摘要
观察丹参-川芎有效成分(丹参素、原儿茶醛、川芎嗪、阿魏酸)配伍对体外原代培养的海马神经元细胞缺糖缺氧(氧糖剥夺)损伤的保护作用,并优选较佳组合。方法原代培养乳鼠海马神经元细胞,免疫组化法进行神经元特异性烯醇化酶(NSE)鉴定,并建立海马神经元细胞氧糖剥夺模型。MTT法确定丹参素、原儿茶醛、川芎嗪、阿魏酸及尼莫地平的非细胞毒性剂量范围,以L9(34)正交表设计安排成分配伍给药。将培养的细胞随机分为12组:对照组、模型组、尼莫地平阳性对照组及正交配伍1～9组。采用比色法、WST-1法、硫代巴比妥酸(TBA)法分别检测细胞上清液中乳酸脱氢酶(LDH)活力、超氧化物歧化酶(SOD)活性及丙二醛(MDA)水平,酶联免疫吸附试验检测细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6水平变化,Hoechst33258荧光染色法观察海马神经元细胞凋亡情况,流式细胞术检测细胞早期凋亡率。采用极差分析法分析正交试验结果。结果药物正交配伍组合均可显著改善缺氧海马神经元细胞的形态,显著减弱LDH活力,增强SOD的活性以及降低MDA的水平,显著抑制细胞TNF-α的释放,降低IL-1β和IL-6的水平,明显减少了细胞的凋亡。丹参-川芎有效成分对LDH活力的影响大小依次为丹参素>川芎嗪>原儿茶醛>阿魏酸,最佳配伍组合为丹参素(120μg/m L)、原儿茶醛(120μg/m L)、川芎嗪(80μg/m L)、阿魏酸(20μg/m L)。对SOD活性的影响主次因素依次为阿魏酸>川芎嗪>丹参素>原儿茶醛,最佳配伍组合为丹参素(120μg/m L)、原儿茶醛(120μg/m L)、川芎嗪(80μg/m L)、阿魏酸(40μg/m L)。对MDA水平的影响主次因素依次为丹参素>原儿茶醛>阿魏酸>川芎嗪,最佳配伍组合为丹参素(60μg/m L)、原儿茶醛(60μg/mL)、川芎嗪(80μg/m L)、阿魏酸(20μg/m L)。对TNF-α水平影响的主次因素依次为川芎嗪>原儿茶醛>丹参素>阿魏�
Objective To observe the protective effect of Salvia miltiorrhizae and Ligusticum chuanxiong effective constituents:danshensu, protocatechuic aldehyde, ligustrazine, and ferulic acid combination on primary cultured hippocampal neurons injured by oxygen glucose deprivation, and find out an optimized combination. Methods Primary cultured rats hippocampal neurons was chosen as research objects by adopting immunohistochemistry of the neuron-specific enolase IgG to authenticate, then the OGD model of the hippocampal neurons injured by oxygen glucose deprivation was established. The non-cytotoxic dose range of danshensu, protocatechuic aldehyde, ligustrazine, ferulic acid, and nimodipine was studied by MTT method. The compatibility of components was arranged by L9(34) orthogonal design. Primary cultured rats hippocampal neurons was divided into 12 groups:control group, model group, Nimodipine positive control group, and orthogonal design 1-9 group. The activity of LDH was measured by colorimetry, the activity of SOD was tested by WST^^-1 and the levels of MDA were examined by TBA. The levels of TNF-α, IL^-1β, and IL-6 in cell culture supernate were examined by ELISA, the apoptosis of hippocampal neurons was detected by Fluorochrome Hoechst33258 staining and the cell early apoptosis rate was detected with flow cytometry. The results of orthogonal test was analyzed by using range analysis method. Results The orthogonal compatibility of reagents played significant roles against the hypoxia damage of hippocampal neurons, improved the cellular morphology obviously, reduced the activity of LDH, increased the activity of SOD and decreased the content of MDA significantly, inhibited the release of TNF-α, and reduced the content of IL^-1β and IL-6, reduced the apoptosis of cells apparently. The effect of active ingredients of S. miltiorrhiza and L.Chuanxiong on LDH activity was danshensu > ligustrazine > protocatechuic aldehyde > ferulic acid, and the best combination was danshensu(120 μg/mL), protocatechuic aldehyde(120 �
作者
周惠芬
万海同
何昱
王慧君
万浩宇
李畅
虞立
杨洁红
ZHOU Hui-fen;WAN Hai-tong;HE Yu;WANG Hui-jun;WAN Hao-yu;LI Chang;YU Li;YANG Jie-hong(Zhejiang Chinese Medical University, Hangzhou 310053, China)
出处
《中草药》
CAS
CSCD
北大核心
2019年第6期1372-1381,共10页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81630105)
国家自然科学基金资助项目(81874366)
浙江省自然科学基金资助项目(LZ17H270001)
浙江省自然科学基金资助项目(LZ18H270001)
浙江省自然科学基金资助项目(LQ19H270001)
关键词
丹参
川芎
配伍
海马神经元细胞
氧糖剥夺
细胞凋亡
丹参素
原儿茶醛
川芎嗪
阿魏酸
Salvia miltiorrhiza Bge.
Ligusticum chuanxiong Hort
compatibility
hippocampal neurons
oxygen glucose deprivation
apoptosis
danshensu
protocatechuic aldehyde
ligustrazine
ferulic acid
