Protective effects of compatibility of effective constituents in Salvia miltiorrhiza and Ligusticum chuanxiong on hippocampal neurons injured by oxygen glucose deprivation / 中草药

ABSTRACT

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 waschosen 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 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on SOD activity was ferulic acid > ligustrazine > danshensu > protocatechuic aldehyde. The best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (40 μg/mL). The order of influence on MDA content was danshensu > protocatechuic aldehyde > ferulic acid > ligustrazine, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on TNF-α content were: ligustrazine > protocatechuic aldehyde > danshensu > ferulic acid, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (40 μg/mL), and ferulic acid (10 μg/mL). The order of influence on the content of IL-1β was ligustrazine > ferulic acid > danshensu > protocatechuic aldehyde, and the best combination was danshensu (30 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on the content of IL-6 was protocatechuic aldehyde > ligustrazine > ferulic acid > danshensu, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (10 μg/mL). The effect on the early apoptosis rate was ferulic acid > protocatechuic aldehyde > ligustrazine > danshensu, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (20 μg/mL), and ferulic acid (40 μg/mL). Conclusion The protective mechanism of the effective constituents of S. miltiorrhizae and L. chuanxiong were relied on reducing the oxidative damage, reducing inflammation damage, and antagonizing cell apoptosis. According to the experimental results, we need to change the prescription ratio and guide clinical medication for different clinical courses.