Effect And Mechanism Of Alda-1 Pretreatment On Intestinal Injury During Deep Hypothermic Circulatory Arres

Background:The rapid improvement of cardiopulmonary bypass(CPB)has greatly promoted the development of cardiac surgery,while the postoperative complications cannot be ignored.The incidence of intestinal injury after cardiac surgery is low,but the mortality is over 50%.Studies have shown that oxidative stress caused by hypothermic ischemia-reperfusion(I/R)during CPB is one of the main factors of postoperative intestinal injury.The accumulation of 4-hydroxynonenal(4-HNE)may be an important factor leading to intestinal mucosal barrier injury.Therefore,this study aims to explore the effect of hypothermic I/R on intestinal epithelial cells and tight junction by establishing in vitro hypothermic I/R model,and to clarify the role of 4HNE in this process.Methods:We used the rat small intestinal crypt epithelial(IEC-6)cells and human colonic cancer(Caco-2)cells to bulid hypothermic oxygen-glucose deprivation/reoxygenation(HOGD/R)model.Apoptosis degree,reactive oxygen species content,tight junction related protein(ZO-1,Occludin and Claudin 1)expression,4-HNE content and ALDH2 expression were detected.Results:Compared with the Control group,the apoptosis rate of IEC-6 cells in HOGD/R group was significantly increased(30.8%±2.1%vs.1.8%±0.1%,p<0.001),the expression level of anti-apoptotic protein Bcl-2 was decreased(p<0.001),and the expression level of pro-apoptotic protein Bax was increased(p<0.001).The expression levels of ZO-1,Claudin-1 and Occludin in Caco-2 cells in HOGD/R group were significantly decreased(p<0.001).The results of western blot and enzyme linked immunosorbent assay showed that the reactive oxygen and 4-HNE were significantly increased in HOGD/R group.ALDH2 expression level was also detected by western blot,and there was no significant difference in ALDH2 expression level between the HOGD/R group and the Control group(p>0.05).ALDH2 activity was detected by enzyme linked immunosorbent assay,and it was significantly lower in the HOGD/R group(p=0.037).Conclusion:HOGD/R significantly increased the apoptosis rate of intestinal epithelial cells and decreased the expression level of tight junction protein,suggesting that the intestinal mucosal barrier was damaged.In addition,HOGD/R also increased intracellular reactive oxygen and 4-HNE.HOGD/R did not affect the expression level of ALDH2,but significantly reduced the activity of ALDH2.Background:Deep hypothermic circulatory arrest(DHCA)provides clear surgical field for complex cardiac surgery and can protect the brain.However,the postoperative complications related to DHCA cannot be ignored,among which the incidence of intestinal injury is only 1.1%,but the prognosis is poor.The Part I results show that hypothermic oxygen-glucose deprivation/reoxygenation(HOGD/R)could increase 4Hydroxynonenal(4-HNE).Alda-1 could up-regulate the activity of Aldehyde dehydrogenase 2(ALDH2)to enhance the scavenging ability of 4-HNE.Therefore,the purpose of this part of the study was to explore the influence of Alda-1 on DHCA intestinal injury.Method:The rat DHCA model was established and the animals were divided into three groups:Sham operation group(Sham group),DHCA group and Alda-1 pretreatment group(Alda-1 group).HOGD/R models of IEC-6 cells and Caco-2 cells were established in vitro and divided into the following three groups:Control group,HOGD/R group,and Alda-1 pretreatment group.The contents of 4-HNE,ALDH2 enzyme activity,mRNA expression,protein expression,and cell activity were detected respectively.Pathological changes were observed by HE staining.Ultrastructure was observed by transmission electron microscopy.Apoptosis of cells,expression of inflammatory factors,and expression of intestinal tight junction protein,and apoptotic protein were also detected.Results:Compared with DHCA group and HOGD/R group,pretreatment with Alda-1 significantly increased the ALDH2 enzyme activity in small intestine tissues and cells(p<0.001),but did not affect the ALDH2 protein expression level(p>0.05).The protein expression level of 4-HNE in DHCA group was significantly higher than that of Sham group(p<0.001),and pretreatment with Alda-1 could significantly reduce the level of 4-HNE(p=0.028).As for IEC-6 cells,the cell activity of HOGD/R group was significantly lower than that of Control group,and Alda-1 pretreatment significantly increased the cell activity(p<0.001).The results of HE and Transmission electron microscope showed that the villi and tight junction structure of the small intestine were severely damaged in DHCA group,and Alda-1 pretreatment could reduce the damage degree of the small intestine tissue structure.TUNEL apoptosis results showed that the apoptosis rate of DHCA group(HOGD/R group)was significantly increased,and Alda1 pretreatment could significantly reduce the apoptotic degree of DHCA(HOGD/R group)(P<0.001).The expression apoptosis-related proteins in small intestine were detected by WB and the results showed that compared with DHCA group,the expression level of pro-apoptotic protein Bax was significantly decreased in Alda-1 group(P<0.001),and the expression level of anti-apoptotic protein Bcl-2 was significantly increased(P<0.001).In addition,DHCA increased the contents of TNFα,IL-6 and IL-1β,and the expression of MPO.The levels of TNF-α and IL-6 were significantly decreased after Alda-1 pretreatment,and the expression of MPO was also decreased.Conclusion:Alda-1 pretreatment increased the activity of ALDH2 enzyme in DHCA small intestine tissue and cells,but did not affect its protein expression level.Alda-1 pretreatment significantly reduced the accumulation of 4-HNE in DHCA intestinal tissues and cells.Alda-1 pretreatment significantly reduced intestinal injury and tightly connected structure damage in DHCA.Pretreatment with Alda-1 significantly reduced apoptosis and inflammatory response of DHCA.Background:Mitophagy plays an important role in the dynamic homeostasis of mitochondria by removing damaged or abnormal mitochondria.Studies have reported that mitophagy is involved in alleviating organ ischemia-reperfusion injury.Aldehyde dehydrogenase 2(ALDH2)mainly distributes in mitochondria,and Alda-1 alleviates intestinal injury after deep hypothermic circulatory arrest(DHCA)by activating ALDH2 activity.Therefore,exploring the mechanism of Alda-1 pretreatment to reduce DHCA intestinal injury is helpful to provide new ideas for clinical intestinal protection strategies.Method:The rat DHCA model and intestinal crypt cell-6(IEC-6)hypothermia oxygen-glucose deprivation/reoxygenation(HOGD/R)model were built.The effects of Alda-1 pretreatment on the mitochondrial function and morphology of small intestinal tissue were detected in DHCA model of rats,and the presence of autophagosome and the expression of autophagy-related proteins in small intestinal tissue were observed under transmission electron microscopy.In HOGD/R cell model,the HOGD/R group and Alda-1 group were stimulated with mitochondrial autophagy inducer(CCCP)and mitochondrial autophagy inhibitor(Mdivi-1)respectively,and the mitochondria were extracted to detect the expression of mitochondrial autophagy proteins PINK1 and Parkin.The apoptosis level,cell activity,ATP content,mitochondrial superoxide(MitoSOX)and mitochondrial membrane potential(JC-1)levels in each group were compared.Results:Transmission electron microscopy(TEM)was used to observe the number and normal morphology of mitochondria in the small intestinal epithelial cells of rats in the sham operation group,while the morphology and swelling of mitochondria in the DHCA group were abnormal,and the mitochondrial crest disappeared.The damage degree of mitochondria in the Alda-1 pretreatment group was lower than that in the DHCA group.In addition,the average ATP content of cells in control group,HOGD/R group and Alda-1 group was(3.89±0.03 vs.1.25±0.04 vs.1.75±0.24 nmol/mg,p<0.001).HOGD/R model showed that compared with the Control group,HOGD/R significantly upregulated the expression of PINK1 and Parkin proteins(p<0.001),and the expression levels of PINK1 and Parkin proteins in mitochondria after Alda-1 pretreatment were significantly higher than those in HOGD/R group(p<0.001).Compared with the Alda-1 group,the protein expression levels of PINK1 and Parkin were significantly decreased after mdivi-1 stimulation after Alda-1(p<0.001),confirming the effectiveness of mdivi-1,a mitochondrial autophagy inhibitor,which can significantly inhibit mitochondrial autophagy.The expression of pro-apoptotic protein Bax was higher in Alda-1+Mdivi-1 group than that in HOGD/R group(p<0.001),while the expression of anti-apoptotic protein Bcl-2 was significantly lower than that in Alda-1 group(p<0.001),and had no difference with HOGD/R group(p>0.05).Compared with HOGD/R group,the protein expression levels of PINK1 and Parkin were significantly increased after CCCP stimulation(p<0.001),which proved that CCCP can effectively improve the level of mitochondrial autophagy,and the protein expression levels of PINK1 and Parkin were comparable of those in Alda-1 group(p>0.05).The expression level of pro-apoptotic protein Bax in HOGD/R+CCCP group was lower than that in HOGD/R group,and the expression level of anti-apoptotic protein Bcl-2 was higher than that in HOGD/R group(p<0.001),and there was no statistical difference in the expression level of these two kinds of apoptotic proteins compared with Alda-1(p>0.05).The results of JC-1 showed that Alda-1 pretreatment significantly increased mitochondrial membrane potential and alleviated mitochondrial damage(p<0.001),while this protective effect was inhibited after the addition of mdivi-1,an inhibitor of mitochondrial autophagy,and the mitochondrial membrane potential level was comparable of that in HOGD/R group(p>0.05).MitoSOX content of each group was detected,and HOGD/R significantly increased MitoSOX production,Alda-1 significantly decreased MitoSOX production of HOGD/R(p<0.001),while MitoSOX production was significantly increased after mdivi-1 stimulates in Alda-1 group(p<0.001).The content of MitoSOX in HOGD/R+CCCP group was significantly lower than that in HOGD/R group and Alda-1+Mdivi-1 group(p<0.001),but there was no significant difference between HOGD/R+CCCP group and Alda-1 group(p>0.05).Conclusion:Alda-1 preconditioning can protect mitochondrial structure and function and significantly enhance mitochondrial autophagy.Inhibition of mitochondrial autophagy reverses the protective effect of Alda-1 preconditioning on HOGD/R injury of IEC-6 cells.Activation of Parkin/PINK1-mediated mitochondrial autophagy has a protective effect comparable to that of Alda-1 preconditioning.