Ginsenoside Rg1Protects Primary Cortical Neurons Against Aβ_25-35 Insult And Promotes Neural Differentiation Of Embryonic Stem Cells Via Selective Steroid Hormone Receptor Activation Pathway

Embryonic stem (ES) cells are extremely attractive for its unique properties of pluripotency with the potential to differentiate into any other cell types in the presence of appropriate stimulatory factors and environmental cues. ES cells have shown its promising prospect in developmental biology, cell-based therapies in regenerative medicine and even drug discovery. The major challenge in differentiation of ES cells is to control and direct the cells toward a specific functionally distinct phenotype of interest1,12,20.Increasing studies showed that ginsenoside Rgl (Rgl), one of the most active constituents in most ginseng species, has extensive neuroprotective and neurotrophic effects, but the underlying mechanisms remain unknown5. Due to the four trans-ring rigid steroid skeleton similar to steroid hormones and the amphiphilic nature, Rgl is assumed to have an ability to bind to intracellular steroid hormone receptors, including estrogen receptor α/β (ERa/β), glucocorticoid receptor (GR) and progesterone receptor (PR). Previous studies also demonstrate that Rgl possesses estrogen-like and glucocorticoid-like activities in vitro7’8, so it is probable that the neuroprotective and neurotrophic effects of Rgl are associated with steroid receptor activation.Junctophilins (JPs) contribute to the formation of junctional membrane structures in excitable cells by interacting with the plasma membrane and spanning the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane18. JP-3and JP-4are expressed in similar discrete neuronal sites in the brain and may collaboratively contribute to junctional membrane complexes (JMC) formation. It was reported that JP-DKO mice showed aberrant behavior and impaired memory. So far, the characteristics of JPs during embryogenesis, and the differences on distribution and functions between JP-3 and JP-4are largely unknown.1. Ginsenoside Rgl Protects Primary Cultured Rat Cortical Neurons against P-amyloid Peptide Insultsβ-amyloid (A(3) was a neurotoxic peptide found in the brains of Alzheimer’s disease (AD) patients. In this experiment, the neuroprotective effects and the underlying mechanisms of Rgl against Aβ25-35insults were explored. The results indicated that Rgl protected primary cortical neurons against Aβ25-35insults in a dose-dependent manner, and20umol/L was the optimal concentration for Rgl. Rgl pretreatment resulted translocation of estrogen receptor a (ERa) and glucocorticoid receptor (GR), rather than estrogen receptor β (ERβ) and progesterone receptor (PR). The IC50of Rgl binding to ERa and GR were8.6μmol/L and12.8μmol/L, respectively, suggesting the activation of ERa and GR by Rgl. The siRNA experiment further demonstrated that knockdown the expression of ERa and GR in primary neurons blocked Rgl neuroprotection, which heralded that the protection of Rgl was associated with ERa and GR activation.Exposure to Aβ25-35induced generation of ROS and NO, decrease of mitochondrial△(?)m and ERK phosphorylation, activation of NF-kB, and finally resulted protein tyrosine nitration and cell apoptosis. All of the injuries could be counteracted or alleviated by Rgl pretreatment. Except for the antioxidant action, the other neuroprotective effects of Rgl were all ERa-and GR-dependent. The antioxidant effect of Rgl might be due to its ability to scavenge free radicals. These results demonstrated that the antiapoptotic effect of Rgl was mediated by ERa and GR.2. Ginsenoside Rgl Facilitates Neural Differentiation of Mouse ES CellsSmall molecules that target specific regulatory processes and control cell fate are therefore valuable tools for probing and manipulating the molecular mechanisms by which stem cells self-renew, differentiate, and arise from somatic cell reprogramming12,20. Ginsenoside Rgl, a steroidal saponin of high abundance in ginseng, is one of the most active components in ginseng. Aside from its attractive neuroprotective effect, Rgl has been found to regulate the proliferation of neural progenitor cells22,23. However, the action mechanisms and potential role of Rgl in neuronal lineage commitment are poorly understood. In this chapter, we explored the ability of Rgl to affect ES behavior as well as the underlying mechanisms.The results indicated that10μmol/L Rgl facilitated the neural differentiation of mouse ES cells and increased the expression of neural specific proteins. Glucocorticoid receptor (GR) existed in ES cells and upregulated during the neurodifferentiation process. GR antagonist RU486effectively blocked the neurogenetic effect of Rgl. Rgl treatment elevated the level of ERK and Akt, and this effect also could be inhibited by RU486. To explore whether ERK and Akt participated in the neurodifferentiation promoted by Rgl, MEK inhibitor U0126and PI3K inhibitor LY294002were applied. The results confirmed the role of these two kinases in the neurogenetic action of Rgl. Interestingly, U0126not only inhibited ERK phosphorylation, but also affected the level of p-Akt. These data implied that Rgl has an ability to initiate differentiation of ES cells towards the neural lineage via GR-MEK-ERKl/2-PI3K-Akt pathway.3. Role of Junctophilin-3,-4in the Neurogenesis of Mouse ES CellsThe expression of JP-3,-4mRNA and protein were detected by qRT-PCR and western blot. The results demonstrated that the expression of JP-3,-4first elevated, and then decreased during the neurodifferentiation process of ES cells. The peak time of JP-3was on d8+5, while that of JP-4happened a little bit earlier, on d8+0. The results of immunocytochemistry and flow cytometric analysis further showed that there were three types of neural cells in derived cultures, including neurons, astrocytes and oligodendrocytes, and the majority of JP-3,-4resided in neurons, rather than neuroglial cells. With respect to the three distinct subtype neurons, the distribution of JP-3,-4was also different, most in glutamatergic neurons, second in cholinergic neurons, and least in GABAergic neurons.To explore whether the JP-3,-4expressed in derived neurons have the ability to maintain the functional crosstalk between cell-surface and intracellular ion channels, the siRNA experiments were performed. Knockdown JP-3,-4expression in derived neurons on d8+10resulted impaired Ca2+-induced Ca2+release (CICR), suggesting that the JP-3,-4proteins in derived neurons were functional to support intracellular Ca2+-mediated communication.Moreover, since the tendency of JP-3,-4was first increase and then decrease, and the transient peak expression happened in the similar time window with that of Nesin, the marker for neuronal precursor cells, indicating that the JP-3,-4transient expression may contribute to the neurodifferentiation of ES cells. To further confirm this hypothesis, the small interfering RNAs (siRNA) were performed to knockdown the expression of JP-3,-4in mouse ES cells. The results exhibited that either the differentiation ratio or the expression of neural marker proteins reduced after JP-3,4siRNA transfection.Conclusion:1. Ginsenoside Rgl possessed a neuroprotective effect on primary cultured rat cortical neurons against Aβ25-35-induced injuries. The underlying mechanisms included upregulation of ERK phosphorylation, inhibition of NF-kB activation, decrease of protein tyrosine nitration and blockage of mitochondrion-mediated apoptosis in an ERa-and GR-dependent manner.2. Ginsenoside Rgl initiated differentiation of ES cells towards the neural lineage via GR-MEK-ERK1/2-PI3K-Akt pathway.3. JP-3,-4expressed in derived neurons from mouse ES cells were functional. They existed mostly in glutamatergic neurons, second in cholinergic neurons, and least in GABAergic neurons. The transient peak expression of JP-3,-4contributed to the neurodifferentiation of ES cells. Compared with JP-3, the role of JP-4for neurodifferentiation was more important.