Nrf2 Regulateds Activation Of A1 Astrocytes Induced By Fine Particulate Matter Exposure

Background:Currently,the impact of air pollution on public health remains one of the main public health problems in China.Ambient fine particulate matter(PM_2.5)is regional and spatial specifity and shows multiple organ effects.Numerous epidemiological and animal experiments have confirmed a significant correlation between PM_2.5exposure and nervous system diseases.Astrocytes play multiple roles in the nervous system,including maintaining the barrier integrity of the blood-brain barrier（BBB）and acting as major immune cells in the nervous system.Astrocytes are highly cellular heterogeneity and show either neuroprotection or neurotoxicity responding to different pathological stress.Animal experiments have shown that PM_2.5exposure induced astrocyte activation.However,the mechanism underlying astrocyte heterogeneity remains unclear in PM_2.5exposure-induced neurotoxicity.Objects:The purpose of this study was to clarify the effect of PM_2.5exposure on the activation of A1 astrocytes in vitro and in vivo,and the regulatory mechanism of Nrf2 pathway in this process by constructing"real-world"and culturing primary astrocyte of PM_2.5exposure models.In addition to the neurotoxicity and mechanism of PM_2.5exposure,the potential protective effects of three phytochemicals（procyanidins,lycium barbarum polysaccharide,and forsythiin）on PM_2.5-induced astrocyte toxicity in mice were evaluated to explore interventions beneficial to public health.Method:1.Long-term fine particulate matter exposure-induced neurotoxic effects in mice1.1 Exposure levels and characterization of PM_2.5in the"Real-world"exposure model.During"real-world"PM_2.5exposure,the concentrations of PM_2.5in the exposure chambers were measured by Microdust Pro real-time mass live dust monitors and compared with mice in the clean-filtered air group;PM_2.5was collected and the particle size and morphology of PM_2.5was evaluated using a nanoparticle analyzer and under a SEM.1.2 Long-term PM_2.5exposure-induced neurobehavioral changes in miceA"real-world"PM_2.5long-term exposure model was established by an online PM_2.5whole-body inhalation exposure system for 15 weeks.Weight of mice were recorded weekly.Open field test and sucrose preference experiment were performed to evaluate the effects of PM_2.5exposure on neurobehavior of mice.1.3 Effects of long-term PM_2.5exposure on the hippocampus histopathologyAfter scarifice,murine brain tissue was harvested and fixed in 4%PFA.Paraffin sections of brain were prepared.H&E and Nissl staining were performed to observe the pathological changes in hippocampus tissue;m RNA of hippocampus tissue was extracted and the m RNA level of S100βwas measured by RT-q PCR;mouse serum samples were collected and the expression of S100βprotein was measured by enzyme-linked immunosorbent assay（ELISA）.2.Astrocyte and Nrf2 pathway activation in mice induced by long-term fine particulate matter exposure2.1 Long-term PM_2.5exposure induces astrocyte activation in hippocampusRT-q PCR was used to measure the m RNA level of Gfap in the hippocampus of control and PM_2.5exposed mice;immunohistochemical staining of GFAP was used to evaluate astrocyte activation in the hippocampus.2.2 Effect of long-term PM_2.5exposure on activation of A1 astrocytes in the hippocampusRT-q PCR,western-blot（WB）,and immunohistochemistry staining were used to detecte C3 levels in control and PM_2.5exposed murine brains.Immunofluorescence colocalization of C3 and GFAP was used to observe the activation of A1 astrocytes.2.3 Activation of Nrf2 pathway induced by long-term PM_2.5exposure in miceUsing RT-q PCR detected the m RNA levels of Nrf2,Ho-1 and Nqo-1 in hippocampus of control and PM_2.5exposed murine hippocampus;WB was used to detecte the protein level of Nrf2 and ROS generation in hippocampus was observed.3.Nrf2 pathway mediates activation of A1 astrocytes following long-term fine particulate matter exposure in vitro3.1 Primary culture and characterization of murine astrocytesNewborn mice within 24 h were selected for astrocyte isolation,and primary cultured astrocytes were purified and identified by immunofluorescence.3.2 Effects of PM_2.5exposure on A1 astrocyte activation and Nrf2 pathway activationThe dose of PM_2.5in subsequent experiments was determined by CCK8;the effect of PM_2.5exposure on the astrocyte cell cycle was determined by flow cytometry;RT-q PCR and WB were used to detecte the levels of Nrf2,HO-1,and Nqo-1 in primarily cultured astrocytes;the expression levels of C3 and Nrf2 in astrocytes were observed by immunofluorescence colocalization,and the levels of ROS generation and activity of antioxidant enzymes including SOD and catalase were tested using commercial kits.3.3 The mechanism of Nrf2 pathway activation in PM_2.5exposure-induced A1 astrocyte activationThe dose of Nrf2 agonist,dimethyl fumarate（DMF）,was determined by CCK8 assay;RT-q PCR and WB assays were used to detecte the m RNA levels of Nrf2,Ho-1,and Nqo-1 in astrocytes;the expression of C3 and Nrf2 in astrocytes was observed by immunofluorescence colocalization,and the levels of ROS generation and activities of antioxidant enzymes including of SOD and catalase were tested using commercial kits.4.Proanthocyanidins improve the neurotoxic effects induced by fine particulate matter exposure4.1 Screening of the three plant compoundsCCK8 and WB assays were used to detect the effects of three phytochemicals including proanthocyanidin（PC）,lycium barbarum polysaccharide（LBP）and forsythia side（Phillyrin）on the activation of A1 astrocytes after PM_2.5exposure.Phytochemicals which could inhibit the activation of A1 astrocytes were screened for subsequent in vivo experiments.The effects of PC on antioxidant enzymes and ROS in astrocytes exposed to PM_2.5were observed.4.2 PC suppress the activation of A1 astrocytes following PM_2.5exposure and underlying mechanisms in vitroA"real-world"PM_2.5exposure combined with PC gavage murine model was constructed.The effects of PC treatment on PM_2.5exposure-induced strocyte cytotoxicity,activation and ROS generationwere evaluated.4.3 PC ameliorates the neurobehavioral changes in mice induced by PM_2.5exposureThe changes of body weight in each group were observed;open field test and sucrose preference experiment were performed to evaluate the effects of PC on the neurobehavior of PM_2.5exposed model mice.Result:1.Long-term fine particulate matter exposure-induced neurotoxic effects in mice1.1 Exposure levels and characterization of PM_2.5in the"Real-world"exposure modelIn the clean air group,the concentration of PM_2.5was close to 0μg/m³,while in PM_2.5group,it was much higher than that in the clean air group,and the average concentration in the exposure chamber was 562.24μg/m³.The particles diameter was about 1μm.1.2 Long-term PM_2.5exposure-induced neurobehavioral changes in miceCompared with the control group,PM_2.5exposure had no effect on the body weight of mice.Results of open field test showed that PM_2.5exposure significantly reduced the total distance（P<0.001）and the time in the central platform（P<0.001）and increased the time in the surrounding area（P<0.001）.Sucrose preference experiment showed the preference rate of sugar was reduced in PM_2.5exposure group compared to the control（P<0.05）.1.3 Effects of long-term PM_2.5exposure on the hippocampus histopathologyH&E staining of brain tissue did not show obvious alterations in the arrangement and morphology of neuron in the hippocampus of mice following PM_2.5exposure on the hippocampus of mice.Compared with the control group,the staining of Nissl body was reduced,and part of Nissl body dissolved,broken,and disappeared in the PM_2.5exposure group.RT-q PCR and ELISA showed that PM_2.5exposure significantly increased S100βm RNA levels in brain tissue（P<0.001）and S100βprotein levels in serum（P<0.05）.2.Astrocyte and Nrf2 pathway activation in mice induced by long-term fine particulate matter exposure2.1 Long-term PM_2.5exposure induces astrocyte activation in hippocampusCompared with the control,PM_2.5exposure increased the m RNA level of Gfap in and promoted the morphological changes of astrocytes in hippocampal area（CA1,CA3,and DG）of mice.Specifically,the number of astrocytes increased,the axon enlarged,and the content of Gfap increased（P<0.001）2.2 Effect of long-term PM_2.5exposure on activation of A1 astrocytes in the hippocampusCompared with the control mice,PM_2.5exposure significantly increased C3 m RNA（P<0.001）and protein levels（P<0.05）.PM_2.5exposure significantly increased the co-location of GFAP and C3 in hippocampal astrocytes of mice.2.3 Activation of Nrf2 pathway induced by long-term PM_2.5exposure in miceCompared with the control mice,PM_2.5exposure significantly increased the levels of Nrf2,Ho-1,NQO-1 m RNA（P<0.05）and Nrf2 protein（P<0.05）.PM_2.5exposure increased ROS generation in hippocampal regions of mice.3.Nrf2 pathway mediates activation of A1 astrocytes following long-term fine particulate matter exposure in vitro3.1 Primary culture and characterization of murine astrocytesAstrocytes with good purity were identified by immunofluorescence.3.2 Effects of PM_2.5exposure on A1 astrocyte activation and Nrf2 pathway activationAccording to CCK-8 assay,0,5,10,and 15μg/m L PM_2.5was selected as the dose for subsequent experiments.Cell cycle results showed that 10 and 15μg/m L PM_2.5exposure induced proliferation of astrocytes.PM_2.5exposure increased the levels of GFAP,C3,Nrf2,HO-1,and NQO-1 expression.Immunofluorescence showed increased co-location of C3 and Nrf2 in astrocytes.Meanwhile,PM_2.5exposure increased the expression levels of two antioxidant enzymes,and the production of ROS.3.3 The mechanism of Nrf2 pathway activation in PM_2.5exposure-induced A1 astrocyte activationAstrocytes were pretreated with 5μg/m L DMF,and the results of RT-QPCR and WB showed that DMF successfully activated Nrf2 pathways and inhibited the expression level of C3 in astrocytes induced by PM_2.5exposure（P<0.001）.The immunofluorescence co-localization also supported this result.Meanwhile,the pretreatment of DMF inhibited the increase of ROS levels(P<0.05 in astrocytes induced by PM_2.5exposure.4.Proanthocyanidins improve the neurotoxic effects induced by fine particulate matter exposure4.1 Screening of the three plant compoundsThe result of CCK8 showed that low-dose forsythiin had significant toxicity to astrocytes.Pre-treatment of PC but not LBP significantly suppressed activation of A1astrocytesd（P<0.001）.PC significantly increased the levels of antioxidant enzymes（P<0.001）and decreased ROS levels（P<0.05）in PM_2.5-exposed astrocytes.4.2 PC suppress the activation of A1 astrocytes following PM_2.5exposure and underlying mechanisms in vitroPC significantly inhibited the activation of A1 astrocytes in the hippocampus（CA1,CA3,and DG）of mice.ROS generationwas decreased in the hippocampus of mice exposed to PM_2.5.4.3 PC ameliorates the neurobehavioral changes in mice induced by PM_2.5exposurePC slightly reduced the weight of body in mice.Open field test and sucrose preference test showed that PC treatment significantly inhibited neurobehavioral changes induced by PM_2.5exposure.Conclusion:1.Long-term exposure to PM_2.5induces neurotoxicity in mice.2.Nrf2 regulates activation of murine A1 astrocytes induced by PM_2.5exposure.3.PC suppress neurotoxicity induced by PM_2.5exposure in mice by activating Nrf2 pathway.PC,a dietary supplement might be one of the means to prevent or delay the occurrence and development of neurological diseases in high-risk population.