RESUMEN
Autophagy is being increasingly recognized as an important regulator of intestinal ischemia-reperfusion(I/R)injury, but its exact role is still debated. Emerging evidence suggests that miR-146a-5p is involved in the initiation and development of I/R injury, but its role in intestinal I/R injury remains unclear. The present study generated an intestinal I/R mouse model and an oxygen glucose deprivation/reoxygenation (OGD/R) Caco-2 cell model and found that autophagy was increased and contributed to the intestinal injury and cell death induced by I/R and OGD/R. In addition, in both I/R and OGD/R models, the miR-146a-5p expression level was decreased and accompanied by an increase in TXNIP expression. By transfecting cells with an miR-146a-5p inhibitor or mimic, we observed that miR-146a-5p inhibits autophagy during OGD/R by targeting TXNIP; this was confirmed by the dual luciferase reporter gene assay. Additionally, through overexpression and knockdown cell lines, we established that TXNIP regulates autophagy during intestinal I/R via the PRKAA/mTOR pathway. The interaction between TXNIP and p-PRKAA was verified by immunofluorescence co-localization and immunoprecipitation assays. Moreover, we confirmed that TXNIP is indispensable for miR-146a-5p-mediated cell protection. Finally, we observed that miR-146a-5p overexpression inhibits autophagy and attenuates intestinal I/R injury via the PRKAA/mTOR pathway by targeting TXNIP in vivo. In conclusion, this study highlights the role of miR-146a-5p in regulating autophagy by targeting TXNIP, suggesting that miR-146a-5p may be a novel drug target for intestinal I/R therapy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Portadoras/biosíntesis , Intestinos/metabolismo , MicroARNs/biosíntesis , Daño por Reperfusión/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Tiorredoxinas/biosíntesis , Animales , Autofagia/fisiología , Células CACO-2 , Humanos , Intestinos/irrigación sanguínea , Intestinos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Transducción de Señal/fisiologíaRESUMEN
Objective:To evaluate the effect of dexmedetomidine on the thioredoxin-interacting protein (TXNIP)/apoptosis signal-regulated kinase 1 (ASK1) signaling pathway in a mouse model of intestinal ischemia-reperfusion (I/R).Methods:Thirty-two SPF healthy adult male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 4 groups ( n=8 each) using a random number table method: sham operation group (Sham group), intestinal I/R group (I/R group), TXNIP inhibitor resveratrol group (Res group) and dexmedetomidine group (Dex group). The mouse model of intestinal I/R injury was developed by clamping the superior mesenteric artery for 45 min followed by 120-min reperfusion in anesthetized animals. Resveratrol 30 mg/kg was intraperitoneally injected before developing the model in Res group, and dexmedetomidine 25 μg/kg was intraperitoneally injected at 30 min before ischemia in Dex group. Blood samples were collected by cardiac puncture at the end of 120-min reperfusion, then the mice were sacrificed, and the small intestine tissues were removed for microscopic examination and for determination of the serum diamine oxidase (DAO) concentration (by enzyme-linked immunosorbent assay) and expression of TXNIP, ASK1 and cleaved-caspase-3 in small intestinal tissues (by Western blot). The apoptosis rate of intestinal epithelial cells was calculated. The intestinal damage was assessed and scored according to Chiu. Results:Compared with group Sham, the Chiu′s score, serum DAO concentrations and apoptosis rate of intestinal epithelial cells were significantly increased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was up-regulated in group I/R ( P<0.05). Compared with group I/R, the Chiu′s score, serum DAO concentration and apoptosis rate of intestinal epithelial cells were significantly decreased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was down-regulated in group Res ( P<0.05). Compared with I/R group, the Chiu′s score, serum DAO concentration and apoptosis rate of intestinal epithelial cells were significantly decreased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was down-regulated in Dex group ( P<0.05). Conclusions:The mechanism by which dexmedetomidine alleviates intestinal I/R injury may be related to inhibition of the TXNIP/ASK1 signaling pathway and reduction of cell apoptosis in mice.
RESUMEN
AIM: To explore the role and mechanism of silent mating type information regulator 2 homolog 3 (SIRT3) in attenuation of intestinal ischemia-reperfusion (I/R) injury by dexmedetomidine in mice. METHODS: Twenty-four healthy male C57BL mice were divided into 4 groups randomly (n=6): sham operation group (Sham group), intestinal ischemia-reperfusion group (I/R group), dexmedetomidine group (Dex group), SIRT3 inhibitor 3-TYP group (3-TYP group). Superior mesenteric artery was clamped for 45 min followed by reperfusion for 2 h to establish intestinal I/R model in I/R group, Dex group, and 3-TYP group. Sham group received sole sham operation. 1 h prior to onset of ischemia, 3-TYP was injected into mice in 3-TYP group intraperitoneally (5 mg/kg, diluted to 0.3 mL), and 0.3 mL normal saline into mice in Dex group intraperitoneally. 30 min prior to onset of ischemia, dexmedetomidine was injected into mice in 3-TYP group and Dex group intraperitoneally (25 μg/kg, diluted to 0.3 mL). 1 h and 30 min prior to onset of ischemia, 0.3 mL normal saline was injected into mice in Sham group and I/R group intraperitoneally, respectively. 2 h of after reperfusion, the mice were sacrificed under anesthesia. Intestinal tissues were took and observed for pathological changes under light microscope after HE staining, and the injury was assessed via the Chiu's score method, and activities of SIRT3 and superoxide dismutase 2 (SOD2) were detected via spectrophotometry, and malondialdehyde (MDA) via spectrophotometry. RESULTS: The pathological injury was exacerbated, and the Chiu's score, the MDA level elevated remarkably, while the activity level of SIRT3 and SOD2 declined remarkably in I/R group, Dex group and 3-TYP group compared to Sham group (P<0.05). The pathological injury was alleviated, and the Chiu's score declined remarkably in Dex group and 3-TYP group compared to I/R group (P<0.05); and the MDA level declined remarkably, while activity level of SIRT3 and SOD2 elevated remarkably in Dex group compared to I/R group (P<0.05); and there was no significant difference both in the activity level of SIRT3 and SOD2 and in the MDA level between 3-TYP group and I/R group. The pathological injury was exacerbated, and the Chiu's score, the MDA level elevated remarkably, while the activity level of SIRT3 and SOD2 declined remarkably in 3-TYP group compared to Dex group (P<0.05). CONCLUSION: SIRT3 and its downstream SOD2 are involved in mediating the effect of attenuation of intestinal ischemia-reperfusion injury through inhibiting oxidative stress response by dexmedetomidine.
RESUMEN
microRNA (miRNA) is a class of 19-25 nucleotide highly conserved single-stranded non-coding RNA that is widely found in plants and animals. Their biological effect is to negatively regulate target gene expression at the post-transcriptional level through complementary pairing with mRNA. Intestinal I/R injury is more common in clinical practice, and ischemia-reperfusion will cause intestinal mucosal barrier damage, and it is related to the occurrence, development, and outcome of many clinical diseases. Many studies have shown that the miRNA subtype genes miR-34a-5p, miR-351-5p, miR-682, miR-21, etc. affect the intestinal I/R injury process to some extent by regulating a series of signal transduction. Therefore, revealing the role of miRNA in intestinal I/R injury and providing a new direction for the diagnosis and treatment of I/R.