Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters
Abstract
Zhi-tao WU1, Xin-ming QI1, Jing-jing SHENG3, Lei-lei MA1, Xuan NI1, Jin REN1, Cheng-gang HUANG2, *, Guo-yu PAN1, *
1Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 3Nanjing Normal University, Nanjing 210000, China
Aim: To investigate the mechanisms underlying the hepatotoxicity of timosaponin A3 (TA3), a steroidal saponin from Anemarrhena asphodeloides, in rats.
Methods: Male SD rats were administered TA3 (100 mg·kg-1·d-1, po) for 14 d, and the blood and bile samples were collected after the final administration. The viability of a sandwich configuration of cultured rat hepatocytes (SCRHs) was assessed using WST-1. Accumulation and biliary excretion index (BEI) of d8-TCA in SCRHs were determined with LC-MS/MS. RT-PCR and Western blot were used to analyze the expression of relevant genes and proteins. ROS and ATP levels, and mitochondrial membrane potential (MMP) were measured. F-actin cytoskeletal integrity was assessed under confocal microscopy.
Results: TA3 administration in rats significantly elevated the total bile acid in serum, and decreased bile acid (BA) component concentrations in bile. TA3 inhibited the viability of the SCRHs with an IC50 value of 15.21±1.73 μmol/L. Treatment of the SCRHs with TA3 (1–10 μmol/L) for 2 and 24 h dose-dependently decreased the accumulation and BEI of d8-TCA. The TA3 treatment dose-dependently decreased the expression of BA transporters Ntcp, Bsep and Mrp2, and BA biosynthesis related Cyp7a1 in hepatocytes. Furthermore, the TA3 treatment dose-dependently increased ROS generation and HO-1 expression, decreased the ATP level and MMP, and disrupted F-actin in the SCRHs. NAC (5 mmol/L) significantly ameliorated TA3-induced effects in the SCRHs, whereas mangiferin (10–200 μg/mL) almost blocked TA3-induced ROS generation.
Conclusion: TA3 triggers liver injury through inducing ROS generation and suppressing the expression of BA transporters. Mangiferin, an active component in Anemarrhena, may protect hepatocytes from TA3-induced hepatotoxicity.
Keywords: timosaponin A3; hepatotoxicity; cholestasis; bile acid; transporter; Cyp7a1; ROS; N-acetyl-L-cysteine; mangiferin
This work was supported by the National Natural Science Foundation of China (Grant No 81302836, 81030065, and 81274055), the Hundred Talents Program of the Chinese Academy of Sciences, Major National Science and Technology Programs (Grant Nos 2012ZX09301001-006 and 2012ZX09302003), and the National High Technology Research and Development Program of China (Grant No 2013AA032202).
* To whom correspondence should be addressed.
E-mail cghuang@mail.shcnc.ac.cn (Cheng-gang HUANG); gypan@cdser.simm.ac.cn (Guo-yu PAN)
Received 2014-03-18 Accepted 2014-05-20
Keywords:
1Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 3Nanjing Normal University, Nanjing 210000, China
Aim: To investigate the mechanisms underlying the hepatotoxicity of timosaponin A3 (TA3), a steroidal saponin from Anemarrhena asphodeloides, in rats.
Methods: Male SD rats were administered TA3 (100 mg·kg-1·d-1, po) for 14 d, and the blood and bile samples were collected after the final administration. The viability of a sandwich configuration of cultured rat hepatocytes (SCRHs) was assessed using WST-1. Accumulation and biliary excretion index (BEI) of d8-TCA in SCRHs were determined with LC-MS/MS. RT-PCR and Western blot were used to analyze the expression of relevant genes and proteins. ROS and ATP levels, and mitochondrial membrane potential (MMP) were measured. F-actin cytoskeletal integrity was assessed under confocal microscopy.
Results: TA3 administration in rats significantly elevated the total bile acid in serum, and decreased bile acid (BA) component concentrations in bile. TA3 inhibited the viability of the SCRHs with an IC50 value of 15.21±1.73 μmol/L. Treatment of the SCRHs with TA3 (1–10 μmol/L) for 2 and 24 h dose-dependently decreased the accumulation and BEI of d8-TCA. The TA3 treatment dose-dependently decreased the expression of BA transporters Ntcp, Bsep and Mrp2, and BA biosynthesis related Cyp7a1 in hepatocytes. Furthermore, the TA3 treatment dose-dependently increased ROS generation and HO-1 expression, decreased the ATP level and MMP, and disrupted F-actin in the SCRHs. NAC (5 mmol/L) significantly ameliorated TA3-induced effects in the SCRHs, whereas mangiferin (10–200 μg/mL) almost blocked TA3-induced ROS generation.
Conclusion: TA3 triggers liver injury through inducing ROS generation and suppressing the expression of BA transporters. Mangiferin, an active component in Anemarrhena, may protect hepatocytes from TA3-induced hepatotoxicity.
Keywords: timosaponin A3; hepatotoxicity; cholestasis; bile acid; transporter; Cyp7a1; ROS; N-acetyl-L-cysteine; mangiferin
This work was supported by the National Natural Science Foundation of China (Grant No 81302836, 81030065, and 81274055), the Hundred Talents Program of the Chinese Academy of Sciences, Major National Science and Technology Programs (Grant Nos 2012ZX09301001-006 and 2012ZX09302003), and the National High Technology Research and Development Program of China (Grant No 2013AA032202).
* To whom correspondence should be addressed.
E-mail cghuang@mail.shcnc.ac.cn (Cheng-gang HUANG); gypan@cdser.simm.ac.cn (Guo-yu PAN)
Received 2014-03-18 Accepted 2014-05-20