本文選題：槲皮素 + 同型半胱氨酸��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2013年博士論文

【摘要】：目的 建立高同型半胱氨酸(Hcy)血癥實驗動物模型,在此基礎(chǔ)上,從代謝和基因表達等途徑的角度探討槲皮素對Hcy合成和分解的影響,尤其是槲皮素對Hcy代謝過程中重要代謝產(chǎn)物和關(guān)鍵酶活性及其基因表達的影響,最終揭示槲皮素調(diào)節(jié)Hcy代謝的分子機制,為植物化學(xué)物應(yīng)用于心血管疾病的防治提供理論依據(jù)。 方法 1.以AIN-93飼料,在此基礎(chǔ)上分別添加1%、2%和3%蛋氨酸(methionine, Met)喂養(yǎng)大鼠21天,采用高效液相色譜(HPLC)法測定血清中Hcy及其相關(guān)代謝產(chǎn)物水平,氨基酸自動分析儀檢測血清中氨基酸含量,確定誘導(dǎo)高Hcy血癥的Met適宜劑量。 2.大鼠分別喂以AIN-93合成飼料或在此基礎(chǔ)上添加1%的Met以及對大鼠進行束縛應(yīng)激,配對喂養(yǎng)6周。采用HPLC法測定血清中Hcy及半胱氨酸(Cys)、谷胱甘肽(GSH)水平,氨基酸自動分析儀檢測血清中其它氨基酸含量,確定束縛應(yīng)激對Met誘導(dǎo)高Hcy血癥模型的影響。 3.大鼠分別給予AIN-93合成飼料或在此飼料中添加1%的Met和0.1、0.5、2.5%的槲皮素(Q)喂養(yǎng)6周。采用HPLC法測定血清中Hcy、Cys和GSH水平,以及血清中槲皮素代謝產(chǎn)物濃度。氨基酸自動分析儀檢測血清中絲氨酸(Ser)、�；撬�(Tau)、甘氨酸(Gly)、胱氨酸含量,同時檢測肝組織和血清中抗氧化指標(biāo)。最終確定槲皮素對Hcy代謝的調(diào)節(jié)作用及適宜劑量。 4.大鼠分別給予AIN-93合成飼料或在此飼料中添加1%的Met和0.5%的槲皮素喂養(yǎng)6周。采用HPLC法測定血清中Hcy及Cys、GSH水平,氨基酸自動分析儀檢測血清中氨基酸含量的變化情況,采用HPLC法測定肝組織中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,采用化學(xué)方法測定胱硫醚-β-合成酶(CBS)、甜菜堿同型半胱氨酸甲基轉(zhuǎn)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法測定SAHH、 CBS、BHMT、MS和CSE mRNA表達水平,最終揭示槲皮素調(diào)節(jié)Hcy代謝的作用靶點。 結(jié)果 1.建立了大鼠高Hcy血癥模型。 2.選擇含1%、2%和3%Met飼料喂飼大鼠3周,結(jié)果飼料中含1%Met可以使血清Hey顯著水平升高(P0.05)目.無生長抑制等毒副作用,含2%和3%Met飼料喂養(yǎng)大鼠后Hcy顯著水平亦升高(P0.05),同時大鼠出現(xiàn)攝食量減少和生長抑制等。各高Met飼料組大鼠血清Cys沒有明顯差異,血清GSH含量雖然高于對照組,但也沒有顯著差異。 3.1%Met組大鼠血清Hcy水平隨著大鼠束縛時間延長不斷增高,在第六周時明顯高于其它各組(P0.05)。而束縛組和束縛+1%Met組隨著時間延長而Hcy水平降低,在第四周和第六周時顯著低于對照組和1%Met組(P0.05)。大鼠血清Cys水平束縛組和束縛+1%Met組顯著低于對照組和1%Met組(P0.05)。血清GSH含量在第六周時束縛組顯著低于其它各組(P0.05)。 4.與正常對照比較,束縛組蘇氨酸(Thr)、Ser、Gly、谷氨酸(Glu)、胱氨酸、酪氨酸(Tyr)、苯丙氨酸(Phe)、賴氨酸(Lys)、精氨酸(Arg)含量顯著降低(P0.05),而Met和Tau則顯著升高(P0.05)。 5.血清中槲皮素和異鼠李素含量隨飼料中槲皮素含量的增加而顯著增加(P0.05)。血清Met和Hcy隨著槲皮素含量增加降低,1%Met+0.5%Q組Hcy含量顯著低于1%Met組(P0.05),對照組血清胱氨酸和Tau含量最低,且顯著低于1%Met組(P0.05)。槲皮素干預(yù)組血清GSH含量隨著槲皮素含量的增加下降,且1%Met+2.5%Q組與1%Met組有顯著差異(P0.05)。Met與槲皮素干預(yù)組血清Ser含量低于對照組,且1%Met+2.5%Q組與對照組有顯著差異(P0.05)。 6.1%Met+0.5%Q組丙二醛(MDA)含量明顯低于對照組、1%Met和1%Met+2.5%Q組(P0.05),而對照組和1%Met組GSH含量明顯高于1%Met+0.1%Q、1%Met+0.5%Q和1%Met+2.5%Q組(P0.05),1%Met+2.5%Q組超氧化物歧化酶(SOD)活性顯著低于正常對照組和1%Met組(P0.05)。FRAP和蛋白質(zhì)羰基化物各組間沒有明顯差異。 7.1%Met+2.5%Q組丙氨酸轉(zhuǎn)氨酶(ALT)、天冬氨酸轉(zhuǎn)氨酶(AST)活性明顯高于其它各組(P0.05),提示2.5%槲皮素可能對肝臟產(chǎn)生一定毒性。 8.與對照組和1%Met比較,1%Met+0.5%Q組SAM顯著升高(P0.05),0.5%Q組SAM含量亦高于對照組,但無統(tǒng)計學(xué)差異。1%Met+0.5%Q組SAH較對照組和槲皮素單獨干預(yù)組顯著升高(P0.05),1%Met組SAM/SAH顯著降低(P0.05)。而0.5%Q組和Met+0.5%Q組SAM/SAH升高。1%Met.0.5%Q和1%Met+0.5%Q組SAHH合成酶活性較對照組顯著升高(P0.05),0.5%Q和1%Met+0.5%Q組SAHH水解酶活性較對照組顯著升高(P0.05)。1%Met及1%Met+0.5%Q組與對照組和0.5%Q組比較,SAHH mRNA基因表達明顯上調(diào)(P0.05),0.5%Q組SAHH mRNA基因表達亦上調(diào)較對照組,但并無統(tǒng)計學(xué)差異。 9.1%Met+0.5%Q組BHMT活性較對照組顯著升高(P0.05),且MS活性顯著高于其它各組(P0.05)。BHMT mRNA表達各組間無顯著差異,與對照組和0.5%Q組比較,1%Met及1%Met+0.5%Q組MS mRNA表達顯著上調(diào)(P0.05),各干預(yù)組CBS酶活性較對照組顯著升高(P0.05),且1%Met+0.5%Q組CBS活性顯著高于0.5%Q組(P0.05),與對照組比較,各干預(yù)組CBS mRNA表達上調(diào)(P0.05),1%Met+0.5%Q組CBS酶mRNA表達較其它各組亦顯著上調(diào)(P0.05)。與對照組比較,各干預(yù)組CSE活性均顯著增加(P0.05),且各干預(yù)組CSE mRNA表達較對照組亦顯著上調(diào)(P0.05)。 結(jié)論 1.1%Met可以成功誘導(dǎo)大鼠高Hcy血癥模型。 2.長時間束縛應(yīng)激可降低血清中Hcy水平,且對氨基酸代謝產(chǎn)生影響。 3.高劑量槲皮素對肝組織不但不具有抗氧化作用,還可能促氧化,對肝組織產(chǎn)生一定毒性。適宜劑量的槲皮素不僅具有抗氧化作用,而且可以明顯降低血清中Hcy水平。 4.槲皮素主要影響Hcy代謝過程中的轉(zhuǎn)硫化途徑,通過提高CBS、CSE活性和mRNA表達從而是降低血清中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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R151.2

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