發(fā)布時(shí)間：2018-09-19 10:56

【摘要】：研究目的 近年來(lái)由于結(jié)核疫情反彈及耐多藥結(jié)核菌和多耐藥結(jié)核菌的出現(xiàn),導(dǎo)致結(jié)核防治情況愈加復(fù)雜,結(jié)核治療療程及用藥等增加,結(jié)核藥物導(dǎo)致的肝臟損傷的發(fā)生率也逐年上升,成為藥物性肝損傷最常見(jiàn)的原因之一,特別是異煙肼(INH)、利福平(RIF)所致的肝損傷居前兩位。雖然關(guān)于利福平、異煙肼等抗結(jié)核藥物誘導(dǎo)產(chǎn)生肝臟毒性的研究很多,但是這些抗結(jié)核藥物的損傷機(jī)制并沒(méi)有得到完全的闡述,目前較多的損傷機(jī)制研究集中在氧化抗氧化機(jī)制,研究發(fā)現(xiàn)該肝臟毒性的產(chǎn)生可能與體內(nèi)氧化抗氧化水平失衡有關(guān)。 大蒜油具有廣泛的藥理作用,于數(shù)千年前就開(kāi)始廣泛應(yīng)用于食藥領(lǐng)域。比如大蒜油有明顯的殺菌消炎作用,特別是近幾年有不少研究發(fā)現(xiàn)大蒜的油狀提取物對(duì)多種結(jié)核桿菌,特別是多耐藥性結(jié)核桿菌、耐多藥結(jié)核桿菌以及一些非抗藥結(jié)核桿菌可以起到很明顯的抑制作用。大蒜油含有很多抗氧化活性的有效成分,它能明顯預(yù)防由于外源物質(zhì)如常見(jiàn)藥物如對(duì)乙酰氨基酚等誘導(dǎo)產(chǎn)生的急性肝臟損傷,但是大蒜油對(duì)利福平和異煙肼所致肝臟損傷是否有保護(hù)作用國(guó)內(nèi)外尚未見(jiàn)報(bào)道。 本次研究參考相關(guān)文獻(xiàn),采用RIF和INH混合灌胃制備結(jié)核藥物肝損傷的動(dòng)物模型,通過(guò)檢測(cè)血清中丙氨酸氨基轉(zhuǎn)移酶(ALT)、天冬氨酸氨基轉(zhuǎn)移酶(AST)、堿性磷酸酶(ALP)和總膽紅素(TB)等指標(biāo)以及觀察肝臟組織病理學(xué)切片,證實(shí)大蒜油是否具有預(yù)防RIF和INH引起的藥物肝損傷的作用,并從機(jī)體氧化抗氧化系統(tǒng)等方面探討大蒜油預(yù)防結(jié)核藥物肝損傷的機(jī)理,為以后進(jìn)一步研究及臨床應(yīng)用提供科學(xué)依據(jù)。 研究方法 1.動(dòng)物模型建立 健康雄性Wistar大鼠SPF級(jí),180-200g,適應(yīng)性喂養(yǎng)7天后,隨機(jī)分為5組：大蒜油低、中、高劑量處理組、RIF+INH模型組、正常對(duì)照組,每組12只。首先大蒜油處理組同時(shí)灌胃給予相應(yīng)濃度的大蒜油(20、40和80mg/kg),正常對(duì)照組灌胃給予等體積的玉米油,2h后RIF+INH模型組和大蒜油處理組均灌胃給予RIF (150mg/kg)+INH (100mg/kg),對(duì)照組灌胃給予等體積的5%淀粉溶液。連續(xù)灌胃28天后處死大鼠,并將血清及剩余肝臟凍存。 2.大蒜油預(yù)防結(jié)核藥物肝損傷的效果評(píng)價(jià) 計(jì)算肝臟系數(shù)；測(cè)定血清中ALT、AST、ALP及TB的活性或含量；制備肝臟病理切片,蘇木素-伊紅(HE)染色,觀察肝損傷情況。 3.大蒜油對(duì)機(jī)體氧化抗氧化系統(tǒng)的影響 測(cè)定肝臟勻漿及血清中超氧化物歧化酶(Superoxide Dismutase, SOD)、過(guò)氧化氫酶(catalase, CAT)、谷胱甘肽過(guò)氧化物酶(glutathione peroxidase, GSH-Px)、谷胱甘肽還原酶(glutathione reductase, GR)、谷胱甘肽硫轉(zhuǎn)移酶(glutathione S-transferase, GST)、丙二醛(malondialdehyde, MDA)、谷胱甘肽(glutathione,GSH、總抗氧化能力(Total Antioxidant Capacity, T-AOC)。 4.統(tǒng)計(jì)學(xué)處理 用IBM SPSS Statistics20統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)分析,采用One-way ANOVA,并進(jìn)行兩兩比較,P0.05為差異有統(tǒng)計(jì)學(xué)意義。 研究結(jié)果 1.血清中ALT、AST、ALP及TB的變化 與正常對(duì)照組大鼠相比,模型組大鼠血清中ALT、AST及ALP的活性明顯升高(P0.05或P0.01),TB的含量明顯升高(P0.01)；與模型組大鼠相比,大蒜油低、中、高劑量處理組血清中ALT、AST、ALP的活性明顯降低(P0.05或P0.01),TB的含量降低。 2.血清及肝臟組織中MDA的變化 與正常對(duì)照組大鼠相比,模型組大鼠血清及肝臟組織中MDA水平明顯升高(P0.05)；與模型組大鼠相比,大蒜油低、中、高劑量處理組大鼠血清中MDA水平分別降低了2.91%、9.30%(P0.05),20.35%(P0.05),肝臟組織中MDA水平明顯降低(P0.05)。 3.血清及肝臟組織中GSH的變化 與正常對(duì)照組大鼠相比,模型組大鼠的血清及肝臟組織勻漿中GSH水平明顯降低(P0.05)；與模型組大鼠相比,大蒜油高劑量處理組大鼠血清中GSH水平明顯升高(P0.05),肝臟組織中GSH水平也明顯升高了27.56%(P0.05)。 4.血清及肝臟組織中GST、GSH-Px和GR的變化 與正常對(duì)照組大鼠相比,模型組大鼠的血清GST水平降低,但差異無(wú)統(tǒng)計(jì)學(xué)意義,模型組大鼠肝臟勻漿中GST水平明顯降低(P0.05)；與模型組大鼠相比,大蒜油低、中、高劑量處理組大鼠血清中GST水平升高,但差異無(wú)統(tǒng)計(jì)學(xué)意義,大蒜油中、高劑量處理組大鼠肝臟組織中GST水平明顯升高(P0.05)。 與正常對(duì)照組大鼠相比,模型組大鼠血清及肝臟組織勻漿中GSH-Px水平明顯降低；與模型組大鼠相比,大蒜油中、高劑量處理組大鼠血清中GSH-Px水平明顯升高(P0.05),大蒜油低、中、高劑量處理組大鼠肝臟組織中GSH-Px水平明顯升高(P0.05)。 與正常對(duì)照組大鼠相比,模型組大鼠血清及肝臟組織勻漿中GR水平?jīng)]有明顯改變,與模型組大鼠相比,大蒜油低、中、高劑量處理組大鼠血清GR水平有所升高,但差異無(wú)統(tǒng)計(jì)學(xué)意義。大蒜油低、中、高劑量處理組大鼠肝臟勻漿組織中GR水平分別升高10.81%、18.92%、29.73%(P0.05)。 5.血清及肝臟組織中SOD和CAT的變化 與正常對(duì)照組大鼠相比,模型組大鼠血清及肝臟組織勻漿中SOD水平明顯降低,與模型組大鼠相比,大蒜油低、中、高劑量處理組大鼠血清SOD水平明顯升高(P0.05),并且大蒜油低、中、高劑量處理組大鼠肝臟組織勻漿中SOD水平也明顯升高(P0.05)。 與正常對(duì)照組大鼠相比,模型組大鼠血清及肝臟勻漿組織中CAT水平明顯降低(P0.05),大蒜油低、中、高劑量處理組大鼠血清中CAT水平分別降低23.96%、19.26%、10.75%,但差異無(wú)統(tǒng)計(jì)學(xué)意義,大蒜油低、中、高劑量處理組大鼠肝臟勻漿中CAT水平明顯下降(P0.05)；與模型組大鼠相比,大蒜油低、中、高劑量處理組大鼠血清中CAT水平分別升高4.13%、10.59%、22.22%,但差異無(wú)統(tǒng)計(jì)學(xué)意義,大蒜油低、中、高劑量組大鼠肝臟組織中CAT水平明顯升高(P0.05)。 6.血清及肝臟組織T-AOC的變化 與正常對(duì)照組大鼠相比,模型組、大蒜油低劑量處理組大鼠血清及肝臟勻漿中T-AOC水平明顯降低(P0.05)；與模型組大鼠相比,大蒜油低、中、高劑量處理組大鼠血清T-AOC水平有所升高,但差異無(wú)統(tǒng)計(jì)學(xué)意義,大蒜油低、中、高劑量處理組大鼠肝臟勻漿中T-AOC水平分別升高1.52%(P0.05)、15.15%(P0.05)、22.22%(P0.05)。 7.肝臟形態(tài)學(xué)的變化 肝臟石蠟切片HE染色結(jié)果顯示模型組肝臟發(fā)生輕微損傷,表現(xiàn)在肝細(xì)胞濁腫,中央靜脈周?chē)糠指闻K細(xì)胞出現(xiàn)氣球樣變。大蒜油各處理組病理?yè)p傷明顯改善,與正常對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義。 結(jié)論： 1.大蒜油對(duì)利福平及異煙肼聯(lián)合所致肝臟毒性有預(yù)防作用。 2.大蒜油預(yù)防利福平及異煙肼聯(lián)合所致肝臟毒性的機(jī)制可能與其抗氧化能力有關(guān)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R965

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