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Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters.

Wu ZT, Qi XM, Sheng JJ, Ma LL, Ni X, Ren J, Huang CG, Pan GY - Acta Pharmacol. Sin. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

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.

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Related in: MedlinePlus

The mRNA and protein levels of genes involved in BA transport and synthesis measured by RT-PCR and Western blot analysis after TA3 (1, 5, and 10 μmol/L) treatment for 24 h. The expression levels of each gene were expressed as values normalized to β-actin expression. (A) Ntcp mRNA expression; (B) Bsep mRNA expression; (C) Mrp2 mRNA expression; (D) Cyp7a1 mRNA expression; (E) Expression of protein levels of Bsep and Cyp7a1. The data are presented as the mean±SD (n=3). bP<0.05, cP<0.01 vs vehicle.
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fig4: The mRNA and protein levels of genes involved in BA transport and synthesis measured by RT-PCR and Western blot analysis after TA3 (1, 5, and 10 μmol/L) treatment for 24 h. The expression levels of each gene were expressed as values normalized to β-actin expression. (A) Ntcp mRNA expression; (B) Bsep mRNA expression; (C) Mrp2 mRNA expression; (D) Cyp7a1 mRNA expression; (E) Expression of protein levels of Bsep and Cyp7a1. The data are presented as the mean±SD (n=3). bP<0.05, cP<0.01 vs vehicle.

Mentions: TA3 significantly decreased the mRNA levels of the influx transporter Ntcp and efflux transporters Bsep and Mrp2. The BA-synthesizing enzyme Cyp7a1 was also downregulated by TA3 (Figure 4A–4D). Western blot analysis showed that the expression levels of Bsep and Cyp7a1 decreased (Figure 4E).


Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters.

Wu ZT, Qi XM, Sheng JJ, Ma LL, Ni X, Ren J, Huang CG, Pan GY - Acta Pharmacol. Sin. (2014)

The mRNA and protein levels of genes involved in BA transport and synthesis measured by RT-PCR and Western blot analysis after TA3 (1, 5, and 10 μmol/L) treatment for 24 h. The expression levels of each gene were expressed as values normalized to β-actin expression. (A) Ntcp mRNA expression; (B) Bsep mRNA expression; (C) Mrp2 mRNA expression; (D) Cyp7a1 mRNA expression; (E) Expression of protein levels of Bsep and Cyp7a1. The data are presented as the mean±SD (n=3). bP<0.05, cP<0.01 vs vehicle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4155534&req=5

fig4: The mRNA and protein levels of genes involved in BA transport and synthesis measured by RT-PCR and Western blot analysis after TA3 (1, 5, and 10 μmol/L) treatment for 24 h. The expression levels of each gene were expressed as values normalized to β-actin expression. (A) Ntcp mRNA expression; (B) Bsep mRNA expression; (C) Mrp2 mRNA expression; (D) Cyp7a1 mRNA expression; (E) Expression of protein levels of Bsep and Cyp7a1. The data are presented as the mean±SD (n=3). bP<0.05, cP<0.01 vs vehicle.
Mentions: TA3 significantly decreased the mRNA levels of the influx transporter Ntcp and efflux transporters Bsep and Mrp2. The BA-synthesizing enzyme Cyp7a1 was also downregulated by TA3 (Figure 4A–4D). Western blot analysis showed that the expression levels of Bsep and Cyp7a1 decreased (Figure 4E).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus