Salidroside ameliorates severe acute pancreatitis-induced cell injury and pyroptosis by inactivating Akt/NF-κB and caspase-3/GSDME pathways

Figures

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  Fig. 1. Sal suppressed SAP-induced cell pyroptosis in vivo. A Representative images of HE stained sections of rat pancreatic tissues from sham, SAP and SAP + Sal group. Image 1 represented lobule structure, Image 2 represented microthrombosis, Image 3 represented red cells and Image 4 represented immune cells. B Quantitative analysis of pancreatic injury score. The histopathologic changes of Fig. 1A were observed under a light microscope. Five non-overlapping visual fields were randomly selected in each section and the pancreatic tissue injury was evaluated by double-blind method and scored based on Schmidt's severity score standard of pancreas as follows: edema (0–4 points), inflammation (0–4 points), glandular cell necrosis (0–4 points) and hemorrhage (0–1 point). C Representative images of TUNEL staining of rat pancreatic tissues from sham, SAP and SAP + Sal group. Blue represents DAPI staining and green represents TUNEL staining. D Quantitative analysis of TUNEL-positive cells. TUNEL-positive cells from 5 randomly selected fields were observed and counted under a microscope. The data were expressed as mean ± standard deviation. E Activity of the serum amylase. F LDH release in the…

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  Fig. 2. Sal suppressed SAP-induced cell injury and pyroptosis in AR42J cells. A Representative morphological images of AR42J cells treated with SAP or SAP + Sal under microscope. Image 4 represented pyroptotic cells. B AR42J cells were treated with SAP or SAP + Sal and then cell viability was determined by CCK-8 assay with an absorbance at 450 nm. C Representative images of Hoechst 33342/PI double staining. Blue represents Hoechst 33342 staining and red represents PI staining. D Quantitative analysis of PI-positive cells. PI-positive cells from 5 randomly selected fields were observed and counted under a fluorescence microscope. The data were expressed as mean ± standard deviation. E Amylase activity in the cultured cell supernatant. F LDH release in the cultured cell supernatant. G IL-1β level in the cultured cell supernatant. H IL-18 level in the cultured cell supernatant. **P < 0.01 compared with control group. ##P < 0.01 compared with SAP group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 3. Sal suppressed activation of Akt/NF-κB and Caspase-3/GSDME pathways. A Ten proteins (Akt1, Akt2, Akt3, Casp3, Epas1, Hif1α, Hif3α, Il10, Npas1 and Npas3) were predicted to be potential targets of Sal in rattus norvegicus by an online tool STITCH. B Four proteins (AKT1, CASP3, HIF1A and IL10) were predicted to be potential targets of Sal in homo sapiens by STITCH. C Effects of Sal on Akt/NF-κB and Caspase-3/GSDME pathways-related proteins were evaluated by Western blot in SAP cell model. D Quantitative analysis of relative protein levels of Fig. 3C. E Effect of Sal on the nuclear translocation of p65 was determined by immunofluorescence. F IHC staining of p-p65 in the rat pancreatic tissues. **P < 0.01 compared with control group. #P < 0.05, ##P < 0.01 compared with SAP group.

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  Fig. 4. Sal ameliorated SAP-induced cell injury and inflammation by inhibiting AKT1 and CASP3 expression. A AR42J cells were transfected with CV-myrAKT1, CV-CAPS3, si-AKT1, si-CASP3 or negative controls. The efficiency of overexpression and downregulation was verified by Western blot. B Quantitative analysis of relative protein levels of Fig. 4A. C Cell viability was detected by CCK-8 assay. D-G AR42J cells were transfected with CV-myrAKT1, CV-CAPS3, si-AKT1, si-CASP3 or negative controls and then treated with SAP and Sal. Amylase activity (D), TNF-α level (E), IL-6 level (F) and IL-8 level (G) in the cultured cell supernatant. For B, **P < 0.01 compared with CV-NC group. For C-G, *P < 0.05, **P < 0.01 compared with SAP group. #P < 0.05, ##P < 0.01 compared with SAP + Sal + CV-NC group. $P < 0.05, $$P < 0.01 compared with SAP + Sal + si-NC group.

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  Fig. 5. Sal suppressed SAP-induced cell pyroptosis through reducing AKT1 and CASP3 expression. AR42J cells were transfected with CV-myrAKT1, CV-CAPS3, si-AKT1, si-CASP3 or negative controls and then treated with SAP and Sal. A Representative morphological images of AR42J cells under microscope. Image 4 represented pyroptotic cells. B Representative images of Hoechst 33342/PI double staining. Blue represents Hoechst 33342 staining and red represents PI staining. C Quantitative analysis of PI-positive cells. D-F LDH release (D), IL-1β level (E) and IL-18 level (F) in the cultured cell supernatant. *P < 0.05, **P < 0.01 compared with SAP group. #P < 0.05, ##P < 0.01 compared with SAP + Sal + CV-NC group. $P < 0.05, $$P < 0.01 compared with SAP + Sal + si-NC group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)