An In Vitro Experiment On The Effect Of Polystyrene Micro/nano-plastics On Glucose Metabolism Of Human Adipocytes Via Oxidative Stress

Objective:With the increasing use and waste of plastic products,microplastic pollution is an epidemic around the world.It has been reported that microplastics can cause glucose metabolism disorders in many organisms and even lead to diabetes in mice.Insulin resistance is the core pathological feature of type 2 diabetes mellitus.As one of the main target organs of insulin,white adipose tissue can regulate the balance of glucose metabolism through various ways.At present,there are few studies on the effect of MPs on adipose tissue,and the mechanism of the disorder of glucose metabolism caused by MPs is still unclear.Therefore,in this study,human adipocytes were used as experimental subjects,and polystyrene was used as representative subjects to evaluate the effects of polystyrene micro/nano-plastics on glucose metabolism of human adipocytes,and to explore the possible role of oxidative stress.Methods: Human adipose-derived mesenchymal stem cells were cultured in vitro to induce the differentiation of human adipocytes,and the adipocyte differentiation was identified by oil red O staining.Human adipocytes were infected with PS-MPs(3μm)and PS-NPs(100nm)with 50μg/m L fluorescence for 1h and 4h,and the entry of PSMPs/NPs into human adipocytes was observed under laser confocal microscope.Human adipocytes were treated with multiple concentrations of 3μm-PS and 100 nmPS for 24 h and 48 h.The effects of these concentrations on adipocyte viability were detected by CCK-8 kit.Three concentrations of 3μm-PS and 100nm-PS,which had no effect on cell viability,were selected for 24 h,and after 3h with/without insulin stimulation,the supernatant of human adipocytes was collected.The glucose content in the supernatant was detected by oxidase method,and the glucose consumption and glucose consumption rate of each group were calculated to evaluate the glucose uptake capacity of human adipocytes.The expression levels of key genes and proteins in glucose uptake signaling pathway were detected by RT-q PCR and Western blot.The levels of oxidative stress related indexes were detected by the kit.Results: 1.The results of confocal laser microscopy showed that after exposure to100nm-PS for 1h,several green fluorescent particles could be seen in human adipocytes.After 4h,the green fluorescent particles persisted in the cells.No green fluorescent particles were observed in the cells after 3μm PS was poisoned for 1 hour.After prolonged exposure time to 4h,a small number of green fluorescent particles were found in the cells.2.The results of CCK-8 detection showed that compared with the control group,3μm-PS treatment significantly decreased the viability of human adipocytes at 2400μg/m L and 1200μg/m L for 24 and 48 h,respectively(P<0.01).3.The results of oxidative stress related indexes showed that after being exposed to 100 nm and 3μm-PS at 75,150 and 300μg/m L for 24 h,300μg/m L of 100nm-PS significantly increased the content of MDA in human adipocytes compared to the control group.The level of SOD was decreased,and the difference was statistically(P<0.05).Compared with the control group,the level of GSH in cells exposed to 3μm-PS at 150 and300μg/m L concentration was decreased(P<0.01),and the level of SOD was also decreased at 3μm PS at 300μg/m L(P<0.05).4.The results of glucose consumption experiment showed that after 100 nm and 3μm-PS intervention in human adipocytes for24 h,the concentrations of 100nm-PS at 300μg/m L and 3μm-PS at 150 and 300μg/m L significantly reduced the intracellular glucose consumption,regardless of whether insulin was added(P<0.01).5.RT-q PCR results showed that compared with control group,after 300μg/m L 100 nm and 3μm-PS infected human adipocytes for 24 h,intracellular GLUT4 gene expression level decreased significantly(P<0.05).6.Western blot results showed that after exposure to 100 nm and 3μm-PS at 300μg/m L for 24 h,the levels of JNK phosphorylated protein in human fat cells and phosphorylation of serine307 in IRS1 were significantly increased(P<0.01).Conclusion:Both PS-MPs/NPs can enter human adipocytes.They may activate JNK protein and enhance phosphorylation of serine 307 in IRS1 by breaking the oxidative stress balance state of human adipocytes,which leads to the blockage of glucose metabolism pathway and the reduction of glucose uptake capacity of cells,ultimately affecting glucose metabolism.