|Alternative Title||Protective Effect and Mechanism of Dihydromyricetin Ammonium Salt on Alcoholic Liver Injury in Mice|
|Place of Conferral||兰州|
|Keyword||二氢杨梅素 二氢杨梅素衍生物 酒精性肝损伤|
方法：1.将SPF级C57BL / 6J小鼠分为5组，即空白对照组（正常小鼠），模型组（酒精组），二氢杨梅素组（5 mg/kg，DHM组），二氢杨梅素铵盐低剂量组（5 mg/kg，低TDHM组），二氢杨梅素铵盐高剂量组（10 mg/kg，高TDHM组）。DHM组及低、高TDHM组均予以相应剂量药物灌胃，空白对照组、酒精组予以等体积生理盐水灌胃，30 min后，酒精组及各药物组予56°白酒（0.25 mL/20 g）灌胃，建立急性酒精性中毒模型。观察各组小鼠的醉酒状态及酒精耐受时间、宿醉时间。6小时后处死小鼠，比色法检测外周血AST、ALT变化，ELISA法检测肝组织中ADH、ALDH和脑组织中β-EP、LENK浓度。2. 将SPF级C57BL / 6J小鼠分为5组，分组情况同上，参照NIAAA模型（Gao-Binge模型），建立慢性酒精性肝病模型。各组均予对照饲料适应饲养5天，接着酒精组与药物组均更换为含5%酒精液体饲料，每日上午各药物组予相应剂量灌胃处理10天，次日清晨酒精组与DHM组及低、高TDHM组均予以31.5%乙醇灌胃，正常空白组予以45.0％麦芽糖糊精溶液灌胃，9小时后处死小鼠。每日观察小鼠的精神状态、体重变化等一般情况，HE、油红O染色观察各组小鼠肝脏组织病理学变化，比色法检测肝组织AST、ALT活力，生化检测TG、ADH、ALDH水平，ELISA检测肝组织SOD、MDA浓度，Western-blot法测定CYP2E1的表达。
（1）行为学观察：低TDHM组、高TDHM组与酒精组相比（41.40±21.03min，50.90±35.83min vs 11.30±8.16min）均可延长小鼠醉酒时间（p＜0.001，p＜0.01），且均较DHM组（41.40±21.03min，50.90±35.83min vs17.70±15.55min）显著（p＜0.05）。高TDHM组小鼠醒酒时间最短（152.40±46.04 min vs 256.71±36.54min，p<0.001），DHM组、低TDHM组也较酒精组小鼠醒酒时间（188.40±49.09min，172.40±45.66min vs 256.71±36.54min）缩短（p＜0.01），各TDHM组与DHM组之间无明显统计学差异。（2）血清ALT、AST水平：DHM组，高TDHM组ALT活力较酒精组（10.70±3.08，8.58±4.83 vs 17.72±6.84）明显下降（p＜0.05），各TDHM组与DHM组间无明显统计学差异。DHM组及低、高TDHM组AST活力较酒精组（45.35±10.17，33.02±2.68，32.21±8.11 vs 64.12±15.00）均明显下降（p＜0.05，p＜0.001，p＜0.01），低、高TDHM药物组均较DHM组作用效果显著（p＜0.05）。（3）肝组织匀浆ADH、ALDH浓度：与酒精组相比DHM组及低、高TDHM组ADH浓度（0.38±0.02，0.51±0.13，0.49±0.06 vs 0.30±0.06）均可提高（p＜0.05，p＜0.01，p＜0.001），且低、高TDHM组较DHM组作用显著（p＜0.01）。低、高TDHM组ALDH浓度较酒精组相比（13.38±3.85，15.99±5.91 vs 7.40±4.47）均明显提高（p＜0.05），且低、高TDHM组较DHM组（13.38±3.85，15.99±5.91 vs 7.86±3.15）作用显著（p＜0.05）。（4）脑组织匀浆β-EP、LENK浓度：与酒精组相比，DHM组及低、高TDHM组（324.82±25.54，309.83±32.55，283.89±38.42 vs 384.28±39.83）均可下调脑组织β-EP水平（p＜0.05，p＜0.01，p＜0.01），各TDHM组与DHM组间无明显统计学差异。各组小鼠LENK浓度之间无明显统计学差异。
2. 二氢杨梅素铵盐对慢性酒精性肝损伤小鼠的保肝作用结果：（1）HE染色及油红O染色病理学改变：与对照组相比酒精组小鼠肝组织发生严重的脂肪变性，DHM组与低、高TDHM组脂肪变性轻于酒精组。（2）肝组织TG含量：与酒精组相比，DHM组、低TDHM组TG水平（0.08±0.007 ，0.08±0.004 vs 0.11±0.02）明显降低（p＜0.05，p＜0.01），且低TDHM组较DHM组降低明显（p＜0.05）。（3）肝组织中ALT、AST水平：酒精组小鼠肝组织ALT、AST水平明显减低，DHM组与低TDHM组ALT水平较酒精组（2.14±0.52，2.12±0.56 vs 1.51±0.17）明显升高（p＜0.05），各TDHM组与DHM组间无明显统计学差异。与酒精组相比，DHM组及低、高TDHM组AST水平（5.00±0.42，5.53±0.38，5.57±0.82 vs 3.97±0.45 ）升高（p＜0.01，p＜0.001，p＜0.01），且低TDHM组较DHM组升高明显（p＜0.05）。（4）肝组织ADH、ALDH活力：与酒精组相比低、高TDHM组ADH水平（25.02±12.13，23.16±13.33 vs 10.18±3.26）均可提高（p＜0.05）。与酒精组相比DHM组及高TDHM组ALDH水平（74.54±14.56，82.77±20.93 vs 56.02±14.17）均升高（p＜0.05），低DHM组升高显著（105.18±25.45 vs 56.02±14.17，p＜0.01），且较DHM组作用明显（p＜0.05）。（5）肝组织MDA、SOD浓度：与酒精组相比，DHM组及低、高TDHM组MDA（40.28±1.48，35.30±4.82，34.88±4.36 vs 47.29±4.35），明显降低（p＜0.01，p＜0.01，p＜0.001）,低、高TDHM组较DHM组降低明显（p＜0.05）。与酒精组相比，低、高TDHM组（8.63±0.98，8.76±2.91 vs 5.55±1.10）可提高SOD水平（p＜0.001，p＜0.05），各TDHM组与DHM组间无明显统计学差异。（6）肝组织CYP2E1水平：与酒精组相比DHM组及低、高TDHM组均可降低CYP2E1水平（p＜0.05，p＜0.01，p＜0.01），低、高TDHM组较DHM组作用明显（p＜0.05，p＜0.01）。
|Other Abstract||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.|
Methods：1.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 with 5% 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 colorietric method 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.
Results：1.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±21.03min, 50.90±35.83min 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±21.03min, 50.90±35.83min 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 vs 256.71±36.54, p<0.001), both DHM group and low TDHM groups(188.40±49.09min, 172.40±45.66min 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 vs 64.12±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 vs 0.30±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 vs 384.28±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).
Conclusions：1.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.
|First Author Affilication||Second Clinical School|
|尹跃霏. 二氢杨梅素铵盐对小鼠酒精性肝损伤的保护作用及机制研究[D]. 兰州. 兰州大学,2019.|
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