The Protective Effects Of Compound Essential Oils And Scorpion Venom Heat-resistant Synthetic Peptide On Brain Injury Caused By PM_2.5 Exposure

Purpose:There is growing evidence that PM_2.5is closely related to nervous system diseases,but the molecular mechanism of PM_2.5-induced neurological diseases is still unclear and there are few drugs that effectively treat PM_2.5-induced diseases.The aim of this study was to investigate the effects of PM_2.5exposure induced brain injury and the therapeutic effects of compound essential oils（CEOs）and scorpion venom heat-resistant synthetic peptide（SVHRSP）on brain injury.CEOs alleviates PM_2.5-induced oxidative stress by inhibiting AMPK/m TOR mediated autophagy,and SVHRSP attenuates PM_2.5-induced microglia polarization by activating PI3K/AKT/NF-κB signaling pathway via TLR4 mediated autophagy.CEOs and SVHRSP play a good neuroprotective effect,which provides a new idea for the treatment of neurodegenerative diseases.Methods:1.Preparation of PM_2.5:After dissolving the filter paper sheet containing PM_2.5in sterile normal saline,the PM_2.5suspension was obtained by ultrasonic vibration and stored in the refrigerator at-4°C for subsequent experiments.2.Experimental grouping and model construction:（1）The first part of the experiment Animal experiment:27 Balb/c mice were randomly divided into three groups（n=9）:1)Control group:Intratracheal instillation of 10μL sterile saline to mice on day 0 and day 2.2)PM_2.5group:Intratracheal instillation of 10μL10mg/m L PM_2.5suspension to mice on day 0 and day 2.3)PM_2.5+CEOs treatment group:statically inhaled 1%CEOs（30 minutes/day）the day before PM_2.5was instilled into the mouse trachea.After 14 days,the model was established,and the brain was separated and stored in a refrigerator at-80°C.Cell experiment:BV2 cells were treated with PM_2.5and CEOs to detect the related indexes.（2）The second part of the experiment Cell experiment:BV2 cells were treated with PM_2.5,SVHRSP,3-MA and TAK-242 to detect the related indexes.3.Detection of related indicators:（1）Autophagy-related index detection:The changes of autophagy in brain tissue and BV2 cells were observed by TEM;q RT-PCR and Western blot were used to detect the gene and protein levels of LC3,P62 and beclin1;immunofluorescence was used to detect the changes in the fluorescence intensity of LC3 in BV2 cells;flow cytometry was used to detect the changes in apoptosis of BV2 cells.（2）Detection of oxidative stress and inflammation-related indicators:HE staining to observe pathological changes in the brain of mice;q RT-PCR and Western blot to detect the gene and protein levels of Nox2,HO-1,IL-1β,and TNF-α;flow cytometry detects the content of ROS in BV2;Malondialdehyde（MDA）and superoxide dismutase（SOD）kits detect the content of MDA and SOD.（3）Detection of microglia polarization related indicators:The gene and protein levels of i NOS and COX-2 in M1 and CD206 and IL-10 in M2 were detected by q RT-PCR and Western blot;The changes of i NOS and ARG1 fluorescence intensity were detected by immunofluorescence method.（4）Detection of pathway related indicators:q RT-PCR and Western blot were used to detect the gene and protein levels of AMPK/m TOR and PI3K/AKT/NF-κB pathway related factors.Results:The first part of the experimental results:（1）The CEOs reduces brain injurycaused by PM_2.5exposure in vivo and in vitro.The results of HE staining showed that the CEOs treatment significantly improved the pathological structure of the irregular cell morphology,loose and disordered arrangement caused by PM_2.5exposure.CCK8 results showed that the therapeutic effect of CEOs on BV2 cells increased in a dose-dependent manner.The results of flow cytometry showed that the CEOs significantly reduced the apoptosis caused by PM_2.5exposure.（2）The CEOs reduces autophagy caused by acute exposure of PM_2.5in vivo and in vitro.The results of q RT-PCR,Western blot and immunofluorescence showed that CEOs treatment significantly reduced the autophagy levels of brain and BV2 cells.TEM results showed that the number of autophagosomes was significantly reduced after the CEOs treatment.（3）CEOs reduces autophagy through AMPK/m TOR signaling pathway.The results of q RT-PCR and Western blot showed that CEOs treatment significantly reduced AMPK levelin the brain and BV2 cells,while increased m TOR level.（4）CEOs alleviates the oxidative stress caused by PM_2.5exposure in vivo and in vitro.The results of q RT-PCR,Western blot and kits showed that CEOs treatment significantly reduced the levels of NF-κB,Nox2 and MDA,while increasing the level of HO-1 and SOD.The second part of the experimental results:（1）SVHRSP inhibits PM_2.5-induced M1 polarization of microglia.It was observed under the microscope that SVHRSP improved the morphological changes of microglia induced by PM_2.5.The results showed that SVHRSP treatment significantly reduced M1 markers and increased M2 markers in BV2 cells.（2）SVHRSP inhibits PM_2.5-induced autophagy in microglia.The results showed that SVHRSP treatment significantly reduced the expression of LC3II and beclin1,but increased the expression of P62.TEM results showed that SVHRSP significantly inhibited the production of autophagosomes.Simultaneously,SVHRSP reduced the level of apoptosis of microglia.（3）Autophagy regulates microglia polarization through PI3K/AKT/NF-κB signaling pathway.After 3-MA inhibited autophagy,the phosphorylation level of PI3K and AKT increased,while the expression of NF-κB decreased,and microglia transformed to M2 Phenotype,SVHRSP plays the similar role as 3-MA.（4）SVHRSP relieves PM_2.5-induced M1 polarization through TLR4-mediated autophagy.After the TLR4 inhibitor TAK-242 inhibits the expression of TLR4,the autophagy level of microglia was reduced while transforming to the M2Phenotype.The phosphorylation level of PI3K and AKT increased while the expression of NF-κB decreased,and SVHRSP plays the similar role as TAK-242.Conclusion:（1）CEOs downregulate PM_2.5-induced autophagy through AMPK/m TOR signaling pathway,thereby alleviating the oxidative stress response of brain tissue and BV2 cells.（2）SVHRPS activates PI3K/AKT/NF-k B signaling pathway through TLR4-mediated autophagy to attenuate PM_2.5-induced M1 polarization of microglia.