Polystyrene Nanoplastics Induces Inflammation Through MAPK/NF-κB Signaling Pathway

Objective In this study,RAW264.7 cells and BALB/c mice were used as models to explore the accumulation and transport rules of polystyrene nanoplastics(PS NPs)as well as the toxic damage caused by them in vivo and in vitro.Additionally,the effect of surface group modification on accumulation and cytotoxicity of PS NPs was also investigated.This study means to provide theoretical support for the study of the metabolic and toxic effects of nanoplastics in the human body.Methods The distribution of PS NPs in mice was detected within 24 h and 21 days after gavage by using the in vivo imaging instrument.Tissue structure damage after 21 days was observed after HE staining,to explore the in vivo metabolism and toxicity of nanoplastics.In the cell model RAW264.7 cells,flow cytometry was used to detect concentration and ROS production of PS,and ELISA was used to detect inflammatory factors IL-6 and TNF-α over 24 h to study the short-term cytotoxicity of nanoplastics.RAW264.7 cells were then exposed to PS NPs and their carboxy and amino-functionalized products(PS,PS-COOH,and PS-NH2),and the concentration of PS NPs within 24 h was detected.Fluorescence microscopy and confocal laser scanning microscope were used to study the effect of functional group modification on the cellular internalization of PS NPs.Polystyrene microplastic(PS-μm)was used as a control,to detect the effects of four types of PS on the generation of ROS and cell apoptosis,and the damage of subcellular structure was observed by transmission electron microscopy.On this basis,Western Blot was used to analyze the activation degree of MAPK and NF-κB pathways,and the intensity of IL-6 and TNF-α was detected by ELISA,to study the effect of functional group modification on the toxicity of PS NPs.Finally,RAW264.7 cells were pretreated with ROS inhibitor NAC and then exposed to PS-COOH for 24 h.The production of ROS,activation of signaling pathways,and secretion of inflammatory factors were detected,to verify that PSCOOH can activate ROS exert cytotoxic effects.Results 1.The results of in vivo experiments showed that within 1 h after gavage,PS transferred from the stomach to the intestinal tract and accumulated in the lung,liver,kidney,intestine,stomach,testis,heart,and brain 21 days later,leading to tissue inflammation.The results of in vitro experiments showed that PS accumulation in cells did not increase linearly within 24 h.The linear increase reached its maximum value within the first 8 hours,after which it decreased until 20 h began to increase again.After PS was internalized into cells,it could cause oxidative stress and an inflammatory response.The growth curve of ROS was sinusoidal and positively correlated with the PS content in cells before 8 h.The expression of TNF-α sharply increased after 1 h and tended to be stable with time.However,IL-6 increased significantly only after 24 h,and the expression of the two cytokines was not related to the intracellular PS concentration.2.The cellular internalization and inflammatory effects of PS were influenced by surface modification.The intracellular accumulation of PS-COOH was greater than that of PS-NH2 and PS,and all three PS NPs mainly accumulated in the cytoplasm,but PS-COOH and PS-NH2 could accumulate in the nucleus.At the same time,PS-COOH had the strongest ROS-inducing potential,starting from a concentration of 100 μg/mL to significantly induce cells to produce ROS,while PS could induce cells to produce ROS only at 1000 μg/mL.PS-μm and PS-NH2 did not cause significant oxidative stress damage.PS-COOH had the strongest cytotoxicity and induced cell apoptosis at a concentration of 100 μg/mL,while PS-NH2 and PS significantly induced apoptosis at 1000 μg/mL,and PS-μm did not induce cell apoptosis.Subsequent studies on the signal transduction mechanism of PS showed that all three kinds of PS NPs could significantly increase the phosphorylation expression levels of p38,ERK,and IκBα proteins in a dose-dependent manner,among which PS-COOH still showed the strongest activation ability.While PS-μm could not up-regulate the phosphorylation expression of IκBα.Further detection of inflammatory factors revealed that all three PS NPs could promote the secretion of TNF-α in RAW264.7 cells.PS-COOH could significantly increase the TNF-α at a concentration only 10μg/mL,but only PS-COOH and PS-NH2 could promote IL-6 secretion.And the effect of PSCOOH on activating MAPK/NF-κB signaling pathway and IL-6 and TNF-α could be inhibited by ROS inhibitor NAC,indicating that PS-COOH achieved cytotoxicity by stimulating cells to produce oxidative stress damage.Conclusion 1.PS can penetrate physiological barriers such as the blood-brain barrier and the blood-testis barrier,accumulate in multiple organs throughout the body,and cause tissue inflammation.In macrophages,PS mainly accumulates in the cytoplasm,and with a non-linear growth pattern.However,carboxy and amino functionalization can increase intracellular accumulation and enable PS to the nucleus.2.PS NPs can activate the MAPK and NF-κB pathways and further stimulate cells to secrete inflammatory factors such as IL-6 and TNF-α,leading to inflammation and apoptosis,with a dose-dependent effect.And the carboxyl functionalization of PS NPs enhanced their cytotoxicity and inflammatory potential,which may be caused by its greater intracellular accumulation and unique activation effect on ROS.