In this research we aimed to investigate the interactions between growth factors (GFs) and dexame... more In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under the following experimental condition: 24h pretreatment with bFGF, subsequent 72h switching in serum-free medium (SFM) and final addition of GFs, only one or more in the last 24h, after a prolonged (60h) DEX treatment. Western blot analysis data showed a marked GFAP expression in cultures submitted to our experimental condition comparing results to untreated or treated controls. In particular, the maximum level of GFAP expression is observed when EGF or INS or both together are added in a prolonged 60h DEX treatment. This finding well correlates with differentiative role played by glucocorticoids interacting with the "competence" factor bFGF and demonstrates as increased GFAP expression mostly depends on maturation, rather than proliferating status of astroglial cells in culture. VIM expression was i...
Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell di... more Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell differentiation. Further, some proteins related to cell cycle including cyclins and p21 are important markers of astrocyte cultures. Aim of investigation was to study the effects of cholinergic precursors (choline, CDP-choline, Acetylcholine and a-Glyceril-Phosphorylcholine) on HO-1 and p21 expression during astroglial cell proliferation and differentiation in primary cultures at 14 and 35 days in vitro (DIV) treated for 24 h with choline metabolites. Our results showed a slight reduction of HO-1 expression (data not statistical significant) in astroglial cell cultures treated with CDP-choline at 14 DIV and 35 DIV. On the contrary, ACh and choline induced a significant increase of HO-1 expression in 14 DIV astrocyte cultures. Surprisingly, choline and ACh dramatically reduced HO-1 expression at 35 DIV. A slight decrease not statistical significant was detectable for a-GPC at 14 DIV and particularly significant at 35 DIV. Data concerning p21 expression, a well known protein inhibiting cell cycle, evidenced a significant increase at 14 and 35 DIV after a-GPC treatment. CDP-choline treatment caused a high increase of p21 expression in 14 DIV astrocyte cultures, but no modification at 35 DIV. Instead, ACh treatment induced a marked increment of p21 expression at 35 DIV. Our data suggest that cholinergic precursors modulate HO-1 and p21 expression during astroglial cell proliferation and differentiation in culture and could be considered a tool to study the induced effects of ischemia and hypoxia diseases in some in vitro models to prevent and reduce its effects after treatment with cholinergic drugs.
The aim of the present investigation is to study the effects of DEX or E(2) treatment during diff... more The aim of the present investigation is to study the effects of DEX or E(2) treatment during differentiation towards neural cell line of rat BM-MSCs in culture. In order to better characterize biochemically our in vitro model, we evaluate by western blotting and immunocytochemical analysis some neural lineage markers (nestin, neurofilament, β-tubulin) and MAP-Kinases. An enhanced expression of the neural markers and MAP-Kinase in DEX-treated BM-MSCs cultures is found. In addition, E(2)-treatment increases MAP-Kinase and β-tubulin expression, but it decreases nestin and neurofilament expression. In conclusion, our findings highlight a significant up and down modulation of nestin, neurofilament, β-tubulin and MAP-Kinases expression in neurosteroids-treated BM-MSCs. In particular, our results clarify the molecular mechanism involved during eventual differentiation of these stem cells treated with DEX and E(2), addressed towards a neural cell line, that may express neurotrophic receptors and release neurotrophines particularly implicated during neurogenesis processes.
In the present study, we evaluated the expression of some proliferation and differentiation marke... more In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 DIV astrocyte cultures pretreated or not with 0.5 mM glutamate for 24 h and than maintained under chronic or acute treatment with 50 μM R(+)enantiomer or raceme alpha-lipoic acid (ALA). GFAP expression significantly increased after (R+)enantiomer acute-treatment and also in glutamate-pretreated ones. Vimentin expression increased after R(+)enantiomer acute-treatment, but it decreased after raceme acute-treatment. Nestin expression drastically increased after acute raceme-treatment in glutamate-pretreated or not cultures, but significantly decreased after (R+)enantiomer acute and chronic-treatments. Cyclin D1 expression increased in raceme acute-treated cultures pretreated with glutamate. MAP-kinase expression slightly increased after (R+)enantiomer acute treatment in glutamate-pretreated or unpretreated ones. These preliminary findings may better clarify antioxidant and metabolic role played by ALA in proliferating and differentiating astrocyte cultures suggesting an interactive cross-talk between glial and neuronal cells, after brain lesions or damages.
Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in gl... more Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in glucose metabolism of different cell types. Choline alphoscerate (a-glyceryl-phosphoryl-choline [aGPC]) is a semisynthetic derivative of phosphatidylcholines representing, among acetilcholine precursors, a cholinergic drug. In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte cultures treated with 50 lM (1)lipoic acid or (1/2)lipoic acid and/or 10 mM aGPC for 24 hr. In addition, we evaluated the possible genoprotective effect by analysis of DNA status detected by alkaline comet assay. The addition of single drugs [(1)lipoic acid, (1/2)lipoic acid, or aGPC] induced an "upward modulation" of the expression of biomarkers used in our study. On the contrary, the cotreatment with either (1)lipoic acid 1 aGPC or (1/2)lipoic 1 aGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned biomarkers. This latter finding demonstrated no additional effect after the cotreatment with both drugs with respect to the single treatments alone. Further studies are necessary to clarify the specific mechanism evoked by the processing of these neuroprotective agents in our in vitro models. Finally, these preliminary findings may represent a good tool with which to clarify the antioxidant and metabolic roles played by lipoic acid in proliferating and differentiating astroglial cell cultures, during an interactive cross-talk between glial and neuronal cells, after brain lesions or damage correlated with oxidative stress that may occur in some degenerative diseases.
This article may be used for research, teaching and private study purposes. Any substantial or sy... more This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
In this research we aimed to investigate the interactions between growth factors (GFs) and dexame... more In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under the following experimental condition: 24h pretreatment with bFGF, subsequent 72h switching in serum-free medium (SFM) and final addition of GFs, only one or more in the last 24h, after a prolonged (60h) DEX treatment. Western blot analysis data showed a marked GFAP expression in cultures submitted to our experimental condition comparing results to untreated or treated controls. In particular, the maximum level of GFAP expression is observed when EGF or INS or both together are added in a prolonged 60h DEX treatment. This finding well correlates with differentiative role played by glucocorticoids interacting with the "competence" factor bFGF and demonstrates as increased GFAP expression mostly depends on maturation, rather than proliferating status of astroglial cells in culture. VIM expression was i...
Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell di... more Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell differentiation. Further, some proteins related to cell cycle including cyclins and p21 are important markers of astrocyte cultures. Aim of investigation was to study the effects of cholinergic precursors (choline, CDP-choline, Acetylcholine and a-Glyceril-Phosphorylcholine) on HO-1 and p21 expression during astroglial cell proliferation and differentiation in primary cultures at 14 and 35 days in vitro (DIV) treated for 24 h with choline metabolites. Our results showed a slight reduction of HO-1 expression (data not statistical significant) in astroglial cell cultures treated with CDP-choline at 14 DIV and 35 DIV. On the contrary, ACh and choline induced a significant increase of HO-1 expression in 14 DIV astrocyte cultures. Surprisingly, choline and ACh dramatically reduced HO-1 expression at 35 DIV. A slight decrease not statistical significant was detectable for a-GPC at 14 DIV and particularly significant at 35 DIV. Data concerning p21 expression, a well known protein inhibiting cell cycle, evidenced a significant increase at 14 and 35 DIV after a-GPC treatment. CDP-choline treatment caused a high increase of p21 expression in 14 DIV astrocyte cultures, but no modification at 35 DIV. Instead, ACh treatment induced a marked increment of p21 expression at 35 DIV. Our data suggest that cholinergic precursors modulate HO-1 and p21 expression during astroglial cell proliferation and differentiation in culture and could be considered a tool to study the induced effects of ischemia and hypoxia diseases in some in vitro models to prevent and reduce its effects after treatment with cholinergic drugs.
The aim of the present investigation is to study the effects of DEX or E(2) treatment during diff... more The aim of the present investigation is to study the effects of DEX or E(2) treatment during differentiation towards neural cell line of rat BM-MSCs in culture. In order to better characterize biochemically our in vitro model, we evaluate by western blotting and immunocytochemical analysis some neural lineage markers (nestin, neurofilament, β-tubulin) and MAP-Kinases. An enhanced expression of the neural markers and MAP-Kinase in DEX-treated BM-MSCs cultures is found. In addition, E(2)-treatment increases MAP-Kinase and β-tubulin expression, but it decreases nestin and neurofilament expression. In conclusion, our findings highlight a significant up and down modulation of nestin, neurofilament, β-tubulin and MAP-Kinases expression in neurosteroids-treated BM-MSCs. In particular, our results clarify the molecular mechanism involved during eventual differentiation of these stem cells treated with DEX and E(2), addressed towards a neural cell line, that may express neurotrophic receptors and release neurotrophines particularly implicated during neurogenesis processes.
In the present study, we evaluated the expression of some proliferation and differentiation marke... more In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 DIV astrocyte cultures pretreated or not with 0.5 mM glutamate for 24 h and than maintained under chronic or acute treatment with 50 μM R(+)enantiomer or raceme alpha-lipoic acid (ALA). GFAP expression significantly increased after (R+)enantiomer acute-treatment and also in glutamate-pretreated ones. Vimentin expression increased after R(+)enantiomer acute-treatment, but it decreased after raceme acute-treatment. Nestin expression drastically increased after acute raceme-treatment in glutamate-pretreated or not cultures, but significantly decreased after (R+)enantiomer acute and chronic-treatments. Cyclin D1 expression increased in raceme acute-treated cultures pretreated with glutamate. MAP-kinase expression slightly increased after (R+)enantiomer acute treatment in glutamate-pretreated or unpretreated ones. These preliminary findings may better clarify antioxidant and metabolic role played by ALA in proliferating and differentiating astrocyte cultures suggesting an interactive cross-talk between glial and neuronal cells, after brain lesions or damages.
Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in gl... more Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in glucose metabolism of different cell types. Choline alphoscerate (a-glyceryl-phosphoryl-choline [aGPC]) is a semisynthetic derivative of phosphatidylcholines representing, among acetilcholine precursors, a cholinergic drug. In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte cultures treated with 50 lM (1)lipoic acid or (1/2)lipoic acid and/or 10 mM aGPC for 24 hr. In addition, we evaluated the possible genoprotective effect by analysis of DNA status detected by alkaline comet assay. The addition of single drugs [(1)lipoic acid, (1/2)lipoic acid, or aGPC] induced an "upward modulation" of the expression of biomarkers used in our study. On the contrary, the cotreatment with either (1)lipoic acid 1 aGPC or (1/2)lipoic 1 aGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned biomarkers. This latter finding demonstrated no additional effect after the cotreatment with both drugs with respect to the single treatments alone. Further studies are necessary to clarify the specific mechanism evoked by the processing of these neuroprotective agents in our in vitro models. Finally, these preliminary findings may represent a good tool with which to clarify the antioxidant and metabolic roles played by lipoic acid in proliferating and differentiating astroglial cell cultures, during an interactive cross-talk between glial and neuronal cells, after brain lesions or damage correlated with oxidative stress that may occur in some degenerative diseases.
This article may be used for research, teaching and private study purposes. Any substantial or sy... more This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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