Fig 2: SP1 regulates the transcriptional activation of CIRP by rs11170510 and rs58123204. (A) A strong transcription factor cluster appeared in the Cirp transcriptional promoter region. The SP1 transcription factor binding site on the Cirp promoter was located in the region chr19:1269276-1269290. (B) HEK293 cells were transfected with a Cirp luciferase reporter construct in the presence or absence of plasmid Sp1 (0.1, 0.5, and 1μg). Cells were subjected to luciferase assays. (C) HEK293 cells were transfected plasmid Sp1 (1 µg) and analyzed using quantitative PCR for Cirp mRNA expression. (D) PBMCs with major A allele or minor G allele of rs11170510 and rs58123204 were subjected to quantitative PCR for Cirp mRNA using its primer (n = 10, major homo AA; n = 10, minor homo GG). Experiments were repeated at least three times. Graphs are indicative of the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 (two-tailed unpaired Mann–Whitney).

Fig 3: CIRP levels are upregulated in multiple organs in the experimental AP model.In WT mice, l-arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg l-arginine. The animals were sacrificed 72 h after the first injection of l-arginine. A, B Western blotting analysis of CIRP levels in the pancreas, intestine, lung, and liver tissues of control and l-arginine-treated AP mice. C Representative images of immunofluorescence staining of CIRP in the pancreas, intestine, lung, and liver tissues. Scale bar: 50 µm. n = 6/group, *p < 0.05 compared with the control group. Data are expressed as mean ± standard deviations.

Fig 4: CIRP deficiency mitigates intestinal tissue damage and edema in the AP mouse model.l-Arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg l-arginine in WT or CIRP KO mice. At 71 h post-first l-arginine injection, mice were intravenously administered with Evans blue (EB) dye through the tail. The animals were sacrificed at 72 h post-first l-arginine injection. The dye was extracted from the intestinal tissues and quantified. A Representative images of hematoxylin and eosin (HE) stained intestinal sections. Scale bar: 100 µm and 50 µm. B Intestinal pathological score. C Quantify intestinal necrosis area. D, E Western blotting analysis of the intestinal levels of RIP-3. F, G Immunohistochemical analysis of MPO, F4/80, and Ly6G in the intestine. H Representative images of EB dye leakage in the intestine. I Intestinal EB dye content (µg/g tissue). J Intestinal wet weight/dry weight ratio. K, L Immunofluorescence analysis of TUNEL. n = 6/group; *p < 0.05 compared with the control group; #p < 0.05 compared with the WT-AP group. Data are expressed as mean ± standard deviations.

Fig 5: CIRP deficiency mitigates hepatic tissue damage and edema in the AP mouse model.l-Arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg l-arginine in WT or CIRP KO mice. At 71 h post-first l-arginine injection, mice were intravenously administered with Evans blue (EB) dye through the tail. The animals were sacrificed at 72 h post-first l-arginine injection. The dye was extracted from the hepatic tissues and quantified. A Representative images of hematoxylin and eosin (HE)-stained hepatic sections. Scale bar: 100 µm and 50 µm. B Hepatic pathological score. C Quantify hepatic necrosis area. D Serum AST levels. E Serum ALT levels. F, G Western blotting analysis of the hepatic levels of RIP-3. H, I Immunohistochemical analysis of MPO, F4/80, and Ly6G in the liver. J Representative images of EB dye leakage in the liver. K Hepatic EB dye content (µg/g tissue). L Hepatic wet weight/dry weight ratio. M, N Immunofluorescence analysis of TUNEL. n = 6/group; *p < 0.05 compared with the control group; #p < 0.05 compared with the WT-AP group. Data are expressed as mean ± standard deviations.