Emodin attenuated severe acute pancreatitis via the P2X ligand‑gated ion channel 7/NOD‑like receptor protein 3 signaling pathway

Abstract

Acute pancreatitis (AP) is an aseptic inflammation characterized with an annual incidence rate, and ~20% patients progressing to severe AP (SAP) with a high mortality rate. Although Qingyi decoction has been frequently used for SAP treatment over the past 3 decades in clinic, the actual mechanism of its protective effects remains unknown. As the major active ingredient of Qingyi decoction, emodin was selected in the present study to investigate the effect of emodin against severe acute pancreatitis (SAP) in rats through NOD‑like receptor protein 3 (NLRP3) inflammasomes. The rats were randomly divided into a sham operation group, an SAP model group induced by a standard retrograde infusion of 5.0% sodium taurocholate into the biliopancreatic duct, and low‑dose (30 mg/kg) and high‑dose (60 mg/kg) emodin‑treated groups. At 12 h after the event, the plasma amylase, lipase, interleukin (IL)‑1β, IL‑18 and myeloperoxidase (MPO) activities were examined. Furthermore, the pathological scores of pancreases were evaluated by hematoxylin and eosin staining. The expression levels of P2X ligand‑gated ion channel 7 (P2X7), NLRP3, apoptosis‑associated speck‑like protein containing a C‑terminal caspase recruitment domain and caspase‑1 were also analyzed by western blot analysis. The data demonstrated that, compared with the SAP group, emodin could significantly relieve the pancreatic histopathology and acinar cellular structure injury, and notably downregulate the plasma amylase and lipase levels, as well as the MPO activities in pancreatic tissues, in a dose‑dependent manner. Furthermore, emodin inhibited the P2X7/NLRP3 signaling pathway followed by the decrease of pro‑inflammatory factors, and the latter is beneficial for the recovery of SAP. Collectively, the data indicated that emodin may be an efficient candidate natural product for SAP treatment.

Figure 1.

Emodin alleviates pancreatic injury in SAP rats. (A) Effects of emodin on hematoxylin and eosin staining of the pancreas in SAP rats. Images are depicted at ×200 magnification (n=3). (B) The histological scores of the pancreases in SAP rats. (C) Effects of emodin on the plasma levels of amylase in SAP rats. (D) Effects of emodin on the plasma levels of lipase in SAP rats. The data are presented as the mean ± standard deviation, n=12. **P<0.01 vs. SO; ^#P<0.05 vs. SAP and ^##P<0.01 vs. SAP. SAP, severe acute pancreatitis; SO, sham operation.

Figure 2.

Emodin attenuates acinar cellular structure injury in SAP rats. Representative images of cell ultrastructure in the (A) SO, (B) SAP, (C) high-dose emodin-treated (60 mg/kg) and (D) low-dose emodin-treated groups. Images were depicted at ×12,000 magnification, n=3. Black arrow indicate granular cytoplasmic reticulum, and adverse changes in the solitary mitochondria and cytoplasmic vacuoles in acinar cells. SAP, severe acute pancreatitis; SO, sham operation.

Figure 3.

Emodin downregulates the expression of MPO in the pancreas of rats with SAP. (A) Effects of emodin on MPO-immunopositive (brown) staining region of the pancreas in SAP rats using an immunohistochemical assay (n=6). Images were depicted at ×20 magnification. (B) The IOD of the immunohistochemical images. (C) Effects of emodin on MPO-immunopositive (red) staining region of the pancreas in SAP rats using an immunofluorescence assay (n=6). Images were depicted at ×20 magnification. (D) The fluorescence density of the immunofluorescence images. The data are presented as the mean ± standard deviation. **P<0.01 vs. SO, ^#P<0.05 vs. SAP and ^##P<0.01 vs. SAP. SAP, severe acute pancreatitis; SO, sham operation; MPO, myeloperoxidase; IOD, integral optic density.

Figure 4.

Emodin inhibits the expression of P2X7 and NLRP3 in the pancreatic tissues. (A) Effects of emodin on the protein expression of P2X7, NLRP3, ASC and caspase-1 in SAP rats. (B) Statistical analysis of the effects of emodin on proteins expression levels (n=3). The data are presented as the mean ± standard deviation, **P<0.01 vs. SO, ^#P<0.05 vs. SAP and ^##P<0.01 vs. SAP. SAP, severe acute pancreatitis; SO, sham operation; P2X7, P2X ligand-gated ion channel 7; NLRP3, NOD-like receptor protein 3; ASC, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain.

Figure 5.

Emodin reduces the plasma concentration of IL-1β and IL-18. (A) Effects of emodin on the plasma concentration of IL-1β in SAP rats. (B) Effects of emodin on the plasma concentration of IL-18 in SAP rats. The data were presented as the mean ± standard deviation. **P<0.01 vs. SO and ^##P<0.05 vs. SAP. IL, interleukin; SAP, severe acute pancreatitis; SO, sham operation.

Figure 6.

The schematic model for the protection of emodin against acute pancreatitis in rats via inhibiting the P2X7/NLRP3 signaling pathway. IL, interleukin; P2X7, P2X ligand-gated ion channel 7; NLRP3, NOD-like receptor protein 3; ASC, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain.