Podophyllotoxin mediates hepatic toxicity via the C5a/C5aR/ROS/NLRP3 and cGMP/PKG/mTOR axis in rats based on toxicological evidence chain (TEC) concept by phosphoproteomic analysis
Background: Podophyllotoxin (PPT), a highly active compound extracted from the rhizome of Dysosma versipellis (DV), has been used as an effective anti-cancer drug clinically since the 1950s. It possesses various biological activities, including antiviral and antitumor effects. However, its clinical...
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Elsevier
2025-01-01
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| Series: | Ecotoxicology and Environmental Safety |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0147651324015173 |
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| author | Chuanxin Liu Xiaobin Huang Jiao Kong Xuejiao Li Yuming Wang Fangfang Zhang Jiajia Duan |
| author_facet | Chuanxin Liu Xiaobin Huang Jiao Kong Xuejiao Li Yuming Wang Fangfang Zhang Jiajia Duan |
| author_sort | Chuanxin Liu |
| collection | DOAJ |
| description | Background: Podophyllotoxin (PPT), a highly active compound extracted from the rhizome of Dysosma versipellis (DV), has been used as an effective anti-cancer drug clinically since the 1950s. It possesses various biological activities, including antiviral and antitumor effects. However, its clinical application is severely limited due to its hepatotoxicity, and the underlying mechanisms remain unclear. This study aims to elucidate the mechanisms of PPT-induced hepatotoxicity using tandem quality tag (TMT) based quantitative proteomics and phosphoproteomics, providing potential targets and directions for developing new therapeutic strategies to facilitate the safe and rational use of podophyllotoxin in clinical settings. Methods: We employed a comprehensive assessment of PPT-induced hepatotoxicity based on the Toxicology Evidence Chain (TEC) concept, originally proposed by our research group in 2018. This approach involves a tiered search for evidence of Harmful Ingredients Evidence (HIE), Injury Phenotype Evidence (IPE), Adverse Outcomes Evidence (AOE), and Toxic Events Evidence (TEE) during the development of PPT-induced hepatotoxicity, thereby constructing a guiding toxicology evidence chain. Sprague-Dawley (SD) rats were administered 20 mg/kg PPT for 4 consecutive days (HIE). Indicators such as hepatic function, oxidative stress, inflammatory factors, as well as the histopathology of liver tissue were evaluated to assess liver damage and synthetic function (AOE). Proteomics and phosphoproteomics were conducted to systematically assess PPT-induced hepatotoxicity at the level of modified proteins and verify the molecular mechanisms of key molecular pathways (TEE1). Furthermore, in vitro THLE-2 cell models were used in conjunction with CCK8, immunofluorescence, and ELISA assays to validate cytotoxicity and its underlying mechanisms (TEE2). Results: Our results showed that after 4 days of PPT administration at 20 mg/kg (HIE), serum levels of AST/ALT, TBA, TP, and ALB in SD rats were significantly increased (P < 0.05), indicating severe liver damage. SOD and T-AOC levels were significantly decreased (P < 0.05), suggesting an oxidative stress state. TNF-α levels were significantly elevated, while IL-10 and IL-3 levels were significantly reduced (P < 0.05), indicating strong activation of the inflammatory response in the liver. Histopathological examination revealed liver sinusoidal congestion in the liver tissue (AOE). Omics analysis revealed that hepatotoxicity primarily affected the complement-pyroptosis and cGMP-PKG-autophagy pathways. Western blot (WB) and RT-qPCR results showed significant upregulation of complement-pyroptosis pathway proteins (C5a, C5aR, NLRP3) and cGMP-PKG-autophagy pathway proteins (PKG, mTOR) in the PPT group (P < 0.05) (TEE1). In vitro cell experiments showed that PPT significantly reduced cell viability (P < 0.05) and increased the expression of proteins associated with pyroptosis and autophagy pathways, including ROS, NLRP3, PKG, and mTOR (P < 0.05) (TEE2). Conclusion: PPT activates the complement system through the C5a/C5aR/ROS/NLRP3 pathway and induces the formation of inflammasomes, promoting pyroptosis. Simultaneously, PPT activates the cGMP-PKG pathway, inhibiting autophagy and further accelerating pyroptosis, ultimately leading to hepatotoxicity. In conclusion, this study comprehensively revealed the underlying mechanisms of PPT-induced hepatotoxicity using the TEC concept. This approach transforms fragmented toxicity indicators into systematic evidence of toxicity, presenting a hierarchical progression of toxicity evidence and avoiding data accumulation in natural drug toxicology. Our findings represent a significant breakthrough in the elucidation of the mechanisms of hepatotoxicity induced by podophyllotoxin. |
| format | Article |
| id | doaj-art-af3c015e20894ef19d60c7ec1e609513 |
| institution | Kabale University |
| issn | 0147-6513 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Ecotoxicology and Environmental Safety |
| spelling | doaj-art-af3c015e20894ef19d60c7ec1e6095132025-01-23T05:25:33ZengElsevierEcotoxicology and Environmental Safety0147-65132025-01-01289117441Podophyllotoxin mediates hepatic toxicity via the C5a/C5aR/ROS/NLRP3 and cGMP/PKG/mTOR axis in rats based on toxicological evidence chain (TEC) concept by phosphoproteomic analysisChuanxin Liu0Xiaobin Huang1Jiao Kong2Xuejiao Li3Yuming Wang4Fangfang Zhang5Jiajia Duan6Luoyang Key Laboratory of Clinical Multiomics and Translational Medicine, Key Laboratory of Hereditary Rare Diseases of Health Commission of Henan Province, Henan Key Laboratory of Rare Diseases, Endocrinology and Metabolism Center, The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology, Luoyang 471003, ChinaSchool of Chinese Materia Medica, Beijing University of Chinese Medicine, Fangshan District, Beijing 102488, ChinaInstitute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, ChinaLuoyang Key Laboratory of Clinical Multiomics and Translational Medicine, Key Laboratory of Hereditary Rare Diseases of Health Commission of Henan Province, Henan Key Laboratory of Rare Diseases, Endocrinology and Metabolism Center, The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology, Luoyang 471003, ChinaSchool of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, ChinaSchool of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, ChinaDepartment of Clinical Laboratory, The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology, Luoyang, China; Corresponding author.Background: Podophyllotoxin (PPT), a highly active compound extracted from the rhizome of Dysosma versipellis (DV), has been used as an effective anti-cancer drug clinically since the 1950s. It possesses various biological activities, including antiviral and antitumor effects. However, its clinical application is severely limited due to its hepatotoxicity, and the underlying mechanisms remain unclear. This study aims to elucidate the mechanisms of PPT-induced hepatotoxicity using tandem quality tag (TMT) based quantitative proteomics and phosphoproteomics, providing potential targets and directions for developing new therapeutic strategies to facilitate the safe and rational use of podophyllotoxin in clinical settings. Methods: We employed a comprehensive assessment of PPT-induced hepatotoxicity based on the Toxicology Evidence Chain (TEC) concept, originally proposed by our research group in 2018. This approach involves a tiered search for evidence of Harmful Ingredients Evidence (HIE), Injury Phenotype Evidence (IPE), Adverse Outcomes Evidence (AOE), and Toxic Events Evidence (TEE) during the development of PPT-induced hepatotoxicity, thereby constructing a guiding toxicology evidence chain. Sprague-Dawley (SD) rats were administered 20 mg/kg PPT for 4 consecutive days (HIE). Indicators such as hepatic function, oxidative stress, inflammatory factors, as well as the histopathology of liver tissue were evaluated to assess liver damage and synthetic function (AOE). Proteomics and phosphoproteomics were conducted to systematically assess PPT-induced hepatotoxicity at the level of modified proteins and verify the molecular mechanisms of key molecular pathways (TEE1). Furthermore, in vitro THLE-2 cell models were used in conjunction with CCK8, immunofluorescence, and ELISA assays to validate cytotoxicity and its underlying mechanisms (TEE2). Results: Our results showed that after 4 days of PPT administration at 20 mg/kg (HIE), serum levels of AST/ALT, TBA, TP, and ALB in SD rats were significantly increased (P < 0.05), indicating severe liver damage. SOD and T-AOC levels were significantly decreased (P < 0.05), suggesting an oxidative stress state. TNF-α levels were significantly elevated, while IL-10 and IL-3 levels were significantly reduced (P < 0.05), indicating strong activation of the inflammatory response in the liver. Histopathological examination revealed liver sinusoidal congestion in the liver tissue (AOE). Omics analysis revealed that hepatotoxicity primarily affected the complement-pyroptosis and cGMP-PKG-autophagy pathways. Western blot (WB) and RT-qPCR results showed significant upregulation of complement-pyroptosis pathway proteins (C5a, C5aR, NLRP3) and cGMP-PKG-autophagy pathway proteins (PKG, mTOR) in the PPT group (P < 0.05) (TEE1). In vitro cell experiments showed that PPT significantly reduced cell viability (P < 0.05) and increased the expression of proteins associated with pyroptosis and autophagy pathways, including ROS, NLRP3, PKG, and mTOR (P < 0.05) (TEE2). Conclusion: PPT activates the complement system through the C5a/C5aR/ROS/NLRP3 pathway and induces the formation of inflammasomes, promoting pyroptosis. Simultaneously, PPT activates the cGMP-PKG pathway, inhibiting autophagy and further accelerating pyroptosis, ultimately leading to hepatotoxicity. In conclusion, this study comprehensively revealed the underlying mechanisms of PPT-induced hepatotoxicity using the TEC concept. This approach transforms fragmented toxicity indicators into systematic evidence of toxicity, presenting a hierarchical progression of toxicity evidence and avoiding data accumulation in natural drug toxicology. Our findings represent a significant breakthrough in the elucidation of the mechanisms of hepatotoxicity induced by podophyllotoxin.http://www.sciencedirect.com/science/article/pii/S0147651324015173PodophyllotoxinHepatotoxicityPhosphorylated proteomicsC5a/C5aR/ROS/NLRP3 axisCGMP/PKG/mTOR/NLRP3 axisToxicological evidence chain (TEC) concept |
| spellingShingle | Chuanxin Liu Xiaobin Huang Jiao Kong Xuejiao Li Yuming Wang Fangfang Zhang Jiajia Duan Podophyllotoxin mediates hepatic toxicity via the C5a/C5aR/ROS/NLRP3 and cGMP/PKG/mTOR axis in rats based on toxicological evidence chain (TEC) concept by phosphoproteomic analysis Ecotoxicology and Environmental Safety Podophyllotoxin Hepatotoxicity Phosphorylated proteomics C5a/C5aR/ROS/NLRP3 axis CGMP/PKG/mTOR/NLRP3 axis Toxicological evidence chain (TEC) concept |
| title | Podophyllotoxin mediates hepatic toxicity via the C5a/C5aR/ROS/NLRP3 and cGMP/PKG/mTOR axis in rats based on toxicological evidence chain (TEC) concept by phosphoproteomic analysis |
| title_full | Podophyllotoxin mediates hepatic toxicity via the C5a/C5aR/ROS/NLRP3 and cGMP/PKG/mTOR axis in rats based on toxicological evidence chain (TEC) concept by phosphoproteomic analysis |
| title_fullStr | Podophyllotoxin mediates hepatic toxicity via the C5a/C5aR/ROS/NLRP3 and cGMP/PKG/mTOR axis in rats based on toxicological evidence chain (TEC) concept by phosphoproteomic analysis |
| title_full_unstemmed | Podophyllotoxin mediates hepatic toxicity via the C5a/C5aR/ROS/NLRP3 and cGMP/PKG/mTOR axis in rats based on toxicological evidence chain (TEC) concept by phosphoproteomic analysis |
| title_short | Podophyllotoxin mediates hepatic toxicity via the C5a/C5aR/ROS/NLRP3 and cGMP/PKG/mTOR axis in rats based on toxicological evidence chain (TEC) concept by phosphoproteomic analysis |
| title_sort | podophyllotoxin mediates hepatic toxicity via the c5a c5ar ros nlrp3 and cgmp pkg mtor axis in rats based on toxicological evidence chain tec concept by phosphoproteomic analysis |
| topic | Podophyllotoxin Hepatotoxicity Phosphorylated proteomics C5a/C5aR/ROS/NLRP3 axis CGMP/PKG/mTOR/NLRP3 axis Toxicological evidence chain (TEC) concept |
| url | http://www.sciencedirect.com/science/article/pii/S0147651324015173 |
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