Involvement of mitochondrial dysfunction in hepatotoxicity induced by Ageratina adenophora in mice#

Involvement of mitochondrial dysfunction in hepatotoxicity induced by Ageratina adenophora in mice # # 紫茎泽兰引起小鼠线粒体功能障碍从而导致肝脏毒性损伤 1 2 first-author 3 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:hychun114@163.com †E-mail:hychun114@163.com hychun114@163.com Key Laboratory of Animal Disease and Environmental Hazards of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China Key Laboratory of Animal Disease and Environmental Hazards of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China Tongren Polytechnic College, Tongren 554300, China Tongren Polytechnic College, Tongren 554300, China Affiliated Sichuan Provincial Rehabilitation Hospital of Chengdu University of TCM, Sichuan Bayi Rehabilitation Center, Chengdu 611135, China Affiliated Sichuan Provincial Rehabilitation Hospital of Chengdu University of TCM, Sichuan Bayi Rehabilitation Center, Chengdu 611135, China Ageratina adenophora is a noxious plant and it is known to cause acute asthma, diarrhea, depilation, and even death in livestock (Zhu et al., 2007 2007 ; Wang et al., 2017 2017 ). A. adenophora grows near roadsides and degraded land worldwide (He et al., 2015b 2015b ). In the areas where it grows, A. adenophora is an invasive species that inhibits the growth of local plants and causes poisoning in animals that come in contact with it (Nie et al., 2012 2012 ). In China, these plants can be found in Yunnan, Sichuan, Guizhou, Chongqing, and other southwestern areas (He et al., 2015a 2015a ) and they have become a dominant species in these local regions. It threatens the native biodiversity and ecosystem in the invaded areas and causes serious economic losses (Wang et al., 2017 2017 ). It has been reported that A. adenophora can grow in the northeast direction at a speed of 20 km per year in China (Guo et al., 2009 2009 ). Because of the damage caused by A. adenophora, it ranks among the earliest alien invasive plant species in China (Wang et al., 2017 2017 ). 目的 2 研究紫茎泽兰造成小鼠肝脏毒性损伤后,肝脏线粒体结构和功能发生变化的情况. 创新点 2 紫茎泽兰可以导致肝脏细胞发生凋亡和焦亡,从而产生中毒损伤,然而尚未有报道其对线粒体超微结构和功能的改变.本研究通过多种手段解决了这一问题. 方法 2 将40只小鼠随机分成4组,分别饲喂不同浓度的紫茎泽兰饲料(对照组、100、200和300 g/kg紫茎泽兰添加饲料组).采用苏木精-伊红染色法(H&E)研究肝脏损伤情况,利用透射电子显微技术研究线粒体超微结构改变.同时,结合实时荧光定量聚合酶链反应(qRT-PCR)、流式细胞术和化学分析方法对线粒体DNA拷贝数、肿胀度和三磷酸腺苷(ATP)酶活性的改变进行探究. 结论 2 紫茎泽兰导致线粒体超微结构的改变,增大了线粒体肿胀度,降低了钠钾ATP酶(Na + K + -ATPase)和钙镁ATP酶(Ca 2+ Mg 2+ -ATPase)活力,同时减少了DNA拷贝数,从而引起肝脏损伤. Ageratina adenophora Hepatotoxicity Mitochondrial dysfunction ATPase Mitochondrial DNA 紫茎泽兰 肝毒性 线粒体功能障碍 三磷酸腺苷(ATP)酶 线粒体DNA Ageratina adenophora is a noxious plant and it is known to cause acute asthma, diarrhea, depilation, and even death in livestock (Zhu et al., 2007 2007 ; Wang et al., 2017 2017 ). A. adenophora grows near roadsides and degraded land worldwide (He et al., 2015b 2015b ). In the areas where it grows, A. adenophora is an invasive species that inhibits the growth of local plants and causes poisoning in animals that come in contact with it (Nie et al., 2012 2012 ). In China, these plants can be found in Yunnan, Sichuan, Guizhou, Chongqing, and other southwestern areas (He et al., 2015a 2015a ) and they have become a dominant species in these local regions. It threatens the native biodiversity and ecosystem in the invaded areas and causes serious economic losses (Wang et al., 2017 2017 ). It has been reported that A. adenophora can grow in the northeast direction at a speed of 20 km per year in China (Guo et al., 2009 2009 ). Because of the damage caused by A. adenophora , it ranks among the earliest alien invasive plant species in China (Wang et al., 2017 2017 ). Previous studies have reported that A. adenophora causes hepatotoxicity in different species of animals. Chronic respiratory disease and exercise intolerance have been reported in horses in Australia due to ingestion of A. adenophora (O'Sullivan, 1985 1985 ). Experimental feeding of A. adenophora samples to cattle from sub-Himalayan region in India has been seen to cause anorexia and photosensitization. Rats administrated with a diet mixed with purified extracts from A. adenophora leaves developed hepatotoxicity and cholestasis (Katoch et al., 2000 2000 ; Kaushal et al., 2001 2001 ). Mitochondria have a pivotal role in the production of adenosine triphosphate (ATP), which is the energy currency of the cell (Koh et al., 2018 2018 ). There are hundreds of mitochondria in each hepatocyte, and each organelle has 2 to 10 copies of mitochondrial DNA (mtDNA) (Robin and Wong, 1988 1988 ). The changes of mass and copy number of mitochondria are associated with cell growth and differentiation (Shay et al., 1990 1990 ), but also can be caused by a variety of noxious stimuli (Nugent et al., 2007 2007 ; Tiao et al., 2007 2007 ). Liver damage is closely related to mitochondrial dysfunction (Shi et al., 2018 2018 ). According to our previous studies, A. adenophora had toxic effects on renal cells, splenocytes, and hepatocytes by activating apoptosis in Saanen goat through mitochondrial pathway (He et al., 2015a 2015a , 2015b 2015b , 2016 2016 ). In addition, apoptosis is closely related with mitochondria (Song et al., 2018 2018 ). These results led us to investigate the changes occurring in the mitochondria during the process of hepatotoxicity caused by A. adenophora . After six weeks of treatment with A. adenophora extract, histopathological analysis was carried out on the liver tissues. Based on the microscopic examination, normal hepatic sinusoid and hepatic lobules can be found clearly in the control and the group treated with a dosage of 100 g/kg A. adenophora (Figs. 1a and 1b 1a and 1b ). However, we observed varying degrees of pathological changes in the liver in the 200 and 300 g/kg dosage groups. Light vacuolar degeneration was seen in the 200 g/kg dosage group (Fig. 1c 1c ). We observed many foci of hepatocytes degeneration mainly in the periportal areas in the high dosage group (Fig. 1d 1d ). Degenerated hepatocytes were swollen with hydropic cytoplasm and unclear nucleus. Inflammatory cells were also universally observed in the necrotic areas (Fig. 1d 1d ). The histological evaluation demonstrated that the livers subjected to medium and high dosages of A. adenophora exhibited destruction of normal hepatic structure to a certain degree. To investigate the changes in the mitochondrial structure induced by A. adenophora , transmission electron microscopy (TEM) was used to measure the ultrastructure in the control and A. adenophora -treated groups. As shown in Fig. 2 2 , the most characteristic change was the cristal abnormalities caused by A. adenophora and these were heterogeneous in sizes and shapes compared to the control group. Partially swollen mitochondria with unusual and sparse cristae were observed in A. adenophora -treated groups (Figs. 2b–2d 2b–2d ). Furthermore, we determined the quantitative degree of mitochondrial swelling using flow cytometry (FCM) with appropriate forward/side scatter (FSC/SSC) plot parameter setting. FSC is closely related to the cell size, while SSC correlates with the granularity and the refractive index. The quantitative degree of mitochondrial swelling was expressed by the ratio of FSC/SSC. The FSC/SSC ratios in A . adenophora -treated (100, 200, and 300 g/kg dosages) groups were 0.850±0.035, 0.941±0.014, and 1.190±0.049, respectively. All of the results obtained were higher than that in the control group (0.770±0.009, P < 0.01; Fig. 3 3 ). To detect the effect of A. adenophora on the mitochondrial function, ATPase activity (Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase) and mtDNA copy number were measured in the control and A. adenophora administration groups. The results showed that treatment with A. adenophora led to the decrease in the contents of Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase in a dose-dependent manner compared with the control group ( P < 0.01; Fig. 4a 4a ). Cellular mtDNA is also a key indicator of mitochondrial function. In an effort to understand the changes in the levels of mtDNA, quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the copy number of mtDNA. As shown in Fig. 4b 4b , the copy number of mtDNA showed a decreasing trend in the A. adenophora -treated groups compared with that in the control group ( P < 0.01). A. adenophora is one of the most harmful invasive plants in China and it is toxic to animals (Yang et al., 2017 2017 ). Previous studies have confirmed that hepatotoxicity could be caused by A. adenophora in mice, rat, and goat (Sani et al., 1992 1992 ; Katoch et al., 2000 2000 ; He et al., 2016 2016 ). Liver, which is the target organ of A. adenophora , plays a vital role in the detoxification of ingested toxin. In the present study, we investigated the effects of A. adenophora in inducing hepatotoxicity in mice that had consumed A. adenophora for 42 d. Mitochondria are the major cellular loci for ATP production (Wang et al., 2009 2009 ). It is known that most liver diseases are closely associated with the depletion of mitochondrial energy production (Koh et al., 2018 2018 ). ATP generated in the cell (including Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase) is an important extracellular ligand in the autocrine signal transduction, intercellular communication and nerve transmission, and has a wide range of physiological and pathophysiological roles (Guo et al., 2018 2018 ; Taruno, 2018 2018 ). Cellular ATP is an indicator of mitochondrial function. Our previous study demonstrated hepatotoxicity caused by A. adenophora through activation of mitochondrial pathway in goat (He et al., 2016 2016 ). However, there is little information about the changes in the structure or function of mitochondria. In the present study, we found that the levels of Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase had a tendency to decrease when induced by A. adenophora in mice liver (Fig. 4a 4a ). This indicated that A. adenophora damaged hepatic function by decreasing ATP availability. Apart from ATP, mtDNA is another indicator of mitochondrial function. Mitochondria are an important source of reactive oxygen species (ROS), but have incomplete DNA repair capacity (Shay et al., 1990 1990 ). The mtDNA can be damaged oxidatively by ROS (Larosche et al., 2010 2010 ). Our recent study showed that ROS was elevated by A. adenophora in mice liver (Sun et al., 2018 2018 ). However, no studies have been reported on the effect of hepatopathy induced by A. adenophora in hepatic mtDNA. In this study, we quantified mtDNA copies by qRT-PCR. A decreasing trend of mtDNA copy number was found in A . adenophora -treated groups (Fig. 4b 4b ). This result was supported by the observation through histopathology (Fig. 1 1 ) and TEM (Fig. 2 2 ) in the present study, which suggests that decreased mtDNA copy numbers are highly associated with liver dysfunction. 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Predicting the spatial distribution of an invasive plant species (Eupatorium adenophorum) in China Compliance with ethics guidelines Wei SUN, Chao-rong ZENG, Dong YUE, and Yan-chun HU declare that they have no conflict of interest. This study was reviewed and approved by the Animal Care and Use Committee of Sichuan Agricultural University (SYXK2014-187), China. All animal operations, collection samples, and procedures were carried out in accordance with the guidelines approved. 10.1631/jzus.B1800645 Project supported by the Science and Technology Support Program of Sichuan Province (No. 2015SZ0201) and the Tongren Science and Technology Project (No. 2018-44), China 2018-12-19 2019-04-04 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 20