硫酸鈹致小鼠肝毒性的線粒體損傷機制
發(fā)布時間：2018-10-10 17:14
【摘要】：目的： 通過整體動物染毒硫酸鈹（BeSO4·4H2O），觀察硫酸鈹對小鼠肝臟的毒性作用，，探討硫酸鈹致小鼠肝毒性的線粒體損傷的可能機制。 方法： 1．選取6周齡健康成年昆明（KM）雄性小鼠30只，隨機分為3組，每組10只，設(shè)陰性對照組和兩個染毒組，對照組按0.1ml/10g（體重）腹腔注射滅菌生理鹽水，低、高劑量染毒組分別腹腔注射1mg/kg和2mg/kg硫酸鈹溶液。隔天染毒，持續(xù)2周。觀察染毒期間小鼠的一般情況變化。 2．斷頸放血法處死小鼠，收集血液，自動生化分析儀檢測小鼠血清谷丙轉(zhuǎn)氨酶（ALT）和谷草轉(zhuǎn)氨酶（AST）含量；分離完整肝臟，計算肝臟臟器系數(shù)；肝組織采用蘇木素-伊紅（HE）染色，光鏡下觀察肝臟病理組織學(xué)變化。 3．小鼠肝線粒體懸液采用差速離心法制備。分別用熒光染料羅丹明123（Rh123）、二氯二氫熒光素-乙酰乙酸酯（DCFH-DA）負載肝線粒體，通過熒光分光光度法測定線粒體膜電位（Δψm）變化和線粒體活性氧（ROS）含量；分別用分光光度法測定線粒體吸光度（A520）值反映線粒體通透性轉(zhuǎn)變孔道（PTP）開放程度、線粒體細胞色素C（Cytc）含量、線粒體丙二醛（MDA）含量和線粒體谷胱甘肽過氧化物酶（GSH-Px）活性；采用紫外分光光度法測定線粒體超氧化物歧化酶（SOD）活性。 結(jié)果： 1．對照組小鼠和染毒組小鼠一般體征無明顯差異；與對照組比較，染毒組體重差異無統(tǒng)計學(xué)意義（P0.05）。 2．與對照組比較，染毒組小鼠肝臟臟器系數(shù)增高、血清ALT和AST水平升高（P0.05）；病理學(xué)檢查發(fā)現(xiàn)，對照組肝細胞結(jié)構(gòu)正常；低劑量染毒組小鼠肝細胞水腫,有灶性壞死等病變；高劑量染毒組小鼠肝細胞胞漿成空泡狀，有廣泛變性、壞死等病理改變。 3．與對照組比較，染毒組線粒體Δψm下降（P0.05）；線粒體SOD活性和GSH-Px活性降低（P0.05）；染毒組線粒體PTP開放明顯（P0.05）；染毒組線粒體Cytc含量、線粒體ROS含量和線粒體MDA含量增高（P0.05）。 結(jié)論： 1．硫酸鈹可致小鼠肝功能異常、肝組織病理形態(tài)學(xué)異常，具有明顯肝毒性。 2．硫酸鈹可致小鼠肝線粒體Δψm下降、PTP開放、Cytc釋放增加、SOD和GSH-Px活性下降，ROS和MDA含量生成增加。 3.肝線粒體功能障礙和氧化損傷可能是硫酸鈹致小鼠肝毒性的主要原因，線粒體可能是硫酸鈹致肝毒性的作用靶點。
[Abstract]:Objective: to observe the toxicity of beryllium sulfate (BeSO4 4H2O) to mice liver and explore the possible mechanism of mitochondrial damage induced by beryllium sulfate. Methods: 1. Thirty healthy male Kunming (KM) mice aged 6 weeks were randomly divided into 3 groups, 10 in each group, with negative control group and two exposure groups. The control group was injected intraperitoneally with sterilized saline according to 0.1ml/10g (body weight). In high dose group, beryllium sulfate solution (1mg/kg) and 2mg/kg (beryllium sulfate) were injected intraperitoneally respectively. Poisoned the next day for 2 weeks. To observe the general changes of mice during exposure. 2. The mice were killed by blood release method, blood was collected, serum alanine aminotransferase (ALT) and alanine aminotransferase (AST) levels were detected by automatic biochemical analyzer, and the intact liver was isolated. Liver tissue was stained with hematoxylin-eosin (HE) and histopathological changes were observed under light microscope. 3. Mouse liver mitochondria suspension was prepared by differential centrifugation. Hepatic mitochondria were loaded with fluorescent dye Rhodamine 123 (Rh123) and dichlorodihydrofluorescein acetate (DCFH-DA). The changes of mitochondrial membrane potential (螖 蠄 m) and the content of reactive oxygen species (ROS) in mitochondria were measured by fluorescence spectrophotometry. The mitochondrial absorbance (A520) was measured by spectrophotometry to reflect the opening degree of mitochondrial permeability transition pore (PTP), mitochondrial cytochrome C (Cytc) content, mitochondrial malondialdehyde (MDA) content and mitochondrial glutathione peroxidase (GSH-Px) activity. The activity of mitochondrial superoxide dismutase (SOD) was determined by ultraviolet spectrophotometry. Results: 1. There was no significant difference in general physical signs between the control group and the exposed group, and there was no significant difference in body weight between the control group and the control group (P0.05). 2. Compared with the control group, there was no significant difference in body weight between the control group and the control group. Liver organ coefficient increased, serum ALT and AST levels increased in the exposed group (P0.05); pathological examination showed that the structure of hepatocytes in the control group was normal; liver cell edema, focal necrosis and other pathological changes were found in the low dose exposure group. Compared with the control group, the mitochondria 螖 蠄 m decreased (P0.05), the activity of mitochondrial SOD and GSH-Px decreased (P0.05). The content of mitochondrial Cytc, mitochondrial ROS and mitochondrial MDA were significantly increased in exposed group (P0.05). Conclusion: 1. Beryllium sulfate can induce abnormal liver function and pathological morphology of liver tissue in mice. 2. Beryllium sulfate could induce the decrease of 螖 蠄 m, the opening of PTP, the increase of Cytc release, the decrease of SOD and GSH-Px activity, and the increase of ROS and MDA production in mouse liver mitochondria. The dysfunction and oxidative damage of liver mitochondria may be the main causes of hepatotoxicity induced by beryllium sulfate in mice, and mitochondria may be the target of hepatotoxicity induced by beryllium sulfate.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114
本文編號：2262613
[Abstract]:Objective: to observe the toxicity of beryllium sulfate (BeSO4 4H2O) to mice liver and explore the possible mechanism of mitochondrial damage induced by beryllium sulfate. Methods: 1. Thirty healthy male Kunming (KM) mice aged 6 weeks were randomly divided into 3 groups, 10 in each group, with negative control group and two exposure groups. The control group was injected intraperitoneally with sterilized saline according to 0.1ml/10g (body weight). In high dose group, beryllium sulfate solution (1mg/kg) and 2mg/kg (beryllium sulfate) were injected intraperitoneally respectively. Poisoned the next day for 2 weeks. To observe the general changes of mice during exposure. 2. The mice were killed by blood release method, blood was collected, serum alanine aminotransferase (ALT) and alanine aminotransferase (AST) levels were detected by automatic biochemical analyzer, and the intact liver was isolated. Liver tissue was stained with hematoxylin-eosin (HE) and histopathological changes were observed under light microscope. 3. Mouse liver mitochondria suspension was prepared by differential centrifugation. Hepatic mitochondria were loaded with fluorescent dye Rhodamine 123 (Rh123) and dichlorodihydrofluorescein acetate (DCFH-DA). The changes of mitochondrial membrane potential (螖 蠄 m) and the content of reactive oxygen species (ROS) in mitochondria were measured by fluorescence spectrophotometry. The mitochondrial absorbance (A520) was measured by spectrophotometry to reflect the opening degree of mitochondrial permeability transition pore (PTP), mitochondrial cytochrome C (Cytc) content, mitochondrial malondialdehyde (MDA) content and mitochondrial glutathione peroxidase (GSH-Px) activity. The activity of mitochondrial superoxide dismutase (SOD) was determined by ultraviolet spectrophotometry. Results: 1. There was no significant difference in general physical signs between the control group and the exposed group, and there was no significant difference in body weight between the control group and the control group (P0.05). 2. Compared with the control group, there was no significant difference in body weight between the control group and the control group. Liver organ coefficient increased, serum ALT and AST levels increased in the exposed group (P0.05); pathological examination showed that the structure of hepatocytes in the control group was normal; liver cell edema, focal necrosis and other pathological changes were found in the low dose exposure group. Compared with the control group, the mitochondria 螖 蠄 m decreased (P0.05), the activity of mitochondrial SOD and GSH-Px decreased (P0.05). The content of mitochondrial Cytc, mitochondrial ROS and mitochondrial MDA were significantly increased in exposed group (P0.05). Conclusion: 1. Beryllium sulfate can induce abnormal liver function and pathological morphology of liver tissue in mice. 2. Beryllium sulfate could induce the decrease of 螖 蠄 m, the opening of PTP, the increase of Cytc release, the decrease of SOD and GSH-Px activity, and the increase of ROS and MDA production in mouse liver mitochondria. The dysfunction and oxidative damage of liver mitochondria may be the main causes of hepatotoxicity induced by beryllium sulfate in mice, and mitochondria may be the target of hepatotoxicity induced by beryllium sulfate.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114
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