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

MLB attenuates TAA-induced liver injury and hepatic fibrosis in rats

The extent of liver injury was assessed by measuring serum aminotransferase levels (Table 1). Eight or 12 weeks of TAA treatment induced significant elevation of AST and ALT levels as compared to the control group (P < 0.05). The levels of AST and ALT were significantly lower in the TAA + LAB 8-week group than in the TAA 8-week group (P < 0.05). However, MLB failed to decrease AST or ALT levels in 12 weeks of TAA treatment.

The extent of hepatic fibrosis was analyzed by morphometric quantitation of Masson's trichrome stained area in liver sections. The representative photomicrographs are shown in Figure 1. Fibrous septa developed in rats treated with TAA for 8 weeks. These septa incompletely surrounded the regenerative parenchyma and a partial nodular formation was present without development of obvious cirrhosis (Figure 2A). The septal fibrosis presented in the TAA 8-week group was attenuated by MLB (Figure 2B). After 12 weeks of TAA treatment, multiple cirrhotic nodules of varying size with fibrous septa were noted (Figure 2C), and these overt cirrhotic changes were also attenuated by MLB (Figures 2D). The morphometric measurement of Masson Trichrome stained area using a computerized imaging analysis system demonstrated that hepatic fibrosis was significantly attenuated by MLB in both the 8-week and 12-week TAA treatment groups (P < 0.05, P < 0.01, respectively) (Figure 2E). Treatment with MLB significantly decreased the amount of hepatic hydroxyproline content in both the 8-week and 12-week TAA treatment groups (P < 0.01, P < 0.01, respectively) (Figure 2F).

MLB decreases activation of HSCs in TAA-treated rats

Activation of HSCs was assessed by measuring the α-SMA stained area in the liver. Immunohistochemistry showed that α-SMA positive cells were evident on the periphery of regenerating nodules in rats treated with TAA for both 8 and 12 weeks (Figures 3A and 3C). Expression of α-SMA was decreased in rats treated with TAA + MLB (Figures 3B and 3D). The morphometric measurement of the α-SMA stained area demonstrated that activation of HSCs was significantly decreased by MLB in both the 8-week and 12-week TAA treatment groups (P < 0.05, P < 0.05, respectively) (Figure 3E).

MLB decreases the hepatic fibrogenic responses in TAA-treated rats

We next evaluated the hepatic expression of fibrosis-related genes in TAA-treated rats. Treatment with TAA increased hepatic expression of fibrosis-related genes including α-SMA, TGF-β1, and collagen α1(I). The expression of these fibrosis-related genes was significantly decreased by MLB in both the 8-week and 12-week TAA treatment groups (Figures 4A-C).

MLB suppresses cell proliferation of HSCs

To investigate the underlying mechanism of the antifibrotic effects of MLB, we performed in vitro studies using immortalized human HSCs, the main fibrogenic cell type in the liver. First, we examined the cytotoxicity of MLB in cultured HSCs. Incubation of HSCs with MLB (≤100 µM) for up to 48 h did not show any discernable cytotoxicity. The estimated cell viability as assessed by MTT assay after 72 h of treatment with 50 µM MLB was 93.4% ± 4.1% (Figure 5A). We then assessed the effect of MLB on HSC proliferation. Cell proliferation measured by MTT assay after 24 hours is shown in Figure 5B. The cell number of HSCs increased significantly after PDGF stimulation (P < 0.01). PDGF-induced HSC proliferation was significantly suppressed by pretreatment with MLB in a dose-dependent manner (P < 0.05).

MLB suppresses NF-κB activation and MCP-1 production in HSCs

We next examined whether MLB has an anti-inflammatory effect on HSCs. NF-κB transcriptional activation was assessed using an NF-κB-dependent luciferase assay and monocyte chemotactic protein-1 (MCP-1) from the culture supernatant was measured using an ELISA assay. As shown in Figures 6A and 6B, MLB inhibited NF-κB activation and MCP-1 production in HSCs in a dose-dependent manner, indicating that MLB has an anti-inflammatory effect on HSCs.

MLB inhibits ROS generation and type I collagen secretion in HSCs

It has been reported that ROS generation stimulates type I collagen production in HSCs. We continuously measured intracellular ROS formation in HSCs using a fluorometer after loading cells with the redox-sensitive dye CM-H₂DCFDA. As shown in Figure 7A, 100 µM of H₂O₂ markedly induced ROS production in HSCs. However, HSCs pre-incubated with MLB (≥50 µM) showed markedly reduced ROS production after stimulation with H₂O₂. Finally, we measured the secreted type I collagen from HSCs in the culture supernatant. Secretion of type I collagen was inhibited by treatment with MLB (≥50 µM) (Figure 7B). However, the expression of α-SMA was not affected by treatment with MLB.

Magnesium lithospermate B preparation

MLB was isolated and purified from Salvia miltiorrhizae as described previously (Jung et al., 2002). Slices of the dried roots of Salviae miltiorrhizae (1.0 kg) were placed in water at 100℃ for two hours. The heating water extract was evaporated to dryness under reduced pressure at 70℃. The heating water extract was suspended in water (500 mL), and the water extract was adjusted to pH 3.5 with hydrogen chloride (HCl) and extracted with saturated butyl alcohol (300 ml × 5 each). The butyl alcohol extract was evaporated to dryness under reduced pressure at 70℃. Then, the butyl alcohol extracts were suspended in water (300 ml) and the water extracts were washed with hexane (300 ml × 3 each). The water extract was evaporated to dryness under reduced pressure at 70℃. The water extract was extracted with ethyl acetate (300 ml × 3 each). The ethyl acetate extract and water extract were evaporated to dryness under reduced pressure at 70℃. The ethyl acetate extract and the water extract were purified by recrystallization and fractional crystallization. The yield of the MLB as amorphous powder was 10 g from Salviae miltiorrhizae (1.0 kg). All spectral data (NMR, 13C-NMR, IR, HR-MS) of MLB were identical to those of literature (Yokozawa et al., 1989). The chemical structure of MLB is illustrated in Figure 1.

Animal experiments of hepatic fibrosis

Six-week-old male Sprague-Dawley rats were prepared. Hepatic fibrosis was induced by an intraperitoneal injection of TAA (Sigma, St. Louis, MO) at a dose of 200 mg/kg twice a week for up to 12 weeks. Rats were fed MLB once daily for up to 12 weeks at a dose of 40 mg/kg using a gavage tube. The rats were randomly assigned to six experimental groups: the control group (n = 5), the control + MLB group (n = 5), the TAA 8-week group (n = 5), the TAA + MLB 8-week group (n = 5), the TAA 12-week group (n = 5), and the TAA + MLB 12-week group (n =5). The TAA 8-week group and the TAA + LAB 8-week groups were sacrificed 8 weeks after the first TAA injection. All of the other rats were sacrificed at week 12. At the time of sacrifice, blood and liver samples were harvested. The livers were fixed in 10% formalin for histological analysis. Portions of liver tissue were immediately frozen in liquid nitrogen for further RNA and protein analysis. All experimental procedures were performed according to the guidelines for the care and use of animals established by Yonsei University College of Medicine.

Assessment of hepatic fibrosis

Four-micrometer-thick sections of formalin-fixed and paraffin-embedded livers from all the experimental groups were processed for hematoxylin and eosin staining (H&E) and Masson's trichrome staining. Tissue sections were immunostained for α-SMA (Dako North America, Inc., Carpinteria, CA), diluted at 1:1000. Stained liver sections were analyzed with the computerized imaging analysis system (MetaMorph version 4.65, Universal Imaging Corp., Downingtown, PA). Hydroxyproline content was quantified colorimetrically from 0.2 g liver samples as previously described (Bataller et al., 2003). The results were expressed as micrograms of hydroxyproline per grams of liver.

Serum biochemistry

Rat whole blood was obtained at the time of sacrifice via vena cava puncture. After centrifugation of each blood sample, the serum was obtained and examined for both aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

RNA isolation and quantitative RT-PCR

Total RNA was extracted from frozen rat liver tissues, using Takara RNA PCR (AMV) Ver 3.0 (Takara Bio Inc., Shiga, Japan) as described in the product protocol, and cDNA was generated from 1 µg of total RNA, using oligo dT-Adaptor primers and Avian Myeloblastosis Virus reverse transcriptase (Life Science, Boston, MA). α-SMA, TGF-β1, collagen α1(I) mRNA in rat liver samples were quantified by quantitative RT-PCR using a Taqman probe (Roche, Mannheim, Germany) and LightCycler® (Roche Diagnostics GmbH, Mannheim, Germany). The PCR primers for α-SMA were 5'-CGATAGAACACGGCATCATCAC-3' sense and 5'-GCATAGCCCTCATAGATAGGCA-3' antisense. The PCR primers for TGF-β1 were 5'-CCTGGAAAGGGCTCAACAC-3' sense and 5'-CAGTTCTTCTCTGTGGAGCTGA-3' antisense. The PCR primers for collagen α1(I) were 5'-CATGTTCAGCTTTGTGGACCT-3' sense and 5'-GCAGCTGACTTCAGGGATGT-3' antisense. The relative expression of target gene mRNA was normalized to the amount of rat porphobilinogen deaminase (PBGD) mRNA in an identical cDNA sample (de Kok et al., 2005). The PCR primers for PBGD were 5'-CACCTGGAATTCAAGAGTATTCG-3' sense and 5'-CCAGGATAATGGCACTGAACT-3' antisense. The relative abundance of the target genes was obtained by calculation using the comparative threshold cycle (Ct) method.

HSCs culture

We used immortalized human HSCs that were established by retroviral expression of human telomerase reverse transcriptase (hTERT) in this study (Schnabl et al., 2002). Telomerase-positive HSCs exibit morphological and functional characteristics of activated HSCs. It was proven that HSCs with hTERT are very useful in researching the biology of activated HSCs (Paik et al., 2006, 2009). HSCs were cultured in a 5% CO₂ humidified incubator at 37℃. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum (FBS) (Invitrogen) and a 1% streptomycin-penicillin mixture.

Cell viability and proliferation assay

Cytotoxicity of MLB in HSCs was assessed using MTT assay. HSCs were seeded at a density of 3 × 10⁴ cells per well in 24-well plates. After 24 h of serum starvation, cells were treated with various concentrations of MLB (0-300 µM). After incubation for up to 72 h, 125 µl of MTT solution (2 mg/ml in PBS) was added to each well and incubated for 4 h. The supernatant was removed and the cells solubilized in DMSO (250 µl) for 30 min. The optical density at 540 nm was measured using an enzyme-linked immunosorbent VERSAmax™ reader (Molecular Devices, Sunnyvale, CA). For the cell proliferation assay, serum-starved HSCs were incubated with PDGF-BB (R&D systems, Minneapolis, MN) with MLB (0-100 µM) for 24 h. The number of viable cells was measured by MTT assay at a wavelength of 540 nm on an ELISA reader.

NF-κB responsive luciferase assay

Recombinant adenoviral vectors expressing a luciferase reporter gene driven by NF-κB transcriptional activation (Ad5NF-κBLuc) were used for the assessment of NF-κB transcriptional activation as described previously (Paik et al., 2003). Briefly, HSCs were infected with Ad5NF-κBLuc (multiplicity of infection 500) for 12 h in DMEM containing 0.5% FBS. After infection, the culture medium was changed to a fresh medium without FBS, and the culture was continued for an additional 8 h. At 20 h post-infection, HSCs were stimulated with TNF-α for 8 h in serum-free conditions with or without pretreatment with MLB. A luciferase assay kit with luciferase cell culture lysis buffer (Promega, Madison, WI) was used to measure NF-κB mediated transcriptional induction according to the manufacturer's protocol. The relative light intensity was measured by a spectrometer and all measurements of luciferase activity were normalized to the protein concentration.

MCP-1 enzyme-linked immunosorbent assay

After 48 h of serum strarvation, HSCs were cultured in medium with 1% FBS for 24 h after pretreatment with MLB for 1 h. Measurements of secreted MCP-1 in the culture supernatant were done by ELISA (R & D Systems, Minneapolis, MN).

Measurement of reactive oxygen species

HSCs were plated at a density of 2 × 10⁴ cells/well in a 96-well plate. After serum starvation for 24 h, cells were preincubated with MLB (0, 50, 100 µM) for 1h. After washing, cells were loaded with a redox-sensitive dye, 10 µM of CM-H₂DCFDA (Molecular Probes, Eugene, OR) at 37℃ for 30 minutes, and then cells were rinsed twice with PBS and stimulated with 100 µM hydrogen peroxide (H₂O₂). CM-H₂DCFDA fluorescence was detected at excitation and emission wavelengths of 488 nm and 520 nm, respectively. ROS formation was measured over a time course of 30 minutes using a multi-well fluorescence scanner (Fluostar Optima, BMG Labtech, Cary, NC).

Western blot

For detection of collagen I secretion, cells were cultured in 100 mm culture dishes (10⁶ cells per dish). Cells were treated with MLB for 48 h, and 4 ml of cell media were precipitated with 0.76 g of sodium sulfite at 4℃ for 3 h and centrifuged at 10,000 g for 30 min. Pellets were resuspended in 0.5 M acetic acid, and 40 µl aliquots were subjected to electrophoresis on a 7.5% acrylamide gel. After blotting, membranes were probed with anti-type I collagen antibody (1:1,000; Biodesign International, Saco, MA), which recognized the heterotrimer of type I collagen. For the detection of cellular α-SMA and α-tubulin, whole-cell extracts were prepared using Triton lysis buffer containing protease and phosphatase inhibitors. Forty micrograms of protein was electrophoresed on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels. The gels were blotted onto a nitrocellulose membrane. Antibodies against α-SMA (Dako) and α-tubulin (Santa Cruz biotechnology), all diluted to 1:1000, were used.

Statistics

The data are shown as mean ± standard deviation. Data were analyzed using the Mann-Whitney U test. Data were considered to be statistically significant when P < 0.05.