Milk fat globule epidermal growth factor 8 alleviates liver injury in severe acute pancreatitis by restoring autophagy flux and inhibiting ferroptosis in hepatocytes

Abstract

Background: Liver injury is common in severe acute pancreatitis (SAP). Excessive autophagy often leads to an imbalance of homeostasis in hepatocytes, which induces lipid peroxidation and mitochondrial iron deposition and ultimately leads to ferroptosis. Our previous study found that milk fat globule epidermal growth factor 8 (MFG-E8) alleviates acinar cell damage during SAP via binding to αvβ3/5 integrins. MFG-E8 also seems to mitigate pancreatic fibrosis via inhibiting chaperone-mediated autophagy.

Aim: To speculate whether MFG-E8 could also alleviate SAP induced liver injury by restoring the abnormal autophagy flux.

Methods: SAP was induced in mice by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine or 7 hly injections of 50 μg/kg cerulein plus lipopolysaccharide. mfge8-knockout mice were used to study the effect of MFG-E8 deficiency on SAP-induced liver injury. Cilengitide, a specific αvβ3/5 integrin inhibitor, was used to investigate the possible mechanism of MFG-E8.

Results: The results showed that MFG-E8 deficiency aggravated SAP-induced liver injury in mice, enhanced autophagy flux in hepatocyte, and worsened the degree of ferroptosis. Exogenous MFG-E8 reduced SAP-induced liver injury in a dose-dependent manner. Mechanistically, MFG-E8 mitigated excessive autophagy and inhibited ferroptosis in liver cells. Cilengitide abolished MFG-E8's beneficial effects in SAP-induced liver injury.

Conclusion: MFG-E8 acts as an endogenous protective mediator in SAP-induced liver injury. MFG-E8 alleviates the excessive autophagy and inhibits ferroptosis in hepatocytes by binding to integrin αVβ3/5.

Keywords: Acute pancreatitis; Autophagy flux; Ferroptosis; Liver injury; Milk fat globule epidermal growth factor 8; αvβ3/5 integrins.

Figure 1

Serum milk fat globule epidermal growth factor 8 concentration was negatively correlated with the inflammatory severity in acute pancreatitis patients. Blood samples from 134 acute pancreatitis patients were collected, and serum milk fat globule epidermal growth factor 8 (MFG-E8) levels were measured. A: Correlation analysis of serum MFG-E8 levels and serum white blood cell levels; B: Correlation analysis of serum MFG-E8 levels and serum procalcitonin levels; C: Correlation analysis of serum MFG-E8 levels and multiple organ failure syndrome scores. WBC: White blood cells; PCT: Procalcitonin; MODS: Multiple organ failure syndrome; MFG-E8: Milk fat globule epidermal growth factor 8.

Figure 2

Milk fat globule epidermal growth factor 8 deficiency aggravated hepatic injury and inflammation in experimental severe acute pancreatitis. In mice, acute pancreatitis (AP) was induced by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine. The animals were sacrificed at 72 h after the first injection of L-arginine. Blood and tissue samples were collected. A: Representative photos of hematoxylin and eosin staining in the liver (400 × or 1000 ×); B: Hepatic injury scores; C: Percentages of necrotic areas; D: Tumor necrosis factor-α levels in the liver; E: Myeloperoxidase levels in the liver; F and G: Representative photos of TdT-mediated dUTP Nick-End Labeling (TUNEL) staining (400 ×) and quantitative of TUNEL staining. n = 3-6/group, error bars indicate the SEM; ^aP < 0.05 vs sham group; ^bP < 0.05 vs vehicle group. SAP: Severe acute pancreatitis; KO: Knockout; WT: Wild type; TNF-α: Tumor necrosis factor-α; MPO: Myeloperoxidase; TUNEL: TdT-mediated dUTP Nick-End Labeling.

Figure 3

Milk fat globule epidermal growth factor 8 deficiency aggravated hepatic autophagy and ferroptosis in experimental severe acute pancreatitis. In mice, severe acute pancreatitis was induced by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine. The animals were sacrificed at 72 h after the first injection of L-arginine. Blood and tissue samples were collected. A: Autophagy related protein 16 like protein 1 (ATG16L1) assists LC3 in the formation of autophagosomes; B-E: Western blot analysis and quantitative of the expression of ATG16L1, P62, LC3, prostaglandin-endoperoxide synthase 2 and glutathione peroxidase 4 in the liver; F: Malondialdehyde levels in the liver; G: Glutathione levels in the liver; H: Representative images of DHE staining in the pancreas. n = 3-6/group, error bars indicate the SEM; ^aP < 0.05 vs sham group; ^bP < 0.05 vs vehicle group. SAP: Severe acute pancreatitis; KO: Knockout; WT: Wild type; ATG16L1: Autophagy related protein 16 like protein 1; PTGS2: Prostaglandin-endoperoxide synthase 2; GPX4: Glutathione peroxidase 4; MDA: Malondialdehyde; GSH: Glutathione.

Figure 4

Milk fat globule epidermal growth factor 8 administration mitigates liver Injury in experimental severe acute pancreatitis. In mice, severe acute pancreatitis was induced by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine. At 2 h after the last injection of L-arginine, normal saline (vehicle) or 5, 10, or 20 μg/kg milk fat globule epidermal growth factor 8 (MFG-E8) was administered through intraperitoneal injection. The animals were sacrificed at 69 h after MFG-E8 treatment (i.e., 72 h after the first injection of L-arginine). Blood and tissue samples were collected. A: Representative photos of hematoxylin and eosin staining in the liver (400 × or 1000 ×); B: Hepatic injury scores; C: Percentages of necrotic areas; D: Tumor necrosis factor-α levels in the liver; E: Myeloperoxidase levels in the liver; F and G: Representative photos of TdT-mediated dUTP Nick-End Labeling (TUNEL) staining (400 ×) and quantitative of TUNEL staining. n = 3-6/group, error bars indicate the SEM; ^aP < 0.05 vs sham group; ^bP < 0.05 vs vehicle group. SAP: Severe acute pancreatitis; TNF-α: Tumor necrosis factor-α; MPO: Myeloperoxidase; TUNEL: TdT-mediated dUTP Nick-End Labeling; MFG-E8: Milk fat globule epidermal growth factor 8.

Figure 5

Milk fat globule epidermal growth factor 8 administration mitigates hepatic autophagy and ferroptosis in experimental severe acute pancreatitis. In mice, arginine-severe acute pancreatitis was induced by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine. At 2 h after the last injection of L-arginine, normal saline (vehicle) or 10, or 20 μg/kg milk fat globule epidermal growth factor 8 (MFG-E8) was administered through intraperitoneal injection. The animals were sacrificed at 69 h after MFG-E8 treatment (i.e., 72 h after the first injection of L-arginine). Blood and tissue samples were collected. A-D: Western blot analysis and quantitative of the expression of autophagy related protein 16 like protein 1, P62, LC3, prostaglandin-endoperoxide synthase 2 and glutathione peroxidase 4 in the liver; E: Malondialdehyde levels in the liver; F: Representative images of DHE staining in the pancreas; G: Glutathione levels in the liver; H: Ultrastructural changes of mitochondria in hepatocytes (electron microscopy). n = 3-6/group, error bars indicate the SEM; ^aP < 0.05 vs sham group; ^bP < 0.05 vs vehicle group. SAP: Severe acute pancreatitis; ATG16L1: Autophagy related protein 16 like protein 1; PTGS2: Prostaglandin-endoperoxide synthase 2; GPX4: Glutathione peroxidase 4; MDA: Malondialdehyde; GSH: Glutathione; MFG-E8: Milk fat globule epidermal growth factor 8.

Figure 6

Milk fat globule epidermal growth factor 8 protects the liver from damage through the integrin αVβ3/5. In mice, severe acute pancreatitis was induced by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine. At 2 h after the last injection of L-arginine, normal saline (vehicle) or 20 μg/kg milk fat globule epidermal growth factor 8 (MFG-E8) was administered through intraperitoneal injection. To determine whether the protective effect of MFG-E8 in liver is mediated through integrin αVβ3/5, 20 mg/kg cilengitide, a specific integrin αVβ3/5 antagonist, was administered intraperitoneally at 1 h before the injection of MFG-E8 (i.e., 1 h after the last injection of L-arginine). The animals were sacrificed at 69 h after MFG-E8 treatment (i.e., 72 h after the first injection of L-arginine). Blood and tissue samples were collected. A: Representative photos of hematoxylin and eosin staining in the liver (400 × or 1000 ×); B: Hepatic injury scores; C: Percentages of necrotic areas; D: Tumor necrosis factor-α levels in the liver; E: Myeloperoxidase levels in the liver; F and G: Representative photos of TdT-mediated dUTP Nick-End Labeling (TUNEL) staining (400 ×) and quantitative of TUNEL staining. n = 3-6/group, error bars indicate the SEM; ^aP < 0.05 vs sham group; ^bP < 0.05 vs vehicle group. SAP: Severe acute pancreatitis; TNF-α: Tumor necrosis factor-α; MPO: Myeloperoxidase; TUNEL: TdT-mediated dUTP Nick-End Labeling; MFG-E8: Milk fat globule epidermal growth factor 8.

Figure 7

Milk fat globule epidermal growth factor 8 mitigates hepatic autophagy and ferroptosis through the integrin αVβ3/5. In mice, severe acute pancreatitis was induced by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine. At 2 h after the last injection of L-arginine, normal saline (vehicle) or 20 μg/kg milk fat globule epidermal growth factor 8 (MFG-E8) was administered through intraperitoneal injection. To determine whether the protective effect of MFG-E8 in liver is mediated through integrin αVβ3/5, 20 mg/kg cilengitide, a specific integrin αVβ3/5 antagonist, was administered intraperitoneally at 1 h before the injection of MFG-E8 (i.e., 1 h after the last injection of L-arginine). The animals were sacrificed at 69 h after MFG-E8 treatment (i.e., 72 h after the first injection of L-arginine). Blood and tissue samples were collected. A-D: Western blot analysis and quantitative of the expression of autophagy related protein 16 like protein 1, P62, LC3, prostaglandin-endoperoxide synthase 2 and glutathione peroxidase 4 in the liver; E: Ultrastructural changes of mitochondria in hepatocytes (electron microscopy); F: Glutathione levels in the liver; G: Malondialdehyde levels in the liver. n = 3-6/group, error bars indicate the SEM; ^aP < 0.05 vs sham group; ^bP < 0.05 vs vehicle group. SAP: Severe acute pancreatitis; ATG16L1: Autophagy related protein 16 like protein 1; PTGS2: Prostaglandin-endoperoxide synthase 2; GPX4: Glutathione peroxidase 4; MDA: Malondialdehyde; GSH: Glutathione; MFG-E8: Milk fat globule epidermal growth factor 8.

Figure 8

Graphical abstract. Exogenous milk fat globule epidermal growth factor 8 restored impaired autophagy, reduced ferroptosis and alleviated liver injury by acting on integrin αVβ3/5. MFG-E8: Milk fat globule epidermal growth factor 8.