Fig 2: OE blocked TLR4/MD2 complex formation in AKI mice. (A–K) Mice were administered OE (100, 200, 400 mg/kg) by gavage for 7 days followed by the intraperitoneal injection of LPS (10 mg/kg) and feeding for another 12 h. TLR4/MD2 (A,B), TLR4/MyD88 (C,D), and TLR4/TRIF (E,F) complex formation in mice kidneys were determined by a co-immunoprecipitation assay. Protein expression of p-JNK, JNK, p-ERK, ERK, p-p38, and p38 (G–J) in mice kidneys was analyzed by a western blotting analysis. The mRNA level of IFN-ß in mice kidney was determined by quantitative real time PCR analysis. Data presented as Means ± SD. * p < 0.05, ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. LPS group; ns, no significance.

Fig 3: Effect of SIL on the ERK and Akt. (A) Protein levels of IDH1 were determined in cell lysates from cells treated with solvent control or IDH1-IN-1 (1 µM or 5 µM) for 24 h by performing immunoblotting. (B) OVCAR3 and SKOV3 cells were exposed to 100 µM SIL for various times. Expression of p-ERK, p-Akt, ERK, and AKT was evaluated by western blot analysis. (C) OVCAR3 and SKOV3 cells were exposed to 100 µM SIL in the presence or absence of 5 mM NAC for 24 h. Expression of p-ERK and p-Akt was evaluated by western blot analysis. (D, F, H, J) Ratios of GSH/GSSG and NADP+/NADPH in cells (n = 5). (E, I) The activity of SOD in cells (n = 5). (G, K) Cells were loaded with DCFH-DA for 1 h and after treatment with 100 µM SIL for 24 h. DCF fluorescence intensity was measured by flow cytometry. Statistical analysis: Student's t-test. Error bars represent SEM, and P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, All experiments were repeated three times

Fig 4: OE inhibited TLR4/MD2 complex formation. (A–F) RAW264.7 macrophages were pretreated with OE (80 µg/mL) for 3 h followed by stimulation with LPS (1 µg/mL) for 2 h. Interaction between TLR4/MD2 (A,B), TLR4/MyD88 (C,D), and TLR4/TRIF (E,F) were determined by a co-immunoprecipitation assay. (G–J) RAW264.7 macrophages were pretreated with OE (20, 40, 80 µg/mL) for 3 h followed by stimulation with LPS (1 µg/mL) for 2 h, and protein expression of p-JNK, JNK, p-ERK, ERK, p-p38, and p38 was determined by a western blotting assay. (K) RAW264.7 macrophages were pretreated with OE (20, 40, 80 µg/mL) for 3 h followed by stimulation with LPS (1 µg/mL) for 6 h. The IFN-ß mRNA level was determined by quantitative real time PCR analysis. Data presented as Means ± SD. ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. LPS group; ns, no significance.