Study On The Reduction Of Bayberry Anthocyanin On Polystyrene Microplastic Toxicities And Its Mechanisms

Microplastics refer to micron or nano-sized plastic particles with the size less than 5 mm.The main compositions of microplastics include polystyrene(PS),polyethylene(PE),polypropylene(PP),etc.,among which PS is widely used in the production of high temperature resistant containers and has attracted more attention.With the detection of microplastics in various foods,human blood and feces,increasing studies have found that exposure to microplastics would induce oxidative stress,inflammation,intestinal toxicity,neurotoxicity and other biological toxicity.However,there is still a lack of research on the reduction of microplastic toxicities.Thus,it is of great significance to explore the effect of berry anthocyanins,which are easy to take in daily diet and have various bioactivities,on reducing microplastic toxicities.Anthocyanins are flavonoid compounds based on flavone nuclei,they are widely found in vegetables and berries to present diverse colors.At present,anthocyanins have been found to possess the effects of relieving oxidative stress and inflammation,regulating the gut bacteria,resisting diabetes and obesity,protecting liver and inhibiting tumor.Using the Caenorhabditis elegans,colonic Caco2 cells and C57BL/6mice as research models,this study evaluated the impacts of anthocyanin cyanin-3-O-glucoside(C3G)extracted from bayberry on reducing the toxicity of microplastic PS,and obtained the following results:(1)Anthocyanin C3G of bayberry can reduce the biotoxicity induced by PS accumulation.Using the soil-growing C.elegans as a model,the effects of berry extracts(mulberry,blueberry,indigo fruit,and bayberry)on PS accumulation and biotoxicities of N2 nematodes induced by high concentrations of PS were evaluated.It was found that bayberry extract had the most significant effect on reducing PS accumulation and toxicities,and the main component in bayberry extract was purified and identified as C3G.Further experiments determined that anthocyanin C3G can significantly reduce PS accumulation,and improve the biotoxicity in C.elegans caused by short-term exposure to high concentrations of PS or long-term exposure to environmental concentrations of PS,indicating that C3G could improve the toxicity of environmental hazards PS to environmental organisms.(2)C3G can enhance the activity of ATP-mediated ABC transporters to reduce PS accumulation in colonic Caco2 cells.Microplastics would accumulate in the gut after being ingested into organisms,the microplastic accumulation is the key factor for cytotoxicity,and reduced intracellular accumulation can directly reduce the microplastic toxicity.After treatment,it was observed that PS treatment would lead to its accumulation in cells,while C3G intervention could reduce PS accumulation in cells and enhance the activity of transmembrane ABC transporters.However,when the activity of ABC transporters was inhibited by the inhibitor MK-571,the reduction of C3G on intracellular PS accumulation was inhibited,confirming that ABC transporters is necessary for C3G to reduce PS accumulation in cells.The activity of ABC transporter is mainly mediated by ATP level,and we found that C3G can significantly increase ATP level in cells.On the one hand,when the number of mitochondria is reduced or their function is impaired,ATP synthesis will be reduced,while C3G can improve the activity of antioxidant enzymes and reduce the mitochondrial damage caused by PS-induced free radicals.On the other hand,C3G increased the expression of AMPK/SIRT1/PGC-1α signaling pathway,which mainly regulates energy synthesis.When the inhibitor of Compound C was used to block the phosphorylation of AMPK,the up-regulation of C3G on intracellular ATP levels was significantly weakened.Then,the effect of C3G on enhancing ABC transporter activity and reducing PS accumulation was significantly inhibited.Therefore,C3G could enhance ABC transporter activity by increasing mitochondrial ATP synthesis,thereby reducing PS accumulation in colonic Caco2 cells.(3)C3G can up-regulate the activity of ABC transporters to reduce colonic PS accumulation and improve PS-induced colonic inflammation in mice.In C57BL/6 mouse model,according to previous reports,0.1 mg of PS and 150 mg/kg·d of C3G were used intragaically for 6 weeks,and found that C3G can reduce PS accumulation in colon of mice.The molecular mechanism involves that C3G can up-regulate the expression of AMPK signaling pathway and increase ATP synthesis,so as to enhance the activity of ABC transporters in colon tissues,which was consistent with the results obtained in colonic Caco2 cells.That is,C3G can reduce PS accumulation in colon and promote PS excretion to feces by enhancing efflux activity of transmembrane ABC transporters.Colonic inflammation is one of the main toxic mechanisms of microplastics.The q RT-PCR and immunohistochemical analysis showed that PS exposure would increase the levels of pro-inflammatory factors TNF-α,IL-1β and CD45 in colon,inducing colonic inflammation.However,C3G intervention can significantly inhibit the expression of inflammatory signaling pathway NF-κB/i NOS-COX-2,down-regulate the level of pro-inflammatory factors,and improve colon inflammation.Therefore,C3G can not only reduce the PS accumulation in colon to decrease the risk of inducing toxicity,but also improve PS-induced colonic inflammation.(4)C3G can regulate the abundance of inflammatory gut bacteria to improve PS-induced colonic inflammation.In addition to the expression of inflammatory proteins,changes in the abundance of inflammatory gut bacteria would also impact the colonic inflammation.After 16 S r RNA sequencing of fecal samples collected from the colon,the changes of gut bacterial abundance were analyzed at the phylum,genus,and species levels.Results showed that PS increased the abundances of pro-inflammatory related bacteria of Desulfovibrio,norank＿f＿Oscillospiraceae,Helicobacter,and Lachnoclostridium;decreased the abundances of anti-inflammatory related bacteria of Dubosiella,Akkermansia,and Alistipes,while C3G could effectively reverse these adverse changes caused by PS.Combined with KEGG pathway enrichment analysis,C3G was predicated to improve the bacterial gene enrichment of anti-inflammatory related tryptophan metabolism,and reduce the pro-inflammatory related bacterial flagella assembly.(5)C3G can regulate the gut bacterial metabolites in anti-inflammatory related metabolic pathways to improve PS-induced colonic inflammation.Changes in the abundance of gut bacteria would lead to changes in the level of metabolites,combined with the non-targeted metabolomics analysis,28 metabolites with significant differences after C3G intervention were screened out.Further analysis found that C3G up-regulated the metabolites in anti-inflammatory pathways,such as L-tryptophan,indole-3-lactic acid,and N-Acetylserotonin in the tryptophan metabolic pathway,as well as 3b-hydroxy-5-cholenoic acid,chenodeoxycholate,taurine,and lithocholic acid in the bile acid metabolic pathway.Finally,6 metabolites of 7,8-dihydro-alpha-ionone,indole-3-lactic acid,L-tryptophan,thioether amide-PC,pantothenate and N-Acetylserotonin,which were highly correlated with the decrease of PS level in colon and the increase of PS level in feces,were identified by correlation analysis,suggesting that the reduction of PS accumulation in colon by C3G was also associated with impacted metabolites levels.In summary,this paper combined different experimental models to study the biotoxicity of environmental pollutants microplastics,and determined that berry anthocyanin intervention can reduce the accumulation of microplastics and reduce the biotoxicity induced by microplastics,jointly reduce the toxic risk of microplastics.This study may provide a new sights for in-depth study on the reduction of natural active components on microplastic toxicities.