Effect Of Silica Nanoparticles On Apoptosis Of HL-7702 Cells

In this study, human hepatic cell line HL-7702 was used as model to detect cell apoptosis after exposure to silica nanoparticles. Morphological changes in HL-7702 cells were observed under light microscopy after Haematoxylin and Eosin (HE) staining, cells apoptosis was detected by both Giemsa staining and flow cytometry (FCM) with AnnexinV-EGFP/PI double staining; mitochondrial membrane potential was examined by FCM with Rh-123 staining; and the expression of apoptosis related proteins were detected by immunocytochemical method. The results were shown as follow:1. Morphological changes in HL-7702 cells induced by silica nanoparticlesIn negative control group, HL-7702 cells were mostly polygonal with little particles in cytoplasm; in exposure groups, cell shape changed from polygonal to spindle length, cell number significantly decrease, more particulate matter were observed resulting in cell transparency decrease.HE staining showed that after HL-7702 cells were treated with 25,50,100 and 200μg/mL of silica nanoparticles, cells exhibit morphological changes: cell number reduced, cell turning shrink, vacuolar degeneration in cytoplasm and nucleus increased,and trachychromatic chromatin and chromatin margination could also be observed.2. Apoptosis in HL-7702 cells induced by silica nanoparticlesAfter HL-7702 cells were treated with 25,50,100 and 200μg/mL of silica nanoparticles, cell apoptosis were observed by light microscopy after Giemsa staining. The results showed silica nanoparticles could induce typical apoptotic changes: blebbing and chromatin condensation. Statistical analysis showed that the percentage of apoptotic cells was increased significantly at all exposed groups compared with control group (P<0.01).FCM with AnnexinV-EGFP/PI double staining was also applied to detected apoptosis in HL7702 cells. After exposure to silica nanoparticles (25,50,100 and 200μg/mL) for 24 h, apoptotic rate significantly increased compared with control group (P<0.01). Although apoptotic rate continuously increased till at 100μg/mL silica treated group, it decreased at 200μg/mL group.3. Changes of mitochondrial membrane potential in HL-7702 induced by silica nanoparticlesFCM with Rh-123 staining was used to detect the changes of mitochondrial membrane potential in HL7702 cells. After exposure to silica nanoparticles (25,50,100 and 200μg/mL) for 24 h, mitochondrial membrane potential decrease with the increasing dose, and there were significant differences between exposure and negative control group (P<0.01).4. Expression of apoptosis related proteins in HL-7702 induced by silica nanoparticles4.1 Expression of Bax,Bcl-2After exposure to silica nanoparticles (25,50,100 and 200μg/mL) for 24 h, the expression of Bax,Bcl-2 enhanced with the increase of silica nanoparticles, there were significant differences between exposure and negative control group(P<0.01), and a decrease in Bcl-2/Bax ratio were also confirmed.4.2 Expression of Cyt C,Caspase-3After exposure to silica nanoparticles (25,50,100 and 200μg/mL) for 24 h, the expressions of Cyt C and Caspase-3 enhanced with the increase of silica nanoparticles, and there were significant differences in Cyt C,Caspase-3 expression between exposure and negative control group(P<0.01).