Ic steatosis in vitro, HepG2 cells had been treated with distinct concentrations of OA (0, 0.1, 0.25, 0.five, 0.75, 1 and two mM). As shown in Figure 1a, OA of less than 1 mM did not minimize cell viability soon after 24 h and 48 h incubation. However, reduction in HepG2 cells viability was observed when OA concentration was enhanced to a lot more than 1 mM (p 0.05). For that reason, OA of 0.5 mM was utilized to induce lipogenesis in HepG2 cells within the following research. Lipid accumulation was investigated by oil red O staining. As shown in Figure 1c,d, massive number of lipid droplets was formed in HepG2 cells after OA exposure for 48 h (p 0.01), compared with untreated cells. Consistent with the results of oil red O staining, TG content material in HepG2 cells was increased after OA incubation (Figure 1b). Moreover, western blot evaluation recommended elevated expression of FAS (p 0.05), a lipogenic protein, in HepG2 cells by OA Caspase 10 Inhibitor Source treatment (Figure 1e,f). In summary, 0.5 mM OA could induce lipid accumulation in HepG2 cells without the need of affecting cell viability. Recent studies recommended that the excess of oxidative anxiety could contribute to cellular injury and also the pathogenesis of NAFLD. Hence, modulating antioxidant enzymes and oxidative anxiety might be significant for NAFLD treatment. SOD is very important peroxidation indexes in NAFLD. As shown in Figure 2a, OA treatment for 48 h significantly enhanced the SOD content material (p 0.01). Concomitantly, HepG2 cells treated with 0.5 mM OA for 48 h prominently enhance the protein levels of Nrf2 and HO-1 (p 0.01, Figure 2b ).Int. J. Mol. Sci. 2021, 22,three ofFigure 1. Induction of steatosis by OA in HepG2 cells. (a) SRB assay of cell viability of HepG2 cells treated with distinct concentration of OA for 24 h and 48 h. (b) Measurement of intracellular TG contents in HepG2 cells immediately after incubation with 0.5 mM OA for 24 h and 48 h. (c) Oil red O staining to detect intracellular lipid droplets in HepG2 cells just after treatment with 0.5 mM OA for 24 h and 48 h. (d) Quantitative analysis of intracellular lipid droplets accumulation in HepG2 cells. (e) Western blot analysis of expression of FAS in HepG2 cells after therapy with 0.5 mM OA for 24 h and 48 h. (f) Quantification FGFR Inhibitor Formulation benefits in the expression of FAS. Information have been expressed as Imply SD of 3 independent experiments (n = 3). p 0.05 and p 0.01, compared with HepG2 cells with out OA therapy (0 h).Int. J. Mol. Sci. 2021, 22,four ofFigure two. Induction of steatosis by OA in HepG2 cells. (a) Measurement of levels of SOD in HepG2 cells following incubation with 0.five mM OA for 24 h and 48 h. (b) Western blot evaluation of expression of Nrf2 and HO-1 in HepG2 cells immediately after therapy with 0.five mM OA for 24 h and 48 h. (c) Quantification benefits of the expression of HO-1. (d) Quantification benefits on the expression of Nrf2. Data had been expressed as Mean SD of three independent experiments (n = 3). p 0.05 and p 0.01, compared with HepG2 cells without OA treatment (0 h).two.2. Effects of Kaempferol and Kaempferide on Cell Viability The structure of kaempferol and kaempferide were presented in Figure 3a,b. As shown in Figure 3c,d, kaempferol and kaempferide much less than 10 did not alter the viability of HepG2 cells. In contrast, kaempferol and kaempferide at 50 and one hundred decreased HepG2 cell viability (p 0.01) just after incubation for 48 h. Moreover, co-incubation of 0.five mM OA with kaempferol and kaempferide (5, ten and 20 ) didn’t trigger reduction in HepG2 cell viability, compared with vehicle-treated cells (Figure 3e,f),.
