A novel compound AB38b attenuates oxidative stress and ECM protein accumulation in kidneys of diabetic mice through modulation of Keap1/Nrf2 signaling

Abstract

Extracellular matrix (ECM) deposition following reactive oxygen species (ROS) overproduction has a key role in diabetic nephropathy (DN), thus, antioxidant therapy is considered as a promising strategy for treating DN. Here, we investigated the therapeutic effects of AB38b, a novel synthetic α, β-unsaturated ketone compound, on the oxidative stress (OS) and ECM accumulation in type 2 diabetes mice, and tried to clarify the mechanisms underlying the effects in high glucose (HG, 30 mM)-treated mouse glomerular mesangial cells (GMCs). Type 2 diabetes model was established in mice with high-fat diet feeding combined with streptozocin intraperitoneal administration. The diabetic mice were then treated with AB38b (10, 20, 40 mg· kg^-1· d^-1, ig) or a positive control drug resveratrol (40 mg· kg^-1· d^-1, ig) for 8 weeks. We showed that administration of AB38b or resveratrol prevented the increases in malondialdehyde level, lactate dehydrogenase release, and laminin and type IV collagen deposition in the diabetic kidney. Simultaneously, AB38b or resveratrol markedly lowered the level of Keap1, accompanied by evident activation of Nrf2 signaling in the diabetic kidney. The underlying mechanisms of antioxidant effect of AB38b were explored in HG-treated mouse GMCs. AB38b (2.5-10 μM) or resveratrol (10 μM) significantly alleviated OS and ECM accumulation in HG-treated GMCs. Furthermore, AB38b or resveratrol treatment effectively activated Nrf2 signaling by inhibiting Keap1 expression without affecting the interaction between Keap1 and Nrf2. Besides, AB38b treatment effectively suppressed the ubiquitination of Nrf2. Taken together, this study demonstrates that AB38b ameliorates experimental DN through antioxidation and modulation of Keap1/Nrf2 signaling pathway.

Keywords: AB38b; ECM; Keap1; Nrf2; antioxidant; diabetic nephropathy; glomerular mesangial cells; oxidative stress; resveratrol.

Fig. 1

Effects of some of the new compounds on cell viability. a Cells were treated with different compounds (20 μM, 10 μM) for 24 h, and cell viability was evaluated by the CCK8 assay. b The protective effect of the compounds on GMCs after H₂O₂-induced cell damage. c Compared with normal glucose (NG), the compounds (10 μM), and Res (10 μM) enhanced the transcription of the antioxidant genes NQO-1, HO-1, and SOD. d The concentration-dependent effect of AB38b on cell viability. e The chemical structure of AB38b. The data are expressed as the mean ± SD, n = 3. *P < 0.05 vs. DMSO; ^#P < 0.05 vs. H₂O₂; ^&P < 0.05, ^&&P < 0.01 vs. NG

Fig. 2

Changes in physiological parameters in diabetic animals after AB38b treatment. a BUN, blood urea nitrogen. b Cr, serum creatinine. c LDL-C, low-density lipoprotein cholesterol. d T-CHO total cholesterol. The data are expressed as the mean ± SD, n = 6. *P < 0.05, **P < 0.01 vs. N; ^#P < 0.05, ^##P < 0.01 vs. DN

Fig. 3

Effects of AB38b on morphological changes and renal fibrosis in the glomeruli of diabetic animals. a PAS staining of the glomerulus. b Sirius red staining of the glomerulus. c TEM analysis. d Immunohistochemical analysis of laminin. e Immunohistochemical analysis of collagen IV. f Relative percentages of PAS staining. g Relative percentages of Sirius red staining. h The quantification of laminin expression. i The quantification of collagen IV expression. The data are expressed as the mean ± SD, n = 6. *P < 0.05, **P < 0.01 vs. N; ^#P < 0.05, ^##P < 0.01 vs. DN

Fig. 4

AB38b reduced oxidative stress while activating Nrf2 in diabetic kidneys. a MDA levels. b LDH activity. c GSH levels. d SOD activity. e CAT levels. f Protein levels of Keap1, Nrf2, NQO-1, and HO-1. g–j The densitometric analysis of the Western blots. The Keap1 g, Nrf2 h, NQO-1 i, and HO-1 j signals were normalized to the β-actin signals for the same samples to determine the fold-change relative to the control, the expression level of which was set as 1. k–m The relative mRNA expression levels of Nrf2 k, NQO-1 l, and HO-1 m in diabetic kidneys. The data are expressed as the mean ± SD, n = 6. *P < 0.05, **P < 0.01 vs. N; ^#P < 0.05, ^##P < 0.01 vs. DN

Fig. 5

AB38b alleviated HG-induced oxidative stress in mesangial cells. Mesangial cells were treated with various concentrations of AB38b (2.5 μM, 5 μM, and 10 μM) and Res (10 μM) with or without normal glucose (NG, 5.6 mM), high glucose (HG, 30 mM) or DMSO (0.05%) for 24 h. The MDA level, GSH level, LDH activity, T-SOD activity, SOD1 activity, SOD2 activity and the intracellular production of ROS in GMCs were detected to evaluate oxidative stress. a MDA levels. b LDH activity. c GSH levels. d T-SOD activity. e SOD1 activity. f SOD2 activity. g The intracellular production of ROS. The data are expressed as the mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs. NG; ^#P < 0.05, ^##P < 0.01 vs. HG

Fig. 6

Effects of AB38b on ECM accumulation in high glucose-induced mesangial cells. a IF analysis of collagen IV in SV40 cells exposed to HG conditions and treated with various concentrations of AB38b. b IF analysis of laminin in SV40 cells exposed to HG conditions and treated with various concentrations of AB38b

Fig. 7

Effects of AB38b on Nrf2 signaling in high glucose-induced mesangial cells. a Western blot analysis of Nrf2 (upper panel), NQO-1, and HO-1 (bottom panel) in the different groups of mesangial cells. b–d The densitometric analysis of the Western blots. The Nrf2 b, NQO-1 c, and HO-1 d signals were normalized to the β-actin signals for the same samples to determine the fold-change relative to the control, the expression level of which was set as 1. e–g The relative mRNA expression levels of Nrf2 e, NQO-1 f, and HO-1 g in the different groups of mesangial cells. h Confocal immunofluorescence images showing the colocalization of Nrf2 and Keap1 in SV40 cells exposed to HG conditions and treated with various concentrations of AB38b. The data are expressed as the mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs. NG; ^#P < 0.05, ^##P < 0.01 vs. HG

Fig. 8

Depletion of Nrf2 partly abrogated the inhibition of ECM components and increase of antioxidant enzymes by AB38b. a Western blot analysis of collagen IV and laminin in the different groups of mesangial cells. b–c The densitometric analysis of the Western blots. The collagen IV b and laminin c signals were normalized to the β-actin signals for the same samples to determine the fold-change relative to the control, the expression level of which was set as 1. d–e CAT activity d, GSH levels e, and SOD activity f in the different groups of mesangial cells. The data are expressed as the mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs. HG + si-Veh; ^#P < 0.05, ^##P < 0.01 vs. HG + si-Nrf2

Fig. 9

AB38b exerted an antioxidant role via reducing cytoplasmic Keap1 and inhibiting the ubiquitination of Nrf2. Mesangial cells were treated with DMSO (0.05%), AB38b (5 μM), SFN (2.5 μM), and MG132 (2.5 μM) for 24 h. a–b The relative mRNA expression levels of Keap1 a and Nrf2 b. c–h The nuclear and cytosolic fractions were extracted from various groups as indicated and were used for immunoblot analysis of Keap1 c–e and Nrf2 f–h. i Co-IP and Western blot analysis showed interactions between Keap1 and Nrf2. j The activity of the 20 S proteasome. k The inhibitory effect of AB38b on ubiquitinated Nrf2 in SV40 cells. Total cell lysates were immunoprecipitated with an anti-Nrf2 antibody and then immunoblotted with an anti-ubiquitin antibody. The data are expressed as the mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs. DMSO