Study Of Silver Nanoparticles Induced-Cell Autophagy And Its Mechanism In HC11 Cells

Backgrond:Silver nanoparticles(AgNPs)are ultramicro particles formed by nano-silvers with strong antibacterial effect and broad-spectrum antibacterial property.In addition to bacteria,silver nanoparticles can also inhibit the growth of molds,fungi,viruses and harmful spores.In recent years,the dairy industry in domestic has developed rapidly,but the breeding conditions and management level are relatively backward,which leads to the high positive rate of dairy cow mastitis and causes great harm to dairy cow production and economic benefits.The results show that the nano-silver breast injector has good curative effect on clinical mastitis and invisible mastitis to different degrees,indicating that nano-silver has good application prospect in the prevention and treatment of cow mastitis.However,it is not known whether nano silver pool or nipple infiltration has any effect on the breast.Objective:To study the effect of silver nanoparticles on the cell viability of mammary epithelial cells,an important part of the mammary gland,and further explain its mechanism in combination with autophagy process,so as to provide scientific basis for the safe application of silver nanoparticles.Methods:HC11 mouse mammary epithelial cells were used as the research object,CellTiterLumiTM Luminescent Cell Viability Assay Kit and LDH Release Assay Kit were used to detect the cytotoxicity of silver nanoparticles,and the appropriate concentration of silver nanoparticles was screened.The autophagy of HC11 cells was detected by transmission electron microscopy,fluorescence staining and Western Blot.The molecular mechanism of autophagy induced by silver nanoparticles was studied by fluorescence staining,flow cytometry,ATP assay kit and Western Blot.Results:1.Study on the cytotoxicity of silver nanoparticles to HC11 cells Compared with the control group,the survival rate of HC11 cells with concentration of 25-100 μg/mL silver nanoparticles with particle size less than 100nm for 24h was significantly decreased(p<0.01)and showed a certain dose dependence.The concentration of LDH in the cell supernatant of 6.25-100 μg/mL silver nanoparticles was significantly increased(p<0.05 or p<0.01)and presented a dose-dependent effect.After comprehensive consideration,12.5,25 and 50 μg/mL concentrations were selected for subsequent tests.2.Study on autophagy of HC11 cells induced by Silver nanoparticles Transmission electron microscopy(TEM)showed typical autophagy characteristics of cells treated with silver nanoparticles(50 μg/mL).Compared with the control group,MDC fluorescence intensity was significantly enhanced in 12.5,25 and 50 μg/mL silver nanoparticles.LC3-Ⅱ and Beclin protein expression were significantly increased(p<0.01),and P62 protein expression was significantly decreased(p<0.01).The red fluorescence intensity of Lyso Tracker was significantly enhanced.The results showed that silver nanoparticles induced autophagy in HC11 cells.3.Study on the molecular mechanism of autophagy induced by silver nanoparticles in HC11 cells Compared with the control group,ROS accumulation occurred in cells of 12.5,25 and 50 μg/mL silver nanoparticles,and DCFH-DA fluorescence intensity was significantly enhanced in the 50 μg/mL silver nanoparticles treatment group(p<0.01).Further application of the total ROS scavenger NAC could reduce the ROS accumulation and expression of LC3Ⅱ in HC11 cells.The total protein expression levels of HO-1,Bach1,Keap1 and Nrf2 were significantly increased(p<0.05 or p<0.01)in a dose-dependent manner.Mitochondrial membrane potential decreased significantly in a dose-dependent manner.ATP content in cells was significantly decreased(p<0.01).The total protein expression levels of PINK1 and Parkin were significantly increased(p<0.01).The expression levels of Akt,p-AMPK and mTOR were significantly increased(p<0.01),while the expression levels of p-Akt,AMPK and p-mTOR were significantly decreased(p<0.05 or p<0.01)in a dose-dependent manner.The results showed that silver nanoparticles induced oxidative stress in HC11 cells,caused mitochondrial damage,activated Akt/AMPK/mTOR signaling pathway,and then induced autophagy.Conclusion:12.5,25 and 50 μg/mL silver nanoparticles with a particle size of less than 100nm could cause decreased cell activity in mouse breast epithelial HC11 cells,initiating the selfprotective mechanism of cells--autophagy.The molecular mechanism of initiating autophagy was as follows:Nano-silver stimulation caused oxidative stress,mitochondrial damage,and activated the Akt/AMPK/mTOR signaling pathway in HC11 cells.It suggests that silver nanoparticles with particle size less than 100nm may cause cytotoxicity in a certain concentration range,and concentration below 12.5 μg/mL can be referred clinically.