INTRODUCTION

Despite the recent successes of various new drugs for liver diseases, development of new drugs for liver diseases is still important because the morbidity of chronic liver diseases (CLDs) is growing worldwide.^1,2 Silymarin has an anti-fibrotic effect through the inhibition of the myofibroblasts activities. It prevents the transformation of HSCs into a myofibroblast-like phenotype and blocks the generation of extracellular matrix (ECM), which induces hepatic fibrosis.^3,4 However, no studies have evaluated the effects of silymarin on liver function with follow-up MRIs. In present study, we investigated the protective effects of silymarin in a TAA-induced animal model longitudinally with MRI. Additionally, we analyzed the correlations between pathological markers and multi-parametric MRI data, and assessed the interpretability of traditional markers and MRI values regarding disease severity.

MATERIALS & METHODS

Six-week-old male Sprague-Dawley rats were used and randomly divided into two groups: the control group (n=9), TAA-injected (n=13; TAA) and TAA-injected and silymarin-treated groups (n=11; TAA-SY). To induce hepatic fibrosis, 200-mg/kg TAA was intraperitoneally injected thrice a week for 8 weeks. After 4 weeks, an intraperitoneal injection of 100-mg/kg silymarin was administered three times a week for 4 weeks. Blood samples were collected from the tail vein every 2 weeks. Serum levels of AST, ALT, TBIL, and ALB were measured. The liver tissue samples were stained with H&E, MT, and iron stains. MRI was performed every 2 weeks using a 9.4-T magnet. MRI parameters were as follows: (1) T₁-weighted dynamic contrast-enhanced (DCE)-MRI with TR/TE=70/2.47 ms, flip angle=35°, averages=1, matrix size=96×96, field-of-view (FOV)=50×50 mm², total scan time=44 min 48 s, and the number of images=400. Further, 25-μmol/kg Gd-EOB-DTPA was intravenously injected after 120 s. The relative enhancement rate (RER) was calculated using the equation RER(t)=[{SI(t)−SI(0)}/SI(0)]×100 (%).⁵ Herein, SI(t) is the signal intensity of the liver at time t, and SI(0) is the average of SI before Gd-EOB-DTPA injection. The area under the curve to maximum time (AUC_tmax) was calculated by the integration of areas from 0 to 6 min. (2) T₂* map with TR/TE=800/2.56 ms, echo=6, flip angle=30°, averages=4, matrix size 128×128, FOV=50×50 mm². Statistical analysis was performed using SPSS 21.0. A one-way ANOVA and independent t-tests was performed to compare the mean between different values, and p<0.05 was considered statistically significant (*p<0.05, **p<0.01, ***p<0.001).

RESULTS AND DISCUSSION

In the TAA group, AST, ALT, and TBIL were gradually increased. In the TAA-SY group, AST, ALT, and TBIL were also seemed to be increased until the fourth week, yet their levels went down with the initiation of the treatment of silymarin (Fig.1A). The NAS scores for lobular inflammation (0.00±0.00 vs. 2.29±0.83 vs. 1.55±0.69) and the Brunt scores for fibrosis degrees (0.22±0.44 vs. 3.29±0.73 vs. 2.36±1.03) were measured; additionally, the differences in the three groups were analyzed. In both scores, the TAA group showed the highest scores compared to the other two groups (Fig.1B). We acquired T1-weighted images with MRI (Fig.2A). In comparison analyses of RER, the TAA group showed lower RER than the control group from the 2nd weeks while the TAA-SY group showed recovery from the 6th week (Fig.2B). The AUC_tmax levels of the TAA group showed a significant decrease from the 4th week and the onward weeks compared to the control. In the TAA-SY group, a significant decrease of the AUC_tmax levels compared to the control group was observed at the 4th week. As the levels of AUC_tmax recovered from the 6^th week, significant differences between the TAA and TAA-SY groups were observed; however, the levels of AUC_tmax did not recover to the levels of the control group at the 8^th week (Fig.2C). In the correlation analyses, we observed negative correlations between AUC_tmax and each of AST, ALT, and TBIL (Fig.2D). We also analyzed T₂* mapping MR images for a multi-parametric analysis (Fig.3A). Similar patterns were observed in the T₂* values compared with T1-weighted images. Between the TAA group and TAA-SY group, no significant differences were observed in the T₂* values; however, the T₂* values of the TAA-SY group became higher from the 6^th week and eventually, it became significantly higher at the 8^th week (Fig.3B). Negative correlations between the T₂* and each of AST, ALT, and TBIL were observed and showed that the correlations were significant (Fig.3C). We further analyzed the iron accumulation in the liver to elucidate the correlative relations between the amounts of iron and T₂* shortening. We observed the iron overload near the portal triad of fibrosis in the liver of both TAA and TAA-SY groups (Fig.4A). We observed significant iron accumulation in both the TAA-treated groups compared to the control group, however, the iron amounts of the TAA-SY group were significantly lower than the TAA group (Fig.4B). The Pearson correlation between the T₂* values and iron accumulated areas (%) in the control, TAA group, and the TAA-SY group at 8 weeks was investigated and the hepatic iron amounts showed strong negative correlations with the T₂* values (Fig.4C).

CONCLUSION

Our results suggest that silymarin treatment has hepatoprotective effects on TAA-induced liver injury and showed the usefulness of multi-parametric MRI as an efficacy evaluation tool for the liver drug development in a preclinical phase.

Acknowledgements

This study was supported by grants of Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education [2018R1A2B2007694].