复方脑康胶囊治疗Alzheimer's病的药效学研究
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摘要
目的：复方脑康胶囊(Compound Naokang Capsule, CNKC)是治疗Alzheimer's病的中药复方制剂。研究CNKC提取物对PC12细胞增殖活性及对NaCN加缺糖造成缺血性样损伤细胞的影响；观察CNKC水提液对Aβ25-35诱导的SH-SY5Y细胞毒性的保护作用；以D-半乳糖老化小鼠为动物模型,研究CNKC水提液对海马神经元的凋亡级联反应的影响,探讨其作用机理,为CNKC的药效学研究和临床应用提供依据。
方法：(1) CNKC提取液对PC12细胞的促进增殖和对缺血样损伤的保护作用。①以PC12细胞为试验模型,试验组加入CNKC提取液适量,用DMEM液配制使浓度分别为0.05mg/mL,0.20mg/mL,0.80mg/mL及2.40mg/mL;对照组加等量DMEM液,观察细胞形态,MTT比色法于540nm处测定吸光度,考察细胞增殖率。②往PC12细胞中每孔加入无糖Earle's液0.5m1和NaCN溶液适量(使NaCN终浓度为20mmol/L),培养15min,制作NaCN加缺糖造成缺血样损伤PC12细胞模型；药物处理组在细胞培养液(无糖Earle's溶液和DMEM培养基)中添加CNKC水提液,使药物终浓度分别为0.05mg/mL、0.20mg/mL、0.80mg/mL及2.40mg/mL,作用4hr,吸去液体,每孔加入无糖Earle's液0.5mL,作用30min；加入NaCN液适量(使NaCN终浓度为20mmol/L),继续培养15min,吸去含NaCN培养液,用D-Hank's溶液洗两次,每孔加入DMEM液0.5mL,作用24h。观察细胞形态,MTT比色法于540nm处测定吸光度(A),同时检测细胞上清液中LDH。计算CNKC水提液对NaCN加缺糖引起PC12细胞损伤的抑制率。
(2) CNKC含药血清对PC12细胞促进增殖和对缺血样损伤的保护作用。取昆明种小鼠112只,称重,随机分为14组,即对照组(8只)、尼莫地平组(8只)、1g/kg CNKC药物组(每个取血点为一组,每组8只,共96只)。对照组给予等体积的生理盐水,尼莫地平组给予16mg/kg的尼莫地平,每日两次连续6天,末次给药后1h摘眼球取血。药物组按给药方案不同分为三个组,分别为：(i)单次给药；(ii)间隔4h给药一次,共给药二次；(iii)前二次间隔4h给药一次,以后每日两次连续3天,共给药8次；各给药方案组于末次给药后0.5h、1h、2h、3h摘眼球取血,分离血清。以NaCN加缺糖造成缺血性样损伤PC12细胞为模型,考察不同给药次数、采血时间的含药血清对模型细胞的影响；在培养液中添加CNKC含药血清、尼莫地平含药血清、空白对照组血清各适量,使血清浓度分别为1%、2%、4%、8%、16%,用于培养PC12模型细胞,MTT法于540nm处测定吸光度(A),计算CNKC药液组PC12细胞的抑制率,考察不同浓度含药血清的药效。
(3) CNKC对β-淀粉样肽诱导SH-SY5Y细胞凋亡的保护作用。①培养SH-SY5Y细胞,加入不同浓度的CNKC提取液,MTT法测定吸光度(A),确定最适试验药物浓度。将SH-SY5Y细胞分为对照组、25μmol/L Aβ25-35组和0.725g/L CNKC水提液+AP25-3525μmol/L组,加药24h后,于倒置显微镜下观察细胞形态。于440nm处测定处的细胞上清液中LDH值；MTT法于波长540nm处测定吸光度(A),分别计算细胞的存活率。②Real-Time PCR检测AIF、 Cyt C、Bax. Bcl-2mRNA含量。将细胞分为对照组、25μmol/LAβ25-35组和0.725g/L CNKC加25μmol/LAβ325-35组。加药后24h,收集细胞,提取细胞总RNA,进行mRNA含量检测。mRNA含量检测步骤为待检测细胞样品的收集、细胞样品Total RNA的抽提、用逆转录酶进行逆转录合成cDNA、Real-time PCR扩增和反应等。以beta actin作为对照,通过扩增曲线与阀值线的交点来计算Ct值,通过Ct值进行数据分析。相对定量的结果通过△Ct法来进行解析,目的基因相对于内参基因的表达量为2ΔCt=2Ctm-Ctn。③Western Blotting检测AIF、Cyt C、 Bax、Bcl-2蛋白含量。将细胞分为对照组、25μmol/L Aβ25-35组和CNKC0.725g/L加25μmol/L Aβ25-35组。加药后培养24h,收集细胞,提取细胞总蛋白,进行蛋白含量检测。④双染法检测细胞凋亡。将细胞分为对照组、25μmol/L Aβ25-35组和CNKC0.725g/L加25μmol/L Aβ25-35组。加药后培养24h,收集细胞。用流式细胞仪检测,激发波长Ex=488nm, Annexin V-FITC的绿色荧光通过FITC通道(FL1)检测,PI红色荧光通过PI通道(FL2)检测。
(4) CNKC水提液对D-半乳糖老化小鼠海马神经元凋亡级联反应的影响。①将筛选合格的健康正常昆明种小鼠100只,随机分为正常对照组(E组),D-半乳糖模型组(D组),CNKC大剂量药物组(A组)、中剂量药物组(B组)、小剂量药物组(C组),每组各20只。A、B、C、D组小鼠每日皮下注射半乳糖60mg/kg, A、B、C3个药物组分别用CNKC水提取液灌胃,给药剂量分别相当于生药量0.65g/kg,0.35g/kg和0.15g/kg,E组每日给动物注射并灌注等量的生理盐水,连续3个月,制作AD模型。对上述各组小鼠进行学习训练实验、定位航行实验。②病理学观察。将小鼠断头处死,取左侧海马经过固定、脱水、石蜡包埋、切片、脱蜡、水化、染色和封片后,光镜下观察海马组织形态结构并计数200倍下海马CA2-CA3区的神经元数量。HE常规染色,光镜观察脑组织形态学的变化。③免疫组织化学染色。取各组试验小鼠的脑组织,经固定处理后,取中段进行石蜡切片,进行免疫组织化学染色。光镜下观察细胞形态并对阳性细胞计数。④取小鼠断头取脑,剥离双侧海马,称重后使用液氮速冻保存,用Real-Time PCR检测AIF、Cyt C、Bax、Bcl-2的mRNA含量。⑤Western Blotting检测AIF、CytC、Bax、Bcl-2蛋白含量。小鼠断头取脑,剥离双侧海马,称重后加入组织裂解液进行组织匀浆,离心,取上清分装,使用BCA法进行总蛋白定量。
(5)数据均以x±s表示,所有数据用SPSS13.0统计分析软件处理。CNKC水提取液对PC12细胞增殖的影响、小鼠学习训练中游泳距离成绩和到达平台时间,采用重复测量数据的方差分析；组间多重比较采用LSD法检验。不同浓度的药物血清对PC12细胞缺血性损伤的影响,采用析因设计资料的方差分析进行统计学分析；组间多重比较采用LSD法检验。其余测量数据均采用单因素方差分析(One-Way ANOVA),如果有显著性差异,则组间两两比较采用LSD法(方差齐性时)或Dunnett T3法(方差不齐时)。显著性水准取α=0.05(双侧),P<0.05为差异有统计学意义。
结果：(1)①细胞形态结果：倒置显微镜下观察,对照组PC12细胞在接种后24h能迅速贴壁生长,大多呈梭形,少数呈三角形,48h时细胞处于对数生长期,贴壁密度约为60%,72h时细胞生长停滞,有的孔内有细胞悬浮。在PC12细胞加入不同浓度的CNKC水提液后,细胞形态无变化,无损伤表现,长势良好,48h时细胞生长旺盛,贴壁密度约为70%(药物终浓度2.40mg/mL组),72h细胞生长速度减缓。将MTT法检测结果进行重复测量数据的方差分析,结果显示,与各自对照组比较,浓度为0.05mg/mL组在作用24小时、48小时、72小时后,吸光度(A)均为无显著差异(F=20.043, P=0.476; F=28.729, P=0.957; F=15.977, P=0.597),未显示促进细胞增殖作用；浓度为0.20mg/mL、0.80mg/mL、2.40mg/mL组在作用24小时、48小时、72小时后,吸光度(A)均有显著差异(P均小于0.05),显示了促进细胞增殖作用。由此可见,CNKC水提取液能不同程度地促进PC12细胞的增殖能力,并随着药物浓度的增加,细胞增殖率也增加。②模型组细胞的突起结构消失,细胞肿胀变圆,折光度下降,部分细胞裂解成碎片。CNKC水提液预处理后能对抗NaCN加缺糖引起的细胞形态学改变,表现为细胞突起结构的消失减少,细胞碎片的形成减少。MTT测定结果显示,与细胞模型组(A为1.52±0.15)相比,正常对照组吸光度(A)为1.98±0.02(Welch F=561.380, P=0.001),存在显著差异,表明模型组细胞活力下降。与模型组相比,浓度为0.05mg/mL浓度CNKC组的吸光度(A)无统计学意义(P=0.976),浓度为0.20mg/mL (P=0.007)、0.80mg/mL (P=0.001)、2.40mg/mL (P=0.000) CNKC组的吸光度(A)均高于模型组,细胞损伤抑制率分别为13.0%、60.9%、80.4%、93.6%,表明CNKC提取液可以改善细胞活力。LDH测定结果显示,与模型组(LDH值为657.2±47.2)相比,正常对照组LDH值为270.8±16.0(Welch F=273.717, P=0.000),低于模型组。与模型组相比,浓度为0.05mg/mL CNKC组的LDH值(633.9±35.8)无显著差异(P=0.978)；而浓度为0.20mg/mL、0.80mg/mL、2.40mg/mL的LDH值均有显著差异(P均小于0.05),细胞损伤抑制率分别为6.0%、28.6%、66.8%、91.8%。PC12细胞损伤后释放至培养基中的LDH增多,模型组LDH值高于对照组,CNKC组可降低细胞LDH的释放,显示出对细胞损伤的保护作用。
(2)①不同给药次数、采血时间的含药血清对PC12细胞缺血性损伤的影响。与对照组比较,模型组的吸光度(A)有显著差异(F=142.627,P=0.000)。与模型组(吸光度(A)为1.43±0.12)比较,单次给药后0.5h.1h、2h.3h的含药血清组的吸光度(A)分别为1.37±0.09、1.41±0.09、1.45±0.10、1.45±0.07(P=0.356,P=0.745,P=0.599,P=0.670),基本未呈现细胞保护作用。二次给药后,取血时间1hr (P=0.000)、2hr (P=0.000)、3hr (P=0.000)的含药血清组,测得的吸光度(A)均有显著差异,高于模型组,显示了细胞保护作用。八次给药后0.5h.1h、2h、3h含药血清组的吸光度(A)均有显著差异(P=0.000, P=0.000, P=0.000, P=0.000),高于模型组,显示了细胞保护作用。②采用析因设计资料的方差分析方法,分析不同浓度的药物血清对PC12细胞缺血性损伤的影响。结果表明,与模型组(A=1.41±0.08)相比,空白对照组的吸光度(A)有显著差异(P=0.000),说明NaCN加缺糖可诱导PC12细胞损伤。与模型组比较,2%CNKC含药血清组测得的吸光度(A)有显著差异(F=106.953, P=0.000),4%CNKC组(F=81.629,P=0.000)和尼莫地平组(P=0.000)的吸光度(A)均有统计学意义,对缺血样损伤的PC12细胞均表现出保护作用。
(3)①SH-SH5Y细胞形态结果显示,Aβ25-35作用72h后,模型组的细胞折光度下降,突触收缩,细胞变圆,聚集,轴突长度和胞体面积小于正常对照组；而CNKC水提液与Aβ25-35共同作用72h后的细胞与模型组比较,细胞形态改善,表现为细胞突触结构消失减少,折光度增强,且细胞分布均匀。MTT法测定结果显示,与对照组比较,Aβ25-35组的A值有显著差异(F=147.104,P=0.000),低于对照组。而CNKC+Aβ25-35组的A值与Aβ25-35组也有显著差异(P=0.000),高于Aβ25-35组。结果显示,Aβ25-35可降低SH-SY5Y细胞的活性,而用CNKC水提液处理培养基,则可减轻Aβ25-35对细胞造成的损伤。LDH漏出率试验结果显示,与对照组比较,模型组(Aβ25-35组)LDH漏出率有显著差异(F=181.790,P=0.000),高于对照组,说明Aβ25-35可至SH-SY5Y细胞损伤；而CNKC+Aβ25-35组与Aβ25-35组比较有显著差异(P=0.007),低于Aβ25-35组,说明CNKC水提液对SHβSY5Y细胞具有很好的保护作用。②mRNA含量检测结果。A260nm/A280nm比值在1.8～2.0之间,同时结合RNA电泳结果,28S和18S RNA二条带清晰可见,说明RNA纯度较高,没有明显降解。对照组、模型组和药物组样品的RNA浓度分别为1140ng/μl、1044ng/μl、1020ng/μl。Real-time PCR结果显示,对照组、模型组和药物组的AIF/actin mRNA相对含量分别为0.0102±0.0027.0.1023±0.0138.0.0252±0.0043；Cyt C/actin分别为0.0256±0.0065.0.0788±0.0062.0.0331±0.0013;Bax/actin分别为0.0203±0.0012、0.1707±0.0096.0.0380±0.0010;Bcl-2/actin分别为0.0409±0.0052、0.0090±0.0015、0.0252±0.0013。与对照组比较,模型组AIF.Cyt C.Bax的mRNA含量均上升(F=60.846,P=0.013；F=89.670,P=0.000；F=420.101,P=0.002),Bcl-2mRNA含量则降低(F=104.859,P=0.012)；Drug+Aβ25-35组与模型组比较,AIF.Cyt C. Bax的mRNA含量均有所降低(P=0.014；P=0.000；P=0.002),而Bcl-2的mRNA则有所升高(P=0.000)。说明CNKC对Aβ25-35诱导的SH-SY5Y细胞损伤可以起到保护作用。③蛋白浓度检测结果显示,对照组、模型组和药物组蛋白浓度分别为16.170μg/μl、16.146μg/μl.16.335μg/μl.通过灰度值分析,对照组、模型组和药物组的AIF含量为0.0145±0.0003、0.0502±0.0003、0.0261±0.0010,Bax含量为0.0252±0.0003、0.0871±0.0008、0.0519±0.0011, Cyt C含量为0.0240+0.0008、0.1045±0.0006、0.0653±0.0007,Bcl-2含量为0.0910±0.0057、0.0144±0.0003、0.0697±0.0010。与对照组相比,模型组的AIF/beta actin、Bax/beta actin、Bcl-2/beta actin、Cyt C/beta actin均有显著差异(Welch F=20950.876, P=0.000; Welch F=13197.318, P=0.000; F=14872.548, P=0.000; Welch F=6704.384; P=0.000), AIF、Bax、Cyt C含量上升,而Bcl-2的含量下降；与模型组比较,Drug+Aβ25-35组的AIF/beta actin、Bax/beta actin、Bcl-2/beta actin、 Cyt C/beta actin均有显著差异(P均为0.000),AIF、Bax、Cyt C含量下降,而Bcl-2的含量上升。这说明CNKC对Aβ25-35诱导的SH-SY5Y细胞损伤可以起到保护作用。④细胞凋亡检测结果显示,对照组、模型组和药物组的细胞凋亡率分别为3.71±1.74、17.12±3.52和7.20±1.15。与对照组相比,模型组的细胞凋亡率有显著差异(F=26.038,P=0.000),细胞凋亡率上升；与模型组比较,Drug+Aβ25-35组的细胞凋亡率有显著差异(P=0.002),凋亡率上升的趋势得到抑制,细胞凋亡率下降。这说明CNKC对Aβ25-35诱导的SH-SY5Y细胞损伤可以起到保护作用。
(4)①将小鼠学习训练成绩进行重复测量数据的方差分析,结果显示,与AD模型组比,在第一象限(NE) CNKC水提液大、中、小剂量组到达安全平台的时间和游泳距离均无显著差异(P均大于0.05),但在第二、三、四象限,到达安全平台的时间和游泳距离均少于AD模型组(P均小于0.05)。实验结果提示,CNKC水提液有促进AD小鼠学习的效应。②HE染色可见,正常对照组海马结构的病理切片显示该处神经元无明显损伤,细胞层次正常、结构清楚。神经元细胞形态完整,排列有序,细胞核体积较大,核仁清晰可见。AD模型组海马结构在光镜下可见组织结构完整,但细胞层次减少,有些神经元形态不完整,排列稀疏、无序,细胞核体积变小,深染,结构不清,神经纤维排列紊乱。CNKC水提液大、中、小剂量组海马结构的病理切片显示该处神经元有轻微损伤,细胞层次尚可、结构较清楚。神经元细胞形态完整,排列有序,细胞核体积较大,核仁清晰可见。③mRNA含量检测结果。RNA的A260nm/A280nm比值在1.8～2.0之间,同时结合RNA电泳结果,28S和18S RNA二条带清晰可见,说明RNA纯度较高,没有明显降解。正常组、AD模型组、药物大剂量组、药物中剂量组、药物小剂量组样品的RNA浓度分别为1248ng/μl、1328ng/μl、1156ng/μl、1220ng/μl、1304ng/μl。Real-time PCR结果显示,正常组、AD模型组、药物大剂量组、药物中剂量组、药物小剂量组的AIF/actin mRNA相对含量分别为0.0036±0.0007、0.0092±0.0008、0.0045±0.0005.0.0051±0.0004、0.0075±0.0012；Cyt C/actin分别为00030±0.0005、0.0128±0.0023、0.0059±0.0015、0.0085±0.0022、0.0102±0.0008;Bax/actin分别为0.0028±0.0003、0.0127±0.0024、0.0050±0.0009、0.0060±0.0014、0.0094±0.0014; Bcl-2/actin分别为0.0281±0.0012、0.0027±0.0002、0.0152±0.0021、0.0103±0.0014、0.0072±0.0015。与对照组比较,模型组AIF、Cyt C、Bax基因mRNA含量均上升,Bcl-2mRNA含量则降低(P值均小于0.05)；药物大、中、小剂量组与模型组比较,AIF、Cyt C、Bax基因mRNA含量均有所降低,而Bcl-2基因mRNA则有所升高(均P值<0.05)。这说明CNKC水提液对D-半乳糖老化小鼠凋亡级联反应有保护作用。④蛋白浓度检测结果显示,正常组、AD模型组、药物大剂量组、药物中剂量组、药物小剂量组蛋白浓度分别为17.995μg/μl、18.082μg/μl、18.388μg/μl、17.901μg/μl、18.239μg/μl。通过灰度值分析,正常组、AD模型组、药物大剂量组、药物中剂量组、药物小剂量组的AIF蛋白含量为0.2978±0.0195、0.7464±0.0389、0.3312±0.0297、0.6133±0.0426、0.5559±0.0393, Bax蛋白含量为01989±0.0120、0.7294±0.0512、0.2617±0.0144、0.3309±0.0235、0.4755±0.0326,Cyt C蛋白含量为0.2292±0.0133、0.8553±0.0501、0.4185±0.0300、0.4928±0.0381、0.5802±0.0394,Bcl-2蛋白含量为0.5673±0.0294、0.1994±0.0115、0.4456±0.0318、0.4304±0.0313、0.1987±0.0119。与对照组相比,模型组的AIF/beta actin、Bax/beta actin、Bcl-2/beta actin、Cyt C/beta actin均有显著差异(F=148.570, P=0.000；Welch F=173.945,P=0.000；F=213.287,P=0.000；F=200.678,P=0.000),AIF、Bax、Cyt C含量上升,而Bcl-2的含量下降；与模型组比较,药物大、中、小剂量组的AIF/beta actin、Bax/beta actin、Bcl-2/beta actin、Cyt C/beta actin均有显著差异(均P=0.000), AIF、Bax、Cyt C含量下降,而Bcl-2的含量上升。⑤Cyt C免疫组织化学染色结果显示,正常对照组的阳性细胞在海马广泛分布,细胞数量与模型组存在有显著差异。模型小鼠海马神经元阳性细胞数比正常对照小鼠增加3.7倍。Bcl-2阳性反应细胞可见,正常对照组着色浅,少有突起,细胞数目少；模型组在皮质与海马可见胞浆及突起,突起分支达三四级,胞浆内有棕黄色阳性反应颗粒,胞核不着色,阳性细胞为梭形或星形。CNKC水提液均不同程度使这二种蛋白质表达的异常有所恢复。
结论：(1) CNKC具有促进PC12细胞增殖的作用,并对NaCN加缺糖造成的缺血性样损伤细胞具有保护作用。(2) CNKC含药血清能减轻NaCN加缺糖造成的缺血性样PC12模型细胞的损伤,使存活细胞数量增加。(3) CNKC对AB25-35诱导的SH-SY5Y细胞损伤可以起到保护作用。(4) CNKC水提液对D-半乳糖老化小鼠凋亡级联反应有保护作用。本实验结果可为CNKC治疗AD提供一定的实验基础和理论参考。
Objective:To study the effects of improving proliferation and ischemia-like injury protection of CNKC on PC12cells, neuroprotective effects of CNKC on SH-SY5Y cell apoptosis induced by Aβ peptide, and the effect of CNKC liquid extract on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose. Moreover, to illustrate elementarily pharmacological mechanism which can provide scientific basis for further development of CNKC.
Methods:(1) The effects of improving proliferation and ischemia-like injury protection of CNKC on PC12cells.①PC12cells were used as experimental models.0.05mg/mL,0.20mg/mL,0.80mg/mL and2.40mg/mL CNKC extracts were prepared with DMEM and added to the PC12cells as experimental group. Equal volume of DMEM were added to PC12cells as control group. Cell morphous were observed and A value was assayed by MTT at540nm to investigate cell proliferation.②Model group:Ischemia-like injury cell model was induced by Earle's solution and NaCN solution(final concentration was20mmol/L). Drug group:0.05mg/mL,0.20mg/mL,0.80mg/mL and2.40mg/mL CNKC extracts were prepared with DMEM and added to the PC12cells, then cultivated for4hours. The liquid was blotted and NaCN solution(final concentration was20mmol/L) was added to the cells, cultivated for15mins. Suck the culture solution, wash with D-Hank's solution twice, add0.5ml DMEM solution and cultivate for24hours. Cell morphous were observed, A value was assayed by MTT at540nm, and LDH in supernatant was assayed. Inhibition ratio of PC12cells was calculated.
(2) The effects of improving proliferation and ischemia-like injury protection of serum contain CNKC on PC12cells.112Kunming mice were randomly divided into14groups:control group, Nimodipine group and CNKC groups(96mice,8mice for every group).Equal volume of solvent was given to control group. And16mg/kg of Nimodipine was given to Nimodipine group, bid for6days. Blood samples were collected one hour after the last dose. CNKC group were divided into three groups:(i) single dose,(ii) the second dose after4hours,(iii)bid for3days, eight dose totally. Blood samples were collected0.5h,lh,2h,3h after last dose, and separated serum. Ischemia-like injury PC12cells induced by NaCN were as experimental model cells. Effect of Serum containing CNKC exacts collected in different administration times and different blood collection time on PC12model cells were investigated.1%,2%,4%,8%,16%serum containing CNKC, Nimodipine, blank blood serum were added to culture media. PC12model cells were cultured by using these culture media. A values were tested by MTT assay. Inhibition ratio of PC12cells was calculated.
(3) Neuroprotective effects of CNKC on SH-SY5Y cell apoptosis induced by Aβ peptide.①SH-SY5Y cell was cultivated. Different concentration of CNKC exacts were added to the cells to optimize the drug concentration for experiment by MTT assay. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol/L) and drug group(0.725g/L CNKC exacts+AP25-3525μmol/L). After cultivated for24hrs, cellular morphous was observed by inverted microscope. LDH was assayed at440nm. A values were determined at540nm by MTT assay and cellular survival rate was calculated.②mRNA levels of AIF,Cyt C,Bax,Bcl-2assayed by Real Time-PCR. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol·L-1) and drug group(0.725g/L CNKC extracts+Aβ25-3525μmol/L). After cultivated for24hrs, the cells were collected and cellular total RNA was extracted to assay the mRNA levels. The steps of mRNA assay were sample collecting, total RNA extracting, cDNA synthesis by reverse transcription and real time PCR amplification. Beta actin was as reference, and Ct was calculated by the intersection point of amplification curve and valve curve. Relative quantitation was analysed by ΔCt. The expression of destination gene was2ΔCt=2Ctm-Ctn.③AIF, Cyt C, Bax, and Bcl-2protein levels were assayed by Western Blotting. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol/L) and drug group(0.725g/L CNKC exacts+Aβ25-3525μmol/L). After cultivated for24hr, the cells were collected and cellular total protein was extracted to assay the protein levels.④Apoptosis assay. SH-SY5Y cells were divided into three groups:control group, model group(A(325-3525μmol/L) and drug group(0.725g/L CNKC extracts+Aβ25-3525μmol/L). After cultivated for24hr, the cells were collected and assayed by flow cytometry. Excitation wave Ex=488nm, green fluorescence of Annexin V-FITC were detected by FITC(FLl) path and red fluorescence(FL2) was detected by PI (FL2) path.
(4) Effect of CNKC extracts on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose.0100valid healthy mice were randomly divided into five groups:normal control group(E), D-galactose model group(D), high dose of CNKC group(A), middle dose of CNKC group(B), and low dose of CNKC group(C).20mice were given to each group. D-galactose was given to the mice of A,B,C,D groups, subcutaneous injection,60mg/kg per day.0.65g/kg, 0.35g/kg and0.15g/kg of CNKC extracts were intragastric administrated to the mice of A,B,C groups respectively. Equal dose of sodium chloride was given to the mice of E group. Continuous rear for three months to obtain AD model. Every group performed the place navigation test and spatial probe test.②Pathology survey. The mice were executed by decapitation. The left hippocampus was taken and carried out the procedure of fixation, dehydration, paraffin imbedding, slice, deparaffinage, hydration, and staining. Morphous of hippocampus tissue was observed under light microscope and neuron amount at CA2-CA3zone of hippocampus was counted. Morphological changes of brain tissue were surveyed after HE staining.③Immunohistochemistry staining. Brain tissue of experimental mice were taken and fixed, midpiece was performed paraffin section and immunohistochemistry staining. Cellular morphous was surveyed and masculine cells were counted under light microscope.④The mice were executed by decapitation. Hippocampus on both sides were separated, weighted and conserved in liquid nitrogen. mRNA levels of AIF, Cyt C, Bax, and Bcl-2were tested by Real Time-PCR.⑤Protein levels of AIF, Cyt C, Bax, and Bcl-2tested by Western Blotting. The mice were executed by decapitation. Hippocampus on both sides were separated and weighted. Lysate was added to tissue homogenate and centrifuge. Total protein quantitation of supernatant was tested by BCA method.
(5)SPSS13.0statisties software was used to analyze the data.The measurement data indicated by the mean value and standard deviation. The results comparison of proliferation effect on PC12cells, mice learning train scores were by repeated-measure variance analysis and multiple comparisons with LSD. The result of PC12cells ischemic injury effected by different concentrations of drug serum was by factorial design univariate and LSD. All of other data were analyzed by One-Way ANOVA and multiple comparisons with LSD(Homogeneity of variance) or Dunnett T3(Heterogeneity of variance). Significance level was a=0.05(bilateral), and P<0.05expressed the difference with statistics significance.
Result:(1)①Cellular Morphous:Observed with inverted microscope. Control group:After cultured for24hrs, the cells grew rapidly. Most of them were fusiform and minority were triangle. After cultured for48hrs, the cells were in logarithmic phase. After cultured for72hrs, the cells grew retardation and there were some floating cells in some holes. Drug group:cellular morphous had no changes and no injury. After cultured for48hrs, cells grew well. After cultured for72hrs, the cells grew slowly. The results of MTT showed that, compared with control group, Absoption values of0.05mg/ml group at24hr,48hr24hr had no significant difference and no cellular proliferation (F=20.043, P=0.476; F=28.729, P=0.957; F=15.977,P=0.597). A values of0.20mg/mL,0.80mg/mL,2.40mg/mL groups at24hr,48hr24hr had significant difference and cellular proliferation (P<0.05).All of them could improve proliferation.②In model group, the cellular apophysis disappeared, cellular swelled, diopter descended and some cells were splited to fragment. Pretreated by CNKC extracts, the morphology of ischemia-like injury cells could change obviously. Fewer cellular apophysis disappeared and cell debris could be observed. The results of MTT showed that, compared with model group(1.52±0.15), A value of normal control group was1.98±0.02(Welch F=561.380, P=0.001) and cellular vigor of model group declined significantly. A values of0.05mg/ml group had no statistical significance (P=0.976). A values of0.20mg/mL (P=0.007),0.80mg/mL (P=0.001),2.40mg/mL (P=0.000) groups were higher than those of model group. Cell injury inhibition ratio were13.0%,60.9%,80.4%, and93.5%. And it showed that CNKC extracts could improve cellular vigor obviously. The results of LDH assay showed that, compared with model group(657.2±47.2), LDH value of normal control group was270.8±16.0(Welch F=273.717, P=0.000) and lower than the value of model group. LDH values of0.05mg/ml group had no statistical significance (P=0.978). LDH values of0.20mg/ml,0.80mg/ml, and2.40mg/ml groups were lower than those of model group. The cell injury inhibition ratio were6.0%,28.6%,66.8%, and91.8%. LDH would increased if cell injuried. LDH of model group was higher than that of normal control group. CNKC extracts could lower the release of LDH and improve cellular damage obviously.
(2)①Effect of serum containing CNKC extracts collected in different administration times and different blood collection time on PC12cells was investigated. Compared with model group(A value was1.43±0.12), control group was significantly different (F=142.627, P=0.000).A values of single dose serum containing CNKC extracts collected0.5hr,1hr,2hrs,3hrs were1.37±0.09,1.41±0.09,1.45±0.10,1.45±0.07(P=1.000), and showed no cytoprotection. A values of twice doses serum containing CNKC extracts collected1hr,2hrs were1.77±0.13, and1.78±0.12(P<0.05), much higher than those of model group and showed obvious cytoprotection. A values of eight doses serum containing CNKC extracts collected0.5hr,1hr,2hrs,3hrs were1.86±0.06,1.90±0.08,1.810±0.09, and1.79±0.11. All of them showed obvious cytoprotection, especially for0.5hr and lhr(P<0.01).②Effect of Serum containing different concentration of CNKC extracts on PC12cells was investigated. Compared with model group(A value was1.41±0.08), A values of serum containing2%CNKC extracts was1.75±0.09and showed cytoprotection. Both serum containing4%CNKC extracts and Nimodipine showed cytoprotection.
(3) The result of cellular morphous showed that after effected by Aβ25-35for72hrs, cellular diopter of the model group dropped, cell rounded and gathered, axon length and cell body were shorter than that of normal control group. However, compared with model group, cellular morphous of drug group was greatly improved. Their cellular synapse and diopter increased and the cells were well-distributed. The results of MTT assay showed that A value of model group(0.590±0.006) was much lower than that of control group(0.631±0.005)(F=147.104, P=0.000), and A value of drug group(0.622±0.004) was higher than that of model group(P=0.000). Cytoactive of SH-SY5Y could be degraded by Aβ25-35, and CNKC extracts could lessen cellular injury. The results of LDH assay showed that LDH of model group(1345.7±98.7) was higher than that of control group(789.1±63.5)(F=181.790, P=0.000), and LDH of drug group(675.7±56.6) was lower than that of model group (P=0.007). This result also showed that Cytoactive of SH-SY5Y could be degraded by Aβ25-35, and CNKC extracts could lessen cellular injury.②Result of mRNA levels. A260nm/A280nm of RNA was between1.8and2.0. Electrophoresis results showed that the straps of28S and18S RNA were very clear. All these illustrated that RNA was with high purity and had no degradation. RNA levels of control group, model group and drug group were1140ng/μl,1044ng/μl, and1020ng/μl respectively. According to the result of real-time PCR, AIF/actin mRNA relative amount of control group, model group and drug group were0.0102+0.0027,0.1023±0.0138,0.0252±0.0043; Cyt C/actin were0.0256±0.0065,0.0788±0.0062,0.0331±0.0013; Bax/actin were0.0203±0.0012,0.1707±0.0096,0.0380±0.0010; Bcl-2/actin were0.0409±0.0052,0.0090±0.0015,0.0252±0.0013。 Compared with control group, AIF,Cyt C,Bax mRNA amounts of model group were increased (F=60.846, P=0.013; F=89.670, P=0.000; F=420.101, P=0.002),and Bcl-2mRNA amount was reduced (F=104.859, P=0.012). Compared with model group, AIF,Cyt C,Bax mRNA amounts of drug group were reduced (P=0.014; P=0.000; P=0.002), and Bcl-2mRNA amount was increased (P=0.000).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by AP25-35.③ Protein concentration of control group, model group and drug group were16.170μg/μl,16.146μg/μl, and16.335μg/μl, respectively. According to the grey values, AIF levels of control group, model group and drug group were AIF levels were0.0145±0.0003,0.0502±0.0003,0.0261±0.0010, Bax levels were0.0252±0.0003,0.0871±0.0008,0.0519±0.0011, Cyt C levels were0.0240±0.0008,0.1045±0.0006,0.0653±0.0007, Bcl-2levels were0.0910±0.0057,0.0144±0.0003,0.0697±0.0010. Compared with control group, AIF/beta actin,Bax/beta actin.Cyt C/beta actin of model group were increased (Welch F=20950.876, P=0.000; Welch F=13197.318, P=0.000; Welch F=6704.384, P=0.000),and Bcl-2/beta actin was reduced(F=14872.548, P=0.000). Compared with model group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of drug group were reduced (P=0.000; P=0.000; P=0.000), and Bcl-2/beta actin was increased(P=0.000).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by AP25-35-.④Apoptosis result showed apoptosis rate of control group, model group and drug group were3.71±1.74,17.12±3.52and7.20±1.15. Compared with control group, apoptosis rate of model group were increased (F=26.038, P=0.000). Compared with model group, apoptosis rate of drug group were reduced (P=0.002).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by Aβ25-35.(4)①ompared with model group, in the first quadrant(NE), there were no differences of the time and distance to safe platform of group C,D,E(P>0.05). However, from the second quadrant, the data was fewer than that of model group(P<0.05). The result showed that CNKC extracts could improve the skill of learning of AD mice.②The result of HE staining showed that there was no obvious impairment in neurons of hippocampal formation (E group), and the cells were normal with clear structure. Morphous of neuron cells were integrity, arranged orderly, with large nucleus and clear nucleoli. Hippocampal formation of AD model group had integrated tissue. However, less cellular layer, incompleted neuron morphous, raritas and disordered cellular arrangement, smaller nucleus, anachromasis, obscure structure and derangement nerve fiber could be seen. Result of pathological section (group A,B,C) showed that there was light impairment in hippocampal formation, and the cellular layer was acceptable with clear structure.③Result of mRNA levels. A260nm/A280nm of RNA was between1.8and2.0. Electrophoresis results showed that the straps of28S and18S RNA were very clear. All these illustrated that RNA was with high purity and had no degradation. RNA levels of group E,D,A,B,C were1248ng/μl,1328ng/μl,1156ng/μl,1220ng/μl, and1304ng/μl. According to the result of real-time PCR, AIF/actin mRNA relative amount of group E,D,A,B,C were0.0036±0.0007,0.0092±0.0008,0.0045±0.0005,0.0051±0.0004,0.0075±0.0012; Cyt C/actin were0.0030±0.0005,0.0128±0.0023,0.0059±0.0015,0.0085±0.0022,0.0102±0.0008; Bax/actin were0.0028±0.0003,0.0127±0.0024,0.0050±0.0009,0.0060±0.0014,0.0094±0.0014; Bcl-2/actin were0.0281±0.0012,0.0027±0.0002,0.0152±0.0021,0.0103±0.0014,0.0072±0.0015. Compared with control group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of model group were increased (P<0.05),and Bcl-2/beta actin was reduced (P<0.05). Compared with model group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of drug groups were reduced (P<0.05), and Bcl-2/beta actin was increased(P<0.05).The result showed that CNKC exacts could protect apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose.④Protein concentration of group E,D,A,B,C were17.995μg/μl,18.082μg/μl,18.388μg/μl,17.901μg/μl,18.239μg/μl, respectively. According to the analysis of grey value, AIF levels of group E,D,A,B,C were0.2978±0.0195,0.7464±0.0389,0.3312±0.0297,0.6133±0.0426,0.5559±0.0393, Bax were01989±0.0120,0.7294±0.0512,0.2617±0.0144,0.3309±0.0235, 0.4755±0.0326, Cyt C were0.2292±0.0133,0.8553±0.0501,0.4185±0.0300,0.4928±0.0381,0.5802±0.0394, Bcl-2were0.5673±0.0294,0.1994±0.0115,0.4456±0.0318,0.4304±0.0313,0.1987±0.0119.⑤Results of immunohistochemistry of Cyt C showed that masculine cells of group E were widespread distributed in hippocampus and cell population was significant deviation from group D. Masculine cell population of model group was3.7times, than that of normal control group. Abnormality of these proteins express could be recovered by CNKC extracts in some degree.
Conclusions:(1)CNKC extracts could improve proliferation and protection on ischemia-like injury PC12cells induced by NaCN.(2) Serum containing CNKC extracts could improve proliferation and protection on ischemia-like injury PC12cells induced by NaCN and increase survival cells.(3) CNKC extracts had neuroprotective effects on SH-SY5Y cell apoptosis induced by Aβ25-35.(4) CNKC extracts had the effect on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose. In addition, the pharmacological mechanism of CNKC could be illustrated elementarily so as to provide scientific basis for further AD clinical treat.
方法：(1) CNKC提取液对PC12细胞的促进增殖和对缺血样损伤的保护作用。①以PC12细胞为试验模型,试验组加入CNKC提取液适量,用DMEM液配制使浓度分别为0.05mg/mL,0.20mg/mL,0.80mg/mL及2.40mg/mL;对照组加等量DMEM液,观察细胞形态,MTT比色法于540nm处测定吸光度,考察细胞增殖率。②往PC12细胞中每孔加入无糖Earle's液0.5m1和NaCN溶液适量(使NaCN终浓度为20mmol/L),培养15min,制作NaCN加缺糖造成缺血样损伤PC12细胞模型；药物处理组在细胞培养液(无糖Earle's溶液和DMEM培养基)中添加CNKC水提液,使药物终浓度分别为0.05mg/mL、0.20mg/mL、0.80mg/mL及2.40mg/mL,作用4hr,吸去液体,每孔加入无糖Earle's液0.5mL,作用30min；加入NaCN液适量(使NaCN终浓度为20mmol/L),继续培养15min,吸去含NaCN培养液,用D-Hank's溶液洗两次,每孔加入DMEM液0.5mL,作用24h。观察细胞形态,MTT比色法于540nm处测定吸光度(A),同时检测细胞上清液中LDH。计算CNKC水提液对NaCN加缺糖引起PC12细胞损伤的抑制率。
(2) CNKC含药血清对PC12细胞促进增殖和对缺血样损伤的保护作用。取昆明种小鼠112只,称重,随机分为14组,即对照组(8只)、尼莫地平组(8只)、1g/kg CNKC药物组(每个取血点为一组,每组8只,共96只)。对照组给予等体积的生理盐水,尼莫地平组给予16mg/kg的尼莫地平,每日两次连续6天,末次给药后1h摘眼球取血。药物组按给药方案不同分为三个组,分别为：(i)单次给药；(ii)间隔4h给药一次,共给药二次；(iii)前二次间隔4h给药一次,以后每日两次连续3天,共给药8次；各给药方案组于末次给药后0.5h、1h、2h、3h摘眼球取血,分离血清。以NaCN加缺糖造成缺血性样损伤PC12细胞为模型,考察不同给药次数、采血时间的含药血清对模型细胞的影响；在培养液中添加CNKC含药血清、尼莫地平含药血清、空白对照组血清各适量,使血清浓度分别为1%、2%、4%、8%、16%,用于培养PC12模型细胞,MTT法于540nm处测定吸光度(A),计算CNKC药液组PC12细胞的抑制率,考察不同浓度含药血清的药效。
(3) CNKC对β-淀粉样肽诱导SH-SY5Y细胞凋亡的保护作用。①培养SH-SY5Y细胞,加入不同浓度的CNKC提取液,MTT法测定吸光度(A),确定最适试验药物浓度。将SH-SY5Y细胞分为对照组、25μmol/L Aβ25-35组和0.725g/L CNKC水提液+AP25-3525μmol/L组,加药24h后,于倒置显微镜下观察细胞形态。于440nm处测定处的细胞上清液中LDH值；MTT法于波长540nm处测定吸光度(A),分别计算细胞的存活率。②Real-Time PCR检测AIF、 Cyt C、Bax. Bcl-2mRNA含量。将细胞分为对照组、25μmol/LAβ25-35组和0.725g/L CNKC加25μmol/LAβ325-35组。加药后24h,收集细胞,提取细胞总RNA,进行mRNA含量检测。mRNA含量检测步骤为待检测细胞样品的收集、细胞样品Total RNA的抽提、用逆转录酶进行逆转录合成cDNA、Real-time PCR扩增和反应等。以beta actin作为对照,通过扩增曲线与阀值线的交点来计算Ct值,通过Ct值进行数据分析。相对定量的结果通过△Ct法来进行解析,目的基因相对于内参基因的表达量为2ΔCt=2Ctm-Ctn。③Western Blotting检测AIF、Cyt C、 Bax、Bcl-2蛋白含量。将细胞分为对照组、25μmol/L Aβ25-35组和CNKC0.725g/L加25μmol/L Aβ25-35组。加药后培养24h,收集细胞,提取细胞总蛋白,进行蛋白含量检测。④双染法检测细胞凋亡。将细胞分为对照组、25μmol/L Aβ25-35组和CNKC0.725g/L加25μmol/L Aβ25-35组。加药后培养24h,收集细胞。用流式细胞仪检测,激发波长Ex=488nm, Annexin V-FITC的绿色荧光通过FITC通道(FL1)检测,PI红色荧光通过PI通道(FL2)检测。
(4) CNKC水提液对D-半乳糖老化小鼠海马神经元凋亡级联反应的影响。①将筛选合格的健康正常昆明种小鼠100只,随机分为正常对照组(E组),D-半乳糖模型组(D组),CNKC大剂量药物组(A组)、中剂量药物组(B组)、小剂量药物组(C组),每组各20只。A、B、C、D组小鼠每日皮下注射半乳糖60mg/kg, A、B、C3个药物组分别用CNKC水提取液灌胃,给药剂量分别相当于生药量0.65g/kg,0.35g/kg和0.15g/kg,E组每日给动物注射并灌注等量的生理盐水,连续3个月,制作AD模型。对上述各组小鼠进行学习训练实验、定位航行实验。②病理学观察。将小鼠断头处死,取左侧海马经过固定、脱水、石蜡包埋、切片、脱蜡、水化、染色和封片后,光镜下观察海马组织形态结构并计数200倍下海马CA2-CA3区的神经元数量。HE常规染色,光镜观察脑组织形态学的变化。③免疫组织化学染色。取各组试验小鼠的脑组织,经固定处理后,取中段进行石蜡切片,进行免疫组织化学染色。光镜下观察细胞形态并对阳性细胞计数。④取小鼠断头取脑,剥离双侧海马,称重后使用液氮速冻保存,用Real-Time PCR检测AIF、Cyt C、Bax、Bcl-2的mRNA含量。⑤Western Blotting检测AIF、CytC、Bax、Bcl-2蛋白含量。小鼠断头取脑,剥离双侧海马,称重后加入组织裂解液进行组织匀浆,离心,取上清分装,使用BCA法进行总蛋白定量。
(5)数据均以x±s表示,所有数据用SPSS13.0统计分析软件处理。CNKC水提取液对PC12细胞增殖的影响、小鼠学习训练中游泳距离成绩和到达平台时间,采用重复测量数据的方差分析；组间多重比较采用LSD法检验。不同浓度的药物血清对PC12细胞缺血性损伤的影响,采用析因设计资料的方差分析进行统计学分析；组间多重比较采用LSD法检验。其余测量数据均采用单因素方差分析(One-Way ANOVA),如果有显著性差异,则组间两两比较采用LSD法(方差齐性时)或Dunnett T3法(方差不齐时)。显著性水准取α=0.05(双侧),P<0.05为差异有统计学意义。
结果：(1)①细胞形态结果：倒置显微镜下观察,对照组PC12细胞在接种后24h能迅速贴壁生长,大多呈梭形,少数呈三角形,48h时细胞处于对数生长期,贴壁密度约为60%,72h时细胞生长停滞,有的孔内有细胞悬浮。在PC12细胞加入不同浓度的CNKC水提液后,细胞形态无变化,无损伤表现,长势良好,48h时细胞生长旺盛,贴壁密度约为70%(药物终浓度2.40mg/mL组),72h细胞生长速度减缓。将MTT法检测结果进行重复测量数据的方差分析,结果显示,与各自对照组比较,浓度为0.05mg/mL组在作用24小时、48小时、72小时后,吸光度(A)均为无显著差异(F=20.043, P=0.476; F=28.729, P=0.957; F=15.977, P=0.597),未显示促进细胞增殖作用；浓度为0.20mg/mL、0.80mg/mL、2.40mg/mL组在作用24小时、48小时、72小时后,吸光度(A)均有显著差异(P均小于0.05),显示了促进细胞增殖作用。由此可见,CNKC水提取液能不同程度地促进PC12细胞的增殖能力,并随着药物浓度的增加,细胞增殖率也增加。②模型组细胞的突起结构消失,细胞肿胀变圆,折光度下降,部分细胞裂解成碎片。CNKC水提液预处理后能对抗NaCN加缺糖引起的细胞形态学改变,表现为细胞突起结构的消失减少,细胞碎片的形成减少。MTT测定结果显示,与细胞模型组(A为1.52±0.15)相比,正常对照组吸光度(A)为1.98±0.02(Welch F=561.380, P=0.001),存在显著差异,表明模型组细胞活力下降。与模型组相比,浓度为0.05mg/mL浓度CNKC组的吸光度(A)无统计学意义(P=0.976),浓度为0.20mg/mL (P=0.007)、0.80mg/mL (P=0.001)、2.40mg/mL (P=0.000) CNKC组的吸光度(A)均高于模型组,细胞损伤抑制率分别为13.0%、60.9%、80.4%、93.6%,表明CNKC提取液可以改善细胞活力。LDH测定结果显示,与模型组(LDH值为657.2±47.2)相比,正常对照组LDH值为270.8±16.0(Welch F=273.717, P=0.000),低于模型组。与模型组相比,浓度为0.05mg/mL CNKC组的LDH值(633.9±35.8)无显著差异(P=0.978)；而浓度为0.20mg/mL、0.80mg/mL、2.40mg/mL的LDH值均有显著差异(P均小于0.05),细胞损伤抑制率分别为6.0%、28.6%、66.8%、91.8%。PC12细胞损伤后释放至培养基中的LDH增多,模型组LDH值高于对照组,CNKC组可降低细胞LDH的释放,显示出对细胞损伤的保护作用。
(2)①不同给药次数、采血时间的含药血清对PC12细胞缺血性损伤的影响。与对照组比较,模型组的吸光度(A)有显著差异(F=142.627,P=0.000)。与模型组(吸光度(A)为1.43±0.12)比较,单次给药后0.5h.1h、2h.3h的含药血清组的吸光度(A)分别为1.37±0.09、1.41±0.09、1.45±0.10、1.45±0.07(P=0.356,P=0.745,P=0.599,P=0.670),基本未呈现细胞保护作用。二次给药后,取血时间1hr (P=0.000)、2hr (P=0.000)、3hr (P=0.000)的含药血清组,测得的吸光度(A)均有显著差异,高于模型组,显示了细胞保护作用。八次给药后0.5h.1h、2h、3h含药血清组的吸光度(A)均有显著差异(P=0.000, P=0.000, P=0.000, P=0.000),高于模型组,显示了细胞保护作用。②采用析因设计资料的方差分析方法,分析不同浓度的药物血清对PC12细胞缺血性损伤的影响。结果表明,与模型组(A=1.41±0.08)相比,空白对照组的吸光度(A)有显著差异(P=0.000),说明NaCN加缺糖可诱导PC12细胞损伤。与模型组比较,2%CNKC含药血清组测得的吸光度(A)有显著差异(F=106.953, P=0.000),4%CNKC组(F=81.629,P=0.000)和尼莫地平组(P=0.000)的吸光度(A)均有统计学意义,对缺血样损伤的PC12细胞均表现出保护作用。
(3)①SH-SH5Y细胞形态结果显示,Aβ25-35作用72h后,模型组的细胞折光度下降,突触收缩,细胞变圆,聚集,轴突长度和胞体面积小于正常对照组；而CNKC水提液与Aβ25-35共同作用72h后的细胞与模型组比较,细胞形态改善,表现为细胞突触结构消失减少,折光度增强,且细胞分布均匀。MTT法测定结果显示,与对照组比较,Aβ25-35组的A值有显著差异(F=147.104,P=0.000),低于对照组。而CNKC+Aβ25-35组的A值与Aβ25-35组也有显著差异(P=0.000),高于Aβ25-35组。结果显示,Aβ25-35可降低SH-SY5Y细胞的活性,而用CNKC水提液处理培养基,则可减轻Aβ25-35对细胞造成的损伤。LDH漏出率试验结果显示,与对照组比较,模型组(Aβ25-35组)LDH漏出率有显著差异(F=181.790,P=0.000),高于对照组,说明Aβ25-35可至SH-SY5Y细胞损伤；而CNKC+Aβ25-35组与Aβ25-35组比较有显著差异(P=0.007),低于Aβ25-35组,说明CNKC水提液对SHβSY5Y细胞具有很好的保护作用。②mRNA含量检测结果。A260nm/A280nm比值在1.8～2.0之间,同时结合RNA电泳结果,28S和18S RNA二条带清晰可见,说明RNA纯度较高,没有明显降解。对照组、模型组和药物组样品的RNA浓度分别为1140ng/μl、1044ng/μl、1020ng/μl。Real-time PCR结果显示,对照组、模型组和药物组的AIF/actin mRNA相对含量分别为0.0102±0.0027.0.1023±0.0138.0.0252±0.0043；Cyt C/actin分别为0.0256±0.0065.0.0788±0.0062.0.0331±0.0013;Bax/actin分别为0.0203±0.0012、0.1707±0.0096.0.0380±0.0010;Bcl-2/actin分别为0.0409±0.0052、0.0090±0.0015、0.0252±0.0013。与对照组比较,模型组AIF.Cyt C.Bax的mRNA含量均上升(F=60.846,P=0.013；F=89.670,P=0.000；F=420.101,P=0.002),Bcl-2mRNA含量则降低(F=104.859,P=0.012)；Drug+Aβ25-35组与模型组比较,AIF.Cyt C. Bax的mRNA含量均有所降低(P=0.014；P=0.000；P=0.002),而Bcl-2的mRNA则有所升高(P=0.000)。说明CNKC对Aβ25-35诱导的SH-SY5Y细胞损伤可以起到保护作用。③蛋白浓度检测结果显示,对照组、模型组和药物组蛋白浓度分别为16.170μg/μl、16.146μg/μl.16.335μg/μl.通过灰度值分析,对照组、模型组和药物组的AIF含量为0.0145±0.0003、0.0502±0.0003、0.0261±0.0010,Bax含量为0.0252±0.0003、0.0871±0.0008、0.0519±0.0011, Cyt C含量为0.0240+0.0008、0.1045±0.0006、0.0653±0.0007,Bcl-2含量为0.0910±0.0057、0.0144±0.0003、0.0697±0.0010。与对照组相比,模型组的AIF/beta actin、Bax/beta actin、Bcl-2/beta actin、Cyt C/beta actin均有显著差异(Welch F=20950.876, P=0.000; Welch F=13197.318, P=0.000; F=14872.548, P=0.000; Welch F=6704.384; P=0.000), AIF、Bax、Cyt C含量上升,而Bcl-2的含量下降；与模型组比较,Drug+Aβ25-35组的AIF/beta actin、Bax/beta actin、Bcl-2/beta actin、 Cyt C/beta actin均有显著差异(P均为0.000),AIF、Bax、Cyt C含量下降,而Bcl-2的含量上升。这说明CNKC对Aβ25-35诱导的SH-SY5Y细胞损伤可以起到保护作用。④细胞凋亡检测结果显示,对照组、模型组和药物组的细胞凋亡率分别为3.71±1.74、17.12±3.52和7.20±1.15。与对照组相比,模型组的细胞凋亡率有显著差异(F=26.038,P=0.000),细胞凋亡率上升；与模型组比较,Drug+Aβ25-35组的细胞凋亡率有显著差异(P=0.002),凋亡率上升的趋势得到抑制,细胞凋亡率下降。这说明CNKC对Aβ25-35诱导的SH-SY5Y细胞损伤可以起到保护作用。
(4)①将小鼠学习训练成绩进行重复测量数据的方差分析,结果显示,与AD模型组比,在第一象限(NE) CNKC水提液大、中、小剂量组到达安全平台的时间和游泳距离均无显著差异(P均大于0.05),但在第二、三、四象限,到达安全平台的时间和游泳距离均少于AD模型组(P均小于0.05)。实验结果提示,CNKC水提液有促进AD小鼠学习的效应。②HE染色可见,正常对照组海马结构的病理切片显示该处神经元无明显损伤,细胞层次正常、结构清楚。神经元细胞形态完整,排列有序,细胞核体积较大,核仁清晰可见。AD模型组海马结构在光镜下可见组织结构完整,但细胞层次减少,有些神经元形态不完整,排列稀疏、无序,细胞核体积变小,深染,结构不清,神经纤维排列紊乱。CNKC水提液大、中、小剂量组海马结构的病理切片显示该处神经元有轻微损伤,细胞层次尚可、结构较清楚。神经元细胞形态完整,排列有序,细胞核体积较大,核仁清晰可见。③mRNA含量检测结果。RNA的A260nm/A280nm比值在1.8～2.0之间,同时结合RNA电泳结果,28S和18S RNA二条带清晰可见,说明RNA纯度较高,没有明显降解。正常组、AD模型组、药物大剂量组、药物中剂量组、药物小剂量组样品的RNA浓度分别为1248ng/μl、1328ng/μl、1156ng/μl、1220ng/μl、1304ng/μl。Real-time PCR结果显示,正常组、AD模型组、药物大剂量组、药物中剂量组、药物小剂量组的AIF/actin mRNA相对含量分别为0.0036±0.0007、0.0092±0.0008、0.0045±0.0005.0.0051±0.0004、0.0075±0.0012；Cyt C/actin分别为00030±0.0005、0.0128±0.0023、0.0059±0.0015、0.0085±0.0022、0.0102±0.0008;Bax/actin分别为0.0028±0.0003、0.0127±0.0024、0.0050±0.0009、0.0060±0.0014、0.0094±0.0014; Bcl-2/actin分别为0.0281±0.0012、0.0027±0.0002、0.0152±0.0021、0.0103±0.0014、0.0072±0.0015。与对照组比较,模型组AIF、Cyt C、Bax基因mRNA含量均上升,Bcl-2mRNA含量则降低(P值均小于0.05)；药物大、中、小剂量组与模型组比较,AIF、Cyt C、Bax基因mRNA含量均有所降低,而Bcl-2基因mRNA则有所升高(均P值<0.05)。这说明CNKC水提液对D-半乳糖老化小鼠凋亡级联反应有保护作用。④蛋白浓度检测结果显示,正常组、AD模型组、药物大剂量组、药物中剂量组、药物小剂量组蛋白浓度分别为17.995μg/μl、18.082μg/μl、18.388μg/μl、17.901μg/μl、18.239μg/μl。通过灰度值分析,正常组、AD模型组、药物大剂量组、药物中剂量组、药物小剂量组的AIF蛋白含量为0.2978±0.0195、0.7464±0.0389、0.3312±0.0297、0.6133±0.0426、0.5559±0.0393, Bax蛋白含量为01989±0.0120、0.7294±0.0512、0.2617±0.0144、0.3309±0.0235、0.4755±0.0326,Cyt C蛋白含量为0.2292±0.0133、0.8553±0.0501、0.4185±0.0300、0.4928±0.0381、0.5802±0.0394,Bcl-2蛋白含量为0.5673±0.0294、0.1994±0.0115、0.4456±0.0318、0.4304±0.0313、0.1987±0.0119。与对照组相比,模型组的AIF/beta actin、Bax/beta actin、Bcl-2/beta actin、Cyt C/beta actin均有显著差异(F=148.570, P=0.000；Welch F=173.945,P=0.000；F=213.287,P=0.000；F=200.678,P=0.000),AIF、Bax、Cyt C含量上升,而Bcl-2的含量下降；与模型组比较,药物大、中、小剂量组的AIF/beta actin、Bax/beta actin、Bcl-2/beta actin、Cyt C/beta actin均有显著差异(均P=0.000), AIF、Bax、Cyt C含量下降,而Bcl-2的含量上升。⑤Cyt C免疫组织化学染色结果显示,正常对照组的阳性细胞在海马广泛分布,细胞数量与模型组存在有显著差异。模型小鼠海马神经元阳性细胞数比正常对照小鼠增加3.7倍。Bcl-2阳性反应细胞可见,正常对照组着色浅,少有突起,细胞数目少；模型组在皮质与海马可见胞浆及突起,突起分支达三四级,胞浆内有棕黄色阳性反应颗粒,胞核不着色,阳性细胞为梭形或星形。CNKC水提液均不同程度使这二种蛋白质表达的异常有所恢复。
结论：(1) CNKC具有促进PC12细胞增殖的作用,并对NaCN加缺糖造成的缺血性样损伤细胞具有保护作用。(2) CNKC含药血清能减轻NaCN加缺糖造成的缺血性样PC12模型细胞的损伤,使存活细胞数量增加。(3) CNKC对AB25-35诱导的SH-SY5Y细胞损伤可以起到保护作用。(4) CNKC水提液对D-半乳糖老化小鼠凋亡级联反应有保护作用。本实验结果可为CNKC治疗AD提供一定的实验基础和理论参考。
Objective:To study the effects of improving proliferation and ischemia-like injury protection of CNKC on PC12cells, neuroprotective effects of CNKC on SH-SY5Y cell apoptosis induced by Aβ peptide, and the effect of CNKC liquid extract on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose. Moreover, to illustrate elementarily pharmacological mechanism which can provide scientific basis for further development of CNKC.
Methods:(1) The effects of improving proliferation and ischemia-like injury protection of CNKC on PC12cells.①PC12cells were used as experimental models.0.05mg/mL,0.20mg/mL,0.80mg/mL and2.40mg/mL CNKC extracts were prepared with DMEM and added to the PC12cells as experimental group. Equal volume of DMEM were added to PC12cells as control group. Cell morphous were observed and A value was assayed by MTT at540nm to investigate cell proliferation.②Model group:Ischemia-like injury cell model was induced by Earle's solution and NaCN solution(final concentration was20mmol/L). Drug group:0.05mg/mL,0.20mg/mL,0.80mg/mL and2.40mg/mL CNKC extracts were prepared with DMEM and added to the PC12cells, then cultivated for4hours. The liquid was blotted and NaCN solution(final concentration was20mmol/L) was added to the cells, cultivated for15mins. Suck the culture solution, wash with D-Hank's solution twice, add0.5ml DMEM solution and cultivate for24hours. Cell morphous were observed, A value was assayed by MTT at540nm, and LDH in supernatant was assayed. Inhibition ratio of PC12cells was calculated.
(2) The effects of improving proliferation and ischemia-like injury protection of serum contain CNKC on PC12cells.112Kunming mice were randomly divided into14groups:control group, Nimodipine group and CNKC groups(96mice,8mice for every group).Equal volume of solvent was given to control group. And16mg/kg of Nimodipine was given to Nimodipine group, bid for6days. Blood samples were collected one hour after the last dose. CNKC group were divided into three groups:(i) single dose,(ii) the second dose after4hours,(iii)bid for3days, eight dose totally. Blood samples were collected0.5h,lh,2h,3h after last dose, and separated serum. Ischemia-like injury PC12cells induced by NaCN were as experimental model cells. Effect of Serum containing CNKC exacts collected in different administration times and different blood collection time on PC12model cells were investigated.1%,2%,4%,8%,16%serum containing CNKC, Nimodipine, blank blood serum were added to culture media. PC12model cells were cultured by using these culture media. A values were tested by MTT assay. Inhibition ratio of PC12cells was calculated.
(3) Neuroprotective effects of CNKC on SH-SY5Y cell apoptosis induced by Aβ peptide.①SH-SY5Y cell was cultivated. Different concentration of CNKC exacts were added to the cells to optimize the drug concentration for experiment by MTT assay. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol/L) and drug group(0.725g/L CNKC exacts+AP25-3525μmol/L). After cultivated for24hrs, cellular morphous was observed by inverted microscope. LDH was assayed at440nm. A values were determined at540nm by MTT assay and cellular survival rate was calculated.②mRNA levels of AIF,Cyt C,Bax,Bcl-2assayed by Real Time-PCR. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol·L-1) and drug group(0.725g/L CNKC extracts+Aβ25-3525μmol/L). After cultivated for24hrs, the cells were collected and cellular total RNA was extracted to assay the mRNA levels. The steps of mRNA assay were sample collecting, total RNA extracting, cDNA synthesis by reverse transcription and real time PCR amplification. Beta actin was as reference, and Ct was calculated by the intersection point of amplification curve and valve curve. Relative quantitation was analysed by ΔCt. The expression of destination gene was2ΔCt=2Ctm-Ctn.③AIF, Cyt C, Bax, and Bcl-2protein levels were assayed by Western Blotting. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol/L) and drug group(0.725g/L CNKC exacts+Aβ25-3525μmol/L). After cultivated for24hr, the cells were collected and cellular total protein was extracted to assay the protein levels.④Apoptosis assay. SH-SY5Y cells were divided into three groups:control group, model group(A(325-3525μmol/L) and drug group(0.725g/L CNKC extracts+Aβ25-3525μmol/L). After cultivated for24hr, the cells were collected and assayed by flow cytometry. Excitation wave Ex=488nm, green fluorescence of Annexin V-FITC were detected by FITC(FLl) path and red fluorescence(FL2) was detected by PI (FL2) path.
(4) Effect of CNKC extracts on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose.0100valid healthy mice were randomly divided into five groups:normal control group(E), D-galactose model group(D), high dose of CNKC group(A), middle dose of CNKC group(B), and low dose of CNKC group(C).20mice were given to each group. D-galactose was given to the mice of A,B,C,D groups, subcutaneous injection,60mg/kg per day.0.65g/kg, 0.35g/kg and0.15g/kg of CNKC extracts were intragastric administrated to the mice of A,B,C groups respectively. Equal dose of sodium chloride was given to the mice of E group. Continuous rear for three months to obtain AD model. Every group performed the place navigation test and spatial probe test.②Pathology survey. The mice were executed by decapitation. The left hippocampus was taken and carried out the procedure of fixation, dehydration, paraffin imbedding, slice, deparaffinage, hydration, and staining. Morphous of hippocampus tissue was observed under light microscope and neuron amount at CA2-CA3zone of hippocampus was counted. Morphological changes of brain tissue were surveyed after HE staining.③Immunohistochemistry staining. Brain tissue of experimental mice were taken and fixed, midpiece was performed paraffin section and immunohistochemistry staining. Cellular morphous was surveyed and masculine cells were counted under light microscope.④The mice were executed by decapitation. Hippocampus on both sides were separated, weighted and conserved in liquid nitrogen. mRNA levels of AIF, Cyt C, Bax, and Bcl-2were tested by Real Time-PCR.⑤Protein levels of AIF, Cyt C, Bax, and Bcl-2tested by Western Blotting. The mice were executed by decapitation. Hippocampus on both sides were separated and weighted. Lysate was added to tissue homogenate and centrifuge. Total protein quantitation of supernatant was tested by BCA method.
(5)SPSS13.0statisties software was used to analyze the data.The measurement data indicated by the mean value and standard deviation. The results comparison of proliferation effect on PC12cells, mice learning train scores were by repeated-measure variance analysis and multiple comparisons with LSD. The result of PC12cells ischemic injury effected by different concentrations of drug serum was by factorial design univariate and LSD. All of other data were analyzed by One-Way ANOVA and multiple comparisons with LSD(Homogeneity of variance) or Dunnett T3(Heterogeneity of variance). Significance level was a=0.05(bilateral), and P<0.05expressed the difference with statistics significance.
Result:(1)①Cellular Morphous:Observed with inverted microscope. Control group:After cultured for24hrs, the cells grew rapidly. Most of them were fusiform and minority were triangle. After cultured for48hrs, the cells were in logarithmic phase. After cultured for72hrs, the cells grew retardation and there were some floating cells in some holes. Drug group:cellular morphous had no changes and no injury. After cultured for48hrs, cells grew well. After cultured for72hrs, the cells grew slowly. The results of MTT showed that, compared with control group, Absoption values of0.05mg/ml group at24hr,48hr24hr had no significant difference and no cellular proliferation (F=20.043, P=0.476; F=28.729, P=0.957; F=15.977,P=0.597). A values of0.20mg/mL,0.80mg/mL,2.40mg/mL groups at24hr,48hr24hr had significant difference and cellular proliferation (P<0.05).All of them could improve proliferation.②In model group, the cellular apophysis disappeared, cellular swelled, diopter descended and some cells were splited to fragment. Pretreated by CNKC extracts, the morphology of ischemia-like injury cells could change obviously. Fewer cellular apophysis disappeared and cell debris could be observed. The results of MTT showed that, compared with model group(1.52±0.15), A value of normal control group was1.98±0.02(Welch F=561.380, P=0.001) and cellular vigor of model group declined significantly. A values of0.05mg/ml group had no statistical significance (P=0.976). A values of0.20mg/mL (P=0.007),0.80mg/mL (P=0.001),2.40mg/mL (P=0.000) groups were higher than those of model group. Cell injury inhibition ratio were13.0%,60.9%,80.4%, and93.5%. And it showed that CNKC extracts could improve cellular vigor obviously. The results of LDH assay showed that, compared with model group(657.2±47.2), LDH value of normal control group was270.8±16.0(Welch F=273.717, P=0.000) and lower than the value of model group. LDH values of0.05mg/ml group had no statistical significance (P=0.978). LDH values of0.20mg/ml,0.80mg/ml, and2.40mg/ml groups were lower than those of model group. The cell injury inhibition ratio were6.0%,28.6%,66.8%, and91.8%. LDH would increased if cell injuried. LDH of model group was higher than that of normal control group. CNKC extracts could lower the release of LDH and improve cellular damage obviously.
(2)①Effect of serum containing CNKC extracts collected in different administration times and different blood collection time on PC12cells was investigated. Compared with model group(A value was1.43±0.12), control group was significantly different (F=142.627, P=0.000).A values of single dose serum containing CNKC extracts collected0.5hr,1hr,2hrs,3hrs were1.37±0.09,1.41±0.09,1.45±0.10,1.45±0.07(P=1.000), and showed no cytoprotection. A values of twice doses serum containing CNKC extracts collected1hr,2hrs were1.77±0.13, and1.78±0.12(P<0.05), much higher than those of model group and showed obvious cytoprotection. A values of eight doses serum containing CNKC extracts collected0.5hr,1hr,2hrs,3hrs were1.86±0.06,1.90±0.08,1.810±0.09, and1.79±0.11. All of them showed obvious cytoprotection, especially for0.5hr and lhr(P<0.01).②Effect of Serum containing different concentration of CNKC extracts on PC12cells was investigated. Compared with model group(A value was1.41±0.08), A values of serum containing2%CNKC extracts was1.75±0.09and showed cytoprotection. Both serum containing4%CNKC extracts and Nimodipine showed cytoprotection.
(3) The result of cellular morphous showed that after effected by Aβ25-35for72hrs, cellular diopter of the model group dropped, cell rounded and gathered, axon length and cell body were shorter than that of normal control group. However, compared with model group, cellular morphous of drug group was greatly improved. Their cellular synapse and diopter increased and the cells were well-distributed. The results of MTT assay showed that A value of model group(0.590±0.006) was much lower than that of control group(0.631±0.005)(F=147.104, P=0.000), and A value of drug group(0.622±0.004) was higher than that of model group(P=0.000). Cytoactive of SH-SY5Y could be degraded by Aβ25-35, and CNKC extracts could lessen cellular injury. The results of LDH assay showed that LDH of model group(1345.7±98.7) was higher than that of control group(789.1±63.5)(F=181.790, P=0.000), and LDH of drug group(675.7±56.6) was lower than that of model group (P=0.007). This result also showed that Cytoactive of SH-SY5Y could be degraded by Aβ25-35, and CNKC extracts could lessen cellular injury.②Result of mRNA levels. A260nm/A280nm of RNA was between1.8and2.0. Electrophoresis results showed that the straps of28S and18S RNA were very clear. All these illustrated that RNA was with high purity and had no degradation. RNA levels of control group, model group and drug group were1140ng/μl,1044ng/μl, and1020ng/μl respectively. According to the result of real-time PCR, AIF/actin mRNA relative amount of control group, model group and drug group were0.0102+0.0027,0.1023±0.0138,0.0252±0.0043; Cyt C/actin were0.0256±0.0065,0.0788±0.0062,0.0331±0.0013; Bax/actin were0.0203±0.0012,0.1707±0.0096,0.0380±0.0010; Bcl-2/actin were0.0409±0.0052,0.0090±0.0015,0.0252±0.0013。 Compared with control group, AIF,Cyt C,Bax mRNA amounts of model group were increased (F=60.846, P=0.013; F=89.670, P=0.000; F=420.101, P=0.002),and Bcl-2mRNA amount was reduced (F=104.859, P=0.012). Compared with model group, AIF,Cyt C,Bax mRNA amounts of drug group were reduced (P=0.014; P=0.000; P=0.002), and Bcl-2mRNA amount was increased (P=0.000).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by AP25-35.③ Protein concentration of control group, model group and drug group were16.170μg/μl,16.146μg/μl, and16.335μg/μl, respectively. According to the grey values, AIF levels of control group, model group and drug group were AIF levels were0.0145±0.0003,0.0502±0.0003,0.0261±0.0010, Bax levels were0.0252±0.0003,0.0871±0.0008,0.0519±0.0011, Cyt C levels were0.0240±0.0008,0.1045±0.0006,0.0653±0.0007, Bcl-2levels were0.0910±0.0057,0.0144±0.0003,0.0697±0.0010. Compared with control group, AIF/beta actin,Bax/beta actin.Cyt C/beta actin of model group were increased (Welch F=20950.876, P=0.000; Welch F=13197.318, P=0.000; Welch F=6704.384, P=0.000),and Bcl-2/beta actin was reduced(F=14872.548, P=0.000). Compared with model group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of drug group were reduced (P=0.000; P=0.000; P=0.000), and Bcl-2/beta actin was increased(P=0.000).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by AP25-35-.④Apoptosis result showed apoptosis rate of control group, model group and drug group were3.71±1.74,17.12±3.52and7.20±1.15. Compared with control group, apoptosis rate of model group were increased (F=26.038, P=0.000). Compared with model group, apoptosis rate of drug group were reduced (P=0.002).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by Aβ25-35.(4)①ompared with model group, in the first quadrant(NE), there were no differences of the time and distance to safe platform of group C,D,E(P>0.05). However, from the second quadrant, the data was fewer than that of model group(P<0.05). The result showed that CNKC extracts could improve the skill of learning of AD mice.②The result of HE staining showed that there was no obvious impairment in neurons of hippocampal formation (E group), and the cells were normal with clear structure. Morphous of neuron cells were integrity, arranged orderly, with large nucleus and clear nucleoli. Hippocampal formation of AD model group had integrated tissue. However, less cellular layer, incompleted neuron morphous, raritas and disordered cellular arrangement, smaller nucleus, anachromasis, obscure structure and derangement nerve fiber could be seen. Result of pathological section (group A,B,C) showed that there was light impairment in hippocampal formation, and the cellular layer was acceptable with clear structure.③Result of mRNA levels. A260nm/A280nm of RNA was between1.8and2.0. Electrophoresis results showed that the straps of28S and18S RNA were very clear. All these illustrated that RNA was with high purity and had no degradation. RNA levels of group E,D,A,B,C were1248ng/μl,1328ng/μl,1156ng/μl,1220ng/μl, and1304ng/μl. According to the result of real-time PCR, AIF/actin mRNA relative amount of group E,D,A,B,C were0.0036±0.0007,0.0092±0.0008,0.0045±0.0005,0.0051±0.0004,0.0075±0.0012; Cyt C/actin were0.0030±0.0005,0.0128±0.0023,0.0059±0.0015,0.0085±0.0022,0.0102±0.0008; Bax/actin were0.0028±0.0003,0.0127±0.0024,0.0050±0.0009,0.0060±0.0014,0.0094±0.0014; Bcl-2/actin were0.0281±0.0012,0.0027±0.0002,0.0152±0.0021,0.0103±0.0014,0.0072±0.0015. Compared with control group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of model group were increased (P<0.05),and Bcl-2/beta actin was reduced (P<0.05). Compared with model group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of drug groups were reduced (P<0.05), and Bcl-2/beta actin was increased(P<0.05).The result showed that CNKC exacts could protect apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose.④Protein concentration of group E,D,A,B,C were17.995μg/μl,18.082μg/μl,18.388μg/μl,17.901μg/μl,18.239μg/μl, respectively. According to the analysis of grey value, AIF levels of group E,D,A,B,C were0.2978±0.0195,0.7464±0.0389,0.3312±0.0297,0.6133±0.0426,0.5559±0.0393, Bax were01989±0.0120,0.7294±0.0512,0.2617±0.0144,0.3309±0.0235, 0.4755±0.0326, Cyt C were0.2292±0.0133,0.8553±0.0501,0.4185±0.0300,0.4928±0.0381,0.5802±0.0394, Bcl-2were0.5673±0.0294,0.1994±0.0115,0.4456±0.0318,0.4304±0.0313,0.1987±0.0119.⑤Results of immunohistochemistry of Cyt C showed that masculine cells of group E were widespread distributed in hippocampus and cell population was significant deviation from group D. Masculine cell population of model group was3.7times, than that of normal control group. Abnormality of these proteins express could be recovered by CNKC extracts in some degree.
Conclusions:(1)CNKC extracts could improve proliferation and protection on ischemia-like injury PC12cells induced by NaCN.(2) Serum containing CNKC extracts could improve proliferation and protection on ischemia-like injury PC12cells induced by NaCN and increase survival cells.(3) CNKC extracts had neuroprotective effects on SH-SY5Y cell apoptosis induced by Aβ25-35.(4) CNKC extracts had the effect on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose. In addition, the pharmacological mechanism of CNKC could be illustrated elementarily so as to provide scientific basis for further AD clinical treat.
引文
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