Chong Zhang1, Lili Li1, Shu Hou1, Zhenghao Shi1, Wenjing Xu1, Qin Wang1, Yinghao He1, Yongfang Gong1, Zhirui Fang1, Yan Yang2. 1. Department of Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Hefei 230032, China. 2. Department of Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Hefei 230032, China. Electronic address: yangyan@ahmu.edu.cn.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus is a medicinal herb used in China for the prevention and treatment of diseases such as diabetes and cancer. As one of the main active ingredients of astragalus, Astragaloside IV (AS-IV) has a wide range of pharmacological effects, including anti-inflammation and anti-cancer effects. AIM OF THE STUDY: Different phosphorylated forms of Smad3 differentially regulate the progression of hepatic carcinoma. The phosphorylation of the COOH-terminal of Smad3 (pSmad3C) and activation of the Nrf2/HO-1 pathway inhibits hepatic carcinoma, while phosphorylation of the linker region of Smad3 (pSmad3L) promotes progression. Thus, pSmad3C/3L and Nrf2/HO-1 pathways are potential targets for drug of anti-cancer development. AS-IV is anti-apoptotic and can inhibit hepatocellular carcinoma cell (HCC) proliferation, invasion, and tumor growth in nude mice. However, it is not clear whether AS-IV has a therapeutic effect on inhibiting the progression of primary liver cancer by regulating the pSmad3C/3L and Nrf2/HO-1 pathway. The purpose of this study is to investigate whether AS-IV inhibits hepatocellular carcinoma by regulating pSmad3C/3L and Nrf2/HO-1 pathway. MATERIALS AND METHODS: primary liver cancer in mice induced by DEN/CCl4/C2H5OH (DCC) and HSC-T6/HepG2 cell models activated by TGF-β1 was investigated for the mechanisms of AS-IV. In vivo assays included liver biopsy, histopathology and post-mortem analysis included immunohistochemistry, immunofluorescent, and Western blotting analysis, and in vitro assays included immunofluorescent, and Western blotting analysis. RESULTS: AS-IV significantly inhibited the development of primary liver cancer, reflecting improved liver biopsy, histopathology. The incidence and multiplicity of primary liver cancer were markedly decreased by AS-IV treatment at the 20th week. AS-IV had observable effects on the TGF-β1/Smad and Nrf2/HO-1 expression in vivo, especially up-regulated pSmad3C, pNrf2, HO-1, and NQO1, while it down-regulated pSmad2C, pSmad2L, pSmad3L, PAI-1, and α-SMA at the 12th week and the 20th week. Furthermore, in vitro analysis further confirmed that AS-IV regulated the expression of pSmad3C/3L and Nrf2/HO-1 pathway in HSC-T6 and HepG2 cells activated by TGF-β1. CONCLUSION: AS-IV administration delays the occurrence of primary liver cancer by continually suppressing the development of fibrosis, the mechanism of the therapeutic effect involving the regulation of the pSmad3C/3L and Nrf2/HO-1 pathways, especially in regulation reversibility and antagonism of pSmad3C and pSmad3L and promoting the phosphorylation of Nrf2.
ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus is a medicinal herb used in China for the prevention and treatment of diseases such as diabetes and cancer. As one of the main active ingredients of astragalus, Astragaloside IV (AS-IV) has a wide range of pharmacological effects, including anti-inflammation and anti-cancer effects. AIM OF THE STUDY: Different phosphorylated forms of Smad3 differentially regulate the progression of hepatic carcinoma. The phosphorylation of the COOH-terminal of Smad3 (pSmad3C) and activation of the Nrf2/HO-1 pathway inhibits hepatic carcinoma, while phosphorylation of the linker region of Smad3 (pSmad3L) promotes progression. Thus, pSmad3C/3L and Nrf2/HO-1 pathways are potential targets for drug of anti-cancer development. AS-IV is anti-apoptotic and can inhibit hepatocellular carcinoma cell (HCC) proliferation, invasion, and tumor growth in nude mice. However, it is not clear whether AS-IV has a therapeutic effect on inhibiting the progression of primary liver cancer by regulating the pSmad3C/3L and Nrf2/HO-1 pathway. The purpose of this study is to investigate whether AS-IV inhibits hepatocellular carcinoma by regulating pSmad3C/3L and Nrf2/HO-1 pathway. MATERIALS AND METHODS: primary liver cancer in mice induced by DEN/CCl4/C2H5OH (DCC) and HSC-T6/HepG2 cell models activated by TGF-β1 was investigated for the mechanisms of AS-IV. In vivo assays included liver biopsy, histopathology and post-mortem analysis included immunohistochemistry, immunofluorescent, and Western blotting analysis, and in vitro assays included immunofluorescent, and Western blotting analysis. RESULTS: AS-IV significantly inhibited the development of primary liver cancer, reflecting improved liver biopsy, histopathology. The incidence and multiplicity of primary liver cancer were markedly decreased by AS-IV treatment at the 20th week. AS-IV had observable effects on the TGF-β1/Smad and Nrf2/HO-1 expression in vivo, especially up-regulated pSmad3C, pNrf2, HO-1, and NQO1, while it down-regulated pSmad2C, pSmad2L, pSmad3L, PAI-1, and α-SMA at the 12th week and the 20th week. Furthermore, in vitro analysis further confirmed that AS-IV regulated the expression of pSmad3C/3L and Nrf2/HO-1 pathway in HSC-T6 and HepG2 cells activated by TGF-β1. CONCLUSION: AS-IV administration delays the occurrence of primary liver cancer by continually suppressing the development of fibrosis, the mechanism of the therapeutic effect involving the regulation of the pSmad3C/3L and Nrf2/HO-1 pathways, especially in regulation reversibility and antagonism of pSmad3C and pSmad3L and promoting the phosphorylation of Nrf2.