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Aim Heat shock factor 1 (HSF1) critically contributes to the host defense, and its reserve is critical for the cell to mobilize enough HSF1.How HSF1 reserve is regulated remains largely unknown.Methods Western blot was used to measure the protein expression.Coimmunoprecipitation was applied by test protein interactions.The mRNA level was measured by real timePCR.Silence of the acetyltransferase p300 was achieved by small interfering RNA.Cell injuries were evaluated by MTT experiments.Chromatin immunoprecipitation was used to measure the binding activity of HSF1 to the Hsp70 promoter.Results Methylene blue (MB), an antioxidant and antiinflammatory agent, was found to increase the amount of the monomeric HSF1 in BV2 microglia, and also in primary microglia, astrocytes, cortical neurons and vital organs.The increased HSF1 contributed to a more increase in nuclear translocation of HSF1, association of HSF1 with Hsp70 promoters, Hsp70 protein levels, and a synergistic protection on the cell injury against oxidative stress in MB/heat shocktreated cells.The MBinduced accumulation of HSF1 was not associated with the change in HSF1 gene transcription and macroautophagy or Hsp70 ATPase activity, but with the proteasomeubiquitin system.The acetyltransferase p300 was found to mediate the effect of MB as: i.inhibition or silence of p300 prevented the MBinduced increase in the monomeric HSFI and the HSF1acetylation level; ii.MB enhanced the interaction between p300 and HSF1.Results also showed that it was PKA but not Akt, mTOR, or AMPK that mediated the effects of MB on p300HSF1 interaction and HSF1 accumulation.Finally, these findings involving PKA and p300 were ascertained in primary microglia, astrocytes, and cortical neurons where inhibition of p300 or PKA prevented the increase in monomeric HSF1 after MB treatment.Conclusions Our results not only provide evidence to illustrate a new pharmacological role of MB, but also pave way to identifiying new agents that can increase HSF1 reserve.