High-Fat Diet Aggravates Acute Pancreatitis via TLR4-Mediated Necroptosis and Inflammation in Rats

Figures

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  Figure 1. HFD aggravated the histological injury and oxidative stress in the pancreas of acute pancreatitis rats. (a) AP was induced in SCD or HFD rats with a standard retrograde infusion of STC into the biliopancreatic duct; the animals were killed for analysis 3 h, 6 h, and 12 h after the STC infusion. (b) HFD rats showed a larger volume of adipocytes in the peripancreatic fat than SCD rats after an 8-week feeding. (c) In the AP group, serious edema (blue arrow), massive acinar necrosis, inflammatory cell infiltration (black arrowhead), and intrapancreatic hemorrhage (yellow arrow) in the pancreas were observed. (d) Pathological score of pancreatic tissues in rats. (e) Percentage of necrotic cells was determined in the pancreas. (f) Serum levels of free fatty acid in rats. (g) Pancreatic levels of MDA. (h) Pancreatic levels of LPO. (i) Pancreatic activities of SOD. (j) Pancreatic levels of GSH. ∗P < 0.05 versus prior subgroup, #P < 0.05 versus SCD group at the same time point. Scale bar = 100 μm.

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  Figure 2. HFD increased the infiltration of inflammatory cell in the pancreas of AP rats. (a) Immunofluorescent staining of MPO-positive neutrophils (green) in the pancreas. (b) Immunofluorescent staining of CD68-positive macrophages (green) in the pancreas. (c) Number of MPO-positive neutrophils. (d) Number of CD68-positive macrophages. ∗P < 0.05 versus prior subgroup, #P < 0.05 versus SCD group at the same time point. Scale bar = 100 μm.

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  Figure 3. HFD exacerbated inflammatory response and necroptosis in the pancreas of AP rats. (a) TLR4 and RIP3 levels of pancreas were analyzed by Western blot. β-Actin served as the loading control. (b) The relative protein expression of TLR4 was expressed as fold changes compared with the control in the blot after normalized to β-actin. (c) The relative protein expression of RIP3 was expressed as fold changes compared with the control in the blot after normalized to β-actin. (d) Immunohistochemistry of NF-κB p65 in the pancreas. (e) The expression of NF-κB p65-positive nuclei in the pancreas. (f) Immunohistochemistry of TNF-α in the pancreas. (g) The mean integrated optical density (IOD) of TNF-α in the pancreas. ∗P < 0.05 versus prior subgroup, #P < 0.05 versus SCD group at the same time point. Scale bar = 50 μm.

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  Figure 4. Inhibition of TLR4 by TAK-242 attenuated pancreatic injury and oxidative stress of HFD rats with AP. (a) TAK-242 or vehicle was injected intraperitoneally 30 minutes before AP modeling in HFD rats, and the animals were killed for analysis 12 h after the STC infusion. (b) In the HAP group, serious edema (blue arrow), massive acinar necrosis, inflammatory cell infiltration (black arrowhead), and intrapancreatic hemorrhage (yellow arrow) in the pancreas were observed. TAK-242 improved the histopathological damage. (c) Pathological score of pancreatic tissues in rats. (d) Percentage of necrotic cells was determined in the pancreas. (e) Pancreatic levels of MDA. (f) Pancreatic levels of LPO. (g) Pancreatic activities of SOD. (h) Pancreatic levels of GSH. ∗P < 0.05 versus HV group, #P < 0.05 versus HAP group. Scale bar = 100 μm.

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  Figure 5. Inhibition of TLR4 attenuated inflammatory response and necroptosis in the pancreas of HFD rats with AP. (a) TLR4 and RIP3 levels of pancreas were analyzed by Western blot. β-Actin served as the loading control. (b) The relative protein expression of TLR4 was expressed as fold changes compared with the control in the blot after normalized to β-actin. (c) The relative protein expression of RIP3 was expressed as fold changes compared with the control in the blot after normalized to β-actin. (d) Immunostaining of NF-κB p65, TNF-α, MPO, and CD68 in the pancreas of HFD rats. (e) The expression of NF-κB p65-positive nuclei in the pancreas. (f) The mean IOD of TNF-α in the pancreas. (g) Number of MPO-positive neutrophils. (h) Number of CD68-positive macrophages. ∗P < 0.05 versus HV group, #P < 0.05 versus HAP group. Scale bar = 100 μm.