Fig 2: Spef2’s suppressive effect on in vivo acute pancreatitis and associated lung injury mediated via the Ampka/Sirt1 axis. Pancreatic and lung tissues and peripheral blood samples were harvested from sham-operated control (SO Ctrl), acute pancreatitis + DMSO vehicle (AP + Veh), acute pancreatitis + scAAV6-delivered Spef2 + control siRNA (AP + scAAV6.Spef2 + siCtrl), acute pancreatitis + scAAV6-delivered Spef2 + Ampka siRNA (AP + scAAV6.Spef2 + siAmpka), and acute pancreatitis + scAAV6-delivered Spef2 + Ampka siRNA + scAAV6-delivered CA-Sirt1 (AP + scAAV6.Spef2 + CA-Sirt1 + siAmpka) rats 24 hours post-induction. (A) Representative H&E staining of pancreatic tissues. (B) Pathological scoring of pancreatic injury. (C) Pancreatic LDH, MDA, LPO, and MPO activity levels. (D) Pancreatic ATP levels. (E) Pancreatic necrosis levels. (F) Representative immunoblots of pancreatic Spef2 and Ampka/Sirt1/NF-?B signaling proteins. (G) Serum CRP, TNF-a, IL-1ß, IL-6, and IL-18 levels from peripheral blood samples. (H) H&E staining of lung tissue showing extensive edema, alveolar congestion, and immune cell in?ltration (scale bar =500 µm). (I) Pathological scoring of lung tissue injury. (J) Lung W/D ratio. (K) Lung MPO activity. (L) BALF TNF-a and IL-1ß levels and (M) BALF protein content. Data represented as means ± SDs. N=6 rats per cohort. *P<0.05, **P<0.01 (one-way ANOVA with Bonferroni post-hoc). Spef2, sperm flagellar 2; Ampka, adenosine monophosphate-activated protein kinase alpha; Sirt1, sirtuin 1; DMSO, dimethyl sulfoxide; siRNA, small-interfering RNA; CA, constitutively active; H&E, hematoxylin and eosin; LDH, lactate dehydrogenase; MDA, malondialdehyde; LPO, lipid peroxide; MPO, myeloperoxidase; ATP, adenosine triphosphate; NF-?B, nuclear factor kappa B; CRP, C-reactive protein; TNF-a, tumor necrosis factor alpha; IL-1ß, interleukin-1 beta; W/D, wet-to-dry; BALF, bronchoalveolar lavage fluid; SD, standard deviation; ANOVA, analysis of variance.

Fig 3: Spef2’s suppressive effect on in vitro pancreatic acinar cell necrosis and in?ammation mediated via the Ampka/Sirt1 axis. 72 hours following scAAV6.NC, scAAV6.Spef2, or scAAV6.Spef2 + CA-Sirt1 infection and 12 hours following siCtrl or siAmpka transfection, AR42J cells were incubated with cerulein to establish an in vitro model of acute pancreatitis. (A) Representative immunoblots of pancreatic Spef2 and Ampka/Sirt1/NF-?B signaling proteins. (B) LDH levels. (C) ATP levels. (D) Cell necrosis levels. (E) Representative TUNEL staining images and (F) associated quantitative measurements of apoptosis. (G) Representative CC-3 fluorescent staining images. CC-3 staining in green and DAPI nuclei staining in blue (scale bar =200 µm). (H) IOD quantitation of fluorescent CC-3 staining. (I) Cell supernatant TNF-a and IL-1ß levels. Data represented as means ± SDs. N=3 biological replicates × 2 technical replicates. *P<0.05, **P<0.01 (one-way ANOVA with Bonferroni post-hoc). Spef2, sperm flagellar 2; Ampka, adenosine monophosphate-activated protein kinase alpha; Sirt1, sirtuin 1; CA, constitutively active; NF-?B, nuclear factor kappa B; LDH, lactate dehydrogenase; ATP, adenosine triphosphate; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; CC-3, cleaved caspase-3; DAPI, 4',6-diamidino-2-phenylindole; TNF-a, tumor necrosis factor alpha; IL-1ß, interleukin-1 beta; SD, standard deviation; ANOVA, analysis of variance; IOD, integrated optical density.