明显增高（P<0.05），西药干预组CREB mRNA表达显著增高（P<0.01），与预干预模型组相比，预干预中药组CREB mRNA表达显著增高（P<0.01），与中药干预组相比，预干预中药组CREB mRNA表达明显增高（P<0.05）；与空白对照组相比，模型组、预干预模型组的BDNF mRNA表达显著性降低（P<0.01），中药干预组、西药干预组的BDNF mRNA表达明显降低（P<0.05），与模型组相比，中药干预组BDNF mRNA表达明显增高（P<0.05），西药干预组BDNF mRNA表达显著增高（P<0.01），与预干预模型组相比，预干预中药组BDNF mRNA表达显著增高（P<0.01），与中药干预组相比，预干预中药组BDNF mRNA表达显著增高（P<0.01），与西药干预组相比，预干预中药组BDNFmRNA表达明显增高（P<0.05）。4.2各组细胞CaMKII、CREB、BDNF蛋白表达的变化4.2.1SABC免疫组化法检测统计结果统计结果显示：与空白对照组相比，模型组、中药干预组、西药干预组、预干预模型组CaMKII蛋白表达显著降低（P<0.01），与模型组相比，中药干预组、西药干预组CaMKII蛋白表达显著增高（P<0.01），预干预模型组相比，预干预中药组的CaMKII蛋白表达显著增高（P<0.01），与中药干预组相比，预干预中药组的CaMKII蛋白表达显著增高（P<0.01），与西药干预组相比，预干预中药组的CaMKII蛋白表达显著增高（P<0.01）；与空白对照组相比，模型组、中药干预组、西药干预组、预干预模型组、预干预中药组的CREB蛋白表达显著降低（P<0.01），与模型组相比，中药干预组、西药干预组的CREB蛋白表达显著增高（P<0.01），与预干预模型组相比，预干预中药组的CREB蛋白表达显著增高（P<0.01），与中药干预组相比，预干预中药组的CREB蛋白表达显著增高（P<0.01）；与空白对照组相比，模型组、中药干预组、西药干预组、预干预模型组BDNF蛋白表达显著降低（P<0.01），与模型组相比，中药干预组、西药干预组的BDNF蛋白表达显著增高（P<0.01），预干预模型组相比，预干预中药组的BDNF蛋白表达显著增高（P<0.01），与中药干预组相比，预干预中药组的BDNF蛋白表达明显增高（P<0.05），与西药干预组相比，预干预中药组的BDNF蛋白表达明显增高（P<0.05）。4.2.2western blot法检测统计结果统计结果显示：与空白对照组相比，模型组、中药干预组、西药干预组、预干预模型组CaMKII蛋白表达显著降低（P<0.01），与模型组相比，中药干预组、西药干预组的CaMKII蛋白表达显著增高（P<0.01），预干预模型组相比，预干预中药组的CaMKII蛋白表达显著增高（P<0.01），与中药干预组相比，预干预中药组的CaMKII蛋白表达显著增高（P<0.01），与西药干预组相比，预干预中药组的CaMKII蛋白表达显著增高（P<0.01）；与空白对照组相比，模型组、预干预模型组CREB蛋白表达显著降低（P<0.01），中药干预组、西药干预组CREB蛋白表达明显降低（P<0.05），与模型组相比，中药干预组、西药干预组的CREB蛋白表达显著增高（P<0.01），与预干预模型组相比，预干预中药组的CREB蛋白表达显著增高（P<0.01），与中药干预组、西药干预组相比，预干预中药组的CREB蛋白表达明显增高（P<0.05）；与空白对照组相比，模型组、中药干预组、西药干预组、预干预模型组BDNF蛋白表达显著降低（P<0.01），与模型组相比，中药干预组、西药干预组的BDNF蛋白表达显著增高（P<0.01），与预干预模型组相比，预干预中药组的BDNF蛋白表达显著增高（P<0.01），与中药干预组、西药干预组相比，预干预中药组的BDNF蛋白表达显著增高（P<0.01）。结论：1不同浓度的KCl不同时间点作用于PC12细胞后，随着KCl的浓度加大及作用时间延长，细胞活力逐渐减弱，即KCl对细胞的损伤程度呈剂量-时间依赖性。最终选取150mmol/LKCl作为细胞损伤浓度，模拟神经细胞钙超载损伤状态；2六味地黄丸含药血清可提高PC12细胞的活力；3六味地黄丸含药血清可明显降低细胞内钙离子浓度，维持和调节细胞内钙稳态；4六味地黄丸含药血清可显著提高信号通路中CaMKII、CREB、BDNF的mRNA和蛋白表达。更多还原

【Abstract】 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.更多还原