Molecular mechanism of panaxydol on promoting axonal growth in PC12 cells.

Nerve growth factor (NGF) promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol promotes neurite outgrowth in PC12 cells, but its molecular mechanism remains unclear. Indeed, although alkynol compounds such as panaxydol can increase intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and the ERK inhibitor U0126 inhibits alkynol-induced axonal growth, how pathways downstream of cAMP activate ERK have not been investigated. This study observed the molecular mechanism of panaxydol-, NGF- and forskolin-induced PC12 cell axon growth using specific signaling pathway inhibitors. The results demonstrated that although the RTK inhibitor SU5416 obviously inhibited the growth-promoting effect of NGF, it could not inhibit the promoting effect of panaxydol on axonal growth of PC12 cells. The adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase inhibitor RpcAMPS could suppress the promoting effect of forskolin and panaxydol on axonal growth. The ERK inhibitor U0126 inhibited axonal growth induced by all three factors. However, the PKA inhibitor H89 inhibited the promoting effect of forskolin on axonal growth but could not suppress the promoting effect of panaxydol. A western blot assay was used to determine the effects of stimulating factors and inhibitors on ERK phosphorylation levels. The results revealed that NGF activates the ERK pathway through tyrosine receptors to induce axonal growth of PC12 cells. In contrast, panaxydol and forskolin increased cellular cAMP levels and were inhibited by adenylyl cyclase inhibitors. The protein kinase A inhibitor H89 completely inhibited forskolin-induced axonal outgrowth and ERK phosphorylation, but could not inhibit panaxydol-induced axonal growth and ERK phosphorylation. These results indicated that panaxydol promoted axonal growth of PC12 cells through different pathways downstream of cAMP. Considering that exchange protein directly activated by cAMP 1 (Epac1) plays an important role in mediating cAMP signaling pathways, RNA interference experiments targeting the Epac1 gene were employed. The results verified that Epac1 could mediate the axonal growth signaling pathway induced by panaxydol. These findings suggest that compared with NGF and forskolin, panaxydol elicits axonal growth through the cAMP-Epac1-Rap1-MEK-ERK-CREB pathway, which is independent of PKA.

Atg5-independent autophagy regulates mitochondrial clearance and is essential for iPSC reprogramming.

Successful generation of induced pluripotent stem cells entails a major metabolic switch from mitochondrial oxidative phosphorylation to glycolysis during the reprogramming process. The mechanism of this metabolic reprogramming, however, remains elusive. Here, our results suggest that an Atg5-independent autophagic process mediates mitochondrial clearance, a characteristic event involved in the metabolic switch. We found that blocking such autophagy, but not canonical autophagy, inhibits mitochondrial clearance, in turn, preventing iPSC induction. Furthermore, AMPK seems to be upstream of this autophagic pathway and can be targeted by small molecules to modulate mitochondrial clearance during metabolic reprogramming. Our work not only reveals that the Atg5-independent autophagy is crucial for establishing pluripotency, but it also suggests that iPSC generation and tumorigenesis share a similar metabolic switch.

Panaxydol and panaxynol protect cultured cortical neurons against Abeta25-35-induced toxicity.

Amyloid beta protein (Abeta), the central constituent of senile plaques in Alzheimer's disease (AD), is known to exert toxic effects on cultured neurons. In the present study, the protective effect of panaxydol (PND) and panaxynol (PNN) on Abeta25-35-induced neuronal apoptosis and potential mechanisms were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN prior to 10 microM Abeta25-35 exposure resulted significantly in elevation of cell survival determined by MTT assay, TUNEL/Hoechst staining and western blot. Furthermore, a marked increase in calcium influx and intracellular free radical generation was found after Abeta25-35 exposure, which could be almost completely reversed by pretreatment of PND or PNN. PND and PNN could also alleviate Abeta25-35-induced early-stage neuronal degeneration. These results indicated that inhibition of calcium influx and free radical generation is a mechanism of the anti-apoptotic action of PND and PNN. Since Abeta plays critical roles in the pathogenesis of AD, these findings raise the possibility that PND and PNN reduce neurodegeneration in AD.

Antiproliferative effect of panaxynol on RASMCs via inhibition of ERK1/2 and CREB.

Panaxynol (PNN) occurs in many foods such as carrot, celery, and several reports have shown that it has neuritogenic and neuroprotective properties. In this study, we have investigated the antiproliferative effect and the mechanism of PNN on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (RASMCs). PNN significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of RASMCs in a concentration-dependent manner. Flow cytometry analysis showed that PNN blocked the cell cycle progression at the G(1)/S phase. Preincubation of RASMCs with 9 microM PNN resulted in a significant inhibition of PDGF-BB-induced extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation expression and PDGF-BB-induced CREB phosphorylation expression. The results indicated that the inhibitory effect of PNN on the PDGF-BB-induced proliferation of RASMCs might be mediated by blocking phosphorylation of ERK1/2 and that of CREB.

Methods for isolating highly-enriched embryonic spinal cord neurons: a comparison between enzymatic and mechanical dissociations.

Spinal cord neuronal culture is a useful system to study normal and abnormal functions of the spinal cord. For many bioassays, obtaining large quantities of highly purified spinal cord neurons is required. However, technical difficulties exist in obtaining these cells reliably and consistently. By comparing two dissociation methods, mechanical and enzymatic dissociations, we found that the enzymatic dissociation of embryonic day 14-15 spinal cords resulted in significantly higher cell yield than the mechanical dissociation (25.40 +/- 5.41 x 10(6) versus 3.43 +/- 0.52 x 10(6) cells per 12 embryos; n = 6/group; p < 0.01). Furthermore, cell viability was significantly higher after the enzymatic than the mechanical dissociation (83.40 +/- 3.08% versus 32.81 +/- 3.49%, n = 4/group; p < 0.01). In both methods, highly purified populations of primary neurons were obtained (mechanical: 85.17 +/- 2.84%; enzymatic: 87.67 +/- 2.52%; n = 3/group). Critical measures that affect culture outcomes include, but not limited to, the age of embryo, cell seeding density, dissociation time, and elimination of non-neuronal cells. Thus, the present study has identified the enzymatic dissociation method to be a preferred method for obtaining large quantity of highly-enriched embryonic spinal cord neurons.

Protective effect of panaxydol and panaxynol on sodium nitroprusside-induced apoptosis in cortical neurons.

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. The protective effect of panaxydol (PND) and panaxynol (PNN) on sodium nitroprusside (SNP)-induced neuronal apoptosis and potential mechanism were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN for 24 h following 1mM SNP, an exogenous NO donor, exposure for 1h, resulted significantly in reduction of cell death induced by SNP determined by MTT assay, LDH release and Hoechst staining. 5 microM PND and PNN also reduced the up-regulation of the pro-apoptotic gene, Bax, down-regulation of the anti-apoptotic gene, Bcl-2. The observations demonstrated that PND and PNN protect neurons against SNP-induced apoptosis via regulating the apoptotic related genes. The results raise the possibility that PND and PNN reduce neurodegeneration in the Alzheimer's brain.

Neuroprotective effect of the stearic acid against oxidative stress via phosphatidylinositol 3-kinase pathway.

Stearic acid is a long-chain saturated fatty acid consisting of 18 carbon atoms without double bonds. In the present study, we reported the neuroprotective effects and mechanism of stearic acid on cortical or hippocampal slices insulted by oxygen-glucose deprivation, NMDA or hydrogen peroxide (H(2)O(2)) in vitro. Different types of models of brain slice injury in vitro were developed by 10 min of oxygen/glucose deprivation, 0.5 mM NMDA or 2 mM H(2)O(2), respectively. After 30 min of preincubation with stearic acid (3-30 microM), cortical or hippocampal slices were subjected to oxygen-glucose deprivation, NMDA or H(2)O(2). Then the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride (TTC) method. Population spikes were recorded in randomly selected hippocampal slices. Stearic acid (3-30 microM) dose-dependently protected brain slices from oxygen-glucose deprivation, NMDA and H(2)O(2) insults. Its neuroprotective effect against H(2)O(2) insults can be completely blocked by wortmannin (inhibitor of PI3K) and partially blocked by H7 (inhibitor of PKC) or genistein (inhibitor of TPK). Treatment of cortical or hippocampal slices with 30 microM stearic acid resulted in a significant increase in PI3K activity at 5, 10, 30 and 60 min. These observations reveal that stearic acid can protect cortical or hippocampal slices against injury induced by oxygen-glucose deprivation, NMDA or H(2)O(2), and its neuroprotective effects are via phosphatidylinositol 3-kinase dependent mechanism.

Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms.

Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.

In vitro anti-coxsackievirus B(3) effect of ethyl acetate extract of Tian-hua-fen.

AIM: To investigation the anti-coxsackievirus B(3) (CVB(3m)) effect of the ethyl acetate extract of Tian-hua-fen on HeLa cells infected with CVB(3m). METHODS: HeLa cells were infected with CVB(3m) and the cytopathic effects (CPE) were observed through light microscope and crystal violet staining on 96-well plate and A(600) was detected using spectrophotometer. The protective effect of the extract to HeLa cells and the mechanism of the effect were also evaluated through the change of CPE and value of A(600). RESULTS: The extract had some toxicity to HeLa cells at a higher concentration while had a marked inhibitory effect on cell pathological changes at a lower concentration. Consistent results were got through these two methods. We also investigated the mechanism of its anti-CVB(3m) effect and the results indicated that the extract represented an inhibitory effect through all the processes of CVB(3m) attachment, entry, biosynthesis and assemble in cells. CONCLUSION: The results demonstrate that the ethyl acetate extract of Tian-hua-fen has a significant protective effect on HeLa cells infected with CVB(3m) in a dose-dependent manner and this effect exists through the process of CVB(3m) attachment, entry, biosynthesis and assemble in cells, suggesting that the ethyl acetate extract of Tian-hua-fen can be developed as an anti-virus agent.