Mechanism And Clinical Application Of ALDH2 In The Regulation Of Autophagy To Protect Acute Liver Injury Caused By Sepsis

Background&AimsSepsis refers to a life-threatening organ dysfunction caused by an infection-induced host response disorder,and organ dysfunction had been an essential component in the diagnosis of sepsis.Sequential Organ Failure Assessment(SOFA)score was used for evaluation of sepsis.Multicenter clinical studies had shown that sepsis with organ dysfunction could lead to higher mortality and readmission rates,and it could significantly affect patients'life quality and long-term prognosis for survivors.Therefore,it is important to explore the molecular regulation mechanism of sepsis leading to organ dysfunction.To find new targets and molecular markers to actively prevent organ dysfunction in patients with sepsis is the key to improve the prognosis of patients with sepsis.Aldehyde dehydrogenase 2(ALDH2)is an important detoxification enzyme secreted by liver cells,it is highly expressed in high-metabolic tissues and organs such as heart and brain tissues.Its sub-organism location is the mitochondria of cells,and it can protect the body against endogenous and exogenous harmful substances.Numerous studies had shown that ALDH2 played a crucial protective role in a range of diseases,including ischemia-reperfusion injury,heart failure,alcoholic fatty liver,neurological dysfunction,and diabetes.As a crucial detoxifying enzyme in the human body,ALDH2 can quickly remove ROS and aldehyde metabolites from the body,inhibit the process of OS,and alleviate mitochondrial damage and ER-Stress,thus protecting cells from damage.Autophagy is a highly conservative self-degradation process in eukaryotic cells,which mainly degrades senescent or damaged organelles in cells.Mitophagy plays an important role in maintaining the normal function and morphology of cells.The role of ALDH2 in autophagy had also been demonstrated in our previous studies.High expression of ALDH2 regulated autophagy and apoptosis by balancing Akt and AMPK and downstream expression of m TOR,STAT3,and Notch1.Therefore,we believe that ALDH2 may protect acute liver injury in sepsis by regulating autophagy.The studies of ALDH2 in sepsis are few,and previous studies were focused on its role in kidney damage,myocardial failure,and lung function damage caused by sepsis.Besides,there is no literature report on the role of ALDH2 and its regulatory mechanisms in liver damage caused by sepsis.As an immune and metabolic organ,the liver is not only a protector,an immune regulator,but also a target organ for damage during the occurrence,development and treatment of sepsis.Therefore,we used ALDH2 transgenic mice to establish a model of acute liver injury in sepsis and collected blood and tissue samples at various stages of the process for detection to explore the two-way regulation mechanism of ALDH2 on autophagy in the process of disease.In addition,we use clinical samples and prognostic information analysis to evaluate the value of ALDH2 as an intervention target for acute liver injury in sepsis and a prognostic evaluation index,which has important scientific and clinical significance.Methods1.The correlation between ALDH2 activity and the patient's disease progression was monitored.The clinically relevant information such as liver injury indicators,inflammation indicators,oxygen saturation was analyzed for clarifying the possible role of ALDH2 in clinical treatment efficacy and prognostic assessment of patients.2.Transgenic technology was applied to construct ALDH2 overexpression mouse models.By inserting the amplified ALDH2 target fragments into the Eco RI cloning site of the vector p Bs CAG-2 downstream of the CAG box,ALDH2 overexpression mouse model was prepared for the follow-up experiments.Western-blot and IHC were performed to verify the overexpression of ALDH2 in liver tissues.3.Cecal ligation and puncture(CLP)method was applied for inducing acute liver injury with ALDH2^+/+mouse.4.After the establishment of CLP induced sepsis with acute liver injury mouse model,blood and liver tissue samples were collected,and the related indicators were detected to check the dynamic changes of ALDH2 with the disease process and its impact on the changes in the disease.5.The prognostic information of each group of mice was collected,and statistical analysis on the collected prognostic information was performed to compare the survival rate among the groups.6.Mouse liver samples at different stages of the above groups were collected.Realtime-PCR,Western-blot,and Haematoxylin-Eosin(H&E)staining were performed to detect the expression of ALDH2 and liver morphology for observation of the dynamic relationship between ALDH2 expression and liver cell morphology among groups;7.ALDH2 activity detection kit and ELISA kit were used to detect the level of ALDH2,the expression of alanine aminotransferase(ALT),alkaline phosphatase(ALP)and aspartate aminotransferase(AST)in the serum of the above groups at different stages.The relationship between the activity level of ALDH2 and the liver damage indicators was compared among the groups;8.The expression of infection indicators-TNF-?and IL-6 in the serum were detected by ELISA in each group.9.The expression and localization of ALDH2 and the expression of TNF-?and IL-6 in liver tissues were detected in each group.10.Liver tissue samples from the mouse models in each time point were collected.WB were applied to detect the expression of the LKB1 and AMPK.Realtime-PCR and WB were applied to detect the expression of the autophagic flux biomarker LC3-II in the liver tissues.Autophagy inhibitors were used to inhibit autophagy in mice,and the apoptosis of liver tissue was observed.11.After the digestion and purification of mouse liver cells,a model of LPS-induced cell inflammation was established.Tunel and LC3II were detected for the measuring of cell apoptosis and autophagy flux.Results1.The white blood cell and procalcitonin(PCT)levels of patients in the ALDH2 high activity group of sepsis were lower than those in the low activity group of ALDH2(P<0.05);patients in the ALDH2 high activity group of sepsis have significantly lower levels of aminotransferase(ALT),aspartate aminotransferase(AST)and total bilirubin(STB)than those in the ALDH2 low activity group(P<0.05).2.ALDH2 overexpression transgenic mouse was obtained,and the expression of ALDH2 in liver of ALDH2^+/+was significant higher than that of control group which was verified by Western-blot and IHC,respectively(P<0.05).3.We have established a ALDH2 overexpression mouse model with cecal ligation,and puncture(CLP-ALDH2^+/+);4.When testing the activity of ALDH2 in the plasma of each group of mice,it was found that the activity of ALDH2 in ALDH2^+/+mice was significantly higher than that of the control group,and the activity of ALDH2 during sepsis was also significantly higher than that of the control group.HE staining of liver tissue indicated that the liver tissue morphology of ALDH2^+/+mice was significantly reduced compared with the control group,and the survival analysis also found that the prognosis of ALDH2^+/+mice was significantly improved than that of the control group(P<0.05);5.Compared with the control group,the CLP-ALDH2^+/+group has significantly lower expression of TNF-?and IL-6 in plasma(P<0.05);6.Immunofluorescence staining was performed on the liver tissues of mice in the CLP sepsis acute liver injury group with mouse macrophage markers(F4/80)and inflammation indicators TNF-?and IL-6.The results suggest that compared to CLP-WT group,the liver inflammation indexes of the CLP-ALDH2^+/+mice were significantly reduced(P<0.05).7.Western blotting results indicate that compared to CLP-WT group,LC3-II in the liver tissue of CLP-ALDH2^+/+mice was significantly increased by 1.5-2.0 times.With the inhibitors of autophagy,the apoptosis of hepotocytes increase.8.ALDH2 overexpression alleviates LPS-induced apoptosis,and inhibition of autophagy increases the apoptosis in hepatocytes.9.ALDH2 activates autophagy through LKB1/AMPK signaling pathway.ConclusionsOur experiments in vivo and in vitro indicate that ALDH2 has a significant protective effect in the process of acute liver injury in sepsis.It can reduce the apoptosis in liver and play a role in the recovery of liver function.Our research suggests that ALDH2 can reduce inflammation by reducing the secretion of IL-6 and TNF-?.In the exploration of the mechanism,we find that ALDH2 can enhance autophagy to protect the function of liver cells in the acute phase of sepsis and reduce the occurrence of liver cell apoptosis.Based on the analysis of clinical samples,we can conclude that ALDH2 has a protective effect in patients with acute liver injury in clinical sepsis,and can significantly reduce the occurrence of acute liver injury.ALDH2 can be used as an intervention target for sepsis and acute liver injury.The value of evaluation indicators for patient prognosis has important scientific research and clinical translation significance.