Effect Of SIRT1 On The Oxidative Damage Of Porcine Intestinal Epithelial Cells By Regulating Mitophagy

Intestinal is the center organ of body’s stress response.Intestinal structure and function are easy to be injured when the body suffers from adverse factors inside and outside environment,which will induce systemic endogenous stress syndrome and then threaten life health seriously.So the healthy and orderly intestinal structure with stability function is of great significance to maintain animal growth,development,reproduction and immune function.Mitochondria as an important place of cell breath and energy generation,its oxidative phosphorylation in intimal respiratory chain is the main source of reactive oxygen free radicals(ROS),meanwhile mitochondrial mediated selective autophagy plays an important role in the degradation of damaged or redundant mitochondria,maintaining the balance of mitochondrial quantity and quality,as well as regulating oxidative damage.It has been found that silent information regulation factor 1(SIRT1)is closely related to mitochondrial autophagy,and it is involved in cell oxidative damage process,but the role of SIRT1 adjusting mitochondria autophagy in intestinal oxidative damage remains to be elucidated.In this study,we mainly explore the relation of SIRT1 and mitophagy in porcine intestinal epithelial cells,and analyze the effect of SIRT1 on porcine intestinal epithelial cells oxidative stress.These results can help to elucidate the molecular mechanism of intestinal oxidative damage and provide new ideas and scientific basis to related antioxidant products research and development.The main research projects are as follows:1.Effects of oxidative stress on oxidative damage、mitophagy and SIRT1/PGC-1α signaling pathway in porcine intestinal epithelial cellsThe IPEC-1 cells were treated with 300 μM H2O2 for 12 h,cells were collected.Firstly,DCFH-DA and JC-1 probe were used to detect reactive oxygen species ROS levels and membrane potential changes in mitochondria;q PCR and Western Blot were used to detect expressions of the tight junction molecule ZO-1,Claudin-1,Occludin m RNA and Claudin-1 proteins;expression levels of autophagy marker genes LC3,Beclin1,p62 and mitophagy PINK1/Parkin pathways molecules;SIRT1 and its downstream PGC-1α expression.The results showed that porcine intestinal epithelial cells under oxidative stress:(1)ROS content increase,mitochondrial membrane potential significantly reduced;the expression of tight junction molecules in the cell monolayer mucosal barrier have been suppressed.(2)The expression levels of autophagy markers LC3,Beclin1,PINK1,Parkin were significantly up-regulated,and the expression level of p62 was decreased.(3)The m RNA and protein expression levels of SIRT1 and its downstream PGC-1α molecules were significantly lower.The above results suggested that oxidative stress let IPEC-1 cells undergo oxidative damage,lead to mitochondrial membrane potential reduced,the integrity of close connection between intestinal epithelial cells have been damaged;activate intracellular mitochondrial autophagy,meanwhile inhibit SIRT1/PGC-1α signaling pathway in IPEC-1 cells.2.Effect of SIRT1 on mitophagy in porcine intestinal epithelial cellsIn order to further study the specific role of SIRT1 molecules,the specific activator SRT 1720 was selected to set the concentration gradient as: 0 μM、1.25 μM、2.5 μM、5 μM.At the same time,we set the concentration gradient of inhibitors EX 527 as: 0 μM、1 μM、5 μM、10 μM、20 μM and treat cells 24 h.Collected cell samples,used q PCR and Western Blot analysis the expression of SIRT1 and downstream PGC-1α molecular,screened for the best effect of activator and inhibitor concentration in IPEC-1 cell.Then used the appropriate concentration of SRT 1720 and EX 527 to treat cells 12 h and incubation with H2O2 for another 12 h,collected cells samples,analyzed the expression level of autophagy marker molecular LC3、ATG5 and mitophagy PINK1/Parkin pathway molecular with q PCR and Western Blot.The results showed:(1)the expression level of SIRT1/PGC-1α pathway molecules respectively rise and decline along with the mounting concentration of EX 527 and SRT 1720,and the activation and inhibition effect is the most significant at the 1.25 μM SRT 1720 and 1 μM EX 527.(2)On the basis of H2O2 treatment,once SIRT1 was activated,the expression levels of mitophagy molecules LC3、ATG5、PINK1 and Parkin were significantly increased,but there was an opposite effect when SIRT1 was inhibited,suggesting that activation of SIRT1 may further enhance oxidative stress-induced mitophagy of IPEC-1 cells.3.Effect of SIRT1 on oxidative damage of porcine intestinal epithelial cellsTreated IPEC-1 cells with activator and inhibitor 12 h,and then co-incubated with H2O2 for another 12 h.Collected cells and used DCFH-DA and JC-1 probe detect ROS levels and membrane potential changes in mitochondria;q PCR and Western Blot were used to detect expressions of the tight junction molecule ZO-1,Claudin-1,Occludin m RNA and Claudin-1 proteins.The experimental results show: under the state of oxidative stress,once SIRT1 was activated,the intracellular ROS level was decreased;the mitochondrial membrane potential recovery;the expression level of tight junction molecules was significantly up-regulated.Simultaneously inhibition of SIRT1 has the opposite effect.The above results indicated that activation of SIRT1 significantly reversed the oxidative damage of porcine intestine epithelial cells,which plays a key role in defensing oxidative stress for porcine intestine cells.In conclusion:1.Oxidative stress induces porcine intestine epithelial cells oxidative damage,which is embodied in that oxidative stress can not only decrease mitochondrial membrane potential and damage the intercellular closed connection integrity but also induce mitophagy and inhibite SIRT1/PGC-1α signaling pathway.2.Activation of SIRT1 may further enhance oxidative stress-induced mitophagy of IPEC-1 cells.3.Activation of SIRT1 may observably repair oxidative damage of porcine intestinal epithelial cells by enhancing the level of mitophagy.