Antioxidant Effects Of Three Vaccinium Ashei In Vitro And Their Protective Mechanism Against Carbon Tetrachloride Induced Acute Liver Injury In Mice

Objective:This study comprehensively evaluated the antioxidant properties of Gardenblue,Britewell and Powderblue from two aspects of antioxidant components and antioxidant capacity,and screened out the varieties with the strongest antioxidant capacity for follow-up animal experimental research.Then,the acute liver injury mouse model was established by CCl4 to study the repairing effect of blueberry juice on acute liver injury in mice and its possible mechanism,so as to offer a theoretical foundation for the comprehensive analysis of the antioxidant effect of blueberry.Methods:In this study,three Vaccinium ashei from Guizhou,including Gardenblue,Britewell and Powderblue were used as raw materials.The anthocyanins,polyphenols,total flavonoids,vitamin C content,SOD activity,three free radical scavenging rates and Oxygen radical absorbance capacity(ORAC)of blueberry fruit,fruit residue and fruit juice were compared under the same conditions.The Vaccinium ashei with rich antioxidant components and high quality were selected and used for subsequent in vivo experiments.After 7 days of adaptive feeding,50 ICR male mice were randomly separated into normal control group(NC),blueberry juice control group(BC),model control group(MC),positive control group(PC)and blueberry juice intervention group(BI),with 10 mice in each group,eating and drinking freely.BI group and BC group were gavaged with blueberry juice,PC group was gavaged with biphenyl diester aqueous solution,and the rest were gavaged with same amount of 0.9%sodium chloride solution.The body weight and food intake were weighed every weekend for 4 weeks.After 4 weeks of intervention,the model of acute liver injury was made by intraperitoneal injection of CCl4.MC group,PC group and BI group were injected with CCl4 peanut oil solution,and the other groups were injected with same amount of 0.9%sodium chloride solution.After modeling,the mice fasted for 16 hours and were killed after weighing,and then collected the blood and liver tissues.(1)Food intake,body weight and liver index were calculated;(2)The content of fasting blood glucose(FBG),triglyceride(TG),total cholesterol(TC),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured;(3)Make pathological sections of liver tissue and take photos for observation;(4)The content of liver product related to oxidative stress:the activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX)and malondialdehyde(MDA)were measured;(5)The content of hepatocyte apoptosis related indexes:glucose regulatory protein 78(GRP78),C/EBP homologous protein(CHOP),ccaspase-3 and Cytochrome c(Cytc);(6)The content of liver fibrosis markers:transforming growth factor?1(TGF-?1),?-smooth muscle actin(?-SMA)and tissue inhibitor of metalloproteinase-1(TIMP-1);(7)Detect the content of inflammatory factors in liver tissue:phosphorylated nuclear factors ?B(p-NF-?B),Interleukin 1?(IL-1?)and interleukin-6(IL-6).Result:1.Comparative analysis of antioxidant components and antioxidant capacity of three kinds of Vaccinium ashei:(1)Anthocyanins,total flavonoids,polyphenols,vitamin C content and pH value of three Vaccinium asheiThe content of anthocyanins,total flavonoids and polyphenols in Gardenblue fruit,fruit residue and fruit juice were significantly higher than those in Britewell and Powderblue(P<0.05);Except the content of total flavonoids between Britewell and Powderblue(P>0.05),others were significantly higher than Powderblue(P<0.05).The content of vitamin C in the fruits and residues of the three Vaccinium ashei was Powderblue,Britewell and Gardenblue from high to low,but it makes no significant difference(P>0.05);In fruit juice,the content vitamin C in Britewell was the highest,followed by Powderblue,Gardenblue was the lowest,and the difference was significant(P<0.05).The pH value of fruit juice from low to high was Britewell,Powderblue and Gardenblue,and the difference was significant(P<0.05).(2)SOD activity of three Vaccinium ashei The SOD activity of Gardenblue fruit,fruit residue and fruit juice was the highest,followed by Powderblue,Britewell was the lowest,and the difference was significant(P<0.05).(3)Free radical scavenging rate and ORAC value of three Vaccinium ashei Among the three Vaccinium ashei,Gardenblue fruit,fruit residue and fruit juice had the highest DPPH free radical scavenging rate and ·OH free radical scavenging rates,and that of Powderblue was the lowest,and the difference was significant(P<0.05);The scavenging rate of ·O2-free radical in Gardenblue fruit,fruit residue and fruit juice was the highest,and it makes no significant difference between Britewell and Powderblue(P>0.05).Among the three Vaccinium ashei,the ORAC value of Gardenblue was the highest and Powderblue was the lowest,regardless of fruit,fruit residue or fruit juice(P<0.05).(4)Correlation analysis of antioxidant components and antioxidant capacity of three Vaccinium ashei Multiple linear regression analysis showed that there was a significant positive correlation between ORAC value and total flavonoids content of three kinds of Vaccinium ashei(P<0.05).2.Detection and analysis results of indexes related to oxidative stress in blood and liver of mice with acute liver injury:(1)Calculation of food intake,body weight and liver index It makes no significant difference in daily food intake among NC group,BC group,MC group and BI group(P>0.05),but the daily food intake of PC group was significantly higher compared with other groups(P<0.05);The weight change trend of mice in each group was the same as that of daily food intake analysis;After modeling,the liver index in MC group was significantly higher compared with NC group and BC group,and the difference was significant(P<0.05).Compared with MC group,the liver index in PC group and BI group was lower,and the liver index in PC group was lower compared with BI group,but it makes no significant difference(P>0.05).(2)Detection of aminotransferase,blood glucose and lipid content Compared with NC group,ALT and AST in MC group significantly increased(P<0.05),indicating the success of modeling.Compared with MC group,ALT and AST in BI group and PC group significantly decreased(P<0.05),and the difference was significant(P<0.05);The analysis results of the content of FBG,TC and TG in mice were the same.MC group was significantly higher than that of NC group and BC group(P<0.05),and MC group>BI group>PC group,and the difference was significant(P<0.05).(3)Detection of antioxidant enzymes and lipid peroxidation products in liver The activity of SOD in MC group was higher than that in NC group,but lower than that in BC group,PC group and BI group,and the differences were significant(P<0.05),but it makes no significant difference between PC and BI group(P>0.05);The results of GSH-PX activity analysis were consistent with SOD;The content of MDA in MC group was significantly increased compared with NC group(P<0.05);The content of MDA in PC and BI group decreased significantly compared with MC group(P<0.05),but it makes no significant difference between NC and BC group,as well as between PC and BI group(P>0.05).(4)Liver tissue section staining and pathological observation HE staining showed that compared with MC group,the hepatocytes of NC group,BC group and PC group had regular morphology,complete structure and dense and uniform arrangement.MC group mice showed disorder of hepatocyte cord arrangement,unclear boundary of hepatocytes,shrinkage of hepatocytes,pyknosis of nuclei,deepening nuclear staining and accumulation of inflammatory factors.The above phenomena were significantly reduced and the structure of hepatocytes was basically normal in BI group.3.Detection and analysis results of liver related signal pathways in mice with acute liver injury:(1)Detection of related indexes of hepatocyte apoptosis Compared with NC group,the content of GRP78 in MC group increased significantly(P<0.05),and decreased significantly in PC group and BI group(P<0.05);and the content of GRP78 in BI group was significantly higher than PC group(P<0.05);Except it makes no significant difference in the content of CHOP between PC group and BI group(P>0.05),the trend of other indicators is the same as GRP78;The difference of caspase-3 was consistent with CHOP;Except that there was no significant difference in the content of CytC between MC and BI group(P>0.05),other differences were the same as GRP78.(2)Detection of hepatic fibrosis markers The content of TGF-?1 in MC group was significantly higher than that in NC group(P<0.05).Compared with MC group,the content of TGF-?1 in PC group was significantly lower(P<0.05),but it makes no difference in BI group(P>0.05).At the same time,it makes no significant difference in the content between NC,BC and PC group(P>0.05);The content of ?-SMA in BI group was significantly lower than that in MC group(P<0.05);The content difference of TIMP-1 is the same as that of ?-SMA.(3)Detection of inflammatory factors in liver tissue Compared with NC group,the content of p-NF-?B in MC group was significantly higher(P<0.05);the content of p-NF-?B in PC group and BI group was lower compared with MC group,and the difference was significant(P<0.05).There were significant differences in the content of p-NF-KB in other groups except BC group and NC group(P<0.05);Compared with NC group,the content of IL-1? in MC group significantly increased(P<0.05),and the content of IL-1? in PC group decreased significantly(P<0.05),but it makes no significant difference between MC group and BI group(P>0.05);The content of IL-6 was the same as that of IL-1? except that there was no significant difference among NC group,PC group and BC group.Conclusion:(1)Gardenblue has the most advantages in both the content of antioxidant active substances and antioxidant capacity.It is a Vaccinium ashei variety with superior antioxidant performance,and the free radical scavenging capacity and antioxidant capacity of blueberry are related to the content of polyphenols,anthocyanins and total flavonoids.Therefore,it shows that the high content of anthocyanins,total flavonoids and polyphenols in blueberry are the main components of its antioxidant ability,and play a decisive role in its antioxidation performance.(2)Blueberry juice can reduce the lipid peroxidation of cell membrane by reducing or eliminating the production of reactive oxygen free radicals and increasing antioxidant enzymes,significantly reduce the degree of liver injury in mice with acute liver injury,and maintain the complete structure and normal physiological and metabolic function of hepatocytes,so as to protect the liver.Blueberry has obvious antioxidant stress effect,which can inhibit hepatocyte apoptosis,liver fibrosis,liver inflammatory factors and other related indicators in varying degrees.Therefore,whether as a daily antioxidant or as an auxiliary food for oxidative damage repair,blueberries have significant effects.