Figure 4 Icariin attenuated profibrotic phenotype and inflammation response of HK-2 cells induced by TGF-β1 . (A) Effect of icariin on the viability of HK-2 cells. ns: not significant compared to the blank control. * p < 0.05 and ** p < 0.01 compared to the blank control. (B) Western blot analysis of collagen I and α-SMA in HK-2 cells. (C-E) Quantitative real-time PCR analysis of IL-1β, TNF-α and IL-6 in HK-2 cells. ns: not significant. * p < 0.05, ** p < 0.01 .
Icariin attenuated oxidative stress and mitochondrial injury in TGF-β1-exposed HK-2 cells
As ROS accumulation is the hallmark of oxidative stress, we firstly evaluated the total ROS level in HK-2 cells using DCFH-DA fluorescence probe. Our results revealed that TGF-β1 significantly stimulated the generation of intracellular ROS, which was reduced by icariin (Figure 5A,B). MitoSOX is an innovative fluorescent dye specifically targeted to mitochondria, and it can serve as a specific indicator of mitochondrial ROS (mtROS) level. Through MitoSOX staining, it was observed that icariin suppressed mtROS overproduction triggered by TGF-β1 (Figure 5A,D). In addition, the application of MitoTracker Red dye revealed that TGF-β1 treatment led to a decrease in mitochondrial length and provoked mitochondrial fragmentation in HK-2 cells (Figure 5C,F); Meanwhile, TGF-β1 stimulation led to mitochondrial depolarization, as evidenced by the reduced ratio of JC-1 aggregates to monomers (Figure 5E,G). These alterations were effectively reversed by icariin treatment, suggesting that icariin improved TGF-β1-induced mitochondrial damage in HK-2 cells.