| Summary: | 碩士 === 國立陽明大學 === 傳統醫藥研究所 === 99 === Activation of hepatic stellate cells (HSCs) plays a major role in liver fibrosis. Angiotensin II (Ang II), a peptide hormone in the renin-angiotensin system, has been implicated in the pathogenesis of hepatic fibrosis. Ang II activates HSCs including cell proliferation, cell migration, reactive oxygen species (ROS) formation, TGF-β synthesis and production of collagen I., The anti-hepato-fibrotic potential of Artemisia capillaris, a Chinese traditional medicine prescribed for jaundice, is yet to be explored. In this study, we investigated if extracts of Artemisia capillaris can inhibit the activation of HSCs by Ang II.
Hepatic stellate cell T6 and LX-2 lines are immortalized rat and human cells, respectively. Cells were pretreated with Artemisia capillaris extracts 2 hours, and thereafter stimulated with 0.01 to 0.1 μM Ang II. MTT assay was performed to test drug toxicity. Chemotaxis of Ang II was evaluated by wound healing assay. Intracellular ROS formation was detected by CM-H2DCFDA dye. Collagen gel contraction assay was performed to evaluate the contractility of HSCs. MAPKs phosphorylation was detected using Western blotting.
Our results showed that 10 and 50-μg/mL ethanolic Artemisia capillaris extracts inhibited Ang II-induced LX-2 and HSC-T6 cell migration, respectively. Furthermore, 25-μg/mL butanol, ethyl acetate, and H2O fractions also inhibited Ang II-induced LX-2 cell migration, but only 35-μg/mL ethyl acetate fraction abolished cell migration in HSC-T6. Further, we used 50% ethyl acetate/hexane and ethyl acetate to wash ethyl acetate fraction and these two subfractions are named EA subfractional 50% E/H and EA subfractional EA, respectively. Both 5-μg/mL EA subfractional 50% E/H and 25-μg/mL EA subfractional EA inhibited Ang II-induced LX-2 cell migration. Moreover, both 5-μg/mL EA subfractional 50% E/H and 6.25-μg/mL EA subfractional EA decreased Ang II-stimulated ERK phosphorylation in LX-2 cells significantly. In addition, ERK phosphorylation inhibitor, PD98059 could prevent Ang II-induced cell migration and ERK activation. EA subfractional EA did not inhibit Ang II-stimulated JNK phosphorylation, but EA subfractional 50% E/H could inhibit JNK activation in LX-2 cells. Addionally, EA subfractional EA decreased Ang II-induced procollagen α1 (I) mRNA expression. But EA subfractional EA could not effectively inhibit intracellular ROS formation. Ang II could not significantly cause cell contraction.
Our study indicated that Artemisia capillaris extracts evidently abolished angiotensin II-induced hepatic stellate cell migration and ERK activation.
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