The Protective Role Of Aldehyde Dehydrogenase 2 In Acute Pancreatitis And The Underlying Mechanisms

Acute pancreatitis(AP)is an acute inflammatory reaction in which digestive proteases are activated in pancreas,which is caused by a variety of causes and result in self-digestion,edema,bleeding,and even necrosis of pancreatic tissue.AP is one of the common causes of acute abdominal pain in the department of emergency,with the characteristics of rapid onset,rapid progress and serious illness.In China,about 400,000 people suffer from AP every year.Most of them are mild acute pancreatitis(MAP),which is self-limited.However,20%to 30%of patients progress to severe acute pancreatitis(SAP).In severe cases,SAP may cause systemic inflammatory response syndrome(SIRS)and multi-organ failure syndrome(MODS),leading to death,with a mortality rate of up to 30%,which seriously endangers patients’ health and living quality of our country.The damage of pancreatic follicular cells is one of the most important causes of MAP progress to SAP.The pancreatic follicular cell accounts for about 80%-85%of the pancreas,which is the functional unit of the exocrine gland of the pancreas.It is the most important cell type of the exocrine part of the pancreas.It can secrete various digestive enzymes,such as trypsinogen,chymotrypsinogen,pancreatic amylase,pancreatic lipase and ribonuclease.Once the trypsin in the pancreatic follicular cells activated,accumulation of inflammatory cells,reactive oxygen species(ROS)and vasoactive substances will eventually lead to a cascade expansion of local inflammation in the pancreas,leading to a conversion from MAP to SAP and having a direct impact on the survival and prognosis of patients.However,the specific mechanism of pancreatic follicular cell damage in AP is not clear.Therefore,it is important to elucidate the specific mechanism of pancreatic follicular cell damage in AP and find effective therapeutic targets.A large amount of ROS production is one of the major pathological changes in the early stage AP,which plays a major role in the pathogenesis of SAP.The pancreas is a digestive gland rich of lipid.The production of ROS in the pancreas causes lipid peroxidation,which leads to an increase in the production of toxic aldehydes.However,the role of lipid peroxidation and toxic aldehydes produced in AP is rarely studied.Aldehyde dehydrogenase 2(ALDH2)is a metabolic enzyme of a variety of toxic aldehydes,such as acetaldehyde,4-HNE and MDA,which can be catalyzed into non-toxic acids.Studies have shown that the increased expression of ALDH2 in rats of heart failure could inhibit the apoptosis of cardiomyocytes,degrade toxic aldehydes and improve left ventricular systolic function.Overexpression of ALDH2 could significantly improve the progression of alcoholic cardiomyopathy and diabetic cardiomyopathy by affecting ASK-1 and GSK-3β signaling pathways.In addition,studies have shown that activation or overexpression of ALDH2 can improve the severity of dilated cardiomyopathy and pulmonary hypertension.Based on the potential role of lipid peroxidation in AP,whether ALDH2 is involved in regulating the occurrence and development of AP as a key enzyme of metabolic toxic aldehydes is a scientific issue that needs to be revealed,which is helpful for the discovery of new therapeutic targets in AP.Therefore,in this study,we will study the accumulation of toxic aldehydes and the protective effect and mechanism of ALDH2 on pancreatic follicular cells in AP.ObjectivesThe main objectives of this experiment are:1.To clarify the accumulation of toxic aldehydes in animal models of AP.2.To exploring the role and mechanism of ALDH2 in AP.3.To demonstrating the role of activation of ALDH2 in the damage of pancreatic follicular cell.Methods(I)In vivo study1.Establishment of cerulean-induced AP model:Male mice 10-week-old,were administered with cerulean(50 μg/kg)1 hour/time for 7 times intraperitoneally;the con group were administered with equal dose of normal saline(NS)intraperitoneally.The mice were sacrificed 24 hours after the last administration.2.Grouping of experimental mice:The mice were randomly divided into 4 groups,each group of 10:Alda-1 control group(Alda-1)and Alda-1 model group(Alda-1+AP),1 h before induction of AP model Alda-1(20 mg/kg)dissolved in DMSO was injected intraperitoneally;the control group(Con)and the AP group were intraperitoneally injected with the same volume of DMSO.Select male C57BL/6J mice(wild type,wild type,WT)mice and transgenic mice(TG)mice over 8 weeks old,randomly divided into 4 groups,each group of 10:control Group(WT),ALDH2 overexpression mouse group(TG),ALDH2 wild-type mouse AP group(WT+AP),ALDH2 gene overexpression mouse AP group(TG+AP),induced AP model.3.Histopathological scoring:Pancreas sections were stained with HE staining.Pancreatic lobular space widening,acinar edema,flaky necrosis,and the infiltration of inflammatory cells in the parenchyma and interstitial tissue were analyzed.Furthermore,the distribution of red blood cells in the pancreas was analyzed.4.Detection of apoptosis of pancreatic follicular cells:Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL),transmission electron microscopy and Western Blot were used to detect the apoptosis of pancreatic follicular cells respectively in cellular,subcellular and molecular level.5.Detection of toxic aldehyde level:Immunohistochemistry and Western Blot was used to detect the levels of 4-HNE and MDA in pancreatic tissue;ELISA and colorimetric method were used to detect the contents of 4-HNE and MDA in human serum.6.ALDH2 expression and enzymatic activity measurement:RT-qPCR and Western Blot were performed to measure the mRNA and protein expression of ALDH2 in pancreatic tissue respectively.The mitochondria isolated from pancreatic tissue and enzymatic activity of ALDH2 was determined by measuring the conversion of NAD+to NADH.(II)In vitro study1.Establishment of pancreatic follicular cell line(AR42J)of AP and aldehyde toxicity model:AR42J cells were administrated with cerulean(10nM,dissolved in normal saline)for 24 h to induce AP model.The Con group was treated with NS.4-HNE(dissolved in DMSO)was used to treat the pancreatic follicular cell for 12 h to establish the aldehyde toxicity model.Before cerulean or 4-HNE treatment,Alda-1(20 μM,dissolved in DMSO)was pretreated for 30 min(the control group administrated equal volume of DMSO).2.Evaluation apoptosis of pancreatic follicular cell:TUNEL and Annexin V-FITC/PI were used to evaluate the apoptosis ratio;Western Blot was used to evaluate the protein expression of mitochondrial apoptotic signal pathway indicators.3.Detection of mitochondrial membrane potential:JC-1 was used to detect the mitochondrial membrane potential.4.Establishment of ALDH2 overexpression/knockout cell lines:AR42J cells were transfected with ALDH2-overexpressed lentivirus.To confirm whether the construction of ALDH2-overexpressed AR42J cell line was successful,the expression efficiency of ALDH2 protein was verified after the selection of puromycin.The ALDH2-knockout gene vector was constructed by CRISPR-Cas9 technology,and puromycin screening was conducted after the knockout efficiency was verified.Sequencing verification and Western Blot were conducted after clone formation selection.5.Detection and activation of AKT phosphorylation level:Western Blot was used to detect the level of phosphorylated AKT and total AKT in pancreatic tissues or follicular cells.AKT phosphorylation activator(SC79)was used to increase the level of phosphorylation of AKT in pancreatic follicular cells.6.Bioinformatics analysis and verification strategy:Combined analysis was conducted on the transcriptome results of two pancreatitis tissues in the published database—GSE109227 and GSE119844.Functional enrichment analysis was performed after the finding of differentially expressed genes in AP.Western Blot was used to validate the differentially expressed genes.Results1.Toxic aldehydes 4-HNE and MDA was accumulated in the pancreatic tissue of APMDA level in serum of AP model mice increased significantly,which was positively correlated with the severity of AP.Using immunohistochemical staining and Western Blot,it was found that the levels of 4-HNE and MDA in the pancreatic tissue was increased significantly in the AP group compared with the Con group.Besides,serum 4-HNE and MDA levels of AP patients were increased significantly compared with that of healthy volunteers.2.Accumulated toxic aldehydes in the pancreatic tissue led to the apoptosis of pancreatic follicular cellBioinformatics analysis showed that the expression of pro-apoptotic gene Bax was increased in the pancreatic tissue and verified by Western Blot.TUNEL staining showed that the proportion of pancreatic follicular cell apoptosis was increased in AP tissue and AP cell model,and the pancreatic follicular cells administrated by 4-HNE in vitro showed a higher proportion of apoptosis compared with the Con group.3.The activity of ALDH2 in pancreatic tissue was inhibited in the pancreatic tissue of APFirstly,ALDH2 mRNA and protein expression was detected.It was found that the expression of ALDH2 in the AP group was not changed significantly compared with the Con group,while ALDH2 activity in AP group was significantly reduced.4.Overexpression of ALDH2 reduced toxic aldehydes accumulation and pancreatic follicular cell apoptosisCompared with the WT group,a significant accumulation of MDA and 4-HNE were detected in the WT+AP group.Compared with the WT+AP group,the accumulation of MDA and 4-HNE in the TG+AP group was significantly reduced.TUNEL staining of pancreatic tissue showed that the apoptosis of pancreatic cells reduced significantly in the Alda-1+AP group compared with the AP group,and it was in coincidence with the trend of mitochondrial apoptotic index.5.Overexpression of ALDH2 reduced AP-induced apoptosis of pancreatic follicular cellCompared with the Con group(NC),the expression of ALDH2 in pancreatic follicular cells transfected with ALDH2 lentivirus(ALDH2-OE group)was increased significantly.Compared with the Con group,the expression of ALDH2 in the ALDH2-KO group was decreased significantly.Overexpression of ALDH2 could reduce the proportion of pancreatic follicular cell apoptosis and knockout of ALDH2 could increase the proportion of pancreatic follicular cell apoptosis compared with that of in the AP group.6.Activation of ALDH2 ameliorated toxic aldehydes accumulation,pancreatic follicular cell apoptosis and pancreatic tissue damageCompared with the AP group,the pancreas weight and pancreas/body weight ratio were decreased significantly in the Alda-1+AP group.HE staining demonstrated that,in the Alda-1+AP group,the pancreatic lobular space widened,acinar edema,flaky necrosis,parenchymal and interstitial inflammatory cell infiltration were ameliorated significantly,as well as decreased red blood cell distribution.Compared with the AP group,cell apoptosis was decreased significantly in the Alda-1+AP group,which was consistent with the change of apoptotic pathway-related proteins.7.Activation of ALDH2 mitigated apoptosis of pancreatic follicular cell by alleviating AKT phosphorylation inhibition induced by toxic aldehydesIn AR42J treated with cerulean,AKT phosphorylation was decreased significantly compared with the Con group.Compared with the AP group,AKT phosphorylation was increased significantly in the Alda-1+AP group.Similarly,after treatment with 4-HNE,it was found that the AKT phosphorylation was decreased significantly,Bax and Cleaved Caspase 3 were increased significantly,while the expression of Bcl-2 was decreased significantly in the AP group compared with the Con group.Compared with the 4-HNE group,AKT phosphorylation was increased significantly in the Alda-1+4-HEN group.In order to clarify the relationship between the AKT phosphorylation and 4-HNE,AKT phosphorylation activator(SC79)in combination with 4-HNE was administrated to AR42J.It was found that the expression of Bax and Cleaved Caspase 3 were decreased and the expression of Bcl-2 was increased in the SC79+4-HNE group compared with the 4-HNE group.Conclusion1.The toxic aldehydes 4-HNE and MDA are accululated in the pathological process of pancreatitis.2.Toxic aldehydes accumulation in AP inhibit phosphorylation of AKT,leading to increased apoptosis of pancreatic follicular cells,thereby aggravating the severity of AP.3.Activation of ALDH2 mitigates apoptosis of pancreatic follicular cell by reversing the decrease of AKT phosphorylation induced by cerulean or 4-HNE.