| Intestinal ischemia-reperfusion(I/R)injury is a common pathophysiological process with a high clinical morbidity and mortality in surgery.It is not only caused by primary mesenteric vessel lesions,but also occurs secondary to courses of various severe diseases,such as hemorrhagic shock,large area burns,etc.Intestinal I/R injury not only causes local damage and destruction of the intestine,but also affects the pathophysiological state of the whole body.It is currently believed that intestinal mucosal barrier function is impaired after I/R,which may lead to increased intestinal permeability and translocation of gut bacteria.Combined with various of pathological factors,intestinal I/R injury can eventually trigger systemic inflammatory response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS),leading to poor prognosis for patients.Therefore,it is of great significance to study the pathogenesis of intestinal I/R injury and to provide timely and effective interventions to alleviate damage.Maintaining the structural and functional integrity of the intestinal mucosal barrier is the key to prevent intestinal barrier dysfunction and subsequent MODS after I/R.The intestinal mucosa epithelial cells(intestinal epithelial cells,IECs)is the important structure basis of intestinal barrier.Previous studies have suggested that apoptosis,necroptosis and other cell death triggered by I/R can cause massive loss of IECs and destroy the integrity of intestinal mucosal barrier.With the in-depth study of cell death,non-apoptotic regulated cell death(RCD),such as pyroptosis and ferroptosis,has been identified and found to be involved in the occurrence and development of many diseases.Therefore,the studies on the role and mechanism of such cell death in intestinal I/R can provide new strategies for the prevention and treatment of in situ and distant organ injuries.In 2012,Dixon et al.reported a new form of cell death---ferroptosis.Ferroptosis is an iron-dependent cell death characterized by accumulation of lipid peroxide,which is different from apoptosis,necroptosis and autophagy in morphology,biochemistry and immunity.Current studies have shown that ferroptosis is closely related to neurodegenerative diseases,tumors and liver fibrosis.It has been reported that inhibition of ferroptosis can alleviate I/R injury of liver,kidney,heart and other organs.However,whether ferroptosis is involved in intestinal I/R injury and its regulatory mechanism have not been studied.Acyl-Co A synthetase long-chain family member 4(ACSL4)is a member of ACSLs family.Different from other family members,ACSL4 can catalyze arachidonic acid(AA)to synthesize arachidonoyl coenzyme A,and then participate in the synthesis of phosphatidylethanolamine(PE).PE is the main component of phospholipids in cell membrane,and also an important component that participates in lipid peroxidation in ferroptosis.Doll et al.confirmed that ACSL4 knockout can significantly inhibit the esterification of AA into PE,thereby reducing the susceptibility of cells to ferroptosis and preventing the occurrence of it.Therefore,inhibition of ACSL4 provides a new feasible treatment for ferroptosis-related diseases.Our preliminary experiment showed that the expression of ACSL4 protein and m RNA levels in intestine were increased after ischemia,suggesting that ACSL4 may be involved in regulating the sensitivity of intestinal tissue to ferroptosis.As the earliest discovered and cloned transcription factor,special protein1(SP1)can specifically bind to the GC box in the promoter region of downstream target genes,thus promotes the transcription and expression of target genes.Studies have confirmed that hypoxia can upregulate the expression of nuclear SP1,leading to cell death.According to the database,the promoter region of ACSL4 gene is rich in GC boxes,which can be recognized specifically by SP1.Therefore,the excessive expression of ACSL4 may be regulated by SP1 during ischemia.In summary,we propose the following hypothesis: ferroptosis is an important mechanism that leads to intestinal I/R injury;The ischemia-induced ACSL4 is an important reason of ferroptosis after reperfusion in the intestine.ACSL4 overexpression may be regulated by SP1 at the transcription level.In order to prove this hypothesis,our study is performed through the following three parts:Part ?: ferroptosis is an important mechanism for intestinal I/R injury;Part ?: inhibition of ACSL4 can reduce ferroptosis and attenuate intestinal I/R injury;Part ?: SP1 binds to the promoter region of ACSL4 gene and regulates the expression of ACSL4.This study provided important mechanism and targets for the occurrence of intestinal I/R injury.Early intervention,which can prevent ferroptosis and the crucial regulators,is the key to protect against in-situ organ injury,SIRS and MODS caused by intestinal I/R.That is of the great significance for improving the prognosis of severe patients.Part ? Ferroptosis is an important mechanism for intestinal I/R injuryBackground: The massive loss of intestinal epithelium is the main reason that causes intestinal I/R injury.Previous studies on cardiac,hepatic and renal I/R showed that ferroptosis can lead to the cell death and serious tissue damage.The administration of ferroptosis inhibitor can significantly attenuate organ injury.However,the relation between ferroptosis and intestinal I/R injury is still unknown.Objective: To investigate the occurrence of ferroptosis after intestinal I/R,and to clarify the effect of ferroptosis inhibition on intestine and remote organ injury after intestinal I/R.Methods:Experiment Set 1: C57BL/6 mice were used to found intestinal ischemia and intestinal I/R models.The sham group underwent superior mesenteric artery(SMA)separation without occlusion.In the ischemic group,the SMA was clamped for 30,45 and 60 minutes.To found I/R model,the SMA was clamped for 45 minutes and then the artery clamp was removed for 15,30,60,120 and 240 minutes.Intestines of each group were collected after ischemia or I/R,and the ferroptosis-related regulators were detected by western blot,lipid peroxidation,transmission electron microscope(TEM),etc.Experiment Set 2: I/R model was established in C57BL/6 mice.Ferroptosis specific inhibitor liproxstatin-1(10mg/kg)was administrated 1 hour prior to operation by intraperitoneal injection(i.p.).Mice were divided into four groups: sham,sham + +lip,I/R and I/R+lip.After reperfusion,samples of each group were collected for subsequent detection: histopathological changes of intestine,liver and lung were evaluated by hematoxylin and eosin(H&E)staining.Expression of ferroptosis-related proteins were detected by western blot.Serum LDH,FD-4,TNF-,and IL-6 levels were used to represent intestinal barrier dysfunction and release of inflammatory mediators.LPO and 12/15-HETE were used to detect lipid peroxidation level.MPO and lung wet/dry ratio were used to detect distant organ injury.Experiment Set 3: Caco-2 cells were used to establish the H/R model.Liproxstatin-1(200n M)was added 12 hours before hypoxia,and cells were divided into four groups: control,control+lip,H/R and H/R+lip.After reoxygenation,samples in each group were collected for subsequent detection: expression of ferroptosis-related proteins were detected by western blot.CCK-8,transepithelium electrical resistant(TEER)and LDH were used to detect cell death and barrier damage.BODIPY 581/591 C11 green fluorescent dye,LPO,12/15-HETE were used to detect lipid peroxidation levels.Results:Experiment Set 1: Compared with sham group,ACSL4 and iron were increased and GPx4,FTH1 and GSH were decreased in intestine at 45 minutes of ischemia.At 30 minutes of reperfusion,the mitochondrion showed more obvious characteristics of ferroptosis,GPx4 expression was decreased,COX2 expression and 12/15-HETE levels were increased.Experiment Set 2: Compared with sham group,the expression of GPx4 in intestine was significantly decreased and COX2 was significantly increased in I/R group.The levels of FD-4,LDH,TNF-and IL-6 in serum were significantly increased,and the levels of 12/15-HETE and LPO were significantly increased.The damage of intestine and distant organs was more serious,and the MPO content was increased.Compared with the I/R group,pretreatment of liproxstatin-1 markedly attenuated the above trend and protected against intestinal I/R injury.Experiment Set 3: Compared with the control group,GPx4 expression was significantly decreased in Caco-2 cells after H/R,and COX2 expression was significantly increased.The survival rate of cells was obviously decreased,and the barrier function was weakened.In H/R group,BODIPY C11 showed obvious green fluorescence,increased 12/15-HETE and LPO levels,and increased LDH content in the medium.Compared with the H/R group,liproxstatin-1 treatment obviously conducted the above protective effects.Conclusion:1.Ferroptosis occurs in the early stage of intestinal I/R.2.Inhibition of ferroptosis can alleviate intestine and remote organ injury after intestinal I/R,improve intestinal barrier function,and reduce lipid peroxidation.Part ? Inhibition of ACSL4 can alleviate intestinal I/R injury caused by ferroptosisBackground: The above results suggested that ferroptosis occurred at the relative early stage of reperfusion.Ferroptosis inhibition mediated by liproxstatins-1 attenuated primary and remote organ injury,decreased lipid peroxidation and promoted intestinal barrier function.The key factor that can regulate ferroptosis under intestinal I/R condition remains to be further explored.Objective: To verify the ACSL4 expression after ischemia in intestine,and to confirm the effect of ACSL4 inhibition on ferroptosis alleviation and intestinal I/R injury protection.Methods:Experiment Set 1: Three patients that were challenged by strangulated intestinal obstruction were selected.One centimeter of ischemic intestines was collected during operation.The lateral margin of the bowel segment was taken as control(The program was approved by the Ethics Committee of the Second Hospital of Dalian Medical University).ACSL4 expression level was detected by western blot.Experiment Set 2: A simple ischemia model was established in C57BL/6 mice.To inhibit ACSL4,rosiglitazone(ROSI,0.4mg/kg)was treated 1 hour prior to surgery by intravenous injection.Mice were divided into four groups: sham,sham+ROSI,ischemia and ischemia +ROSI.ACSL4 was detected in each group after ischemia.Experiment Set 3: I/R model was established in C57BL/6 mice.Rosiglitazone(ROSI,0.4mg/kg)was administrated 1 hour before operation.Mice were divided into four groups: sham,sham+ROSI,I/R and I/R +ROSI.After reperfusion,samples were taken from each group for subsequent detection: H&E staining was used to detect pathological changes in the intestines,serum FD-4 and LDH were used to detect intestinal barrier dysfunction and injury,western blot was used to detect ferroptosis-related proteins,and 5/12/15-HETE and LPO were used to detect lipid peroxidation levels.Experiment Set 4: Caco-2 cells were used to establish the H/R model and pretreated with ACSL4 RNA interference(RNAi),being divided into four groups: control+ si-NC,control+ si-ACSL4,H/R+ si-NC,and H/R+ si-ACSL4.After reoxygenation,samples in each group were collected for subsequent detection: expression of ferroptosis-related proteins were detected by western blot.CCK-8,TEER and LDH were used to detect cell injury and intestinal barrier damage.BODIPY 581/591 C11 green fluorescent dye,5/12/15-HETE,LPO were used to detecte lipid peroxidation levels.Results:Experiment Set 1: ACSL4 expression was increased in ischemic intestines compared with normal ones.Experiment Set 2: Compared with sham group,the activity of ACSL4 in ischemic intestine was significantly increased.Compared with ischemia group,the activity of ACSL4 in ischemia+ROSI group was significantly decreased.Experiment Set 3: Compared with sham group,pathological damage of intestine was more obvious in I/R group.Serum level of FD-4 was increased,GPx4 expression in intestine was significantly decreased,COX2 expression was significantly increased,5/12/15-HETE and LPO levels were increased,and serum LDH content was increased.Compared with the I/R group,ROSI pretreatment obviously relieved the above trend and protected intestine against I/R injury.Experiment Set 4: Compared with the control group,GPx4 expression was significantly reduced in Caco-2 cells in the H/R group,in contrast to COX2 expression.Cell survival was significantly reduced,barrier function was weakened,BODIPY C11 green fluorescence intensity,5/12/15-HETE and LPO levels were increased,and LDH content in the medium was increased.Compared with the H/R group,ACSL4 inhibition by RNAi significantly reduced ferroptosis and H/R-induced cell injury.Conclusion:1.After ischemia,the expression and activity of ACSL4 in intestine are induced.2.Inhibition of ACSL4 before reperfusion can reduce intestinal I/R injury,improve intestinal barrier function,inhibit ferroptosis and decrease lipid peroxidation.Part ? SP1 binds to the promoter region of ACSL4 gene and regulates the expression of ACSL4Background: The above results showed that inhibition of ferroptosis can significantly reduce intestinal I/R injury.ACSL4 is an important factor that induced ferroptosis after reperfusion.The inhibition of ACSL4 after ischemia reduced the occurrence of ferroptosis and protected the intestine.The underlying mechanism that induced ACSL4 is still unknown.Objective: To verify the regulatory effect of transcription factor SP1 on ACSL4 and to further confirm the binding sites.Methods:Experiment Set 1: Ischemic intestines from patients,45 min ischemic intestines from mice and hypoxic Caco-2 cells were collected for ACSL4 m RNA assay by q PCR.Meanwhile,hypoxic cells were also used to detect nuclear SP1 expression and location by western blot and immunofluorescence(IF).Experiment Set 2: Hypoxic Caco-2 cells were pretreated with SP1 RNAi,which were divided into four groups: control+ si-NC,control+ si-SP1,H/R+ si-NC and H/R+ si-SP1.SP1 overexpression plasmid(pc DNA)was used as the treatment and cells were divided into four groups: control+pc DNA-NC,control+pc DNA-SP1,H/R+pc DNA-NC and H/R+pc DNA-SP1.The protein expressions of SP1 and ACSL4 were detected by western blot,and the m RNA expression levels of ACSL4 was detected by q PCR.Experiment Set 3: The SP1 overexpressed plasmid(pc DNA)and dual-luciferase reporter gene labeled ACSL4 promoter cloned plasmid(ACSL4-prom S)were cotransfected to Caco-2 cells normoxia and hypoxia,and cells were divided into four groups respectively : control+pc DNA-NC+ACSL4-vector,control+pc DNA-SP1+ACSL4-vector,control+pc DNA-NC+ACSL4-prom S,control+pc DNAsp1+ACSL4-prom S;hypoxia+pc DNA-NC+ACSL4-vector,hypoxia+pc DNASP1+ACSL4-vector,hypoxia+pc DNA-NC+ACSL4-prom S,hypoxia+pc DNAsp1+ACSL4-prom S.Dual-luciferase reporter gene assay was performed in each group after hypoxia.Experiment Set 4: Under the condition of normoxia and hypoxia,cells were grouped into SP1 group(experimental group),RNA Polymerase ? group(positive control).Ch IP experiment was carried out in each group after hypoxia.According to the database prediction,primers of three candidate sites(GL1,GL2,GL3)were designed,and functional sites were screened by agarose gel electrophoresis.Experiment Set 5: In Caco-2 cells,co-transfected SP1 overexpressed plasmid(pc DNA)and dual-luciferase reporter gene labeled ACSL4 promoter(wild type specific locus deletion)plasmids were used for transfection with normoxia and hypoxia,and cells were divided into three groups respectively: control+pc DNA-SP1+WT,control+pc DNASP1+Del-GL2,control+pc DNA-SP1+Del-GL3);hypoxia+pc DNA-SP1+WT,hypoxia+pc DNA-SP1+Del-GL2,hypoxia+pc DNA-SP1+Del-GL3.Dual-luciferase reporter gene assay was performed in each group after hypoxia.Results:Experiment Set 1: Compared with normal/sham group,the levels of ACSL4 m RNA in ischemic intestinal of human and mice was increased.Compared with the control group,the level of ACSL4 m RNA in hypoxic cells increased,and the expression and distribution of SP1 protein in the nucleus was also increased.Experiment Set 2: Compared with the control+si-NC group,SP1 and ACSL4 protein expressions and ACSL4 m RNA levels were decreased in the control+si-SP1 group.Compared with the hypoxia+ si-NC group,the protein expression of SP1 and ACSL4 was decreased and the m RNA level of ACSL4 was reduced in the hypoxia+si-SP1 group.Compared with the control+pc DNA-NC group,SP1 and ACSL4 protein expressions and ACSL4 m RNA levels were increased in the control+pc DNA-SP1 group.Compared with the hypoxia+pc DNA-NC group,the protein expression of SP1 and ACSL4 of the hypoxia+pc DNA-SP1 group increased and the m RNA level of ACSL4 increased.Experiment Set 3: Compared with the control+pc DNA-SP1+ACSL4-vector group,the dual-luciferase reporter gene activity was enhanced in the control+pc DNASP1+ACSL4-prom S group.Dual-luciferase reporter activity of the hypoxia+pc DNASP1+ACSL4-vector group was increased compared with that of the hypoxia+pc DNASP1+ACSL4-prom S group.Experiment Set 4: Under normoxic and hypoxic conditions,sites GL(2,3)were amplified apparent bands compared with site GL1.Experiment Set 5: Under normoxic condition,compared with control+pc DNASP1+WT group,dual-luciferase reporter gene activity of control+pc DNA-SP1+Del-GL2 group was decreased,while control+pc DNA-SP1+Del-GL3 group had no significant changes.Dual-luciferase reporter activity of the hypoxia+pc DNA-SP1+Del-GL2 group was decreased compared with that of the hypoxia+pc DNA-SP1+Del-GL3 group,while that of the hypoxia+pc DNA-SP1+Del-GL3 group was not significantly changed under hypoxia.Conclusion:1.Transcription factor SP1 positively regulates the transcription level and the protein expression of ACSL4.2.Transcription factor SP1 regulates ACSL4 by binding to the GL2 site in the promoter region.In summery,our study reveals the function of ferroptosis in intestinal I/R injury and demonstrates that the inhibition of ferroptosis can ameliorate in situ and remote organ injury.ACSL4,a protein vital to ferroptosis,is induced after ischemia and is involved in I/R injury.Sp1 can upregulate ACSL4 expression by binding to the ACSL4 promoter region.These findings indicate a potential mechanism that lead to intestinal I/R injury at early stage,and guide us to extend ferroptosis inhibition to intestinal I/R treatment. |
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