Fig 1: Interleukin (IL)‐22 treatment ameliorated bleomycin (BLM)‐induced inflammation and collagen deposition. (A) Experimental schematic of the animal. Mice were intratracheally injected with 50 μl normal saline (NS) or BLM (5 mg/kg) to induced fibrosis at day 0. Intranasal instillation of 50 μl recombinant IL‐22 (100 ng) was used on day 0, day 7, and day 14 after the treatment with BLM, parts of mice were sacrificed at day 7, day 14, and day 21. Each test was repeated three times. Six mice per group were used. (B1–3) The pathological changes of lung tissues on days 7, 14, and 21 in NS treated mice by HE staining (×200). (B4–6) The pathological changes of lung tissues on days 7, 14, and 21 in BLM treated mice by HE staining (×200). (B7–9) The pathological changes of lung tissues on days 7, 14, and 21 in both BLM and IL‐22 treated mice by HE staining (×200). (C1–3) The pathological changes of lung tissues at day 7, 14, and 21 days in NS treated mice by MS staining (×200). (C4–6) The pathological changes of lung tissues at day 7, 14, and 21 days in BLM treated mice by MS staining (×200). (C7–9) The pathological changes of lung tissues at day 7, 14, and 21 days in both BLM and IL‐22 treated mice by MS staining (×200). (D) Comparison of the pathological scores of alveolitis at 7 days in BLM (n = 6) and both BLM and IL‐22 treated mice (n = 6). (E) Comparison of the pathological scores of fibrosis at 21 days in BLM (n = 6) and both BLM and IL‐22 treated mice (n = 6). (F–G) The mRNA expression of transforming growth factor (TGF)‐β1 and TGF‐βR2 in lung tissues of BLM, NS, and both BLM and IL‐22 treated mice at day 21 by real‐time PCR. (H) The protein expression of TGF‐β1 and TGF‐βR2 in lung tissues of BLM, NS, both BLM and IL‐22 treated mice by WB. (I–J) The quantification of (G). (K) The protein expression of P‐smad2/3 in lung tissues of BLM, NS, and both BLM and IL‐22 treated mice by WB. (L) The quantification of (K). (N) The protein expression of α‐SMA, Collagen‐I, and fibronectin (FN) in lung tissues of BLM, NS, and both BLM and IL‐22 treated mice by WB. (M, O–P) The quantification of (N)
Fig 2: IL‐22 treatment repressed the transforming growth factor (TGF)‐β pathway and the production of collagen in vitro. Samples were collected at 24, 48, and 72 h after treatment cells with 10 ng/ml human interleukin (IL)‐22 and 5 ng/ml TGF‐β1 for mRNA or protein analysis (n = 3). (A–B) In both A549 and mouse primary AT2 cells, the expressions of TGF‐βR2, α‐smooth muscle actin (α‐SMA), Collagen‐I, and E‐cad after treatment with TGF‐β1 and/or IL‐22 by WB. (C–D) In both HELF and human primary fibroblasts (FB), the expressions of TGF‐βR2, α‐SMA, Collagen‐I and Vimentin after treatment with TGF‐β1 and/or IL‐22 by WB. (E–L) The mRNA expressions of TGF‐βR2, α‐SMA, Collagen‐I and E‐cad after treatment with TGF‐β1 and/or IL‐22 by real‐time PCR in both type II AECs (A549 cell line and mouse primary AT2). (M–T) The mRNA expressions of TGF‐βR2, α‐SMA, Collagen‐I, and Vimentin after treatment with TGF‐β1 and/or IL‐22 in both fibroblasts (HELF cell line and human primary FB) by real‐time PCR
Fig 3: The effects of BAY 60-7550 on Aβ-induced changes in IL-17 and IL-22 expression. IL-22 and IL-17 expression after BAY (i.c.v.) treatment was tested in the cortex (A,B) and in the hippocampus (C,D) of mice (mean ± SEM, n = 10). **p < 0.01, ***p < 0.001, vs vehicle-treated control group. #p < 0.05, ##p < 0.01, vs vehicle-treated Aβ group. $p < 0.05, vs BAY (10 μg)-treated Aβ group.
Fig 4: Effects of IL-22 expression on the IC50 value of DDP in MG63/DDP and MG63 cells. Different concentrations of cisplatin (2.5, 5.0, 10, 20, 40 and 80 µg/ml) were used to treat the MG63/DDP cells transfected with IL-22 siRNA or control siRNA and MG63 cells transfected with IL-22 overexpression plasmid or control plasmid. The MTT assay was used to detect cell viability and the IC50 value was calculated. (A) The protein expression of IL-22 in MG63/DDP cells transfected with IL-22 siRNA or control siRNA was detected, and the IL-22/β-actin ratio was calculated. (B) mRNA expression of IL-22 in MG63/DDP cells transfected with IL-22 siRNA or control siRNA. (C) Cell viability inhibition rate of DDP in MG63/DDP cells transfected with IL-22 siRNA or control siRNA. (D) IC50 of DDP in MG63/DDP cells transfected with IL-22 siRNA or control siRNA. (E) The protein expression of IL-22 in MG63 cells transfected with the IL-22 overexpression plasmid or control plasmid was determined, and the IL-22/β-actin ratio was calculated. (F) The mRNA expression of IL-22 in MG63 cells transfected with the IL-22 overexpression plasmid or control plasmid. (G) The cell viability inhibition rate of DDP in MG63 cells transfected with the IL-22 overexpression plasmid or control plasmid. (H) IC50 of DDP in MG63 cells transfected with the IL-22 overexpression plasmid or control plasmid. Experiments were repeated three times. Data are reported as the mean ± standard deviation. **P<0.01 vs. control plasmid/siRNA group. IL, interleukin; IC50, half maximal inhibitory concentration; DDP, cisplatin; siRNA, small interfering RNA.
Fig 5: IL-22 expression in osteosarcoma tissues and cell lines. Western blotting and reverse transcription-quantitative PCR were used to detect the expression of IL-22 in osteosarcoma tissues and cell lines. (A) The protein expression of IL-22 in 2 osteosarcoma tissues and adjacent normal tissues was measured using western blotting, and the IL-22 protein/β-actin ratio was calculated. (B) The mRNA expression of IL-22 in 30 osteosarcoma tissues and adjacent normal tissues. (C) The protein expression of IL-22 in normal osteoblast hFOB1.19 cells and osteosarcoma cell lines was measured using western blotting, and the IL-22 protein/β-actin ratio was calculated. (D) The mRNA expression of IL-22 in normal osteoblast hFOB1.19 cells and osteosarcoma cell lines. Experiments were repeated three times. Data are reported as the mean ± standard deviation. **P<0.01 vs. normal tissues. ##P<0.01 vs. hFOB1.19 cells. IL, interleukin.
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