BACKGROUND/OBJECTIVE: Orostachys japonicus A. Berger (Crassulaceae) has been used in traditional herbal medicines in Korea and other Asian countries to treat various diseases, including liver disorders. In the present study, the anti-fibrotic effects of O. japonicus extract (OJE) in cellular and experimental hepatofibrotic rat models were investigated. MATERIALS/METHODS: An in vitro hepatic stellate cells (HSCs) system was used to estimate cell viability, cell cycle and apoptosis by MTT assay, flow cytometry, and Annexin V-FITC/PI staining techniques, respectively. In addition, thioacetamide (TAA)-induced liver fibrosis was established in Sprague Dawley rats. Briefly, animals were divided into five groups (n = 8): Control, TAA, OJE 10 (TAA with OJE 10 mg/kg), OJE 100 (TAA with OJE 100 mg/kg) and silymarin (TAA with Silymarin 50 mg/kg). Fibrosis was induced by treatment with TAA (200 mg/kg, i.p.) twice per week for 13 weeks, while OJE and silymarin were administered orally two times per week from week 7 to 13. The fibrotic related gene expression serum biomarkers glutathione and hydroxyproline were estimated by RT-PCR and spectrophotometry, respectively, using commercial kits. RESULTS: OJE (0.5 and 0.1 mg/mL) and silymarin (0.05 mg/mL) treatment significantly (P < 0.01 and P < 0.001) induced apoptosis (16.95% and 27.48% for OJE and 25.87% for silymarin, respectively) in HSC-T6 cells when compared with the control group (9.09%). Further, rat primary HSCs showed changes in morphology in response to OJE 0.1 mg/mL treatment. In in vivo studies, OJE (10 and 100 mg/kg) treatment significantly ameliorated TAA-induced alterations in levels of serum biomarkers, fibrotic related gene expression, glutathione, and hydroxyproline (P < 0.05-P < 0.001) and rescued the histopathological changes. CONCLUSIONS: OJE can be developed as a potential agent for the treatment of hepatofibrosis.
Animals ; Apoptosis ; Asian Continental Ancestry Group ; Biomarkers ; Cell Cycle ; Cell Survival ; Fibrosis* ; Flow Cytometry ; Gene Expression ; Glutathione ; Hepatic Stellate Cells* ; Humans ; Hydroxyproline ; In Vitro Techniques ; Korea ; Liver ; Liver Cirrhosis ; Models, Animal ; Rats* ; Rats, Sprague-Dawley ; Silymarin ; Spectrophotometry ; Thioacetamide

Objective: To investigate the antifibrotic effects of Chrysanthemum indicum ethanol extract (CIEE) against activated hepatic stellate cells (HSC) and thioacetamide (TAA)-induced hepatofibrosis in rats. Methods: Cell viability and proliferation of HSC-T6 cells were measured using MTT assay. Primary HSCs were used to study morphology. TAA (200 mg/kg) was used to induced hepatic fibrosis in rats. CIEE (100 and 500 mg/kg) and silymarin (50 mg/kg) were administered orally. Liver functions including alanine transaminase, aspartate transaminase, glutathione, and hydroxyproline levels were measured using commercial kits. Liver sections and fibrotic biomarker expression were measured using hematoxylin and eosin staining and real-Time polymerase chain reaction. Results: In vitro study revealed that CIEE (0.1, 0.25, and 0.5 mg/mL) inhibited the proliferation of activated HSCs exposed to transforming growth factor (TGF)-β and restored the activated primary HSC morphology. In in vivo studies, TAA-induced increase in liver/body weight ratio (5.46 ± 0.26) was significantly reduced (4.13 ± 0.22) by CIEE (P<0.05 at 500 mg/kg). CIEE (100 and 500 mg/kg) improved the liver functions by significantly attenuating changes in alanine transaminase, aspartate transaminase, glutathione, and hydroxyproline levels (P<0.05). Further, CIEE (100 and 500 mg/kg) ameliorated the histological changes in liver tissue and TGF-β expression significantly (P<0.05) in TAA-induced rats. Conclusions: CIEE significantly protects against TAA-induced liver damage in rats and can be used in the treatment of liver fibrosis.