槲皮素调节同型半胱氨酸代谢的作用及机制分析

发布时间：2018-06-09 06:13

本文选题：槲皮素 + 同型半胱氨酸　；参考：《中国人民解放军军事医学科学院》2013年博士论文

【摘要】：目的建立高同型半胱氨酸(Hcy)血症实验动物模型,在此基础上,从代谢和基因表达等途径的角度探讨槲皮素对Hcy合成和分解的影响,尤其是槲皮素对Hcy代谢过程中重要代谢产物和关键酶活性及其基因表达的影响,最终揭示槲皮素调节Hcy代谢的分子机制,为植物化学物应用于心血管疾病的防治提供理论依据。方法 1.以AIN-93饲料,在此基础上分别添加1%、2%和3%蛋氨酸(methionine, Met)喂养大鼠21天,采用高效液相色谱(HPLC)法测定血清中Hcy及其相关代谢产物水平,氨基酸自动分析仪检测血清中氨基酸含量,确定诱导高Hcy血症的Met适宜剂量。 2.大鼠分别喂以AIN-93合成饲料或在此基础上添加1%的Met以及对大鼠进行束缚应激,配对喂养6周。采用HPLC法测定血清中Hcy及半胱氨酸(Cys)、谷胱甘肽(GSH)水平,氨基酸自动分析仪检测血清中其它氨基酸含量,确定束缚应激对Met诱导高Hcy血症模型的影响。 3.大鼠分别给予AIN-93合成饲料或在此饲料中添加1%的Met和0.1、0.5、2.5%的槲皮素(Q)喂养6周。采用HPLC法测定血清中Hcy、Cys和GSH水平,以及血清中槲皮素代谢产物浓度。氨基酸自动分析仪检测血清中丝氨酸(Ser)、牛磺酸(Tau)、甘氨酸(Gly)、胱氨酸含量,同时检测肝组织和血清中抗氧化指标。最终确定槲皮素对Hcy代谢的调节作用及适宜剂量。 4.大鼠分别给予AIN-93合成饲料或在此饲料中添加1%的Met和0.5%的槲皮素喂养6周。采用HPLC法测定血清中Hcy及Cys、GSH水平,氨基酸自动分析仪检测血清中氨基酸含量的变化情况,采用HPLC法测定肝组织中S-腺苷蛋氨酸(SAM)、S-腺苷同型半胱氨酸(SAH)含量以及S-腺苷同型半胱氨酸水解酶(SAHH)水解活性和合成活性,采用化学方法测定胱硫醚-β-合成酶(CBS)、甜菜碱同型半胱氨酸甲基转移酶(BHMT)、蛋氨酸合成酶(MS)和胱硫醚-γ-裂解酶(CSE)活性。采用Real-time PCR法测定SAHH、 CBS、BHMT、MS和CSE mRNA表达水平,最终揭示槲皮素调节Hcy代谢的作用靶点。结果 1.建立了大鼠高Hcy血症模型。 2.选择含1%、2%和3%Met饲料喂饲大鼠3周,结果饲料中含1%Met可以使血清Hey显著水平升高(P0.05)目.无生长抑制等毒副作用,含2%和3%Met饲料喂养大鼠后Hcy显著水平亦升高(P0.05),同时大鼠出现摄食量减少和生长抑制等。各高Met饲料组大鼠血清Cys没有明显差异,血清GSH含量虽然高于对照组,但也没有显著差异。 3.1%Met组大鼠血清Hcy水平随着大鼠束缚时间延长不断增高,在第六周时明显高于其它各组(P0.05)。而束缚组和束缚+1%Met组随着时间延长而Hcy水平降低,在第四周和第六周时显著低于对照组和1%Met组(P0.05)。大鼠血清Cys水平束缚组和束缚+1%Met组显著低于对照组和1%Met组(P0.05)。血清GSH含量在第六周时束缚组显著低于其它各组(P0.05)。 4.与正常对照比较,束缚组苏氨酸(Thr)、Ser、Gly、谷氨酸(Glu)、胱氨酸、酪氨酸(Tyr)、苯丙氨酸(Phe)、赖氨酸(Lys)、精氨酸(Arg)含量显著降低(P0.05),而Met和Tau则显著升高(P0.05)。 5.血清中槲皮素和异鼠李素含量随饲料中槲皮素含量的增加而显著增加(P0.05)。血清Met和Hcy随着槲皮素含量增加降低,1%Met+0.5%Q组Hcy含量显著低于1%Met组(P0.05),对照组血清胱氨酸和Tau含量最低,且显著低于1%Met组(P0.05)。槲皮素干预组血清GSH含量随着槲皮素含量的增加下降,且1%Met+2.5%Q组与1%Met组有显著差异(P0.05)。Met与槲皮素干预组血清Ser含量低于对照组,且1%Met+2.5%Q组与对照组有显著差异(P0.05)。 6.1%Met+0.5%Q组丙二醛(MDA)含量明显低于对照组、1%Met和1%Met+2.5%Q组(P0.05),而对照组和1%Met组GSH含量明显高于1%Met+0.1%Q、1%Met+0.5%Q和1%Met+2.5%Q组(P0.05),1%Met+2.5%Q组超氧化物歧化酶(SOD)活性显著低于正常对照组和1%Met组(P0.05)。FRAP和蛋白质羰基化物各组间没有明显差异。 7.1%Met+2.5%Q组丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)活性明显高于其它各组(P0.05),提示2.5%槲皮素可能对肝脏产生一定毒性。 8.与对照组和1%Met比较,1%Met+0.5%Q组SAM显著升高(P0.05),0.5%Q组SAM含量亦高于对照组,但无统计学差异。1%Met+0.5%Q组SAH较对照组和槲皮素单独干预组显著升高(P0.05),1%Met组SAM/SAH显著降低(P0.05)。而0.5%Q组和Met+0.5%Q组SAM/SAH升高。1%Met.0.5%Q和1%Met+0.5%Q组SAHH合成酶活性较对照组显著升高(P0.05),0.5%Q和1%Met+0.5%Q组SAHH水解酶活性较对照组显著升高(P0.05)。1%Met及1%Met+0.5%Q组与对照组和0.5%Q组比较,SAHH mRNA基因表达明显上调(P0.05),0.5%Q组SAHH mRNA基因表达亦上调较对照组,但并无统计学差异。 9.1%Met+0.5%Q组BHMT活性较对照组显著升高(P0.05),且MS活性显著高于其它各组(P0.05)。BHMT mRNA表达各组间无显著差异,与对照组和0.5%Q组比较,1%Met及1%Met+0.5%Q组MS mRNA表达显著上调(P0.05),各干预组CBS酶活性较对照组显著升高(P0.05),且1%Met+0.5%Q组CBS活性显著高于0.5%Q组(P0.05),与对照组比较,各干预组CBS mRNA表达上调(P0.05),1%Met+0.5%Q组CBS酶mRNA表达较其它各组亦显著上调(P0.05)。与对照组比较,各干预组CSE活性均显著增加(P0.05),且各干预组CSE mRNA表达较对照组亦显著上调(P0.05)。结论 1.1%Met可以成功诱导大鼠高Hcy血症模型。 2.长时间束缚应激可降低血清中Hcy水平,且对氨基酸代谢产生影响。 3.高剂量槲皮素对肝组织不但不具有抗氧化作用,还可能促氧化,对肝组织产生一定毒性。适宜剂量的槲皮素不仅具有抗氧化作用,而且可以明显降低血清中Hcy水平。 4.槲皮素主要影响Hcy代谢过程中的转硫化途径,通过提高CBS、CSE活性和mRNA表达从而是降低血清中Hcy含量。
[Abstract]:objective
The experimental animal model of Hyperhomocysteine (Hcy) was established. On this basis, the effects of quercetin on the synthesis and decomposition of Hcy were discussed from the aspects of metabolism and gene expression, especially the effects of quercetin on the important metabolites and key enzyme activities and the expression of key enzymes in the metabolic process of Hcy. Finally, quercetin was revealed to regulate the Hcy generation. The molecular mechanism of Xie provides a theoretical basis for the application of phytochemicals in the prevention and treatment of cardiovascular diseases.
Method
1. on the basis of AIN-93 feed, 1%, 2% and 3% methionine (methionine, Met) were fed to rats for 21 days. The level of Hcy and its related metabolites in serum was measured by high performance liquid chromatography (HPLC). Amino acid automatic analyzer was used to detect the content of amino acid in serum, and the appropriate dosage of Met to induce hypercholesterolemia was determined.
The 2. rats were fed with AIN-93 synthetic feed or on this basis, adding 1% Met and binding stress to rats, paired feeding for 6 weeks. The serum levels of Hcy and cysteine (Cys), glutathione (GSH) were measured by HPLC, and the amino acid content of the serum was detected by amino acid automatic analyzer, and the binding stress was determined to induce high Hcy blood in Met. The impact of the disease model.
3. rats were given AIN-93 synthetic feed or 1% Met and 0.1,0.5,2.5% of quercetin (Q) fed in this feed for 6 weeks. The serum level of Hcy, Cys and GSH, and the concentration of quercetin metabolites in serum were measured by HPLC. The amino acid automatic analyzer was used to detect sera (Ser), taurine (Tau), glycine (Gly), cystine content. At the same time, the antioxidant indexes in liver tissue and serum were detected. The regulation of quercetin on Hcy metabolism and appropriate dose were determined.
The 4. rats were given AIN-93 synthetic feed or 1% Met and 0.5% quercetin for 6 weeks. The changes of serum Hcy, Cys, GSH level, amino acid automatic analyzer in serum were measured by HPLC method, and HPLC method was used to determine S- adenosylmethionine (SAM) and S- adenosine homocysteine (adenosine homocysteine). SAH) content and the hydrolytic activity and synthetic activity of S- adenosine homocysteine hydrolase (SAHH), the activity of cystionine - beta synthase (CBS), betaine homocysteine methyltransferase (BHMT), methionine synthetase (MS) and cystthioether - lyase (CSE) activity by chemical method. SAHH, CBS, BHMT, BHMT, etc. were determined by Real-time The level of NA expression reveals the target of quercetin regulating Hcy metabolism.
Result
1. the rat model of hyperHcy was established.
2. the rats were fed with 1%, 2% and 3%Met feed for 3 weeks. The results showed that the diet containing 1%Met could increase the level of serum Hey significantly (P0.05). There was no growth inhibition and other toxic side effects. The significant level of Hcy in rats fed with 2% and 3%Met feed was also increased (P0.05). At the same time, the decrease of food intake and growth inhibition in rats. The serum Cys in the high Met feed rats There was no significant difference, although serum GSH level was higher than that of the control group, but there was no significant difference.
The level of serum Hcy in the group 3.1%Met rats increased with the prolonged binding time, which was significantly higher than the other groups at sixth weeks (P0.05). The binding group and the bound +1%Met group decreased with the time prolonged and the Hcy level was significantly lower in the fourth week and the sixth week (P0.05). The serum Cys level binding group and the binding +1%Me in the rat serum were significantly lower in the fourth and sixth weeks. Group t was significantly lower than that of control group and group 1%Met (P0.05). Serum GSH content was significantly lower than that of other groups (GSH) at sixth weeks.
4. compared with normal control, the binding group of threonine (Thr), Ser, Gly, glutamic acid (Glu), cystine, tyrosine (Tyr), phenylalanine (Phe), lysine (Lys), and arginine (Arg) significantly decreased (P0.05), while Met and Tau increased significantly (P0.05).
5. the content of quercetin and isomarin in serum increased significantly with the increase of quercetin content in the diet (P0.05). Serum Met and Hcy decreased with the increase of quercetin content, Hcy content in group 1%Met+0.5%Q was significantly lower than that in 1%Met group (P0.05), and the serum cystine and Tau content was lowest in the control group, and significantly lower than that in the 1%Met group (P0.05). The content of SH decreased with the increase of quercetin content, and there was significant difference between the 1%Met+2.5%Q group and the 1%Met group (P0.05) and the serum Ser content in the.Met and quercetin intervention group was lower than that of the control group, and there was a significant difference between the 1%Met+2.5%Q group and the control group (P0.05).
The content of malondialdehyde (MDA) in group 6.1%Met+0.5%Q was significantly lower than that of control group, 1%Met and 1%Met+2.5%Q group (P0.05), while the content of GSH in the control group and 1%Met group was significantly higher than that of 1%Met+0.1%Q, 1%Met+0.5%Q and 1%Met+2.5%Q groups (P0.05). There was no significant difference between the two groups.
7.1%Met+2.5%Q group alanine aminotransferase (ALT), aspartate aminotransferase (AST) activity was significantly higher than other groups (P0.05), suggesting that 2.5% quercetin may have a certain toxicity to the liver.
8. compared with the control group and 1%Met, the SAM in the 1%Met+0.5%Q group was significantly higher (P0.05), and the SAM content in the 0.5%Q group was also higher than that in the control group, but there was no statistical difference between the.1%Met+0.5%Q group and the control group and the quercetin alone group (P0.05), and the SAM/SAH significantly decreased in the 1%Met group (P0.05). The activity of SAHH synthase in the group Q was significantly higher than that in the control group (P0.05). The activity of SAHH hydrolase in 0.5%Q and 1%Met+0.5%Q groups was significantly higher than that in the control group (P0.05), and the SAHH mRNA gene expression was up to up significantly compared with the control group and the 0.5%Q group, and the expression of the SAHH mRNA gene was up to be up to the control group, but there was no statistical difference.
The activity of BHMT in group 9.1%Met+0.5%Q was significantly higher than that in the control group (P0.05), and the activity of MS was significantly higher than that of the other groups (P0.05), and there was no significant difference in the expression of.BHMT mRNA. Compared with the control group and 0.5%Q group, the expression of MS mRNA in 1%Met and 1%Met+0.5%Q groups was significantly up, and the activity of the intervention group was significantly higher than that of the control group. The activity of CBS was significantly higher than that of the 0.5%Q group (P0.05). Compared with the control group, the expression of CBS mRNA in the intervention group was up (P0.05). The mRNA expression of CBS enzyme in the 1%Met+0.5%Q group was also significantly up (P0.05). The CSE activity in the intervention groups was significantly increased (P0.05), and the expression of each intervention group was also significantly up.
conclusion
1.1%Met can successfully induce high Hcy model in rats.
2. long term restraint stress can reduce serum Hcy level and affect amino acid metabolism.
3. high dose quercetin not only does not have antioxidant effect on liver tissue, but also may promote oxidation and produce certain toxicity to liver tissue. The suitable dose of quercetin not only has antioxidant effect, but also can obviously reduce the level of Hcy in serum.
4. quercetin mainly affects the sulfidation pathway in Hcy metabolism process. By increasing CBS, CSE activity and mRNA expression, it decreases serum Hcy level.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R151.2

【参考文献】