Follistatin-like 1 ameliorates severe acute pancreatitis associated lung injury via inhibiting the activation of NLRP3 inflammasome and NF-κB pathway

Abstract

Objective: Severe acute pancreatitis (SAP) is one of the most common abdominal conditions of digestive system that usually causes acute lung injury through systemic inflammation. Follistatin-like 1 (FSTL-1) has been reported to have anti-inflammatory and anti-apoptotic effects in a variety of diseases. The aim of this study was to investigate the effects of FSTL-1 on SAP-associated lung injury (SAPALI) and the underlying mechanism.

Methods: SAP model was induced by intraperitoneal injection of the L-arginine in C57BL/6 mice. The haematoxylin and eosin (H&E) staining was applied to determine the severity of lung and pancreatic injury. ELISA kits were used to determine serum amylase and inflammatory cytokines levels. TUNEL staining was carried out to measure cell apoptosis. Western blotting was applied to analyze the related proteins of NLRP3 inflammasome and NF-κB pathways.

Results: FSTL-1 was significantly increased in the lung of SAP mice. Knockout of FSTL-1 ameliorated pancreatic injury, lung injury, inflammation and apoptosis in mice with SAP. Moreover, the protein levels of NLRP3, ASC, Caspase-1, p-p65 and p-IκBα were obviously reduced in the FSTL-1 KO+SAP group in comparison with SAP group, suggesting that inhibition of FSTL-1 repressed the activation of the NLRP3 inflammasome and NF-κB pathway.

Conclusion: This study helps us understand the mechanism of FSTL-1 in SAPALI and might provide a potential new strategy for the treatment of SAPALI.

Keywords: Follistatin-like 1; SAP-associated lung injury; Severe acute pancreatitis; apoptosis; inflammation.

Figure 1

FSTL-1 was increased in the lung tissues of SAP mice. A: FSTL-1 mRNA was tested in SAP mice by RT-PCR; B: The protein level of FSTL-1 was measured in SAP mice by western blotting; C: FSTL-1 level was measured by IHC in SAP mice (200× magnification, Scale bars = 50 μm). n = 6 for each group. Compared with control, *P < 0.05, **P < 0.01, ***P < 0.001. FSTL-1: Follistatin-like 1; SAP: severe acute pancreatitis; IHC: immunohistochemical.

Figure 2

Silence of FSTL-1 attenuated inflammation and pancreatic injury in SAP mice. A: The mRNA and protein level of FSTL-1 in SAP mice; B: HE staining of the pancreas tissues (200× magnification, Scale bars = 50 μm); C: Overall histopathological score; D: Acinar cell necrosis; E: Inflammatory cell infiltration; F: Tissue edema; G-I: ELISA kits analysis of the levels of amylase, lipase, TNF-α, IL-6, and IL-1β. n = 6 for each group. Compared with control, **P < 0.01, ***P<0.001; compared with FSTL-1 KO, #P < 0.05, ##P < 0.01. FSTL-1: Follistatin-like 1; SAP, severe acute pancreatitis; SAP: severe acute pancreatitis; HE: haematoxylin and eosin; TNF-α: tumor necrosis factor-α; IL-6: Interleukin-6; IL-1β: Interleukin-1β.

Figure 3

Silence of FSTL-1 attenuated inflammation and lung damage in SAPALI mice. A: HE staining of the lung tissues (200× magnification, Scale bars = 50 μm). B: Scoring of the alveolar septal capillary density in lung tissues. C-E: The level of TNF-α, IL-6 and IL-1β were analyzed by ELISA kits; F, G: The numbers of the Ly6G and CD11b detected by Immunofluorescence (200× magnification, Scale bars = 50 μm). n = 6 for each group. Compared with control, *P < 0.05, **P < 0.01, ***P < 0.001; compared with FSTL-1 KO, #P < 0.05. FSTL-1: Follistatin-like 1; SAP, severe acute pancreatitis; SAPALI: SAP-associated lung injury; HE: haematoxylin and eosin; TNF-α: tumor necrosis factor-α; IL-6: Interleukin-6; IL-1β: Interleukin-1β.

Figure 4

Silence of FSTL-1 repressed cell apoptosis in SAPALI lung tissues. A: TUNEL staining of the apoptotic cells in the lung tissues (200× magnification, Scale bars = 50 μm; B: Western blotting analysis of the cleaved-caspase-3 and cleaved-caspase-8 levels in SAPALI lung tissues. n = 6 for each group. Compared with control, *P < 0.05, **P < 0.01, ***P < 0.001; compared with FSTL-1 KO, #P < 0.05, ##P < 0.01. FSTL-1: Follistatin-like 1; SAP, severe acute pancreatitis; SAPALI: SAP-associated lung injury.

Figure 5

Silence of FSTL-1 inhibited NLRP3 inflammasome and NF-κB pathway activation in SAPALI mice. A: Western blotting assay was applied to analyze the levels of Caspase-1, NLRP3, and ASC in SAPALI lung tissues; B: p-p65 and p-IκBα levels were detected in SAPALI lung tissues. n = 6 for each group. Compared with control, *P < 0.05, **P < 0.01; compared with FSTL-1 KO, #P < 0.05. SAP, severe acute pancreatitis; FSTL-1: Follistatin-like 1; SAPALI: SAP-associated lung injury.

Figure 6

Knockout of FSTL-1 repressed SAPALI via suppressing the activation of NF-κB pathway, thereby inhibiting the inflammation and apoptosis in mice after SAP. FSTL-1: Follistatin-like 1; SAP, severe acute pancreatitis; SAPALI: SAP-associated lung injury.