大蒜油对正己烷代谢的影响及机制

发布时间：2018-01-15 19:27

本文关键词：大蒜油对正己烷代谢的影响及机制　出处：《山东大学》2012年硕士论文　论文类型：学位论文

更多相关文章： 正己烷 2 5-已二酮 大蒜油 CYP2E1 ADH

【摘要】：近年来,在我国有机溶剂诱发的职业中毒事件时有发生。工业生产过程中,正已烷(n-hexane)常被用做有机溶剂,虽属无毒类,但由于其高挥发性、高脂溶性,且有蓄积作用和神经系统的毒性作用等特点,被认为是高危害性毒物。在工业生产中,正已烷主要经呼吸道、消化道、皮肤等途径进入人体,进入人体的正已烷主要分布在脑、肾、肝、脾、睾丸等脂肪含量相对较高的组织。正已烷的急、慢性中毒具表现为不同的特征：急性正已烷中毒主要表现为急性脑病、皮肤及粘膜刺激症；而慢性正已烷中毒则以神经毒性为主,主要表现为远端粗大神经纤维轴突退变导致的感觉-运动型周围神经病。值得注意的是,目前对正已烷引起的神经毒作用尚无特效的治疗方法。大蒜油(garlic oil, GO)作为一种植物提取物是从大蒜中提取的一类与水不相混溶的油状成分,含有多种含硫化合物,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。研究证实,大蒜油具有抗菌、抗肿瘤、解毒、增强机体免疫能力、降低血脂等功能,同时有研究显示大蒜油能够抑制肝脏CYP2E1表达,降低CYP2E1活性。文献报道显示正已烷是通过代谢形成2,5-已二酮(2,5-hexanedione,2,5-HD)引起周围神经损伤的,而这一活化代谢过程主要是由肝脏中的CYP2E1、CYP2B1以及ADH等酶完成。2,5-HD作为正已烷的活性代谢产物,被认为是正已烷引起神经毒性的终毒物。本实验室已经证实,在正已烷一次给药过程中,大蒜油能够显著抑制正已烷代谢活化为2,5-HD。但是大蒜油对正已烷慢性中毒的拮抗效果和保护的确切机制尚不清楚,并且国内外尚未见报道。本实验欲通过建立正已烷慢性中毒模型并同时给予大蒜油,观察大蒜油对正已烷染毒大鼠肝脏中CYP2E1、CYP2B1及ADH等正已烷代谢酶的影响,并且同时监测大鼠血清中2,5-HD含量的变化,从而探讨大蒜油拮抗正已烷中毒的机制,为正已烷中毒的防治提供依据。本次实验中,给予Wistar雄性大鼠连续10周经口灌胃正已烷2000mg/kg. bw染毒,复制大鼠慢性正已烷中毒致神经毒性病变模型,在正已烷灌胃前1小时对大鼠分别灌胃给予40mg/kg. bw、80mg/kg. bw的大蒜油进行提前性干预。灌胃期间没两周测定一次各组大鼠神经行为学改变包括平衡指数和步态评分。在实验结束时测定大鼠肝脏中CYP2E1、CYP2B1及ADH的含量及活性变化；测定大鼠血清中2,5-HD的含量,以观察正已烷在大鼠体内的代谢活化情况。结果 1.体重变化正常对照组与大蒜油对照组大鼠体重稳定增长,两组间无统计学差异；正已烷模型组体重增长缓慢,甚至出现负增长,模型组大鼠1周后体重显著低于正常对照大鼠(P0.05)；而大蒜油低剂量组和大蒜油高剂量组大鼠体重分别在9、5周后显著高于模型大鼠(P0.05或P0.01)。 2.大鼠神经行为学改变 (1)步态及评分：10周试验结束时,模型组大鼠70%完全瘫痪(4分),出现足后翻现象,证明模型建立成功。大蒜油低、高剂量组到实验结束时,出现不同程度步态异常,后肢间距增宽,但均无严重瘫痪症状出现,其中步态3分大鼠分别占30%和10%。大蒜油组步态评分优于模型组(P0.05)。 (2)大鼠转棒试验：除正常对照组和大蒜油对照组外,其余各组停留时间均有明显降低趋势。与对照组相比,模型组停留时间从第四周开始明显下降(P0.01)；与模型组相比,大蒜油低高剂量组大鼠停留时间明显延长(P0.05或P0.01)。 3.肝脏中氧化抗氧化指标改变与正常对照组相比,模型组大鼠肝脏GSH-PX, T-AOC和抑制羟基自由基能力水平下降,MDA含量上升,并且具有统计学意义(P0.05或P0.01),GSH含量的差异无统计学意义。与模型组相比,大蒜油低、高剂量组大鼠肝脏GSH-PX, GSH, T-AOC及抑制羟基自由基能力水平明显上升,MDA含量显著下降,差异有统计学意义(P0.05或P0.01)。 4.大鼠肝脏中CYP2B1、CYP2E1、ADH蛋白水平的变化 (1)肝脏中CYP2B1蛋白水平的变化：与正常组相比,大蒜油对照组肝脏中CYP2B1相对含量升高了96.0%,差异具有统计学意义(P0.01)；与正常组相比,模型组肝脏中CYP2B1相对含量升高了318.3%,差异有统计学意义(P0.01)；与模型组相比,大蒜油低、高剂量组肝脏中CYP2B1相对含量分别升高了3.3%和57.2%,低剂量组差异不具有统计学意义(P0.05),但高剂量组差异具有统计学意义(P0.01)。(2)肝脏中CYP2E1蛋白水平的变化：与正常组相比,大蒜油对照组肝脏中CYP2E1相对含量下降了83.1%,差异具有统计学意义(P0.01)；与正常组相比,模型组肝脏中CYP2E1相对含量上升了122.5%,差异有统计学意义(P0.01)；与模型组相比,大蒜油低、高剂量组肝脏中CYP2E1相对含量分别下降了32.9%和39.1%,且差异均具有统计学意义(P0.01)。(3)肝脏中ADH蛋白水平的变化：与正常组相比,其余各组ADH蛋白含量变化无显著差异,无统计学意义(P0.05)。 5.大鼠肝脏中CYP2B1、CYP2E1、ADH活性水平的变化 (1)肝脏中CYP2B1活性水平的变化：与正常组相比,大蒜油组对照组肝脏中CYP2B1活性升高了126.3%,差异具有统计学差异(P0.05)；与正常组相比,模型组肝脏中CYP2B1活性升高了293.1%,差异具有统计学差异(P0.01)；与模型组相比,大蒜油低、高剂量组肝脏中CYP2B1活性分别升高了13.1%和52.6%,低剂量组差异不具有统计学意义(P0.05),但高剂量组差异具有统计学意义(P0.01)。(2)肝脏中CYP2E1活性水平的变化：与正常组相比,大蒜油组对照组肝脏中CYP2E1活性下降了48.3%,差异具有统计学差异(P0.01)；与正常组相比,模型组肝脏中CYP2E1活性升高了72.2%,差异具有统计学差异(P0.01)；与模型组相比,大蒜油低、高剂量组肝脏中CYP2E1活性分别降低了27.4%和44.5%,且差异均具有统计学意义(P0.01)。(3)肝脏中ADH活性水平的变化：与正常对照组相比,大蒜油对照组ADH活性升高了9.3%,差异无统计学意义(P0.05)；与正常对照组相比,模型组ADH活性升高了49.8%,差异具有统计学意义(P0.01)；与模型组相比,大蒜油低、高剂量组ADH活性分别降低了25.3%和86.0%；差异均具有统计学意义(P0.01)。 6.大鼠血清中2,5-已二酮含量的变化正常组与大蒜油对照组大鼠血清中未检测出2,5-HD。与模型组相比,大蒜油低、高剂量组血清中2,5-HD含量分别降低了47.7%和78.7%,且均差异具有统计学意义(P0.01)。结论 (1)大蒜油可有效拮抗正已烷所致的大鼠外周神经组织损伤,改善大鼠神经行为功能。 (2)在慢性正已烷染毒大鼠体内,大蒜油能够有效抑制正已烷代谢活化为终毒物2,5-HD。 (3)大蒜油能够显著抑制正已烷染毒大鼠肝脏CYP2E1的表达及活性,同时抑制ADH的活性,这可能是大蒜油拮抗正已烷毒性的重要机制之一。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R135

【参考文献】