Gardenoside Increases Astrocyte's Neuroprotective Effect On Dopaminergic Neurons By Inhibiting LPS-induced Secretion Of Inflammatory Factors

Part I Objective The purpose is to obtain neuron-enriched system including the more purified dopaminergic neurons and settle background for further study.Methods The ventral mesencephallic tissue of E12inbred Sprague-Dawley rat embryos was harvested and cultured in the medium supplemented with basic fibroblast growth factor /F12/N2/ L-ascorbic acid-2-phosphate sesquimagnesium salt for five days,and subsequently promoted to differentiate into dopaminergic neurons in special free basic fibroblast growth factor medium containing L-ascorbic acid-2-phosphate sesquimagnesium salt during the later four days .Lastly,we inhibited the survival of glia by neurobasal/ arabinosylcytosin.The cells was identified and counted by immunohistochemical staining with anti- rat tyrosine hydroxylase and anti-ratβ-tubulinⅢantibodies.Results The mesencephalic progenitor cells expanded well under the medium supplemented with basic fibroblast growth factor /F12/ N2/ L-ascorbic acid-2- phosphate sesquimagnesium salt.Within five to seven days in vitro,the cell number multiplied with about a (16.54±1.25)-fold.Then the survival of glia was inhibited when neurobasal/ arabinosylcytosin medium was added.12 days later,neurons constitute above 94% of total cells, about (35.56±4.13)% of the total cell population differentiated into dopamine neurons.Conclusion Neuron-enriched system comprising high percentage dopamine neurons could be obtained through primary culture of mesencephalic crogenitor cells of E12 Rats combining basic fibroblast growth factor/L-ascorbic acid-2- phosphate sesquimagnesium salt and neurobasal/arabinosylcytosin. Part IIObjective The purpose was to explore the protective effect and probable mechanism of gardenoside on the damage of dopaminergic neurons induced by lipopolysaccharide.Methods Both neuron-enriched cultures and neuron-astrocyte cultures were pretreated with vehicle or gardenosides (10,20 and 40mg/L) for 30min at 37°C. Subsequently the culture media were renewed in order to remove gardenosides, and lipopolysaccharide was added into all culture media at a final concentration of 10mg/L. 24h later,the culture media were collected for measurement of tumor necrosis factor-alpha, nitric oxide, interleukin-6, GDNF and matrix metallopeptidase -9;the cells were collected for counting the number of cells labeled with an antibody against tyrosine hydroxylase and assessment of expression of tyrosine hydroxylase mRNA by Reverse Transcription-Polymerase Chain Reaction.Results Astrocytes play a neuroprotective role on dopaminergic neurons.Gardenoside didn't promoted the survival of dopaminergic neurons in neuron-enriched culture, but significantly increased the survival of dopaminergic neurons in neuron-astrocyte culture by 57%.The amount of tumor necrosis factor-alpha, nitric oxide and GDNF released from the neuron-astrocyte cultures after 24h of addition of lipopolysaccharide was not changed significantly,while the expression of interleukin-6,matrix metallopeptidase-9 was increased significantly.In this study,we found that gardenoside concentration-dependently attenuated the lipopolysaccharide-induced increase in expression of interleukin-6 and matrix metallopeptidase -9.Conclusions Astrocytes play a neuroprotective role on dopaminergic neurons and lipopolysaccharide could decrease this role by inducing the secretion of pro-inflammatory factors.Gardenoside may protect dopaminergic neurons from lipopolysaccharide-induced injury in an astrocyte-dependent manner and it is inhibition in production of proinflammatory factors,not promotion of secretion of GDNF that contributes to the effect. In view of the well-documented very low toxicity of gardenosides,this report may point the way to an important new direction for developing therapeutic interventions for inflammation-related diseases such as Parkinson's disease.