[Protective effect of notoginsenoside R1 on neuron injury induced by OGD/R through ATF6/Akt signaling pathway].

Notoginsenoside R1(NGR1),a critical compound in traditional herb Panax notoginseng, is a kind of estrogen receptor agonist.It is reported to exhibit anti-apoptotic,anti-oxidative and anti-inflammatory properties activity, so it is widely used for treatment of various diseases.In order to investigate the potential neuroprotective effect of NGR1 in hypoxic-ischemic brain damage(HIBD), primary cortical neurons were used in this study to establish oxygen-glucose deprivation/reoxygenation(OGD/R) injury models. They were treated with NGR1 and estrogen receptor inhibitor ICI-182780 respectively, then the neuronal survival, cell membrane integrity and apoptosis were assessed by MTT assay,lactate dehydrogenase test(LDH) and Hoechst 33342 stain respectively, while the protein expression levels of ATF6α,p-Akt,Akt,Bax and Cleaved Caspase-3 were measured by Western blotting. Results indicated that as compared with the blank control group,OGD/R could induce cell injury and apoptosis(P<0.05), reduce relative integrity of cell membrane(P<0.05), decrease protein expression of ATF6α,p-Akt(P<0.05), and increase protein expression of Bax and Cleaved Caspase-3(P<0.05) in the primary cortical cells. After NGR1 treatment, the expression levels of ATF6α,p-Akt were obviously increased, and the expression levels of Bax and Cleaved Caspase-3 and the apoptosis of neuron were decreased(P<0.05). However, these neuroprotective properties of NGR1 against ODG/R-induced cell damage could be blocked by ICI-182780. This finding indicated that NGR1 may protect the primary cortical neurons against OGD/R induced injury,and the mechanism may be associated with accelerating the activation of the ATF6/Akt signaling pathway via estrogen receptors.

[Study on molecular target promoting human neural stem cells of ginsenoside Rg1 by gene chip].

OBJECTIVE: To screen out main molecular target promoting human neural stem cells (NSCs) of ginsenoside Rg1 by using the gene chip technology. METHOD: First, MTT assay was adopted to screen out the optimal concentration of Rg1-promoted NSC proliferation (120 mg x L(-1)). Then, on the 7th day after the Rg1-promoted NSC proliferation, the expression of target genes was observed by the gene chip technology. The most important target gene and signal transduction pathways were screened out through the data calculations. RESULT: On the 7th day after the Rg1-promoted NSC proliferation, obtained 440 differential genes, 266 significantly upregulated genes and 174 significantly down-regulated genes. HES1 gene, CAMP (cyclic adenosine monophosphate)-PKA (protein kinase A) and PI3K (phosphatidylinositol 3 kinase)-AKT signal transduction pathways were closely related to the NSC proliferation. CONCLUSION: The differentially expressed genes screened out by gene chip may provide new clues for studies on molecular mechanism of ginsenoside Rg1-promoted NSCs proliferation.

[Effect of ginsenoside Rg1 on functional expression of human neural stem cells: a patch clamp study].

OBJECTIVE: To observe the effects of ginsenoside Rg1 on the functional expression of human neural stem cells (hNSCs). METHOD: The membrane electrophysiological properties and sodium and potassium ion channels in the hNSCs induced by Rg1 were analyzed using the whole-cell patch-clamp. RESULT: On the 7th day, the neuron-like cells derived from ginsenoside Rg1 (20 mg x L(-1))-induced NSCs show: (1) The resting membrane potential: (-45.70 +/- 2.63) mV, the membrane capacitance: (26.89 +/- 1.91) pF, the membrane input impedance: (877.51 +/- 20.44) MH (P < 0.05 compared with the control group, respectively); (2) The detection rate of inward sodium current which is rapidly activated and inactivated in voltage-dependence was 50%, and its average peak value was (711.48 +/- 158.03) pA (P < 0.05 compared with the control group); (3) The outward potassium currents were composed of rapidly activated and inactivated transient outward potassium current and delayed rectifier outward potassium current, and its average peak value was (1 070.42 +/- 177.18) pA (P < 0.05 compared with the control group). CONCLUSION: Ginsenoside Rg1 can promote the functional expression and maturity of hNSCs.

[The effect of TSPG in vivo on transplantation of neural stem cells in treatment of Parkinson's disease mouse].

AIM: To observe the effect for the model of PD and the transplantation of NSCs after injection of TSPG into mouse in advance. METHODS: Firstly, we divided the mouse into 5 groups. For the group of 1-4, we established the model of PD with MPTP. For the group of 5, before the establishment of the model, we injected TSPG into mouse in advance for prevention. And then, we evaluated the effect by paralysis agitans score standard and praxiology marker. Secondly, we obtained the NSCs from the 7-12 week embryo cerebral cortex. Then we transplanted NSCs which pretreated by TSPG into the striatum of the 5 groups. After 60 days, we obtained the brain section, and detected the TH by ICC to analyse the differentiation status of NSCs. RESULTS: The prevention of TSPG could decrease the neural cells damage by MPTP, and could protect the nervous system. After we transplanted NSCs into the striatum of Parkinson' s disease mouse, we found that for the group of 5, the paralysis agitans, auto-activity and memory function had the most distinct amelioration. And the number of dopaminergic neurons increased most transparently in brain section, and the neurons contact was the most enriched with the adjacent nervous cells. CONCLUSION: TSPG can decrease the neural cells damage and can produce a marked effect in treatment of PD by transplanting NSCs invivo.

[Effect of TSPG on proliferation and differentiation of human embryonic neural stem cell into dopaminergic neuron].

OBJECTIVE: To investigate the effects of total saponins of panax ginseng (TSPG) on proliferation and differentiation of human embryonic neural stem cell (NSC) into dopaminergic neuron. METHOD: Isolation, cultivation and identification of human embryonic NSC from cerebral cortex of 7-12 week abortus. By using flow cytometry and MTT assay, the effects of various concentration of TSPG and TSPG cooperating with cytokines( EGF, bFGF) in NSC culture media for 3 days on proliferation of human embryonic NSC has studied. By employing immunocytochemistry assay of the expression of tyrosine hydroxylase (TH), the effect of different dilution of TSPG and TSPG cooperating with IL-1 on induced differentiation of human embryonic NSC into dopaminergic neuron has researched. RESULT: TSPG can significantly promote the proliferation of NSC. When TSPG cooperating with EGF and bFGF, the proliferation of NSC is much stronger than that of only using FGF and bFGF. TSPG also induces NSC to differentiate into dopaminergic neuron, especially when TSPG is cooperating with IL-1. CONCLUSION: TSPG can not only obviously accelerate the proliferation of NSC, but also significantly induce differentiation of NSC into dopaminergic neuron.

[Synergistic effects of total saponins of panax ginseng in combination with hematopoietic growth factor on proliferation and differentiation of CD34(+) cells ex vivo].

To investigate the effects of total saponins of panax ginseng (TSPG) in combination with hematopoietic growth factors (HGF) on proliferation and differentiation of CD34(+) cells ex vivo, the purified CD34(+) cells from cord blood and bone marrow were expanded by various concentrations of TSPG with combination of cytokines in liquid culture systems and the expanded cell number, CD34(+) cell number, CD33(+) cell ratio, the numbers of total CFC and hematopoietic progenitor cell number were detected. The results showed that TSPG (10 - 70 microg/ml) could raise the expanded cell number, CD34(+) cell number, and the numbers of total CFC, TSPG 50 microg/ml was identified as the most potent stimulating concentration, and increased total nucleated cells to (2470.5 +/- 79.96) x 10(3), CFC to (53.96 +/- 4.286) x 100% and CD34(+) cells to (21.86 +/- 3.094) x 100%; TSPG (10 - 50 microg/ml) could raise the colony formation rate of CFU-GM, TSPG (20 microg/ml) induced the best effect on granulocytopoietic differentiation committed of CD34(+) cells. It is concluded that the optimal concentration of TSPG can promote CD34(+) cells to proliferate and differentiate by cooperating with hematopoietic growth factors.

[Experimental study on expression of GM-CSF from human endothelial cells and monocytes induced by total saponins of panax ginseng].

OBJECTIVE: To study the effect of total saponins of Panax ginseng (TSPG) on the expression of granulocytemacrophage colony-stimulating factor (GM-CSF) from human endothelial cells and monocytes and the relationship between TSPG and human granulocytopoiesis and monocytopoiesis modulation. METHODS: Adopting the hematopoietic progenitor cells culture in vitro, hematopoietic growth factor biological assay, immunocytochemistry and nucleic acid in situ hybridization, the GM-CSF expression in the endothelial cells and monocytes were detected. RESULTS: The conditioned cultural media of endothelial and monocytes induced and prepared by TSPG, could significantly promote the proliferation and differentiation of human colony forming unit-granulocyte macrophage (CFU-GM), and enhance the protein and mRNA expression of GM-CSF in endothelial cells and monocytes. CONCLUSION: TSPG could possibly through direct or indirect route, promote hematopoietic, induce endothelial cells and monocytes in the microenvironment to synthetize and secrete GM-CSF, so as to further promote the proliferation and differentiation of human CFU-GM.

[Modulation of expression of human GM-CSF and GM-CSFRalpha by total saponins of Panax ginseng].

The purpose of the present study was to investigate the biological mechanism for modulating granulocytopoiesis by Panax ginseng. The techniques of culture of hematopoietic progenitor cells and hematopoietic stromal cells in vitro, biological assay of hematopoietic growth factor (HGF), immunocytochemistry, in situ hybridization of nucleic acid, immunoprecipitation and Western blot were used to explore the effect of total saponins of Panax ginseng (TSPG) on the expression of human granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-macrophage colony stimulating factor receptor alpha (GM-CSFRalpha). The results indicated that (1) bone marrow stromal cell (BMSC), thymocyte (TC), splenocyte (SC), endothelial cells (EC), and monocyte (MO) conditioned media prepared with TSPG (50 microg/ml) could significantly enhance the proliferation of CFU-GM; (2) the expressions of GM-CSF in protein and mRNA level in BMSC, TC, SC, EC and MO induced by TSPG (50 microg/ml) were much higher than that of the control; (3) the expression of GM-CSFRalpha protein in hematopoietic cells induced by TSPG (50 microg/ml) was stronger than that of the control; (4) TSPG (50 microg/ml) could stimulate the transient tyrosine phosphorylation of GM-CSFR and Shc protein. We speculate that TSPG may directly and/or indirectly promote the stromal cells and lymphocytes to produce GM-CSF and other cytokine and induce bone marrow hematopoietic cells to express GM-CSF receptors (GM-CSFRalpha), leading to the regulation of the GM-CSFR-mediated signals transduction pathway and the proliferation of human CFU-GM.