Study On The Protective Effect Of Ethanol Extract Of Propolis On Alcoholic Fatty Liver In Mice

Alcoholic liver disease is an important disease that concerns public health.In the liver injuries caused by alcohol,free radical-mediated oxidative stress plays a dominant role as well as the production of a series of pro-inflammatory cytokines that promote multiple abnormalities in liver functions.Chinese propolis,rich in flavonoids,is a natural antioxidant.Many flavonoid compounds present in propolis have been demonstrated to exhibit significant hepatoprotective effects in eliminating liver inflammation,preventing liver fibrosis,protecting and repairing hepatocytes.However,the hepato-protective effects of propolis in alcohol-induced liver injuries and its related mechanisms have not yet been explored.In this study,ethanolic extracts of Chinese propolis(EECP)were studied.In C57BJ/6L mice,liver injuries were induced by continuous intragastric administration of alcohol for 8 weeks.On the one hand,EECP was co-administered with alcohol to observe the intervening effects of propolis on alcohol-induced liver injuries;on the other hand,after liver injuries had been induced by continuous administration of alcohol for 8 weeks,EECP was administrated for 4 further weeks to evaluate the therapeutic effects of EECP.The experimental mice were assigned as follows:group A fed with high-fat diet and continuously gavaged with alcohol for 8 weeks;group B(intervention group)fed with high-fat diet and intragastric administration of alcohol and EECP for 8 weeks.55° Red Star Erguotou was used as the source of alcohol.During the first two experimental weeks,a dose of 4 mL and 6 mL per kilogram of body weight per day was gavaged to the mice in group A and group B.From the third week the dose increased to 8 mL per kilogram of body weight per day and continued till the end of the eighth experimental week.Mice in group B received at the same time a dose of 10 mL EECP per kilogram of body weight per day.At the first time-point(the end of 8th experimental week),blood and liver tissue samples were collected from 6 mice from group A and group B,respectively.From the beginning of the 9th experimental week,the remaining mice in group A were switched to commercial pellet feeds and randomly divided into subgroups,group A1 and group A2.They were continued to be fed for 4 further weeks.Among them,group A1 was the naturally recovered group.Group A2 was daily gavaged by 10 mL of EECP per kg body weight until the end of the 12th week.Blood and liver tissue samples were also collected from the mice.The protective effects of EECP on alcohol-induced liver injuries were evaluated by measuring the triglyceride(TG)and total cholesterol(TC)levels in both serum and liver tissue,the transaminase levels in serum,and observing frozen sections of the liver tissue.RNA-Seq was also used to analyze genes that were differentially expressed in the liver tissue between the alcohol-treated group A and the EECP-intervened group(group B).The biological processes these genes involved in under the influence of EECP intervention were also identified,which helped to reveal the possible pathways and underlying mechanisms of EECP’s hepato-protective effects on alcohol-induced liver injuries.The main findings are as follows:1)A total of 24 components,including flavonoids,fatty acids,aromatic acids,and alcohols were identified in EECP using GC-MS.The total flavonoid content was 20.7%.2)The TG and TC levels in both the serum and the liver tissue of the EECP intervention group(group B)were significantly lower than those of group A treated with alcohol.Likewise,the TG and TC contents in the liver tissue of group A2 were significantly lower than the naturally recovered group(group A1).3)The serum levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in group B were significantly lower than those in group A.The serum levels AST and ALT in of group A2 were significantly lower than those in the natural recovered group(group A1).4)The results of liver cryosections showed that fat accumulation in liver cells of mice treated with EECP(group B)was significantly attenuated than that of mice in group A.5)RNA-Seq results showed that EECP could down-regulate the expression of genes involved in lipogenesis in both female and male mice,and thus reduce the accumulation of fat in the liver.Furthermore,EECP could significantly modulate the cytokines and cytokine receptor interaction pathway in female mice,which helped to relieve hepatic inflammation.6)The qRT-PCR results verified the results of RNA-Seq with respect to the transcriptional expression of Idi1、Cyp51、Fabp5 and G6pc between group A and group B.Our present results showed that EECP could effectively alleviate the increase of serum and hepatic TG and TC levels caused by alcohol intake,decrease serum levels of liver transaminases reflecting liver injuries,reduce lipid synthesis in the liver tissue by down-regulating the expression of lipogeneis-related genes,and thus attenuated the accumulation of lipids in the liver tissue and ameliorate the formation of fatty liver.