4-octyl itaconate inhibits high glucose induced renal tubular epithelial cell fibrosis through TGF-β-ROS pathway

Figures & data

Figure 1. High glucose induces an increase in TGF-β production and promotes its pro-fibrotic mediated by TGF-β receptors.

HK2 cells were cultured with 5 mM (NC) or 30 mM glucose (HG) for 48h. (a) The levels of TGF-β in the supernatant of HK2 was detected by ELISA; (b) The protein expressions of Fibronectin in HK2 were detected by western blot in HK2 cells sitimulated with 30 mM glucose with or without TGF-β receptor blocker LY2109761; (c) The protein expressions of collagen I protein in HK2 was detected by immunofluorescence. (*P < 0.05, vs NC; ^#P < 0.05, vs HG).

Figure 2. 4-OI inhibits the profibrotic effect and EMT induced by high glucose in HK2 cells.

HK2 cells were co-cultured with 30mM glucose and 100μM 4-OI (HG + OI). (a) The proteins expressions of Fibronectin, α - SMA and Vimentin in HK2 were detected by western blot; (b) The protein expressions of collagen I protein in HK2 was detected by immunofluorescence from each group. (*P < 0.05, vs NC; ^#P < 0.05, vs HG).

Figure 3. 4-OI reduces the TGF-β production induced by high glucose and inhibited the pro-fibrotic effect of TGF-β.

(a) High glucose stimulated HK2 cells were cultured with or without 100μM 4-OI (HG + OI). The levels of TGF-β in the supernatant of HK2 was detected by ELISA; (b) The HK2 cells were administrated with 25 μM, 50 μM, or 100 μM 4-OI respectively, and simultanously co-cultured with 10 ng/ml TGF-β. The protein expressions of Fibronectin in HK2 were detected by western blot. (*P < 0.05, vs NC; NS P ≥ 0.05, vs TGF-β; ^#P < 0.05, vs TGF-β).

Figure 4. 4-OI exerts its anti-fibrotic effect by inhibiting the excessive production of ROS induced by high glucose and TGF-β.

HK2 cells were treated with 10 mM NAC or 100 μM 4-OI, and simultaneously stimulated with 10 ng/ml TGF-β or 30 mM glucose. (a)(b) Fluorescence images of ROS within HK2 detected using the DCFH-DA probe after the different treatments; (c) The proteins expressions of Fibronectin, α - SMA and Vimentin in HK2 were detected by western blot from each group; (d) The protein expressions of collagen I protein in HK2 was detected by immunofluorescence from each group. (*P < 0.05, vs NC; ^#P < 0.05, vs TGF-β).

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.