Melatonin Attenuates Sepsis-Induced Intestinal Injury Via SIRT3-mediated Autophagy

Study backgroundSepsis is an organ dysfunction caused by the body’s imbalance response to infection.Intestinal is not only one of the target organs of sepsis damage,but also the initial cause to aggregate the disease into MODS.Intestinal injury caused by sepsis is characterized by intestinal ischemic necrosis,which resulted in weakened barrier function,thus,intestinal toxins and bacteria translocate into capillaries with blood flow,disseminating to the whole body,leading to inflammation and damage to distant organs,which is also called MODS.Mitochondrial dysfunction aggravated intestinal injury.Under physiological conditions,there would be a small amount of reactive oxygen species(ROS)and it is worth mentioning that moderate ROS level helps to maintain cell autophagy level to periodically eliminate damaged cells/organelles such as mitochondria or misfolded protein,which is beneficial to keep the normal function of mitochondria and cells.Mitochondrial autophagy/mitophagy is a phenomenon of cells selectively removing senescence or damaged mitochondria.Autophagy related protein LC3 Ⅱ and autophagic substrate p62 are commonly used as indicators of autophagy levels.As we know,cellular oxygen utilization is impaired during sepsis,resulting in impaired mitochondrial function,characterized by decreased mitochondrial membrane potential,disintegration of mitochondria,release of large amounts of mitochondrial content into the cytoplasm,followed by inhibition of autophagy/mitophagy,and increase in apoptosis and necrosis,ultimately causes serious tissue damage.Relative research has revealed that the degree of mitochondrial injury is correlated with mortality of sepsis.However,the relationship between mitochondrial dysfunction and autophagy/mitophagy in the intestinal injury of sepsis is still not clear,the specific pathogenesis has not been fully elucidated,besides,due to the lack of typical biomarkers of intestinal injury and nonspecific therapeutic protocols,treatment in sepsis remains to be a challenge.Therefore,to explore the mechanisms of sepsis and to search for potential therapeutic drugs is of important clinical significance.Some studies have shown that mitochondrial protective agents such as resveratrol and polydatin protect against intestinal damage after sepsis.In this study,we intend to use mitochondrial protective physiological hormone,melatonin,to explore its antioxidant effects on cell autophagy,thus providing experimental basis for the development of pathogenesis and drug targets of sepsis intestinal injury.The sirtuins family,which was encoded by the silent information regulator(SIR)gene,is evolutionarily highly conserved,and plays a role in cell energy as well as redox reactions.SIRT3 is mainly located in the mitochondria,whose function is to deacetylate the mitochondrial related target protein such as SOD2 and thus affecting their function;our previous studies have shown that in hemorrhagic shock mice related intestinal injury,the expression and activity of SIRT3 play an important role.Polydatin,as a mitochondrial protective agent,stimulates the deacetylase activity of SIRT3,decreases the ratio of AcSOD2/SOD2 in the mitochondria,increases the activity of SOD2,and then inhibits the accumulation of ROS in the mitochondria and stabilizes mitochondrial function.Therefore,SIRT3 is involved in the regulation of mitochondrial antioxidant enzymes.Melatonin(Mel)is a hormone secreted by the human pineal gland,chromosomal cells of the digestive tract mucosa can also secreted Mel,and the melatonin concentration in gastrointestinal tissues is higher.Melatonin not only has anti-inflammatory and immunomodulatory effects,but also is a powerful free radical scavenger agents in the body to play an anti-oxidant effect,some studies also confirmed that melatonin mainly locates in the mitochondria,on the one hand to directly scavenge oxygen free radicals,on the other hand to play an antioxidant effect through the antioxidant enzymes such as SOD2.In addition,melatonin also plays a role in regulating cell autophagy when cells are damaged.However,there are still little studies on the mechanism of its role in gut protection yet.Whether melatonin,as a potential mitochondrial protective agent,plays an antioxidant role through the activation of SIRT3?If so,then what is the specific mechanism?ObjectiveMice model of cecal ligation puncture procedure(CLP)and intestinal epithelial cell model of LPS stimulation were used to simulate the development of sepsis,to explore the antioxidant and autophagy improvement role of Mel and its specific mechanisms in sepsis intestinal injury,providing a new theoretical basis for clinical application of melatonin in intestinal injury during sepsis.Method1.Models and groups1.1 Animal model of sepsis and groupsThe C57/BL mice were used as the study object and CLP operation was used to establish severe sepsis model.The time points of 0h,2h,4h,8h and 12h after CLP were observed respectively.8h was selected for the time point of the experiment,mice were randomly divided into 4 groups:①sham group,②CLP+Vehicle group,③CLP+Mel group,④CLP+Mel+3-TYP group.1.2 Cell model of sepsis and groupsThe rat intestinal crypt epithelial cell line IEC-6 was used as the study object,and 1 μg/ml of LPSwas used to stimulate IEC-6 cells.The time points of 0h,2h,4h,8h and 12h after LPS were observed respectively.8h was selected as the time point of this part of the experiment,the cells are divided into the following five groups:①control group,②LPS group,③LPS+MeI group,④LPS+Mel+3-TYP group,⑤LPS+3-TYP group.2.Tested indices2.1 Evaluation of intestinal injury in mice after sepsisThe pathological changes of the small intestine tissue at 0h,2h,4h,8h,and 12h after CLP were observed by Hematoxylin and Eosin(HE)staining.The protein expression of mitochondrial protein ATP5B in the intestine was detected by Western blot.The intestinal histopathological changes and content of ATP5B at different time points were compared to evaluate the degree of damage.2.2 Effect of Mel on intestinal injury in miceTo verify the effect of drug pretreatment on the intestinal damage of sepsis,the intestinal change was measured by HE staining.2.3 Effect of Mel on mitochondria in intestinal epithelial cellsDCFH-DA dye was used to evaluate the ROS level in different groups under fluorescence microscope.The stronger green fluorescence,the higher level of ROS was.The mitochondrial membrane potential of each group was detected by immunofluorescence with JC-1 probe.When the ratio of red/green light decreased,mitochondrial membrane potential decreased,indicating mitochondrial damage.2.4 Effect of Mel on intestinal tissue/cell SIRT3 and SOD2 protein expression and activitiesThe expression of SIRT3 and SOD2 in each group was detected by Western Blot.The level of acetylation in the intestine was detected by immunohistochemistry.Total SOD activity in the intestine was measured using the total SOD enzyme activity assay kit,and IP was used to detect acetylated-SOD2(Ac-SOD2)at the animal level.By comparing the protein expression and activity of SIRT3 and SOD2 and the Ac-SOD2/SOD2 ratio,we assessed the effect of melatonin on SIRT3 and SOD2.2.5 Effect of Mel on autophagy after sepsisWestern Blot was used to detect and analyze autophagy-related proteins LC3 II and p62,mitochondrial autophagy-related proteins PINK1 and PARKIN,and immunofluorescence technique was used to observe the amount and distribution of LC3 Ⅱ protein by confocal microscope.Results1.The establishment of sepsis intestinal injury modelIn CLP-induced sepsis mice model,as time prolonged,intestinal injury progressively aggravated,and intestinal tissue was significantly damaged at 8h after CLP,while mitochondrial protein level of ATP5B decreased after CLP,and was also significantly decreased at 8h.Therefore,we chose 8h as our experiment time.2.Mel relieved intestinal injuryCompared with control group,CLP mice showed obvious damage in small intestine.Mel pretreatment could greatly reduce the degree of damage in small intestine after sepsis.3.Effects of Mel on mitochondrial injury after sepsisCompared with control group,intracellular ROS level and mitochondrial membrane potential decreased and oxidative stress increased after LPS stimulation,whereas Mel reduced ROS production,decreased oxidative stress and maintained mitochondrial membrane potential,however,3-TYP partially suppressed these effects.4.Effects of Mel on intracellular SIRT3 and SOD2 protein expression and activitiesCompared with control group,the protein expression of SIRT3 in the sepsis group slightly reduced,while the activity of deacetylase significantly increased.Besides,the level of SOD2 protein was not changed,but the enzyme activity significantly decreased.Mel pretreatment reduced CLP/LPS effects on SIRT3,SOD2 protein expression and enzyme activity.5.Effect of Mel on autophagy after sepsisCompared with control group,the protein expression of LC3 Ⅱ,PINK1,PARKIN decreased in the intestine,and the content of p62 increased after sepsis,indicating that the level of autophagy decreased.However,Mel could promote autophagy,while 3-TYP partially inhibited the effect of melatonin on autophagy.Conclusion1.Small intestine was damaged and mitochondrial protein content was decreased after sepsis.2.Mel reduced sepsis-associated intestinal injury.3.Mel reduced the level of ROS and maintained mitochondrial membrane potential,thus ameliorating mitochondrial damage.4.Intestinal protection of Mel may be associated with SIRT3 mediated SOD2 pathway,reducing ROS level and promoting autophagy/mitochondrial autophagy.5.Melatonin is a potential agonist of SIRT3.