Nutr Res Pract.  2017 Dec;11(6):470-478. 10.4162/nrp.2017.11.6.470.

Anti-fibrotic effects of Orostachys japonicus A. Berger (Crassulaceae) on hepatic stellate cells and thioacetamide-induced fibrosis in rats

• Koppula S
• Yum MJ
• Kim JS
• Shin GM
• Chae YJ
• Yoon T
• Chun CS
• Lee JD
• Song M

• KMID: 2395301
• DOI: http://doi.org/10.4162/nrp.2017.11.6.470

Abstract

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.

Keyword

Liver; glutathione; hydroxyproline; apoptosis; silymarin

MeSH Terms

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
Biomarkers
Glutathione
Hydroxyproline
Silymarin
Thioacetamide

Figure

• 

  Fig. 1 Cell viability assay of HSC-T6/Chang liver cells and morphological changes in primary HSCs in response to treatment with OJE. (A) HSC-T6 and Chang liver cells were incubated with OJE at the indicated concentrations for 24 h, after which the cell viability was determined by MTT assay. Primary HSCs were cultivated for 1 week (B) and exposed to OJE at 0.5 mg/mL for 24 h (C). Pictures were taken before and after 24 h of treatment with OJE. Magnification was 100×. Arrows indicate HSCs. The data are expressed as the means ± SEM (n = 8), which were compared using one-way analysis of variance (ANOVA) followed by Student's t-test. NS, not significant and ***P < 0.001 compared to the control group. OJE, O. japonica extract; HSC, hepatic stellate cells; DMSO, dimethyl sulfoxide.

• 

  Fig. 2 Effect of OJE on the cell cycle in HSC-T6 cells. DNA contents in different phases of the cell cycle were measured by flow cytometry using propidium iodide. The cell cycle distribution for each treatment group and the percentage of the cell cycle distribution are represented by graphs (A) and histogram (B), respectively. OJE: O. japonica extract.

• 

  Fig. 3 Effect of OJE on apoptosis and fibrosis expression in HSC-T6 cells. A: Control cells. Flow cytometric data indicate apoptosis in HSC-T6 cells after incubation with silymarin (B), OJE 0.5 mg/mL (C), or OJE 0.1 mg/mL (D) for 24 h. E: Data show the apoptotic (Annexin V+ and PI-) and late apoptotic (Annexin V+ and PI+) cells. F: Fibrosis related gene expression. The data are presented as the means ± SEM (n = 8) which were compared using one-way analysis of variance (ANOVA) followed by Student's t-test. * P < 0.05 and ** P < 0.01 compared to the control group. OJE: O. japonica extract.

• 

  Fig. 4 Effect of OJE on AST/ALT levels, total glutathione (GSH) contents and hydroxyproline levels in TAA-induced liver fibrosis rats. A: HSC-T6 cells were incubated with 10 and 50 mg/mL OJE for 24 h. Expression of fibrosis related genes in HSC-T6 cells was determined by real-time PCR. The results are expressed as normalized fold values relative to the control. Levels of ALT (A) and AST (B) in serum were measured by spectrophotometry. (C) Total GSH contents in liver tissue (D) and hydroxyproline levels of TAA-induced liver tissue of rats were measured using spectrophotometry. TAA, thioacetamide-induced liver fibrosis rats; Silymarin, positive control rats; OJE 10, TAA plus OJE 10 mg/kg treated rats; OJE 50, TAA plus OJE 50 mg/kg treated rats. Data are expressed as means ± SEM (n = 8) which were compared using one-way analysis of variance (ANOVA) followed by Student's t-test. ###P < 0.001 compared to the control group, *P < 0.05, **P < 0.01, ***P < 0.001 compared with TAA group. OJE, O. japonica extract; TAA, thioacetamide; ALT, alanine transaminase; AST, aspartate transaminase; GSH, glutathione.

• 

  Fig. 5 Effect of OJE on TAA-induced morphology of liver tissues by Hematoxylin and Eosin (H&E) staining. At the end of the experiment, all animals were sacrificed and livers were fixed in Bouin's solution. After staining with H&E, liver sections were observed by light microscopy. Control, naive rats (A); TAA, TAA-induced liver fibrosis rats (B); Silymarin, Positive control rats (C); OJE 50, TAA plus OJE 50 mg/kg treated rats (D); OJE 10, TAA plus OJE 10 mg/kg treated rats (E). Scale bar = 200 µm. OJE, O. japonica extract; TAA, thioacetamide.

• 

  Fig. 6 Effect of OJE on TAA-induced fibrosis by Masson's trichrome staining of liver tissues. This stain was performed as described for H&E staining. Control, naive rats (A); TAA, TAA-induced liver fibrosis rats (B); Silymarin, Positive control rats (C); OJE 50, TAA plus OJE 50 mg/kg treated rats (D); OJE 10, TAA plus OJE 10 mg/kg treated rats. Percentage of fibrosis area plot (F). Scale bar = 200 µm. Quantification was accomplished using ImageJ. Values are presented as the means ± SEM (n = 8) which were compared using one-way analysis of variance (ANOVA) followed by Student's t-test. #P < 0.05 compared to the control group, **P < 0.01, ***P < 0.001 compared to the TAA group. TAA, Thioacetamide; OJE, O. japonica extract.

• 

  Fig. 7 Effect of OJE on fibrosis related gene expression in TAA-induced liver tissues. Expression of fibrosis related genes in liver tissue was measured by real-time PCR. (A) TGF-β, (B) α-SMA, (C) Col1α1. The results are expressed as normalized fold values relative to the control. Values are presented as the means ± SEM (n = 8) which were compared using one-way analysis of variance (ANOVA) followed by Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the TAA group. TAA, Thioacetamide; OJE, O. japonica extract; α-SMA, alpha-smooth muscle actin; Col1α1, collagen type1 alpha 1; TGF-β1, transforming growth factor β1.

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