The protein expressions of neurofilaments, including NF68, NF160 and NF200, under the herbal treatment were revealed by western blot. The determination of neurite outgrowth in cultured PC12 cells upon the treatment of herbal extracts was performed by light microscope equipped with a phase-contrast condenser and SPOT imaging software. The protective effect against tBHP-induced cytotoxicity under the herbal treatment was measured by MTT assay. A luciferase reporter construct carrying four repeats of anti-oxidant response element (ARE) and a downstream luciferase reporter gene luc2P was transfected into PC12 cells to study the transcriptional activation of ARE. The mRNA expression of antioxidant enzymes under the herbal treatment was analyzed by quantitative real-time PCR.
These jujube-containing decoctions processed similar neuro-protective and brain beneficial properties. The herbal treatment induced the protein expression of neurofilaments. Neurite outgrowth was observed under the herbal treatment. In parallel, the pre-treatment of herbal extracts protected PC 12 cells against oxidative stress-induced apoptosis in a dose-dependent manner. Moreover, the herbal treatments triggered the mRNA expressions of relevant anti-oxidation genes, i.e. glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modulatory subunit (GCLM), glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase (NQO1) via the activation of anti-oxidant response element (ARE).
The results therefore demonstrated neuro-protective and differentiating properties of the three closely related decoctions, and which subsequently illustrated the enhancement function of jujube within a multi-herbal decoction.