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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 6  |  Issue : 4  |  Page : 461-468

Mitochondria are main targets of time/dose-dependent oxidative damage-based hepatotoxicity caused by rhizoma dioscoreae bulbiferae in mice


Atomization Inhalation Research Center, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Date of Submission20-Apr-2020
Date of Acceptance08-Sep-2020
Date of Web Publication16-Dec-2020

Correspondence Address:
Prof. Zu-Guang Ye
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100029
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_72_20

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  Abstract 


Background: Rhizoma Dioscoreae bulbiferae could cause liver damage, which limited its application in the clinic. Aims and Objectives: To explore the mechanism of hepatotoxicity of Rhizoma Dioscoreae bulbiferae in mice. Materials and Methods: In the present study, the water extraction of Rhizoma Dioscoreae bulbiferae (W.E.R) was administrated via intragastrical with Low (19.6 g/kg), Middle (28.0 g/kg), and High (40.0 g/kg) dose in mice. At each time point 14 days, 21 days, and 28 days, the body weight, liver coefficient, indexes of liver function alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate transaminase (AST), various biochemical biomarkers of liver tissue and mitochondria were detected and analyzed. Results: We found that W.E.R could decrease the body weight, increase the liver coefficient and the expression levels of indexes of liver function in mice. Next, we investigated that W.E.R could increase the expression levels of reactive oxygen species (ROS) and malondialdehyde (MDA), decrease the expression levels of adenosine triphosphate (ATP) and improve the enzyme activities of total superoxide dismutase (SOD). Third, we found that W. E. R could increase the enzyme activities of manganese-superoxide dismutase, decrease the enzyme activities of sodium-potassium adenosine triphosphatase (Na + -K + -ATPase) and calcium-magnesium adenosine triphosphatase (Ca 2+ -Mg 2+ -ATPase). Finally, we analyzed that there were significant negative correlations between body weight, expression level of ATP, activity of Na + -K + -ATPase, activity of Ca 2+ -Mg 2+ -ATPase and time, dose. There were significant positive correlations between liver coefficient, ALP, ALT, AST, expression levels of ROS, MDA and time, dose. Conclusion: All the results above indicated that W.E.R could cause the hepatotoxicity based on the oxidative damage in mice, which and mitochondria might be the main targets.

Keywords: Correlation, hepatotoxicity, mitochondria, oxidative damage, Rhizoma Dioscoreae bulbiferae


How to cite this article:
Hou HP, Zhang GP, Li H, Chen TF, Gao YH, Song L, Zhang ZX, Ye ZG. Mitochondria are main targets of time/dose-dependent oxidative damage-based hepatotoxicity caused by rhizoma dioscoreae bulbiferae in mice. World J Tradit Chin Med 2020;6:461-8

How to cite this URL:
Hou HP, Zhang GP, Li H, Chen TF, Gao YH, Song L, Zhang ZX, Ye ZG. Mitochondria are main targets of time/dose-dependent oxidative damage-based hepatotoxicity caused by rhizoma dioscoreae bulbiferae in mice. World J Tradit Chin Med [serial online] 2020 [cited 2021 Jan 18];6:461-8. Available from: https://www.wjtcm.net/text.asp?2020/6/4/461/303543




  Introduction Top


Recently, the reports of adverse reactions caused by traditional Chinese medicine made the public worry about the safety of Chinese Medicine. Every coin had two sides, even Traditional Chinese Medicine. However, the value of Traditional Chinese Medicine in clinic should not be ignored because of the occurrence of adverse reactions. While the toxicity mechanisms should be explored with modern scientific research methods, which will provide new targets and ideas for reducing the occurrence of adverse reactions.

Rhizoma Dioscoreae bulbiferae, also named Dioscorea bulbifera L., was originally recorded in Thousand Golden Prescriptions of Simiao Sun. It was cool-natured, bitter in taste, which could eliminate the mass and relieve swelling, cool the blood and stop bleeding, clear heat, and detoxicate. Rhizoma Dioscoreae bulbiferae, also named Dioscorea bulbifera L., was originally recorded in Thousand Golden Prescriptions of Simiao Sun.[1],[2],[3],[4] According to modern pharmacological researches, Rhizoma Dioscoreae bulbiferae had various different roles in the clinic, such as significant hydroxyl radical scavenging ability and strong anti-lipid peroxidation capacity,[5],[6] promoting tumor cell degradation and increasing the body's response to tumor cells,[7] significantly inhibiting the proliferation of gastric cancer cells clonal formation and migration ability,[8],[9] enhancing the activity of natural killer cells and the number of antibody-forming cells,[10] anti-inflammatory effect,[11] and treatment of thyroiditis,[12] and so on. In recent years, with the wide usage in the clinic, more and more researches were reported the hepatotoxicity caused by Rhizoma Dioscoreae bulbiferae,[13],[14],[15],[16] which greatly limited to play its unique clinical efficacy.

What were the following pathways and mechanisms of the hepatotoxicity caused by Rhizoma Dioscoreae bulbiferae? At present, domestic and foreign researches were mainly focused on inflammation,[17] cytokines,[18] cholestasis, cytochrome P450,[19] oxidative damage, etc. Meanwhile, what roles do mitochondria play as the targets for oxidative factor production and primary attack? Did they play key roles in the occurrence and development of hepatotoxicity of Rhizoma Dioscoreae bulbiferae? To this end, combined with modern gene expression technology and molecular biology guiding concepts, the possible pathways and mechanisms of hepatotoxicity caused by Rhizoma Dioscoreae bulbiferae were deeply explored, especially the role of mitochondria, which could provide new targets and ideas for the following mechanism and had the significant value and significance in clinical safety applications.


  Materials and Methods Top


Ethics statement

All animal procedures were carried out in compliance with the guidelines for scientific animal procedures approved by the ethics committee of the China Academy of Chinese Medical Sciences.

Preparation of water extraction of Rhizoma Dioscoreae bulbiferae (W.E.R)

Rhizoma Dioscoreae bulbiferae were brought from Beijing Qiancao Herbal Pieces Co. Ltd., originated in Hunan Province (Lot No. 140120002), which was identified as Rhizoma Dioscoreae bulbiferae by the Medicine Pharmacies, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

Preparation of W.E.R was improved, followed previous reports.[20] Details were shown: The dried tubers of Rhizoma Dioscoreae bulbiferae were cut into pieces of about 0.5–1 cm, and eight times the volume of water (mass, kg: volume, L) was added. The mixture was immersed in water for 4 h at room temperature and heated for 20 min, following filtered to obtain the filtrate with four layers of gauze. The remaining dregs were added to five times the volume of water and boiled for 20 min, which was filtered to obtain the filtrate with four layers of gauze. The above filtrates were combined and concentrated to a concentration of 2 kg/L, which was stored at 4°C.

Animals

A total of 108 male (Institute of Cancer Research [ICR]) weighing 20–22 g were used in this study, with nine animals being used in each experiment group at each time point. Mice were divided into four groups: normal animal (water only, control), low-dose group (low, 19.6 g/kg W.E.R), middle-dose group (middle, 28.0 g/kg W.E.R), and high-dose group (high, 40.0 g/kg W.E.R). In each group, subgroups were set up, 14 days, 21 days and 28 days. All animals were injected with corresponding liquid, via intragastrical administration.

Body weight, liver coefficient, and liver function

Detection of body weight, liver coefficient, and liver function

At each time point (14 days, 21 days, 28 days), the fasting weight and liver weight of all mice in four groups were weighed. Then, the liver coefficient was calculated as the following formula:

Liver coefficient = Liver weight (g)• Body weight (g)−1.

The mice were sacrificed and the serum was collected by centrifugation at 3000 r/min, lasting for 15 min at 4°C. Then the expression levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate transaminase (AST) were detected with Automatic Biochemical Analyzer.

Tissue preparation

At each time point (14 days, 21 days, 28 days), the big leaves of the liver were taken out and immersed with 4% paraformaldehyde for hematoxylin-eosin (HE) staining, and the others were prepared for the detection of biochemical expression levels of liver and mitochondria of liver.

Hematoxylin-eosin staining

HE staining was performed following previous reports. Briefly, 2–3 μm liver samples were washed with phosphate-buffered saline (PBS) three times for 5 min. Moreover, the sections were stained with hematoxylin for 5 min and eosin for 5 min, following gradient alcohol and xylene, and sealed for observation. All observations and experiments were double blind.

Detection of oxidative damage in liver

The indicators of oxidative damages in liver tissue mainly include reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), and total superoxide dismutase (T-SOD). The detection kits of ROS (Lot No. E004), MDA (Lot No. A003-1), ATP (Lot No. A095-1) and T-SOD (Lot No. A001-1) in the liver were brought from Nanjing Jiancheng Bioengineering Institute. Moreover, the procedures were strictly followed the manuals.

Detection of mitochondrial damage

Extraction of mitochondria

The extraction kits of mitochondria were bought from Beyotime Biotechnology (Lot No. C3606). The procedures were followed as: Pieces of livers (about 60 mg) in mice in each group were taken out and washed with PBS, which were then homogenated with 600 μL precooled Reagent A. All procedures were performed on ice. Then, the homogenate was centrifuged at 600 g for 5 min at 4°C. The supernatant was carefully transferred to another centrifuge tube and centrifuged at 11,000 g for 10 min at 4°C. The supernatant was carefully removed, and the resulting precipitate was the isolated mitochondria.

Detection of mitochondrial damage in liver

The indicators of mitochondrial damages in liver tissue mainly include manganese-superoxide dismutase (Mn2+-SOD), sodium-potassium adenosine triphosphatase (Na+-K+-ATPase), and calcium-magnesium adenosine triphosphatase (Ca2+-Mg2+-ATPase). The detection kits of Mn2+-SOD (Lot No. A001-1), Na+-K+-ATPase (Lot No. A016-1) and Ca2+-Mg2+-ATPase (Lot No. A016-1) in the liver were bought from Nanjing Jiancheng Bioengineering Institute. Moreover, the procedures were strictly followed the manuals.

Statistical analysis

Operators and detectors were double-blinded for all quantitative and qualitative processes. All images were analyzed using Image-Pro Plus software (MEDIA CYBERNETICS, USA). Data are expressed as means ± variance. Data comparisons were performed through analysis of variance using SPSS 20.0 statistical software (Chicago,IL,USA), and the Chi-square test was used to assess differences in count data. When comparing the homogeneity of variance in pairs between groups, least significant difference method was used to analyze it. Tamhane's method was used to analyze when the difference was uneven. Pearson correlation coefficient was used to analyze the time and dose correlation between various biochemical indexes, administration time, and dose. The correlation coefficient r > 0 indicated a positive correlation, and r < 0 indicated a negative correlation. Differences were considered statistically significant at P < 0.05.


  Results Top


Effect of W.E.R on liver weight and liver function indexes

After the mice were orally administered with W.E.R, the bodyweight of each group was reduced. Compared with the control group, the body weights of the low and middle dose groups were not statistically significant at 14 days, 21 days and 28 days (P > 0.05). However, there were statistically significant differences in the high-dose groups at each time point (P < 0.05). At 14 days, 21 days, and 28 days, there were significant differences between high-dose and low-dose groups (P < 0.05) [Figure 1].
Figure 1: Body weight in control, low-, middle- and high-dose groups at each time point 14 days, 21 days, and 28 days after administration of W.E.R. The body weight was decreased at each group at each time point. And compared with control group, there were significant differences in high-dose groups at 14 days, 21 days, and 28 days. At 14 days, 21 days, and 28 days, there were significant differences between high-dose and low-dose groups. Values are means ± standard error (*Compared with control group;aAt the same dose, compared with 14 days at the 28 days;bAt the same dose, compared with 21 days at the 28 days;cAt the same dose, compared with 14 days at the 21 days;dAt the same time, compared with low-dose group at the high-dose group;eAt the same time, compared with middle-dose group at the high-dose group;fAt the same time, compared with low-dose group at the middle-dose group; n = 9 per each group)

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After the mice were intragastrically administered with W.E.R, the liver coefficients of the animals in each group were increased. Compared with the control group, the liver coefficient of the low-, medium-, and high-dose groups at 21 days and 28 days, and the middle- and high-dose groups at 14 days was statistically significant (P < 0.05). At 14 days, 21 days, and 28 days, there were significant differences between high-dose and low-dose groups (P < 0.05) [Figure 2].
Figure 2: Liver coefficients in control, low-, middle- and high-dose groups at each time point 14 days, 21 days, and 28 days after administration of W.E.R. The liver coefficient was increased at each group at each time point. And compared with control group, there were significant differences in all groups at each time point, excluding in low-dose group at 14 days. Values are means ± standard error (*Compared with control group;aAt the same dose, compared with 14 days at the 28 days;bAt the same dose, compared with 21 days at the 28 days;cAt the same dose, compared with 14 days at the 21 days;dAt the same time, compared with low-dose group at the high-dose group;eAt the same time, compared with middle-dose group at the high-dose group;fAt the same time, compared with low-dose group at the middle-dose group; n = 9 per each group)

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The enzyme activities of ALP, ALT, and AST in each group were increased at different time points, especially in the high-dose group at 14 days, 21 days, and 28 days. Compared with the control group, the difference was statistically significant (P < 0.05) [Figure 3].
Figure 3: Enzyme activities of biomarkers of liver function such as alkaline phosphatase, alanine aminotransferase, and aspartate transaminase in control, low-, middle- and high-dose groups at each time point 14 days, 21 days and 28 days after administration of W.E.R. The expression levels of alkaline phosphatase, alanine aminotransferase, and aspartate transaminase were increased at each group at each time point. At 14 days, compared with control group, there were significant differences in high-dose groups in the expression levels of alkaline phosphatase, alanine aminotransferase, and aspartate transaminase. At 21 days, compared with control group, there were significant differences in high-dose groups in the expression levels of alkaline phosphatase and aspartate transaminase. There were significant differences in middle- and high-dose groups in the expression level of alanine aminotransferase. At 28 days, compared with control group, there were significant differences in middle- and high-dose groups in the expression level of alkaline phosphatase. There were significant differences in low, middle and high-dose groups in the expression level of alanine aminotransferase. There were significant differences in high-dose group in the expression level of aspartate transaminase. Values are means ± standard error (*Compared with control group;aAt the same dose, compared with 14 days at the 28 days;bAt the same dose, compared with 21 days at the 28 days;cAt the same dose, compared with 14 days at the 21 days;dAt the same time, compared with low-dose group at the high-dose group;eAt the same time, compared with middle-dose group at the high-dose group;fAt the same time, compared with low-dose group at the middle-dose group; n = 9 per each group)

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Finally, we performed the HE staining to ensure the hepatotoxicity of W.E.R in mice at each time group. Then, we investigated that in the control group, the structure of liver tissue and cells was normal after administration of W.E.R. After 14 days, there was no structural abnormality in the hepatocytes at the low-dose group. There were small granulation swellings in the liver tissue at the middle-dose group. At high-dose group, some of the liver cells did not have the normal morphology, accompanied by necrosis and mild fibrosis. After 21 days, at low-dose group, there was lymphocyte hyperplasia in the portal area. At middle-dose group, there were some lymphocyte hyperplasia and neutrophil infiltration. At high-dose group, including the above pathological changes, there were hepatocellular focal swelling, cytoplasmic cavitation, necrosis, and fibrous hyperplasia. After 28 days, at low-, middle-, and high-dose groups, there was more serious damage to the cells and tissues of the liver.

Effect of aqueous extract of W.E.R on oxidative damage of liver tissue in mice

At each time point, there were different degrees of increase in the expression levels of ROS. Compared with the control group, there were significant differences at high-dose group at each time point 14 days, 21 days, and 28 days (P < 0.05). At 21 days and 28 days, there were significant differences between high-dose and low-dose groups. At 28 days, there were significant differences between middle-dose and low-dose groups. In the middle-dose group at 28 days, there were significant differences compared with 14 days [Figure 4]. At each time point, there were different degrees of increase in the expression levels of MDA. Compared with control group, there were significant differences at each group at each time point 14 days, 21 days and 28 days (P < 0.05), excluding the low-dose group at 14 days [Figure 5]. At each time point, there were different degrees of decrease in the expression levels of ATP. Compared with control group, there were significant differences at high-dose group at 14 days, at low-, middle- and high-dose groups at 21 days and 28 days (P < 0.05) [Figure 6]. At each time point, there were different degrees of increase in the expression levels of T-SOD. Compared with control group, there were significant differences at high-dose group at 14 days and 21 days, at all groups at 28 days (P < 0.05) [Figure 7].
Figure 4: The production of reactive oxygen species in control, low, middle and high-dose groups at each time point 14 days, 21 days and 28 days after administration of W.E.R. The expression levels of reactive oxygen species were increased at each group at each time point. Compared with control group, there were significant differences in high-dose groups at 14 days, 21 days and 28 days. Values are means ± standard error (*Compared with control group;aAt the same dose, compared with 14 days at the 28 days;bAt the same dose, compared with 21 days at the 28 days;cAt the same dose, compared with 14 days at the 21 days;dAt the same time, compared with low-dose group at the high-dose group;eAt the same time, compared with middle-dose group at the high-dose group;fAt the same time, compared with low-dose group at the middle-dose group; n = 9 per each group)

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Figure 5: Expression levels of malondialdehyde in control, low, middle and high-dose groups at each time point 14 days, 21 days and 28 days after administration of W.E.R. The expression levels of malondialdehyde were increased at each group at each time point. Compared with control group, there were significant differences in each group at 14 days, 21 days, and 28 days, excluding in low-dose group at 14 days. Values are means ± standard error (*Compared with control group;aAt the same dose, compared with 14 days at the 28 days;bAt the same dose, compared with 21 days at the 28 days;cAt the same dose, compared with 14 days at the 21 days;dAt the same time, compared with low-dose group at the high-dose group;eAt the same time, compared with middle-dose group at the high-dose group;fAt the same time, compared with low-dose group at the middle-dose group; n = 9 per each group)

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Figure 6: Expression levels of adenosine triphosphate in control, low, middle and high-dose groups at each time point 14 days, 21 days and 28 days after administration of W.E.R. The expression levels of adenosine triphosphate were decreased at each group at each time point. Compared with control group, there were significant differences in high-dose group at 14 days. There were significant differences in low-, middle- and high-dose groups at 21 days and 28 days. Values are means ± standard error (*Compared with control group;aAt the same dose, compared with 14 days at the 28 days;bAt the same dose, compared with 21 days at the 28 days;cAt the same dose, compared with 14 days at the 21 days;dAt the same time, compared with low-dose group at the high-dose group;eAt the same time, compared with middle-dose group at the high-dose group;fAt the same time, compared with low-dose group at the middle-dose group; n = 9 per each group)

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Figure 7: Enzyme activities of total superoxide dismutase in control, low, middle and high-dose groups at each time point 14 days, 21 days and 28 days after administration of W.E.R. The enzyme activities of total superoxide dismutase were higher at each group at each time point. Compared with control group, there were significant differences in high-dose groups at 14 days and 21 days. There were significant differences in low, middle and high-dose groups at 28 days. Values are means ± standard error (*Compared with control group;aAt the same dose, compared with 14 days at the 28 days;bAt the same dose, compared with 21 days at the 28 days;cAt the same dose, compared with 14 days at the 21 days;dAt the same time, compared with low-dose group at the high-dose group;eAt the same time, compared with middle-dose group at the high-dose group;fAt the same time, compared with low-dose group at the middle-dose group; n = 9 per each group)

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Effect of W.E.R on mitochondrial function indexes

At each time point, there were different degrees of increase in the enzyme activities of Mn2+-SOD. Compared with control group, there were significant differences at each group at each time point 14 days, 21 days and 28 days (P < 0.05), except the low-dose group at 21 days [Figure 8]. At each time point, there were different degrees of decrease in the enzyme activities of Na+-K+ ATPase. Compared with control group, there were significant differences at each group at each time point 14 days, 21 days and 28 days (P < 0.05) [Figure 9]. At each time point, there were different degrees of decrease in the enzyme activities of Ca2+-Mg2+ ATPase. Compared with control group, there were significant differences at each group at each time point 14 days, 21 days, and 28 days (P < 0.05), except the low-dose group at 14 days [Figure 10].
Figure 8: Enzyme activities of manganese-superoxide dismutase in control, low, middle and high-dose groups at each time point 14 days, 21 days and 28 days after administration of W.E.R. The enzyme activities of manganese-superoxide dismutase were higher at each group at each time point. Compared with control group, there were significant differences in each group at each time point, excluding in low-dose group at 21 days (*Compared with control group;aAt the same dose, compared with 14 days at the 28 days;bAt the same dose, compared with 21 days at the 28 days;cAt the same dose, compared with 14 days at the 21 days;dAt the same time, compared with low-dose group at the high-dose group;eAt the same time, compared with middle-dose group at the high-dose group;fAt the same time, compared with low-dose group at the middle-dose group; n = 9 per each group)

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Figure 9: Enzyme activities of sodium potassium adenosine triphosphatase in control, low , middle and high dose groups at each time point 14 days, 21 days and 28 days after administration of W.E.R. The enzyme activities of sodium potassium adenosine triphosphatase were lower at each group at each time point. Compared with control group, there were significant differences in each group at each time point. Values are means ± standard error (*compared with control group; a At the same dose, compared with 14 days at the 28 days; b At the same dose, compared with 21 days at the 28 days; c At the same dose, compared with 14 days at the 21 day; d At the same time, compared with low dose group at the high dose group; e At the same time, compared with middle dose group at the high dose group; f At the same time, compared with low dose group at the middle dose group; n =9 per each group)

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Figure 10: Enzyme activities of calcium magnesium adenosine triphosphatase in control, low, middle and high dose groups at each time point 14 days, 21 days and 28 days after administration of W.E.R. The enzyme activities of calcium magnesium adenosine triphosphatase were lower at each group at each time point. Compared with control group, there were significant differences in each group at each time point, excluding in Low dose group at 14 days. Values are means ± standard error (*Compared with control group; a At the same dose, compared with 14 days at the 28 days; b At the same dose, compared with 21 days at the 28 days; c At the same dose, compared with 14 days at the 21 days; d At the same time, compared with low dose group at the high dose group; e At the same time, compared with middle dose group at the high dose group; f At the same time, compared with low dose group at the middle dose group; n =9 per each group)

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Correlation between time, dose, and various biochemical indicators

To evaluate the correlation between the various biochemical markers and time, dose, we analyzed the correlation with the Pearson correlation coefficient. The results showed that there were significant negative correlations between body weight, expression level of ATP, activity of Na+-K+-ATPase, activity of Ca2+-Mg2+-ATPase and time, dose (P < 0.05). There were significant positive correlations between liver coefficient, ALP, ALT, AST, expression levels of ROS, MDA and time, dose (P < 0.05) [Table 1].
Table 1: Correlation analysis between biochemical markers and time, dose

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  Discussion Top


In recent years, the irrational use of drugs has led to the emergence of drug-induced liver injury, and its mortality has increased year by year, which is also the main reason for clinical trial failure and drug recall. The liver toxicity of Rhizoma Dioscoreae bulbiferae has greatly limited its clinical application. The diterpene lactones of Rhizoma Dioscoreae bulbiferae are the main substances that play a medicinal value clinically, but they are also the main components of the liver toxicity caused by Rhizoma Dioscoreae bulbiferae.[21] Niu et al. found that ethyl acetate extraction of Rhizoma Dioscoreae bulbiferae can cause severe liver damage in ICR mice.[22] Further studies have shown that the administration of ethyl acetate extraction of can causes a significant increase in the levels of ALT and AST.[20],[23]

In our research, the weight loss of the mice, the increase of the liver coefficient, and the abnormal liver function indexes indicated the hepatotoxicity of Rhizoma Dioscoreae bulbiferae. As the dose was increased and the bodyweight of the mice was decreased significantly, the correlation analysis showed that the bodyweight of the mice was significantly negatively correlated with the administration time and the dose. The liver coefficients were increased with increasing dose and prolonged dosing period. Correlation analysis showed that the liver coefficient of the mice was significantly positively correlated with the time of administration and the dose administered. The activity of ALP, ALT, and AST in serum was increased significantly with the increase of dosage and the prolongation of the drug administration period. Correlation analysis showed that ALP, ALT, and AST levels were significantly positively correlated with administration time and dose. The above results indicated that W.E.R of Rhizoma Dioscoreae bulbiferae could cause significant liver damage in mice. The results of HE staining showed that the liver tissue structure of mice showed obvious pathological changes, such as fibrosis, lymphocytic infiltration, hepatocyte necrosis, etc., and the liver tissue damage was more serious with the increase of dosage and cycle, which further confirmed that liver damage had formed.

In the previous researches, the acetoacetate extraction of Rhizoma Dioscoreae bulbiferae can cause the increase of the expression of 10 cytokines in serum, the decrease of the expression of one cytokine.[18],[22] CD30 L expression was increased and interleukin-3 expression was decreased by ELISA, which may be potential biomarkers of the liver caused by Rhizoma Dioscoreae bulbiferae. In the study of Le Shi, it was found that the total saponin extraction of Rhizoma Dioscoreae bulbiferae could cause L-02 cell damage and cholestasis in rats,[24] while oxidative damages were increased and cytochrome P450 expressions were increased,[19] suggesting that oxidative damage was involved in liver toxicity caused by Rhizoma Dioscoreae bulbiferae.[13],[25]

In the present research, the detection of oxidative damage showed that the ATP synthesis ability was decreased significantly with the increase of the dose of the drug and the prolongation of the drug administration period, which was significantly negatively correlated with the administration time and the dose. It is speculated that Rhizoma Dioscoreae bulbiferae may induce liver damage by inhibiting ATP synthesis and affecting the energy metabolism of liver cells. With the prolonged dose and period of administration, the contents of ROS and MDA in the liver were increased, and it was significantly positively correlated with the time of administration and the dose. All the results indicated that the W.E.R of Rhizoma Dioscoreae bulbiferae can cause hepatotoxicity based on oxidative damage in mice.

Mitochondria are prevalent in cells of animals and plants, which not only could produce ATP and the main parts of aerobic respiration, energy metabolism in the aerobic cells, but also the primary targets of ROS production and attack. Recent researches reported that the occurring and development of many diseases were involved in mitochondrial damage, such as Parkinson disease,[26],[27] Alzheimer's disease,[28],[29] diabetes,[30],[31] tumors,[32],[33] and so on.

Na+-K+-ATPase and Ca2+-Mg2+-ATPase are present in the mitochondrial inner membrane, and Na+-K+-ATPase can maintain the sodium and potassium ion gradients inside and outside the mitochondrial membrane. The role of Ca2+-Mg2+-ATPase is to pump Mg2+ into the mitochondria, providing a substrate for the synthesis of ATP,[34],[35],[36] while pumping Ca2+ out of the mitochondria to maintain Ca2+ stability in the mitochondria. When Ca2+-Mg2+-ATPase activity is inhibited, Ca2+ in the mitochondria cannot be transported outward, which would cause Ca2+ to remain in the mitochondria, causing mitochondrial swelling and mitochondrial structure. The decrease in Mg2+ content in mitochondria could cause inhibition of ATP synthesis. In this study, the W.E.R of Rhizoma Dioscoreae bulbiferae significantly reduced the activity of mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in hepatocytes, which would lead to the disorder of the homeostasis of mitochondrial membrane ions and the retention of sodium water in mitochondria, leading to Ca2+ overload, Mg2+ decreased, which in turn affects the synthesis of ATP. The results demonstrated that the W.E.R of Rhizoma Dioscoreae bulbiferae could cause oxidative damage in liver tissue, inhibit the synthesis of ATP by inhibiting the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase, and further destroy the structure and function of mitochondria, eventually leading to liver toxicity.


  Conclusion Top


In the present research, it was found that the W.E.R of Rhizoma Dioscoreae bulbiferae can induce time- and dose-dependent hepatotoxicity in mice. Further researches suggested Rhizoma Dioscoreae bulbiferae could induce excessive ROS in liver tissue, and lipid peroxidation production MDA, which caused oxidative damage and reduces mitochondrial ATPase activity in vitro and in vivo, inhibiting mitochondrial synthesis of ATP, destroying mitochondrial structure and function, and causing liver damage. Mitochondria are important targets for oxidative damage based-liver toxicity caused by he W.E.R of Rhizoma Dioscoreae bulbiferae.

Acknowledgments

This research was supported by The National Key Research and Development Plan 2019YFC1712403, Major New Drug Discovery Science and Technology Major Projects 2017ZX09201002-006 and the Research Project of Capital Health and Development 2018-4-4231.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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