| Objective:Proteomics,genomics and metabonomics were used to reveal the pathogenesis of acute and chronic alcoholic liver disease from the whole,tissue and molecular level,and to study the mechanism of Mongolian medicine Carthamus tinctorius water extract on acute and chronic alcoholic liver injury.Methods:?1?Preparation of experimental drug?The Carthamus tinctorius water extract was soaked in 10 times water for 30 minutes,and extracted at 60?for three times,30min each time.The filtrate was collected and concentrated at 65?and pressure of-0.07MPa.The extract was obtained by freeze-drying.?2?Preparation of animal model of acute alcoholic liver injury:rats were randomly divided into four groups,There were 10 rats in the normal group and three dose groups of the model,There were 10 rats of 6 g/kg-1,8 g/kg-1 and 10 g/kg-1respectively,40 rats in total.After 7 days of adaptive feeding,The normal group was given 10 g/kg-1 physiological saline,and the three dose groups were respectively given 40%alcohol?with Niulanshan Erguotou liquor,alcohol degree:56°?.The first day of the experiment was given once to simulate acute alcoholic liver injury;Preparation of the animal model of chronic alcoholic liver injury:The rats were randomly divided into four groups,20 rats in the normal group and three different dose groups in the model group,20 rats in 6 g/kg-1,8 g/kg-1 and 10 g/kg-1 respectively,80 rats in total.After 7 days of adaptive feeding,The normal group was given 10ml/kg-1 physiological saline,and the three dose groups were respectively given 40%alcohol?with Niulanshan Erguotou liquor,alcohol degree:56°?.The drug was administered once a day for 28 days.The rats were weighed twice a week and the dosage was adjusted according to their weight.?3?Experimental study on long-term medication safety of Carthamus tinctorius water extract:The rats were randomly divided into four groups:normal group?10 rats?and Carthamus tinctorius water extract group?3 doses?:low dose group?clinical dosage 0.4767g/kg?,medium dose group?clinical dosage 9 times:4.2903g/kg?,high dose group?clinical dosage 18 times:8.5806g/kg?.Normal group was given physiological saline,The three dosage groups of the aqueous extract of Carthamus tinctorius water extract administrated to stomach respectively with the corresponding dosage.Once a day for three months.Blood was collected from the tail every two weeks and the corresponding indexes were observed.?4?Experimental study on prevention of acute alcoholic liver injury by aqueous extract of Carthamus tinctorius water extract:rats were randomly divided into six groups:normal group,model group,positive group?Hugan tablet?,low?0.4767g/kg?,medium?1.4301g/kg?and high dose?4.2903g/kg?.The normal group was given the corresponding normal saline,the positive control group was given the liver protecting tablet,the other groups were given the corresponding dose of Carthamus tinctorius water extract once a day for 14 days.On the 15th day,alcohol was given to gavage?simulating the liver injury caused by drinking a large amount of alcohol?to prepare the acute alcoholic liver injury model of rats?the concentration of alcohol was determined according to the model experiment of alcoholic liver injury?.Fasting can not help water for 16 hours,anesthesia,blood collection,serum separation,-80?refrigerator storage,standby.In addition,the same part of the right lobe of the liver was taken and fixed in 10%formaldehyde solution for he pathological section.?5?The experiment of Carthamus tinctorius water extract against chronic alcoholic liver injury:the normal group was given physiological saline,the other groups were given alcohol once a day for 28 days.Alcohol was stopped after 28 days,and Carthamus tinctorius water extract was given from 29th day.The normal group was given the corresponding normal saline,the positive control group was given the liver protecting tablet,the other groups were given the corresponding Carthamus tinctorius water extract once a day for 21 days.After the last administration,the animals in each group were fasted for 16 hours,anesthetized,collected blood,separated serum,and stored in a refrigerator at-80?for later use.In addition,the same part of the right lobe of liver was taken and fixed in 10 solution for histopathological examination.?6?Based on metabonomics,the protective mechanism of Carthamus tinctorius water extract against acute and chronic alcoholic liver injury in rats was studied:The serum metabonomics of Mongolian medicine Carthamus tinctorius water extract for the prevention of acute and chronic alcoholic liver injury in rats was studied by using UPLC-MS,cluster analysis,PCA and opls-da.To explore the endogenous biomarkers of Mongolian medicine Carthamus tinctorius water extract on acute and chronic alcoholic liver injury in rats.?7?Study on the protective mechanism of Carthamus tinctorius water extract based on serum exosomes?EVS?against chronic alcoholic liver injury in rats:Extraction,isolation and identification of EVs in rat serum.The proteomic differences of serum EVs were measured by label free quantitative technique,and the changes of serum EVs mi RNA were measured by gene chip screening.Results:?1?The extracted Carthamus tinctorius water extract recovered and concentrated under reduced pressure,and the recovery was 37.08%by freeze-drying.?2?The alcohol concentration of acute alcoholic liver injury model was 8ml/kg-1,and that of chronic alcoholic liver injury model was 8ml/kg-1.?3?The results of long-term drug safety experiment of Carthamus tinctorius water extract were as follows:there were three dose groups of Carthamus tinctorius water extract:low dose group?clinical dosage 0.4767g kg?,medium dose group?clinical dosage 9 times:4.2903g/kg?,high dose group?clinical dosage 18 times:8.5806g kg?without liver injury for three months.There was no significant difference between the serum ALT group and the normal group on the 14d,28d,42d,56d,70d and 84day.There was no significant difference between the serum ast group and the normal group on the 14d,28d,42d,56d,70d and 84d day after administration,and there was significant difference between the low dose group and the blank group on the 14 day?P<0.05?.There was no statistical significance between the serum TG group and the normal group on the 14d,28d,42d,56d,70d and 84 days after the treatment,and there was statistical significance between the middle dose group and the blank group on the 28 day?P<0.05?.There was no significant difference between the serum LDH group and the normal group on the 14d,28d,42d,56d,70d and 84day.There was no significant difference between the serum CK group and the normal group on the14d,28d,42d,56d,70d and 84 day.There was no statistical significance between the serum ureal group and the normal group on the 14d,28d,42d,56d,70d and 84 days after the treatment,and there was statistical significance between the low dose group and the blank group on the 42d days?P<0.05?.There was no significant difference between the serum grep group and the normal group on the 14d,28d,42d,56d,70d and 84 days after the treatment,there was significant difference between the high dose group and the blank group on the 42d days?P<0.05?,and there was significant difference between the low dose group and the blank group on the 56 days?P<0.05?.?4?The aqueous extract of Carthamus tinctorius water extract can prevent acute alcoholic liver injury.Compared with the model group,serum ALT and AST administration group had statistical significance?P<0.05?.?5?The effect of Carthamus tinctorius water extract on chronic alcoholic liver injury.Compared with the model group,the low dose and middle dose of serum ALT were statistically significant?P<0.05?,and the ast group was statistically significant?P<0.01?.?6?The results of metabonomics showed that there were 11 up-regulated endogenous metabolites in the serum of rats with acute liver injury,including eicosapentaenoic acid,ethyl arachidonate,docosaenoic acid,docosahexaenoic acid,tocopherol acid,eicosapentaenol ethanolamide,phospholipid acid,lysophosphatidylcholine,?-linolenic acid,all trans retinoic acid,ethyl furfuraldehyde,etc.;down regulated endogenous metabolites There are two original metabolites,i.e.linolenic triglyceride and monoacyl triglyceride.After Carthamus tinctorius water extract,11 endogenous metabolites and 2 endogenous metabolites were down regulated;3 endogenous metabolites were up regulated in serum of rats with chronic liver injury,namely triglyceride,palmitic acid,linolenic acid triglyceride,etc.;8 endogenous metabolites were down regulated,namely lysophosphatidylcholine,palmitoyl ethanolamide,adrenaline,sphingosine,lysophosphatidylcholine,?-linolenic acid,docosahexaenoic acid,eicosapentaenoic acid and other endogenous metabolites.After Carthamus tinctorius water extract,the above three endogenous metabolites were down regulated and the above eight endogenous metabolites were up regulated.Among them,there are 5 endogenous metabolites in the serum of acute and chronic liver injury model and Carthamus tinctorius water extract treatment,i.e.linolenic triglyceride,eicosapentaenoic acid,docosahexaenoic acid,?-linolenic acid and lysophosphatidic acid.?7?The experimental results showed that there were 188 differential proteins in the model group,135 in the low dose group,193 in the middle dose group and 239 in the high dose group.After the model group compared with the normal group,each administration group compared the model group(P A:Rno-mir-3068-5p,rno-mir-1949,rno-mir-153-3p,rno-mir-322-3p,rno-mir-743a-5p,rno-mir-150-5p,rno-mir-122-5p,rno-mir-3065-3p,and 22 related regulatory proteins were obtained after prediction of the target gene protein.Conclusion:?1?The mortality rate of acute and chronic alcoholic liver injury model rats with alcohol concentration of 10 g/kg-1 was too high?so it was not used in later experiments?.6 g/kg-1 and 8 g/kg-1 can cause liver injury,but this experiment is to simulate the alcoholic liver injury caused by drinking large alcohol once and for a long time.Therefore,8 g/kg-1 alcohol is selected as the model in later experiments.Although the amount of alcohol in acute and chronic alcoholic liver injury model is the same,the liver injury will recover within 48 hours without medication.However,the chronic alcoholic liver injury can not be repaired automatically 21 days after the model was stopped.?2?According to the observation of serum ALT,AST,TG,CK,LDH,grep,ureal and histopathology,it was found that the water extract of Carthamus tinctorius water extract had no side effects on liver,heart and kidney.?3?The water extract of Mongolian medicine Carthamus tinctorius water extract can prevent and treat alcoholic liver injury.Although the dosage of Carthamus tinctorius water extract was the same in the low,middle and high dose groups in the acute and chronic alcoholic liver injury experiment,the concentration of Carthamus tinctorius water extract in the range of 0.4767g/kg-4.2903g/kg in the acute alcoholic liver injury experiment had good prevention and treatment effect.However,the concentration of Carthamus tinctorius water extract in the range of0.4767g/kg-1.4301g/kg has a good therapeutic effect on chronic alcoholic liver injury.?4?In the study of serum metabolomics of acute alcoholic liver injury,34compounds were found,13 of which were statistically significant between the model group and the blank group,and between the administration group and the model group.Among them,triglyceride linolenate and triglyceride monoacyl showed a downward trend in the state of alcoholic liver injury,and the prevention and treatment group with Carthamus tinctorius water extract extract showed an upward trend.Eleven compounds,such as eicosapentaenoic acid,arachidonic acid ethyl ester,docosaenoic acid,docosahexaenoic acid,tocopherol acid,eicosapentaenol ethanolamide,phospholipid acid,lysophosphatidic acid,?-linolenic acid,all trans retinoic acid,grape furfural acid ethyl ester and so on,showed an increasing trend in the state of alcoholic liver injury and a decreasing trend in the prevention and treatment group with Carthamus tinctorius water extract;chronic liver injury In the study of serum metabolomics of alcoholic liver injury,20 compounds were found,11of which were statistically significant between the model group and the blank group,and between the administration group and the model group.Among them,lysophosphatidylcholine,palmitoyl ethanolamide,adrenaline,sphingosine,lysophosphatidylcholine,?-linolenic acid,docosahexaenoic acid,eicosapentaenoic acid and other eight compounds showed a downward trend in the state of alcoholic liver injury,and an upward trend after treatment with Carthamus tinctorius water extract.Three compounds,triglyceride,palmitic acid and linolenic acid triglyceride,showed an increasing trend in the state of alcoholic liver injury,and a decreasing trend after treatment with Carthamus tinctorius water extract;there were seven compounds in the serum of acute and chronic alcoholic liver injury,including triglyceride linolenate,eicosapentaenoic acid,docosahexaenoic acid,?-linolenic acid,lysophospholipin,etc Under the premise that the model group is statistically significant compared with the blank group,the administration group is statistically significant compared with the model group.?5?The proteomic results of exosomes showed that the anti chronic alcoholic liver injury of Carthamus tinctorius water extract was related to antigen processing and presentation pathway,protein processing in endoplasmic reticulum,phagocyte and other pathways,and aldh1l1,Marco,Prox1,PON1,ass1,unc13d,p4hb,SOD1,Copa,rpl6,pdia3,katnal2,CRP,snd1,TFG,a were involved in these pathways Rpc4,Carr and other 17 proteins.Among them,aldh1l1 is the highest expression in the liver,which is related to the detoxification function of the liver;pdia3 plays an important role in the regulation of inflammation,apoptosis and oxidative stress under various disease conditions.In this study,it was found that the regulation of aldh1l1 and pdia3protein was one of the mechanisms of Carthamus tinctorius water extract in the treatment of chronic alcoholic liver injury;The results of nucleic acid detection showed that rno-mir-3068,rno-mir-1949,rno-mir-153,rno-mir-322,rno-mir-743a,rno-mir-150,rno-mir-122 and rno-mir-3065 were involved in the treatment of Carthamus tinctorius water extract.Among them,rno-mir-122 has the highest expression of mi RNA in liver tissue,which is involved in the lipid metabolism of liver tissue.The results showed that the aqueous extract of Carthamus tinctorius water extract could inhibit the chronic alcoholic liver injury by regulating the expression of rno-mir-122 and regulating the endogenous biomarkers such as triglyceride,palmitic acid,linolenic acid triglyceride and lysophosphatidyl.In conclusion,regulating the expression of exosomes,such as rno-mir-122,pdia3 and aldh1l1,is a new target for Carthamus tinctorius water extract to play the role of antioxidation,lipid metabolism and alcohol. |
PDF Full Text Request