Emodin alleviates severe acute pancreatitis-associated acute lung injury by decreasing pre-B-cell colony-enhancing factor expression and promoting polymorphonuclear neutrophil apoptosis

Abstract

The present study aimed to evaluate the protective effects of emodin on severe acute pancreatitis (SAP)‑associated acute lung injury (ALI), and investigated the possible mechanism involved. SAP was induced in Sprague‑Dawley rats by retrograde infusion of 5% sodium taurocholate (1 ml/kg), after which, rats were divided into various groups and were administered emodin, FK866 [a competitive inhibitor of pre‑B‑cell colony‑enhancing factor (PBEF)] or dexamethasone (DEX). DEX was used as a positive control. Subsequently, PBEF expression was detected in polymorphonuclear neutrophils (PMNs) isolated from rat peripheral blood by reverse transcription‑quantitative polymerase chain reaction and western blotting. In addition, histological alterations, apoptosis in lung/pancreatic tissues, apoptosis of peripheral blood PMNs and alterations in the expression of apoptosis‑associated proteins were examined by hematoxylin and eosin staining, terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling assay, Annexin V/propidium iodide (PI) assay and western blotting, respectively. Serum amylase activity and wet/dry (W/D) weight ratios were also measured. An in vitro study was also conducted, in which PMNs were obtained from normal Sprague‑Dawley rats and were incubated with emodin, FK866 or DEX in the presence of lipopolysaccharide (LPS). Apoptosis of PMNs and the expression levels of apoptosis‑associated proteins were examined in cultured PMNs in vitro by Annexin V/PI assay and western blotting, respectively. The results demonstrated that emodin, FK866 and DEX significantly downregulated PBEF expression in peripheral blood PMNs. In addition, emodin, FK866 and DEX reduced serum amylase activity, decreased lung and pancreas W/D weight ratios, alleviated lung and pancreatic injuries, and promoted PMN apoptosis by regulating the expression of apoptosis‑associated proteins: Fas, Fas ligand, B‑cell lymphoma (Bcl)‑2‑associated X protein, cleaved caspase‑3 and Bcl‑extra‑large. In addition, the in vitro study demonstrated that emodin, FK866 and DEX significantly reversed the LPS‑induced decrease of apoptosis in PMNs by regulating the expression of apoptosis‑associated proteins. In conclusion, the present study demonstrated that emodin may protect against SAP‑associated ALI by decreasing PBEF expression, and promoting PMN apoptosis via the mitochondrial and death receptor apoptotic pathways.

Figure 1.

Effects of emodin, FK866 and DEX on PBEF expression in PMNs isolated from rat peripheral blood. PMNs were harvested from rat peripheral blood following various treatments. (A) The mRNA expression levels of PBEF were detected by reverse transcription-quantitative polymerase chain reaction. (B) Protein expression levels of PBEF were detected by western blotting. Data are presented as the mean ± standard deviation. **P<0.01 compared with the control group; ^#P<0.05, ^##P<0.01 compared with the SAP group. DEX, dexamethasone; PBEF, pre-B-cell colony-enhancing factor; PMN, polymorphonuclear neutrophils; SAP, severe acute pancreatitis.

Figure 2.

Representative HE-stained sections of lung and pancreatic tissues. (A) Lung and (B) pancreatic tissues were excised from rats in each group following various treatments and histological alterations were evaluated by HE staining. Scale bar, 100 µm. DEX, dexamethasone; HE, hematoxylin and eosin; SAP, severe acute pancreatitis.

Figure 3.

Effects of emodin, FK866 and DEX on serum amylase activity and W/D weight ratio. (A) Serum amylase activity was measured; values are expressed as U/dl. (B) Lung and (C) pancreatic W/D weight ratios. Data are presented as the mean ± standard deviation. **P<0.01 compared with the control group; ^#P<0.05, ^##P<0.01 compared with the SAP group. DEX, dexamethasone; SAP, severe acute pancreatitis; W/D, wet/dry.

Figure 4.

Representative TUNEL-stained sections of lung and pancreatic tissues. (A) Lung and (B) pancreatic sections were subjected to TUNEL staining, in order to detect apoptotic cells. Representative images are shown. Scale bar, 50 µm. DEX, dexamethasone; SAP, severe acute pancreatitis; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling.

Figure 5.

Effects of emodin, FK866 and DEX on PMN apoptosis in SAP rats. (A) PMN apoptosis was examined by Annexin V/propidium iodide staining and flow cytometry. (B) Protein expression levels of Fas, FasL, Bax, cleaved caspase-3 and Bcl-xL were detected by western blotting. β-actin served as an internal control. Data are presented as the mean ± standard deviation. **P<0.01 compared with the control group; ^#P<0.05, ^##P<0.01 compared with the SAP group. Bax, B-cell lymphoma 2-associated X protein; Bcl-xL, B-cell lymphoma-extra-large; DEX, dexamethasone; FasL, Fas ligand; PMN, polymorphonuclear neutrophils; SAP, severe acute pancreatitis.

Figure 6.

Effects of emodin, FK866 and DEX on apoptosis of LPS-stimulated PMNs in vitro. PMNs were isolated from normal rats and cultured in vitro. Subsequently, PMNs were stimulated with LPS and incubated with emodin, FK866 and DEX for 16 h. (A) Flow cytometric analysis of PMN apoptosis, using Annexin V/propidium iodide staining. (B) Protein expression levels of Fas, FasL, Bax, cleaved caspase-3 and Bcl-xL were examined by western blotting. Data are presented as the mean ± standard deviation. **P<0.01 compared with the control group; ^#P<0.05, ^##P<0.01 compared with the SAP group. Bax, B-cell lymphoma 2-associated X protein; Bcl-xL, B-cell lymphoma-extra-large; DEX, dexamethasone; FasL, Fas ligand; LPS, lipopolysaccharide; PMN, polymorphonuclear neutrophil.