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.