Abstract:

Objective To discuss the effect of Pueraria lobata-derived exosome-like nanovesicles （PL-ELNs） on senescence of L929 mouse fibroblasts induced by D-galactose （D-gal）， and to clarify the possible mechanism of action. Methods The PL-ELNs were extracted from Pueraria lobata by differential centrifugation combined with sucrose density gradient centrifugation. Transmission electron microscope （TEM） was used to observe the morphology of PL-ELNs； nanoparticle tracking analysis （NTA） was used to identify the particle size of PL-ELNs； kit method was used to quantify the protein concentration of PL-ELNs. The L929 cells were divided into control group， D-gal group， and 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups. The PKH26-labeled PL-ELNs were used to treat the L929 cells， and the uptake of PL-ELNs by L929 cells was observed. D-gal was used to establish the senescence model of L929 cells. CCK-8 method was used to screen the optimal concentrations of D-gal and PL-ELNs and to detect the activities of the cells in various groups； SA-β-gal staining was used to observe the staining of L929 cells in various groups and to calculate the percentage of SA-β-gal staining positive cells. EdU staining was used to detect the percentages of EdU positive cells in L929 cells in various groups； flow cytometry was used to detect the intracellular reactive oxygen species （ROS） levels and apoptotic rates of L929 cells in various groups； Western blotting method was used to detect the expression levels of collagen type Ⅰ （ColⅠ）， collagen type Ⅲ （ColⅢ）， P21 and P16 proteins in L929 cells in various groups. Results The PL-ELNs with a saucer-like morphology and a median particle size of 142.3 nm were isolated from Pueraria lobata， and could be effectively taken up by L929 cells. D-gal at 20 g·L?1 successfully induced senescence of L929 cells. The CCK-8 assay results showed that compared with control group， the activity of L929 cells in D-gal group was significantly decreased （P<0.01）； compared with D-gal group， the activities of L929 cells in 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly increased （P<0.01）. The SA-β-gal staining results showed that compared with control group， the percentage of SA-β-gal staining positive L929 cells in D-gal group was significantly increased （P<0.01）； compared with D-gal group， the percentages of SA-β-gal staining positive L929 cells in 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly decreased （P<0.05 or P<0.01）. The Western blotting results showed that compared with control group， the expression levels of P21 and P16 proteins in L929 cells in D-gal group were significantly increased （P<0.01）； compared with D-gal group， the expression levels of P21 and P16 proteins in L929 cells in 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly decreased （P<0.05 or P<0.01）. The EdU staining results showed that compared with control group， the percentage of EdU positive L929 cells in D-gal group was significantly decreased （P<0.01）； compared with D-gal group， the percentages of EdU positive L929 cells in 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly increased （P<0.05 or P<0.01）. The flow cytometry results showed that compared with control group， the ROS levels in L929 cells in D-gal group was significantly increased （P<0.01）； compared with D-gal group， the ROS levels in L929 cells in 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly decreased （P<0.05 or P<0.01）. Compared with control group， the apoptotic rate of L929 cells in D-gal group was significantly increased （P<0.01）； compared with D-gal group， the apoptotic rates of L929 cells in 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly decreased （P<0.01）. The Western blotting results showed that compared with control group， the expression levels of ColⅠ and ColⅢ proteins in L929 cells in D-gal group were significantly decreased （P<0.01）； compared with D-gal group， the expression levels of ColⅠ and ColⅢ proteins in L929 cells in 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly increased （P<0.05 or P<0.01）. Conclusion PL-ELNs can ameliorate D-gal-induced senescence of L929 cells， and the mechanism may be related to increasing the proliferation activity of senescent cells， reducing apoptosis， and decreasing the ROS level and the expression levels of P21 and P16 proteins in the cells.

Key words: Pueraria lobata, Exosome, Nanovesicle, Cell senescence, D-galactose, L929 cells