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. 2011 Jun 30;43(6):341-9.
doi: 10.3858/emm.2011.43.6.037.

Antifibrotic effects of magnesium lithospermate B on hepatic stellate cells and thioacetamide-induced cirrhotic rats

Yong Han Paik  1 , Young Joon Yoon, Hyun Chul Lee, Man Kil Jung, So Hee Kang, Sook In Chung, Ja Kyung Kim, Jae Yong Cho, Kwan Sik Lee, Kwang Hyub Han
Affiliations

Antifibrotic effects of magnesium lithospermate B on hepatic stellate cells and thioacetamide-induced cirrhotic rats

Yong Han Paik et al. Exp Mol Med. .

Abstract

Magnesium lithospermate B (MLB) is one of the major active components of Salvia miltiorrhizae. The anti-oxidative effects of Salvia miltiorrhizae have been previously reported. The aim of this study was to investigate the effect of purified MLB on hepatic fibrosis in rats and on the fibrogenic responses in hepatic stellate cells (HSCs). Hepatic fibrosis was induced in rats by intraperitoneal thioacetamide (TAA) injections over a period of 8 or 12 weeks. MLB was orally administered daily by gavage tube. Serum AST and ALT levels in TAA+ MLB group were significantly lower than those in TAA only group at week 8. Hepatic fibrosis was significantly attenuated in TAA+MLB group than in TAA only group at week 8 or 12. Activation of HSCs was also decreased in TAA+MLB group as compared to TAA only group. Hepatic mRNA expression of α-smooth muscle actin (α-SMA), TGF-β1, and collagen α1(I) was significantly decreased in TAA+MLB group as compared to TAA only group. Incubation with HSCs and MLB (>or=100 μM) for up to 48 h showed no cytotoxicity. MLB suppressed PDGF-induced HSC proliferation. MLB inhibited NF-ΚB transcriptional activation and monocyte chemotactic protein 1 (MCP-1) production in HSCs. MLB strongly suppressed H(2)O(2)-induced reactive oxygen species (ROS) generation in HSCs, and MLB inhibited type I collagen secretion in HSCs. We concluded that MLB has potent antifibrotic effect in TAA-treated cirrhotic rats, and inhibits fibrogenic responses in HSCs. These data suggest that MLB has potential as a novel therapy for hepatic fibrosis.

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Figures

Figure 1
Figure 1
The chemical structure of magnesium lithospermate B.
Figure 2
Figure 2
MLB suppresses hepatic fibrosis in TAA-treated rats. The extent of hepatic fibrosis was assessed by Masson's trichrome staining (A, B, C, and D, original magnification × 40); (A) TAA 8-week, (B) TAA 8-week + MLB, (C) TAA 12-week, (D) TAA 12-week + MLB. (E) Masson's trichrome-stained area was quantified using a computerized imaging analysis system. (F) Hepatic hydroxyproline content was colorimetrically quantified from liver samples. *P < 0.05 compared with TAA 8-week, **P < 0.01 compared with TAA 8-week, †P < 0.01 compared with TAA 12-week.
Figure 3
Figure 3
MLB inhibits HSC activation in TAA-treated rats. Activation of HSCs was assessed by α-SMA staining (A, B, C, and D, original magnification × 40); (A) TAA 8-week, (B) TAA 8-week + MLB, (C) TAA 12-week, (D) TAA 12-week + MLB. (E) The α-SMA-stained area was quantitated using a computerized imaging analysis system. *P < 0.05 compared with TAA 8-week, **P < 0.05 compared with TAA-12 week.
Figure 4
Figure 4
Hepatic mRNA expression of fibrosis-related genes in TAA-treated rats. (A) α-SMA, (B) TGF-β1, (C) collagen α1(I) mRNA levels in rat livers were quantified by quantitative RT-PCR using Taqman probe after reverse transcription. The mRNA expression of these molecules was normalized with rat PBGD expression. Data represent the mean ± SD of two independent experiments. †P < 0.01 compared with TAA-8 week, *P < 0.01 compared with TAA-12 week, **P < 0.05 compared with TAA 12-week.
Figure 5
Figure 5
Effects of MLB on HSCs viability and proliferation. (A) Cytotoxicity by MLB was tested in HSCs. HSCs were treated with various concentrations (0-300 µM) of MLB for up to 72 h in a serum-free condition. Cytotoxicity was assessed by MTT assay. Data represent the mean ± SD of two independent experiments. (B) Effect of MLB on cell proliferation was tested in HSCs. HSCs were stimulated with PDGF (20 ng/ml) for 24 h with various concentrations (0-100 µM). Data represent the mean ± SD of two independent experiments. †P < 0.01 between no treatment and PDGF only, *P < 0.05 compared with PDGF only.
Figure 6
Figure 6
MLB attenuates NF-κB transcriptional activation and inflammatory chemokine MCP-1 production in HSCs. (A) HSCs were infected with the Ad5NF-κBLuc (MOI 500) for 12 h in DMEM containing 0.5% FBS. At 20 h post-infection, HSCs were treated with TNF-α (10 ng/ml) for 8 h with pretreatment of various concentrations of MLB (0-100 µM) for 1 h. Cells were lysed and NF-κB-mediated luciferase activity was quantified. Data represent the mean ± SD of 2 independent experiments and are expressed as fold-increase over unstimulated cells. All measurements of luciferase activity were normalized to the protein concentration. †P < 0.01 between no treatment and TNF-α only, ‡P < 0.01 compared with TNF-α alone. (B) Secreted MCP-1 in culture medium was quantified by ELISA. After 48 h serum starvation, HSCs were cultured in medium with 1% FBS for 24 h with various concentrations of MLB (0-100 µM). Data represent the mean ± SD of two independent experiments. *P < 0.05 compared with no MLB treatment, **P < 0.01 compared with no MLB treatment.
Figure 7
Figure 7
MLB causes decreases in ROS production and type I collagen secretion in HSCs. (A) Serum starved HSCs were pre-incubated with various concentrations of MLB (0-100 µM). After loading redox-sensitive dye CM-H2DCFDA (10 µM) into HSCs, cells were stimulated with 100 µM H2O2 and fluorescence was measured with a spectrometer. (B) HSCs were treated with various concentrations of MLB (0-100 µM) for 48 h. Western blot of the precipitated proteins from the culture supernatant probed for type I collagen; Western blot of cellular proteins (20 µg/ml) probed for α-SMA or α-tubulin.
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