| Part 1: Study on nutritional components of blueberry in Majiang of GuizhouObjective: To investigate nutritional components in Guizhou blueberry cultivation in2016,and compare the nutritional components of the same species of blueberry with 10 years ago.Methods: To detecte the sugar,fat and protein of blueberries by direct titration method,Soxhlet extractor fat determination method and spectrophotometry respectively.The contents of vitamin A,C,D and E of it were determined by reverse chromatography,acid titration,forward chromatography and reverse chromatography.The content of SOD was determined by the method of self-oxidation of phenol by three.The method of high performance liquid chromatography(HPLC-MS)was applied to the extraction of blueberry anthocyanins by acidic solvent extraction.Results: The content of protein and total sugar in blueberries at 2016,compared with 2007 reduced(P < 0.05),the content of fat,total acid and the sugar-acid ratio compared with the 2007's without change(P >0.05);the content of SOD in 2016,compared with the 2007's decreased(P< 0.05),the content of anthocyanin in 2016,compared with the 2007's increased(P< 0.01),the content of Na+ and Ca2+ in 2016 was increased,compared with the 2007's(P<0.01),the content of K+ and Mg2+ in 2016 was no change,compared with the 2007's(P>0.05).The content of Vitamin A and D in 2016 was increased,compared with the 2007's(P<0.05).There are at least 16 kinds of anthocyanin,we used the cyanidin-3-Omolecular glucoside to the flowing experiment.Conclusion: We show that the blueberry of Majiang in Guizhou province with high nutritional value,the three nutrient content planting blueberry decreased after 10 years,it may be related to rainfall increased and fruit age increasing;the content of anthocyanins,vitamin C,vitamin E was high,and was significantly higher than the content of related indicators in 2007,the cause may be related to sodium and calcium level in mature mature trees blueberry anthocyanin accumulation and stability increase of blueberry anthocyanin and the the hyperchromic effect.Part 2: Effects of blueberry on histone acetylation and extracellular matrix inObjective: To investigate the effects of blueberry on liver function and hepatic fibrosis in rats with hepatic fibrosis induced by(CCl4),and the levels of histone acetylation modification of ac H3K9,ac H3K14 and ac H3K18 in liver tissue.Methods: 50 male SD rats were randomly divided into five groups,Control group,Hepatic fibrosis group,Blueberry treatment group,Blueberry intervention group,and Natural recovery group,10 rats in each group.Except for normal group,the other groups hypodermic injection the 40% peanut oil and CCl4 at 0.3 ml/100 g,rat model of hepatic fibrosis was prepared by injection once every three days,each rat of hepatic fibrosis was prepared by injection once every three days,but the normal used the peanet oil.The normal and the liver fibrosis group were killed at the end of 12 weeks in this study,the other groups were killed at the end of 16 weeks.The liver function and levels of hepatic fibrosis were measured using the serum of each group,measured the liver index,the left liver tissue were used to observe the pathological changes by HE and Masson staining.The expression of ?-SMA,TIMP-1,collagen I and the levels of ac H3K9,ac H3K14 and ac H3K18 were detected by Western blot in right hepatic tissue of rats in each group.Results: 1.The liver index increased significantly in the liver fibrosis group(P<0.01)vs nomal group,but the blueberry treatment and prevention group decreased(P<0.05)vs the natural recovery group.2.The pathology examination showed that infiltration of inflammatory cell of the injection of CCl4 peanut oil solution of the hepatic fibrosis rats in liver tissue,liver fibrous connective tissue increased,part of the regional formation of pseudolobuli was significantly higher than that in normal rats,Masson staining showed that the expression of extracellular matrix of collagen in the liver fibrosis group was significantly higher than that in the normal group(P<0.05),the two result were to illustrate the success of liver fibrosis.3.The AST showed liver fibrosis group increased significantly(P<0.01)vs the nomal group,but the blueberry treatment and prevention group decreased(P<0.05)vs the natural recovery group.The ALT showed the same result as AST,the liver fibrosis group increased significantly(P<0.01)vs the nomal group,but theblueberry treatment and prevention group decreased(P<0.05)vs the natural recovery group.4.About the index of liver fibrosis(HA,?-C)showed that liver fibrosis group increased significantly(P<0.01)vs the nomal group,but the blueberry treatment and prevention group decreased(P<0.05)vs the natural recovery group,and the blueberry prevention group was more obvious.the index of liver fibrosis of LN showed that liver fibrosis group increased significantly(P<0.01)vs the nomal group,the blueberry treatment and prevention group decreased(P<0.05)vs the natural recovery group.5.Compared with the normal group,the expression levels of ?-SMA?TIMP-1 and collagen ? in liver tissue of rats with hepatic fibrosis were increased(P<0.01).But the blueberry treatment and prevention group decreased(P<0.05)vs the natural recovery group.6.Compared with the normal group,the expression levels of ac H3K9,ac H3K14 and ac H3K18 in liver tissue of rats with hepatic fibrosis were decreased(P<0.01).Compared with the natural recovery group,the expression levels of ac H3K9 ? ach3K14 ? ac H3K18 in liver tissue of blueberry treatment group and blueberry prevention group were decreased(P<0.05),and the blueberry prevention group was more obvious.Compared with the natural recovery group,the expression levels of ac H3K9,ac H3K14 and ac H3K18 in liver tissue of blueberry treatment group and blueberry prevention group were increased(P<0.05).Conclusion: We truly showed that the blueberry fruit can significantly improve liver fibrosis in rats,improved the extracellular matrix deposition and metabolism in the liver,and improved the liver fibrosis pathological changes,reduced serum liver function indexes of ALT,AST and serum fibrosis index,Preventive use of blueberries can improve liver fibrosis.The modification of histone acetylation at ac H3K9,ac H3K14 and ac H3K18 may play an important role in the occurrence and development of fibrosis.Part 3:Effects of anthocyanins extracted from blueberry on the acetylation of ratObjective: To investigate the effects of blueberry on the apoptosis of HSCs-T6 and the level of histone acetylation modification of ac H3K9,ac H3K14 and ac H3K18.Methods: Purification of anthocyanins from blueberry by pharmacological method(HPLC-MS),and HSCs-T6 routine resuscitated,then added the anthocyanins with 50ug/m L,100ug/m L,150ug/m L,200ug/m L.The effects of different concentrations of anthocyanins used by HSCs-T6 in 72 h to observe the proliferation by the real-time x CELLigence(RTCA)dynamically and to determine the appropriate concentration of anthocyanin and the time.According to the concentration and the time,used the Annexin V-FITC/PI,observed the changes of HSCs-T6 cells treated with different concentrations of blueberry anthocyanins by the fluorescence microscope,detected the apoptosis of HSCs-T6 cells treated with different concentrations of anthocyanins by flow cytometry.The expression of-SMA,TIMP-1,collagen I and the levels of ac H3K9,ac H3K14 and ac H3K18 of cultured blueberry anthocyanin concentration treated with HSCs-T6 were detected by Western blot.Results: Compared with the control group,the treatment of HSCs-T6 cells with different concentrations of blueberry anthocyanins could significantly inhibit the proliferation of HSCs-T6 cells in a dose-dependent manner,and the expression of 36 h was significantly inhibited,while used the 50 mol/L concentration,the cell viability was less than 50%(P<0.05).With the increase of blueberry anthocyanin concentration,the number of HSCs-T6 treated with 36 h,more and more HSCs-T6 nuclei were stained red by PI,which was concentration-dependent.The flow cytometry was used to detect the apoptosis,compared with the control group,after treatment with different concentrations of blueberry anthocyanin HSCs-T6 cells with 36 h,with the increasing of concentration,the apoptosis rate increased,and the apoptosis rate was positively correlated with the concentration,the difference was statistically significant(P<0.01).Compared with the control group,the expression of ?-SMA,collagen I and TIMP-1 protein was significantly decreased after treatment with 50ug/m Lconcentration of blueberry anthocyanins(36h)in HSCs-T6 cells(P<0.05);Compared with the control group,the expression of ac H3K9,ac H3K14,and ac H3K18 increased after 36 h treated with 50ug/m L blueberry anthocyanin concentration(HSCs-T6)(P<0.01).Conclusion: We showed that blueberry anthocyanins can inhibit the proliferation of rat activated HSCs-T6,promoted cell apoptosis,regulated ECM and catabolism.Blueberry anthocyanin is one of the most important components of blueberry,it can improve liver fibrosis by upregulating the activated HSC cell line HSCs-T6 histone acetylation at ac H3K9,ac H3K14 ac H3K18,one of the mechanisms for improving liver fibrosis was that it may be changed the histone acetylation modification,made the ECM metabolism related protein expression to reduce the deposition of ECM. |
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