Fig 1: Exogenous irisin restores gut barrier function after gut IR. Irisin (250 µg/kg in 0.5 mL saline, a single dose, iv) was administered immediately after reperfusion. Anti-irisin (4 mg/kg, Abcam) blocking antibodies were administered at 24 h before gut IR. Four hours after reperfusion, mice were sacrificed, and tissue samples were collected. A, Serum irisin levels; (B,C) Western blot analysis of irisin expression; (D,E) immunofluorescence staining of irisin (green) and the corresponding nuclear counterstaining (blue) in gut tissues; (F) gut injury score; (G) haematoxylin and eosin (H&E) staining; (H) water content of gut; (I) serum FITC-dextran levels; (J,K) colony-forming units (CFUs) from mesenteric lymph node (MLN) and lung tissues; (L,M) serum levels of LDH and lactate; and (N,O) serum TNF-a and CIRP levels. n = 6 per group, mean ± SEM, *P < .05 vs the sham group, # P < .05 vs the gut IR group
Fig 2: Treatment with exogenous irisin downregulates inflammatory responses after ischemia-reperfusion (I/R). Irisin treatment in mice was conducted by intravenous administration (250 µg/kg, a single dose) at the beginning of reperfusion. The liver tissues and blood samples were harvested at 24 h after reperfusion. A, Myeloperoxidase (MPO) immunofluorescence staining (green), the corresponding nuclear counterstaining (blue) and both channels merged of representative liver sections (magnification 400×); B, analysis of MPO fluorescence intensity; C, serum tumor necrosis factor a (TNF-a) levels; D, Serum cold-inducible RNA binding protein (CIRP) levels in each group. n = 6, mean ± SEM, *P < 0.05 versus sham group, #P < 0.05 versus vehicle group.
Fig 3: Interaction between CIRP and XA-Aptamers or extracellular domain of TLR4(A) Overlay of 2D 1H-15N HSQC spectra of the CIRP without (black) and with 5 M equivalents of X-Aptamer (red). The residues experiencing most peak broadening effect are indicated. This characterized peak broadening in the spectrum of the complex indicates intermediate-exchange timescale for NMR or intermediate interaction.(B) ITC result for CIRP and X-Aptamer. The KD value for the interaction was calculated to be 1.71 µM.(C) Overlay of 2D 1H-15N HSQC spectra of the CIRP without (black) and with 5 M equivalents of extracellular domain of TLR4 (red). The residues experiencing most peak broadening effect are indicated. This characterized peak broadening in the spectrum of the complex indicates intermediate-exchange or intermediate timescale for NMR interaction.(D) ITC result for CIRP and extracellular domain of TLR4. The KD value for the interaction was calculated to be 3.72 µM.(E) The interface for CIRP and X-Aptamer interaction. Orange areas indicate the NMR-derived interface, in which peaks experienced the most broadening effect in NMR titrations.(F) The interface for CIRP and extracellular domain of TLR4. Orange areas indicate the NMR-derived interface, in which peaks experienced the most broadening effect in NMR titrations. See also Figures S3–S5.
Fig 4: CIRP deficiency reduces mitochondrial damage and endoplasmic reticulum stress in AP miceL-Arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg L-arginine in wild type or CIRP knockout mice. The animals were sacrificed at 72 h after the first injection of L-arginine. (A and I) Ultrastructural alterations in the pancreas (electron microscopy). Scale bars:2µm. (B–F) Western blot analysis of PGC-1a, Tfam, PINK1, and Mfn-2 in the pancreas. (G) The SOD levels in pancreas. (H) The GSH levels in pancreas. (J-M) Western blot analysis of GRP78, PDI, IRE1a, and pIRE1a in the pancreas. (N and O) Western blot analysis of BAX, Bcl-2 in the pancreas; n = 6/group, *p < 0.05 versus sham group; #p < 0.05 versus L-arginine WT group. Data are expressed as means ± SEM.
Fig 5: CIRP expression is increased in AP mice and recombination CIRP directly damages pancreatic acinar cells(A) Pancreatic CIRP immunofluorescence in sham and AP mice (200X). Scale bars:50µm.(B) Pancreatic CIRP immunohistochemical analysis in sham and AP mice (400X). Scale bars:20µm.(C) Serum levels of CIRP in sham and AP mice. L-Arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg L-arginine. The animals were sacrificed at 72 h after the first injection of L-arginine. n = 8/group, *p < 0.05 versus sham group.(D and E) Western blot analysis of PGC-1a, Tfam and Mfn-2 in AR42J cells. The pancreatic AR42J cells (5×105/well) were treated with 250, 500, or 1000 ng/mL recombination murine CIRP for 72 h n = 4/group, *p < 0.05 versus 0-ng/mL group.(F and G) Western blot analysis of PGC-1a, Tfam and Mfn-2 in AR42J cells. The pancreatic AR42J cells (5×105/well) were treated with 1000 ng/mL recombination murine CIRP for 24, 48 or 72 h n = 4/group, *p < 0.05 versus 0-h group.(H and I) Western blot analysis of GRP78, PDI, IRE1a, and pIRE1a in AR42J cells. The pancreatic AR42J cells (5×105/well) were treated with 250, 500, or 1000 ng/mL recombination murine CIRP for 72 h. (n = 4/group, *p < 0.05 versus 0-ng/mL group).(J and K) Western blot analysis of GRP78, PDI, IRE1a, and pIRE1a in AR42J cells. The pancreatic AR42J cells (5×105/well) were treated with 1000 ng/mL recombination murine CIRP for 24, 48, or 72 h n = 4/group, *p < 0.05 versus 0-h group. (L) Calcein/PI cell viability/cytotoxicity assay staining of AR42J cells. The pancreatic AR42J cells (5×105/well) were treated with 250, or 1000 ng/mL recombination murine CIRP for 72 h. Scale bars:20µm. (M) Cell counting kit-8 assay of AR42J cells. The pancreatic AR42J cells (5×103/well) were treated with 250, 500, or 1000 ng/mL recombination murine CIRP for 24, 48 or 72 h n = 8/group, *p < 0.05 versus control group.(N and O) Western blot analysis of RIP-3 in AR42J cells. The pancreatic AR42J cells (5×105/well) were treated with 250, 500, or 1000 ng/mL recombination murine CIRP for 72 h n = 4/group, *p < 0.05 versus 0-ng/mL group.(P and Q) Western blot analysis of RIP-3 in AR42J cells. The pancreatic AR42J cells (5×105/well) were treated with 1000 ng/mL recombination murine CIRP for 24, 48, or 72 h n = 4/group, *p < 0.05 versus 0-h group. Data are expressed as means ± SEM. See also Figures S1 and S2.
Supplier Page from CUSABIO Technology LLC for Mouse Cold-inducible RNA-binding protein(CIRBP) ELISA Kit