Figure legends:
Fig. 1 Nrf2 KO mice exhibited more severe renal injury and iron metabolism disruption after cisplatin stimulation than wild-type mice.C57BL/6 mice with or without Nrf2 gene KO were treated by an intraperitoneal injection of cisplatin (20 mg/kg) or vehicle. Three days later, the mice were sacrificed, and their serum and renal tissues were collected. The serum BUN (A) and SCr (B) levels were then detected. (C) and (D) HE staining indicated renal tissue damage. (E) The serum contents of iron ions were measured to assess the iron metabolism in the mice. (F to H) The protein levels of Nrf2, NQO1, HO-1, FTH-1, FTL and TFR in renal tissues were analyzed by Western blot. All experiments were repeated 5 times, P* ≤ 0.05 and P**≤ 0.01 versus the control group, P# ≤ 0.05 and P## ≤ 0.01 versus the WT CDDP group. WT, wild-type C57BL/6 mice; KO, Nrf2 KO C57BL/6 mice.
Fig. 2 Cisplatin-stimulated Nrf2 KO mice exhibited more severe lipid peroxidation than wild-type mice. Renal tissues were collected for the measurement of (A) MDA and (B) GSH. (C) and (D) The protein expression levels of GPX4 and xCT in renal tissues were analyzed by Western blot. (E) and (F) The mRNA levels of GPX4 and SLC7A11 in renal tissues were detected by real-time quantitative PCR. All experiments were repeated 5 times, P* ≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P## ≤ 0.01 versus the WT CDDP group.
Fig. 3 Leonurine protects HK-2 cells from ferroptosis. HK-2 cells were treated with gradient concentrations of erastin (A) or RSL3 (B) for 24 h, and the cell survival rate was detected by the CCK-8 assay. (C) and (D) After 24 h of treatment with or without leonurine (100 µM), HK-2 cells were stimulated with erastin (30 µM) or RSL3 (0.4 µM), and the cell survival rate was detected by the CCK-8 assay at 24 h. (E) The cell iron content was then measured by calcein-AM staining and measurement of the OD value. (F) The above mentioned cells were stained with FerroOrange and detected by confocal microscopy. (G and H) The protein levels of TFR, FTL and FTH-1 in HK-2 cells were analyzed by Western blot. All experiments were repeated 5 times, P* ≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P##≤ 0.01 versus the RSL3-treated group.
Fig. 4 Leonurine alleviates lipid peroxidation induced by RSL3 in HK-2 cells. (A) The ROS content in HK-2 cells was measured by DCFH-DA. (B) Liperfluo staining, as observed by confocal microscopy, was used to detect lipid peroxidation in HK-2 cells. HK-2 cells were harvested, and the levels of (C) GSH and (D) MDA were detected. (E to G) The protein levels of xCT, GPX4, Nrf2, NQO1 and HO-1 in HK-2 cells were analyzed by Western blot. All experiments were repeated 5 times, P* ≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P##≤ 0.01 versus the RSL3-treated group.
Fig. 5 The inhibitory effect of leonurine on HK-2 cell ferroptosis is dependent on Nrf2 gene expression. (A and B) The efficiency of Nrf2 knockdown in HK-2 cells was analyzed by Western blot. (C) The CCK-8 assay was performed to determine the survival rate of cells. The contents of (D) GSH and (E) MDA in HK-2 cells were detected by appropriate kits. (F) The ROS level in HK-2 cells was measured by DCFH-DA. (G) The iron content in HK-2 cells was detected by calcein-AM staining. (H to J) The protein levels of FTH-1, TFR, xCT and GPX4 in HK-2 cells were analyzed by Western blot. All experiments were repeated 5 times, P* ≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P## ≤ 0.01 versus the RSL3-treated group. NS, no specificity.
Fig. 6 Leonurine alleviates cisplatin-induced acute renal injury in vivo. The animals were grouped based on the treatment administered. On the fourth day, the mice were sacrificed, and their blood and kidneys were harvested. The levels of (A) BUN and (B) SCR in renal tissues were detected. (C-D) HE staining was performed to determine the degree of renal injury, and the renal injury indices were calculated according to the standard method. (E-F) The protein levels of KIM1 and NGAL in renal tissues were analyzed by Western blot. (G) F4/80 staining was performed to determine the degree of renal tissue inflammation. All experiments were repeated 5 times, P* ≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P## ≤ 0.01 versus the WT CDDP group.
Fig. 7 Leonurine reverses the cisplatin-induced iron metabolism disruption in the mouse kidney. (A) DAB staining was performed to detect the iron deposition in renal tissues. (B) The iron content in renal tissues was detected by an iron assay kit. (C and D) The protein levels of TFR, FTL and FTH-1 in renal tissues were analyzed by Western blot. (E and F) The mRNA levels of FTH-1 and TFR in renal tissues were measured by real-time quantitative PCR. All experiments were repeated 5 times, P* ≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P## ≤ 0.01 versus the WT CDDP group.
Fig. 8 Leonurine alleviates cisplatin-induced lipid peroxidation via the Nrf2 pathway. The levels of (A) MDA, (B) GSH and (C) SOD in renal tissues were detected by the appropriate assay kits. (D-F) The protein levels of GPX4, xCT, Nrf2, NQO1 and HO-1 in renal tissues were analyzed by Western blot. The mRNA levels of (G) GPX4 and (H) SLC7A11 in renal tissues were measured by real-time quantitative PCR. (I) The mitochondrial structure of renal cells was observed under an electron microscope. All experiments were repeated 5 times, P*≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P## ≤ 0.01 versus the WT CDDP group.
Fig. 9 Leonurine alleviates cisplatin-induced renal injury in vivo, and this phenomenon is dependent on Nrf2 gene expression. C57BL/6 mice with or without Nrf2 gene KO were subjected to the same treatments as described for the abovementioned animal experiments. (A and B) The levels of BUN and SCr in mouse kidneys were detected. (C and D) HE staining was performed to detected changes in renal tissue damage based on the evaluation of renal injury criteria. (E to G) The protein levels of Nrf2, NQO1, HO-1, KIM1 and NGAL in renal tissues were analyzed by Western blot. All experiments were repeated 5 times, P* ≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P##≤ 0.01 versus the WT CDDP group. NS, no specificity.
Fig. 10 The effect of leonurine on alleviating cisplatin-induced ferroptosis in vivo depends on Nrf2 gene expression. The levels of (A) Fe2+, (B) GSH and (C) MDA in renal tissues were measured by the appropriate assay kits. (D-F) The protein levels of FTH-1, TFR, GPX4 and xCT in renal tissues were analyzed by Western blot. (G) The iron deposition in renal tissues was detected by DAB staining. (H) Morphological changes in renal cell mitochondria were observed by electron microscopy. All experiments were repeated 5 times, P* ≤ 0.05 and P** ≤ 0.01 versus the control group, P# ≤ 0.05 and P##≤ 0.01 versus the WT CDDP group, NS, no specificity.