Role Of Dopamine D₂-like Receptors In The Regulation Of Autophagy And Inflammation In Astrocytes

Objective:To study the role and molecular mechanism of activated dopamine D2-like receptor in the regulation of autophagy and neuroinflammation in astrocytes.Methods:Primary astrocytes of cortical cortex in wild-type and DRD3 receptor knockout mice were used as the research object.Dopamine D2-like receptor agonist Pramipexole(PPX)or Quinpirole was used as a tool.Q-PCR was used to detect the transcription levels of DRD2 receptor and DRD3 receptor in astrocytes.CCK8 detects cell viability.The levels of pro-inflammatory factors TNF-?,IL-1?,IL-6 and iNOS mRNA were detected by Q-PCR.Then,on the normal astrocytes,the cells inhibited by Chloroquine(CQ)and the astrocytes with the loss of DRD3 receptor,the cells were pretreated with LPS for 5 h and treated with PPX for 1 h.Finally,Adenosine triphophate(ATP)was added to stimulate the cells for 30 minutes.The supernatant and intracellular inflammatory protein IL-1? and caspase-1 were detected by Western Blot.And the contents of IL-1 in the supernatant were detected by ELISA.Western Blot was used to detect the expression of autophagy related proteins and molecules of the phosphorylation signaling in astrocytes.The GFP-LC3 plasmid was transfected and the number of GFP-LC3 punctate aggregates in the cells was observed by the confocal microscopy.Results:Astrocytes expressed DRD2 and DRD3 receptors,and the mRNA level of DRD3 receptor was about twice that of DRD2 receptor.The DRD2 receptor agonist Quinpirole did not affect cell viability at 200 ?M,and the DRD3 receptor agonist PPX had no effect on cells at 800 ?M.Compared with the LPS group,the DRD2 receptor agonist Quinpirole inhibited the increase of transcription levels of LPS-induced pro-inflammatory cytokines TNF-?,IL-1?,IL-6,iNOS in a dose range of 10-100 ?M.There is no concentration dependence within the range.DRD3 receptor agonist PPX does not inhibit LPS-induced(?)scription of pro-inflammatory cytokin s TNF-? and IL-1? at a dose range of 100-400 ?M.Compared with the control group,PPX inhibited the expression and release of mature IL-1? and caspase-1 in the supernatant of LPS+ATP-induced astrocytes at a dose range of 50-200 ?M in a concentration-dependent manner.After knocking out the DRD3 receptor or CQ(40 ?M)completely inhibit autophagy,the effect of PPX(50,200 ?M)on the maturation and release of inflammatory proteins IL-1? and caspase-1 disappeared compared with the LPS group was disappeared,Compared with the control group,the DRD2 receptor agonist Quinpirole did not observe any change in the autophagy proteins LC3-? and BECN 1 at a dose(10-200 ?M)and time(1-12 h)range.Compared with the control group,the DRD3 receptor agonist PPX promoted the increase of LC3-? and BECN 1 protein in astrocytes in a time-dependent manner(1.5-24 h)and dose-dependent manner(100-400 ?M)within a certain dose range.PPX does not alter the phosphorylation levels of astrocyte mammalian rapamycin target(S2248)(mTOR(S2248))and Unc-51-like kinase 1(S555)(ULK1(S555)),what indicated that mTOR signaling pathway is not involved in the regulation of autophagy activity of astrocytes by PPX.Confocal observed PPX processing 12 h,intracellular GFP-LC3 dots increased significantly.These results suggest that DRD3 receptor activation and autophagy activity increase the inhibition of PPX on inflammasome.Conclusion:The dopamine D2-like receptor agonist Quinpirole and PPX regulate the inflammatory response through different mechanisms.The selective DRD2 receptor agonist Quinpirole inhibits the transcription of pro-inflammatory cytokines,whereas the DRD3 receptor agonist PPX does not affect the transcription of pro-inflammatory cytokines,but increases the autophagy activity of astrocytes through the DRD3 receptor,inhibits caspase-1 activation of inflammasome,and thereby reduces IL-1? maturation and release.