Novel Agar Membrane Developed For The Applications On The Effect Study Of Fine Atmospheric Particulates On Human Hepatotoxicity

Air pollution is formed of different components produced by nature and human activities.Particulate matter(PM)is one of the main air pollutants.It is also one of the primary pollutants affecting the air quality in China.Atmospheric particulate pollution has already become an important factor threatening human health,especially PM2.5 with aerodynamic diameter less than 2.5μm,which is considered to be closely related to the occurrence and development of many major diseases.At present,the most commonly used methods for the collection of atmospheric particulate matter are the membrane filtration and the impactor collection.The sampling membranes commonly used for collecting atmospheric particulates are made of polytetrafluoroethylene(Teflon),glass fiber,polycarbonates,quartz and other materials.However,the commonly used sampling membranes have the disadvantages of strong background interference,low extraction efficiency and other defects.They are not fully applicable to cell experiments.Considering the shortcomings of the current sampling membranes,this experiment explored the replacement of the conventional membranes of the impactor sampler with a membrane made of biocompatible agar medium,so that the atmospheric particles hit and stayed on the soft agar membrane.The agar membranes which contain atmospheric particles directly exposed to the cells.According to the above,we developed a new low-disturbed,low-destructive sampling method of cell experiments.In this study,human hepatoma cell HepG2 and human normal cell HL7702 were used as in-vitro models to observe the influence of fine particulate matters on hepatocyte viability,oxidative stress,apoptosis and proteomics sampled on the agar membrane and Teflon membrane under the same conditions.The effects of fine particulate matters on the toxicity of both hepatocytes were evaluated.The main contents are as follows:1)Agar sampling membrane was prepared by freeze-drying method.The optimal membrane preparation conditions were 1%agar gel concentration and then slowly freezing in refrigerator.The structural characteristics of agar sampling membrane were characterized by SEM,ATR-FTIR and other characterization methods.Using SEM-EDX,ATR-FTIR,ion chromatography,ICP-OES,GC-MS,and other equipment to characterize the particulate matter.,the consistency of the atmospheric particulate collection efficiency of agar sampling membrane and traditional sampling membrane(Teflon membrane)was verified.The unique advantages of agar sampling membrane in the collection of atmospheric particulate matter were explored and the feasibility of agar sampling membrane in the collection of atmospheric particulate matter was verified.2)Using agar membrane and Teflon membrane to collect fine particulate matters PM2.1 in a certain area of Nanjing.Firstly,CCK-8 method was used to detect the viability of HepG2 cells and HL7702 cells with different treatments of PM2.1on both membranes.The stimulation concentrations of 50,100,200μg/mL were determined for the agar group,and 100,200,400μg/mL for Teflon group.iTRAQ was used to study the fold changes in the overall proteomics of the cells after stimulation,together with further analysis of the advantages and disadvantages of the two membranes and the toxic effects of PM2.1 on the cells.The apoptosis of cells after 24 hours of PM2.1 stimulation was detected by Annexin V-FITC/PI double staining method.The mRNA and protein expression levels of Bax,Bcl-2 and Caspase 3 were detected by Real-time PCR and Western Blotting.The reason for the hepatocyte apoptosis may be the change in the ratio of Bcl-2/Bax and the activation of Caspase3,which may be caused by high oxidative stress in the cells.Using flow cytometry DCFH-DA fluorescent probe method to detect PM2.1 stimulation for 24 hours,it was found that with the increase of PM2.1 concentration,the composition of reactive oxygen species also increased.The total SOD,CAT activity decreased and MDA content increased in the cells after 24 hours of PM2.1 stimulation,which were detected by enzyme-labeled method,indicating that the cells induced oxidative stress after PM2.1 stimulation.Real-time PCR and Western Blotting were used to detect the mRNA and protein expression of Nrf2,HO-1 and KEAP1 in the two cells after 24 hours of PM2.1 stimulation.It was found that the cells up-regulated HO-1 and self-ubiquitinated KEAP-1 pathway to deal with PM2.1 oxidative stress.Comparing the results of cell viability and apoptosis,it was found that the agar group was more toxic than the Teflon group at the same concentration and HL7702 was more likely to be apoptotic than HepG2 in the same concentration group.The reason why the agar group is more toxic may be related to the direct extraction method and the agar membrane itself,which is easy to adsorb organic substances and endotoxin in the atmosphere.HL7702,as a human normal cell,is often more sensitive than human liver cancer cells.