| Significant oxidative stress occurs in the intestine of piglets during the weaning process,which is an important cause of intestinal barrier damage.How to alleviate the oxidative stress of weaned piglets has become the focus of common concern in academia and industry.Energy is essential for the renewal and normal function of intestinal epithelial cells,and the process of energy production is accompanied by the production of mitochondrial oxidative respiratory chain ROS.As a cell energy factory,mitochondria are not only the main place for the production of reactive oxygen species,but also an important target for reactive oxygen species to attack.In order to protect cells from oxidative damage,cells form a delicate self-protection mechanism,that is,to selectively degrade damaged mitochondria before functionally damaged mitochondria induce cell death,which is called mitophagy.However,so far,there is no research report on the role of mitophagy in the repair of oxidative stress-induced piglet intestinal barrier damage and its regulatory mechanism.Curcumin is a natural phenolic pigment extracted from the rhizomes of plants in the Zingiberaceae or Araceae family,and is a natural antioxidant.This thesis aims to explore the role of mitophagy in piglet oxidative stress and the regulation mechanism of curcumin,and provide new ideas and directions for the nutritional regulation of piglet intestinal barrier damage repair,which has important theoretical significance and application value.1.Weaning disrupts intestinal antioxidant status,impairs intestinal barrier and mitochondrial function,and triggers mitophagy in pigletsIn this trial,we investigated the influence of weaning on antioxidant status,intestinal integrity,mitochondrial function and the mitophagy level in piglets(weaned at 21d)during the 1 week after weaning.The redox status was measured by antioxidant enzymes activities,related genes expression and malondialdehyde(MDA)content in jejunum.The intestinal barrier function was assessed by the Ussing chamber and expression of tight junction proteins in the jejunum.The function of intestine mitochondria was measured by mitochondria DNA(mtDNA)content and activities of mitochondria oxidative phosphorylation complexes.The levels of light chain 3-1(LC3-?),light chain 3-?(LC3-?),PTEN-induced putative kinase 1(PINK1)and Parkin were determined to investigate whether mitophagy is involved in the weaning process.The results showed that,as compared with the preweaning phase(d 0),weaning suppressed(P<0.05)the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)on d 3 and d 7 postweaning,decreased(P<0.05)the expression of copper and zinc superoxide dismutase(Cu/Zn-SOD),manganese-containing superoxide dismutase(Mn-SOD)on d 3 postweaning,declined(P<0.05)the level of glutathione peroxidase 1(GPX-1),glutathione peroxidase 4(GPX-4)on d 3 and d 7 postweaning,and increased(P<0.05)MDA content in jejunum on d 3 and d 7 postweaning.The jejunal TER and levels of occludin,claudin-1 and Zonula occludens-1 on d 3 and 7 postweaning were reduced(P<0.05),and paracellular flux of fluorescein isothiocyanate dextran(4 kDa)on d 3 and 7 postweaning was increased(P<0.05).Weaning induced mitochondrial dysfunction,as demonstrated by decreased(P<0.05)content of mtDNA on d 3 and d 7 postweaning and declined(P<0.05)activities of mitochondria complexes(?,?,?,?)in jejunum on d 1,d 3 and d 7 postweaning.Weaning led to an increased(P<0.05)expression level of mitophagy related proteins,PINK1 and Parkin in the intestinal mitochondria,as well as an enhancement(P<0.05)of the ratio of LC3-? to LC3-? content in the jejunal mucosa on d 1,d 3 and d 7 postweaning.These results suggest that weaning disrupted intestinal oxidative balance,and this imbalance may impair intestinal barrier and mitochondrial function,and trigger mitophagy and active AMPK in piglets.2.Diquat-induced oxidative stress increases intestinal permeability,impairs mitochondrial function and triggers mitophagy in pigletsWe studied the effects of diquat-induced oxidative stress on piglet intestinal barrier,mitochondrial function and mitophagy.Twelve 35-day-old healthy weaned piglets(average weight of 9.6 kg)were selected and randomly divided into two groups:control group and stress group.Each group has 6 repetitions,and each repetition has 1 piglet.Pigs in the stress group were injected with 10 mg/kg diquat,and piglets in the control group were injected with an equivalent amount of 0.9%(w/v)NaCl solution.The results showed that:(1)Diquat significantly reduced the average daily feed intake and average daily gain of piglets(P<0.05),and significantly inhibited superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in the jejunum(P<0.05)significantly increased the content of malondialdehyde in the jejunum(P<0.05).(2)Diquat significantly reduced the transmembrane resistance of porcine jejunal epithelial cells and the expression of tight junction proteins(Claudin-1,occludin,zonula occludens-1)(P<0.05),and significantly improved the permeability of jejunum FD4(P<0.05).(3)Diquat treatment resulted in obvious swelling,vacuolization,matrix concentration of mitochondria in the piglet's jejunum,and reduced or broken mitochondrial cristae;Diquat significantly increased the production of reactive oxygen species in the intestinal mitochondria(P<0.05)and significantly reduced the intestine mitochondrial membrane potential(P<0.05),which indicates that diquat causes mitochondrial dysfunction(P<0.05).(4)Diquat significantly reduced the expression of genes related to mitochondrial biogenesis and function,PGC-1?,SIRT-1,NRF-1,TFAM,mtSSB,mtpolr,glucokinase,CS,ATPS and CcOX ? and ? in the jejunum(P<0.05).(5)Diquat significantly increased the expression of mitophagy-related proteins PINK1 and Parkin in the intestinal mitochondria(P<0.05),and significantly increased the ratio of LC3-?to LC3-? content in the jejunal mucosa(P<0.05).(6)Diquat treatment significantly increased the expression of pAMPK/AMPK.These results indicate that oxidative stress disrupts the intestinal barrier,causes mitochondrial dysfunction,triggers mitophagy,and activates the AMPK signaling pathway.3.AMPK-PINK1/Parkin-mediated mitophagy is involved in the regulation of oxidative stress-induced intestinal epithelial barrier damage and mitochondrial energy metabolism disorders in pigletsThe purpose of the experiment is to study the effects of H2O2-induced oxidative stress on intestinal epithelial cell oxidation balance,intestinal epithelial barrier function and mitochondrial energy metabolism,and clarify that AMPK-PINK1/Parkin-mediated mitophagy is involved in oxidative stress-induced intestinal barrier damage in piglets The role of repair,to analyze the role and regulatory mechanism of mitophagy in the repair of oxidative stress-induced intestinal barrier damage in piglets.The experiment was conducted to construct a H2O2-induced oxidative stress model of piglet intestinal epithelial IPEC-J2 cells to study the effects of H2O2-induced oxidative stress on the oxidative balance of intestinal epithelial cells,intestinal epithelial barrier function,mitochondrial energy metabolism,mitophagy and AMPK signaling pathway.The results showed that:(1)Compared with the control group,600 ?M H2O2 treatment significantly increased the expression level of p-AMPK(P<0.05).H2O2 treatment at 600 ?M and 800 ?M significantly increased the expression levels of PINK 1,Parkin and the ratio of LC3?/?(P<0.05),indicating that H2O2-induced oxidative stress activated AMPK signaling pathway and PINK 1-Parkin-mediated mitophagy.(2)Compared with the control group,H2O2 treatment significantly reduced the SOD and CAT activities of intestinal epithelial cells(P<0.05),significantly increased the MDA content(P<0.05),and significantly reduced the anti-oxidative stress-related genes Cu/Zn-SOD,Mn-SOD,GPX-1,GPX-4 expression(P<0.05).Compared with the oxidative stress group,the addition of Compound C and Mdivi-1 significantly reduced the activity of SOD(P<0.05),and the addition of both at the same time intensified the reduction of SOD activity induced by H2O2(P<0.05).(3)Compared with the control group,the oxidative stress group significantly increased the mitochondrial active oxygen content(P<0.05).Compared with the oxidative stress group,the content of mitochondrial active oxygen in the Compound C+H2O2 group,Mdivi-1+H2O2 group and Compound C+Mdivi-1+H2O2 group also increased significantly(P<0.05).(4)Compared with the control group,H2O2treatment significantly reduced the expression of TER and Claudin in intestinal epithelial cells(P<0.05),and significantly increased the permeability of FD4(P<0.05).Compared with the oxidative stress group,Compound C+H2O2 and Mdivi-1+H2O2 treatments significantly reduced the TER of intestinal epithelial cells(P<0.05),but had no significant effect on FD4 permeability and tight junction protein expression(P<0.05).(5)Compared with the control group,H2O2 induced oxidative stress significantly reduced the ATP production of intestinal epithelial cells,mitochondrial membrane potential and the activity of mitochondrial respiratory chain complex ?,?,and ?(P<0.05).Compared with the oxidative stress group,the addition of Compound C and Mdivi-1 significantly reduced the activity of mitochondrial respiratory chain complex ?(P<0.05),but had no effect on ATP production,mitochondrial membrane potential,and complex ?,?.Significant influence(P<0.05).Compared with the Compound C+H2O2 group and the Mdivi-1+H2O2 group,adding both at the same time significantly reduced the mitochondrial membrane potential and the activity of mitochondrial respiratory chain complex ? and ?(P<0.05).Compared with the control group,oxidative stress group,Compound C+H2O2 group,Mdivi-1+H2O2 group,Compound C+Mdivi-1+H2O2 group can all observe obvious mitochondrial swelling,respiratory ridge breakage and mitochondrial vacuolization.phenomenon.(6)Compared with the control group,the oxidative stress group,Compound C+H2O2 group,Mdivi-1+H2O2 group,and Compound C+Mdivi-1+H2O2 group can observe a decrease in the overall oxygen consumption of cells.Compared with the control group,oxidative stress significantly reduced the cell's basic respiratory oxygen consumption,maximum respiratory oxygen consumption and ATP production(P<0.05).Compared with the oxidative stress group,the addition of Compound C and Mdivi-1 significantly reduced the maximum respiratory oxygen consumption of cells(P<0.05).Compared with the oxidative stress group,the simultaneous addition of Compound C+Mdivi-1+H2O2 significantly reduced the cell's maximum respiratory oxygen consumption and standby respiratory capacity(P<0.05).(7)Compared with the control group,H2O2 treatment significantly increased the expression of PINK1,Parkin,Beclin-1 and the ratio of LC3?/? expression(P<0.05).Compared with the oxidative stress group,Compound C+H2O2 and Mdivi-1+H2O2 significantly inhibited the protective mitophagy response induced by oxidative stress(P<0.05),which was specifically manifested as a significant reduction in PINK1,Parkin,and Beclin-1 The ratio of the expression level of LC3?/?.Compared with the Compound C+H2O2 group and the Mdivi-1+H2O2 group,the expression of Parkin in the Compound C+Mdivi-1+H2O2 group was significantly lower(P<0.05).(8)Compared with the control group,the oxidative stress group can observe the phenomenon that the damaged mitochondria are wrapped by autophagic vesicles in the double membrane.Compared with the oxidative stress group,there were fewer mitochondrial autophagosomes in the Compound C+H2O2 group.There were almost no mitochondrial autophagosomes in the Mdivi-1+H2O2 group and Compound C+Mdivi-1+H2O2 group.(9)To directly visualize the activation of mitophagy in live IPEC-J2 cells,we transfected Ad-GFP-LC3 and Ad-HB AD-Mito-dsred to monitor the formation of mitophagy in EPEC-J2 cells in real time.H2O2 treatment significantly increased the colocalization of Ad-GFP-LC3 and Ad-HBAD-Mito-dsred in EPEC-J2 cells,while colocalization in the Compound C+H2O2 group,Mdivi-1+H2O2 and Compound C+MMdivi-1+H2O2 group Significantly reduced.The results suggest that the H2O2-induced oxidative stress model can lead to intestinal epithelial barrier damage,increased mitochondrial reactive oxygen content,mitochondrial vacuolation and mitochondrial energy metabolism disorders;H2O2-induced oxidative stress in intestinal epithelial cells can activate AMPK signaling pathway and PINK 1-Parkin-mediated mitophagy;inhibition of AMPK signaling pathway and mitophagy aggravated H2O2-induced oxidative stress,intestinal barrier damage and mitochondrial energy metabolism disorders,confirming AMPK signaling pathway and PINK 1-Parkin-mediated mitophagy play an important regulatory role in oxidative stress-induced intestinal damage.4.Curcumin ameliorates oxidative stress-induced intestinal barrier injury and mitochondrial damage by promoting Parkin dependent mitophagy through AMPK-TFEB signal pathwayThe intestinal epithelium is the most critical barrier against harmful antigens and pathogens.Oxidative stress is closely related to the disorder of intestinal barrier function.Curcumin has received widespread attention due to its anti-oxidative stress function.However,whether curcumin can relieve intestinal damage and mitochondrial damage caused by oxidative stress is still unclear.This trial aims to explore whether curcumin can alleviate oxidative stress-induced intestinal barrier damage in piglets by protecting mitochondrial function and promoteing mitophagy.We tend to clarify that whether curcumin can relieve the intestinal barrier induced by oxidative stress based on AMPK-PINK1/Parkin pathway.We found that adding curcumin to the diet of the diquat-induced porcine oxidative stress model can protect the intestinal barrier function,improve the redox state,reduce mitochondrial damage,trigger mitophagy and affect the AMPK-TFEB signaling pathway.Furthermore,we wtilized the in vitro oxidative stress model and found that curcumin can effectively alleviate the oxidative stress of IPEC-J2 cells induced by H2O2 in a PINK1-Parkin-mitophagy-dependent manner.Furthermore we found that siParkin inhibited the anti-oxidative stress effect of curcumin on IPEC-J2,and protective effect of curcumin on the intestinal barrier and mitochondrial function of IPEC-J2 cells induced by H2O2.In cells transfected with GFP-Parkin?UBL(a mutant Parkin protein encoding E3 ubiquitin ligase activity),the protective effect of curcumin was inhibited,indicating that the protective effect of curcumin requires Parkin's E3 ubiquitin ligase activity.On the other hand,we also found that after interference with PRKAA1,the protective function of curcumin on H2O2 treated IPEC-J2 cells was weakened.Immunofluorescence and luciferase experiments showed that curcumin significantly enhanced the nuclear transport and transcription activity of TFEB in IPEC-J2 cells treated with H2O2,but this effect was inhibited by Compound C,which indicated that curcumin promotes TFEB transcription through AMPK signaling pathway.In summary,these results reveal that curcumin induces Parkin-dependent mitophagy nuclear translocation of TFEB by activating AMPK,thereby improving oxidative stress and enhancing intestinal barrier function and mitochondrial function.In summary,weaning disrupts the oxidative balance of the intestines and cause oxidative stress in the intestines.Both weaning and Diquat-induced oxidative stress cause damage to the intestinal barrier function and mitochondrial dysfunction,and activate the intestinal mitochondria,which means AMPK and mitophagy may be involved in the repair of intestinal barrier damage after weaning stress and oxidative stress.Moreover,simultaneous inhibition of AMPK signaling pathway and mitophagy in an in vitro oxidative stress model induced by H2O2 aggravated H2O2-induced oxidative stress,intestinal barrier damage,and mitochondrial energy metabolism disorder,confirming that AMPK signaling pathway and mitophagy play a role in oxidative stress and intestinal injury.We found that curcumin regulates Parkin's E3 ubiquitin ligase-mediated mitophagy through AMPK-TFEB signaling pathway,thereby alleviating the intestinal barrier damage and mitochondrial function damage induced by oxidative stress. |
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