Study On The Mechanism Of Dopamine And Salidroside In Regulation Of Parkinson’s Disease And Depression Through Activation Of Microglia

Background:Microglia,the immune competent cells of the central nervous system（CNS）,become activated in response to brain injury,infection,and a variety of neuroinflammatory stimuli.They are the first non-neuronal cells to respond to various acute brain injury.Chemokines,cytokines,and other immunomodulatory molecules will be secreted after microglial activation.These molecules play a significant role in degenerative encephalopathy,primary and secondary brain damage.Overactivity of microglia often leads to neurotoxicity and some neurodegenerative diseases,including Parkinson’s disease（PD）and depression.The most important pathological changes of PD are the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain,which leads to the significant change of dopamine（DA）content in the striatum.Many studies have suggested that depression is highly associated with chronic stress stimulation,involving chronic inflammation and microglia activation in CNS.Salidroside（SLDS）has long time been used to treat a variety of inflammatory diseases.By reducing excessive inflammatory response,it has a strong protective effect on hepatitis,colitis,myocardial injury and cerebral ischemia.At present,the mechanism related to the inflammatory activation of microglia with PD and depression is still unclear,and the mechanism of SLDS regulating microglia inflammatory activation and thus alleviating depression symptoms is also not fully understood.Research Objectives:1.To explore the effects of DA on microglia cells in CNS and its cellular and molecular mechanisms.2.To explore the role of salidroside（SLDS）in the treatment of depression,and its cellular and molecular mechanism in regulation of microglia.Experimental Methods:1.M1 phenotype microglia model was established using BV2 mouse microglia cell line with 200 ng/m L lipopolysaccharide（LPS）.DA（2μM）was used to stimulate resting microglia and M1 microglia.The expression and distribution of F-actin and vimentin were determined by immunofluorescence staining.By transfecting Paxillin-GFP,we visualized the assembly and disassembly of focal adhesions.Moreover,we investigated the signaling pathways about the differential regulation of DA in resting microglia and M1 phenotype microglia,how DA transducted the signals in to the cells by Western blotting（WB）.2.The depression model was established by chronic stress strategy.The effects of SLDS were determined via forced swimming test（FST）,Morris water maze（MWM）and open field test（OFT）.Immunofluorescence staining of the expression of Iba-1 and CD68 were detected in the hippocampus of mice.The effects of SLDS on the activation of ERK1/2,P38 MAPK and p65 NF-κB were conducted by WB.The expression of IL-1β,IL-6,IL-18 and TNF-αwere tested by RT-PCR and ELISA.Finally,changes in apoptosis of PC12 cells cultured with conditioned medium were measured by flow cytology and CCK-8 assay.Results and Conclusion:1.DA differentially regulated the morphology,cytoskeleton,the assembly of focal adhesions,and the phagocytic activity of resting and activated microglia.In resting microglia,DA exhibited no effects on ERK1/2 activity but activated P38MAPK.However,DA inhibited the phosphorylation of ERK1/2 and P38 MAPK in activated microglia.In resting microglia,DA signaling is not transmitted by dopamine receptors but through dopamine transporters and monoamine transporters.The activation of P38 MAPK further regulated the phosphorylation of paxillin-Ser⁸³.The differential regulation of DA on microglia cells under different activation states provides a theoretical basis for us to understand the signal transduction changes caused by the progression of PD disease.2.The results of FST,MWM and OFT showed that SLDS treatment alleviated depressive behavior in stressed mice.SLDS significantly reduced the number of Iba-1and CD68 both positive microglia which was a typical M1 phenotype activation in the hippocampus of stressed mice.SLDS inhibited the activation of microglia through inhibiting the activation of ERK1/2,P38 MAPK and p65 NF-κB,and reducing the expression and release of neuroinflammatory cytokines in stressed mice.These conclusions were also verified by LPS-induced primary microglia.SLDS reduced the phagocytic activity,changed their morphology and attachment state.SLDS may play a neuroprotective role against PC12 cells by inhibiting the release of inflammatory cytokines from pirmary microglia.SLDS can improve the progression of depression and play a neuroprotective role by inhibiting the activation of microglia.