Yijia Lou

Involvement of p38MAPK and reactive oxygen species in icariin-induced cardiomyocyte differentiation of murine embryonic stem cells in vitro.

We previously reported that treatment of icariin could significantly induce cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro. In the present study, the exact activity initiated by icariin was further confirmed and the underlying molecular mechanism was investigated. We found that cardiomyocyte differentiation was efficiently stimulated only if icariin was administrated between days 5 and 8 in differentiation course, which indicated with elevated percentage of embryoid bodies (EB) and with beating areas and up- regulated expression of alpha-actinin and troponin T. Exposure of icariin triggered intracellular reactive oxygen species (ROS) generation of EBs in 3 h, which was abolished in the presence of either NADPH oxidase inhibitor DPI or antioxidant Trolox. Meanwhile, expression of NOX4, a membrane combined enzyme responsible for ROS generation, was promoted by icariin in a dose-dependent manner. Although p38MAPK (mitogen-activated protein kinase), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal protein kinase (JNK) were spontaneously activated in early differentiation, only the phosphorylation of p38MAPK was enhanced and prolonged when icariin was present, whereas both ERK and JNK showed no response to icariin treatment. Moreover, the inducible effect of icariin was blunted by SB203580, a specific inhibitor of p38MAPK. On the contrary, neither UO126 nor SP600125, the specific inhibitor of ERK and JNK, could abolish icariin-stimulated differentiation. Nuclear location of MEF2C, which played a critical role in cardiomyocyte differentiation and could be activated by p38MAPK, was stimulated after icariin exposure. Taken together, these results suggest that ROS generation and the subsequent activation of p38MAPK are essential for the inducible function of icariin on cardiomyocyte differentiation of murine embryonic stem cells in vitro.

Reactive oxygen species involved in prenylflavonoids, icariin and icaritin, initiating cardiac differentiation of mouse embryonic stem cells†

The significant promoting effects of some prenylflavonoids on cardiac differentiation of mouse embryonic stem (ES) cells via reactive oxygen species (ROS) signaling pathway were investigated. The most effective differentiation was facilitated by icariin (ICA), followed by icaritin (ICT), while desmethylicaritin (DICT) displayed the weakest but still significant inducible effect. Contrarily, DICT demonstrated the strongest anti‐oxidative activity while ICA displayed only little in vitro, which was well matched with the hydroxyl (OH) numbers and the positions in the molecular structures. Therefore, ROS signaling cascades were assumed to be involved in prenylflavonoids induced cardiomyogenesis. Treatment with ICA, intracellular ROS in embryoid bodies was rapidly elevated, which was abolished by the NADPH‐oxidase inhibitor apocynin; elimination of intracellular ROS by vitamin E or pyrrolidine dithiocarbamate (PDTC) inhibited ICA induced cardiomyogenesis; ROS‐sensitive extracellular‐regulated kinase 1, 2 (ERK1, 2) and p38 activation were further observed, the cardiomyogenesis was significantly inhibited in the presence of ERK1, 2 or p38 inhibitor U0126 or SB203580, indicating the roles of NADPH‐ROS‐MAPKs signaling cascades in prenylflavonoids induced cardiac differentiation. There was no difference in Nox4 NADPH oxidase expression between ICA and ICT treatments, however, ROS concentration in EBs after ICT administration was lower than that after ICA treatment, followed by less activation of ERK1, 2, and p38. These results revealed that the significant promoting effects of prenylflavonoids on cardiac differentiation was at least partly via ROS signaling cascades, and the facilitating abilities preferentially based on the nature of prenylflavonoids themselves, but anti‐oxidative activity determined by the OH numbers and the positions in the structures do influence the cardiomyogenesis in vitro. J. Cell. Biochem. 103: 1536–1550, 2008.

Anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma A549 cell line

Anti-tumor activity of bakuchiol, an analogue of resveratrol, was investigated on human lung adenocarcinoma A549 cell line. MTT assay revealed that IC(50) of bakuchiol at 72h was 9.58+/-1.12 micromol/l, much lower than that of resveratrol (33.02+/-2.35 micromol/l). Bakuchiol but not resveratrol elevated intracellular reactive oxygen species. Bakuchiol reduced mitochondrial membrane potential (Psim) of cells in a concentration- and time-dependent manner, showing a more potent effect than that of resveratrol. More apoptotic cells were induced by bakuchiol, compared with resveratrol. Subsequently, S phase arrest, caspase 9/3 activaton, p53 and Bax up-regulation, as well as Bcl-2 down-regulation were observed in bakuchiol-treated A549 cells. The results point toward that S phase-related cell cycle regulation, more importantly reactive oxygen species-related apoptosis might contribute to the anticancer properties of bakuchiol, which will strongly support the further development of bakuchiol against non-small-cell lung cancer.

Asiatic acid protects primary neurons against C2-ceramide-induced apoptosis.

Ceramides derived from sphingosine contribute to the apoptotic processes of neuronal cells in neurodegenerative disorders including Alzheimers disease. This study investigates the potential neuroprotective effects of Asiatic acid, a triterpenoid derived from Centella asiatica, against C(2)-ceramides-induced cell death in primary cultured rat cortical neuronal cells. In primary neurons, Asiatic acid (0.01 to 1.0 μmol/l) reduced C(2)-ceramide-induced cell death and mitochondria membrane potential loss in a concentration-dependent manner. Furthermore, Asiatic acid decreased cellular production of reactive oxygen species following C(2)-ceramide treatment. At a maximal concentration of 1.0 μmol/l, Asiatic acid partly counteracted the pro-apoptotic effects of the C(2)-ceramide by reducing the cytosolic release of HtrA2/Omi, the upregulation of Bax and caspase 3, as well as the dephosphorlyation of ERK1/2. Taken together, these data suggest that Asiatic acid protects neurons from C(2)-ceramide-induced cell death by antagonizing mitochondria-dependent apoptosis.

Enhanced co-expression of β-tubulin III and choline acetyltransferase in neurons from mouse embryonic stem cells promoted by icaritin in an estrogen receptor-independent manner

A previous small molecule screen demonstrated that some prenylflavonoids can promote neuronal differentiation from mouse embryonic stem (ES) cells based on morphologic criteria. Here we build on this observation and examine the neuronal subtypes induced by icaritin, a compound screened, and the molecular events underlying the differentiation. In the presence of icaritin, the number of neural rosettes in embryoid bodies (EBs) expressing nestin efficiently increased and the neuroectodermal gene Fgf5 expression upregulated during germ layer formation. The neural progenitors generated from icaritin-treated EBs were further differentiated into the neurons (marked by beta-tubulin III) and also enhanced the choline acetyltransferase (ChAT) expression upon terminal differentiation. A suppression of p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and sustained extracellular signal-regulated protein kinase (ERK) phosphorylation existed simultaneously without estrogen-like activities involved. Taken together, enhanced co-expression of beta-tubulin III and choline acetyltransferase in neuronal differentiation from mouse ES cells is promoted by icaritin via estrogen receptor-independent action.

Effects of icariin combined with Panax notoginseng saponins on ischemia reperfusion‐induced cognitive impairments related with oxidative stress and CA1 of hippocampal neurons in rat

Previous studies suggest that treatment with icariin (ICA) combined with Panax notoginseng saponins (PNS) improved behavior and cholinergic system disorders followed by amyloid β‐peptide25−35 lateral ventricle injection in rats. The present study investigated whether administration of ICA + PNS had preventive and therapeutic effects on bilateral common carotid arteries (CCA) occlusion‐induced cerebral ischemia‐reperfusion (IR) injury in rats. Male Sprague‐Dawley rats were divided randomly as follows: sham‐operated, i.g. vehicle, ICA (5 mg/kg), PNS (40 mg/kg), ICA + PNS (2.5 + 20, 5 + 40 or 10 + 80 mg/kg), and ergoloid mesylate as a positive control (0.45 mg/kg) in model rats. Treatment was performed once a day for 7 days prior to ischemia. The rats were subjected to transient global IR induced by CCA occlusion in combination with intraperitoneal injection of sodium nitroprusside (2.0 mg/kg), then treated with ICA + PNS for another 14 days continuously. ICA + PNS significantly improved the rat passive avoidance task in step‐down paradigms, and spatial cognition in the eight‐arm radial maze, concomitant with an improvement of blood viscosity. Increased lipid peroxidation in brain after IR injury was observed, MDA being 0.56 ± 0.10 nmol/mg prot vs 0.48 ± 0.06 nmol/mg prot in the vehicle control (p < 0.05). Treatment with ICA + PNS 2.5 + 10, 5 + 40, 10 + 80 mg/kg produced a marked reduction in the MDA level to 0.46 ± 0.06, 0.42 ± 0.09 and 0.45 ± 0.08 nmol/mg prot, respectively vs 0.56 ± 0.10 nmol/mg prot in IR injury only control (p < 0.05, p < 0.01). A decrease in superoxide dismutase activity was observed in the brain of IR rats (the SOD activity being 72.75 ± 4.62 U/mg prot vs 80.97 ± 6.06 U/mg prot in control, p < 0.05). ICA + PNS 5 + 40 mg/kg prevented the IR injury mediated fall in superoxide dismutase activity being 78.90 ± 6.61 U/mg prot versus 72.75 ± 4.62 U/mg prot (p < 0.05). ICA + PNS tended to attenuate apoptosis in hippocampal CA1 pyramidal neurons. Either ICA or PNS treatment alone did not obviously improve cognitive impairment (except that lipid peroxidation was reduced by PNS‐treatment). The results indicated that ICA + PNS may ameliorate learning and memory deficit and blood viscosity by protecting neurons from oxidative stress in ischemic brain. Copyright

Activating glucocorticoid receptor-ERK signaling pathway contributes to ginsenoside Rg1 protection against β-amyloid peptide-induced human endothelial cells apoptosis.

The deposition of β-amyloid (Aβ) in neurons and vascular cells of the brain has been characterized in Alzheimers disease. Ginsenoside Rg1 (Rg1) is an active components in Panax ginseng, a famous traditional Chinese medicines recorded in Compendium of Materia Medica. Present study attempted to evaluate the potential mechanisms of Aβ-mediated insult and the protective effects of Rg1 on human endothelial cells. Rg1 attenuated the Aβ25-35-associated mitochondrial apoptotic events, accompanied by inhibiting HIF-1α expression followed by intracellular reactive nitrogen species generation, and protein nitrotyrosination. These protective effects were abolished by glucocorticoid receptor (GR) antagonist RU486 or p-ERK inhibitor U0126 rather than estrogen receptor α antagonist ICI 82,780. Taken together, our results suggested that Rg1 protected against Aβ25-35-induced apoptosis at least in part by two complementary GR-dependent ERK phosphorylation pathways: (1) down-regulating HIF-1α initiated protein nitrotyrosination, and (2) inhibiting mitochondrial apoptotic cascades. These data provided a novel insight to the mechanisms of Rg1protective effects on Aβ25-35-induced endothelial cells apoptosis, suggesting that GR-ERK signaling pathway might play an important role in it.

Ginsenoside Rg1 protection against β-amyloid peptide-induced neuronal apoptosis via estrogen receptor α and glucocorticoid receptor-dependent anti-protein nitration pathway.

Ginsenoside Rg1 (Rg1) acts as a neuroprotective agent against various insults, however, the underlying mechanism has not been fully elucidated yet. Here, we report that Rg1 protects primary rat cerebrocortical neurons against β-amyloid peptide₂₅₋₃₅ (Aβ₂₅₋₃₅) injury via estrogen receptor α (ERα) and glucocorticoid receptor (GR)-dependent anti-protein nitration pathway. In primary rat cerebrocortical neuron cultures under basal conditions, Rg1 leads to nuclear translocation of ERα and GR, induces related responsive gene PR, pS₂ and MKP-1, SGK transcription. Meantime, Rg1 also increases the basal level of ERK1/2 phosphorylation. In the presence of toxic level of Aβ₂₅₋₃₅, Rg1 maintains ERK1/2 phosphorylation, attenuates iNOS expression, NO production, and inhibits NF-κB nuclear translocation, protein nitration and cell death. The antiapoptotic effects of Rg1 via both ERα and GR were abolished by small interfering RNAs (siRNA). ERK1/2 phosphorylation inhibitor U0126 can block downstream iNOS expression and NO generation. Interestingly, the anti-protein nitration effect of Rg1 is well matched with ERα and GR activation, although its anti-ROS production effect is in an ERα- and GR-independent manner. These results suggest that Rg1 ameliorates Aβ₂₅₋₃₅-induced neuronal apoptosis at least in part by two complementary ERα- and GR-dependent downstream pathways: (1) upregulation of ERK1/2 phosphorylation followed by inhibiting iNOS expression, NO generation and protein tyrosine nitration. (2) reduction NF-κB nuclear translocation. These data provide new understanding into the mechanisms of Rg1 anti-apoptotic functions after Aβ₂₅₋₃₅ exposure, suggesting that ERα and GR-dependent anti-protein tyrosine nitration pathway might take an important role in the neuroprotective effect of Rg1.

Protective effects of asiatic acid against D-galactosamine/lipopolysaccharide-induced hepatotoxicity in hepatocytes and kupffer cells co-cultured system via redox-regulated leukotriene C4 synthase expression pathway.

Asiatic acid is a triterpenoid component possessing antioxidative, anti-inflammatory and hepatoprotective activity. In this issue, we explored the protective effects of asiatic acid and the relative mechanism in the D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced hepatotoxicity in hepatocytes and kupffer cells co-cultured system. The cultures were pretreated with asiatic acid for 12 h, followed by D-GalN/LPS exposure for 12 h. Asiatic acid reduced aspartate aminotransferase and lactate dehydrogenase generation and increased cell viability in a concentration-dependent manner. Meanwhile, the effects of asiatic acid in leukotriene C(4) synthase (LTC(4)S) expression and cellular redox status including reactive oxygen species and GSH content were detected. The results showed that D-GalN/LPS induced the increase of reactive oxygen species followed by extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear factor-kappaB (NF-kappaB) activation. Treatment with ERK 1/2 specific inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) abolished the ERK1/2 protein phosphorylation and blunted LTC(4)S expression. Reactive oxygen species signaling pathway inhibitor pyrrolidine dithiocarbamate (PDTC) inhibited reactive oxygen species generation and NF-kappaB activation, which in turn blocked LTC(4)S expression and attenuated the injury. Asiatic acid can protect the hepatocytes against D-GalN/LPS-induced hepatotoxicity. During which, the cell redox was ameliorated and increased expression of LTC(4)S was reversed by the pretreatment of asiatic acid. Taken together, asiatic acid can protect against D-GalN/LPS-induced hepatotoxicity partly via redox-regulated LTC(4)S expression pathway.

Ginsenoside Rg1 facilitates neural differentiation of mouse embryonic stem cells via GR-dependent signaling pathway.

Ginsenoside Rg1, a steroidal saponin of high abundance in ginseng, possesses the neuroprotective effects. In this study, we tried to explore the effect of Rg1 on promoting differentiation of mouse embryonic stem (ES) cells towards the neuronal lineage and its potential role involved in glucocorticoid receptor (GR) activation. Rg1 treatment induced a remarkable increase in the population of neuron-like cells in a time-dependent manner. More than 80% of Rg1-treated embryoid bodies (EBs) differentiated into neuron-like cells on d 8+10. Furthermore, the gradually increased protein expression of neurofilament (NEFM) and β-tubulin III (a neuronal specific protein) was determined. GR expression gradually increased during the differentiation course. RU486, an antagonist of GR, could efficiently block the neurogenesis-promoting activity of Rg1. On the other side, Rg1 stimulated the phosphorylation of ERK1/2 and Akt at different time points through GR activation-dependent mechanisms. Treatment of both U0126 (an inhibitor of MEK) and LY294002 (an inhibitor of PI3 K), hampered the neuronal differentiation induced by Rg1. Meantime, U0126 further decreased Rg1-induced p-Akt expression. In conclusion, Rg1 possesses the effects on inducing differentiation of mouse ES cells into neurons in vitro via the GR-MEK-ERK1/2-PI3 K-Akt signaling pathway.