An-Guo Wu1, Jin-Feng Teng2, Vincent Kam-Wai Wong3, Xiao-Gang Zhou4, Wen-Qiao Qiu2, Yong Tang5, Jian-Ming Wu6, Rui Xiong2, Rong Pan2, Yi-Ling Wang2, Bin Tang3, Tian-Yi Ding3, Lu Yu7, Wu Zeng3, Da-Lian Qin8, Betty Yuen-Kwan Law9. 1. State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau SAR, China; School of Pharmacy, Southwest Medical University, Luzhou 646000, China; Key Laboratory of Medical Electrophysiology of Ministry of Education, Sichuan Key Medical Laboratory of New Drug Discovery and Drugability Evaluation, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, Southwest Medical University, Luzhou 646000, China. Electronic address: wuanguo@swmu.edu.cn. 2. School of Pharmacy, Southwest Medical University, Luzhou 646000, China; Key Laboratory of Medical Electrophysiology of Ministry of Education, Sichuan Key Medical Laboratory of New Drug Discovery and Drugability Evaluation, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, Southwest Medical University, Luzhou 646000, China. 3. State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau SAR, China. 4. School of Pharmacy, Southwest Medical University, Luzhou 646000, China. Electronic address: zxg@swmu.edu.cn. 5. School of Pharmacy, Southwest Medical University, Luzhou 646000, China; Key Laboratory of Medical Electrophysiology of Ministry of Education, Sichuan Key Medical Laboratory of New Drug Discovery and Drugability Evaluation, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, Southwest Medical University, Luzhou 646000, China. Electronic address: tangy1989@yeah.net. 6. School of Pharmacy, Southwest Medical University, Luzhou 646000, China; Key Laboratory of Medical Electrophysiology of Ministry of Education, Sichuan Key Medical Laboratory of New Drug Discovery and Drugability Evaluation, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, Southwest Medical University, Luzhou 646000, China. Electronic address: jianmingwu@swmu.edu.cn. 7. State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau SAR, China; School of Pharmacy, Southwest Medical University, Luzhou 646000, China; Key Laboratory of Medical Electrophysiology of Ministry of Education, Sichuan Key Medical Laboratory of New Drug Discovery and Drugability Evaluation, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, Southwest Medical University, Luzhou 646000, China. Electronic address: yulu863@sina.com. 8. School of Pharmacy, Southwest Medical University, Luzhou 646000, China; Key Laboratory of Medical Electrophysiology of Ministry of Education, Sichuan Key Medical Laboratory of New Drug Discovery and Drugability Evaluation, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, Southwest Medical University, Luzhou 646000, China. Electronic address: dalianqin@swmu.edu.cn. 9. State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau SAR, China. Electronic address: yklaw@must.edu.mo.
Abstract
BACKGROUND: Emerging evidences indicate the important roles of autophagy in anti-oxidative stress, which is closely associated with cancer, aging and neurodegeneration. OBJECTIVE: In the current study, we aimed to identify autophagy inducers with potent anti-oxidative effect from traditional Chinese medicines (TCMs) in PC-12 cells and C. elegans. METHODS: The autophagy inducers were extensively screened in our herbal extracts library by using the stable RFP-GFP-LC3 U87 cells. The components with autophagic induction effect in Trillium tschonoskii Maxim. (TTM) was isolated and identified by using the autophagic activity-guided column chromatography and Pre-HPLC technologies, and MS and NMR spectroscopic analysis, respectively. The anti-oxidative effect of the isolated autophagy inducers was evaluated in H2O2-induced PC-12 cells and C. elegans models by measuring the viability of PC-12 cells and C. elegans, with quantitation on the ROS level in vitro and in vivo using H2DCFDA probe. RESULTS: The total ethanol extract of TTM was found to significantly increase the formation of GFP-LC3 puncta in stable RFP-GFP-LC3 U87 cells. One novel steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-Deoxytrillenogenin, (Deoxytrillenoside CA, DTCA) and one known steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-O-acetyl-epitrillenogenin (Epitrillenoside CA, ETCA) were isolated, identified and found to have novel autophagic effect. Both DTCA and ETCA could activate autophagy in PC-12 cells via the AMPK/mTOR/p70S6K signaling pathway in an Atg7-dependent. In addition, DTCA and ETCA could increase the cell viability and decrease the intracellular ROS level in H2O2-treated PC-12 cells and C. elegans, and the further study demonstrated that the induced autophagy contributes to their anti-oxidative effect. CONCLUSION: Our current findings not only provide information on the discovery of novel autophagy activators from TTM, but also confirmed the anti-oxidative effect of the components from TTM both in vitro and in vivo.
BACKGROUND: Emerging evidences indicate the important roles of autophagy in anti-oxidative stress, which is closely associated with cancer, aging and neurodegeneration. OBJECTIVE: In the current study, we aimed to identify autophagy inducers with potent anti-oxidative effect from traditional Chinese medicines (TCMs) in PC-12 cells and C. elegans. METHODS: The autophagy inducers were extensively screened in our herbal extracts library by using the stable RFP-GFP-LC3 U87 cells. The components with autophagic induction effect in Trillium tschonoskii Maxim. (TTM) was isolated and identified by using the autophagic activity-guided column chromatography and Pre-HPLC technologies, and MS and NMR spectroscopic analysis, respectively. The anti-oxidative effect of the isolated autophagy inducers was evaluated in H2O2-induced PC-12 cells and C. elegans models by measuring the viability of PC-12 cells and C. elegans, with quantitation on the ROS level in vitro and in vivo using H2DCFDA probe. RESULTS: The total ethanol extract of TTM was found to significantly increase the formation of GFP-LC3 puncta in stable RFP-GFP-LC3 U87 cells. One novel steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-Deoxytrillenogenin, (Deoxytrillenoside CA, DTCA) and one known steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-O-acetyl-epitrillenogenin (Epitrillenoside CA, ETCA) were isolated, identified and found to have novel autophagic effect. Both DTCA and ETCA could activate autophagy in PC-12 cells via the AMPK/mTOR/p70S6K signaling pathway in an Atg7-dependent. In addition, DTCA and ETCA could increase the cell viability and decrease the intracellular ROS level in H2O2-treated PC-12 cells and C. elegans, and the further study demonstrated that the induced autophagy contributes to their anti-oxidative effect. CONCLUSION: Our current findings not only provide information on the discovery of novel autophagy activators from TTM, but also confirmed the anti-oxidative effect of the components from TTM both in vitro and in vivo.