Fig 2: N-3 PUFAs inhibit IL-11 production by downregulating ERK1/2 pathway. (A-C) Hepatocytes were isolated from WT and fat-1 transgenic mice (n=5) at the indicated time after 400 mg/kg APAP administration. (A) The mRNA expression of Fra-1 was evaluated by reverse transcription-quantitative PCR and expressed as a ratio to GAPDH. (B) The protein level of Fra-1 was determined by western blotting analysis. (C) The protein levels of pERK, ERK and GAPDH were detected by western blotting analysis. (D-F) Primary hepatocytes were isolated from WT and fat-1 mice (n=5) and stimulated with 20 mM APAP for 24 h in vitro. In some cases, the cells were pretreated with 50 µM U0126 for 2 h before APAP stimulation. (D) Cellular apoptosis was measured by Annexin V-PI staining. (E) The culture supernatants were collected for evaluating IL-11 production through ELISA. (F) The protein level of Fra-1 in hepatocytes was confirmed by immunoblotting analysis. **P<0.01. NS, not significant; N-3 PUFAs, omega-3 polyunsaturated fatty acids; APAP, acetaminophen; WT, wild-type; Fra-1, Fos-like-1; p, phosphorylated.

Fig 3: N-3 PUFAs inhibit IL-11 production in hepatocytes. (A-D) WT and fat-1 transgenic mice were injected with 400 mg/kg APAP. (A) The serum levels of IL-11, IL-6, IL-13 and IL-22 were determined at the indicated time points. (B) Liver mononuclear cells and (C) hepatocytes were isolated from WT and fat-1 transgenic mice (n=5) at the indicated time after APAP (400 mg/kg) injection. The levels of IL-11 and IL-6 expression were measured by reverse transcription-quantitative PCR and expressed as a ratio to GAPDH. (D) The protein level of IL-11 in liver tissues was detected by immunohistochemical staining 6 h post-APAP injection. Scale bar, 50 µm. (E) Protein levels of IL-11 and GAPDH were determined by western blotting analysis. (F) Primary hepatocytes were isolated from WT or fat-1 transgenic mice and treated with 20 mM APAP for 24 h in vitro. The supernatants were collected, and the secreted levels of IL-11 and IL-6 were evaluated by ELISA. **P<0.01. NS, not significant; N-3 PUFAs, omega-3 polyunsaturated fatty acids; APAP, acetaminophen; WT, wild-type.

Fig 4: IL11 inhibited the expression of IDO1. (A) mRNA expression estimated by RT-PCR for IDO1 after supplementing rmIL11 in NIH3T3 and L929 cells. (B) Western blot analysis and densitometric analysis of IDO1 protein levels after supplementing rmIL11 in NIH3T3 and L929 cells; GAPDH was used as the loading control. (C) Representative immunocytochemistry staining of IDO1 and cell nuclei (Hoechst) after supplementing rmIL11 in NIH3T3 and L929 cells. (D) IDO1 content estimated by ELISA from treated cell lysate. (E) The relative KYN level was detected by Ehrlich assay after inhibiting IL11 in NIH3T3 and L929 cells. OGD—oxygen–glucose deprivation; CON—control. The results are presented as the means ± SDs, and Student’s t-test was used to determine the p-value. * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with CON group in NIH3T3 cells; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with CON group in L929 cells.

Fig 5: OGD inhibits the expression of IDO1/KYN in fibroblasts via IL11. Western blot analysis and densitometric analysis of collagen I and collagen III protein levels after supplementing 50 µM KYN and 25 ng/mL rmIL11 in NIH3T3 and L929 cells; GAPDH was used as the loading control. OGD—oxygen–glucose deprivation; CON—control. The results are presented as the means ± SDs, and Student’s t-test was used to determine the p-value. * p < 0.5 and ** p < 0.01, compared with CON group; # p < 0.05 and ## p < 0.01, compared with IL11 group; & p < 0.05 and && p < 0.01, compared with IL11 + OGD group.