Alleviation of acute pancreatitis-associated lung injury by inhibiting the p38 mitogen-activated protein kinase pathway in pulmonary microvascular endothelial cells

Abstract

Background: Previous reports have suggested that the p38 mitogen-activated protein kinase signaling pathway is involved in the development of severe acute pancreatitis (SAP)-related acute lung injury (ALI). Inhibition of p38 by SB203580 blocked the inflammatory responses in SAP-ALI. However, the precise mechanism associated with p38 is unclear, particularly in pulmonary microvascular endothelial cell (PMVEC) injury.

Aim: To determine its role in the tumor necrosis factor-alpha (TNF-α)-induced inflammation and apoptosis of PMVECs in vitro. We then conducted in vivo experiments to confirm the effect of SB203580-mediated p38 inhibition on SAP-ALI.

Methods: In vitro, PMVEC were transfected with mitogen-activated protein kinase kinase 6 (Glu), which constitutively activates p38, and then stimulated with TNF-α. Flow cytometry and western blotting were performed to detect the cell apoptosis and inflammatory cytokine levels, respectively. In vivo, SAP-ALI was induced by 5% sodium taurocholate and three different doses of SB203580 (2.5, 5.0 or 10.0 mg/kg) were intraperitoneally injected prior to SAP induction. SAP-ALI was assessed by performing pulmonary histopathology assays, measuring myeloperoxidase activity, conducting arterial blood gas analyses and measuring TNF-α, interleukin (IL)-1β and IL-6 levels. Lung microvascular permeability was measured by determining bronchoalveolar lavage fluid protein concentration, Evans blue extravasation and ultrastructural changes in PMVECs. The apoptotic death of pulmonary cells was confirmed by performing a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis and examining the Bcl2, Bax, Bim and cle-caspase3 levels. The proteins levels of P-p38, NFκB, IκB, P-signal transducer and activator of transcription-3, nuclear factor erythroid 2-related factor 2, HO-1 and Myd88 were detected in the lungs to further evaluate the potential mechanism underlying the protective effect of SB203580.

Results: In vitro, mitogen-activated protein kinase (Glu) transfection resulted in higher apoptotic rates and cytokine (IL-1β and IL-6) levels in TNF-α-treated PMVECs. In vivo, SB2035080 attenuated lung histopathological injury, decreased inflammatory activity (TNF-α, IL-1β, IL-6 and myeloperoxidase) and preserved pulmonary function. Furthermore, SB203580 significantly reversed changes in the bronchoalveolar lavage fluid protein concentration, Evans blue accumulation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cell numbers, apoptosis-related proteins (cle-caspase3, Bim and Bax) and endothelial microstructure. Moreover, SB203580 significantly reduced the pulmonary P-p38, NFκB, P-signal transducer and activator of transcription-3 and Myd88 levels but increased the IκB and HO-1 levels.

Conclusion: p38 inhibition may protect against SAP-ALI by alleviating inflammation and the apoptotic death of PMVECs.

Keywords: Acute lung injury; Acute pancreatitis; Apoptosis; P38; Pulmonary microvascular endothelial cells; SB203580.

Figure 1

p38 mitogen-activated protein kinase overactivation aggravates tumor necrosis factor-alpha-induced apoptosis and inflammatory cytokine expression in pulmonary microvascular endothelial cells. A: Western blotting confirmed that mitogen-activated protein kinase kinase (Glu) transfection constitutively activated p38 mitogen-activated protein kinase in pulmonary microvascular endothelial cells; B: Apoptotic rate in null and mitogen-activated protein kinase kinase (Glu) pulmonary microvascular endothelial cells stimulated with or without tumor necrosis factor-alpha for 24 h; C: Representative images of flow cytometry analysis for apoptosis; D: The expression level of interleukin-6 in each group; E: The expression level of interleukin-1β in each group. Data are expressed as mean ± SE of 3-4 samples per group; ^aP < 0.05; ^bP < 0.01. IL: Interleukin; MKK: Mitogen-activated protein kinase kinase; TNF-α: Tumor necrosis factor-alpha.

Figure 2

Effects of SB203580 on pancreatic histopathological changes and histopathological severity scores. A: Representative hematoxylin and eosin images of pancreatic sections (magnification 200 ×); I: Sham group; II: Severe acute pancreatitis (SAP) group; III: SAP + SB2.5 group; IV: SAP + SB5 group; V: SAP + SB10 group; B: Total scores; C: Edema scores; D: Inflammatory infiltration scores; E: Hemorrhage scores; F: Necrosis scores. B-F: Pancreatic histopathology scores in different groups; Data were expressed as mean ± SE; ^aP < 0.05 vs sham group; ^bP < 0.05 vs SAP group; ^cP < 0.05 vs SAP + SB2.5 group. SAP: Severe acute pancreatitis.

Figure 3

Effects of SB203580 on pulmonary histopathological changes and histopathological severity scores. A: Representative hematoxylin and eosin images of pulmonary sections (magnification 200 ×); I: Sham group; II: Severe acute pancreatitis (SAP) group; III: SAP + SB2.5 group; IV: SAP + SB5 group; V: SAP + SB10 group; B: Total scores. C: Edema scores; D: Inflammatory infiltration scores. B-D: Pulmonary histopathology scores in different groups; Data are expressed as mean ± SE of the mean; ^aP < 0.05 vs sham group; ^bP < 0.05 vs SAP group; ^cP < 0.05 vs SAP + SB2.5 group; ^dP < 0.05 vs SAP + SB5 group. SAP: Severe acute pancreatitis.

Figure 4

Effects of SB203580 on pulmonary severity indices of severe acute pancreatitis in rats. A: Bronchoalveolar interleukin-1β (IL-1β); B: Bronchoalveolar tumor necrosis factor alpha (TNF-α); C: Serum IL-1β; D: Serum TNF-α; E: Lung myeloperoxidase activity; F: Partial pressure of oxygen; G: Oxygen saturation; H: Representative western blotting analysis results for IL-1β in lung tissues; I: Representative western blotting analysis results for IL-6 in lung tissues; J: Representative western blotting analysis results for TNF-α in lung tissues. Data are expressed as mean ± SE of the mean; ^aP < 0.05 vs sham group; ^bP < 0.05 vs severe acute pancreatitis group; ^cP < 0.05 vs severe acute pancreatitis + SB2.5 group. IL: Interleukin; MPO: Myeloperoxidase; PaO₂: Pressure of oxygen; SaO₂: Oxygen saturation; SAP: Severe acute pancreatitis; TNF-α: Tumor necrosis factor-alpha.

Figure 5

Effects of SB203580 on pulmonary capillary injury of severe acute pancreatitis rats. A: Evans blue extravasation in lung tissues; B: Bronchoalveolar protein concentration; C: Representative electron microscope photomicrographs of pulmonary microvascular endothelial cells in lung tissues; Sham group: Magnification 1500 × and 4000 ×, respectively; Severe acute pancreatitis (SAP) group: Magnification 1500 × and 5000 ×, respectively; SAP + SB10 group: Magnification 1500 × and 5000 ×, respectively; White arrows: Dense endothelial cell–cell junctions; White arrowheads: Low electron density clouds with an irregular thickness indicating basal membrane edema; Yellow arrows: Dissolution, rupture and debris of capillary endothelial; Orange star: Red blood cell. Data were expressed as mean ± SE; ^aP < 0.05 vs sham group; ^bP < 0.05 vs SAP group; ^cP < 0.05 vs SAP + SB2.5 group. SAP: Severe acute pancreatitis.

Figure 6

Effect of SB203580 on apoptosis of lung tissues in severe acute pancreatitis. A: Representative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling images of pulmonary sections (200 ×); B: Cle-caspase3; C: Bcl2; D: Bax; E: Bim. B-E: Representative western blotting analysis results for apoptosis-related proteins in lung tissues; Data were expressed as mean ± SE; ^aP < 0.05 vs sham group; ^bP < 0.05 vs severe acute pancreatitis group. SAP: Severe acute pancreatitis.

Figure 7

Effect of SB203580 on proinflammatory and proapoptotic signaling pathways. Representative Western blotting analysis results for protein expression in pulmonary tissue; A: P-p38; B: NFκB; C: IκB; D: HO-1; E: Nrf2; F: P-STAT3; G: Myd88. Data are expressed as mean ± SE; ^aP < 0.05 vs sham group; ^bP < 0.05 vs severe acute pancreatitis group; ^cP < 0.05 vs severe acute pancreatitis + SB2.5 group; ^dP < 0.05 vs severe acute pancreatitis + SB5 group. Nrf2: Nuclear factor erythroid 2-related factor 2; STAT3: Signal transducer and activator of transcription-3.