HMGB1 regulates ferroptosis through Nrf2 pathway in mesangial cells in response to high glucose

Abstract

Ferroptosis, a novel type of programmed cell death, is involved in inflammation and oxidation of various human diseases, including diabetic kidney disease. The present study explored the role of high-mobility group box-1 (HMGB1) on the regulation of ferroptosis in mesangial cells in response to high glucose. Compared with healthy control, levels of serum ferritin, lactate dehydrogenase (LDH), reactive oxygen species (ROS), malonaldehyde (MDA), and HMGB1 were significantly elevated in diabetic nephropathy (DN) patients, accompanied with deregulated ferroptosis-related molecules, including long-chain acyl-CoA synthetase 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2), NADPH oxidase 1 (NOX1), and glutathione peroxidase 4 (GPX4). In vitro assay revealed that erastin and high glucose both induced ferroptosis in mesangial cells. Suppression of HMGB1 restored cellular proliferation, prevented ROS and LDH generation, decreased ACSL4, PTGS2, and NOX1, and increased GPX4 levels in mesangial cells. Furthermore, nuclear factor E2-related factor 2 (Nrf2) was decreased in DN patients and high glucose-mediated translocation of HMGB1 in mesangial cells. Knockdown of HMGB1 suppressed high glucose-induced activation of TLR4/NF-κB axis and promoted Nrf2 expression as well as its downstream targets including HO-1, NQO-1, GCLC, and GCLM. Collectively, these findings suggest that HMGB1 regulates glucose-induced ferroptosis via Nrf2 pathway in mesangial cells.

Figure 1. Elevated levels of ferroptosis in DN patients

Levels of serum ferritin (A) and LDH (B) in DN patients and healthy controls. Levels of serum HMGB1 (C) and mRNA expression of HMGB1 (D) in DN patients and healthy controls. Real-time PCR was used to detect the expression of ferroptosis-related proteins, including ACSL4, PTGS2, NOX1, and GPX4 (E). **P<0.01, ***P<0.001, compared with control.

Figure 2. High glucose induced ferroptosis in mesangial cells

SV40 MES 13 cells were cultured and treated with eratin and DFX alone or combined with high glucose. Production of LDH was detected in each group (A). Real-time PCR was performed to determine levels of ACSL4, PTGS2, NOX1, and GPX4 (B). *P<0.05, **P<0.01, compared with control. ^#P<0.05, ^##P<0.01, compared with HG group.

Figure 3. HMGB1 mediates the glucose-induced ferroptosis in mesangial cells

SV40 MES 13 cells were cultured and transfected with HMGB1 siRNA or scramble siRNA (Scr-siRNA), followed by high glucose (25 mM) treatment. (A) Determination of protein levels of HMGB1 by Western blot. (B) Cell viability in each group was determined with CCK-8 assay. (C) ELISA was used to detect the production of LDH. (D) Extracellular HMGB1 protein levels were determined by Western blot. (E) Real-time PCR was performed to determine levels of ACSL4, PTGS2, NOX1, and GPX4. *P<0.05, **P<0.01, compared with control. ^#P<0.05, ^##P<0.01, compared with HG group.

Figure 4. HMGB1 regulates ferroptosis-induced oxidative stress upon exposure to high glucose

Levels of ROS (A) and MDA (B) in DN patients and healthy controls. SV40 MES 13 cells were transfected with HMGB1 siRNA and stimulated with high glucose. The production of ROS (C), IL-6 (E) and TNF-α (F) was detected. Furthermore, SV40 MES 13 cells were treated with eratin and DFX alone or combined with high glucose, and the levels of ROS were detected (D). *P<0.05, **P<0.01, ***P<0.001, compared with control. ^##P<0.01, compared with HG group.

Figure 5. HMGB1 regulates glucose-induced ferroptosis via Nrf2 signaling pathway

ELISA and real-time PCR assays were performed to determine levels of Nrf2 in DN patients and healthy controls (A,B). SV40 MES 13 cells were transfected with HMGB1 siRNA or scramble siRNA and stimulated with high glucose (25 mM). Western blot and immunofluorescence staining were performed to detect expression of HMGB1 in the cytoplasm and nucleus in glucose-treated SV40 MES 13 cells (C,D). (E) The protein levels of Nrf2, TLR4, and NF-κB p65 were determined using Western blot. (F,G) Relative mRNA levels of Nrf2 and its downstream targets, including HO-1, NQO-1, GCLC and GCLM, were detected using real-time PCR in SV40 MES 13 cells after siRNA treatment. *P<0.05, **P<0.01, ***P<0.001, compared with control. ^#P<0.05, ^##P<0.01, compared with HG group.