| Background and objectiveAlcoholic liver disease(ALD)is a liver disease caused by long-term heavy drinking.It is the second common cause of liver injury in China and a serious public health problem to be solved worldwide.At present,there is no specific therapeutic drug for ALD,and alcohol withdrawal and nutritional support are the most important and fundamental interventions.ACY1215 is a selective inhibitor of histone deacetylases 6(HDAC6),which has been proved to improve acute liver injury and liver failure due to its strong anti-inflammatory effect.So far,the effect of ACY1215 on ALD has not been reported.Therefore,this study aims to explore the effect and mechanism of ACY1215 on ALD,so as to provide new strategies for the treatment of ALD.Method1.AML12 cells and Hep G2 cells were cultured in ethanol medium for 24 hours,respectively.The morphological changes of AML12 cells and Hep G2 cells were observed by oil red O(ORO)staining.The expression levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)were detected by enzyme linked immunosorbent assay(ELISA)to evaluate the effect of ethanol on hepatocytes.2.Mice were intragastrically administered with ethanol for 14 days or three times to establish liver injury.The morphological changes of liver were observed by ORO and hematoxylin-eosin(H&E)staining.The levels of ALT,AST,total cholesterol(TC)and triglyceride(TG)in serum of mice were detected by ELISA to evaluate the effect of acute ethanol intragastric administration on liver of mice.3.AML12 cells and Hep G2 cells were co-cultured with ACY1215 and ethanol,respectively.The morphological changes of cells were observed by ORO staining,and the expression levels of ALT and AST were detected by ELISA to evaluate the effect of ACY1215 on ethanol-induced hepatocyte injury.4.After intragastric administration with ethanol for 6 hours,ACY1215 was given to mice for 14 days,or ACY1215 was given to mice for 14 days,then ethanol was given to mice for three times.The morphological changes of liver were observed by ORO and H&E staining.The expression levels of ALT,AST,TC and TG in serum of mice were detected by ELISA to evaluate whether ACY1215 has therapeutic or preventive effects on ethanol-induced liver injury in mice.5.AML12 cells induced by ethanol for 24 hours and AML12 cells co-cultured with 10 μM ACY1215 and ethanol for 24 hours were selected for transcriptome sequencing.Based on the sequencing results,the signaling pathway of ACY1215 to improve ethanol-induced AML12 cells injury was screened.6.Wesern blotting,q RT-PCR and immunofluorescence and immunohistochemistry were used to verify whether ACY1215 could alleviate ethanol-induced liver injury through the signaling pathway screened by transcriptome sequencing in AML12 cells,Hep G2 cells and mice.Results1.Ethanol-induced red lipid droplets were observed in AML12 cells and Hep G2 cells,and the levels of ALT and AST increased.2.Ethanol gavage for 14 days or three times resulted in liver structural damage,inflammatory cell infiltration and lipid deposition,and elevated levels of ALT,AST,TC and TG in serum of mice.3.ACY1215 reduced ethanol-induced lipid droplet formation and decreased the levels of ALT and AST in hepatocytes.4.ACY1215 reduced ethanol-induced liver structural damage,inflammatory cell infiltration and lipid deposition,and reduced serum ALT,AST,TC and TG levels in mice.5.Transcriptome sequencing showed that TLR4/My D88/p38MAPK/NF-κB signaling pathway was the key pathway for ACY1215 to improve ethanol-induced liver injury.6.ACY1215 inhibited the expression of TLR4/My D88/p38MAPK/NF-κB pathway-related molecules in AML12 cells,Hep G2 cells and mouse liver induced by ethanol.Conclusion1.Ethanol can cause damage to AML12 cells and Hep G2 cells,and can also damage the liver of mice.2.ACY1215 had protective effects on ethanol-induced AML12 cells injury,Hep G2 cells injury and mice liver injury.3.ACY1215 improved ethanol-induced liver injury and mice liver injury by inhibiting TLR4/My D88/p38 MAPK/NF-κB signaling pathway. |
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