Study On The Biotoxic Effects Of Microplastics Based On Caco-2 Cells And Mouse Intestinal Model

As a contaminant of interest in recent years,microplastics(< 5mm in diameter)have been increasingly reported to be detected in food products such as beer,honey,and salt.Due to their tiny size,microplastics can enter organisms along the food chain and cause various adverse effects.There are limited studies on the harmful effects of microplastics that accumulate in the intestine after entering the human body,especially in mammals.In this study,we investigated the effects of microplastics on the intestine through in vitro and in vivo experiments.In vitro experiments were conducted to study the toxicity mechanism of different sizes of polystyrene(PS)on Caco-2 cells.For in vivo experiments,polystyrene(PS),polyethylene(PE),polyethylene terephthalate(PET),polyvinyl chloride(PVC),and nylon 66(PA66)were exposured to C57BL/6 mice at two doses(3,30 mg particles/kg body weight)to study the effects of different types of microplastics on serum lipid metabolism,intestinal immunity,and intestinal microbiota in mice.The details and results of the study are as follows.1.After co-incubation of 20 nm PS(PS20)and 200 nm PS(PS200)with Caco-2cells for 6 h,a significant decrease in cell viability was observed at a PS20 concentration of 80 ?g/m L,and no significant change in cell viability was observed at a PS200 concentration of 100 ?g/m L.Results of reactive oxygen species(ROS)levels,lactate dehydrogenase(LDH)release,and glutathione(GSH)content indicated that PS20 and PS200 had a concentration-dependent effect on Caco-2 cell toxicity,and PS20 had a greater effect on cells than PS200.The entry of PS into the lysosomes of cells was observed by immunofluorescence assay,and a large amount of PS20 was subsequently found to co-localize with mitochondria.JC-1 fluorescent probe was applied to detect the mitochondrial membrane potential of the cells,and the experimental results showed that PS20 induced severe depolarization of the mitochondrial membrane potential.The flow cytometry results were consistent with the mitochondrial membrane potential assay,with early apoptosis induced by PS20 at a concentration of 20 ?g/m L and late apoptosis at concentration of 80 ?g/m L2.6-week-old male(star dose age)C57BL/6 mice were randomly grouped and gavaged with microplastics for 4 weeks.Body weight of PET,PVC,and PA66 groups was significantly decreased.Coefficients of hepatic,renal,and splenic were not significantly affected by PS,PE,PET,PVC and PA66 treatments.Besides,PS,PE,PET,PVC,and PA66 caused significant effects on serum total triglyceride(TC),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)metabolic disorders.3.Based on host gene expression,transcript levels of ileum and colon mucin genes(MUC2,MUC3),microbial recognition receptors(TLR2,TLR4,NOD2),regulatory factors(Fox P3,GPR43),inflammatory cytokines(IL-10),and transforming growth factor(TGF-?)were analyzed to determine the effects of different types of microplastics on the intestine.PS,PE,PET,PVC,and PA66-induced damage to the ileum mucosa exhibited a down-regulation of MUC3 expression.Exposured of microplastics causes activation of the intestinal immune system in mice.The down-regulation of TLR2,TLR4,and NOD2 transcript levels in the ileum was generally greater than in the colon by the microplastics used in this study.The down-regulation of Fox P3,IL-10,and TGF-?transcript levels indicated that microplastics have the potential to impair intestinal immune function.4.The effects of different kinds of microplastics on the intestinal microbiota of mice were investigated by 16 S r DNA high-throughput sequencing.The results showed that PS,PE,PET,PVC and PA66 altered the Alpha diversity and Beta diversity of mouse intestinal microorganisms and significantly reduced the relative abundance of Lactobacillus.The relative abundance of Firmicutes in the mouse intestine was significantly reduced after 4 weeks of treatment.At the species level,high doses of PS,PET,PVC,and PA66 groups significantly increased the relative abundance of Akkermansia?muciniphila.