| Research Backgrounds:"Alcohol abuse" and "alcohol dependence" are the major risk factors for the high morbidity and mortality of alcoholic liver disease,which has become the second largest liver disease next only to viral liver disease and seriously harmful to human health.Alcoholic liver disease is a metabolic disease of the liver caused by acute or chronic drinking.In the early stage,it is mainly simple fatty liver disease,and then develops to fatty hepatitis,liver fibrosis,liver cirrhosis,hepatic carcinoma and other irreversible damage.Although alcoholic liver disease has a far-reaching consequences on the global health and economic development,there is little progress in the research of drugs for the treatment of the disease,and there is no satisfactory therapeutic strategy in addition to abstinence and nutritional support.Alcoholic liver disease belongs to the category of "alcoholic jaundice","alcoholic consumptive disease","alcoholophilia","alcoholic tympanites","liquor addiction","hypochondriac pain" and "alcohol intoxication" in traditional Chinese medicine.The acute alcoholic liver injury is mainly due to the lack of moderation or restraint for binge-drinking,alcoholic toxicity with syndrome of dampness-heat in middle jiao,the liver maladjustment,air-resistance lag,dysfunction of spleen in transportation,water and dampness retention,stagnation and blockade of phlegm-damp,qi stagnation and phlegm coagulation,blood stasis block,phlegm retention and blood stasis.The location of disease is liver,involving the gall bladder,spleen and stomach.The alcoholic toxicity with syndrome of[accumulation and binding of]damp-heat in the body is the fundamental pathogenesis.The Flower of Lobed Kudzuvine[Pueraria lobata(Willd.)Ohwi;Gehua in Chinese;GH]and Japanese Raisin Tree Seed(Hovenia dulcis Thunnb.;Zhijuzi in Chinese;ZJZ)are the traditional Chinese medicine for the treatment of alcohol intoxication and liver diseases in China.Gehua diffuses and penetrates,dispersing the dampness-heat from the muscle surface;Zhijuzi penetrates and diuresis,and promoting the damp-heat pathogens from the urine.The combination of GH and ZJZ is scattered infiltration,eliminate dampness,and improve the occasion according to the situation,so that the pathogenic qi of liquor dampness is discharged from the body,which plays the role of relieving alcoholism and protecting liver.The research group has completed the treatment of acute alcohol toxicity by Gehua or Zhijuzi in the early stage,and has carried out the experimental study of different proportion of Gehua and Zhijuzi on chronic alcoholic liver injury.The study confirmed that the combination of Gehua and Zhijuzi has certain prevention and treatment effect on alcoholic liver injury,and can improve the relevant indicators in different degrees,and play the role of relieving alcoholism and protecting liver.Research Objectives:Alcoholic liver injury is one of the most urgent public health problems in the world.It is a progressive disease that is difficult to cure in the late stage of the disease.If it can be timely treated in the early stage,it can be prevented from developing into irreversible diseases.Acetaldehyde-mediated toxicity,oxidative stress and lipid metabolic imbalance are generally considered to be closely related to the incidence of alcoholic liver disease.Transcription factor Nrf2 is a key therapeutic target for the prevention and treatment of oxidative stress-mediated diseases,and the Keapl-Nrf2-ARE signaling pathway is a key regulator for the regulation of cellular protective enzymes,playing an important role in the early stage of alcoholic liver injury.On the basis of the preliminary research,this paper takes the compatibility of Gehua and Zhijuzi as the research object to carry out the research on the protection of them to the acute alcoholic liver injury.The animal model was made by intragastric administration of white spirits,and the liver injury was observed dynamically.By using technologies such as biochemistry,pathology,molecular biology methods,explore whether the composition can activate the Keapl-Nrf2-ARE pathways induced antioxidant enzyme activity and protect liver.According to the theory of traditional Chinese medicine,the occurrence of acute alcoholic liver injury is mainly due to the accumulation of heat and dampness caused by alcohol,which leads to the dysfunction of spleen in transport,liver dysfunction,the stagnation of qi and phlegm coagulation,the heat caused by lingering phlegm,the stasis of blood,and the accumulation of phlegm and blood stasis.More and more evidences show that the normal expression of AQPs of aquaporin may be the molecular biological basis for water metabolism and homeostasis in vivo,and the abnormal expression of AQP may be one of the pathological mechanisms of damp-heat syndrome.The oxidative stress induced by alcohol in the early stages of acute alcoholic liver disease plays an important role,is causing abnormal lipid metabolism,promote the lipid peroxidation,the key factors that cause liver lipid accumulation,glycerin water channel protein AQP9 is involved in the glycerol and triglyceride metabolism important channel proteins,involved in lipid metabolism of liver cells and fatty degeneration.AQP9 plays an important role in regulating lipid metabolism and maintaining glycerol metabolic balance.At the same time try to combine the traditional Chinese medicine pathogenesis,to explore both can influence the expression of water-glycerin channel protein AQP9 regulates fat metabolism in liver,and provide the scientific evidence for clinical medicine,and to carry out other alcohol related disease research to provide ideas and reference.Research Methods:1.The effects of Gehua,Zhijuzi and their compatibility on ethanol metabolism in mice with acute alcoholic liver injury:According to body weight,mice were randomly divided into model group,positive control group(Silymarin),Gehua group,Zhijuzi group,GH-ZJZ combination ?(1:1),GH-ZJZ combination ?(1:2)and GH-ZJZ combination ?(2:1),a total of 7 groups,15 mice in each group.The animal model of acute alcoholic liver injury was replicated with 56%(V/V)red star er guo-tou liquor,one large dose of gavage(Model group ?)and several consecutive gavages(Model group ?).According to the metabolism characteristics of alcohol in vivo,samples were collected at 1 hour,4 hours and 10 days after drinking,and the concentration of alcohol in blood and the activity of alcohol dehydrogenase in liver were measured by gas chromatography and UV-Vis spectrophotometer.The concentration of alcohol in blood and the activity of alcohol dehydrogenase in liver were detected by gas chromatograph and ultraviolet-visible spectrophotometer.2.The effects of Gehua,Zhijuzi and their compatibility on Hepatic function,Serum lipid level and Liver pathology conformation in mice with acute alcoholic liver injury:According to body weight,mice were randomly divided into model group,positive control group(Silymarin),Gehua group,Zhijuzi group,GH-ZJZ combination ?(1:1),GH-ZJZ combination ?(1:2)and GH-ZJZ combination ?(2:1),a total of 8 groups,15 mice in each group.The animal model of acute alcoholic liver injury was replicated with 56%(V/V)red star er guo-tou liquor,one large dose of gavage(Model group ?)and several consecutive gavages(Model group ?).According to the characteristics of serum enzymology,liver function(ALT,AST,ALP,ALB and TP content),serum lipid(TG and CHO content)and morphologic changes of liver tissue were measured at 4 hours,6 hours,12 hours,16 hours and 10 days after drinking.3.The effects of Gehua,Zhijuzi and their compatibility on Hepatic antioxidant enzyme activities in mice with acute alcoholic liver injury:The mice in each group were killed at 4 hours,6 hours,12 hours,16 hours and 10 days after drinking 56%(V/V)red star er guo-tou liquor,and their livers were quickly removed for testing.Then using ultraviolet spectrophotometer and fluorospectro photometer to calculated the absorbance and activity of SOD,MDA,GSH and ROS in the liver tissue,respectively.4.The effects of Gehua,Zhijuzi and their compatibility on protective mechanism in mice with acute alcoholic liver injury:The gene and protein expression of Keap1,Nrf2 and AQP9 were detected by RT-PCR and Western blot.Research Results:1.The effects of Gehua,Zhijuzi and their compatibility on the blood ethanol concentration and the liver alcohol-metabolizing enzymes in mice with acute alcoholic liver injury:?Alcoholic liver injury model of mice I(1 hours or 4 hours after drinking):Compared with the model group,the Gehua,Zhijuzi and their compatibility groups(1:1,1:2 and 2:1)were significantly reduced the concentration of ethanol in the blood and increased the activity of alcohol dehydrogenase in the liver.Among them,Gehua-Zhijuzi(2:1)group has a better therapeutic effect in promoting the metabolism of ethanol and alleviating the toxicity of ethanol to the liver,showing a better effect of alcohol detoxification.?Alcoholic liver injury model of mice ?(10 days after drinking):Compared with the model group,Gehua,Zhijuzi and their compatibility groups(1:1,1:2 and 2:1)all significantly decreased the concentration of ethanol in the blood,elevated the activity of alcohol dehydrogenase in the liver,thus alleviating the liver injury induced by ethanol,showing a certain effect of anti temulence and liver protection.In addition,Gehua-Zhijuzi(2:1)group showed a better therapeutic effect in alleviating the toxic effect of ethanol on liver,and showed a better effect of alcohol detoxification.2.The effects of Gehua,Zhijuzi and their compatibility on liver function and pathomorphology change in liver tissues in mice with acute alcoholic liver injury:?Alcoholic liver injury model of mice ?(4 hours,6 hours,12 hours and 16 hours after drinking):Compared with the blank group,the serum levels of AST,ALT and ALP in the model group were significantly increased,while the contents of TP and ALB were significantly decreased.Under the light microscope,there was diffuse intracytoplasmic vacuolar degeneration in the liver tissue,accompanied by inflammatory cell infiltration and the formation of fat droplets Compared with the model group,Gehua,Zhijuzi and their compatibility groups(1:1,1:2 and 2:1)can significantly decreased the levels of AST,ALT and ALP in serum of mice,and the contents of TP and ALB in serum of mice were increased,so as to improve the liver function of mice.The GH-ZJZ(2:1)group was superior to the other groups in the improvement of liver serum transaminase and liver protein synthesis dysfunction caused by alcohol(12 hours after alcohol consumption).In addition,each administration group improved the alcohol-induced microvesicular steatosis,diffuse intracytoplasmic vacuolar degeneration,lipid droplet accumulation and other pathological changes to varying degrees,which had a protective effect on alcohol-induced acute liver injury.?Alcoholic liver injury model of mice II(10 days after drinking):Compared with the blank group,the serum levels of ALT,AST and ALP in the model group were significantly increased,while the levels of TP and ALB were significantly reduced,indicating that alcohol induced liver function disorder in the mice and caused acute liver injury Compared with the model group,the liver function and protein synthesis were improved to varying degrees by Gehua,Zhijuzi and their combinations(1:1,1:2 and 2:1).The GH-ZJZ(2:1)group was better than the other groups in improving liver function and protein synthesis function,that is,the combination group had a better protective effect on alcohol-induced liver injury.In addition,the hepatocyte swelling,microvesicular steatosis,inflammatory cell infiltration,lipid droplet accumulation and other pathological changes induced by alcohol were improved to varying degrees in each administration group3.The effects of Gehua,Zhijuzi and their compatibility on Serum lipid and functional mechanism in mice with acute alcoholic liver injury:?Alcoholic liver injury model of mice I(4 hours,6 hours,12 hours and 16 hours after drinking):Compared with the blank group,serum TG and CHO levels in the model group were significantly elevated,that is,abnormal lipid function was caused by alcohol.Compared with the model group,Gehua,Zhijuzi and their combinations(1:1,1:2 and 2:1)significantly improved the serum lipid metabolism disorder The GH-ZJZ(2:1)group was superior to the other dosing groups in reducing the elevation of serum TG and CHO induced by alcohol(12 hours after alcohol consumption).In addition,the expression of AQP9 was inhibited by Gehua,Zhijuzi and their combination groups(1:1,1:2 and 2:1),which reduced the intake of glycerin in hepatocytes,improved lipid metabolism and protected the liver.There was no significant difference in regulating the expression of AQP9 and alleviating the abnormal lipid metabolism caused by alcohol.?Alcoholic liver injury model of mice II(10 days after drinking):The serum TG and CHO levels in the model group were significantly higher than those in the blank group,that is to say,acute alcohol intake caused lipid metabolism disorder in mice.Gehua Zhijuzi and their compatibility groups(1:1,1:2 and 2:1)all significantly decreased the level of serum TG and CHO,and inhibited the expression of AQP9,which had a certain improvement on the abnormal blood lipid caused by alcohol and played a role in protecting the liver.GH-ZJZ(2:1)group is better than other groups in improving the abnormal lipid metabolism caused by acute alcohol intake,showing a better therapeutic effect.4.The effects of Gehua,Zhijuzi and their compatibility on hepatic oxidative and antioxidative balance and functional mechanism in mice with acute alcoholic liver injury:?Alcoholic liver injury model of mice ?(4 hours,6 hours,12 hours and 16 hours after drinking):Compared with the blank group,the levels of SOD and GSH in the liver of the model group were significantly decreased,while the activities of MDA and ROS were significantly increased,indicating that single large dose of alcohol gavage could cause oxidative stress injury in mice Compared with the model group,Gehua,Zhijuzi and their combination groups(1:1,1:2 and 2:1)could enhance antioxidant enzyme activity,promote the scavenge of lipid peroxidation products by hepatocytes,and thereby alleviate the oxidative damage caused by alcohol.Among them,the GH-ZJZ(2:1)group was better than the other groups in alleviating oxidative stress injury caused by alcohol(12 hours after drinking),showing better liver protection effect.In addition,Gehua,Zhijuzi and their combination group(1:1,1:2 and 2:1)could effectively inhibit the expression of Keapl induced by alcohol(12 hours after drinking),promote the expression of Nrf2,alleviate the oxidative stress response in the liver of mice,and improve the acute liver injury.There was no significant difference in regulating the expression of the Keapl-Nrf2-ARE signaling pathway in liver tissue and alleviating the oxidative stress injury caused by alcohol in all drug administration groups.?Alcoholic liver injury model of mice ?(10 days after drinking):After 10 days of continuous intragastric administration of alcohol in the model group,the activity of SOD and GSH in the liver decreased significantly,while the content of MDA and ROS increased significantly,indicating that continuous alcohol intake would cause oxidative stress injury in mice.Compared with the model group,Gehua,Zhijuzi and their combination group(1:1,1:2 and 2:1)can activate the Keap1-Nrf2-ARE signal pathway,enhance the activity of antioxidant enzymes(SOD and GSH)in the liver,accelerate the catabolism of lipid peroxides(MDA and ROS),and reduce the damage of alcohol to the liver tissues of mice.In addition,the GH-ZJZ(2:1)was superior to other groups in inhibiting alcohol-induced oxidative stress in liver tissues,improving the antioxidant capacity of mice,and reducing lipid peroxidation.Research Conclusions:In conclusion,this study confirmed that Gehua,Zhijuzi and their compatibility groups(1:1,1:2 and 2:1)have protective effects on alcohol-induced acute liver injury.First of all,each administration group can enhance the activity of antioxidant enzymes,reduce the lipid peroxidation,improve the cell damage induced by oxidative stress by activating keapl-nrf2-are signal pathway,so as to alle-viate the liver damage caused by alcohol and play the role of liver protection.Secondly,by regulating the expression of AQP9,the drug groups can reduce the permeability of glycerin,reduce the intake of glycerin,and improve the abnormal lipid metabolism of liver,so as to alleviate the oxidative stress and fatty degeneration of liver induced by alcohol and protect the liver cells.It should be noted that Gehua-Zhijuzi(2:1)group showed a positive effect in promoting alcohol metabolism,improving liver function,reducing liver steatosis and improving antioxidant capacity of mice,that is,the compatibility group had a better protective effect on alcohol induced liver injury.Therefore,this study confirmed that the combination of Gehua-Zhijuzi showed a certain prevention and treatment effect on acute alcoholic liver injury,and made it clear that the combination of the two had the function of mutual use and synergism,which had the effect of enhancing alcohol and liver protection,was a natural alcohol and liver protection drug for the treatment of alcoholic liver disease,and provided a scientific basis for the clinical application of Gehua-Zhijuzi to prevent and treat acute alcoholic liver injury. |
PDF Full Text Request