The Protective Effect Of Qige Granule On Alcoholic Liver Injury And Its Metabolomic Study

BackgroundRegarding,the improvement of people' s living standards and global environmental and climate change such as declining of air quality,radiation,unhealthy diet,virus infection and improper using of drugs treatment,excessive drinking of alcohol etc.All factors affect human health.Especially alcohol use,that is part of Chinese History.It still appears in almost all social Practices.In a modern society,liquor is the main drink and commonly consumed to show happiness or respect in many social events such as birthday parties,wedding ceremonies,business meetings etc.Consequently,not only the consumption of alcohol is rapidly increasing but also the diseases caused by drinking.Drinking heavily can cause a variety of health conditions,including alcoholism,liver damage and various cancers.When alcohol is consumed,it enters the stomach,intest ine and other organs,almost 80 percent of metabolism is processed by the liver,it converted alcohol into a substance with less toxicity substance of human body and discharged.Regardless of how much we consumed,the body can only metabolized a certain amount of alcohol each hour.As previous studies,Qige Granules may have the protective effect on gastric mucosa and liver injury.However,the pharmacological study has not been further explored.The previous animal experiments have found that liver injury caused by ethanol can be alleviated by Astragalus-Pueraria root.Therefore,the study of the mechanism and efficacy evaluation of Qige granules to preventing and treating liver injury in rats was discussed and synthesized in domestic and foreign research including the previous results of group research.ObjectiveThe object of this study is to study the protective effect of Qige Granules on alcohol-induced liver injury in rats.The effects of Qige Granules on metabolites in liver,plasma and urine of alcoholic liver injury was studied by ultra-high pressure liquid chromatography(UPLC-Q-TOF-MS).In order to clarify the overall regulation mechanism of Qige Granules on liver protection,the effects of Qige Granules on metabolites in liver,plasma and urine were studied.Methods1.Establishment of alcohol-induced subacute alcoholic liver injury model in rats.SD rats were randomly divided into 3 groups,8 rats per each group,namely,normal group,model group and sample group.The sample group was given Qige Granules daily,the normal group and the model group were given saline daily.And every day both the model Group and the sample group were given 50 percent of ethanol for 7 days,60 percent of ethanol for 3 days,70 percent of ethanol for 2 days and 80 percent of ethanol for 2 days respectively after 4 hours of administration,for 14 consecutive days.9 hours before the last gastric perfusion,the rats all group were given intraperitoneal injection of 3 percent of pentobarbital sodium salt anesthetics,and blood samples from abdominal aorta were taken to observe the effects of ethanol on biochemical indexes.Liver tissues were taken,weighed and left lobes of each group were taken for pathological HE staining to observe the tissue damage.Data collected for statistical analysis,The P value less than 0.05 will be considered to test the difference in statistical significance.2.Protective effect of Qige Granules on alcohol-induced liver injury in rats.Establishment of alcoholic liver injury model in rats induced by ethanol.SD female rats,SPF grade,weighing 180-220 grams.They were randomly divided into 6 groups,namely,normal group,liver injury model group,Silymarin(positive control group),LQG(low dose of Qige granules group),MQG(medium dose of Qige granules group),and HQG(high dose of Qige granules group).In group LQG,MQG and HQG(low,medium and high dose of Qige granules)were given orally every day.normal Group and model group were given normal saline(12ml/kg)by gastric perfusion every day.Silymarin Group(positive control group)was given 0.5percent Silymarin by gavage.The animals were weighed every 3 days to adjust the dosage of the sample.Every day except Group A(normal group),the other groups were given 50percent of ethanol for 7 days,60percent of ethanol for 3 days,70percent of ethanol for 2 days and 80percent of ethanol for 2 days respectively after 4 hours of administration,for 14 consecutive days.The urine of each group was collected in metabolic cage for 24 hours on the 7th and 14th days of the experiment.The urine was frozen in a refrigerator at-80?.9 hours after the last gastric perfusion,fasting and water intolerance,weighing the final body mass,giving intraperitoneal injection of 3percent of sodium pentobarbital salt anesthetics,and abdominal aorta blood collection,liver tissue,blood collection at room temperature for 1 hour,blood stratification,centrifugation,supernatant,-20? preservation,the detection of various indicators and pathological examination.3.Metabonomics UPLC-Q-TOF-MS was used to identify potential biomarkers in liver,plasma and urine of alcoholic liver injury model induced by ethanol.To establish a pharmacodynamic evaluation system of Qige Granules based on biomarkers for alcoholic liver injury in rats,and to determine the key metabolic pathway of Qige Granules in the treatment of liver injury.Data were processed by Marker Lynx 4.1 software.In the first stage,PCA analysis(principal component analysis)was used to analyze the difference of metabolic profiles between model group and each group.Then,through OPLS-DA analysis(supervised orthogonal partial least squares discriminant analysis),the VIP values(variable weights)obtained by model group were found for screening differential metabolites.The metabolite whose VIP value exceeds 1 indicates that the metabolite plays an important role in inter-group differences.The signi f'icance of the difference was tested by nonparametric test of two independent samples in t test,and the threshold of statistical significance was set to P<0.05.Finally,compounds with VIP>1 and P<0.05 were selected as marker metabolites.Results1.The liver of rats was severely damaged by ethanol stimulation,and all rats were drunk,such as drowsiness.Observing the liver with naked eyes,we found that the liver size of model group rats was bigger than that of non-ethanol group,dark red and glossy.The mean liver wet weight and liver index were higher than those of other groups.Serum liver function test was significantly higher than that of other groups.HE staining and pathological section results showed that liver injury tissue was obvious in the model group,with a large number of steatosis,most large and small fat vesicles,disordered arrangement of hepatocyte cord,a little congestion and hemorrhage in hepatic sinus,and obvious swelling of stem cells.2.After administration of Qige Granule,feces and drunkenness were found in a few rats,but the body hair,diet and mental status were improved in different degrees compared with the liver injury model group.The levels of AST,ALT and TG in serum of each dose group of Qige Granule were lower than those of liver injury model group,and the difference between high dose group and liver injury model group was significant.HE staining and pathological section results showed that there was no obvious abnormality in the structure of hepatic lobules in the low dose group of Qige granule,and the number of inflammatory cells and fat vesicles was reduced compared with the model group;the hepatocytes in the middle dose group of Qige granule occasionally scattered in hepatocytes steatosis,and the degree of swelling was reduced;the hepatocytes in the high dose group of Qige granule were slightly normal,hepatocyte swelling was significantly reduced,steatosis and inflammation were reduced.The degree of symptomatic cells decreased,and the degree of pathological changes in liver tissue was significantly reduced.3.To confirm the biomarkers of alcoholic liver injury model in rats induced by ethanol,to analyze the effect of Qige Granule on the biomarkers of alcohol-induced liver injury model in rats,and to explore the protective mechanism of Qige Granule.(1)Ten potential biomarkers of liver metabonomics were obtained,including docosahexaenoic acid,choline phosphate,ethyl glucuronide,linoleic acid,docosahexaenoic acid(22n-6,22n-3),lysophosphatidylcholine(LysoPC)(20:3/18:0/17:0),lysophosphatidyl ethanolamine(LysoPE)(20:5),to evaluate the liver injury model induced by Qige granule.The intervention effect of Qige Granule on 7 biomarkers,including docosahexaenoic acid,docosahexaenoic acid(22n-6/22n-3),linoleic acid and lysophosphatidylcholine(20:3/18:0/17:0),was found to have regulatory and restorative effects.Among them,three biomarkers,lysophosphatidylcholine(20:3),linoleic acid and docosahexaenoic acid(22n-6),had significant regulatory effects(P<0.05).Linoleic acid metabolism pathway is the key pathway for Qige Granule to interfere with the pharmacodynamics of liver injury model rats.(2)Potential biomarkers of serum metabonomics eventually yielded 27 variables(25 model biomarkers),including ethyl glucuronide,docosahexaenoic acid,oleic amide,21-hydroxypropenedione,L-acetylcarnitine,tetrahydrodeoxy cortisol,docosapentaenoic acid(22n-6),adrenaline,tryptophan,linoleic acid,5-hydrovalerate,arachidone.Acid,lysophosphatidylcholine(16:1/20:1/20:3/16:0/18:0),lysophosphatidylethanol amine(22:1/16:0/22:6/18:0)and tryptophan were used to evaluate the intervention effect of Qige Granule on liver injury model.It was found that Qige granule had eight biomarkers,lysophosphatidylcholine(20:1),docosahexaenoic acid,tetrahydrodeoxycortisol and lysophosphatidyl ethanolamine(22:1/16:0).Adrenaline acid,linolenic acid and oleic acid had regulatory and restorative effects,and one of them had significant regulatory effects(P<0.05).The key pathways of Qige Granule intervention on the pharmacodynamics of liver injury model rats are glycerol phospholipid metabolism,tryptophan metabolism and arachidonic acid metabolism.(3)Potential biomarkers of urinary metabonomics were eventually identified as 10 variables(9 model biomarkers),deoxycortisol,Cervonoyl ethanolamide,17alpha-hydroxypregenone,16alpha-hydroxypregenone,1H-indole-3-acetyl-inositol,7-keto deoxycholic acid,deoxyinosine,3-aminosalicylic acid,pyrogallol-2-0-glucuronate as evaluation variables.The intervention effect of Ge Granule on liver injury model showed that Qige Granule could regulate and restore six biomarkers,pyrogallol-2-0-glucuronate,deoxycortisol,17alpha-hydroxyphenolone,16alpha-hydroxyphenolone,1H-indol-3-ylacetyl-myo-inositol and deoxyinosine,four of which were deoxycortisol,17alpha-hydroxyphenolone and 16alpha-hydroxyphenolone.Pregnanolone and 1H-indole-3-acetyl-muscle-inositol had significant regulatory effects(P<0.05).The key pathway of Qige Granule' s intervention on the pharmacodynamics of liver injury model rats is the biosynthetic pathway of steroid hormones.Conclusion1.During alcoholic liver injury induced by ethanol,fatty degeneration of liver tissue,disordered arrangement of hepatocyte cord,slight congestion and hemorrhage of hepatic sinus,and obvious cell swelling occurred in rats.Serum AST,ALT,TG and liver index increased significantly,indicating that the model was successful.2.Qige Granule can alleviate liver enlargement and hepatocyte injury caused by ethanol,and it can reduce serum AST,ALT and TG.This experiment shows that Qige Granule can protect alcoholic liver injury induced by ethanol in rats.3.Qige Granule can regulate alcoholic liver injury.Qige Granule regulates the potential biomarkers in rat liver,serum and urine.Its mechanism is related to linoleic acid metabolism,glycerol phospholipid metabolism,tryptophan metabolism,arachidonic acid metabolism and steroid hormone biosynthesis metabolism.The metabolic pathways were classified and analyzed.The common metabolic pathways in liver and serum were glycerol phospholipid metabolism and unsaturated fatty acid biosynthesis pathway.The metabolic pathway coexisting in serum and urine is steroid hormone biosynthesis metabolic pathway.Indicates imbalance of lipid metabolism and bile acid metabolism in rats with alcoholic liver injury.