Effect Of Progesterone On The Expression Of Inflammatory Cytokines In Microglia Induced By A?_25-35 And It's Role In Mediating Toll-like Receptors

Alzheimer's disease?AD?is a kind of progressive neurodegenerative disease.It is characterized by extracellular accumulation of amyliod-??A??and the formation of amyloid plaques and intracellular height Phosphorylation.In addition,the role of neuroinflammation in the pathogenesis of AD can not be underestimated.Microglia,the innate immune system in the central nervous system,forms the first line of defense in the brain against invading pathogens.A large number of microglial cells infiltrate around the plaque formed by A?,and microglia activate and release a large number of pro-inflammatory factors such as TNF-?,IL-1?and IL-6,as well as neurotoxicity such as NO and ROS.Substances influence the pathogenesis of AD and even increase the progression of AD.Studies have shown that A?can activate the microglial inflammatory response through direct binding to Toll-like receptors?TLRs?.In many subtypes of TLRs,TLR3 and TLR4expression levels are significantly increased during the onset of AD.TLRs can activate glial inflammatory responses through two signaling pathways:MyD88-dependent and non-MyD88-dependent signaling pathways.TLR4 can activate downstream signaling pathways either by relying on MyD88 or by relying on TRIF.TLR3 can only rely on TRIF to activate the downstream signaling pathway.In the course of AD inflammation,MyD88 and TRIF play a key role as adapters for the TLRs signaling pathway.Progesterone is one of many neurosteroids and has neuroprotective and anti-inflammatory effects.Like many steroid hormones,progesterone also plays an important role by binding to its corresponding specific receptor.The receptors that mediate the effects of progesterone are progesterone classical receptor?PR?and progesterone membrane-associated protein 1?PGRMC1?.Studies have shown that classical receptors for progesterone are not present in microglia,but progesterone membrane-bound protein 1 is ubiquitous in microglia and astrocytes.Part I Effects of progesterone on the expression of inflammatory factors in microglia induced by A?_25-35Objective:To investigate the effect of progesterone on the expression of inflammatory cytokines IL-1?and TNF-?in microglia by activating the primary microglia formed by A?active fragment A?_25-35.Methods:Neonatal SD neonatal mice were sacrificed within 24 hours and the mixed glial cells of cerebral cortex cells were cultured for 9-12 days.After purification of the microglial cells by"right-hand-hand method",the purity of microglia was identified by double staining with Iba1microglia-specific antibody and Hoechst 33258.Activation of microglial inflammatory response using A?active fragment A?_25-355-35 established a cell model of AD.The effects of different concentrations of A?_25-355-35 on the activity of microglial cells were detected by MTT,and the effects of different concentrations of A?_25-355-35 and progesterone on the expression of inflammatory cytokines IL-1?and TNF-?in microglia were examined.Result:1.Purity of primary microgliaMicrobubble cell morphologies were clear?green marks?after immunofluorescence specific staining with Iba1 marker;morphologies of the nuclei stained with Hoechst 33258 were clear?blue marks?,and the purity of the primary microglial cells after detection coincided with?95%.2.A?_25-355-35 dose-dependently affects microglia activityThe results of MTT assay showed that compared with the solvent control group?100%?,the cell survival rate?87.69±9.7?was not statistically different after 10?M A?_25-355-35 treatment for 24 h?P>0.05?.The survival rate of cells treated with 20?M A?_25-355-35 for 24 h was?66.69±9.3?%?P<0.01?in the solvent control group;the survival rate of cells treated with 40?M A?_25-355-35 for 24 h was in the solvent control group?23.04±6.2?%?P<0.01?.In the above concentration range,according to the toxicity of A?_25-355-35 and cell survival rate,20?mol/L A?_25-355-35 was selected for 24 h to establish an AD cell model.3.The effect of dose-dependent A?_25-355-35 on the expression of inflammatory cytokines IL-1?and TNF-?in microgliaELISA The results showed that compared with the control group,10?M A?_25-355-35 increased the expression of TNF-?and IL-1?in microglia?P<0.05?;20?MA?_25-355-35 made microglial cells TNF-?and IL-1?.The expression level was significantly up-regulated?P<0.01?;40?M A?_25-355-35 increased the expression of TNF-?and IL-1?in microglia?P<0.05?.4.Effect of progesterone dose-dependence on the expression of inflammatory cytokines IL-1?and TNF-?in microgliaELISA results showed that compared with the control group,the expression levels of inflammatory factors in microglia at the concentrations of1?M and 5?M were not statistically different?P>0.05?;the concentration was lower at 20?M.The expression level of glial inflammatory cytokines was down-regulated?P<0.05?.5.Effect of progesterone dose-dependence on the up-regulation of IL-1?and TNF-?expression in microglia induced by A?_25–35ELISA results showed that compared with the control group,the expression of TNF-?and IL-1?in the A?_25-355-35 model group was significantly increased?P<0.01?.Compared with the model group,the levels of TNF-?and IL-1?in the PROG-protected group were decreased?P<0.05?in the medium?10?M?and high?20?M?groups.Part II Effects of progesterone on the expression of TLRs in A?_25-35 induced microglial cellsObjective:This part of the experiment was to investigate the effect of PGRMC1 on TLRs-mediated A?_25-355-35 microglial inflammatory signaling pathway.Methods:Western blot was used to detect the effects of progesterone on the expression of TLR4,TLR3,MyD88,TRIF and NF-?B in A?_25-355-35 induced microglia injury and the inhibitor of PGRMC1 on A?_25-355-35 induced microglial injury.The effects of TLR4,TLR3,and NF-?B expression are regulated.Result:1.Progesterone dose-dependently down-regulates the expression of TLR3and TLR4 in A?_25–35-induced microglial cellsWestern Blot showed that the expression levels of TLR3 and TLR4 were significantly up-regulated in the A?_25-355-35 model group compared with the control group?P<0.01?.Compared with the model group,the expression of TLR3 and TLR4 was down-regulated in PROG protection group after PROM concentration was 5?M?P<0.05?.2.Progesterone dose-dependently down-regulates MyD88 and TRIF expression levels in A?_25–35-induced microglial cellsWestern Blot results showed that compared with the control group,the expression levels of MyD88 and TRIF were significantly upregulated in the A?_25-355-35 model group?P<0.01?.Compared with the model group,the levels of TRIF and MyD88 were down-regulated in PROG-protected group after the concentration of PROG reached 5?M?P<0.05?.3.Progesterone dose-dependently down-regulates the expression of NF-?B in A?_25–35-induced microglial cellsWestern Blot showed that compared with the control group,the expression of NF-?B?P65?was significantly up-regulated in the A?_25-355-35 model group?P<0.01?.Compared with the model group,the PROG protection group had a PROG concentration of 5?M after The expression of NF-?B?P65?was down-regulated?P<0.05?.4.AG205 inhibits the expression of TLR3,TLR4 and NF-?B when progesterone down-regulates microglial damage induced by A?_25–35Western Blot showed that the expression levels of TLR3,TLR4,and NF-?B were decreased in the progesterone-protected group compared with the A?_25-355-35 model group?P<0.05?.Compared with the progesterone-protected group,the TLR3,TLR4,and NF-?B in the AG205-treated group were significantly lower than those in the progesterone-protected group.The expression of NF-?B was up-regulated?P<0.05?.Conclusion:1.A?_25-355-35 activated microglial cells up-regulated the expression of IL-1?and TNF-?.2.Progesterone inhibits the up-regulation of IL-1?and TNF-?expression induced by A?_25-355-35 activated microglia.3.Progesterone inhibits the expression of TLR3 and TLR4 in TLRs signaling pathway and may inhibit the inflammatory response induced by A?_25-355-35 microglia.4.Progesterone may further inhibit the inflammatory response of A?_25-35induced microglial cells by inhibiting the expression of MyD88 and TRIF,which are important adaptors of the TLRs signaling pathway.5.Progesterone may play a neuroprotective role partly through PGRMC1inhibiting the microglial inflammatory response induced by A?_25-35.