Research On The Effect And Mechanism Of Combined Exposure To Micro-/Nanoplastics Polystyrene And Plasticizer DEHP On Learning And Memory Function Of Mice

BackgroundAs an artificial material,plastic is widely used in all walks of life because of its low cost,durability and easy production.Micro-/nano-plastics（MNPs）are ubiquitous in natural ecosystems such as terrestrial,aquatic and atmospheric systems.Due to their large specific surface area and strong surface hydrophobicity,MNPs can easily adsorb and enrich heavy metals and organic pollutants.In addition,most plastic products incorporate some plastic additives during the molding process to modify the properties of the polymer and make the plastic product more durable and sustainable.Di-（2-ethylhexyl）phthalate（DEHP）is a plasticizer widely used in the production of plastic products.It is used in plastic products,cosmetics,toys and medical appliances and other products.These plastic additives can be released into the environment in the form of microplastics during the crushing process of plastic products.Polystyrene（PS）has high yield,wide distribution and strong adsorption capacity for hydrophobic organic pollutants.Therefore,PS and DEHP are likely to cause compound pollution in the ecological environment.However,the research on the joint exposure toxicity of PS and DEHP is still very limited.Whether PS can carry and release DEHP into the body and whether the joint exposure will bring greater harm to the nervous system are still unclear.ObjectivesIn this study,three kinds of micro-and nano-scale PS particles and DEHP were selected according to the environmental exposure concentration.By establishing a subchronic long-term oral exposure model in mice,the effects of combined exposure of PS and DEHP on neurobehavior in mice were evaluated,and the molecular mechanism of their toxic effects was explored,so as to provide research data and reference for health risk assessment of combined exposure of MNPs and environmental pollutants.Methods1.Characterization of PS material and its adsorption to DEHP.Scanning electron microscope was used to observe the morphology of PS particles,nano-particle size analyzer was used to detect the hydrodynamic diameter and Zeta potential of PS particles,Fourier infrared spectroscopy was used to detect the composition of PS materials,and confocal microscope was used to shoot fluorescence signals to determine whether Nile red labeled PS particles can produce stable fluorescence.Secondly,we used gas chromatography-mass spectrometer（GC-MS）to detect the adsorption capacity of DEHP on PS.2.Establishment of subchronic exposure animal model of PS and DEHP combined exposure.Mice were randomly divided into 9 groups:blank control group（group C）,solvent control group（group SC）,DEHP group,50 nm/500 nm/5μm PS group（PS50/500/5000）,PS50/500/5000 combined exposure group with DEHP（PS50/500/5000+DEHP）.The doses of PS and DEHP were 50 mg/kg·bw/day and 0.5 mg/kg·bw/day,respectively,and the rats were poisoned by gavage for 90 days.3.Distribution and overall toxic effect of PS and DEHP combined exposure in mice.The accumulation of fluorescent PS particles in brain,intestine,liver,kidney,testis and blood of mice was detected by confocal microscope and fluorescence colorimetry.The accumulation of DEHP and its primary metabolite Mono（2-ethylhexyl）phthalate（MEHP）in the brain,intestine,liver,kidney,testis and blood of mice was detected by GC-MS.Automatic blood cell analyzer was used to detect blood routine,and automatic biochemical analyzer was used to detect blood biochemical indexes in serum.Serum inflammatory indexes,stress hormones and androgens were detected by ELISA.The level of oxidative stress in serum was detected according to the kit.4.The neurotoxic effect of PS and DEHP combined exposure.After exposure,the behavior and learning and memory abilities of mice were evaluated by open field,water maze and dark avoidance experiments.The structural changes of hippocampus and cortex in mice were observed by HE staining,Nissl staining and electron microscope.The permeability of blood-brain barrier in mice was detected by FITC-Dextran staining.Immunofluorescence method was used to detect the expression of tight junction protein,microglia and astrocyte activation markers and sex hormone receptor-related proteins in hippocampus.The expression of inflammatory factors in hippocampus was detected by immunohistochemistry.The level of reactive oxygen species in hippocampus was detected by fluorescence colorimetry and the level of oxidative stress was detected according to the kit.The apoptosis rate of hippocampus was detected by TUNEL experiment.The NF-κB pathway,apoptosis and autophagy related proteins in hippocampus were detected by WB experiment.Targeted metabonomics and ELISA were used to detect the level of neurotransmitters in hippocampus.5.Analysis and verification of phosphorylated protein.We used the phosphorylation modified proteomics techniques to analyze the effects of PS50 and DEHP exposure alone or in combination on the proteomics of mouse hippocampus,and used WB experiment to detect the protein expression of Alzheimer’s disease（AD）-related pathways.Silver staining with glycine was used to detect senile plaques and nerve tangles in hippocampus,and the levels of related neurotransmitters,namely Glu and D-Serine,were detected by colorimetry and ELISA,and the level of Ca²⁺was detected by colorimetry.6.Establishment of exposure model of mouse hippocampal neurons（HT22）in vitro.HT22 cells were intervened by 50 nm PS and MEHP for 24 hours,the cell viability was detected by CCK8 method,the uptake of fluorescent 50 nm PS by HT22 cells was observed by confocal microscope,the uptake of MEHP by HT22 cells was detected by GC-MS,the ROS level of HT22 cells was detected by fluorescence colorimetry and the oxidative stress level was detected according to the kit.HT22 cells were cultured with or without N-methyl-D-aspartic acid receptor（NMDAR）inhibitor Memantine（Me M）and inositol triphosphate receptor（Inositol 1,4,5-triphate receptor,IP3R）inhibitor,Xestosponginc（Xe C）,and then exposed to corresponding concentrations of PS50 and MEHP alone or in combination,the apoptosis was detected by Annexin V-FITC/PI double staining,the contents of Ca²⁺and D-Ser were detected by colorimetry,Glu was detected by ELISA,and AD pathway related proteins were detected by WB.7.Establishment of microglia（BV2）and its co-culture model with HT22 cells.BV2cells were intervened by 50 nm PS and MEHP for 24 hours,the cell viability was detected by CCK8 method,the uptake of fluorescent 50 nm PS by BV2 cells was observed by confocal microscope,the uptake of MEHP by BV2 cells was detected by GC-MS,the inflammatory factors of BV2 were detected by ELISA,and the apoptosis of co-cultured cells was detected by Annexin V-FITC/PI double staining method.The content of Ca²⁺in co-cultured cells was detected by colorimetry,and the expression of p-NF-κB p65（Ser536）in BV2 cells and AD marker protein in HT22/BV2 co-cultured cells was detected by WB method.8.Discussion on the mechanism of gut-brain axis.The changes of colon structure in mice were observed by HE staining and electron microscope.The intestinal barrier and sex hormone receptor-related proteins were detected by immunofluorescence,and the levels of colitis factors and important neurotransmitters were detected by ELISA.The changes of intestinal flora were detected by 16S r DNA gene sequencing,and the content of short-chain fatty acids was detected by targeted metabonomics.Results1.PS particles with three particle sizes can enter the blood of mice and accumulate in tissues,which is related to the particle size.PS can carry DEHP,which can promote DEHP and MEHP to enter the blood and accumulate in various tissues after combined exposure.MEHP accumulates most in the brain,suggesting that the brain is an important target organ.The content of DEHP and MEHP in the body is closely related to the particle size of PS,and the smaller the particle size,the higher the content.2.Neurotoxic effects of PS and DEHP exposure alone and in combination.The combined exposure of PS and DEHP can affect the motor activity of mice,leading to anxiety behavior and the decline of learning and memory ability.Both PS and DEHP exposure alone and in combination can damage the structure of hippocampus and cortex in mice,increase the permeability of blood-brain barrier in hippocampus,induce neuroinflammation,affect the level of oxidative stress,lead to the increase of apoptosis and autophagy in hippocampus,reduce the level of sex hormone receptors,and also lead to the changes of various neurotransmitters in hippocampus.Joint exposure will increase the neurotoxicity induced by single exposure,and its influence will gradually weaken with the increase of particle size.3.The mechanism of AD-like lesions induced by PS and DEHP alone or in combination in mice.By analyzing the peptide of differentially phosphorylated protein,we screened out the AD signaling pathway,and found that senile plaques and nerve fibers were entangled into knots,and up-regulated the AD marker protein Aβ_1-42 and APP,Tau,p-Tau（Ser 396）and p-Tau（Thr 231）.NMDAR Ca²⁺channel-related neurotransmitters Glu and D-Ser and Grin2b,p-Grin2b（Tyr 1472）,Grin1 and p-Grin1（Ser 890）proteins,IP3R Ca²⁺channel-related proteins GPCR and IP3R,Ca²⁺and downstream kinases Ppp3cb and p-ERK1/2（Thr 202/Tyr 204）proteins,The levels of endoplasmic reticulum stress-related proteins Bip,p-PERK（Thr 982）,p-el F2α（Ser 51）,ATF4,CHOP and C-Caspase12 down-regulated the level of p-GSK3β（Ser 9）.Compared with PS50 and DEHP exposure alone,the combined exposure of PS50 and DEHP led to more significant changes in the above indexes.3.The mechanism of AD-like lesions induced by PS and DEHP alone or in combination in mice.By analyzing the peptide of differentially phosphorylated protein,we screened out the AD signaling pathway,and found that senile plaques and nerve fibers were entangled into knots,and up-regulated the AD marker protein Aβ_1-42 and APP,Tau,p-Tau（Ser 396）and p-Tau（Thr 231）.NMDAR Ca²⁺channel-related neurotransmitters Glu and D-Ser and Grin2b,p-Grin2b（Tyr 1472）,Grin1and p-Grin1（Ser 890）proteins,IP3R Ca²⁺channel-related proteins GPCR and IP3R,Ca²⁺and downstream kinases Ppp3cb and p-ERK1/2（Thr 202/Tyr 204）proteins,The levels of endoplasmic reticulum stress-related proteins Bip,p-PERK（Thr 982）,p-el F2α（Ser51）,ATF4,CHOP and C-Caspase12 down-regulated the level of p-GSK3β（Ser 9）.Compared with PS50 and DEHP exposure alone,the combined exposure of PS50 and DEHP led to more significant changes in the above indexes.4.The direct molecular mechanism of AD lesions induced by the combined exposure of PS and DEHP.We studied the mechanism by establishing an HT22 cell model.The results showed that PS50+MEHP could lead to a decrease in the viability of HT22 cells,the entry of 50 nm PS into cells,the increase of MEHP content,affect the level of oxidative stress,and up-regulate Aβ_1-42,an important marker protein of AD,as well as APP and p-Tau（Ser 396）.NMDAR Ca²⁺channel-associated neurotransmitters Glu and D-Ser and p-Grin2b（Tyr 1472）and p-Grin1（Ser 890）proteins,IP3R Ca²⁺associated GPCR and IP3R proteins,Ca²⁺and downstream kinases Ppp3cb,GSK3βand p-ERK1/2proteins.Endoplasmic reticulum stress-related proteins Bip,p-PERK（Thr 982）,p-el F2α（Ser 51）,ATF4,CHOP and C-Caspase12,apoptosis rate and protein expression levels of apoptotic proteins Bax,C-Caspase9 and C-Caspase3,and autophagy-related proteins Beclin1 and LC3;The protein expression levels of kinase p-GSK3β（Ser 9）,apoptotic protein Bax and autophagy protein p62 were down-regulated.Exposure to 50 nm PS in combination with MEHP resulted in more marked changes in these parameters compared to PS and MEHP exposure alone.Moreover,after intervention with NMDAR inhibitor MEM and IP3R inhibitor Xe C,the expressions of AD-like lesion-related proteins and signaling pathways induced by the combined exposure of PS50 and MEHP were significantly alleviated.5.Indirect molecular mechanisms of action of PS and DEHP combined exposure induction of AD lesions.Through the establishment of BV2 cells and HT22/BV2cocultured cell model to study the indirect mechanism,we found that the joint exposure of PS50 and MEHP can lead to the decrease of BV2 cell viability,the entry of PS50 into cells and the increase of intracellular MEHP content,induce the inflammation of BV2cells,and up-regulate the apoptosis rate and Ca²⁺level of HT22/BV2 cocultured cells as well as Aβ_1-42 and p-Tau（Ser 396）protein levels.Compared with PS50 and MEHP exposure alone,the joint exposure of PS50 and MEHP resulted in more significant changes in the above indicators.6.Exploration on the mechanism of intestine-brain axis.Both PS and DEHP exposure alone and in combination could damage the structure of the colon of mice,increase the permeability of the intestinal barrier,induce intestinal inflammation,reduce the level of sex hormone receptors and change the level of neurotransmitters.The combined exposure enhanced the toxic effects of the individual exposure,and the toxic effects gradually increased with the decrease of PS particle size.At the same time,some intestinal flora（such as Ileibacterium,Acetitomaculum,Desulfovibrio and Oligella）and its metabolites undergo specific changes and have significant correlation with neurobehavioral abnormalities,blood-brain barrier damage,neuroinflammation,neurotransmitter and hormone levels.Conclusions1.PS can enter the blood of mice and accumulate in tissues,which is related to the particle size.PS can carry DEHP,which can promote DEHP accumulation in brain,intestine,liver,kidney,testis and blood,and MEHP accumulation in brain after combined exposure.MEHP accumulates most in the brain,suggesting that the brain is an important target organ.The content of DEHP and MEHP in the body is closely related to the particle size of PS,and the smaller the particle size,the higher the content.2.The exposure of PS and DEHP alone or in combination can induce inflammatory injury,oxidative stress and hormone level disorder,and the toxic effect of combined exposure is more obvious,and the effect of small particle size is obviously greater than that of large particle size.3.The experiment in vivo and in vitro proved that PS and DEHP have neurotoxicity,and the smaller the particle size of PS,the higher the toxic effect.Ca²⁺overload is caused by NMDAR and IP3R Ca²⁺channels,and finally AD-like lesions are induced in mice and neurons.The toxic effect caused by combined exposure is higher than that caused by single exposure.4.The combined exposure of PS and MEHP can activate NF-κB signaling pathway of microglia,promote them to release inflammatory factors,and then induce neuronal damage.5.The combined exposure of PS and DEHP can change the intestinal flora and its metabolites in mice,destroy the intestinal barrier and exert neurotoxic effects through the intestinal brain axis.