Figure 5 Icariin attenuated oxidative stress and mitochondrial
injury in TGF-β1-exposed HK-2 cells. (A, B, D) Representative images
and quantitative data of DCFH-DA staining and MitoSOX staining. (C, F)
Representative confocal microscope images of mitochondria stained by
MitoTracker Red and relative mitochondrial length in different groups of
HK-2 cells. The results were normalized to the mitochondrial length of
control HK-2 cells. (E) Representative images of mitochondrial membrane
potential (MMP) assay using JC-1 staining. (G) Fluorescence intensity
ratio of JC-1 aggregates to monomers. ns: not significant. * p
< 0.05, ** p < 0.01.
Nrf2 signaling is important for the protective effect of
icariin
Considering that Nrf2/HO-1 plays a vital role in inhibiting oxidative
stress and maintaining mitochondrial homeostasis, we further detected
the protein levels of Nrf2 and HO-1 in response to icariin both in
vivo and in vitro . Figure 6A illustrated the results of western
blot analysis, which indicated a significant reduction in protein levels
of Nrf2 and HO-1 in the obstructed kidneys from UUO group. Whereas,
icariin treatment rescued Nrf2 and HO-1 expression. Additionally, in
HK-2 cells subjected to various treatments, western blot analysis of
nuclear and cytoplasmic cell extracts showed that icariin treatment
markedly promoted nuclear translocation of Nrf2 and elevated protein
expression of HO-1 (Figure 6B). To further examine the role of Nrf2
activation in mediating the protective effects of icariin, we pretreated
TGF-β1-induced HK-2 cells with ML385, a Nrf2 inhibitor, and subsequently
re-evaluated the profibrotic molecules, inflammatory factors, and
mitochondrial injury markers of HK-2 cells again. Consequently, these
protective effects of icariin on TGF-β1-treated HK-2 cells were
significantly counteracted by ML385 pretreatment (Figure 4B-E and Figure
5). Taken together, these observations provided strong evidence that
icariin ameliorated renal interstitial fibrosis and inflammation, at
least in part, through Nrf2-dependent attenuation of mitochondrial
function.