| Alcohol is a major cause of alcohol-related liver disease and is associated with high morbidity and mortality.According to the World Health Organization(WHO),3.0 million people die each year from alcohol-related causes.Long-term alcoholism can lead to a range of physical illnesses.Acute or chronic alcohol intake can lead to liver disease(alcoholic hepatitis,steatosis,steatohepatitis,fibrosis and cirrhosis).Drinking can increase fat production and inhibit the oxidation of fatty acids,among which alcoholic hepatic steatosis,more than 90%,is the most common,characterized by excessive accumulation of lipid droplets in hepatocytes,of which 35%develop into steatohepatitis,fibrosis,and cirrhosis.After ingesting alcohol,some of the ethanol is first metabolized by the action of alcohol dehydrogenase in the gastric mucosa.Most of the ethanol is mainly metabolized in the liver,and the liver is the main site of ethanol metabolism.There are three main metabolic pathways of ethanol in hepatocytes:dehydrogenase oxidative metabolism;cytochrome P450 enzyme system metabolism,the most common is CYP2E1;peroxidase system metabolism.Dehydrogenase in the liver is the main enzyme responsible for alcohol metabolism.Alcohol is first metabolized to acetaldehyde by alcohol dehydrogenase(ADH)and then metabolized to acetic acid by aldehyde dehydrogenase(ALDH).Alcoholic liver disease is associated with hepatotoxicity of alcohol and the resulting toxic acetaldehyde.ALDH2 has the highest affinity for acetaldehyde and is the most important enzyme for the metabolism of acetaldehyde.It catalyzes the irreversible dehydrogenation of various endogenous and exogenous aldehydes into corresponding non-toxic carboxylic acids.Approximately 40%of the East Asian population has semi-dominant polymorphisms of ALDH2*2,which are essentially inactive in vivo.In this experiment,ethyl acetate was used as the extractant to extract the air-dried high quality Sedum sylvestris whole grass.The obtained solvent extract was concentrated by a rotary vacuum evaporator,and then separated by silica gel column chromatography.Initially identified by thin layer chromatography,fractions having similar Retardation factor values(Rf)were combined and the solvent was again evaporated to obtain subcomponents.The identified extract was further analyzed by high performance liquid chromatography with a component having a specific Rf of 0.58,and the obtained extract was determined to be Taraxerone.Secondly,an acute mouse drunkenness model and a chronic mouse model of alcoholic liver injury were established.Using metadoxine as a positive control,the obtained extract(Taraxerone)was used for the treatment of mice to observe the hangover effect.The experimental results showed that the liver-to-human ratio of the mice in the alcohol group was calculated to be 0.0988%,which was significantly higher than the control group by 0.0530%,which was 1.86 fold higher than that of the control group.After treatment with Metadoxine and Taraxerone,the mice were compared.The liver index was 0.0753%and 0.0802%,respectively,which was 1.42 fold and 1.51 fold higher than that of the control group,indicating that Metadoxine and Taraxerone have ameliorating effects on alcohol-induced liver hypertrophy.In the acute alcohol test of mice,the activity of ADH and ALDH enzymes in the liver gradually decreased within 1 h,and then gradually recovered,indicating that a large amount of alcohol inhibited the activity of ADH and ALDH enzymes,and Metadoxine and Taraxerone treatment increased ADH and ALDH enzymes.Activity to accelerate alcohol metabolism.Compared with the control group,the ADH and ALDH activities in the alcohol group of chronic alcohol mice were 32.37 U/mgprot and 40.68 U/mgprot,respectively,which were ADH(20.63 U/mgprot)and ALDH(17.18 U/mgprot)in the control group,respectively.1.56 fold and 2.37 fold,the ADH activities of mice treated with Metadatin and Taraxerone were 42.53 U/mgprot and 40.06 U/mgprot,respectively,which were 2.06 fold and 1.94 fold higher than the control group,respectively,and the ALDH activities were 53.64 U/mgprot and47.02 U/mgprot,respectively,increased by 3.12 fold and 2.74 fold respectively.The liver pathological section of the mice showed that the liver of the mice in the alcohol group was severely damaged,the hepatic cord arrangement was disordered,the macrobubble was fatty-like,and the liver fibrosis was observed.After treatment with Metadoxine and Taraxerone,The liver sections of the treated mice showed that the liver plates were arranged neatly and the liver cells were not steatotic.Acute alcoholic gavage and chronic alcoholic liver injury in mice increased the levels of TG,AST,ALT and AKP in serum.Metadoxine and Taraxerone reduced the levels of TG,AST,ALT and AKP,which were statistically significant.The TG content of the mouse liver slurry in the alcohol group was 2.27 mmol/gprot,which was 1.52 fold that of the control group(1.49 mmol/gprot).After treatment with Metadoxine and Taraxerone,the TG content was 1.76 mmol/gprot and 1.82 mmol/gprot,respectively.They were 1.18 fold and 1.22 fold to the control group,respectively.It shows that metadoxine and Taraxerone can reduce TG content in liver.Then,alcohol,Metadoxine and Taraxerone were used to treat immortalized human normal liver cell HL7702 cells.It was observed that the number of HL7702 cells in the alcohol group was small,and the number of other groups was higher.The cells were evenly distributed in a single layer,indicating that alcohol has HL7702 cell growth.Inhibition,Metadoxine and Taraxerone slow down the inhibition of alcohol by cells.Oil red O staining results showed that there was a large amount of lipid deposition in the alcohol group,and the orange-red lipid droplets were clearly seen,and the lipid droplets in the cells were arranged in a beaded shape with uniform distribution and clear boundaries.After treatment with Metadoxine and Taraxerone,there are a small amount of lipid droplets in the cells,which are unevenly distributed and disorderly arranged.It shows that Metadoxine and Taraxerone can reduce the formation of lipid droplets.The TG content of HL7702 cells in the alcohol group was 1.18 mmol/gprot,which was significantly increased by 1.93 fold compared with the control group of 0.61 mmol/gprot,which was statistically significant.After treatment with Metadoxine and Taraxerone,the intracellular TG content was relatively reduced,0.86mmol/gprot and 0.91 mmol/gprot,respectively,with significant differences.It shows that Metadoxine and Taraxerone have the effect of inhibiting intracellular TG synthesis.Finally,based on this experiment,the molecular docking method was used to explore the mechanism,and the binding energy of small molecule to protein was-8.02 kcal/mol(Taraxerone),-4.24 kcal/mol(PCA),-4.03 kcal/mol(VB6).Observing the interaction between small molecules and acetaldehyde dehydrogenase,the results show that the small molecule of the ligand is in the hydrophobic cavity of the protein molecule,and the interaction between the protein and the protein is mainly hydrogen bonding interaction and water transport.The above experiments prove that the extracted Taraxerone has the effect of hangover,can increase the activity of dehydrogenase in the liver,reduce the activity of transaminase in serum,and reduce the formation of lipid droplets to reduce liver damage.The results of molecular docking indicate that there is a hydrophobic interaction and hydrogen bonding between Taraxerone and acetaldehyde dehydrogenase.Taraxerone acts on the coenzyme NAD~+by hydrogen bonding to maintain the stability of acetaldehyde dehydrogenase,thereby increasing the activity of acetaldehyde dehydrogenase.The interaction of Taraxerone with protein water delivery and hydrogen bonding activates the activity of the enzyme. |
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