Fraxin Promotes the Activation of Nrf2/ARE Pathway via Increasing the Expression of Connexin43 to Ameliorate Diabetic Renal Fibrosis

Abstract

Diabetic nephropathy (DN) is quickly becoming the largest cause of end-stage renal disease (ESRD) in diabetic patients, as well as a major source of morbidity and mortality. Our previous studies indicated that the activation of Nrf2/ARE pathway via Connexin43 (Cx43) considerably contribute to the prevention of oxidative stress in the procession of DN. Fraxin (Fr), the main active glycoside of Fraxinus rhynchophylla Hance, has been demonstrated to possess many potential pharmacological activities. Whereas, whether Fr could alleviate renal fibrosis through regulating Cx43 and consequently facilitating the activation of Nrf2/ARE pathway needs further investigation. The in vitro results showed that: 1) Fr increased the expression of antioxidant enzymes including SOD1 and HO-1 to inhibit high glucose (HG)-induced fibronectin (FN) and inflammatory cell adhesion molecule (ICAM-1) overexpression; 2) Fr exerted antioxidant effect through activating the Nrf2/ARE pathway; 3) Fr significantly up-regulated the expression of Cx43 in HG-induced glomerular mesangial cells (GMCs), while the knock down of Cx43 largely impaired the activation of Nrf2/ARE pathway induced by Fr; 4) Fr promoted the activation of Nrf2/ARE pathway via regulating the interaction between Cx43 and AKT. Moreover, in accordance with the results in vitro, elevated levels of Cx43, phosphorylated-AKT, Nrf2 and downstream antioxidant enzymes related to Nrf2 were observed in the kidneys of Fr-treated group compared with model group. Importantly, Fr significantly improved renal dysfunction pathological changes of renal fibrosis in diabetic db/db mice. Collectively, Fr could increase the Cx43-AKT-Nrf2/ARE pathway activation to postpone the diabetic renal fibrosis and the up-regulation of Cx43 is probably a novel mechanism in this process.

Keywords: AKT; Connecxin43; Nrf2/ARE pathway; diabetic nephropathy; fraxin; oxidative stress.

FIGURE 1

Fr suppressed the development of inflammatory fibrosis factors by boosting the expression of antioxidant enzymes in high glucose induced GMCs. (A-B) The protein expression of FN, ICAM-1, Nrf2, HO-1 and SOD1 in GMCs were measured by western blot assay, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. 0 h. (C) Cell survival analysis through MTT assay of GMCs was treated with different concentrations of Fr for 24 h, ^*** p < 0.001 vs. Ctrl. (D) The cellular distribution of FN and ICAM-1 was untreated or treated through Immunofluorescence (magnification ×400). Bar: 40 μm. The red and green fluorescence represent the localization of FN and ICAM-1 respectively. (E-F) The protein levels of FN, ICAM-1, Nrf2, HO-1 and SOD1 in GMCs were measured through western blot assay, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. Ctrl; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. HG. Independent experiments were performed at least three times with similar results.

FIGURE 2

Fr inhibited oxidative stress by activating the Nrf2/ARE pathway in HG-induced GMCs. (A) The western blot results showed the expression of Nrf2 in the nucleus and cytoplasm. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. 0 h. (B) Western blot analysis of nuclear and cytoplasm Nrf2 in GMCs treated with HG and Fr for 24 h ^* p < 0.05, ^** p < 0.01 vs. Ctrl; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. HG. (C) The subcellular distribution of Nrf2 in GMCs was measured by immunofluorescent staining (×600 magnification). Bar: 40 μm. Green fluorescence indicated localization of Nrf2. (D) Intracellular H₂O₂ level was provoked by high glucose and Fr treatment for 12 h *p < 0.05 vs. Ctrl; ^### p < 0.001 vs. HG. (E) Transcriptional activity of Nrf2 was measured by luciferase reporter assay. ^* p < 0.05 vs. Ctrl; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. HG. (F) Fluorescent probe DHE was applied to detect the intracellular ROS levels for 12 h ^** p < 0.01 vs. Ctrl; ^# p < 0.05, ^## p < 0.01 vs. HG. Independent experiments are performed at least three times with similar results.

FIGURE 3

Interfering with Cx43 attenuated high glucose induced activation of the Nrf2/ARE signaling pathway in GMCs. (A) The effects of Cx43-siRNA on Cx43 expression in GMCs under 30 mM HG (24 h) conditions was measured by the western blot assay. ^*** p < 0.001 vs. (NG + NC); ^### p < 0.001 vs. (HG + NC). (B-C) The effects of Cx43-siRNA on the expression of FN, ICAM-1, Nrf2, HO-1, and SOD1was induced by HG (30 mM, 24 h). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. (NG + NC); ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. (HG + NC). (D) The subcellular distribution of Nrf2 in GMCs was measured by immunofluorescent staining under HG (6 h) conditions (×600 magnification). Bar: 40 μm. Green fluorescence indicated localization of Nrf2. Independent experiments are performed at least three times with similar results.

FIGURE 4

Fr promoted Nrf2 nuclear translocation by increasing Cx43 expression in HG-induced GMCs. (A-B) Expression of Cx43 were detected by the western blot assay in GMCs. (C-D) The protein levels of Cx43, FN, ICAM-1, Nrf2, HO-1 and SOD1 were detected by the western blot assay. (E) The subcellular distribution of Nrf2 in GMCs was measured by immunofluorescent staining (×600 magnification). Bar: 40 μm. Green fluorescence indicated localization of Nrf2. (F) The western blot results shown the expression of Nrf2 in the nucleus and cytoplasm. (G) The H₂O₂ levels in GMCs were detected. (H) The intracellular superoxide levels were detected by using the DHE probe. (I) Transcriptional activity of Nrf2 was determined by luciferase reporter assay. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. Ctrl; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. HG; ^{^} p < 0.05, ^{^^} p < 0.01, ^{^^^} p < 0.001 vs. HG with Fr. Independent experiments are performed at least three times with similar results.

FIGURE 5

Fr promoted the activation of Nrf2/ARE pathway by regulating the interaction between Cx43 and AKT. (A) Immunoprecipitation results of Cx43 suggested that Cx43 interacted with AKT. (B) IF results showed that AKT and Cx43 co-localized in the cytoplasm of the GMCs (1000× magnification). Green fluorescence indicated localization of AKT and red fluorescence indicated localization of Cx43. Bar: 20 μm. (C) The expression of p-AKT were treated with Cx43-siRNA in GMCs. (D) The western blot analysis of the p-AKT and Nrf2in GMCs was treated with MK-2206 and/or Fr. (E) The subcellular distribution of Nrf2 in GMCs was measured by immunofluorescent staining (×600 magnification). Bar: 40 μm. Green fluorescence indicated localization of Nrf2. (F) The western blot analysis of the HO-1 and SOD1 in GMCs was treated with MK-2206 and/or Fr. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. Ctrl; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. HG; ^{^} p < 0.05, ^{^^} p < 0.01, ^{^^^} p < 0.001 vs. HG with Fr. Independent experiments are performed at least three times with similar results.

FIGURE 6

Fr alleviated renal injury and improved renal function in db/db mice. (A) Body weight of experimental animals. (B) KW/BW, kidney weight/body weight ratio, n = 6 (C–K) HbA1c, GSP, BUN, Up, Cr, SOD and MDA of experimental mice. Data were expressed as ±SEM, n = 6, Ctrl: control group; DN: diabetic nephropathy group; DN + Fr (L): Fr treatment group (low dose: 25 mg/kg); DN + Fr (M): Fr treatment group (medium dose: 50 mg/kg); DN + Fr (H): Fr treatment group (high dose: 100 mg/kg); DN + Val: valsartan treatment group (10 mg/kg). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. Ctrl; ns, no significance; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. DN.

FIGURE 7

Fr reduced the degree of renal fibrosis in db/db mice by inhibiting the protein expression of FN and ICAM-1. (A) Glomerular histopathology analysis was performed by PAS, HE and Masson staining (×400 magnification). Bar: 25 μm, n = 6 (B) Statistical analysis of mesangial index. (C) The expression of FN in the glomeruli were shown by immunohistochemical staining (×200 magnification). Bar: 50 μm, n = 6. (D) Statistical analysis of FN protein expression. (E) The levels of FN and ICAM-1 in kidney tissues of db/db mice were detected by the western blot assay. Independent experiments were performed at least three times with similar results. ^* p < 0.05, ^*** p < 0.001 vs. Ctrl; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. DN.

FIGURE 8

Fr stimulated the activation of Nrf2/ARE signaling pathway by up-regulating Cx43 expression in kidney tissues. (A) The expression of Cx43 and Nrf2 in the glomeruli were shown by immunohistochemical staining (×200 magnification). Bar: 50 μm, n = 6. (B-C) Statistical analysis of Cx43 and Nrf2 protein expression. (D) The levels of Cx43, p-AKT, Nrf2, HO-1, and SOD1 in kidney tissues of db/db mice were detected by the western blot assay. Independent experiments were performed at least three times with similar results. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. Ctrl; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. DN.