Calycosin plays a protective role in diabetic kidney disease through the regulation of ferroptosis

Figures & data

Table

GPX4	Primer F	5′-GTTACTCCCTGGCTCCTG-3′
	Primer R	5′-CTCCCAGTGAGGCAAGAC-3′
NCOA4	Primer F	5′-TGATCTCCAACCTTTTCC-3′
	Primer R	5′-CTTACATACCCAGCACCG-3′
β-actin	Primer F	5′-GATGACCCAGATCATGTTTGAG-3′
	Primer R	5′-TAATGTCACGCACGATTTCC-3′

Figure 1. Calycosin plays a protective role in HG-induced renal tubule injury. (A–D) The levels of LDH (A), MDA (B), GSH (C) and lipid ROS (D) were measured in HK-2 cells treated with NG or HG. ***p < 0.001 vs. control. (E) The cell viability was measured in HK-2 cells treated with HG and different concentrations (0, 5, 10, 20, 40 and 80 μM) of calycosin. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; ##p < 0.01, ###p < 0.001 vs. HG. (F–I) The levels of LDH (F), MDA (G), GSH (H) and lipid ROS (I) were measured in HK-2 cells treated with HG and different concentrations (0, 10, 20 and 40 μM) of calycosin. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. HG. (J, K) mRNA (J) and protein (K) levels of GPX4 and NCOA4.

Figure 2. Calycosin elicits its effects through the inhibition of ferroptosis. (A–C) HK-2 cells were treated with erastin (35 μM) and/or calycosin (40 μM). Then, the cell viability (A), lipid ROS levels (B) and NOCA4 protein levels (C) were detected. ***p < 0.001 vs. vehicle; ##p < 0.01, ###p < 0.001 vs. erastin. (D) Three knockdown lentiviruses against GPX4 (shGPX4-1, shGPX4-2 and shGPX4-3) were transfected to HK-2 cells, and GPX4 expression level was measured by real-time PCR and Western blot. ***p < 0.001 vs. shNC. (E–G) HK-2 cells were transfected with shGPX4 lentivirus and/or calycosin (40 μM). Then, the cell viability (E), lipid ROS levels (F), and NOCA4 protein levels (G) were detected. **p < 0.01, ***p < 0.001 vs. shNC; #p < 0.05, ###p < 0.001 vs. shGPX4.

Figure 3. Calycosin increases cell viability in HG-induced renal tubule injury via inhibiting ferroptosis. (A–C) HK-2 cells were treated with erastin (35 μM) and/or calycosin (40 μM) upon pre-treatment with HG. Then, the cell viability (A), lipid ROS levels (B) and NOCA4 protein levels (C) were detected. **p < 0.01, ***p < 0.001 vs. vehicle; #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. HG + Cal + Erastin.

Figure 4. Calycosin plays a protective role in db/db mouse through the inhibition of ferroptosis. (A) Levels of BUN and Cr. (B) Levels of LDH, MDA and GSH. ***p < 0.001 vs. db/m; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. db/db vehicle. (C, D) HE (C) and Masson’s Trichrome (D) staining of kidney sections. (a) db/m mice; (b) db/db mice; (c) db/db mice treated with 10 mg/kg of calycosin; (d) db/db mice treated with 10 mg/kg of calycosin. (E, F) The mRNA (E) and protein (F) levels of GPX4 and NCOA4 were detected. *p < 0.05, **p < 0.01, ***p < 0.001 vs. db/m and ##p < 0.01, vs. db/db vehicle.