Blood flow to the target organ is reduced or completely blocked, resulting in tissue hypoxia, which is defined as ischemia (Li, Hu et al. 2016). Significant lack of blood supply to the intestine can be induced by thrombosis or obstruction of the mesenteric arteries caused by emboli related to cardiac illness (Puleo, Arvanitakis et al. 2011). Reduced blood flow interrupts the oxygen supply required for cell activity, resulting in cell damage and death (Li, Hu et al. 2016, Lillo-Moya, Rojas-Sole et al. 2021). Restoring blood flow is crucial to rescue and preserve normal cellular function. Nevertheless, ischemia promotes the creation of toxic compounds as well as the generation of inflammatory and epithelial cell apoptosis and necrosis (Gonzalez, Moeser et al. 2015, Liu, Chen et al. 2019). Pyroptosis is a type of pro-inflammatory programmed cell death that differs from apoptosis and necrosis (Shi, Gao et al. 2017, Frank and Vince 2019, Bertheloot, Latz et al. 2021). It is defined as the production of membrane pores mediated by cystease cutting Gasdermin D, resulting in membrane rupture and cell content leakage, as well as the release of a significant number of inflammatory mediators, such as IL-18, IL-1β, and others (He, Wan et al. 2015, Broz, Pelegrin et al. 2020). Multiple studies have discovered the function of pyroptosis in the incidence and development of inflammatory illnesses, immunological tumors, and other disorders (Chen, Shi et al. 2019, Wu, Lin et al. 2019, Shen, Wang et al. 2021). Pyroptosis shows its critical role in the genesis, progression, and prognosis of several digestive organ illnesses, such as hepatitis cirrhosis, inflammatory bowel disease, and intestinal inflammation (Huang, Feng et al. 2019, Zuo, Zhou et al. 2020, Wang, Zhou et al. 2021). And in previous studies, the presence of pyroptosis in intestinal I/R injury has been demonstrated (Rathinam, Vanaja et al. 2012, He, Wan et al. 2015, Jia, Cui et al. 2020, Wang, Fan et al. 2020). In this study, we verified the presence of pyroptosis with LDH kit detection and Hoechst 33342/PI double staining. Furthermore, we detected Gasdermin D, IL-18, and IL-1β, which are indicators of pyroptosis. It was found that at both mRNA level and protein level, pyroptosis mainly increased after 45 min ischemia and 90 min reperfusion in C57BL/6 mice, while in IEC-6 cells, pyroptosis mainly increased after 4 h hypoxia and 120 min reoxygenation. Both in vivo and in vitro, the number of pyroptosis decreased with the extension of reperfusion or reoxygenation time after fixed ischemia or hypoxia time, and after reperfusion for 360 min or reoxygenation for 1440 min, the number of pyroptosis almost returned to the level of the sham operation group or control group.
miRNAs are endogenous non-coding RNA with a length of 19–22 nucleotides that are induced in the nucleus by RNA polymerase II (Lee, Ahn et al. 2003, Mendell and Olson 2012, Xue, Yang et al. 2021). These miRNAs are subsequently transported to the cytoplasm by exportin-5 and further processed into double-stranded mature miRNAs by the RNase III endonuclide Dicer (Lee, Ahn et al. 2003). It has been proposed that certain miRNAs have a pathogenic function in intestinal I/R injury. For example, by modulating miR-351-5p/MAPK13-mediated inflammation and apoptosis, dioscin reduces intestinal I/R injury in rats (Zheng, Han et al. 2019). Autophagy is inhibited by activating miRNA-182/Deptor/mTOR axis to minimize intestinal I/R injury in mice (Li, Luo et al. 2020). In addition to intestinal I/R injury, mir-122a has also been found to play a role in the regulation of other diseases. An investigation on human HCC shows cyclin G1 is a gene target of miR-122a, which is regularly down-regulated in this cell line (Gramantieri, Ferracin et al. 2007). In gastrointestinal malignancies, miR-122a performs as a new tumor suppressor downstream of APC (Wang, Lam et al. 2009). Nevertheless, the effect of miR-122a on I/R injury has yet to be documented. In this study, we investigated the potential pathogenic mechanism of miR-122a in intestinal I/R injury. Results indicated that the mRNA expression of miR-122a was considerably higher in the H/R or I/R groups compared to the normal control or sham operation groups, but reduced when reoxygenation or reperfusion time was prolonged. Furthermore, using several databases, we discovered that miR-122a had binding sites with EGFR, and the results were confirmed using the Dual-luciferase reporter assay. The protein and mRNA expression levels of EGFR were considerably higher when the EGFR-overexpression plasmid was transfected into IEC-6 alone compared to the control group in the co-transfection experiment. However, co-transfection of miR-122a mimic and EGFR-overexpression plasmid with IEC-6 led to lower expression levels of EGFR protein and mRNA than that only EGFR-overexpression plasmid was used. These results suggest that miR-122a directly targets EGFR.
Intestinal I/R injury is one of the most dangerous conditions in clinic (Kip, Grootjans et al. 2021). It is caused by hemorrhagic shock, trauma, strangulation ileus, acute mesenteric ischemia, and other pathological conditions (Matsuda, Yang et al. 2014, Shoup 2018, Qasim, Li et al. 2020). Until now, there is no established treatment for this injury in clinic. Most of the drugs discovered have been designed to reduce the complications associated with it (Li, Xu et al. 2017, Hu, Mao et al. 2018, Chen, Mohr et al. 2020, Jia, Cui et al. 2020). According to research, the most serious outcome of intestinal I/R injury is mucosal barrier failure, which can lead to SIRS, MODS, and ultimately death (Rutgeerts, Sandborn et al. 2005, Duranti, Vivo et al. 2018, Zhang, Wu et al. 2020). This work demonstrated that pyroptosis is a vital factor in the development of SIRS and MODS following intestinal I/R injury. We performed a miR-122a inhibitor transfection experiment to further investigate the regulating mechanism of miR-122a in intestinal I/R injury. Our findings revealed that miR-122a worsened pyroptosis by suppressing EGFR protein expression and then enhancing NLRP3 protein expression, consequently aggravating intestinal I/R injury. The above results indicate that the miR-122a-EGFR-NLRP3 signaling pathway may be a priority attraction for the advancement of novel medicines to treat intestinal I/R injury.
EGFR signaling pathway appears on epithelial, fibroblast, glial, keratinocyte, and other cell surfaces. Its function is involved in cell growth, proliferation, differentiation, and other physiological activities (Zhu, Shimizu et al. 2011, Sasada, Azuma et al. 2016, Purba, Saita et al. 2017). Patients with epithelial-derived cancers show high levels of EGFR expression, which suggests that EGFR might be utilized as a target for anticancer drug delivery systems (Ohno, Takanashi et al. 2013, Arkhipov, Shan et al. 2014). Another study on I/R-induced acute renal injury in mice shows that cathelicidin-related antimicrobial peptides inhibit NLRP3 inflammasome activation, which requires the participation of EGFR (Pan, Liang et al. 2020). However, no reports indicate how EGFR participates in intestinal I/R injury. As a result, we investigated the changes on EGFR expression under H/R and I/R conditions compared to normal control or sham operation groups in this research. By using qPCR, western blotting, and immunofluorescence assays, we discovered that EGFR expression was considerably lower in H/R or I/R groups contrasted to normal control or sham operation groups. However, contrasted to the H/R group, the corresponding pyroptosis indicators were ameliorated in H/R group under transfection with EGFR-overexpression plasmid condition, resulting in improvement of H/R-induced cell injury. Based on these findings, it could be suggested that EGFR plays a significant regulatory role in I/R-induced intestinal injury. However, whether EGFR can be selected as a biomarker in I/R-induced intestinal injury remains to be clarified.
This study found that miR-122a targeted EGFR-NLRP3 signaling pathway to affect intestinal I/R injury by regulating pyroptosis. We hope that this mechanism can be a beneficial therapeutic target for the identification and treatment of intestinal I/R injury in the future.