Effects Of Advanced Oxidized Protein Products On Proliferation Of MRC-5 Cells And Expression Of FN And Col I

Objective: To investigate the effects of advanced oxidized protein products(AOPP)on human embryonic lung fibroblasts(MRC-5),reactive oxygen species(ROS),Fibronectin(FN)and collagen I(Col I).Methods: 1.MRC-5 cells were divided into three groups: human serum albumin(HSA)group,AOPP time group(12,24,48 and 72 hours),concentration group(50,100,200 and 400?g/ml),NADPH oxidase inhibitor Apocynin(APO)group(7)AOPP + APO).2.Different concentrations of AOPP and MRC-5 cells mentioned above were cocultured for different time,and the viability of the cells was detected by MTT assay.3.ELISA was used to detect the concentration of FN,Col I and transforming growth factor-?1(TGF-?1)in the supernatant of the cells.and their m RNA expressions were detected by RT-PCR.According to the MTT test results,the optimal concentration of AOPP were used to stimulate MRC-5 cells at optimal time point.Flow cytometry and fluorescence microscopy were used to detect the expression of reactive oxygen free radical(ROS).5.NADPH oxidase inhibitor(APO)and MRC-5 cells were preincubated for 1 hour,and 200?g/ml AOPP was added to stimulate for 48 hours to observe the changes of intracellular ROS.The concentration of FN,Col I and TGF-?1 were investigated,their gene expression detected.Results: 1.AOPP(50,100,200,400?g/ml)was used to treat MRC-5 cells at 12,24,48 and 72 hours,and it was found that AOPP had significant proliferation of MRC-5 cells(P<0.05),and it reached the peak at the concentration of 200?g/ml and 48 h.2.In terms of protein secretion and gene expression,the levels of FN,Col I and TGF-?1 were upregulated in concentration and time dependent manner,and reached the peak at the concentration of 200?g/ml and 48 h.While HSA had no significant effect on them(P>0.05).3.Compared with the control group,the intracellular ROS levels were higherin cells when MRC-5 cells were incubated with 200 ?g/ml of AOPP for 48 hours.(P<0.05);4.MRC-5 cells were preincubated with APO for 1 hour,and then cocultured with 200?g/ml AOPP for 48 hours.It is found that the levels of FN,Col I and TGF-?1 protein in the supernatant of cells were decreased and m RNA expression was downregulated(P<0.05).The levels of ROS in MRC-5 cells decreased(P<0.05).HSA had no significant effect on the expression of them(P> 0.05).Conclusions: AOPP can promote the proliferation of MRC-5 cells,and up-regulate the expression of FN,Col I and TGF-?1,perhaps through ROS activated by NADPH.