Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway

Abstract

Intestinal ischemia-reperfusion (I/R) injury is a life-threatening vascular emergency and has long been a disturbing problem for surgeons. Oxidative stress is considered a vital factor in I/R injury. Metformin has anti-oxidative properties and protects against I/R injury. The present study aimed to investigate whether Metformin protects against intestinal I/R injury and reveal the protective mechanism of Metformin. I/R injury was induced in mice by temporary superior mesenteric artery occlusion, and Caco-2 cells were subjected to OGD/R to establish an in vitro model. Different doses of Metformin were administered in vivo and in vitro. We found that I/R injury led to intestinal barrier disruption and cell death by examining histopathological results and the intestinal barrier index, including TER, tight junction proteins and serum biomarkers. We confirmed the existence of pyroptosis in intestinal I/R injury. Moreover, we confirmed the role of pyroptosis in intestinal I/R injury by silencing the gasdermin D (GSDMD). Then, we confirmed that Metformin treatment protected barrier function against intestinal I/R injury and reduced oxidative stress and the inflammatory response. Importantly, Metformin reduced pyroptosis-related proteins, including NLRP3, cleaved caspase-1, and the N-terminus of GSDMD. Knocking down the GSDMD could reversed the protective effects of Metformin, which showed pyroptosis was one of the major cell death pathways controlled by Metformin treatment in setting of intestinal I/R injury. We also discovered that Metformin suppressed the expression of TXNIP and the interaction between TXNIP and NLRP3. We performed siRNA knockdown and found that the protective effects were abolished, which further confirmed our findings. In conclusion, we believe that Metformin protects against intestinal I/R injury in a TXNIP-NLRP3-GSDMD-dependent manner.

Keywords: Intestinal I/R injury; Metformin; Pyroptosis; TXNIP.

Fig. 1

Intestinal ischemia-reperfusion injury induced intestinal barrier disruption and epithelial cell injury. Mice were performed with a temporary occlusion of SMA with an atraumatic vascular for different period and then release the clamp to allow a 120-min reperfusion to build an I/R model. (A) The experimental design of the present work. To evaluate the intestinal injury of different ischemia period, the animals were divided into four groups: the different ischemia period (30,60 and 90 min) group and sham group(n = 6). To determine the protective effect of metformin, another set of animals were administrated with metformin (20 or 40 mg/kg) or saline immediately at the end of a 60-min ischemia period. CY-04 (20 mg/kg), a specific inhibitor of NLRP3 inflammasomes, was used as a positive control for metformin. The OGD/R model was launched in Caco-2 cells. si-Txnip/Gsdmd were applied to revealed the roles of pyroptosis and TXNIP/NLRP3/GSDMD axis in the protective effect of metformin. (B)Survival rates were calculated in different groups (n = 10). (C-D)The integrity of the intestinal barrier was evaluated with the serum I-FABP levels and TER. (E–F) the histopathological damage was estimated with the H&E staining and the Chiu's score classification of small intestine injury (Table 1) was applied to grade the histological score. (G–H) The expressions of the tight junction protein, ZO-1 and occludin, were analyzed by Western blot. Different OGD/R models were induced in Caco-2 cells as mentioned in Methods. (I)The releasing levels of LDH were detected. (J) Cell viability was measured with CCK-8 assay. The values were showed as the mean ± SEM in Fig. 1(C–J) (n = 6) and Fig. 1B(n = 10). *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group.

Fig. 2

Intestinal ischemia-reperfusion injury induced intestinal inflammation and activation of the NLRP3-related pyroptosis. The animal model was launched as mentioned. (A–C) The inflammatory factors in intestinal tissues, including IL-6, IL-1β and TNF-α, were evaluated. (D–E) Pyroptosis-related proteins in intestinal tissues, including NLRP3, cleaved Caspase-1, IL-1β and IL-18, were examined by Western blot. (F–G) GSDMD in intestinal tissues was detected by Western blot. Pyroptosis was suppressed by knockdown the GSDMD with si-Gsdmd in vitro before the OGD/R. (H) the releasing levels of LDH were detected (I) Cell viability was measured with CCK-8 assay. The values were showed as the mean ± SEM(n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group, ^#p < 0.05 compared with si-Con group and ^&p < 0.05 compared with si-Con+OGD/R group.

Fig. 3

Metformin protected against intestinal ischemia-reperfusion injury induced intestinal barrier disruption and cell injury. The animal models were launched with a 60-min ischemia period as mentioned. Different doses of metformin (20 or 40 mg/kg) were used to treat the mice as pervious mentioned. (A) The histological damage was evaluated with H&E staining. (B) The histological scores were calculated with the Chiu's score classification of small intestine injury (Table 1). (C) The levels of I-FABP in serum were detected. (D) The TER of gut mucosae was measured. (E–F) The expressions of the tight junction protein, ZO-1 and occludin, were analyzed by Western blot. The cells were kept in a low-oxygen/glucose condition for 4 h then induced an OGD/R models as mentioned. Metformin were subjected at different dose (1 or 2 mM) (G) The releasing levels of LDH were detected. (H) Cell viability was measured with CCK-8 assay. The values were showed as the mean ± SEM(n = 5–6). *p < 0.05 compared with no-treatment group and ^#p < 0.05 compared with low dose group.

Fig. 4

Metformin protected against intestinal ischemia-reperfusion injury induced intestinal inflammation and pyroptosis. The I/R models were established as mentioned and the CY-09 was administered at 20 mg/kg as a positive control. (A–C) The inflammatory factors in intestinal tissues, including IL-6, IL-1β and TNF-α, were evaluated. (D–E) Pyroptosis-related proteins in intestinal tissue, including NLRP3, cleaved Caspase-1, IL-1β and IL-18, were examined by Western blot. (F–G) The expression of GSDMD in vivo was detected by Western blot. (H–I) The expression of NLRP3 was also detected in vitro by immunofluorescence. si-Gsdmd was used to inhibit the pyroptosis and revealed the relationship between metformin and pyroptosis (J) the releasing levels of LDH were detected (K) Cell viability was measured with CCK-8 assay. The values were showed as the mean ± SEM(n = 5–6). *p < 0.05 compared with no-treatment group and ^#p < 0.05 compared with metformin treatment group. ^$p < 0.05 compared with si-Con group and ^&p < 0.05 compared with si-Con+OGD/R group.

Fig. 5

Metformin protected against intestinal ischemia-reperfusion injury induced oxidative stress and TXNIP-NLRP3 interaction in vitro. Caco-2 cells were induced to an OGD/R model as mentioned. Metformin treated models at different dose (1 or 2 mM). (A–C) Oxidative stress was measured by SOD, GSH and MDA. (D–E) ROS was detected with a DHE staining and the ROS-positive cells were calculated by two random observers. (F–G) the expression of TXNIP was examined by Western blot. (H–I) The expression of TXNIP was also detected in vitro by immunofluorescence. (J) the co-localization of TXNIP and NLRP3 was showed by immunofluorescence. The values were showed as the mean ± SEM(n = 5–6). *p < 0.05 compared with no-treatment group and ^#p < 0.05 compared with low dose group.

Fig. 6

Knockdown of TXNIP abolished metformin-inhibited pyroptosis in vitro. Caco-2 cells were transfected with TXNIP-targeting siRNA or control siRNA. Then cells were induced as OGD/R models and received metformin treatment at different dose (1 or 2 mM). (A–B) The expression of TXNIP and pyroptosis-related proteins were detected by Western blot. (C) The levels of LDH releasing were detected. The values were showed as the mean ± SEM(n = 5–6). *p < 0.05 compared with si-Con group and #p < 0.05 compared with si-Con+OGD/R group.

Fig. 7

The mechanism map of the protective properties of metformin against intestinal ischemia-reperfusion injury via a TXNIP-NLRP3-GSDMD pathway.