Fig 1: Cardiomyocytes are responsible for producing IL-38 in infarcted heart. A, Representative images of infarcted heart sections stained with antibodies against cardiomyocyte marker a-actinin (green), macrophage marker CD68 (green), and the cytokine IL-38(red), and a nuclear stain (blue). Scar bar: 50 µm (each time point n = 8). B, IL-38 mRNA expression levels in cultured cardiomyocytes with H2O2 stimulation for 6h and 12 h. C, IL-38 protein expression levels in cultured cardiomyocytes with H2O2 stimulation for 6 and 12 h (control n = 6, 6 h n = 8, 12 h n = 8). D, Real-time analysis of Bcl-2 and Bax mRNA levels after incubation with rIL-38 (50 ng/mL) for the indicated times. The results are expressed as the Bax/Bcl-2 ratio (control n = 6, 6 h n = 8, 12 h n = 8). *P < .05 vs sham; **P < .01 vs sham
Fig 2: IL-38 deficiency exacerbates aortic valve lesions in mice. (A) Old mice (16 mo old) were fed a standard diet or HFD for 4 mo. Representative Von Kossa staining images show that IL-38 KO-HFD mice displayed greater aortic valve leaflet thickening and calcification compared to WT-HFD mice. Values are mean ± SEM, n = 3 mice per group. *P < 0.05 and **P < 0.01 versus WT-standard diet; #P < 0.05 and ##P < 0.01 versus WT-HFD; &P < 0.05 and &&&P < 0.001 versus IL-38 KO-standard diet. (Scale bar, 100 µm). (B) Mouse AVICs from WT or IL-38 KO mice were treated with vehicle or matrilin-2 (2 µg/mL) for 72 h. Representative immunoblots (Left) and densitometric data (Right) show that AVICs from IL-38 KO mice exhibited higher spontaneous inflammatory and osteogenic activities in comparison to AVICs from WT mice. In addition, AVICs from IL-38 KO mice had moderately greater responses to matrilin-2 stimulation, but the difference between AVICs from IL-38 KO mice and AVICs from WT mice did not reach statistical significance. Values are mean ± SEM, n = 4 mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001 versus WT vehicle. (C) Human AVICs from non-CAVD valves were pretreated with either scrambled shRNA or IL-38 shRNA prior to matrilin-2 (2 µg/mL) treatment for 72 h. Representative immunoblots (Left) and densitometric data (Right) show that knockdown of IL-38 markedly increased the inflammatory and osteogenic responses to matrilin-2 stimulation. Values are mean ± SEM, n = 4 donors. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus scrambled shRNA alone; #P < 0.05 and ##P < 0.01 versus scrambled shRNA + matrilin-2; &P < 0.05 and &&P < 0.01 versus IL-38 shRNA alone.
Fig 3: The regulatory mechanism of the long noncoding RNA (lncRNA) H19/tumour protein p53 (TP53)/interleukin (IL)-38 axis involved in osteoarthritis (OA). lncRNA H19 was upregulated in OA. H19 upregulated the expression of IL-38 by recruiting TP53 to the IL-38 promoter region, which promoted binding of IL-38 with IL-36 receptor and inhibited the inflammatory response in OA. IL-36R, interleukin-36 receptor; mRNA, messenger RNA.
Fig 4: Exogenous overexpression of interleukin (IL)-38 attenuates inflammatory response of osteoarthritis (OA) mice. a) Messenger RNA (mRNA) expression of IL-38 in knee joint cartilage tissues from OA mice and sham-operated mice in response to lentivirus vector (LV)-IL-38 or LV-negative control (NC), as determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). *p < 0.05 versus sham-operated mice injected with LV-NC; #p < 0.05 versus OA mice injected with LV-NC. b) IL-38 protein level in the knee joint cartilage tissues (left) and synovial fluid (right) from OA mice and sham-operated mice in response to LV-IL-38 or LV-NC as measured using western blot analysis and enzyme-linked immunosorbent assay (ELISA). *p < 0.05 versus sham-operated mice injected with LV-NC; #p < 0.05 versus OA mice injected with LV-NC. c) Levels of inflammation-related factors (IL-6, IL-8, IL-17, IL-22, tumour necrosis factor (TNF)-a, interferon (IFN)-?, and cartilage oligomeric matrix protein (COMP)) in synovial fluid from OA mice and sham-operated mice in response to LV-IL-38 or LV-NC, tested using ELISA. d) The Osteoarthritis Research Society International (OARSI) score of cartilage damage in OA mice and sham-operated mice treated with LV-IL-38 or LV-NC identified by safranin O-fast green staining. e) Chondrocyte apoptosis in OA mice and sham-operated mice treated with LV-IL-38 or LV-NC detected using Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. In panels c) to f), *p < 0.05 versus OA mice with injection of LV-NC. The measurement data were expressed as mean (standard deviation). The cell experiment was repeated three times independently. Comparison between two groups was conducted by independent-samples t-test; n = 10.
Fig 5: IL-38 inhibited cardiomyocyte apoptosis and cardiac fibrosis in vivo. A, Representative images of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-stained heart sections from different experimental groups 1 and 28 d post-MI. TUNEL (green) and 4, 6-diamidino-2-phenylindole (blue) staining of nuclei in apoptotic cardiomyocytes (red) in the peri-infarct zone. Scar bar: 100 µm. B, Quantitative analysis of the percentages of TUNEL-positive nuclei (each group n = 6). C, Real-time PCR determined mRNA expression levels of Bax and Bcl-2 in the in infarcted heart on day 1 after MI. The results were also expressed as ratio of Bax/Bcl-2 (n = 4). D, Representative Masson’s trichrome staining images of collagen deposition (blue) in both infarct and remote areas on day7 post-MI. E, The extent of fibrosis, as assessed by the fibrotic area/left ventricle and interstitial fibrosis, was compared among the different groups (each group n = 6). **P < .01 vs sham; # P < .05 vs MI+PBS; ## P < .01 vs MI+PBS
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