Dioscin Improves Pyroptosis in LPS-Induced Mice Mastitis by Activating AMPK/Nrf2 and Inhibiting the NF-κB Signaling Pathway

Figure 5

Dioscin activates AMPK-Nrf2 in LPS+ATP-induced mMECs. Dioscin (400 ng/ml) was pretreated for mMECs for different times (0, 0.5 h, 1 h, 3 h, 6 h, and 12 h). (a) The protein expressions of HO-1, p-AMPK, and AMPK were determined by Western blot. (b) Cytoplasmic, (c) nuclear, and (d) total levels of Nrf2 were determined by Western blot. (e) The phosphorylation ratio of AMPK was quantified. (f) HO-1 was quantified by comparison with β-actin. (g) The ROS content was determined by ROS detection kits. The mMECs were pretreated with dioscin (400 ng/ml) for 2 h, and LPS (1 μg/ml) was stimulated for 6 h, and 30 min before LPS stimulation was completed, ATP (5 mM) was added for stimulation. (h) Nuclear translocation of Nrf2 protein was detected by immunofluorescence; the scale bar represents 200 μm. The green arrow represents the nuclear translocation of Nrf2 protein. (i) The protein of nuclear (j) Nrf2 and (k) HO-1 was detected by Western blot. ^# and ^## compared to the control group; and compared to the LPS+ATP group.

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