Role And Mechanism Of A1-type Astrocytes In PM_0.2-induced Neurotoxicity

Objective:To investigate the effects of quasi-nanoparticle(PM_0.2)-exposure-induced A1-reactive astrocytes on neuronal synapses and to delve into the key regulatory role played by microglia in order to provide a scientific basis for revealing the key mechanisms of PM_0.2-induced synaptic damage.Methods:Transmission electron microscopy was coupled with EDS energy spectrum analyzer to observe the morphology and size of PM_0.2 and to analyze its components.Lipopolysaccharide kit to detect endotoxin in PM_0.2.Mixed glial cells and primary neurons were obtained from the cerebral cortex of newborn 24 h ICR mice and cultured for a period of time for cell experiments.The mixed glial cells were divided into control（0μg/m L）and low,medium and high dose exposure groups(3,10 and 30μg/m L PM_0.2)by dose and time,with exposure times of 24 and 48 h.The high-dose exposure group was selected to add inhibitors(30μg/m L PM_0.2+60μM microglia inhibitor-minocycline or 30μg/m L PM_0.2+3m M ROS inhibitor NAC).Then supernatants from the high-dose exposure and intervention groups were taken to act on neurons（supernatant:neuronal basal medium=2:1）.Neurons were pretreated with C3 receptor inhibitor（C3a RA）for 2 h and then co-cultured with supernatant from the mixed glial cell high-dose exposure group（supernatant:neuronal basal medium=2:1）.CCK-8 assay for the survival of mixed glial cells and primary neurons.Immunofluorescence assay to identify microglial cell markers（CD68）.ELISA kits detect inflammatory factors TNFαand IL-1β.RT-PCR was performed to detect changes in A1/A2 type-specific genes,cytokines and neuronal synapse-associated genes.western blot was used to determine C3,S100a10,PSD95,SYP,IL-1αand C1q protein expression.Results:1 PM_0.2 activates microglia to induce A1sThe collected PM_0.2 samples were observed by electron microscopy and analyzed by energy spectrum,and seven major elements were found in PM_0.2:C,O,Si,Cu,Cr,Ca and Fe,with the largest proportion of C elements（>70%）,which far exceeded the content of other elements.Meanwhile,LPS analysis showed that endotoxin levels increased with increasing PM_0.2 dose,but there was no statistically significant difference（P>0.05）.A1s-specific genes（Serping1,C3,Amigo2,Psmb8,Ggta1,Srgn,H2-T23,Ligp1,Ugt1a,Gbp2,and H2-D1）and proteins（C3）increased significantly（P<0.05）with increasing PM_0.2exposure dose and time;only two genes（Fkbp5 and Fbln5）did not change（P>0.05）.While only CD14,Ptx3 and Ptgs2 were expressed at higher levels among A2s-specific genes than in the control group（P<0.05）,the remaining genes（S100a10,Cd109,Emp1,Tm4sf1,Clcf1,B3gnt5,Slc10a6 and Sphk1）and proteins（S100a10）were not statistically different（P>0.05）.These results suggest that PM_0.2 exposure may cause conversion of astrocytes to A1s.Further studies revealed that PM_0.2exposure activated microglia（CD68）,while m RNA and protein levels of cytokines IL-1α,TNF-αand C1q were significantly increased（P<0.05）.Pretreatment of mixed glial cells with the microglia inhibitor-minocycline significantly improved the expression of some A1s-specific genes（C3,Serping1,Ggta1,Amigo2 and Fkbp5）induced by PM_0.2 exposure（P<0.05）,suggesting that microglia are involved in PM_0.2-induced A1s typing.2 PM_0.2-induced A1s induce synaptic damage in neuronsAstrocytes induce neuronal excitatory synapse formation by secreting phosphatidylinositol proteoglycans（Gpc4/6）and thrombospondin（Thbs1/2）.We further investigated whether A1s still produce these factors.After 48 h of PM_0.2exposure,m RNA expression of Sparcl1 in mixed glial cells decreased（P<0.05）,while m RNA expression of Thbs1 and Thbs2 increased（P<0.05）,and m RNA levels of Gpc4,Gpc6 and Sparc did not change（P>0.05）.In primary neurons,the m RNA levels of PSD95 and SYP decreased after48 h of PM_0.2 exposure（P<0.05）,while PSD95 protein levels decreased（P<0.05）,but SYP protein levels did not change（P>0.05）.Subsequently,we intervened in primary neurons using C3 receptor antagonists and microglia inhibitors and found that C3a RA and minocycline reversed the PM_0.2-induced decrease in PSD95 protein and m RNA（P<0.05）.In conclusion,PM_0.2-induced A1s caused neuronal synaptic damage through the complement C3 pathway.3 NAC ameliorates PM_0.2-induced synaptic damage,independent of A1 differentiationOxidative stress is one of the common mechanisms in most CNS diseases.To verify whether oxidative stress is involved in A1s typing,we intervened in mixed glial cells for 48h using antioxidant-NAC.The results showed that the m RNA levels of most A1s-specific genes（C3,Ligp1,Amigo2,Serping1,H2-D1,H2-T23,Fkbp5 and Ggta1）did not change significantly（P>0.05）with the intervention of NAC,and only the expression of two genes,Srgn and Ugt1a,was changed（P<0.05）.Treatment of primary neurons with mixed glial cell supernatants with or without NAC revealed significant recovery of PM_0.2-induced m RNA levels of PSD95 and SYP（P<0.05）.The above results suggest that oxidative stress derived from PM_0.2 exposure does not affect A1s typing,but directly induces synaptic damage.Conclusions:1 PM_0.2-activated microglia induced A1s by secreting Il-1α,TNF-αand C1q;2 PM_0.2-induced A1s lost the ability to promote neuronal synaptogenesis and induced synaptic damage in neurons by secreting C3;3 Minocycline ameliorated PM_0.2-induced synaptic damage by blocking A1s differentiation;4 NAC ameliorated PM_0.2-induced synaptic damage,but not with A1s differentiation.