Studies of Herbal Pure Compounds Tanshinone IIA and Osthole against Hepatic Fibrosis in Rats and Hepatic Stellate Cell Activation

• Main Authors: Ya-Wei Liu, 劉亞薇
• Other Authors: Yi-Tsau Huang
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/84044215206360952234

博士 === 國立陽明大學 === 傳統醫藥研究所 === 103 === Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in many patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Preventing activation and inflammation of hepatic stellate cells (HSCs) is the major strategy to treat hepatic fibrosis. Clinical therapies for hepatic diseases remain unsatisfactory. In the present studies, two approaches were used to investigate the potentials of Traditional Chinese Medicines (TCM) in treating hepatic fibrosis and HSC activation. Previously, our group has taken years to study Salvia miltiorrhiza Bae. (S. miltiorrhiza) which is a common herb used in the prescriptions of treating hepatic disease. It was found that Sal B exerts inhibitory ability in HSC activation and hepatic injury. I attempted to explore whether the Tanshinone IIA (Tan IIA, C19H18O3, MW = 294.34), one of lipophilic diterpene, in S. miltiorrhiza could inhibit HSC activation. Another approach in our lab was that we screened hundreds pure compounds isolated from herbs, we discovered that osthole, an active component contained in the fruit of Cnidium monnieri (L.) Cusson (C.monnieri (L.) Cusson), could inhibit HSC activation effectively in preliminary screening. Therefore, I further conducted both in vitro and in vivo studies to investigate the therapeutic effects of osthole on rat liver fibrosis and HSC activation. We used lipopolysaccharide (LPS) (100 ng/ml) to stimulate rat hepatic stellate cells (HSC-T6). Tan IIA (10 uM) was used to inhibit LPS-induced HSC activation and compared with Sal B (200 uM). All concentrations of Tan IIA (1 to 10 uM) and Sal B (200 uM) showed no cytotoxicity against HSC-T6 cells. LPS stimulated NF-κB luciferase activities, nuclear translocation of NF-κB-p65, and phosphorylations of ERK, JNK and p38, were suppressed by Tan IIA. In addition, Tan IIA significantly inhibited LPS-induced HSCs chemotaxis, in both wound-healing and trans-well invasion assays. Moreover, Tan IIA attenuated LPS-induced mRNA expressions of CCL2, CCL3, CCL5. Furthermore, Tan IIA also attenuated LPS-induced fibrosis-related mRNA expressions, including IL-1, TNF-, iNOS, ICAM-1, and IL-6. In addition, the protein and gene expression of a-SMA were both downregulated by Tan IIA in HSC-T6 cells. For the investigation of osthole (C15H16O3, MW = 244.29), I established the thioacetamide (TAA)-model of Sprague-Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and a-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects on inflammation-related genes and chemokine production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole (10 ug/ml) attenuated TGF-b1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-a-triggered NF-κB activities significantly. Besides, osthole attenuated TGF-b1- or ET-1-induced HSC contractility. Our results demonstrated that Tan IIA decreased LPS-induced HSC activation. In addition, osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats, and significantly suppressed HSCs activation in vitro. According to the study results, Tan IIA from S. miltiorrhiza could inhibit HSC activation, and osthole from C.monnieri Cusson could improve rat liver injury and also attenuate HSC activation. This thesis provides the evidences for using S. miltiorrhiza and C.monnieri (L.) Cusson in the clinical and the academic of TCM.