本文關(guān)鍵詞：大蒜油對正己烷代謝的影響及機制　出處：《山東大學》2012年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 正己烷 2 5-已二酮 大蒜油 CYP2E1 ADH

【摘要】：近年來,在我國有機溶劑誘發(fā)的職業(yè)中毒事件時有發(fā)生。工業(yè)生產(chǎn)過程中,正已烷(n-hexane)常被用做有機溶劑,雖屬無毒類,但由于其高揮發(fā)性、高脂溶性,且有蓄積作用和神經(jīng)系統(tǒng)的毒性作用等特點,被認為是高危害性毒物。在工業(yè)生產(chǎn)中,正已烷主要經(jīng)呼吸道、消化道、皮膚等途徑進入人體,進入人體的正已烷主要分布在腦、腎、肝、脾、睪丸等脂肪含量相對較高的組織。正已烷的急、慢性中毒具表現(xiàn)為不同的特征：急性正已烷中毒主要表現(xiàn)為急性腦病、皮膚及粘膜刺激癥；而慢性正已烷中毒則以神經(jīng)毒性為主,主要表現(xiàn)為遠端粗大神經(jīng)纖維軸突退變導(dǎo)致的感覺-運動型周圍神經(jīng)病。值得注意的是,目前對正已烷引起的神經(jīng)毒作用尚無特效的治療方法。 大蒜油(garlic oil, GO)作為一種植物提取物是從大蒜中提取的一類與水不相混溶的油狀成分,含有多種含硫化合物,主要包括二烯丙基硫醚、二烯丙基二硫、二烯丙基三硫、烯丙基甲基三硫、烯丙基甲基二硫醚和烯丙基甲基硫醚等。研究證實,大蒜油具有抗菌、抗腫瘤、解毒、增強機體免疫能力、降低血脂等功能,同時有研究顯示大蒜油能夠抑制肝臟CYP2E1表達,降低CYP2E1活性。文獻報道顯示正已烷是通過代謝形成2,5-已二酮(2,5-hexanedione,2,5-HD)引起周圍神經(jīng)損傷的,而這一活化代謝過程主要是由肝臟中的CYP2E1、CYP2B1以及ADH等酶完成。2,5-HD作為正已烷的活性代謝產(chǎn)物,被認為是正已烷引起神經(jīng)毒性的終毒物。本實驗室已經(jīng)證實,在正已烷一次給藥過程中,大蒜油能夠顯著抑制正已烷代謝活化為2,5-HD。但是大蒜油對正已烷慢性中毒的拮抗效果和保護的確切機制尚不清楚,并且國內(nèi)外尚未見報道。本實驗欲通過建立正已烷慢性中毒模型并同時給予大蒜油,觀察大蒜油對正已烷染毒大鼠肝臟中CYP2E1、CYP2B1及ADH等正已烷代謝酶的影響,并且同時監(jiān)測大鼠血清中2,5-HD含量的變化,從而探討大蒜油拮抗正已烷中毒的機制,為正已烷中毒的防治提供依據(jù)。本次實驗中,給予Wistar雄性大鼠連續(xù)10周經(jīng)口灌胃正已烷2000mg/kg. bw染毒,復(fù)制大鼠慢性正已烷中毒致神經(jīng)毒性病變模型,在正已烷灌胃前1小時對大鼠分別灌胃給予40mg/kg. bw、80mg/kg. bw的大蒜油進行提前性干預(yù)。灌胃期間沒兩周測定一次各組大鼠神經(jīng)行為學改變包括平衡指數(shù)和步態(tài)評分。在實驗結(jié)束時測定大鼠肝臟中CYP2E1、CYP2B1及ADH的含量及活性變化；測定大鼠血清中2,5-HD的含量,以觀察正已烷在大鼠體內(nèi)的代謝活化情況。 結(jié)果 1.體重變化 正常對照組與大蒜油對照組大鼠體重穩(wěn)定增長,兩組間無統(tǒng)計學差異；正已烷模型組體重增長緩慢,甚至出現(xiàn)負增長,模型組大鼠1周后體重顯著低于正常對照大鼠(P0.05)；而大蒜油低劑量組和大蒜油高劑量組大鼠體重分別在9、5周后顯著高于模型大鼠(P0.05或P0.01)。 2.大鼠神經(jīng)行為學改變 (1)步態(tài)及評分：10周試驗結(jié)束時,模型組大鼠70%完全癱瘓(4分),出現(xiàn)足后翻現(xiàn)象,證明模型建立成功。大蒜油低、高劑量組到實驗結(jié)束時,出現(xiàn)不同程度步態(tài)異常,后肢間距增寬,但均無嚴重癱瘓癥狀出現(xiàn),其中步態(tài)3分大鼠分別占30%和10%。大蒜油組步態(tài)評分優(yōu)于模型組(P0.05)。 (2)大鼠轉(zhuǎn)棒試驗：除正常對照組和大蒜油對照組外,其余各組停留時間均有明顯降低趨勢。與對照組相比,模型組停留時間從第四周開始明顯下降(P0.01)；與模型組相比,大蒜油低高劑量組大鼠停留時間明顯延長(P0.05或P0.01)。 3.肝臟中氧化抗氧化指標改變 與正常對照組相比,模型組大鼠肝臟GSH-PX, T-AOC和抑制羥基自由基能力水平下降,MDA含量上升,并且具有統(tǒng)計學意義(P0.05或P0.01),GSH含量的差異無統(tǒng)計學意義。與模型組相比,大蒜油低、高劑量組大鼠肝臟GSH-PX, GSH, T-AOC及抑制羥基自由基能力水平明顯上升,MDA含量顯著下降,差異有統(tǒng)計學意義(P0.05或P0.01)。 4.大鼠肝臟中CYP2B1、CYP2E1、ADH蛋白水平的變化 (1)肝臟中CYP2B1蛋白水平的變化：與正常組相比,大蒜油對照組肝臟中CYP2B1相對含量升高了96.0%,差異具有統(tǒng)計學意義(P0.01)；與正常組相比,模型組肝臟中CYP2B1相對含量升高了318.3%,差異有統(tǒng)計學意義(P0.01)；與模型組相比,大蒜油低、高劑量組肝臟中CYP2B1相對含量分別升高了3.3%和57.2%,低劑量組差異不具有統(tǒng)計學意義(P0.05),但高劑量組差異具有統(tǒng)計學意義(P0.01)。(2)肝臟中CYP2E1蛋白水平的變化：與正常組相比,大蒜油對照組肝臟中CYP2E1相對含量下降了83.1%,差異具有統(tǒng)計學意義(P0.01)；與正常組相比,模型組肝臟中CYP2E1相對含量上升了122.5%,差異有統(tǒng)計學意義(P0.01)；與模型組相比,大蒜油低、高劑量組肝臟中CYP2E1相對含量分別下降了32.9%和39.1%,且差異均具有統(tǒng)計學意義(P0.01)。(3)肝臟中ADH蛋白水平的變化：與正常組相比,其余各組ADH蛋白含量變化無顯著差異,無統(tǒng)計學意義(P0.05)。 5.大鼠肝臟中CYP2B1、CYP2E1、ADH活性水平的變化 (1)肝臟中CYP2B1活性水平的變化：與正常組相比,大蒜油組對照組肝臟中CYP2B1活性升高了126.3%,差異具有統(tǒng)計學差異(P0.05)；與正常組相比,模型組肝臟中CYP2B1活性升高了293.1%,差異具有統(tǒng)計學差異(P0.01)；與模型組相比,大蒜油低、高劑量組肝臟中CYP2B1活性分別升高了13.1%和52.6%,低劑量組差異不具有統(tǒng)計學意義(P0.05),但高劑量組差異具有統(tǒng)計學意義(P0.01)。(2)肝臟中CYP2E1活性水平的變化：與正常組相比,大蒜油組對照組肝臟中CYP2E1活性下降了48.3%,差異具有統(tǒng)計學差異(P0.01)；與正常組相比,模型組肝臟中CYP2E1活性升高了72.2%,差異具有統(tǒng)計學差異(P0.01)；與模型組相比,大蒜油低、高劑量組肝臟中CYP2E1活性分別降低了27.4%和44.5%,且差異均具有統(tǒng)計學意義(P0.01)。(3)肝臟中ADH活性水平的變化：與正常對照組相比,大蒜油對照組ADH活性升高了9.3%,差異無統(tǒng)計學意義(P0.05)；與正常對照組相比,模型組ADH活性升高了49.8%,差異具有統(tǒng)計學意義(P0.01)；與模型組相比,大蒜油低、高劑量組ADH活性分別降低了25.3%和86.0%；差異均具有統(tǒng)計學意義(P0.01)。 6.大鼠血清中2,5-已二酮含量的變化 正常組與大蒜油對照組大鼠血清中未檢測出2,5-HD。與模型組相比,大蒜油低、高劑量組血清中2,5-HD含量分別降低了47.7%和78.7%,且均差異具有統(tǒng)計學意義(P0.01)。 結(jié)論 (1)大蒜油可有效拮抗正已烷所致的大鼠外周神經(jīng)組織損傷,改善大鼠神經(jīng)行為功能。 (2)在慢性正已烷染毒大鼠體內(nèi),大蒜油能夠有效抑制正已烷代謝活化為終毒物2,5-HD。 (3)大蒜油能夠顯著抑制正已烷染毒大鼠肝臟CYP2E1的表達及活性,同時抑制ADH的活性,這可能是大蒜油拮抗正已烷毒性的重要機制之一。
[Abstract]:In recent years, in our country occupation poisoning induced by organic solvents have occurred. In the process of industrial production, hexane (n-hexane) is often used as an organic solvent, is non-toxic, but because of its high volatile, high fat soluble, and has the characteristics of accumulation and nervous system toxicity, are considered the high risk of the poison. In industrial production, n-hexane mainly through respiratory tract, gastrointestinal tract, skin and other ways to enter the body, into the body of n-hexane is mainly distributed in the brain, kidney, liver, spleen, testis, fat content is relatively high. The organization of n-hexane acute and chronic poisoning with different characteristics: acute n-hexane poisoning mainly for acute encephalopathy, skin and mucous membrane irritation; chronic n-hexane poisoning is mainly mainly peripheral neurotoxicity, distal axonal degeneration leads to thick nerve fibers in the sensory motor neuropathy. Worthy of note It is the current treatment methods of n-hexane induced neurotoxicity is not specific.
Garlic oil (garlic oil GO) is a kind of plant extracts extracted from garlic is a kind of mixture compositions, containing a variety of sulfur compounds, including diallyl sulfide, diallyl diallyl sulfide sulfur two, three, three allyl methyl sulfide, allyl methyl sulfide two ether and allyl methyl sulfide. The study confirmed that garlic oil has antibacterial, antitumor, detoxification, enhancing immunity, reducing blood lipid and other functions, at the same time, studies have shown that garlic oil can inhibit the expression of CYP2E1 in liver tissue, reduce the activity of CYP2E1. The report shows n-hexane is the formation of 2,5- has two ketone through metabolism (2,5-hexanedione 2,5-HD), caused by peripheral nerve injury, and the activation of metabolic processes in the liver is mainly composed of CYP2E1, CYP2B1 and ADH.2,5-HD as enzyme active metabolite of n-hexane, is believed to be caused by n-hexane neurotoxicity The end of poison. The laboratory has confirmed that in hexane administered once in the process of garlic oil can inhibit the metabolic activation of n-hexane was 2,5-HD. but the exact mechanism of antagonistic effect of garlic oil on chronic n-hexane poisoning and the protection is not clear, and there is no report. This experiment is to establish experiment model of chronic poisoning and also given garlic oil, garlic oil on liver CYP2E1 were observed in rats exposed to n-hexane, effects of n-hexane metabolic enzyme CYP2B1 and ADH, and while monitoring the changes of 2,5-HD content in serum of rats, and to explore the mechanism of garlic oil against n-hexane poisoning, and provide evidence for the prevention of n-hexane poisoning. In this experiment, Wistar male rats for 10 weeks by gavage of n-hexane 2000mg/kg. BW exposure, the rats of chronic n-hexane poisoning caused by neurotoxic lesions in n-hexane model before gavage 1 Hours of rats were orally given 40mg/kg. BW, 80mg/kg. BW of garlic oil for early intervention. During the two weeks by gavage once the rats neurobehavioral changes including balance index and gait score. The determination of CYP2E1 in rat liver at the end of the experiment, change the content and activity of CYP2B1 and ADH; Determination of the content of 2,5-HD in serum of rats, to observe the activation of n-hexane metabolism in rats.
Result
1. body weight change
The normal control group and the control group of garlic oil and stable growth of the body weight of rats, no significant difference between the two groups; n-hexane model group weight slow growth or even negative growth after 1 weeks, body weight of rats in model group were significantly lower than those in normal control rats (P0.05); and the weight of garlic oil in low dose group and high dose of garlic oil groups of rats were significantly higher than in 9,5 weeks after rats (P0.05 or P0.01).
Neurobehavioral changes in 2. rats
(1) gait and score: the end of the 10 week trial, completely paralyzed rats in the model group 70% (4 points), with a phenomenon that model was established successfully. The garlic oil low, high dose group to the end of the experiment, different degrees of abnormal gait, limb spacing widened, but there were no serious paralysis the symptoms, gait 3 rats respectively accounted for 30% and 10%. garlic oil group gait score better than the model group (P0.05).
(2) rat rotarod test: in addition to the normal control group and garlic oil group, the other groups were significantly decreased. The residence time compared with the control group, model group, residence time decreased significantly from week 4 (P0.01); compared with the model group, garlic oil and low residence time of rats in high dose group significantly extended (P0.05 or P0.01).
Changes of oxidation and antioxidant index in 3. liver
Compared with the normal control group, model group rat liver GSH-PX, T-AOC and inhibition of hydroxyl radical ability decreased, MDA content increased, and was statistically significant (P0.05 or P0.01), there were no significant differences in GSH content. Compared with the model group, garlic oil low, high dose group rat liver GSH-PX, GSH T-AOC, and inhibition of hydroxyl radical ability levels were significantly increased, MDA content decreased significantly, the difference was statistically significant (P0.05 or P0.01).
Changes of CYP2B1, CYP2E1 and ADH protein levels in the liver of 4. rats
(1) changes of CYP2B1 protein level in the liver: compared with normal group, control group CYP2B1 relative content of garlic oil in the liver increased 96%, the difference was statistically significant (P0.01); compared with the normal group, the relative content of CYP2B1 of liver in the model group increased 318.3%, the difference was statistically significant (P0.01); compared with the model group, garlic oil low, the relative content of CYP2B1 in liver of high dose group were increased by 3.3% and 57.2%, difference between the low dose group was not statistically significant (P0.05), but the difference between the high dose group was statistically significant (P0.01). (2) the change of CYP2E1 protein level in the liver: compared with normal group, garlic oil the relative content of CYP2E1 in liver of control group decreased by 83.1%, the difference was statistically significant (P0.01); compared with the normal group, the relative content of CYP2E1 of liver in the model group increased by 122.5%, the difference was statistically significant (P0.01); compared with the model group, large Garlic oil is low, the relative content of CYP2E1 in liver of high dose group decreased by 32.9% and 39.1%, and the differences were statistically significant (P0.01). (3) the changes of ADH protein in the liver: compared with normal group, no significant difference in other groups ADH protein content was not statistically significant (P0.05).
Changes in the activity level of CYP2B1, CYP2E1 and ADH in the liver of 5. rats
(1) the changes of CYP2B1 activity level in the liver: compared with normal group, the control group of garlic oil group in the liver CYP2B1 activity increased by 126.3%, a statistically significant difference (P0.05); compared with the normal group, the activity of CYP2B1 in liver in the model group increased 293.1%, the difference has statistical difference (P0.01); compared with the model garlic oil group, low and high dose group liver CYP2B1 activity were increased by 13.1% and 52.6%, difference between the low dose group was not statistically significant (P0.05), but the difference between the high dose group was statistically significant (P0.01). (2) the changes of CYP2E1 activity level in the liver: compared with normal group, garlic oil group CYP2E1 the activity of liver in group decreased 48.3% in control, the difference has statistics difference (P0.01); compared with the normal group, the activity of CYP2E1 in liver in the model group increased by 72.2%, a statistically significant difference (P0.01); compared with the model group, high dose of garlic oil is low. Group of liver CYP2E1 activity were decreased by 27.4% and 44.5%, and the differences were statistically significant (P0.01). (3) the changes of ADH activity level in the liver: compared with normal control group, garlic oil control group increased the activity of ADH was 9.3%, the difference was not statistically significant (P0.05); compared with the normal control group, model the activity of ADH increased by 49.8%, the difference was statistically significant (P0.01); compared with the model group, garlic oil low, high dose group ADH activity were decreased by 25.3% and 86%; the differences were statistically significant (P0.01).
Changes of the content of 2,5- two ketone in the serum of 6. rats
In normal group and garlic oil control group, serum 2,5-HD. was not detected in the rats. Compared with the model group, the 2,5-HD content in the serum of garlic oil low and high dose group decreased by 47.7% and 78.7%, respectively, and the difference was statistically significant (P0.01).
conclusion
(1) the garlic oil can effectively antagonize the n-hexane induced rat peripheral nerve tissue injury, improve the neurological behavior in rats.
(2) in vivo in rats with chronic n-hexane poisoning, garlic oil can effectively inhibit the metabolic activation of n-hexane as the final 2,5-HD. poison
(3) garlic oil can significantly inhibit the expression and activity of CYP2E1 in liver of n-hexane exposed rats, and inhibit the activity of ADH, which may be an important mechanism of garlic oil against n-hexane toxicity.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R135

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