The intestine is considered as one of the immune barrier systems of the body, so as the source of bacteria and endotoxins in the body[11].Therefore, the intestine is extremly sensitive to stress and it is pretty difficult for it to recover back to physiological homeostasis once it hurt.Intestinal I/R injury is a pathophysiological process that occurs locally but can affect the whole body under the action of inflammatory cells and mediators, leading to serious consequences such as multiple organ failure.During intestinal I/R, a large amount of ROS releases,which activates inflammatory factors and increases the permeability of the intestinal wall, thus resulting in the displacement of the intestinal flora.Intestinal I/R can not only lead to intestinal structural and functional changes, but also cause damage to kidney, liver and even lead to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), and even death [12–14] in severe cases.The incidence and fatality rate of intestinal I/R injury are increasing year by year, which is a problem that the medical community has been concerned about and needs to be solved urgently. However, there is no effective treatment.
Ghrelin is an active peptide composed of 28 amino acids. It is a member of the G protein-coupled receptor family, which can activate the growth hormone secretin-stimulating hormone receptor (GHSR) and promote the secretion of growth hormone (GH)[15].It has a diverse and a wide range of biological activities.It plays an important role in the regulation of food intake, energy balance, growth and development, regulating gastrointestinal function and other aspects[16–17].IL-6 is often used to assess the extent of intestinal inflammation.Ghrelin exert a potent anti-inflammatory effect by inhibiting the expression of proinflammatory cytokines such as IL-6[18].In I/R-induced intestinal mucosal injury, central or peripheral application of Ghrelin significantly increased the resistant of intestinal tissue[19–20].This suggests that at intestinal I/R, Ghrelin improves the intestinal barrier dysfunction and reduces organ damage by inhibiting proinflammatory cytokine release and infiltration.This study showed that the intestinal mucosal tissue of rats in the I/R group was severely damaged, with increased release of inflammatory factors and largely damaged mitochondria.However, exogenous administration of Ghrelin treatment before I/R can reverse the appeal performance, confirming that Ghrelin has the function of anti-inflammatory, antioxidant and protective mitochondrial, thus exerting its protective effect on intestinal I/R.
Studies suggest that the anti-inflammatory and other biological functions of Ghrelin are related to AMPK.AMPK can be activated by the reduction of adenine nucleoside triphosphate (ATP) levels and the accumulation of adenosine monophosphate (AMP).In this experiment, the expression of AMPK of the rats given Ghrelin before I/R was higher compared with the other three groups, suggesting that Ghelin could activate AMPK expression, thus protecting the intestinal epithelial cells from I/R damage.But how are the relevant pathways initiated after Ghrelin activation of AMPK? We further visually examined the mitochondria-related indicators in each group.In our study, the number of autophagosomes increased in the intestinal epithelial cells after exogenous administration of Ghrelin, so do the expression levels of autophagy proteins such as AMPK, FUNDC1, and LC3.Therefore, we speculated that Ghrelin activated AMPK and further promote the occurrence of mitophagy.
As is well known, mitochondria is the main place of redox metabolism in animal body.If the mitochondria disorders, which more likely to produces more ROS, will seriously affect the cell function and induce microcirculation and mitochondrial distress syndrome(MMDS), further aggravate the inflammatory response, and eventually lead to tissue and organ damage.Mitophagy can sustaine self-renewal by selectively removing impaired or dysfunctional mitochondria, thus maintaining intracellular environmental stability [21]Mitophagy can inhibit the production of excessive ROS as well as mtDNA and then reduce or even inhibit the release of inflammatory factors and chemical chemokines, and avoid MMDS, SIRS and MODS.Therefore, the regulation of mitophagy and the timely clearance of excessive ROS are important for the treatment of I/R damage.
In our experiment, after administing FUNDC1 antagonist and examining the expression of autophagy proteins such as AMPK and FUNDC1 in the intestinal epithelial tissues of rats, we found that the level of proteins was basically similar to that of the I/R group, suggesting that Ghrelin promotion of mitophagy may be mediated through the AMPK/FUNDC1 pathway.The FUNDC1 receptor protein is the protein localized to the outer mitochondrial membrane, in which the LIR motif on FUNDC1 binds to LC3 to initiate autophagy.FUNDC1 mainly involved in mitophagy induced by ischemia, hypoxia and reduced mitochondrial membrane potential [22] .In the case of ischemia, hypoxia, and decreased mitochondrial membrane potential, AMPK is activated and thus promotes FUNDC1-LC3II binding to mediate mitochondrial autophagy[4–5] and remove damaged mitochondria.Numerous studies have demonstrated that AMPK/ FUNDC1-mediated mitophagy plays an important protective role in nervous system [6], cardiovascular system [7–8], and urinary system I/R.Our experiment can further clarify the existence of AMPK/FUNDC1, a mitophagy pathway, in intestinal I/R, to protect the intestinal tract and distant organs.
After intestinal I/R, acute lung injury occurs as a major complication, leading to high mortality rate[23].The damaged intestine releases mtDNA, accelerating the release of inflammatory cytokines and chemical chemokines,whicjh would enter the circulation and trigger a systemic inflammatory response, mainly affecting in distant organs such as the lung, liver and kidney[24].In our experiment, it was concluded that Ghrelin could reduce oxidative stress after intestinal I/R by promoting mitophagy, thus reducing the release of inflammatory factors and thus avoiding lung injury by the pathological observation of HE staining and the detection of IL-6 and mtDNA levels.
In conclusion, our study demonstrated that Ghrelin has a protective effect against intestinal I/R injury and its distal septal organ damage, which is achieved through mitophagy.Further studies showed that Ghrelin could exert its protective effect through the AMPK/FUNDC1 pathway.Our study revealed the role of mitophagy in intestinal I/R and clarified the mechanism of Ghrelin in intestinal protection, laiding the foundation for its application of Ghrelin in clinical practice.However, the specific molecular mechanism of the upregulation of FUNDC1 protein expression by AMPK still needs to be further clarified.