Shikonin Activates Nrf2 via Mst1 Downregulation

Next, we explored the precise mechanism by which shikonin activated Nrf2. One of the essential elements of the Hippo signaling system, which regulates cell survival and death to control tissue growth, is Mst1^{30 (link)}. Mst1 is well known for being a pro-apoptotic molecule, and by being suppressed, it reduces the generation of ROS, which lessens cell apoptosis^{31 (link)}. Recent research has revealed that Mst1 controls both autophagy and apoptosis in cardiomyocytes^{32 (link),33 (link)}. We detected the expression of Mst1 and found that the high expression of Mst1 induced by DOX was significantly decreased after shikonin administration (Fig. 7A,B). Compared with the control groups, mice treated with DOX displayed decreased phosphorylation of AMPK and AKT in the hearts, but shikonin couldn’t increase the phosphorylation of AMPK and AKT (Fig. 7A,C). To Verify the hypothesis that shikonin activated Nrf2 via Mst1, NRCMs were infected with adenovirus to overexpress Mst1 (Fig. S2C). In the cells infected with GFP, shikonin could reverse the low expression of Nrf2 induced by DOX, but in the cells overexpressing Mst1, shikonin had no effect on the low expression of Nrf2 induced by DOX (Fig. 7D,E). Further detection of ROS level showed that shikonin decreased the level of ROS induced by DOX, and overexpression of Mst1 completely offsets the protective effect of shikonin on cardiomyocyte oxidative stress (Fig. 7F). In addition, NRCMs exposed to DOX had decreased cell viability and after shikonin administration the cell viability was increased. However, overexpression of Mst1 abolished the protection of shikonin against DOX-induced cell death (Fig. 7G).Figure 7

Mst1 downregulation was responsible for shikonin-mediated activation on Nrf2. (A–C) Western blot and quantitative analysis showing the protein levels of p-AMPK, t-AMPK, p-AKT, t-AKT and Mst1 in vivo (n = 6). Original blots/gels are presented in Supplementary Fig. S8. (D,E) Western blot and quantitative analysis showing the Nrf2 expression after Ad-Mst1administration (n = 6). Original blots/gels are presented in Supplementary Fig. S9. (F) ROS level (n = 6). (G) CCK-8 assay for cell viability (n = 6). **p < 0.01, ****p < 0.0001, significantly different as indicated. ns not significant.