The Role And Mechanism Of PDIA3 In Alcoholic Liver Injury

Background: alcoholic liver disease(ALD)is one of the most frequent liver diseases in many countries around the world,even in China.The manifestation of early alcoholic liver disease is that alcoholic fatty liver gradually evolves into alcoholic steatohepatitis,liver fibrosis,and in severe cases,liver cirrhosis and hepatocellular carcinoma.It is important to understand the pathogenesis of alcoholic liver disease.Endoplasmic reticulum stress(ER stress)is involved in the production of many diseases,including ALD.As one of the pathogenesis,it is of great significance to explore the detailed role of ER stress in alcoholic liver disease.PDI family is related to endoplasmic reticulum stress.As a resident factor in the endoplasmic reticulum,it is mainly located in the endoplasmic reticulum.It may play a role in many liver-related diseases and is one of the important roles.However,the role of PDI family in alcoholic liver disease and the specific related molecular mechanism have not been studied and explored in detail.Therefore,this article intends to explore the role and related molecular mechanism of PDI family factors in alcoholic liver disease.Objective:To explore the role and mechanism of PDIA3 in ALD,ALD models were constructed in vivo and in vitro.To investigate whether PDIA3 can affect ALD disease progression by regulating lipid metabolism through endoplasmic reticulum stress.Methods:1 A mouse model of alcoholic liver NIAAA was established in vivoTwenty male C57BL6 mice were divided into model blank group and model blank group,and alcoholic liver model was established in the model group.The mice were fed with liquid diet for 5 days,and then fed with 5% to 6% alcohol liquid diet for 10 consecutive days.On the morning of the 11 th day,the mice were given a high dose of alcohol by gavage,and the animals were treated about 9 hours later.Blood and liver samples were collected for experiments.The liver tissues of mice were taken and placed on clean filter paper for photography,weighing and recording.The liver was placed in Paraformaldehyde to fix the tissue specimen,and hematoxylin-eosin H&E and oil red O staining were used to check the changes in the mouse liver.2 Check expression of PDIA3 in NIAAA alcoholic liver model miceWestern blotting to check expression of PDIA3.3 Alcoholic hepatocyte model was constructed in vitro to detect the expression level of PDIA3 factorMouse liver parenchymal cells were cultured in six-well plate for about 24 hours,and the cell density and state in the six-well plate were observed until the cell density reached about 70%,and the alcoholic liver model was constructed for about 24 hours.Western blotting to check expression of PDIA3.4 To explore the role of PDIA3 in endoplasmic reticulum stress4.1 Silencing PDIA3Mouse liver parenchymal cells were cultured in six-well plates for about 24 hours,and the cell density and state in six-well plates were observed until the cell density reached about 40%.Cultured mouse liver parenchymal cells were randomly divided into four groups: blank group;Blank + silencing PDIA3(si-PDIA3)group;Alcohol model group;Alcohol model + silencing PDIA3(si-PDIA3)group;Western blotting to check expression of CHOP,GRP78 and ATF4.4.2 Overexpression of PDIA3Mouse liver parenchymal cells were cultured in six-well plates for 24 hours,and the cell density and state in six-well plates were observed until the cell density reached about 30%.The cultured mouse liver parenchymal cells were randomly divided into four groups: blank group(pc DNA3.1);Blank + PDIA3 overexpression group(pc DNA-PDIA3);ETOH group(ETOH+pc DNA3.1);Alcohol model + PDIA3 overexpression group(pc DNA-PDIA3);Western blotting to check expression of CHOP,GRP78 and ATF4.5 To explore the role of PDIA3 in lipid metabolism5.1 Silencing PDIA3Mouse liver parenchymal cells were cultured in six-well plates for 24 hours,and the cell density and state in six-well plates were observed until the cell density reached about 40%.The cultured hepatocytes were randomly divided into four groups: blank group;Blank + silencing PDIA3(si-PDIA3)group;Alcohol model group;Alcohol model + silencing PDIA3(si-PDIA3)group;Western blotting to check expression of PPARα,CPT1αand SREBP1 c.5.2 Overexpression of PDIA3Mouse liver parenchymal cells were cultured in six-well plates for about 24 hours,and the cell density and state in six-well plates were observed until the cell density reached about 30%.The cultured hepatocytes were randomly divided into four groups:blank group(pc DNA3.1);Blank + PDIA3 overexpression group(pc DNA-PDIA3);ETOH group(ETOH+pc DNA3.1);Alcohol model + PDIA3 overexpression group(pc DNA-PDIA3);Western blotting to check expression of PPAR α,CPT1 α and SREBP1 c.6 To explore the relationship between PDIA3 and endoplasmic reticulum stress and lipid metabolismHepatic parenchymal cells were cultured in six-well plates for 24 hours,and the cell density and state in six-well plates were observed until the cell density reached about 40%.Hepatocytes were randomly divided into four groups: blank group;Alcohol model group;Alcohol model + silencing PDIA3(si-PDIA3)group;Alcohol + silencing PDIA3(si-PDIA3)+ endoplasmic reticulum stress inhibitor group;The expression levels of ATF4,GRP78 and CHOP-related endoplasmic reticulum stress markers and lipid metabolism-related indicators CPT1α,PPARα and SREBP1 c were detected.Results: The expression of PDIA3 in the liver tissue of mice with alcoholic liver model decreased and the tissue had obvious lesions.Oil red O staining showed that there was fat accumulation in the liver of the mice in the alcoholic liver model group,and HE staining showed that the liver tissue of the mice in the alcoholic liver model group had obvious damage.After ALD was induced in vitro,the expression level of PDIA3 was decreased,and the endoplasmic reticulum stress markers CHOP,GRP78 and ATF4 were significantly increased,while CPT1α and PPARα,which were involved in lipid metabolism,were decreased.In addition,SREBP1 c,which is involved in lipid synthesis,was significantly increased under ethanol induction.The expression of CHOP,GRP78,ATF4 and SREBP1 c was further increased,while the expression of CPT1α and PPARα was significantly decreased after PDIA3 was knocked down in AML12 mice hepatocytes in vitro.After overexpression of PDIA3,the expression of endoplasmic reticulum stress and lipid metabolism related indicators were improved to a certain extent.These results suggest that PDIA3 may affect ALD disease progression through endoplasmic reticulum stress and lipid metabolism.After knocking down PDIA3 in vitro and giving a certain dose of ER stress inhibitor,the expression levels of ER stress and lipid metabolism related indicators were improved compared with PDIA3 knockdown group.These results suggest that PDIA3 may affect the progression of ALD by affecting lipid metabolism through PERK pathway of endoplasmic reticulum stress.Conclusion: This is the first study to demonstrate the protective role of PDIA3 in alcoholic liver disease.PIDIA3 can regulate lipid metabolism-related factors by affecting the PERK pathway in the UPR pathway of endoplasmic reticulum stress,thereby affecting the disease process of alcoholic liver disease.