Inhibition of angiotensin II type 1 receptor reduces oxidative stress damage to the intestinal barrier in severe acute pancreatitis

Abstract

Intestinal barrier injury is a common complication of severe acute pancreatitis (SAP), which is often accompanied by intestinal mucosal barrier injury and results in serious consequences. However, the exact mechanism remains unclear. We aimed to investigate whether angiotensin II type 1 receptor (AT1)-mediated oxidative stress is involved in SAP intestinal barrier injury and assessed the effects of inhibiting this pathway. The SAP model was established by retrograde bile duct injection of sodium taurocholate (5%). The rats were divided into three groups: the control group (SO), the SAP group (SAP), and the azilsartan intervention group (SAP + AZL). Serum amylase, lipase, and other indexes were measured to evaluate SAP severity in each group. Histopathological changes in the pancreas and intestine were evaluated by HE staining. The oxidative stress of intestinal epithelial cells was detected by superoxide dismutase and glutathione. We also detected the expression and distribution of intestinal barrier-related proteins. The results showed that the serum indexes, the severity of tissue damage, and the level of oxidative stress in the SAP + AZL group were significantly lower than in the SAP group. Our study provided hitherto undocumented evidence of AT1 expression in the intestinal mucosa, confirming that AT1-mediated oxidative stress is involved in SAP intestinal mucosal injury, and inhibiting this pathway could effectively reduce intestinal mucosal oxidative stress injury, providing a new and effective target for the treatment of SAP intestinal barrier injury.

Keywords: AT1; intestinal mucosal barrier; oxidative stress; severe acute pancreatitis.

FIGURE 1

Serum biochemical parameters and histological changes. The activities of serum amylase (A) and lipase (B) and the indicators of TNF‐α(C), endotoxin (D), IL‐6 (E), IL‐10 (F), and DAO (G), It was obviously higher in SAP group than in SO group. Compared with SAP group, the activities of serum amylase (A) and lipase (B), TNF‐α (C), endotoxin (D), IL‐6 (E), IL‐10 (F), and DAO (G) in SAP + AZL group were significantly decreased. Typical sections of the ileum stained with H&E (H) (light magnification ×200). These Values are expressed as the mean ± SD (n = 8). *p < 0.05 and **p < 0.01 versus the SO group; #p < 0.05 and ##p < 0.01 versus the SAP group.

FIGURE 2

The contents of SOD (A), GSH (B), MDA (C), and ROS (D) in ileum tissues were detected. Compared with SO group, SOD activity and GSH content of SAP group were decreased, while MDA and ROS indexes were increased. Compared with SAP group, SOD and GSH were significantly increased, MDA and ROS were significantly decreased in SAP + AZL group. These values are expressed as the mean ± SD (n = 8). *p < 0.05 and **p < 0.01 versus the SO group; #p < 0.05 and ##p < 0.01 versus the SAP group.

FIGURE 3

Using western‐blot method to detect the ileum tissue AT1 and downstream protein NOX2 expression (A). Compared with the SO group, the expression of AT1 and NOX2 in the SAP group was significantly increased, while the expression of AT1 and NOX2 in the SAP + AZL group was decreased (B,C). The results of immunohistochemistry showed that AT1 was significantly expressed in IECs of SAP group. Values are expressed as the mean ± SD (n = 8). **p < 0.01 versus the SO group; #p < 0.05 versus the SAP group.

FIGURE 4

Effect of AZL treatment on histopathology of pancreas and ileum in rats with severe acute pancreatitis. H&E staining images of pancreas and ileum sections were selected, and intestinal mucosal villus destruction, lamina propria exposure, pancreatic cell edema and necrosis, and inflammatory cell infiltration were observed in SAP group (light microscope multiple, ×200). Terminal ileum (A). The pancreas (C). Comparison of pathological scores between ileum and pancreas (B, D). These values are expressed as the mean ± SD (n = 8). *p < 0.05 and **p < 0.01 versus the SO group; #p < 0.05 and ##p < 0.01 versus the SAP group.

FIGURE 5

Western‐blot of TJ protein imprinting in ileum (A), was used to determine the expression levels of ZO‐1, occludin, claudin‐1, and (B–D). Data are expressed as the mean ± SD (n = 8). *p < 0.05 and **p < 0.01 versus the SO group; #p < 0.05 versus the SAP group.

FIGURE 6

The expressions of AT1 and TJ proteins in the ileum were observed by confocal laser scanning microscopy. Intestinal sections of SO group, SAP group and SAP + AZL group were stained with rabbit anti‐AT1 (green), rabbit anti‐ZO‐1 (green), rabbit anti‐occludin (green) and rabbit anti‐claudin‐1 (green), respectively. Nuclei were stained with DAPI (blue). Light lens multiple ×400.