Sipjeondaebo-tang Alleviates Oxidative Stress-Mediated Liver Injury through Activation of the CaMKK2-AMPK Signaling Pathway

Figure 4

The effect of SDT on CaMKK2 activation. (a) Effect of SDT on intracellular Ca²⁺ level. Fluo-4 stained HepG2 cells were incubated with SDT (300 μg/mL), and fluorescence intensity for 15 min was monitored (left). Changes in fluorescence intensity for 10 min were calculated as AUC (right). (b) CaMKK2 phosphorylation by SDT. CaMKK2 phosphorylation was determined by using HepG2 cell lysates that had been treated with SDT (300 μg/mL) for indicated times. Equal protein loading was verified by β-actin immunoblotting (upper). Intensity of phosphorylated CaMKK2 was quantified by scanning densitometry (lower). (c) Effect of SDT on HeLa cells. HeLa cells were treated with 300 μg/mL SDT for 0.5 h, and AMPK and ACC phosphorylation were determined by immunoblot analysis. Phenotype of HeLa cells was verified by LKB1 immunoblotting. (d) Effect of STO-609 on the inhibition of AA plus iron-mediated H₂O₂ production by SDT. SDT-dependent phosphorylation of AMPK (upper) and reduction of H₂O₂ production (lower) were monitored in STO-609 (1 μg/mL, 1 h) pretreated cells. (e) Effect of STO-609 on SDT-mediated cytoprotection. The relative cell viability was determined using MTT assay. All values represent mean ± SD of three separated experiments; significant versus untreated control, ^∗∗P < 0.01 and ^∗P < 0.05; significant versus AA plus iron, ^##P < 0.01; NS, not significant.

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