Saikosaponins induced hepatotoxicity in mice via lipid metabolism dysregulation and oxidative stress: a proteomic study

Abstract

Background: Radix Bupleuri (RB) has been popularly used for treating many liver diseases such as chronic hepatic inflammation and viral Hepatitis in China. Increasing clinical and experimental evidence indicates the potential hepatotoxicity of RB or prescriptions containing RB. Recently, Saikosaponins (SS) have been identified as major bioactive compounds isolated from RB, which may be also responsible for RB-induced liver injury.

Methods: Serum AST, ALT and LDH levels were determined to evaluate SS-induced liver injury in mice. Serum and liver total triglyceride and cholesterol were used to indicate lipid metabolism homeostasis. Liver ROS, GSH, MDA and iNOS were used to examine the oxidative stress level after SS administration. Western blot was used to detect CYP2E1 expression. A 8-Plex iTRAQ Labeling Coupled with 2D LC - MS/MS technique was applied to analyze the protein expression profiles in livers of mice administered with different doses of SS for different time periods. Gene ontology analysis, cluster and enrichment analysis were employed to elucidate potential mechanism involved. HepG2 cells were used to identify our findings in vitro.

Results: SS dose- and time-dependently induced liver injury in mice, indicated by increased serum AST, ALT and LDH levels. According to proteomic analysis, 487 differentially expressed proteins were identified in mice administrated with different dose of SS for different time periods. Altered proteins were enriched in pathways such as lipid metabolism, protein metabolism, macro molecular transportation, cytoskeleton structure and response to stress. SS enhanced CYP2E1 expression in a time and dose dependent manner, and induced oxidative stress both in vivo and in vitro.

Conclusion: Our results identified hepatotoxicity and established dose-time course-liver toxicity relationship in mice model of SS administration and suggested potential mechanisms, including impaired lipid and protein metabolism and oxidative stress. The current study provides experimental evidence for clinical safe use of RB, and also new insights into understanding the mechanism by which SS and RB induced liver injury.

Fig. 1

HPLC chromatograms. a Standard mix and b Alcohol elution of SS. SSa and SSd were indicated by “A” and “D”, respectively

Fig. 2

SS-induced liver toxicity. a and e liver index, Serum level of b and f AST, c and g ALT and d and h LDH after different time and dose of SS administration. All the data are presented as mean ± S.E. (n = 8), * p < 0.05, **p < 0.01, ***p < 0.001, compared to time 0 h or vehicle control group, respectively

Fig. 3

GO analysis for SS-induced differentiated expressed proteins. GO annotation enriched biological pathways for (a) time-toxicity study and b dose-toxicity study

Fig. 4

Cluster analysis of SS-regulated proteins in time-toxicity study. a Cluster analysis and profiles. Legend (left) indicates fitness to cluster center. Numbers of protein counts in each cluster were plotted with cluster number (NF indicates non-fit proteins). b Enrichment of GO biological pathways to cluster profiles

Fig. 5

Cluster analysis of SS-regulated lipid metabolism related proteins. a The list of SS-regulated proteins involved in lipid metabolism. Legend indicates fold changes (Log). b GO annotation enriched biological pathways based on selected proteins. c Cluster analysis and profiles for selected proteins. d Enrichment of GO biological pathways to cluster profiles

Fig. 6

Effect of SS on lipid contents in serum and liver. a TG and b total cholesterol in serum, and c TG, d total cholesterol level in liver. All results are presented as mean ± S.E. (n = 8), * p < 0.05, **p < 0.01, ***p < 0.001, compared to vehicle control group

Fig. 7

SS-induced oxidative stress in mice liver. Proteomic results for CYP2E1 expression in a time-toxicity study and b dose-toxicity study. * p value < 0.05. Proteins levels of CYP2E1 in c time-toxicity study and d dose-toxicity study were determined by western blot. Representative images of immunoblots are shown and analyzed using GAPDH as loading control. Effects of different dose of SS administration on e ROS, f GSH, g MDA and h iNOS level in liver. All results are presented as mean ± S.E. (n = 8),* p < 0.05, **p < 0.01, ***p < 0.001, compared to vehicle control group

Fig. 8

Effects of SS on HepG2 cells. a Effects of SS on cell viability of HepG2 cells after 24 h treatment. Effects of 12 h treatment of SS on b ROS, c GSH and d iNOS level in HepG2 cells. e Effects of SS on morphology changes of HepG2 cells after 12 h treatment. All results are presented as mean ± S.E. (n = 3), **p < 0.01, ***p < 0.001, compared to vehicle control group