| Part ? The role of histone acetylation in the expression and modification of neurocytes' transcription factor Nrf2 induced by manganese chlorideObjective In this study, PC12 cell were selected as cell model of dopaminergic neurons. And histone acetylase inhibitor anacardic acid(AA) and histone deacetylase inhibitor trichostatin A(TSA) were selected as the pre-treatment reagent. The protein expression level of Nrf2, the m RNA expression level of Nrf2 and Nrf2 acetylation level were measured to explore the relationship between histone acetylation and Nrf2 protein high level expression activated by manganese chloride in PC12 cell.Methods PC12 cells were treated with AA(8.5 ?M) for 1 hour or TSA(100 ng/ml) for 6 h,followed by exposure to three doses(0 ?M, 100 ?M and 300 ?M) of Mn Cl2 for 24 h,respectively. After that, nuclear protein and cytoplasmic protein were extracted from PC12 cells and examined the protein level of Nrf2 by Western Blot; The total m RNA were extracted to examine m RNA level of Nrf2 by RT-PCR; The total protein extracted to examine the level of Nrf2 acetylation by immunoprecipitation and western blot.Results(1) A. PC12 cells were treated with three doses(0 ?M, 100 ?M and 300 ?M) of Mn Cl2 for 24 h, respectively. The result indicated that the Mn group(300 ?M Mn Cl2 for24 h) had significant higher protein level of nuclear/cytoplasm Nrf2 compared to the group with normal saline treatment(P<0.05). B. PC12 cells were incubated with or without TSA 6 h, followed by exposure to three doses(0 ?M, 100 ?M and 300 ?M)Mn Cl2 for 24 h, respectively. The Mn group(300 ?M Mn Cl2 for 24 h) with TSA treatment had significant higher nuclear transition of Nrf2 and m RNA level of Nrf2 compared to the Mn group(100 ?M Mn Cl2 for 24 h) without TSA treatment(P<0.05).C. PC12 Cells were incubated with or without AA 1h, followed by exposure to three doses(0 ?M, 100 ?M and 300 ?M) of Mn Cl2 for 24 h, respectively.The result indicated that: a. The AA treatment groups showed higher m RNA level of Nrf2 compared to the normal saline groups,(P<0.01); b. The Mn group(100 ?M Mn Cl2 for 24 h) with AA treatment had significant higher m RNA level of Nrf2 compared to the Mn group(100?M Mn Cl2 for 24 h) without AA treatment(P<0.01); c. The Mn group(300 ?M Mn Cl2 for 24 h) with AA treatment also had higher m RNA level of Nrf2 compared to the Mn group(300 ?M Mn Cl2 for 24 h) without AA treatment(P<0.01).(2) PC12 cells were treated with AA(8.5 ?M) for 1 hour or TSA(100 ng/ml) for 6h, followed by exposure to three doses(0 ?M, 100 ?M and 300 ?M) of Mn Cl2 for 24 h,respectively. The result indicated that all the treatment groups showed no difference in Nrf2 acetylation compared to the control group(P>0.05).Conclusions Pre-treatment with TSA could promote the effectt that the nuclear import of the transcription factor Nrf2 which was induced by Mn Cl2 in a certaint condition. In a certaint condition, both AA and TSA increase the m RNA level of Nrf2 that treated with Mn Cl2. The results indicated that histone acetylation inhibitor(TSA or AA) and Mn Cl2 treatment might not affect the Nrf2 acetylation in PC12 cell.Part ? The role of histone acetylation in Nrf2/HO-1 pathway activation by manganese chloride in nerve cellsObjective Preliminary study results show that HDACs inhibitors TSA pretreatment can promote nuclear transfer of Nrf2 protein induced by manganese chloride. In this study,histone acetylase inhibitor anacardic acid(AA) and histone deacetylase inhibitor trichostatin A(TSA) were selected as the pre-treatment reagent. Nrf2-ARE complex activity, the protein expression level of HO-1 and the m RNA expression level of HO-1were measured to explore the relationship between histone acetylation and Nrf2/HO-1signaling pathway in PC12 cell.Methods PC12 cells were treated in the same way. After that, nuclear protein were extracted from PC12 cells and examined the binding activity of Nrf2-ARE by ELISA Kit; The RNA were extracted to examine m RNA level of HO-1 by q RT-PCR; The total protein were extracted to examine protein level of HO-1 by Western Blot.Results(1) PC12 cells were treated with three doses(0 ?M, 100 ?M and 300 ?M) of Mn Cl2 for 24 h, respectively. The result indicated that: A. The Mn group(100 ?M Mn Cl2 for 24 h) had significant higher binding activity of Nrf2 and ARE and protein level of HO-1 compared to the group without Mn Cl2 treatment(P<0.01); B. The Mn group(300 ?M Mn Cl2 for 24 h) had significant higher m RNA level of HO-1 and protein level of HO-1compared to the control group(P<0.01).(2) PC12 cells were incubated with or without TSA 6 h, followed by exposure to three doses(0 ?M, 100 ?M and 300 ?M) Mn Cl2 for 24 h, respectively. The result indicated that: A. The Mn group(100 ?M Mn Cl2 for 24 h) with TSA treatment had significant higher binding activity of Nrf2 and ARE, m RNA level of HO-1 and protein level of HO-1 compared to the Mn group(100 ?M Mn Cl2 for 24 h) without TSA treatment(P<0.01); B.The Mn group(300 ?M Mn Cl2 for 24 h) with TSA treatment also had higher m RNA level of HO-1 and protein level of HO-1 compared to the Mn group(300 ?M Mn Cl2 for 24 h) without TSA treatment(P<0.01).(3) PC12 Cells were incubated with or without AA 1h, followed by exposure to three doses(0 ?M, 100 ?M and 300 ?M) of Mn Cl2 for 24 h, respectively. The result indicated that: A. the Mn group(100 ?M Mn Cl2 for 24 h) with AA treatment had significant reduce the binding activity of Nrf2 and ARE and enhenced m RNA level of HO-1 compared to the Mn group(100 ?M Mn Cl2 for 24 h) without AA treatment(P<0.01); B. the Mn group(300 ?M Mn Cl2 for 24 h) with AA treatment had significant reduce the binding activity of Nrf2 and ARE and enhenced m RNA level of HO-1compared to the Mn group(300 ?M Mn Cl2 for 24 h) without AA treatment(P<0.01).Conclusions TSA could promote binding activity of Nrf2 and ARE induced by Manganese chloride, while AA could restrain binding activity of Nrf2 and ARE induced by Manganese chloride in a certaint condition. Also, both AA and TSA promote the increase of m RNA level of HO-1 induced by Manganese chloride, but only TSA could promote the increase of protein level of HO-1 induced by Manganese chloride. In conclusion, hyperacetylation could have protective effect in the Neurotoxic effect of manganese.Part ? The role of histone acetylation in increasing ROS generation and reducing the role of GSH levels induced by Mn Cl2Objective Preliminary study results show that HDACs inhibitors TSA pretreatment could promote the level of binding activity of Nrf2 and ARE induced by manganese chloride.In this study, histone acetylase inhibitor anacardic acid(AA) and histone deacetylase inhibitor trichostatin A(TSA) were selected as the pre-treatment reagent. The accumulation of reactive oxygen species(ROS) and consumption of reduced glutathione(GSH) were measured to explore the relation among histone acetylation,ROS and GSH in PC12 cell.Methods PC12 cells were treated in the same way. After that, PC12 cells were examined the ROS level by DCFH-DA and the GSH content by Microplate Reader.Results(1) PC12 cells were treated with three doses(0 ?M, 100 ?M and 300 ?M) of Mn Cl2 for 24 h, the result indicated that: A. The Mn group(100/300?M Mn Cl2 for 24 h)had significant enhenced ROS level compared to the contol group(P<0.01). B. The Mn group(300 ?M Mn Cl2 for 24 h) had significant reduced GSH compared to the contol group(P<0.01).(2) PC12 cells were incubated with or without TSA 6 h, followed by exposure to three doses(0 ?M, 100 ?M and 300 ?M) Mn Cl2 for 24 h, respectively. The result indicated that: A. The Mn group(100 ?M Mn Cl2 for 24 h) with TSA treatment had significant lower ROS level compared to the Mn group(100 ?M Mn Cl2 for 24 h) without TSA treatment(P<0.01). B. The Mn group(300 ?M Mn Cl2 for 24 h) with TSA treatment had significant lower GSH compared to the Mn group(300 ?M Mn Cl2 for 24h) without TSA treatment(P<0.05).Conclusions TSA could restrain the effect that the ROS level increased which induced by Manganese chloride in a certaint condition. TSA could restrain the effect that GSH content descended which induced by manganese chloride in a certaint condition. |
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