Figure 1. OE alleviated LPS-induced inflammation in macrophages. (A,B) Cell viability of TE (A) and fraction B (B) on RAW264.7 macrophages. (C,D) RAW264.7 macrophages were pretreated with the indicated concentration of various substances for 3 h followed by LPS (1 μg/mL) stimulation for 24 h. The medium was collected to determine the NO release level. (E–I) RAW264.7 macrophages were pretreated with OE (20, 40, 80 μg/mL) for 3 h followed by LPS (1 μg/mL) stimulation for 24 h. Total protein expression level of iNOS, COX-2, TNF-α, and IL-6 were detected by western blotting assay. (J–N) RAW264.7 macrophages were pretreated with OE (20, 40, 80 μg/mL) for 3 h followed by LPS (1 μg/mL) stimulation for 6 h. The mRNA level of iNOS (J), COX-2 (K), TNF-α (L), IL-6 (M), and MCP-1 (N) were determined by quantitative real time PCR analysis. CTR: control. Data presented as Means ± SD. ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. LPS group; ns, no significance.

Figure 1. OE alleviated LPS-induced inflammation in macrophages. (A,B) Cell viability of TE (A) and fraction B (B) on RAW264.7 macrophages. (C,D) RAW264.7 macrophages were pretreated with the indicated concentration of various substances for 3 h followed by LPS (1 μg/mL) stimulation for 24 h. The medium was collected to determine the NO release level. (E–I) RAW264.7 macrophages were pretreated with OE (20, 40, 80 μg/mL) for 3 h followed by LPS (1 μg/mL) stimulation for 24 h. Total protein expression level of iNOS, COX-2, TNF-α, and IL-6 were detected by western blotting assay. (J–N) RAW264.7 macrophages were pretreated with OE (20, 40, 80 μg/mL) for 3 h followed by LPS (1 μg/mL) stimulation for 6 h. The mRNA level of iNOS (J), COX-2 (K), TNF-α (L), IL-6 (M), and MCP-1 (N) were determined by quantitative real time PCR analysis. CTR: control. Data presented as Means ± SD. ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. LPS group; ns, no significance.

Figure 2. OE suppressed NF-κB activation in macrophages. (A) RAW264.7 macrophages were pretreated with OE (80 μg/mL) for 3 h followed by stimulation with LPS (1 μg/mL) for 2 h. p65 nuclear translocation was detected by immunofluorescent staining. Scale bars: 20 μm. (B–F) RAW264.7 macrophages were pretreated with OE (20, 40, 80 μg/mL) for 3 h followed by LPS (1 μg/mL) stimulation for 2 h. Nuclear and cytoplasmic protein expression of p65 (B,C) and the protein expression level of IκB, p-p65, and p65 (D–F) were determined by a western blotting assay. Data presented as Means ± SD., ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. LPS group; ns, no significance.

Figure 2. OE suppressed NF-κB activation in macrophages. (A) RAW264.7 macrophages were pretreated with OE (80 μg/mL) for 3 h followed by stimulation with LPS (1 μg/mL) for 2 h. p65 nuclear translocation was detected by immunofluorescent staining. Scale bars: 20 μm. (B–F) RAW264.7 macrophages were pretreated with OE (20, 40, 80 μg/mL) for 3 h followed by LPS (1 μg/mL) stimulation for 2 h. Nuclear and cytoplasmic protein expression of p65 (B,C) and the protein expression level of IκB, p-p65, and p65 (D–F) were determined by a western blotting assay. Data presented as Means ± SD., ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. LPS group; ns, no significance.

Figure 3. 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-β 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.

Figure 3. 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-β 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.

Figure 4. The anti-inflammatory effect of OE was counteracted by siTLR4. (A,B) RAW264.7 macrophages were transfected with siNC (100 nM) and siTLR4 (100 nM). The total protein expression of TLR4 was determined by a western blotting assay. (C–K) After transfecting siNC (100 nM) and siTLR4 (100 nM), RAW264.7 macrophages were pretreated with OE (80 μg/mL) for 3 h followed by stimulation with LPS (1 μg/mL) for 2 h. Nuclear and cytoplasmic protein expression of p65 (C,D) and the protein expression of IκB, p-p65, p65, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 (E–K) were determined by a western blotting assay. Data presented as Means ± SD. & p < 0.05, && p < 0.01; ns, no significance.

Figure 4. The anti-inflammatory effect of OE was counteracted by siTLR4. (A,B) RAW264.7 macrophages were transfected with siNC (100 nM) and siTLR4 (100 nM). The total protein expression of TLR4 was determined by a western blotting assay. (C–K) After transfecting siNC (100 nM) and siTLR4 (100 nM), RAW264.7 macrophages were pretreated with OE (80 μg/mL) for 3 h followed by stimulation with LPS (1 μg/mL) for 2 h. Nuclear and cytoplasmic protein expression of p65 (C,D) and the protein expression of IκB, p-p65, p65, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 (E–K) were determined by a western blotting assay. Data presented as Means ± SD. & p < 0.05, && p < 0.01; ns, no significance.

Figure 5. OE diminished ROS production by inhibiting NOX2 endocytosis. (A) RAW264.7 macrophages were pretreated with OE (20, 40, 80 μg/mL) for 3 h or with Dyn (40 μM) for 30 min followed by stimulation with LPS (1 μg/mL) for 6 h. ROS production was measured by a ROS detection kit via flow cytometry. (B–D) RAW264.7 macrophages were pretreated with OE (80 μg/mL) for 3 h or with Dynasore (40 μM) for 30 min followed by stimulation with LPS (1 μg/mL) for 4 h. Co-localization between NOX2 and EEA1 was determined by immunofluorescent staining (B). Scale bars: 20 μm. The cytoplasmic and membranal protein expression of NOX2 were determined by a western blotting assay (C,D). (E,F) After transfecting siNC (100 nM) or siTLR4 (100 nM), RAW264.7 macrophages were pretreated with OE (80 μg/mL) for 3 h followed by stimulation with LPS (1 μg/mL) for 4 h. Cytoplasmic and membranal protein expression of NOX2 were determined by a western blotting assay. (G–J) Mice were administered OE (100, 200, 400 mg/kg) by gavage for 7 days followed by intraperitoneal injection of LPS (10 mg/kg) and feeding for another 12 h. The total protein expression of NOX2 (G,H) and ROS production (I,J) in mice kidneys were determined by a western blotting assay and DHE staining, respectively. Scale bars: 50 μm. CTR: control. Data presented as Means ± SD. ** p < 0.01 vs. control group; ## p < 0.01 vs. LPS group; && p < 0.01; ns, no significance.

Figure 5. OE diminished ROS production by inhibiting NOX2 endocytosis. (A) RAW264.7 macrophages were pretreated with OE (20, 40, 80 μg/mL) for 3 h or with Dyn (40 μM) for 30 min followed by stimulation with LPS (1 μg/mL) for 6 h. ROS production was measured by a ROS detection kit via flow cytometry. (B–D) RAW264.7 macrophages were pretreated with OE (80 μg/mL) for 3 h or with Dynasore (40 μM) for 30 min followed by stimulation with LPS (1 μg/mL) for 4 h. Co-localization between NOX2 and EEA1 was determined by immunofluorescent staining (B). Scale bars: 20 μm. The cytoplasmic and membranal protein expression of NOX2 were determined by a western blotting assay (C,D). (E,F) After transfecting siNC (100 nM) or siTLR4 (100 nM), RAW264.7 macrophages were pretreated with OE (80 μg/mL) for 3 h followed by stimulation with LPS (1 μg/mL) for 4 h. Cytoplasmic and membranal protein expression of NOX2 were determined by a western blotting assay. (G–J) Mice were administered OE (100, 200, 400 mg/kg) by gavage for 7 days followed by intraperitoneal injection of LPS (10 mg/kg) and feeding for another 12 h. The total protein expression of NOX2 (G,H) and ROS production (I,J) in mice kidneys were determined by a western blotting assay and DHE staining, respectively. Scale bars: 50 μm. CTR: control. Data presented as Means ± SD. ** p < 0.01 vs. control group; ## p < 0.01 vs. LPS group; && p < 0.01; ns, no significance.

Figure 6. OE ameliorated LPS-induced AKI in mice. (A–I) Mice were administered OE (100, 200, 400 mg/kg) by gavage for 7 days followed by an intraperitoneal injection of LPS (10 mg/kg) and feeding for another 12 h. Kidney morphology (A) was observed. Serum CRE (B) and BUN (C) levels were measured by kits according to the manufacturer’s instructions. The mRNA level of Kim-1 in mice kidney was determined by quantitative real time PCR (D). Histopathology of kidneys was analyzed by HE staining (E). Arrows indicate the infiltration of inflammatory cells. Scale bars: 50 μm. Immunochemical analysis was applied to determine expression of CD68, F4/80, and MCP-1 in mice kidneys (F–I). Scale bars: 50 μm. Data presented as Means ± SD. ** p < 0.01 vs. control group; ## p < 0.01 vs. LPS group; ns, no significance.

Figure 6. OE ameliorated LPS-induced AKI in mice. (A–I) Mice were administered OE (100, 200, 400 mg/kg) by gavage for 7 days followed by an intraperitoneal injection of LPS (10 mg/kg) and feeding for another 12 h. Kidney morphology (A) was observed. Serum CRE (B) and BUN (C) levels were measured by kits according to the manufacturer’s instructions. The mRNA level of Kim-1 in mice kidney was determined by quantitative real time PCR (D). Histopathology of kidneys was analyzed by HE staining (E). Arrows indicate the infiltration of inflammatory cells. Scale bars: 50 μm. Immunochemical analysis was applied to determine expression of CD68, F4/80, and MCP-1 in mice kidneys (F–I). Scale bars: 50 μm. Data presented as Means ± SD. ** p < 0.01 vs. control group; ## p < 0.01 vs. LPS group; ns, no significance.

Figure 7. OE exhibited anti-inflammatory activity in AKI mice. (A–M) 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. The total protein expression of iNOS, COX-2, TNF-α, and IL-6 (A–E) in mice kidneys was determined by a western blotting assay. The mRNA level of iNOS (F), COX-2 (G), TNF-α (H), IL-6 (I), and MCP-1 (J) in mice kidneys was determined by quantitative real time PCR analysis. The protein expression level of IκB, p-p65, and p65 in mice kidneys was determined by a western blotting assay. 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.

Figure 7. OE exhibited anti-inflammatory activity in AKI mice. (A–M) 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. The total protein expression of iNOS, COX-2, TNF-α, and IL-6 (A–E) in mice kidneys was determined by a western blotting assay. The mRNA level of iNOS (F), COX-2 (G), TNF-α (H), IL-6 (I), and MCP-1 (J) in mice kidneys was determined by quantitative real time PCR analysis. The protein expression level of IκB, p-p65, and p65 in mice kidneys was determined by a western blotting assay. 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.

Figure 8. 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-β 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.

Figure 8. 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-β 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.