蓝莓营养成分及其改善大鼠肝纤维化机制中组蛋白乙酰化修饰的研究
发布时间：2018-08-28 08:48
【摘要】：第一部分贵州麻江蓝莓营养成分的研究目的:1.探讨2016年贵州麻江蓝莓营养成分,并比较与10年前同品种蓝莓营养成分的比较的情况。方法:以直接滴定法、索氏脂肪提取器测定法、分光光度法及酸碱滴定法分别对蓝莓中的糖、脂肪、蛋白质及酸进行检测;以反向色谱分离法、酸性滴定法、正向色谱分离法、反向色谱分离法分别对蓝莓中维生素A、C、D、E的含量进行检测;改良的Marklund方法(改良的邻苯三酚自氧化法)检测SOD含量;以溶剂浸提法提取后高效液相色谱法(HPLC-MS)对蓝莓花青素进行鉴定。结果:贵州省麻江县2016年种植的圆蓝品种的蓝莓蛋白质、总糖的含量以及糖酸比均降低,差异有统计学意义(P0.05),而脂肪、总酸的含量无明显变化,差异无统计学意义(P0.05)。与2007年比较,2016年贵州省麻江县种植的圆蓝品种的SOD,蓝莓花青素、维生素C、维生素E的含量有显著性增加,差异有统计学意义(P0.05),而维生素A、维生素D的含量无明显变化,差异无统计学意义(P0.05);与2007年比较,2016年蓝莓中所含的Na+、Ca2+均显著性增加,差异有统计学意义(P0.05),而K+及Mg2+的含量无差异,无统计学意义(P0.05)。结论:研究表明,贵州省麻江圆蓝品种蓝莓具有较高营养价值,种植10年后蓝莓的三大营养物质成分含量有所降低,与当年降雨量增多及果树树龄增大有关;麻江圆蓝蓝莓中的花青素、维生素C、维生素E的含量较高,且明显高于2007年栽种初产果实中相关指标的含量,这与蓝莓成熟后花青素富集及成熟蓝莓鲜果中钠、钙离子水平增加对蓝莓花青素稳定性增加而发生的增色效应有关。第二部分蓝莓对肝纤维化大鼠肝脏组蛋白乙酰化修饰及细胞外基目的:探讨蓝莓对四氯化碳(CCl4)致大鼠肝纤维化后大鼠血清的肝功能、肝纤维化三项,肝组织中细胞外基质及组蛋白乙酰化修饰ac H3K9、ac H3K14、ac H3K18位点修饰水平的影响。方法:50只雄性SD大鼠,180±20g,随机分为对照组(Control group)、模型组(Hepatic Fibrosis group)、蓝莓治疗组(Blueberry Treatment group)、蓝莓预防组(Blueberry Prevention group)、自然恢复组(Natural Recovery group),每组10只。除正常组外,其余各组用40%CCl4花生油溶液0.3 m L/100 g皮下注射,间隔三天注射一次以制备大鼠肝纤维化模型,正常组大鼠皮下注射等量花生油溶液,实验12周末处死正常组与肝纤维化组大鼠,实验16周末处死剩余组大鼠;取各组大鼠血清检测其肝功能、肝纤维化三项指标;取各组大鼠肝脏,测定肝脏指数;取左肝叶组织常规固定,HE染色、Masson染色观察组织病理改变情况;蛋白质免疫印迹法(Western blot,WB)检测并比较各组大鼠右肝叶组织α-SMA、TIMP-1、Ⅰ型胶原表达情况和ac H3K9、ac H3K14、ac H3K18修饰水平变化。结果:1.关于肝指数,与正常组比较,肝纤维化组大鼠肝脏指数增加(P0.05),与肝纤维化组比较,经蓝莓汁灌胃后,蓝莓治疗组与蓝莓预防组大鼠的肝指数降低,差异有统计学意义(P0.05);与自然恢复组比,蓝莓预防组肝指数降低,有显著性差异(P0.05),而蓝莓治疗组无明显变化,无明显差异(P0.05);与蓝莓治疗组比较,蓝莓预防组肝指数降低,有显著性差异(P0.05)。2.根据肝纤维化分级,病理学检查提示注射CCl4花生油溶液的肝纤维化组大鼠肝组织内大量炎性细胞浸润,肝脏纤维结缔组织明显增多,部分区域有假小叶形成,明显高于正常组大鼠,Masson染色观察提示CCl4花生油溶液注射后,肝纤维化组大鼠肝脏中胶原等细胞外基质表达明显增加,说明肝纤维化造模成功;与模型组比较,蓝莓治疗组及蓝莓预防组大鼠肝脏纤维化分级显著性降低,有显著性差异(P0.01);与自然恢复组比较,蓝莓预防组大鼠肝脏纤维化分级显著性降低,有显著性差异(P0.01),而蓝莓治疗组大鼠肝脏纤维化分级无明显变化,无明显统计学差异(P0.05)。与蓝莓治疗组比较,蓝莓预防组肝脏纤维化分级有明显变化,有显著性差异(P0.05)。3.关于肝功能:(1)与正常组比较,模型组AST显著增高(P0.01);与模型组比较,蓝莓治疗组及蓝莓预防组AST显著降低(P0.01);与自然恢复组比较,蓝莓预防组显著降低(P0.01),蓝莓治疗组降低(P0.05);与蓝莓治疗组比较,蓝莓预防组有降低(P0.05);(2)与正常组比较,模型组ALT显著增高(P0.01);与模型组比较,蓝莓治疗组及蓝莓预防组ALT显著下降(P0.01);与自然恢复组比较,蓝莓预防组ALT显著降低(P0.01),蓝莓治疗组无显著性差异(P0.05);与蓝莓治疗组比较,蓝莓预防组ALT降低(P0.05)。3.关于肝纤维化指标,(1)与正常组比较,模型组HA显著增高(P0.01);与模型组比较,蓝莓治疗组及蓝莓预防组HA显著降低(P0.01);与自然恢复组比较,蓝莓预防组显著降低(P0.01),蓝莓治疗组降低(P0.05);与蓝莓治疗组比较,蓝莓预防组降低(P0.05);(2)与正常组比较,余各组大鼠LN显著性增加(P0.05)。(3)与正常组比较,模型组Ⅳ-C显著增高(P0.01);与模型组比较,蓝莓治疗组及蓝莓预防组Ⅳ-C显著降低(P0.01);与自然恢复组比较,蓝莓治疗组及蓝莓预防组显著降低(P0.05);与蓝莓治疗组比较,蓝莓预防组降低(P0.05);4.与正常组比较,模型组肝组织中的α-SMA、?型胶原和TIMP-1蛋白表达均增加,有显著性差异(P0.01);与模型组比较:蓝莓治疗组、蓝莓预防组及自然恢复组肝组织中的α-SMA、?型胶原和TIMP-1表达降低,有显著性差异(P0.01);与自然恢复组比较,蓝莓治疗组及蓝莓预防组肝组织中的α-SMA、?型胶原和TIMP-1表达降低,有显著性差异(P0.05),以蓝莓预防组降低较为明显。与蓝莓治疗组比较,蓝莓预防组肝组织中的的α-SMA、?型胶原和TIMP-1表达降低。5.与正常组比较,模型组肝组织中的ac H3K9、ac H3K14、ac H3K18修饰水平均降低,有显著性差异(P0.01);与模型组比较,蓝莓治疗组、蓝莓预防组及自然恢复组肝组织中的ac H3K9、ac H3K14、ac H3K18修饰水平均升高,有显著性差异(P0.01);与自然恢复组比较,蓝莓治疗组及蓝莓预防组的ac H3K9、ac H3K14、ac H3K18修饰水平均升高,有显著性差异(P0.05),以蓝莓预防组升高较为明显。与蓝莓治疗组比较,蓝莓预防组肝组织中的ac H3K9、ac H3K14、ac H3K18修饰水平升高。结论:证实了口服蓝莓可以显著改善肝纤维化病理改变,降低血清肝功能指标ALT、AST及血清肝纤维化相关指标,改善肝脏内细胞外基质沉积和代谢,预防性使用蓝莓对肝纤维化的改善作用更好。ac H3K9、ac H3K14、ac H3K18组蛋白乙酰化修饰改变可能参与了肝纤维化的发生及发展,并与蓝莓改善肝纤维化发生机制有关。第三部分蓝莓提取的花青素在体外对激活型大鼠肝星状细胞株组蛋白乙酰化修饰的影响和增殖凋亡的作用目的:探讨蓝莓花青素对大鼠肝星状细胞株(HSCs-T6)增殖与凋亡的影响,并对蓝莓花青素处理后的HSCs-T6细胞外基质蛋白及组蛋白乙酰化修饰ac H3K9、ac H3K14、ac H3K18位点修饰水平的影响。方法:采用药理学的方法提纯蓝莓中的花青素。HSCs-T6常规复苏培养后,分别加入蓝莓花青素50ug/m L、100ug/m L、150ug/m L、200ug/m L。使用实时无标记细胞分析仪(RTCA x CELLigence)动态观察不同浓度蓝莓花青素(50ug/m L、100ug/m L、150ug/m L、200ug/m L)处理HSCs-T6 72 h后细胞增殖的影响,确定合适的花青素浓度及作用时间;根据(RTCA)实验结果选择合适的时间后加入Annexin V-FITC/PI双染后,荧光显微镜下观察不同浓度蓝莓花青素处理后的HSCs-T6细胞形态变化;流式细胞仪检测不同浓度花青素处理HSCs-T6细胞凋亡的情况;western blotting检测蓝莓花青素最适浓度处理HSCs-T6细胞36 h,细胞的α-SMA、Ⅰ型胶原及TIMP-1表达,western blotting检测HSCs-T6细胞ac H3K9、ac H3K14、ac H3K18修饰水平变化。结果:与对照组相比,不同浓度蓝莓花青素处理HSCs-T6细胞36h,均能明显抑制HSCs-T6细胞的增殖,呈剂量依赖性;浓度为50μmol/L时,HSCs-T6细胞的存活率低于50%(P0.05);免疫荧光实验显示,随着蓝莓花青素浓度增加并处理HSCs-T6 36 h,HSCs-T6凋亡数量逐渐增加,更多的HSCs-T6细胞核被PI染成红色,且呈浓度依赖性。流式细胞仪检测的凋亡实验中,与对照组相比,不同浓度的蓝莓花青素处理HSCs-T6细胞36 h,随着蓝莓花青素浓度增加凋亡率增加,差异有统计学意义(P0.01);与对照组相比,50ug/m L浓度的蓝莓花青素处理HSCs-T6细胞36h,细胞的α-SMA、Ⅰ型胶原、TIMP-1表达明显降低(P0.05);与对照组相比,50 ug/m L浓度的蓝莓花青素处理HSCs-T6细胞36 h,细胞的ac H3K9、ac H3K14、ac H3K18修饰水平增加(P0.01);结论:蓝莓花青素可以抑制大鼠激活型HSC细胞株HSCs-T6增殖,促进细胞凋亡,调节ECM合成和分解代谢。蓝莓花青素是蓝莓改善肝纤维化的重要有效成分之一;蓝莓花青素可上调HSCs-T6细胞中ac H3K9、ac H3K14、ac H3K18组蛋白乙酰化修饰水平,通过改变其组蛋白乙酰化修饰对ECM代谢相关蛋白表达来达到减少ECM沉积是蓝莓花青素改善肝纤维化的机制之一。
[Abstract]:Objective: 1. To study the nutritional components of blueberry in Majiang, Guizhou in 2016, and compare the nutritional components with that of the same variety of blueberry 10 years ago. Methods: Sugar, fat and protein in blueberry were determined by direct titration, Soxhlet fat extractor, spectrophotometry and acid-base titration, respectively. The contents of vitamin A, C, D and E in blueberry were determined by reverse chromatography, acidic titration, forward chromatography and reverse chromatography, respectively; the content of SOD was detected by improved Marklund method (improved pyrogallol autoxidation method); the content of SOD was determined by solvent extraction followed by high performance liquid chromatography (HPLC-MS). Results: The contents of protein, total sugar and sugar-acid ratio of blueberries planted in 2016 in Majiang County of Guizhou Province were significantly lower than those in 2016 (P 0.05), while the contents of fat and total acid had no significant difference (P 0.05). The contents of SOD, blueberry anthocyanin, vitamin C and vitamin E increased significantly (P 0.05), but the contents of vitamin A and vitamin D did not change significantly (P 0.05); compared with 2007, the contents of Na +, Ca2 + in 2016 blueberry increased significantly (P 0.05), while the contents of K + increased significantly (P 0.05). There was no significant difference in the contents of Mg2 + and Mg2 + (P 0.05). Conclusion: The results showed that the blueberry varieties of Majiang Round Blue had high nutritional value, and the contents of the three nutrients of blueberry decreased after planting for 10 years, which was related to the increase of rainfall and the age of fruit trees. The content of vegetable E was higher than that of the related indexes in the primary fruits in 2007, which was related to the enrichment of anthocyanin and the increase of sodium and calcium in the fresh fruits of mature blueberries. Objective: To investigate the effects of blueberry on serum liver function, liver fibrosis, extracellular matrix and histone acetylation modified AC H3K9, AC H3K14, AC H3K18 loci in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4). Mol group, Hepatic Fibrosis group, Blueberry Treatment group, Blueberry Prevention group, Natural Recovery group, 10 rats in each group. Rats in the normal group were subcutaneously injected with the same amount of peanut oil solution, rats in the normal group and the liver fibrosis group were sacrificed at the end of the 12th week and rats in the remaining group at the end of the 16th week. E staining and Masson staining were used to observe the histopathological changes; Western blot (WB) was used to detect and compare the expression of alpha-SMA, TIMP-1, type I collagen and the changes of modification levels of AC H3K9, AC H3K14 and AC H3K18 in the right hepatic lobe of rats in each group. Results: 1. Compared with the normal group, the liver index of rats in the liver fibrosis group increased. In addition (P 0.05), compared with the liver fibrosis group, the liver index of the blueberry treatment group and the blueberry prevention group decreased significantly (P 0.05); compared with the natural recovery group, the liver index of the blueberry prevention group decreased significantly (P 0.05), while the blueberry treatment group did not change significantly (P 0.05). Compared with the control group, the liver index of the blueberry prevention group decreased significantly (P 0.05). 2. According to the liver fibrosis grading, pathological examination showed that a large number of inflammatory cells infiltrated in liver tissue, fibrous connective tissue increased significantly, and pseudolobules formed in some areas of the liver of the rats injected with CCl4 peanut oil solution, which was significantly higher than that of the normal group, Ma. Son staining showed that the expression of extracellular matrix (ECM) such as collagen in liver of rats with hepatic fibrosis was significantly increased after CCl4 peanut oil injection, indicating that the model of hepatic fibrosis was successfully established. Compared with the blueberry group, the grade of liver fibrosis in the blueberry prevention group decreased significantly (P 0.01), but the grade of liver fibrosis in the blueberry treatment group did not change significantly (P 0.05). Compared with the blueberry treatment group, the grade of liver fibrosis in the blueberry prevention group changed significantly (P 0.05). 3. Can: (1) compared with the normal group, the model group AST significantly increased (P 0.01); compared with the model group, the blueberry treatment group and the blueberry prevention group AST significantly decreased (P 0.01); compared with the natural recovery group, blueberry prevention group significantly decreased (P 0.01), blueberry treatment group decreased (P 0.05); compared with the blueberry treatment group, blueberry prevention group decreased (P 0.05); (2) compared with the normal group, blueberry prevention group decreased (P 0.05); (2) compared with the blueberry Compared with the model group, ALT was significantly increased (P 0.01); compared with the model group, ALT in the blueberry treatment group and the blueberry prevention group was significantly decreased (P 0.01); compared with the natural recovery group, ALT in the blueberry prevention group was significantly decreased (P 0.01), but there was no significant difference in the blueberry treatment group (P 0.05); compared with the blueberry treatment group, ALT in the blueberry prevention group was significantly decreased (P 0.05). 3. (1) Compared with the normal group, the HA of the model group increased significantly (P 0.01); compared with the model group, the HA of the blueberry treatment group and the blueberry prevention group decreased significantly (P 0.01); compared with the natural recovery group, the blueberry prevention group decreased significantly (P 0.01), the blueberry treatment group decreased (P 0.05); compared with the blueberry treatment group, the blueberry prevention group decreased (P 0.05); and (2) compared with the normal group, the rest of each group. LN increased significantly (P 0.05). (3) Compared with the normal group, model group IV-C increased significantly (P 0.01); compared with model group, blueberry treatment group and blueberry prevention group IV-C decreased significantly (P 0.01); compared with natural recovery group, blueberry treatment group and blueberry prevention group decreased significantly (P 0.05); compared with blueberry treatment group, blueberry prevention group decreased (P 0.05); 4. Compared with the normal group, the expression of alpha-SMA, collagen and TIMP-1 protein in the liver tissue of the model group increased significantly (P 0.01); Compared with the model group, the expression of alpha-SMA, collagen and TIMP-1 protein in the liver tissue of the blueberry treatment group, the blueberry prevention group and the natural recovery group decreased significantly (P 0.01); Compared with the natural recovery group, the expression of collagen and TIMP-1 protein in the blueberry was significantly lower (P 0.01). The expression of alpha-SMA, collagen and TIMP-1 in the liver tissue of the treatment group and the blueberry prevention group was significantly lower than that of the blueberry prevention group (P 0.05). Compared with the blueberry treatment group, the expression of alpha-SMA, collagen and TIMP-1 in the liver tissue of the blueberry prevention group was significantly lower. Compared with the model group, the levels of ACH3K9, ACH3K14 and ACH3K18 in the liver tissues of the blueberry treatment group, the blueberry prevention group and the natural recovery group were significantly higher (P 0.01); compared with the natural recovery group, the levels of ACH3K9, ACH3K14, ACH3 K18 in the blueberry treatment group and the blueberry prevention group were significantly higher (P 0.01). Compared with the blueberry treatment group, the levels of ACH3K9, ACH3K14 and ACH3K18 in the liver tissue of the blueberry prevention group increased. Conclusion: Oral blueberry can significantly improve the pathological changes of liver fibrosis and reduce the serum ALT, AST and blood levels of liver function indicators. The acetylation modification of ACH3K9, ACH3K14 and ACH3K18 histones may be involved in the occurrence and development of liver fibrosis and may be related to the mechanism of blueberry improving liver fibrosis. Effects of anthocyanin extracted from blueberry on histone acetylation and apoptosis of activated rat hepatic stellate cell line in vitro Objective: To investigate the effects of blueberry anthocyanin on proliferation and apoptosis of rat hepatic stellate cell line (HSCs-T6), and to modify the extracellular matrix protein and histone acetylation of HSCs-T6 treated with blueberry anthocyanin. Methods: Anthocyanins in blueberries were purified by pharmacological methods. HSCs-T6 was cultured by routine resuscitation. Anthocyanins 50ug/m L, 100ug/m L, 150ug/m L, 200ug/m L were added to blueberries respectively. Real-time labeless cell analyzer (RTCA x CELLigence) was used to observe the dynamic changes of blueberry flowers at different concentrations. The effect of anthocyanin (50ug/m L, 100ug/m L, 150ug/m L, 200ug/m L) on the proliferation of HSCs-T6 cells after 72 h treatment was determined to determine the appropriate anthocyanin concentration and action time. According to the experimental results of RTCA, the morphology of HSCs-T6 cells treated with different concentrations of blueberry anthocyanin was observed under fluorescence microscope after adding Annexin V-FITC/PI double staining. Flow cytometry was used to detect the apoptosis of HSCs-T6 cells treated with different concentrations of anthocyanin; Western blotting was used to detect the expression of alpha-SMA, collagen type I and TIMP-1 in HSCs-T6 cells treated with blueberry anthocyanin at the optimum concentration for 36 hours; Western blotting was used to detect the changes of the modification levels of AC H3K9, AC H3K14 and AC H3K18 in HSCs-T6 cells. Compared with HSCs-T6 cells treated with different concentrations of blueberry anthocyanin for 36 hours, the proliferation of HSCs-T6 cells was significantly inhibited in a dose-dependent manner; the survival rate of HSCs-T6 cells was less than 50% (P 0.05) when the concentration of blueberry anthocyanin was 50 micromol/L; immunofluorescence assay showed that with the concentration of blueberry anthocyanin increased and treated with HSCs-T6 for 36 hours, the number of apoptosis of HSCs-T6 cells increased gradually, and more. The nuclei of HSCs-T6 cells were stained red by PI in a concentration-dependent manner. In the apoptosis test of flow cytometry, compared with the control group, the apoptosis rate of HSCs-T6 cells treated with blueberry anthocyanin at different concentrations for 36 hours increased with the increase of blueberry anthocyanin concentration, and the difference was statistically significant (P 0.01); compared with the control group, the 50 ug/ml concentration of blueberry anthocyanin increased the apoptosis rate (P 0.01). The expression of alpha-SMA, collagen type I and TIMP-1 in HSCs-T6 cells treated with blueberry anthocyanin decreased significantly after 36 hours (P 0.05); compared with the control group, the expression of ACH3K9, ACH3K14 and ACH3K18 in HSCs-T6 cells treated with blueberry anthocyanin at 50 ug/ml for 36 hours increased (P 0.01); Conclusion: blueberry anthocyanin can inhibit the increase of HSCs-T6 in rat activated HSC cells. Blueberry anthocyanin is one of the important components of blueberry to improve liver fibrosis; blueberry anthocyanin can up-regulate the acetylation level of AC H3K9, AC H3K14 and AC H3K18 histones in HSCs-T6 cells, and can be achieved by altering the acetylation of histone to the expression of ECM metabolism-related proteins. Reducing ECM deposition is one of the mechanisms of blueberry anthocyanin improving liver fibrosis.
【学位授予单位】：贵州医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.2
本文编号：2208894
[Abstract]:Objective: 1. To study the nutritional components of blueberry in Majiang, Guizhou in 2016, and compare the nutritional components with that of the same variety of blueberry 10 years ago. Methods: Sugar, fat and protein in blueberry were determined by direct titration, Soxhlet fat extractor, spectrophotometry and acid-base titration, respectively. The contents of vitamin A, C, D and E in blueberry were determined by reverse chromatography, acidic titration, forward chromatography and reverse chromatography, respectively; the content of SOD was detected by improved Marklund method (improved pyrogallol autoxidation method); the content of SOD was determined by solvent extraction followed by high performance liquid chromatography (HPLC-MS). Results: The contents of protein, total sugar and sugar-acid ratio of blueberries planted in 2016 in Majiang County of Guizhou Province were significantly lower than those in 2016 (P 0.05), while the contents of fat and total acid had no significant difference (P 0.05). The contents of SOD, blueberry anthocyanin, vitamin C and vitamin E increased significantly (P 0.05), but the contents of vitamin A and vitamin D did not change significantly (P 0.05); compared with 2007, the contents of Na +, Ca2 + in 2016 blueberry increased significantly (P 0.05), while the contents of K + increased significantly (P 0.05). There was no significant difference in the contents of Mg2 + and Mg2 + (P 0.05). Conclusion: The results showed that the blueberry varieties of Majiang Round Blue had high nutritional value, and the contents of the three nutrients of blueberry decreased after planting for 10 years, which was related to the increase of rainfall and the age of fruit trees. The content of vegetable E was higher than that of the related indexes in the primary fruits in 2007, which was related to the enrichment of anthocyanin and the increase of sodium and calcium in the fresh fruits of mature blueberries. Objective: To investigate the effects of blueberry on serum liver function, liver fibrosis, extracellular matrix and histone acetylation modified AC H3K9, AC H3K14, AC H3K18 loci in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4). Mol group, Hepatic Fibrosis group, Blueberry Treatment group, Blueberry Prevention group, Natural Recovery group, 10 rats in each group. Rats in the normal group were subcutaneously injected with the same amount of peanut oil solution, rats in the normal group and the liver fibrosis group were sacrificed at the end of the 12th week and rats in the remaining group at the end of the 16th week. E staining and Masson staining were used to observe the histopathological changes; Western blot (WB) was used to detect and compare the expression of alpha-SMA, TIMP-1, type I collagen and the changes of modification levels of AC H3K9, AC H3K14 and AC H3K18 in the right hepatic lobe of rats in each group. Results: 1. Compared with the normal group, the liver index of rats in the liver fibrosis group increased. In addition (P 0.05), compared with the liver fibrosis group, the liver index of the blueberry treatment group and the blueberry prevention group decreased significantly (P 0.05); compared with the natural recovery group, the liver index of the blueberry prevention group decreased significantly (P 0.05), while the blueberry treatment group did not change significantly (P 0.05). Compared with the control group, the liver index of the blueberry prevention group decreased significantly (P 0.05). 2. According to the liver fibrosis grading, pathological examination showed that a large number of inflammatory cells infiltrated in liver tissue, fibrous connective tissue increased significantly, and pseudolobules formed in some areas of the liver of the rats injected with CCl4 peanut oil solution, which was significantly higher than that of the normal group, Ma. Son staining showed that the expression of extracellular matrix (ECM) such as collagen in liver of rats with hepatic fibrosis was significantly increased after CCl4 peanut oil injection, indicating that the model of hepatic fibrosis was successfully established. Compared with the blueberry group, the grade of liver fibrosis in the blueberry prevention group decreased significantly (P 0.01), but the grade of liver fibrosis in the blueberry treatment group did not change significantly (P 0.05). Compared with the blueberry treatment group, the grade of liver fibrosis in the blueberry prevention group changed significantly (P 0.05). 3. Can: (1) compared with the normal group, the model group AST significantly increased (P 0.01); compared with the model group, the blueberry treatment group and the blueberry prevention group AST significantly decreased (P 0.01); compared with the natural recovery group, blueberry prevention group significantly decreased (P 0.01), blueberry treatment group decreased (P 0.05); compared with the blueberry treatment group, blueberry prevention group decreased (P 0.05); (2) compared with the normal group, blueberry prevention group decreased (P 0.05); (2) compared with the blueberry Compared with the model group, ALT was significantly increased (P 0.01); compared with the model group, ALT in the blueberry treatment group and the blueberry prevention group was significantly decreased (P 0.01); compared with the natural recovery group, ALT in the blueberry prevention group was significantly decreased (P 0.01), but there was no significant difference in the blueberry treatment group (P 0.05); compared with the blueberry treatment group, ALT in the blueberry prevention group was significantly decreased (P 0.05). 3. (1) Compared with the normal group, the HA of the model group increased significantly (P 0.01); compared with the model group, the HA of the blueberry treatment group and the blueberry prevention group decreased significantly (P 0.01); compared with the natural recovery group, the blueberry prevention group decreased significantly (P 0.01), the blueberry treatment group decreased (P 0.05); compared with the blueberry treatment group, the blueberry prevention group decreased (P 0.05); and (2) compared with the normal group, the rest of each group. LN increased significantly (P 0.05). (3) Compared with the normal group, model group IV-C increased significantly (P 0.01); compared with model group, blueberry treatment group and blueberry prevention group IV-C decreased significantly (P 0.01); compared with natural recovery group, blueberry treatment group and blueberry prevention group decreased significantly (P 0.05); compared with blueberry treatment group, blueberry prevention group decreased (P 0.05); 4. Compared with the normal group, the expression of alpha-SMA, collagen and TIMP-1 protein in the liver tissue of the model group increased significantly (P 0.01); Compared with the model group, the expression of alpha-SMA, collagen and TIMP-1 protein in the liver tissue of the blueberry treatment group, the blueberry prevention group and the natural recovery group decreased significantly (P 0.01); Compared with the natural recovery group, the expression of collagen and TIMP-1 protein in the blueberry was significantly lower (P 0.01). The expression of alpha-SMA, collagen and TIMP-1 in the liver tissue of the treatment group and the blueberry prevention group was significantly lower than that of the blueberry prevention group (P 0.05). Compared with the blueberry treatment group, the expression of alpha-SMA, collagen and TIMP-1 in the liver tissue of the blueberry prevention group was significantly lower. Compared with the model group, the levels of ACH3K9, ACH3K14 and ACH3K18 in the liver tissues of the blueberry treatment group, the blueberry prevention group and the natural recovery group were significantly higher (P 0.01); compared with the natural recovery group, the levels of ACH3K9, ACH3K14, ACH3 K18 in the blueberry treatment group and the blueberry prevention group were significantly higher (P 0.01). Compared with the blueberry treatment group, the levels of ACH3K9, ACH3K14 and ACH3K18 in the liver tissue of the blueberry prevention group increased. Conclusion: Oral blueberry can significantly improve the pathological changes of liver fibrosis and reduce the serum ALT, AST and blood levels of liver function indicators. The acetylation modification of ACH3K9, ACH3K14 and ACH3K18 histones may be involved in the occurrence and development of liver fibrosis and may be related to the mechanism of blueberry improving liver fibrosis. Effects of anthocyanin extracted from blueberry on histone acetylation and apoptosis of activated rat hepatic stellate cell line in vitro Objective: To investigate the effects of blueberry anthocyanin on proliferation and apoptosis of rat hepatic stellate cell line (HSCs-T6), and to modify the extracellular matrix protein and histone acetylation of HSCs-T6 treated with blueberry anthocyanin. Methods: Anthocyanins in blueberries were purified by pharmacological methods. HSCs-T6 was cultured by routine resuscitation. Anthocyanins 50ug/m L, 100ug/m L, 150ug/m L, 200ug/m L were added to blueberries respectively. Real-time labeless cell analyzer (RTCA x CELLigence) was used to observe the dynamic changes of blueberry flowers at different concentrations. The effect of anthocyanin (50ug/m L, 100ug/m L, 150ug/m L, 200ug/m L) on the proliferation of HSCs-T6 cells after 72 h treatment was determined to determine the appropriate anthocyanin concentration and action time. According to the experimental results of RTCA, the morphology of HSCs-T6 cells treated with different concentrations of blueberry anthocyanin was observed under fluorescence microscope after adding Annexin V-FITC/PI double staining. Flow cytometry was used to detect the apoptosis of HSCs-T6 cells treated with different concentrations of anthocyanin; Western blotting was used to detect the expression of alpha-SMA, collagen type I and TIMP-1 in HSCs-T6 cells treated with blueberry anthocyanin at the optimum concentration for 36 hours; Western blotting was used to detect the changes of the modification levels of AC H3K9, AC H3K14 and AC H3K18 in HSCs-T6 cells. Compared with HSCs-T6 cells treated with different concentrations of blueberry anthocyanin for 36 hours, the proliferation of HSCs-T6 cells was significantly inhibited in a dose-dependent manner; the survival rate of HSCs-T6 cells was less than 50% (P 0.05) when the concentration of blueberry anthocyanin was 50 micromol/L; immunofluorescence assay showed that with the concentration of blueberry anthocyanin increased and treated with HSCs-T6 for 36 hours, the number of apoptosis of HSCs-T6 cells increased gradually, and more. The nuclei of HSCs-T6 cells were stained red by PI in a concentration-dependent manner. In the apoptosis test of flow cytometry, compared with the control group, the apoptosis rate of HSCs-T6 cells treated with blueberry anthocyanin at different concentrations for 36 hours increased with the increase of blueberry anthocyanin concentration, and the difference was statistically significant (P 0.01); compared with the control group, the 50 ug/ml concentration of blueberry anthocyanin increased the apoptosis rate (P 0.01). The expression of alpha-SMA, collagen type I and TIMP-1 in HSCs-T6 cells treated with blueberry anthocyanin decreased significantly after 36 hours (P 0.05); compared with the control group, the expression of ACH3K9, ACH3K14 and ACH3K18 in HSCs-T6 cells treated with blueberry anthocyanin at 50 ug/ml for 36 hours increased (P 0.01); Conclusion: blueberry anthocyanin can inhibit the increase of HSCs-T6 in rat activated HSC cells. Blueberry anthocyanin is one of the important components of blueberry to improve liver fibrosis; blueberry anthocyanin can up-regulate the acetylation level of AC H3K9, AC H3K14 and AC H3K18 histones in HSCs-T6 cells, and can be achieved by altering the acetylation of histone to the expression of ECM metabolism-related proteins. Reducing ECM deposition is one of the mechanisms of blueberry anthocyanin improving liver fibrosis.
【学位授予单位】：贵州医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.2
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