Octreotide attenuates intestinal ischemia/reperfusion mischief in rats through modulation of Nrf2/PRX2/ASK1/JNK signaling pathway

Abstract

Intestinal ischemia/reperfusion (IIR) is a substantial cause of mortality and morbidity worldwide. Octreotide (OCT) has been proven to be effective against various organ insults. However, the exact mechanism by which it exerts protective effect against IIR is still obscure. Thus, the aim was to unveil the potential role of octreotide in an IIR model and decipher its mechanism of action. The rats were allocated into sham-operated, IIR, and OCT groups. Histopathological changes were performed to assess the intestinal injury. Immunohistochemical analysis was used to estimate the NF-κB, Bcl2, caspase-3, IL-17, LC3B, and beclin-1. The mRNA of TNF-α and IL-17 were examined using real time PCR. The levels of p-Nrf2, PRX2, p-JNK, ASK1, and LC3 were assessed using western blot technique. The levels of total antioxidant capacity and SOD were measured using appropriate kits. Furthermore, the protein expressions of Bax, caspase-3, ASK1, and Nrf2 were assessed using proper ELISA kits. Additionally, the comet assay was determined to investigate the effect on apoptosis. At the molecular level, OCT administration upregulated TAC and SOD levels, demonstrating its antioxidant effect. The anti-apoptotic effect was signified by the upregulation of Bcl2 and downregulation of Bax and caspase-3, which was confirmed by comet assay. Furthermore, OCT decreased the levels of TNF-α, NF-κB, and IL-17, confirming its anti-inflammatory effect. OCT pre-treatment triggered autophagy, as evidenced by the upregulation of beclin-1 and LC3B. These effects were accomplished by increasing p-Nrf2 and PRX2 and decreasing ASK1 and p-JNK. Consequently, this impeded the necrosis of intestinal cells and improved the intestinal histoarchitecture abnormalities. Ultimately, OCT successfully ameliorated IIR injury via modulating the Nrf2/PRX2/ASK1/JNK signaling trajectory, leading to autophagic, antioxidant, anti-apoptotic, and anti-inflammatory effects.

Keywords: Antioxidant; Apoptosis; Autophagy; IIR; Inflammation; Octreotide.

Fig. 1

Effect of OCT on histopathological changes in rats subjected to intestinal ischemia/reperfusion injury. The representative photomicrographs of sham rats (A) showed normal intestinal villi with numerous goblet cells (white arrowhead). The IIR intestinal sections (B) showed severe hemorrhagic necrotic changes along the entire intestinal villi (white arrowhead) mixed with severe hemorrhage within the deep mucosa (black arrowhead). The OCT intestinal sections (C) showed mild desquamative changes that were mostly within the apical portion of the intestinal villi (white arrowhead). The panel (D) demonstrated the Chiu’s scoring. OCT (50 μg/kg, i.p.) was injected 30 min prior to intestinal IR (60 min/2 h). Results are expressed as mean ± SD (n = 6). Statistical analysis was performed using Kruskal–Wallis, followed by Dunn’s post hoc test (P < 0.05), as compared with the SO (*) and IIR (#) groups. IIR, intestinal ischemia/reperfusion; OCT, octreotide; SO, sham-operated. Scale Bar = 50 µm for all photomicrographs

Fig. 2

Effect of OCT on A p-Nrf2, B Nrf2, C PRX2, D p-ASK1, E ASK1, and F p-JNK in rats subjected to intestinal ischemia/reperfusion injury. OCT (50 μg/kg, i.p.) was injected 30 min prior to intestinal IR (60 min/2 h). Results are expressed as mean ± SD (n = 3). A statistically significant difference at P < 0.05 was highlighted with * and # as compared with the SO and IIR groups, respectively. Panel G shows the photomicrograph of the western blot assay for the expressions p-Nrf2, p-ASK1, p-JNK, and PRX2, respectively. ASK1, apoptosis signal regulating kinase; JNK, c-Jun N-terminal kinase; IIR, intestinal ischemia/reperfusion; Nrf2, nuclear factor-erythroid 2-related factor 2; OCT, octreotide; PRX2, peroxiredoxin 2; SO, sham-operated

Fig. 3

Effect of OCT on A NF-кB/P65, B TNF-α, and C, D IL- 17 in rats subjected to intestinal ischemia/reperfusion injury. Panels A (I, II, and III) and C (I, II, and III) represent the immunohistochemical photomicrographs for NF-кB/P65 and IL- 17, respectively, where I, II, and III represent SO, IIR, and OCT groups, respectively. Panels B and D represent the gene expressions of TNF-α and IL- 17, respectively. OCT (50 μg/kg, i.p.) was injected 30 min prior to the intestinal IR (60 min/2 h) injury. Results are expressed as mean ± SD (n = 6 for PCR analysis and n = 4 for immunohistochemical analysis, and arrowheads indicate positive immunostaining). A statistically significant difference at P < 0.05 was highlighted with * and # as compared with the SO and IIR groups, respectively. IIR, intestinal ischemia/reperfusion; IL- 17, interleukin- 17; NF-кB, nuclear factor-kappa B 2; OCT, octreotide; SO, sham-operated; TNF-α, tumor necrosis factor alpha. Scale Bar = 50 µm for all photomicrographs

Fig. 4

Effect of OCT on A, B caspase− 3, C Bax, and D Bcl2 in rats subjected to intestinal ischemia/reperfusion injury. Panels A (I, II, and III) and C (I, II, and III) represent immunohistochemical photomicrographs for caspase- 3 and Bcl2, respectively. Panels B, C, and D-V represent the protein expression of caspase-3, Bax, and Bcl2 using ELISA, respectively. OCT (50 μg/kg, i.p.) was injected 30 min prior to intestinal IR (60 min/2 h). Results are expressed as mean ± SD (n = 6 for ELISA and n = 4 for immunohistochemical analysis, and white arrowheads indicate positive immunostaining). A statistically significant difference at P < 0.05 was highlighted with * and # as compared with the SO and IIR groups, respectively. Bcl- 2, B cell lymphoma 2; Bax, Bcl- 2-associated X protein; IIR, intestinal ischemia/reperfusion; OCT, octreotide; SO, sham-operated. Scale Bar = 50 µm for all photomicrographs

Fig. 5

Effect of OCT on A TAC and B SOD in rats subjected to intestinal ischemia/reperfusion injury. OCT (50 μg/kg, i.p.) was injected 30 min prior to intestinal IR (60 min/2 h). Results are expressed as mean ± SD (n = 6). A statistically significant difference at P < 0.05 was highlighted with * and # as compared with the SO and IIR groups, respectively. IIR, intestinal ischemia/reperfusion; OCT, octreotide; SO, sham-operated; SOD, superoxide dismutase; TAC, total antioxidant capacity

Fig. 6

Effect of OCT on A beclin- 1 and B, C LC3 in rats subjected to intestinal ischemia/reperfusion injury. Panels A (I, II, and III) and B (I, II, and III) represent the immunohistochemical photomicrographs for beclin- 1 and LC3B, respectively (arrowheads indicate positive immunostaining). Panel C represents the protein expression of LC3 using western blot technique. OCT (50 μg/kg, i.p.) was injected 30 min prior to intestinal IR (60 min/2 h). Results are expressed as mean ± SD (n = 4). A statistically significant difference at P < 0.05 was highlighted with * and # as compared with the SO and IIR groups, respectively. IIR, intestinal ischemia/reperfusion; LC3B, light chain; OCT, octreotide; SO, sham-operated. Scale Bar = 50 µm for all photomicrographs

Fig. 7

Effect of OCT on intestinal DNA damage in intestinal ischemic reperfused rats. Comet assay showing the degree of DNA damage and tail length in intestinal tissues (A-I). Normal control group showing no DNA damage (A-II). Intestinal ischemia reperfusion injury group revealing high percent of DNA damage; DNA head at white arrow and DNA tail at white straight line (A-III). Octreotide-treated group revealing intact DNA with low DNA damage. Scale Bar = 50 nm. B showing histogram statistical analysis of tail length. Results are expressed as mean ± SD (n = 3). A statistically significant difference at P < 0.05 was highlighted with * and # as compared with the SO and IIR groups, respectively. IIR, intestinal ischemia/reperfusion; OCT, octreotide; SO, sham-operated

Fig. 8

The proposed mechanism of OCT against intestinal ischemia/reperfusion injury. ASK1, apoptosis signal regulating kinase; Bcl- 2: B cell lymphoma 2; Bax: Bcl- 2-associated X protein; IIR, intestinal ischemia/reperfusion; IL- 17, interleunin- 17; JNK, c-Jun N-terminal kinase; Keap 1, Kelch-like ECH-associated protein 1; LC3, light chain; Nrf2, nuclear factor-erythroid 2-related factor 2; NF-кB, nuclear factor-kappa B; ROS, reactive oxidative stress; TNF-α, tumor necrosis factor alpha; TNFR, tumor necrosis factor receptor