大蒜油对利福平及异烟肼所致肝脏毒性保护作用的实验研究
发布时间：2018-09-19 10:56
【摘要】：研究目的 近年来由于结核疫情反弹及耐多药结核菌和多耐药结核菌的出现,导致结核防治情况愈加复杂,结核治疗疗程及用药等增加,结核药物导致的肝脏损伤的发生率也逐年上升,成为药物性肝损伤最常见的原因之一,特别是异烟肼(INH)、利福平(RIF)所致的肝损伤居前两位。虽然关于利福平、异烟肼等抗结核药物诱导产生肝脏毒性的研究很多,但是这些抗结核药物的损伤机制并没有得到完全的阐述,目前较多的损伤机制研究集中在氧化抗氧化机制,研究发现该肝脏毒性的产生可能与体内氧化抗氧化水平失衡有关。 大蒜油具有广泛的药理作用,于数千年前就开始广泛应用于食药领域。比如大蒜油有明显的杀菌消炎作用,特别是近几年有不少研究发现大蒜的油状提取物对多种结核杆菌,特别是多耐药性结核杆菌、耐多药结核杆菌以及一些非抗药结核杆菌可以起到很明显的抑制作用。大蒜油含有很多抗氧化活性的有效成分,它能明显预防由于外源物质如常见药物如对乙酰氨基酚等诱导产生的急性肝脏损伤,但是大蒜油对利福平和异烟肼所致肝脏损伤是否有保护作用国内外尚未见报道。 本次研究参考相关文献,采用RIF和INH混合灌胃制备结核药物肝损伤的动物模型,通过检测血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)和总胆红素(TB)等指标以及观察肝脏组织病理学切片,证实大蒜油是否具有预防RIF和INH引起的药物肝损伤的作用,并从机体氧化抗氧化系统等方面探讨大蒜油预防结核药物肝损伤的机理,为以后进一步研究及临床应用提供科学依据。 研究方法 1.动物模型建立 健康雄性Wistar大鼠SPF级,180-200g,适应性喂养7天后,随机分为5组：大蒜油低、中、高剂量处理组、RIF+INH模型组、正常对照组,每组12只。首先大蒜油处理组同时灌胃给予相应浓度的大蒜油(20、40和80mg/kg),正常对照组灌胃给予等体积的玉米油,2h后RIF+INH模型组和大蒜油处理组均灌胃给予RIF (150mg/kg)+INH (100mg/kg),对照组灌胃给予等体积的5%淀粉溶液。连续灌胃28天后处死大鼠,并将血清及剩余肝脏冻存。 2.大蒜油预防结核药物肝损伤的效果评价 计算肝脏系数；测定血清中ALT、AST、ALP及TB的活性或含量；制备肝脏病理切片,苏木素-伊红(HE)染色,观察肝损伤情况。 3.大蒜油对机体氧化抗氧化系统的影响 测定肝脏匀浆及血清中超氧化物歧化酶(Superoxide Dismutase, SOD)、过氧化氢酶(catalase, CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)、谷胱甘肽还原酶(glutathione reductase, GR)、谷胱甘肽硫转移酶(glutathione S-transferase, GST)、丙二醛(malondialdehyde, MDA)、谷胱甘肽(glutathione,GSH、总抗氧化能力(Total Antioxidant Capacity, T-AOC)。 4.统计学处理 用IBM SPSS Statistics20统计软件进行数据分析,采用One-way ANOVA,并进行两两比较,P0.05为差异有统计学意义。 研究结果 1.血清中ALT、AST、ALP及TB的变化 与正常对照组大鼠相比,模型组大鼠血清中ALT、AST及ALP的活性明显升高(P0.05或P0.01),TB的含量明显升高(P0.01)；与模型组大鼠相比,大蒜油低、中、高剂量处理组血清中ALT、AST、ALP的活性明显降低(P0.05或P0.01),TB的含量降低。 2.血清及肝脏组织中MDA的变化 与正常对照组大鼠相比,模型组大鼠血清及肝脏组织中MDA水平明显升高(P0.05)；与模型组大鼠相比,大蒜油低、中、高剂量处理组大鼠血清中MDA水平分别降低了2.91%、9.30%(P0.05),20.35%(P0.05),肝脏组织中MDA水平明显降低(P0.05)。 3.血清及肝脏组织中GSH的变化 与正常对照组大鼠相比,模型组大鼠的血清及肝脏组织匀浆中GSH水平明显降低(P0.05)；与模型组大鼠相比,大蒜油高剂量处理组大鼠血清中GSH水平明显升高(P0.05),肝脏组织中GSH水平也明显升高了27.56%(P0.05)。 4.血清及肝脏组织中GST、GSH-Px和GR的变化 与正常对照组大鼠相比,模型组大鼠的血清GST水平降低,但差异无统计学意义,模型组大鼠肝脏匀浆中GST水平明显降低(P0.05)；与模型组大鼠相比,大蒜油低、中、高剂量处理组大鼠血清中GST水平升高,但差异无统计学意义,大蒜油中、高剂量处理组大鼠肝脏组织中GST水平明显升高(P0.05)。 与正常对照组大鼠相比,模型组大鼠血清及肝脏组织匀浆中GSH-Px水平明显降低；与模型组大鼠相比,大蒜油中、高剂量处理组大鼠血清中GSH-Px水平明显升高(P0.05),大蒜油低、中、高剂量处理组大鼠肝脏组织中GSH-Px水平明显升高(P0.05)。 与正常对照组大鼠相比,模型组大鼠血清及肝脏组织匀浆中GR水平没有明显改变,与模型组大鼠相比,大蒜油低、中、高剂量处理组大鼠血清GR水平有所升高,但差异无统计学意义。大蒜油低、中、高剂量处理组大鼠肝脏匀浆组织中GR水平分别升高10.81%、18.92%、29.73%(P0.05)。 5.血清及肝脏组织中SOD和CAT的变化 与正常对照组大鼠相比,模型组大鼠血清及肝脏组织匀浆中SOD水平明显降低,与模型组大鼠相比,大蒜油低、中、高剂量处理组大鼠血清SOD水平明显升高(P0.05),并且大蒜油低、中、高剂量处理组大鼠肝脏组织匀浆中SOD水平也明显升高(P0.05)。 与正常对照组大鼠相比,模型组大鼠血清及肝脏匀浆组织中CAT水平明显降低(P0.05),大蒜油低、中、高剂量处理组大鼠血清中CAT水平分别降低23.96%、19.26%、10.75%,但差异无统计学意义,大蒜油低、中、高剂量处理组大鼠肝脏匀浆中CAT水平明显下降(P0.05)；与模型组大鼠相比,大蒜油低、中、高剂量处理组大鼠血清中CAT水平分别升高4.13%、10.59%、22.22%,但差异无统计学意义,大蒜油低、中、高剂量组大鼠肝脏组织中CAT水平明显升高(P0.05)。 6.血清及肝脏组织T-AOC的变化 与正常对照组大鼠相比,模型组、大蒜油低剂量处理组大鼠血清及肝脏匀浆中T-AOC水平明显降低(P0.05)；与模型组大鼠相比,大蒜油低、中、高剂量处理组大鼠血清T-AOC水平有所升高,但差异无统计学意义,大蒜油低、中、高剂量处理组大鼠肝脏匀浆中T-AOC水平分别升高1.52%(P0.05)、15.15%(P0.05)、22.22%(P0.05)。 7.肝脏形态学的变化 肝脏石蜡切片HE染色结果显示模型组肝脏发生轻微损伤,表现在肝细胞浊肿,中央静脉周围部分肝脏细胞出现气球样变。大蒜油各处理组病理损伤明显改善,与正常对照组差异无统计学意义。 结论： 1.大蒜油对利福平及异烟肼联合所致肝脏毒性有预防作用。 2.大蒜油预防利福平及异烟肼联合所致肝脏毒性的机制可能与其抗氧化能力有关。
[Abstract]:research objective
In recent years, due to the rebound of tuberculosis epidemic and the emergence of multi-drug resistant tuberculosis bacteria and multi-drug resistant tuberculosis bacteria, the situation of tuberculosis prevention and treatment has become more complex, the course of tuberculosis treatment and drug use have increased, the incidence of liver injury caused by tuberculosis drugs has also increased year by year, becoming one of the most common causes of drug-induced liver injury, especially isoniazid (INH), Rif Rifampicin, isoniazid and other anti-tuberculosis drugs induce hepatotoxicity, but the mechanism of these anti-tuberculosis drugs damage has not been fully elaborated. At present, more research focused on the mechanism of oxidative and antioxidant, studies found that the production of hepatotoxicity. It may be related to the imbalance of oxidation and antioxidation in vivo.
Garlic oil has a wide range of pharmacological effects and has been widely used in food and drug fields for thousands of years. For example, garlic oil has obvious bactericidal and anti-inflammatory effects. Especially in recent years, a number of studies have found that garlic oil extracts have a variety of Mycobacterium tuberculosis, especially multidrug-resistant Mycobacterium tuberculosis, multidrug-resistant Mycobacterium tuberculosis and some Non-drug-resistant nodules There are many antioxidant active ingredients in garlic oil, which can obviously prevent acute liver injury induced by exogenous substances such as paracetamol. However, whether garlic oil has protective effect on liver injury induced by rifampicin and isoniazid is still unknown at home and abroad. No report.
In this study, the animal model of liver injury induced by tuberculosis drugs was established by RIF and INH, and the liver histopathological sections were observed to confirm the existence of garlic oil by detecting alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TB) in serum. It can prevent RIF and INH-induced drug-induced liver injury, and explore the mechanism of garlic oil preventing tuberculosis drug-induced liver injury from the aspects of oxidative and antioxidant system, so as to provide scientific basis for further research and clinical application.
research method
1. animal models
After 7 days of adaptive feeding, healthy male Wistar rats were randomly divided into 5 groups: low, medium and high doses of garlic oil treatment group, RIF+INH model group, normal control group, 12 rats in each group. The RIF+INH model group and garlic oil treatment group were given RIF(150mg/kg)+INH(100mg/kg) by gastric lavage, while the control group was given 5% starch solution of the same volume. The rats were sacrificed after 28 days of continuous gastric lavage, and the serum and remaining liver were frozen.
2. garlic oil to prevent liver injury caused by tuberculosis drugs
The liver coefficient was calculated, the activities or contents of ALT, AST, ALP and TB in serum were determined, and the liver pathological sections were prepared and stained with hematoxylin-eosin (HE).
3. garlic oil on the oxidation and antioxidant system
Determination of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST), malondialdehyde (MDA) in liver homogenate and serum De, MDA), glutathione (glutathione, GSH, total antioxidant capacity (Total Antioxidant Capacity, T-AOC).
4. statistical processing
The data were analyzed by IBM SPSS Statistics 20 software, and one-way ANOVA was used. The difference was statistically significant in P 0.05.
Research results
1. the changes of ALT, AST, ALP and TB in serum
Compared with the normal control group, the activity of ALT, AST and ALP in the serum of the model group increased significantly (P 0.05 or P 0.01), and the content of TB increased significantly (P 0.01); Compared with the model group, the activity of ALT, AST and ALP in the serum of the middle and high dose groups decreased significantly (P 0.05 or P 0.01), and the content of TB decreased significantly.
2. changes of MDA in serum and liver tissues
Compared with the normal control group, the level of MDA in the serum and liver tissue of the model group increased significantly (P 0.05); compared with the model group, the level of MDA in the serum of the rats in the garlic oil treatment group decreased by 2.91%, 9.30% (P 0.05), 20.35% (P 0.05), and the level of MDA in the liver tissue decreased significantly (P 0.05).
3. changes of GSH in serum and liver tissues
Compared with the normal control group, the level of GSH in serum and liver tissue homogenate of the model group decreased significantly (P 0.05); compared with the model group, the level of GSH in serum of the high dose garlic oil treatment group increased significantly (P 0.05), and the level of GSH in liver tissue increased by 27.56% (P 0.05).
4. the changes of GST, GSH-Px and GR in serum and liver tissues
Compared with the normal control group, the serum GST level of the model group decreased, but the difference was not statistically significant. The liver homogenate GST level of the model group decreased significantly (P 0.05); Compared with the model group, the serum GST level of the rats in the garlic oil low, medium and high dose groups increased, but the difference was not statistically significant. The level of GST in the liver tissue of the treated group increased significantly (P0.05).
Compared with the normal control group, the levels of GSH-Px in the serum and liver homogenate of the model group rats were significantly lower; compared with the model group rats, the levels of GSH-Px in the serum of the rats in the high-dose garlic oil treatment group were significantly higher (P 0.05), the levels of GSH-Px in the liver tissue of the rats in the middle-dose treatment group were significantly higher (P 0.05).
Compared with the normal control group, the GR levels in the serum and liver homogenate of the model group rats did not change significantly. Compared with the model group rats, the levels of GR in the serum of the rats treated with garlic oil were lower, the levels of GR in the liver homogenate of the rats treated with garlic oil were higher in the middle and high dose groups, but the difference was not statistically significant. Do not increase 10.81%, 18.92%, 29.73% (P0.05).
5. the changes of SOD and CAT in serum and liver tissues
Compared with the normal control group, the level of SOD in the serum and liver tissue homogenate of the model group decreased significantly, and compared with the model group, the level of SOD in the serum of the rats treated with garlic oil was lower, the level of SOD in the serum of the rats treated with middle and high doses of garlic oil was significantly higher (P 0.05), and the level of SOD in the liver tissue homogenate of the rats treated with garlic oil was also significantly higher (P 0.05).
Compared with the normal control group, the level of CAT in the serum and liver homogenate of the model group decreased significantly (P 0.05). The level of CAT in the liver homogenate of the middle and high dose groups decreased by 23.96%, 19.26% and 10.75% respectively, but the difference was not statistically significant. Compared with the model group, the levels of CAT in the serum of the rats treated with garlic oil were 4.13%, 10.59% and 22.22% respectively, but there was no significant difference between the two groups.
6. changes of T-AOC in serum and liver tissue
Compared with the normal control group, the level of T-AOC in the serum and liver homogenate of the rats in the model group was significantly lower (P 0.05); compared with the rats in the model group, the level of T-AOC in the serum of the rats in the garlic oil low dose treatment group was significantly lower, but the level of T-AOC in the serum of the rats in the middle and high dose treatment group was higher, but the difference was not statistically significant. The level of T-AOC in the homogenate increased by 1.52% (P0.05), 15.15% (P0.05), 22.22% (P0.05), respectively.
7. changes in liver morphology
HE staining of liver paraffin section showed slight hepatic injury in the model group, including hepatocyte turbid swelling and balloon-like changes in some hepatocytes around the central vein.
Conclusion:
1. garlic oil has a preventive effect on liver toxicity induced by rifampicin and isoniazid.
2. The mechanism of garlic oil preventing hepatotoxicity induced by rifampicin and isoniazid may be related to its antioxidant capacity.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
本文编号：2249929
[Abstract]:research objective
In recent years, due to the rebound of tuberculosis epidemic and the emergence of multi-drug resistant tuberculosis bacteria and multi-drug resistant tuberculosis bacteria, the situation of tuberculosis prevention and treatment has become more complex, the course of tuberculosis treatment and drug use have increased, the incidence of liver injury caused by tuberculosis drugs has also increased year by year, becoming one of the most common causes of drug-induced liver injury, especially isoniazid (INH), Rif Rifampicin, isoniazid and other anti-tuberculosis drugs induce hepatotoxicity, but the mechanism of these anti-tuberculosis drugs damage has not been fully elaborated. At present, more research focused on the mechanism of oxidative and antioxidant, studies found that the production of hepatotoxicity. It may be related to the imbalance of oxidation and antioxidation in vivo.
Garlic oil has a wide range of pharmacological effects and has been widely used in food and drug fields for thousands of years. For example, garlic oil has obvious bactericidal and anti-inflammatory effects. Especially in recent years, a number of studies have found that garlic oil extracts have a variety of Mycobacterium tuberculosis, especially multidrug-resistant Mycobacterium tuberculosis, multidrug-resistant Mycobacterium tuberculosis and some Non-drug-resistant nodules There are many antioxidant active ingredients in garlic oil, which can obviously prevent acute liver injury induced by exogenous substances such as paracetamol. However, whether garlic oil has protective effect on liver injury induced by rifampicin and isoniazid is still unknown at home and abroad. No report.
In this study, the animal model of liver injury induced by tuberculosis drugs was established by RIF and INH, and the liver histopathological sections were observed to confirm the existence of garlic oil by detecting alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TB) in serum. It can prevent RIF and INH-induced drug-induced liver injury, and explore the mechanism of garlic oil preventing tuberculosis drug-induced liver injury from the aspects of oxidative and antioxidant system, so as to provide scientific basis for further research and clinical application.
research method
1. animal models
After 7 days of adaptive feeding, healthy male Wistar rats were randomly divided into 5 groups: low, medium and high doses of garlic oil treatment group, RIF+INH model group, normal control group, 12 rats in each group. The RIF+INH model group and garlic oil treatment group were given RIF(150mg/kg)+INH(100mg/kg) by gastric lavage, while the control group was given 5% starch solution of the same volume. The rats were sacrificed after 28 days of continuous gastric lavage, and the serum and remaining liver were frozen.
2. garlic oil to prevent liver injury caused by tuberculosis drugs
The liver coefficient was calculated, the activities or contents of ALT, AST, ALP and TB in serum were determined, and the liver pathological sections were prepared and stained with hematoxylin-eosin (HE).
3. garlic oil on the oxidation and antioxidant system
Determination of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST), malondialdehyde (MDA) in liver homogenate and serum De, MDA), glutathione (glutathione, GSH, total antioxidant capacity (Total Antioxidant Capacity, T-AOC).
4. statistical processing
The data were analyzed by IBM SPSS Statistics 20 software, and one-way ANOVA was used. The difference was statistically significant in P 0.05.
Research results
1. the changes of ALT, AST, ALP and TB in serum
Compared with the normal control group, the activity of ALT, AST and ALP in the serum of the model group increased significantly (P 0.05 or P 0.01), and the content of TB increased significantly (P 0.01); Compared with the model group, the activity of ALT, AST and ALP in the serum of the middle and high dose groups decreased significantly (P 0.05 or P 0.01), and the content of TB decreased significantly.
2. changes of MDA in serum and liver tissues
Compared with the normal control group, the level of MDA in the serum and liver tissue of the model group increased significantly (P 0.05); compared with the model group, the level of MDA in the serum of the rats in the garlic oil treatment group decreased by 2.91%, 9.30% (P 0.05), 20.35% (P 0.05), and the level of MDA in the liver tissue decreased significantly (P 0.05).
3. changes of GSH in serum and liver tissues
Compared with the normal control group, the level of GSH in serum and liver tissue homogenate of the model group decreased significantly (P 0.05); compared with the model group, the level of GSH in serum of the high dose garlic oil treatment group increased significantly (P 0.05), and the level of GSH in liver tissue increased by 27.56% (P 0.05).
4. the changes of GST, GSH-Px and GR in serum and liver tissues
Compared with the normal control group, the serum GST level of the model group decreased, but the difference was not statistically significant. The liver homogenate GST level of the model group decreased significantly (P 0.05); Compared with the model group, the serum GST level of the rats in the garlic oil low, medium and high dose groups increased, but the difference was not statistically significant. The level of GST in the liver tissue of the treated group increased significantly (P0.05).
Compared with the normal control group, the levels of GSH-Px in the serum and liver homogenate of the model group rats were significantly lower; compared with the model group rats, the levels of GSH-Px in the serum of the rats in the high-dose garlic oil treatment group were significantly higher (P 0.05), the levels of GSH-Px in the liver tissue of the rats in the middle-dose treatment group were significantly higher (P 0.05).
Compared with the normal control group, the GR levels in the serum and liver homogenate of the model group rats did not change significantly. Compared with the model group rats, the levels of GR in the serum of the rats treated with garlic oil were lower, the levels of GR in the liver homogenate of the rats treated with garlic oil were higher in the middle and high dose groups, but the difference was not statistically significant. Do not increase 10.81%, 18.92%, 29.73% (P0.05).
5. the changes of SOD and CAT in serum and liver tissues
Compared with the normal control group, the level of SOD in the serum and liver tissue homogenate of the model group decreased significantly, and compared with the model group, the level of SOD in the serum of the rats treated with garlic oil was lower, the level of SOD in the serum of the rats treated with middle and high doses of garlic oil was significantly higher (P 0.05), and the level of SOD in the liver tissue homogenate of the rats treated with garlic oil was also significantly higher (P 0.05).
Compared with the normal control group, the level of CAT in the serum and liver homogenate of the model group decreased significantly (P 0.05). The level of CAT in the liver homogenate of the middle and high dose groups decreased by 23.96%, 19.26% and 10.75% respectively, but the difference was not statistically significant. Compared with the model group, the levels of CAT in the serum of the rats treated with garlic oil were 4.13%, 10.59% and 22.22% respectively, but there was no significant difference between the two groups.
6. changes of T-AOC in serum and liver tissue
Compared with the normal control group, the level of T-AOC in the serum and liver homogenate of the rats in the model group was significantly lower (P 0.05); compared with the rats in the model group, the level of T-AOC in the serum of the rats in the garlic oil low dose treatment group was significantly lower, but the level of T-AOC in the serum of the rats in the middle and high dose treatment group was higher, but the difference was not statistically significant. The level of T-AOC in the homogenate increased by 1.52% (P0.05), 15.15% (P0.05), 22.22% (P0.05), respectively.
7. changes in liver morphology
HE staining of liver paraffin section showed slight hepatic injury in the model group, including hepatocyte turbid swelling and balloon-like changes in some hepatocytes around the central vein.
Conclusion:
1. garlic oil has a preventive effect on liver toxicity induced by rifampicin and isoniazid.
2. The mechanism of garlic oil preventing hepatotoxicity induced by rifampicin and isoniazid may be related to its antioxidant capacity.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
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