METHODS: The hESCs were differentiated into neural stem cells (NSCs), and NSC-DECM was extracted from confluent monolayers of NSCs through treatment with deionized water. DFSCs seeded on NSC-DECM, Geltrex, and tissue culture polystyrene (TCPS) were subjected to neural induction during a period of 21 days. Expression of early/intermediate (Musashi1, PAX6, NSE, and βIII-tubulin) and mature/late (NGN2, NeuN, NFM, and MASH1) neural markers by DFSCs was analyzed at the 7-, 14-, and 21-day time points with quantitative real-time polymerase chain reaction. Immunocytochemistry for detection of βIII-tubulin, PAX6, and NGN2 expression by DFSCs on day 7 of neural induction was also carried out.
RESULTS: Quantitative RT-PCR showed that expression of PAX6, Musashi1, βIII-tubulin, NSE, NGN2, and NFM by DFSCs was enhanced on NSC-DECM versus either the Geltrex or TCPS groups. Immunocytochemistry showed that DFSCs in the NSC-DECM group displayed more intense staining for βIII-tubulin, PAX6, and NGN2 expression, together with more neurite outgrowths and elongated morphology, as compared with either Geltrex or TCPS.
CONCLUSIONS: DECM derived from neurogenesis of hESCs can enhance the neurogenic potential of DFSCs.
MATERIALS AND METHODS: Fresh specimens of P. australis were freeze-dried and subjected to ethanol extraction. The ethanol extract (PAEE) was evaluated for its protective effects against 1 µg/ml LPS-stimulated neuroinflammation in BV2 microglial cells.
RESULTS: LPS reduced the viability of BV2 microglia cells and increased the levels of nitric oxide (NO), prostaglandin E2 (PGE2), intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). However, the neuroinflammatory response was reversed by 0.5-2.0 mg/ml PAEE in a dose-dependent manner. Analysis of liquid chromatography-mass spectrometry (LC-MS) of PAEE subfractions revealed five compounds; methyl α-eleostearate, ethyl α-eleostearate, niacinamide, stearamide, and linoleic acid.
CONCLUSION: The protective effects of PAEE against LPS-stimulated neuroinflammation in BV2 microglial cells were found to be mediated by the suppression of excess levels of intracellular ROS and pro-inflammatory mediators and cytokines, denoting the protective role of P. australis in combating continuous neuroinflammation. Our findings support the use of P. australis as a possible therapeutic for neuroinflammatory and neurodegenerative diseases.