Glabridin, a bioactive component of licorice, ameliorates diabetic nephropathy by regulating ferroptosis and the VEGF/Akt/ERK pathways

Abstract

Background: Glabridin (Glab) is a bioactive component of licorice that can ameliorate diabetes, but its role in diabetic nephropathy (DN) has seldom been reported. Herein, we explored the effect and underlying mechanism of Glab on DN.

Methods: The bioactive component-target network of licorice against DN was by a network pharmacology approach. The protective effect of Glab on the kidney was investigated by a high-fat diet with streptozotocin induced-diabetic rat model. High glucose-induced NRK-52E cells were used for in vitro studies. The effects of Glab on ferroptosis and VEGF/Akt/ERK pathways in DN were investigated in vivo and in vitro using qRT-PCR, WB, and IHC experiments.

Results: Bioinformatics analysis constructed a network comprising of 10 bioactive components of licorice and 40 targets for DN. 13 matching targets of Glab were mainly involved in the VEGF signaling pathway. Glab treatment ameliorated general states and reduced FBG, HOMA-β, and HOMA-insulin index of diabetic rats. The renal pathological changes and the impaired renal function (the increased levels of Scr, BUN, UREA, KIM-1, NGAL, and TIMP-1) were also improved by Glab. Moreover, Glab repressed ferroptosis by increasing SOD and GSH activity, and GPX4, SLC7A11, and SLC3A2 expression, and decreasing MDA and iron concentrations, and TFR1 expression, in vivo and in vitro. Mechanically, Glab significantly suppressed VEGF, p-AKT, p-ERK1/2 expression in both diabetic rats and HG-induced NRK-52E cells.

Conclusions: This study revealed protective effects of Glab on the kidney of diabetic rats, which might exert by suppressing ferroptosis and the VEGF/Akt/ERK pathway.

Keywords: Bioinformatics; Diabetic nephropathy; Ferroptosis; Glabridin; VEGF signaling pathway.

Fig. 1

Bioinformatics analysis of licorice in diabetic nephropathy. A Common targets between putative predicted targets of licorice and targets associated with DN in the GeneCards and DisGeNET databases. B Bioactive component-target network of licorice against DN. The green nodes denote the bioactive components in licorice, and the red nodes denote the corresponding targets of the components. C GO and (D) KEGG enrichment analysis of 40 targets from the network. E Among the 40 targets, the genes in red rectangles were targets of Glab, which were mainly enriched in the VEGF signaling pathway in DN

Fig. 2

Animal experimental design. After adaptive feeding for a week, the DM model was established by a 3-week HFD followed 10-days intraperitoneal injection 40 mg/kg/day STZ. The control group (n = 5) received the citrated buffer only. DM model rats were randomly divided into three groups received diverse treatments: the DM group (n = 5), the DM + Glab group (n = 5), and the DM + Rosi group (n = 5). The latter two groups the corresponding treatments for a total of four weeks (from week 0 to week 4)

Fig. 3

Glab attenuates diabetic symptoms of DM rats. A Fasting blood glucose (FBG) level. B Fasting insulin (FINS) level. C HOMA-β index. D HOMA-insulin index. E H&E staining of pancreatic tissues (magnification 200 × ; bar = 100 μm.). (^***P < 0.005, vs. the control group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.005, vs. the DM group)

Fig. 4

The effect of Glab on renal function. A H&E, B PAS, and C Sirius red staining of renal tissues (magnification 200 × ; bar = 100 μm). D Alloknesis score was recorded to evaluate mechanical itch behavior. E Kidney weight and (F) index. The renal function was assessed by detecting (G) serum creatinine (Scr), (H) blood urea nitrogen (BUN), and (I) urinary albumin excretion rate (UAER). J Kidney damage biomarkers (KIM-1, NGAL, and TIMP-1) in urinary excretion were detected by western blot (^***P < 0.005, vs. the control group; ^##P < 0.01 and ^###P < 0.005, vs. the DM group)

Fig. 5

- Effects of Glab on the level of oxidative stress associated parameters, AGEs, and ferroptosis markers in the kidneys of the DM rats. The levels of (A) advanced glycation end products (AGEs), (B) reactive oxygen species (ROS), (C) malondialdehyde (MDA), (D) catalase (CAT), (E) glutathione (GSH), (F) superoxide dismutase (SOD), and (G) iron concentration determined by commercial kits. The expression of ferroptosis markers (GPX4, SLC7A11, SLC3A2, and TFR1) at (H) mRNA and (I) protein levels were detected by RT-qPCR and western blot, respectively (^***P < 0.005, vs. the control group; ^##P < 0.01 and ^###P < 0.005, vs. the DM group)

Fig. 6

Effects of Glab on high glucose (HG) induced oxidative stress and ferroptosis in vitro. The condition of DN in NRK-52E cells was induced by 30 mM glucose. A Cell apoptosis was detected by annexin V-PI double staining. B Cell viability detected by CCK-8 assay. C Lipid peroxidation was determined by the fluorescent probe C11 BODIPY 581/591. Green and blue colors indicate peroxidated lipids and nucleus respectively. The levels of (D) SOD, (E) CAT, (F) MDA, as well as (G) iron concentration were measured by commercial kits. The expression of ferroptosis markers (GPX4, SLC7A11, SLC3A2, and TFR1) at (H) mRNA and (I) protein levels were detected by RT-qPCR and western blot, respectively (^***P < 0.005, vs. the control group; ^#P < 0.05 and ^###P < 0.005, vs. the HG group)

Fig. 7

Effects of Glab on the VEGF/Akt/ERK pathway in DN in vitro and in vivo. Western blot was conducted to analyze the protein expression of VEGF, Akt, p-Akt, ERK1/2, and p-ERK1/2 in (A) HG-induced NRK-52E cells and (B) the kidney of DM rats. C Representative images and quantitation of immunohistochemistry for VEGF, p-Akt, and p-ERK1/2 in kidney tissues from rats of diverse groups (^***P < 0.005, vs. the control group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.005, vs. the DM group or the HG group)