| Neural degenerative disease and neuronal injuries are commonly encountered disease in nervous system. The prognosis of these diseases is close related to survival of nerve cell, neuronal differentiation and functional recovery of neurons. Neuronal differentiation is affected by the activation or inhibition of related genes. Topoisomerase Ⅱβ(TopoⅡβ) is a critical nuclear enzyme for neural development and neuronal differentiation. Tetramethylpyrazine(TMP), also called 2,3,5,6-Tetramethylpyrazine or Ligustrazine, was an active alkaloidal extracted and purified from traditional Chinese medicinal herb Chuanxiong. Recent studies have shown that TMP induces differentiation of neural stem cells or neurogenesis after nervous injury. However, the mechanisms under these functions are unclear. In this study, we use the SH-SY5 Y cell model to detect the neuronal differentiation induced by TMP. We further examined the changes of TopoⅡβ in this process to determin the critical role of Topo Ⅱ β in the TMP-induced neuronal differentiation. Finally, the molecular mechanisms under how TMP induces the expression of Topo Ⅱ β to promote neuronal differentiation were investigated. There are three parts in this study.Part 1 The effect of TMP on the growth and neuronal differentiation of SH-SY5 Y cellObjective: To study TMP’s effects on the growth and neuronal differentiation of SH-SY5 Y cells.Methods: 1. Cell culture: SH-SY5 Y cells were cultured with the DMEM culture medium in humidified incubator with 5% CO2. 2. Cell viability was measured by MTT assay. 3. Lactate dehydrogenase(LDH) release assay was used to detect the percentage of LDH release to evaluate cytotoxicity of TMP on SH-SY5 Y cells. 4. Cell cycle and apoptosis rate detection: Cells were collected on 0, 3 and 5 days after TMP treatment. Flow cytometry was used to analyze the percentage of G0/G1, S and G2/M phase, and the apoptotic rate was also evaluated. 5. Nuclear staining by DAPI: The cells were continually incubated with 80 μmol/L TMP. Nuclear staining was conducted on 0, 1, 3 and 5 days by DAPI and karyomorphology was observed. 6. Caspase-3 activity detection: The cells were continually incubated with 80 μmol/L TMP. Cells were collected on 0, 1, 3 and 5 days after TMP treatment and Caspase-3 activity was examined. 7. Morphological properties and the percentage of differentiated cells: The cell numbers and neurite outgrowth were evaluated and analyzed. Cells were considered differentiated when the total neurite length was longer than 100 μm for each cell. Differentiated cells percentages were calculated. 8. Western blot was used to detect the protein expression of p21 and the neuronal markers MAP2 and tau. 9. Statistical analysis: The results were expressed as mean±standard deviation(SD) and evaluated by analysis of variance(One-Way ANOVA).P<0.05 was considered statistically significant.Results: 1 Cell proliferation tested by MTT assayThe proliferation of SH-SY5 Y cell was inhibited by TMP under concentrations of 40, 80, 120, 160, 320 and 400 μmol/L respectively(P<0.05). However, TMP treatment with 20 μmol/L showed no inhibition effects on the cell(P>0.05). These data indicated TMP could inhibit the cell proliferation in a time and dose dependent manner.2 Cytotoxicity detected by LDH release assay The percentage of LDH release showed no statistical significant difference between 40,80 and 120μmol/L TMP treated cell on 1, 3 and 5 days respectively(P>0.05). The LDH release in 160 μmol/L TMP-treated cells for 5 days was 16.1±1.50%, with significant difference from 11.2±1.20% in the control cells(P<0.05). LDH release of 240 μmol/L TMP-treated cells was14.54±2.00%, 16.63±2.00% and 19.53±3.32% on 1, 3 and 5 days, respectively, whereas the results for control cells were 11.7±1.34%, 11.1±1.56, 11.2±1.20%. There are statistical differences between the two groups on each time point(P<0.05). The data suggested that TMP with low concertrations(40,80 and 120 μmol/L) have no cytotoxic effect on the cells.3 TMP’s effects on cell cycle and apoptosis The percentage of G0/G1 phase of the TMP(80 μmol/L) treated cell increased significantly in contrast to that of the control cell after 3 or 5 days treatment(P<0.05), indicating cell cycle arrest at G0/G1 phase. As revealed by flow cytometry, there are no apoptotic peak before the diploid peak both in TMP-treated and control cells. No nuclear morphologic changes were found after SH-SY5 Y cells were treated with TMP for 5 days. Followed by DAPI staining, there were no nuclear fragmentation, apoptotic body and other apoptotic changes. Both the Caspase-3 activity and active-caspase-3 expression showed no significant difference between TMP-treated cells and the control cells(P>0.05). These results indicated that TMP(80 μmol/L) induced neuronal differentiation of SH-SY5 Y cell without leading to apoptosis.4 Morphological changes and percentage of differentiation of SH-SY5 Y cells SH-SY5 Y cell growth was slowed down by TMP. Neurite outgrowth and percentage of differentiation increase significantly upon TMP treatment except the cells treat with 20 μmol/L TMP. The cells exhibited a neuron-like phenotype with neurite extending more than 100 μm in average length for each cell. The neurite outgrowth of cells treated with 40 μmol/L TMP started to be obvious as early as 2 days. The neurite outgrowth of SH-SY5 Y cells treated with 80 μmol/L and 120 μmol/L are the longest among that of all treated cell, and are no significant difference between these two groups(P>0.05).5 Neuronal differentiation identified by neuronal markers The neuronal markers, microtubule-associated protein 2(MAP2) and tau increased significantly after TMP(80 μmol/L) treatment for 3 and 5 days. There are significant differences in comparing with the control cells(P<0.05).Conclusions: TMP(80 μmol/L) can induce G0/G1 cell cycle arrest and promote neuronal differentiation of SH-SY5 Y cell with no cytotoxicity. We select 80 μmol/L of TMP in the subsequent experiments.Part 2 The epigenetic mechanisms of TMP-induced differentiation of SH-SY5 Y cellsObjective: To evaluate the expression of Topo Ⅱ β correlated with TMP-induced neuronal differentiation of SH-SY5 Y cell and the related epigenetic mechanisms.Methods: 1. Cell culture: SH-SY5 Y cells were cultured with the DMEM culture medium in humidified incubator with 5% CO2. 2. Morphological properties and the percentage of differentiated cells: The cell numbers and neurite outgrowth were evaluated. Cells were considered differentiated when the total neurite length was longer than 100 μm for each cell. Differentiated cells percentages were calculated. 3. Protein expression of TopoⅡα, TopoⅡβ, histone H3 and H4, acetylated histone H3(Ac-H3) and acetylated histone H4(Ac-H4) by Western blot. 4. Reverse transcription PCR(RT-PCR) analyzes the m RNA expression. 5. Chromatin Immunoprecipitation(Ch IP)assay to examine the association of Ac-H3 and Ac-H4 to TopoⅡβ promoter. 6. Statistical analysis: same as part 1.Results: 1 The expression of TopoⅡβ and TopoⅡα after TMP treatment The TopoⅡα protein expression in SH-SY5 Y cells was down-regulated after TMP treatment(80 μmol/L) after 3 and 5 days. On the contrary, the expression of TopoⅡβ was up-regulated by TMP(P<0.05).2 RT-PCR detection of TopoⅡβ m RNA expression The expression of TopoⅡβ m RNA increased significantly after 3 and 5 days upon TMP treatment in contrast to the control cell(P<0.05).3 TopoⅡβ inhibitor ICRF-193 inhibited the TMP-induced neuronal differentiation of SH-SY5 Y cellsThe expression of TopoⅡβ and MAP2 protein increased significantly after 5 days of TMP treatment. However, their expressions were both attenuated by Topo Ⅱ inhibitor ICRF-193 in a dose-dependent manner(P<0.05). Neurite outgrowth was repressed by ICRF-193 both in quantity and length(P<0.05), suggesting that TopoⅡβ palys a pivotal role in TMP-induced neuronal differentiation.4 TMP induced hyperacetylation of histone H3 and H4 in SH-SY5 Y cellsThe expression of Ac-H3 was up-regulated from the third day after TMP treatment(P<0.05). However, Ac-H4 expression was promoted from the fifth day of TMP treatment(P<0.05). The results reflected that both Ac-H3 and Ac-H4 could be induced by TMP, while it occurred earlier in Ac-H3 than in Ac-H4.5 TMP induced neuronal differentiation through enhancing the acetylation of histone H3 and H4 in the TopoⅡβ gene promoter.As confirmed by ChIP assay, Ac-H3 and Ac-H4 were more tightly associated with the Topo Ⅱ β gene promoter during TMP induction differentiation of SH-SY5 Y cells. Moreover, the association of Ac-H3 started at an early time than that of Ac-H4.Conclusions: It was demonstrated for the first time that TMP induces neuronal differentiation of SH-SY5 Y cell through up-regulation of TopoⅡβ. The associations of Ac-H3 and Ac-H4 with TopoⅡβ promoter region play pivotal roles in TMP-induced Topo Ⅱ β expression and neuronal differentiation.Part 3 Effects of PI3K/Akt signal pathway on TMP-induced neuronal differentiation of SH-SY5 Y cellObjective: To study the effects of cell signal molecules PI3K/Akt and ERK1/2 on the expression of TopoⅡβ and the underlying mechanisms in the process of TMP-induced neuronal differentiation.Methods: 1. Cell culture: SH-SY5 Y cells were cultured with the DMEM culture medium at 37 ℃ in humidified incubator with 5% CO2. 2. Morphological properties and the percentage of differentiated cells: The cell numbers and neurite outgrowth were evaluated. Cells were considered differentiated when the total neurite length was longer than 100 μm for each cell. Differentiated cells percentages were calculated. 3. Protein expressions were detected by Western blot. 4. RT-PCR analyzes the m RNA expression of TopoⅡα and TopoⅡβ. 5. Ch IP assay to detect the association of NF-Y and Sp1 with TopoⅡβ gene promoter. 6. Statistical analysis: same as part 1.Results: 1 The expressions of p-Akt, p-ERK1/2, TopoⅡα, TopoⅡβ, Sp1 and NF-YA proteins after TMP treatmentThe proteins were detected on 0, 1, 3 and 5 days of TMP treatment. Results demonstrated that TopoⅡα was decreased, in contrast, TopoⅡβ protein was upregulated which was consistent with p-Akt and Sp1(P<0.05). The expression of p-ERK1/2 and NF-YA proteins did not alter significantly after TMP treatment(P>0.05).2 Effects of PI3K/Akt pathway inhibitor LY294002 on the expressions of p-AKT, p-ERK1/2, TopoⅡα, TopoⅡβ, Sp1 and NF-YA proteinsWestern blot showed that the expressions of Topo Ⅱ α and NF-YA induced by TMP were not altered by LY294002(P>0.05). However, the up-regulated expressions of p-Akt, Sp1 and Topo Ⅱ β were remarkable decreased by LY294002(P<0.05).3 LY294002 inhibited TMP-induced neurite extention and neuronal marker expressionThe neurite length was decreased from 150.97±6.62 μm to 91.80±7.82 μm and the differentiated cells percentage declined from 80.04±3.26% to 46.2±33.56%(P<0.05) after treatment of LY294002. Meanwhile, LY294002 inhibited the TMP-induced expression of neuronal marker MAP2. However,the MEK/ERK inhibitor U0126 could not alter the morphological changes and neuronal marker MAP2 expression induced by TMP. The data indicated that PI3K/Akt may participate in TMP-induced neuronal differentiation of SH-SY5 Y cell.4 The changes of TopoⅡ m RNA expression under TMP treatment with or without LY294002PI3K/Akt inhibitor LY294002 reduced the TMP-induced Topo Ⅱ β m RNA expression(P<0.05), while it had no effect on the expression of TopoⅡα m RNA(P>0.05). TMP-induced TopoⅡβ m RNA expression could be affected by RNA polymerase Ⅱinhibitor α-amanitin, not be affected by protein synthesis inhibitor Cycloheximide(CHX), indicating that TMP activated TopoⅡβ expression on the transcriptional level.5 The association of transcription factor Sp1 and NF-YA with TopoⅡβ gene promoterThe association of transcription factor Sp1 and NF-YA with TopoⅡβ gene promoter was promoted by TMP treatment(P<0.05). The association of Sp1 could be inhibited by LY294002, in contrast, the association of NF-YA with TopoⅡβ gene promoter could not be affected significantly by LY294002(P>0.05).Conclusions: PI3K/Akt signal pathway was activated during the neuronal differentiation process induced by TMP. Activated PI3K/Akt enhanced the association of Sp1 to Topo Ⅱβ gene promoter and resulted in up-regulating Topo Ⅱβ gene expression. PI3K/Akt/Sp1/TopoⅡβ signaling pathway regulates TMP-induced neuronal differention of SH-SY5 Y cell. |
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