| Objective To study the effect of the new drug ammonium dihydromyricetin on hangover and liver protection in mice with acute and chronic alcoholic liver injury,and to explore its mechanism.Methods1.SPF C57BL/6J mice were divided into 5 groups,then with normal mice as a blank control,model group(alcohol group),dihydromyricetin group(5 mg/kg,DHM group),low dose group of ammonium dihydromyricetin(5 mg/kg,low TDHM group),high dose group of ammonium dihydromyricetin(10 mg/kg,high TDHM group).First,each drug group was given the corresponding dose of drugs intragastrically,and the blank control group and alcohol group were given the same volume of normal saline intragastrically,after 30 minutes,the alcohol group and the drug group were gavaged with 56° liquor(0.25 mL/20 g)to establish an acute alcoholism model.Observe the drunkenness and alcohol tolerance time of each group of mice.6 hours when the mice were killed,the colorimetric method was used to detect the changes of AST and ALT in peripheral blood.Concentration of ADH and ALDH in liver tissue and β-EP and LENK in brain tissue were detected by ELISA.2.The SPF-class C57BL/6J mice were divided into 5 groups,and the grouping was the same as above.The model of chronic alcoholic liver disease was established by referring to the NIAAA model(Gao-Binge model).Each group was fed to the control diet for 5 days,then the alcohol group and the drug group were replaced with5% alcohol liquid feed.Each morning,each drug group was given the corresponding dose for 10 days.The next morning alcohol group and DHM group,the low and high TDHM groups were intragastrically administered with 31.5% ethanol.The normal blank group was intragastrically administered with 45.0% maltodextrin solution,and the mice were killed 9 hours later.Observe the general state of mental state and body weight of mice every day,through HE and oil red O staining,differences of fatty change among liver tissues of mice from various groups are observed.the colorietricmethod is used to detect the AST,ALT levels of liver tissue,the biochemical assay is used to detect the TG,ADH and ALDH levels,the ELISA is used to detect SOD,MDA levels of liver tissue,and the Western-blot method is used to detect the expression level of CYP2E1.Results1.Results of the hangover alleviating and hepatoprotective effect of ammonium dihydromyricetin on model mice with acute alcoholism:(1)Behavioral observation: both low and high TDHM groups(41.40±21.03 min,50.90±35.83 min vs 11.30±8.16min)could prolong the drunkenness time of mice(p<0.001,p<0.01),both were significantly higher(p<0.05)than that of DHM group(41.40±21.03 min,50.90±35.83 min vs 17.70±15.55min).The sobering time of mice in high TDHM group was shorter than that in alcohol group(152.40±46.04 vs256.71±36.54,p<0.001),both DHM group and low TDHM groups(188.40±49.09 min,172.40±45.66 min vs 256.71±36.54min)were also shorter than that in alcohol group(p<0.01),but there was no significant difference between TDHM group and DHM group.(2)Serum ALT and AST levels: The activity of ALT in DHM group and high TDHM group(10.70±3.08,8.58±4.83 vs 17.72±6.84)were significantly lower than that in alcohol group(p<0.05),but there was no significant difference between TDHM group and DHM group.The activity of AST in blood of mice in DHM group,low and high TDHM group(45.35±10.17,33.02±2.68,32.21±8.11 vs64.12±15.00)was lower than that in alcohol group(p<0.05,p<0.001,p<0.01),both low and high TDHM groups were significantly lower than that in DHM group(p<0.05).(3)Concentration of ADH and ALDH in liver:Compared with alcohol group,DHM group,low and high TDHM groups(0.38±0.02,0.51±0.13,0.49±0.06 vs0.30±0.06)could increase the concentration of ADH(p<0.05,p<0.01,p<0.001),and the effect of low and high TDHM groups were more significant than that of DHM group(p<0.01).The concentration of ALDH in low and high TDHM groups(13.38±3.85,15.99±5.91 vs 7.40±4.47)were significantly higher than that in alcohol group(p<0.05),all TDHM groups were significantly higher than that in DHM group(13.38±3.85,15.99±5.91 vs 7.86±3.15,p<0.05).(4)Concentrations of β-EP and LENK in brain:Compared with alcohol group,the level of β-EP in DHM group,low and high TDHM groups(324.82±25.54,309.83±32.55,283.89±38.42 vs 384.28±39.83)was decreased(p<0.05,p<0.01,p<0.01),low and high TDHM groups were significantly decreased(309.83±32.55 vs384.28±39.83,p<0.01;283.89±38.42 vs 384.28±39.83,p<0.01);DHM and TDHM groups were not significantly different.There was no significant difference in LENK concentrations between the groups of mice.2.Hepatoprotective effects of ammonium dihydromyricetin on mice with chronic alcoholic liver injury:(1)Pathological changes of HE staining and oil red O staining: Compared with the control group,the liver tissue of the alcohol group had severe steatosis,and the DHM group and the low and high TDHM group had milder steatosis than the alcohol group.(2)The TG content in liver:Compared with alcohol group,the level of TG in DHM group and low TDHM group(0.08±0.007,0.08±0.004 vs 0.11±0.02)decreased significantly(p<0.05,p<0.01),and the level of TG in low TDHM group was significantly lower than that in DHM group(p<0.05).(3)Levels of ALT and AST in liver:The levels of ALT and AST in liver of mice in alcohol group were significantly lower.the levels of ALT in DHM group and low TDHM group(2.14±0.52,2.12±0.56 vs 1.51±0.17)were significantly higher than those in alcohol group(p<0.05),but there was no significant difference between TDHM group and DHM group.Compared with alcohol group,the levels of AST in DHM group,low and high TDHM group(5.00±0.42,5.53±0.38,5.57±0.82 vs 3.97±0.45)were significantly higher(p < 0.01,p < 0.001,p < 0.01),low TDHM was more significantly higher than DHM group(p<0.05).(4)Activities of ADH and ALDH in liver:Compared with alcohol group,both low and high TDHM groups(25.02±12.13,23.16±13.33 vs 10.18±3.26)all could increase the level of ADH(p<0.05).Compared with alcohol group,the level of ALDH in DHM group and high DHM group(74.54±14.56,82.77±20.93 vs 56.02±14.17)were higher than that in alcohol group(p<0.05),and the level of ALDH in low TDHM group was significantly higher(105.18±25.45 vs 56.02±14.17,p<0.01),and the effect of low TDHM group was more obvious than that in DHM group(p<0.05).(5)MDA and SOD concentration in liver: Compared with alcohol group,MDA in DHM group,low and high TDHM groups(40.28±1.48,35.30±4.82,34.88±4.36 vs 47.29±4.35)and decreased significantly(p<0.01,p<0.01,p<0.001),low and high TDHM groups decreased significantly compared with DHM group(p<0.05).Compared with alcohol group,SOD in low and high TDHM groups(8.63±0.98,8.76±2.91 vs 5.55±1.10)could increase(p<0.001,p<0.05),but there was no significant difference between TDHM group and DHM group.(6)The level of CYP2E1 in liver: compared with alcohol group,the level of CYP2E1 in DHM group low and high TDHM groups were lower than that in alcohol group(p<0.05,p<0.01,p<0.01),and the level of CYP2E1 in low and high TDHM group was significantly lower than that in DHM group(p<0.05,p<0.01).Conclusions1.Like DHM,TDHM can relieve alcohol and promote awakening in mice with acute alcoholism.The mechanism may be related to reduceing liver cell damage,increaseing metabolism and reduceing brain tissue β-EP,and the effect of hangover and brain-wakening is more significant than DHM.2.Like DHM,TDHM has protective effect on chronic alcoholic liver disease.its mechanism may be related to reducing hepatocyte destruction,increaseing metabolism,reducing TG,MDA and CYP2E1 levels,anti-oxidation and reducing oxygen free radicals,and its protective effect is better than DHM.3.TDHM,as a water-soluble derivative of DHM,can improve the bioavailability of drugs and is expected to become a new generation of preventive and therapeutic drugs for ALD. |
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