Effects of Snake-Derived Phospholipase A2 Inhibitors on Acute Pancreatitis: In vitro and in vivo Characterization

Abstract

Objective: We aimed to investigate the effects of snake-derived phospholipase A2 inhibitor (PLA2) from Sinonatrix percarinata and Bungarus multicinctus on acute pancreatitis in vivo and in vitro and assess the mechanisms.

Methods: The levels of platelet-activating factor (PAF) and tumor necrosis factor (TNF)-α were detected by ELISA, and the characteristics of autophagy were detected by transmission electron microscopy and Western blotting (LC3, p62, and ATG5).

Results: In vitro experiments showed that PLA2 treatment caused obvious formation of autophagic bodies. By contrast, Sinonatrix and Bungarus peptides reduced the number of autophagic bodies. The concentrations of PAF and TNF-α, and the expressions of p62, autophagy-related 5 (ATG5), and microtubule-associated protein 1A/1B-light chain 3 (LC3)II/LC3I in the PLA2-treated group were significantly higher than in the control group (P<0.05). The concentrations of PAF and TNF-α, and the expressions of p62, ATG5, and LC3II/LC3I in the Sinonatrix or Bungarus peptide treatment groups were significantly lower than in the PLA2-treated cells (P<0.05). In the pancreatic tissue, autophagic bodies were observed in the model group; autophagic bodies were remarkably reduced in Sinonatrix or Bungarus peptide-treated groups compared with the model group. In vivo experiments also showed that the levels of PAF and TNF-α, and the expressions of p62, ATG5, and LC3II/LC3I were significantly higher in the model group than in the control group (P<0.05). The levels of PAF and TNF-α in the model group, and the expressions of p62, ATG5, and LC3II/LC3I in Sinonatrix or Bungarus peptide-treated groups were significantly lower than in the model group (P<0.05).

Conclusion: Sinonatrix or Bungarus peptide could ameliorate the features of acute pancreatitis, likely through regulating autophagy.

Keywords: PLA2; acute pancreatitis; autophagy; snake-derived PLI.

Figure 1

Determining the effect of PLA2 treatment by flow cytometry. (A) Representative images of flow cytometry; (B) Quantitative apoptosis data. Data were expressed as mean and standard deviation. N=6 in each group (Bonferroni test). *P<0.05: compared with the control group; ^#P<0.05: compared with 0.5 h.

Figure 2

Effects of snake-derived PLIs on PAF and TNF-α levels in pancreatic tissue. (A) PAF level; (B) TNF-α level. Data were expressed as mean and standard deviation. N=6 in each group (Bonferroni test). *P<0.05: compared with the control group; ^#P<0.05: compared with the model group.

Figure 3

Snake-derived PLI reduced PAF and TNF-α levels in PLA 2-treated cells and AP mice model. (A, B) The level of PAF and TNF-α in the pancreatic acinar cells; (C, D) The level of PAF and TNF-α in vivo. Data were expressed as mean and standard deviation. N=6 in each group (Bonferroni test). *P<0.05: compared with the control group; ^#P<0.05: compared with the model group.

Figure 4

Snake-derived PLI reduced autophagic body formation in PLA 2-treated cells and AP mice model. (A) TEM results of cells; (B) TEM results of pancreatic tissue. Arrows indicate the autophagic bodies. *P<0.05: compared with the control group; ^#P<0.05: compared with the model group.

Figure 5

Snake-derived PLI reduced the expression of LC3, p62, and ATG5 in PLA 2-treated cells and AP mice model. (A–C) LC3, p62, and ATG5 in PLA 2-treated cells; (D–F) LC3, p62, and ATG5 in AP mice model. The original blots are shown in Supplementary Figure S1. Data were expressed as mean and standard deviation. N=6 in each group (Bonferroni test). *P<0.05: compared with the control group; ^#P<0.05: compared with the model group.