摘要
目的考察蔷薇红景天总黄酮提取物(TFE)对β淀粉样蛋白25-35(Aβ_(25-35))诱导的PC12细胞损伤的保护作用及其活性部位筛选。方法采用20μmol·L^(-1)Aβ_(25-35)诱导PC12细胞损伤,经AB-8大孔吸附树脂色谱法分离蔷薇红景天TFE,得水及20%、40%、60%、80%、95%乙醇洗脱物(即TFE-0%、TFE-20%、TFE-40%、TFE-60%、TFE-80%和TFE-95%)。计算不同体积分数乙醇洗脱物浸膏得率(赋予0.3权重),紫外分光光度法检测不同体积分数乙醇洗脱物总黄酮含量(赋予0.7权重),综合评价不同体积分数乙醇洗脱物。MTT法检测不同体积分数乙醇洗脱物对损伤后PC12细胞活力的影响;采用倒置显微镜观察细胞形态;采用2,4-二硝基苯肼显色法检测乳酸脱氢酶(LDH)的活性;采用黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)的含量;酶联免疫测定(ELISA)法分别检测细胞上清中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、环氧合酶-2(COX-2)和白细胞介素-1β(IL-1β)的分泌水平。结果TFE-40%总黄酮浓度和浸膏得率的综合得分高于其他不同体积分数乙醇洗脱物。与空白组比较,模型组细胞间隙变大,数目减少,出现细胞碎片,突触明显减少,存活率和SOD水平明显降低(P<0.05),LDH水平明显升高(P<0.05);与模型组比较,TFE-40%、TFE-60%洗脱物和盐酸多奈哌齐组细胞形态均有一定程度改善,数目增多,突触增加,细胞形态较完整;TFE-40%、TFE-60%、TFE-80%及TFE-95%组的细胞上清液中IL-6、TNF-α、COX-2和IL-1β的水平均明显降低(P<0.05)。结论蔷薇红景天TFE-40%洗脱物对Aβ_(25-35)诱导的PC12细胞损伤具有较好保护作用,为最佳活性部位,其机制可能与拮抗Aβ_(25-35)的神经毒性和抑制细胞炎症的发生有关。
Objective To determine the protective effect of total flavonoids extract of Rhodiola rosea L.(TFE)on PC12 cell damage induced by amyloidβ-protein 25-35(Aβ_(25-35))and screen its active parts.Methods PC12 cell damage was induced by 20μmol·L^(-1)Aβ_(25-35),and TFE was isolated by AB-8 macroporous adsorption resin chromatography.Water and 20%,40%,60%,80%and 95%ethanol elution(TFE-0%,TFE-20%,TFE-40%,TFE-60%,TFE-80%and TFE-95%)were obtained.The extract yield of ethanol elution with different volume fractions(0.3 weight)was calculated.The total flavonoids content of ethanol elution with different volume fractions(0.7 weight)was detected by UV spectrophotometry,and the ethanol elution with different volume fractions was evaluated comprehensively.The effects of different volume fractions of ethanol elution on the viability of injured PC12 cells were detected by MTT method.The morphology of cells was observed by inverted microscope,the activity of lactate dehydrogenase(LDH)was detected by 2,4-dinitrophenylhydrazine colorimetric method,and the content of superoxide dismutase(SOD)by xanthine oxidase method.The levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),cyclooxygenase-2(COX-2)and interleukin-1β(IL-1β)in the supernatant were detected by enzyme-linked immunosorbent assay.Results The comprehensive scores of total flavonoids concentration and extract yield of TFE-40%eluates were higher than those of other ethanol eluents with different volume fractions.Compared with the blank group,the intercellular space of the model group was larger,the number of cells decreased,the cell fragments appeared,the synapse decreased significantly,the survival rate and the level of SOD decreased significantly(P<0.05),and the level of LDH increased significantly(P<0.05).Compared with the model group,the cell morphology of TFE-40%,TFE-60%and Donepezil hydrochloride groups improved to some extent,the number of synapses increased,with more complete cell morphology.The levels of IL-6,TNF-α,COX-2,and IL-1βin the supernatant of TFE-40%,TFE-60%,TFE-80%and TFE-95%groups were significantly decreased(P<0.05).Conclusion The TFE-40%eluent of Rhodiola rosea L.has a good protective effect on the injury of PC12 cells induced by Aβ_(25-35),which is the best active part.The mechanism may be related to the neurotoxicity and inhibition of cellular inflammation of antagonism of Aβ_(25-35).
作者
刘忠毅
王启文
阿孜古丽·阿里木
王晓梅
胡君萍
王新玲
LIU Zhong-yi;WANG Qi-wen;Aziguli Alimu;WANG Xiao-mei;HU Jun-ping;WANG Xin-ling(College of Pharmacy,Xinjiang Medical University,Urumqi 830011)
出处
《中南药学》
CAS
2024年第2期409-415,共7页
Central South Pharmacy
基金
新疆维吾尔自治区“十三五”重点学科建设项目(No.XJDX17132016)。
