Scutellariae radix suppresses LPS-induced liver endothelial cell activation and inhibits hepatic stellate cell migration

Journal of Ethnopharmacology 150 (2013) 835–842

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Scutellariae radix suppresses LPS-induced liver endothelial cell activation and inhibits hepatic stellate cell migration Hong-Jhang Chen a, Tzu-Ming Liang b, I-Jung Lee a, Yi-Tsau Huang a,b, Yun-Lian Lin a,c,n a

National Research Institute of Chinese Medicine, Taipei, Taiwan Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan c School of Pharmacy, National Taiwan University, Taipei, Taiwan b

art ic l e i nf o

a b s t r a c t

Article history: Received 18 February 2013 Received in revised form 8 August 2013 Accepted 26 August 2013 Available online 11 September 2013

Ethnopharmacological relevance: Liver fibrosis is the result of long-term liver damage and the woundhealing process, in which the hepatic stellate cell (HSC) plays a crucial role during fibrogenesis. The liver sinusoidal endothelial cell (LSEC) is a liver-resident scavenger, contributing to sinusoidal remodeling, HSC activation and liver fibrosis. Lipopolysaccharide (LPS) causes an inflammatory reaction associated with portal circulation and LSECs signaling. Scutellariae radix, the root of Scutellaria baicalensis Georgi, is a Chinese herb widely used for liver diseases. However, its effect on LSEC activation and HSC migration in liver fibrosis has not been investigated yet. Aim of this study: LPS-induced rat LSEC (rLSEC) activation was used as a model to screen and explore the active components of Scutellariae radix. The anti-fibrotic effect of Scutellariae radix on rLSEC activation and rHSC migration was further investigated. Materials and methods: LPS-induced rLSEC mRNA expression, including VEGF, VEGFR, MCP-1, and TGF-β1, were examined by real-time PCR analyses. MCP-1 protein levels were measured by an ELISA kit. rLSEC conditioned medium on rHSC migration was measured by wound-healing assay and transwell chemoattraction assay. Results: Results showed LPS-induced rLSEC activation with upregulated MCP-1 mRNA and protein expressions, and that rLSEC-condition medium enhanced rHSC migration. Both baicalein and wogonin from the active subfraction significantly reduced MCP-1 expression, but only baicalein markedly inhibited rHSC migration in rLSEC conditioned medium. Conclusion: This study demonstrated that Scutellariae radix attenuates LPS-induced rLSEC activation and HSC migration with downregulation of MCP-1 expression. The results provide supporting evidence that Scutellariae radix may be beneficial for the amelioration of liver fibrosis. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Scutellariae radix Rat liver endothelial cell (rLSEC) MCP-1 rLSEC conditioned medium Rat hepatic stellate cell (rHSC) Liver fibrosis

1. Introduction Liver fibrosis is a major cause of morbidity and mortality from hepatic diseases. It is a wound-healing response following chronic liver inflammation, including viral hepatitis B and C, alcoholic hepatitis, non-alcoholic steatohepatitis, and autoimmune hepatitis, and it is characterized by the accumulation of extracellular matrix following liver injury (1). During chronic liver injury, activated

Abbreviations: (rHSCs), rat hepatic stellate cells; (MCP-1), monocyte chemotactic protein 1; (rLSECs), rat liver sinusoidal endothelial cells; (Sal B), salvianolic acid B; (SBE), ethanolic extracts of Scutellariae radix; (TGF-β1), transforming growth factorbeta 1; (VEGF), vascular endothelial growth factor; (VEGFR), vascular endothelial growth factor receptor n Corresponding author at: National Research Institute of Chinese Medicine, Taipei, Taiwan. Tel.: þ 88 622 820 1999x6531; fax: þ 88 622 825 0743. E-mail address: [email protected] (Y.-L. Lin). 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.08.049

hepatic stellate cells (HSCs), which are key players nearby liver sinusoidal endothelial cells (LSECs) in liver fibrogenesis, migrate to the sites of inflammation and produce extracellular matrix to fill the gaps created by massive parenchymal cell death. Persistent injury may lead to permanent scarring and cirrhosis of the liver (Hernandez-Gea and Friedman, 2011). LSECs are unique cells with non-diaphragm fenestration organized on a sieve plate (Braet and Wisse, 2002). The plates filter the fluids that are exchanged between hepatocytes and the portal vein, thereby playing a key role in liver fibrosis, particularly in the early stages before HSCs undergo myofibroblastic differentiation (Lee et al., 2007; Deleve et al., 2008). During liver inflammation, LSECs are activated and secrete monocyte chemotactic protein 1 (MCP-1), further stimulating HSCs to migrate to the site of inflammatory cells (Marra et al., 1999). Lipopolysaccharide (LPS), a gram-negative bacterial cell wall component that is enriched within the intestinal lumen and portal circulation, has been demonstrated to have a potential role in LSEC

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(Uhrig et al., 2005; Dauphinee and Karsan, 2006; Jagavelu et al., 2010) and HSC (Seki and Schnabl, 2012) signaling, which is related to liver fibrosis. Currently, an effective therapeutic agent for liver fibrosis remains to be discovered (Zois et al., 2008). However, Chinese herbal medicine such as Sho-saiko-to (TJ-9) and Salvia miltiorrhiza have long been used to alleviate liver diseases, and have demonstrated the ability to attenuate liver fibrosis (Shimizu et al., 1999; Sakaida et al., 2004; Yang et al., 2008). Compounds of plant origin have also been claimed to have potential effects on the activated HSCs and liver fibrosis. Such plants include curcumin (Bisht et al., 2011), resveratrol (Bishayee et al., 2010), salvianolic acid B (Tsai et al., 2010), epigallocatechin-3-gallate (EGCG) (Zhen et al., 2006), etc. Scutellariae radix (Huang Qin), (Huang qi), the root of Scutellaria baicalensis Georgi (Labiatae) (SB), is one of the most widely used plants in traditional Chinese medicine (TCM) for treating liver diseases (Chien et al., 2011). More than 30 flavonoids, including baicalin, baicalein, wogonin, and oroxylin-A have been identified in Scutellariae radix (Li et al., 2004). The effects of a variety of the pharmacological activities of its extract or isolated compounds on liver diseases have been reported, such as preventing liver damage from bile duct ligation or carbon tetrachloride (Nan et al., 2002), chemical-induced injury (Jang et al., 2003), H2O2-induced injury (Zhao et al., 2006), ischemia/reperfusion injury (Chan et al., 2011), and HSC activation (Pan et al., 2012). However, the mechanisms underlying Scutellariae radix mediated LSEC activation, and the interactions between LSEC and HSC migration have yet to be investigated. In this study, lipopolysaccharide (LPS) was used to investigate the effect of the mRNA expression of fibrogenesisrelated genes including VEGF, VEGFR, MCP-1, and TGF-β1 in rLSECs. MCP-1 was selected as a target gene for the following screening platform. LPS-induced MCP-1 gene and protein expression in rLSECs, as well as the role of a rLSEC conditioned medium in rHSC migration, were examined to explore the active components and the anti-fibrotic potential of Scutellariae radix.

2. Materials and methods

based on dry raw materials) was sequentially partitioned with ethyl acetate (EtOAc) and n-butanol (BuOH) to make it fraction soluble in EtOAc (SBE-EtOAc-) (28.6 g, 0.95% of raw materials), n-butanol- (SBE-BuOH) (158.6 g, 5.29% of raw materials) and water- (SBE-H2O) (482.0 g, 16.1% of raw materials), respectively. The SBE-EtOAc fraction was chromatogramed by a silica gel column (5.5  60 cm) and eluted with a Hexane/EtOAc gradient. The fraction of 30–50% EtOAc elute was flavones-enriched and bioactive. The subfraction was further purified over Sephadex LH-20 (3.8  40 cm, 50% EtOAc/MeOH elution) and then silica gel columns (2.8  30 cm, 30% or 40% EtOAc/Hexane) to give baicalin (3.44 g), baicalein (6.88 g), wogonin (2.06 g), oroxylin-A (0.64 g), scutellarein (0.38 g), and 5,7,2′-trihydroxy-6-methoxyflavone (6, 0.13 g) (Fig. 1). The SBE-BuOH fraction was repeatedly purified by Sephadex LH-20 column chromatography with methanol elution to give two major components, baicalin and scutellarein. All of the isolated compounds were mainly identified by 1D- and 2D-NMR and MS, and further compared with published data. For bioassay, the extracts and isolated pure compounds were all dissolved in dimethyl sulfoxide (DMSO) and diluted with a medium to give a final DMSO concentration of less than 0.1%. A vehicle control group containing 0.1% DMSO and salvianolic acid B (Sal B), a polyphenol from Salvia miltiorrhiza, was used as a positive control (Tsai et al., 2010).

2.3. Preparation of rat liver endothelial cells (rLSECs) and hepatic stellate cells (rHSCs) Sprague-Dawley (SD) rats (8–12 weeks old, 350–450 g) were purchased from National Yang-Ming University (Taipei, Taiwan) and kept in an air-conditioned, pathogen-free room at 237 2 1C on a regulated 12 h light/dark cycle. They were housed individually and fed a standard laboratory diet (Lab Rodent Chow Die 5001, Ralston Purina, St. Louis, MO, USA) ad libitum. Animals were cared for and handled following the guidelines set forth by the Animal Care Committee of National Yang-Ming University and the NIH Guidelines for the Care and Use of Laboratory Animals.

2.1. Materials and equipments Silica gel (230–400 mesh) and Sephadex™ LH-20 (Amersham Biosciences, Uppsala, Sweden) were used for column chromatography. Solvents (analytical grade) were purchased from Merck. Nuclear magnetic resonance (NMR) spectra were run in CDCl3 or DMSO-d6 on a Varian unity INOVA-500 or VNMRS 600 (Varian, Palo Alto, CA, USA) using standard pulse sequences. Mass spectra (ESIMS) were recorded on a Finnigan MAT LCQ ion trap mass spectrometer system (Thermoquest, San Jose, CA, USA). Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco BRL (Carlsbad, CA, USA). FBS was purchased from Gibco BRL (Gaithersburg, MD, USA). All other chemicals were of analytical grade and purchased from commercial suppliers. 2.2. Preparation of Scutellariae radix extract and isolation of pure compounds Scutellariae radix was purchased from an herbal retailer in Taipei. A voucher herbarium specimen has been deposited at the Herbarium of National Research Institute of Chinese Medicine (NRICM) (NHP 0365) and was identified by Dr. I-Jung Lee, the herbarium leader of NRICM. The dried slices of Scutellariae radix (3 kg) were extracted three times at 60 1C by aqueous ethanol (80% ethanol/H2O) overnight. The extract was concentrated with a vacuum rotary evaporator under reduced pressure and dried in a vacuum oven (690.4 g). The dried sample (SBE, about 23.0% yield

Fig. 1. Structures of isolated pure compounds from Scutellariae radix.

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The isolation of rat LSECs (rLSECs) and rat HSCs (rHSCs) were mainly carried out according to Friedman and Roll's method (Friedman and Roll, 1987). In brief, cells were isolated from SD rats using sequential pronase-collagenase digestion followed by Percoll (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation. Either rLSECs or rHSCs were cultured in a DMEM supplement with 10% FBS. Cells between three to five passages were used for experiments. The purity of rLSECs and rHSCs were estimated by immunostaining with the von Willebrand factor (Abcam, Cambridge, MA, USA) (Knittel et al., 1995) and desmin (Lin et al., 2006), respectively. 2.4. mRNA expression of VEGF, VEGFR, MCP-1 and TGF-β1 by real-time polymerase chain reaction (PCR) rLSECs were seeded into 6-well culture plates at a density of 2.5  105 cells/well. After incubation overnight, cells were washed with PBS and the medium was replaced with a serum-free medium. After 24 h starvation, cells were pretreated with extract or a pure compound of Scutellariae radix (25 μg/mL or 10 μg/mL, respectively) for 1 h, and then stimulated with LPS (100 ng/mL, Sigma) for another 6 h. After washing with cold phosphatebuffered saline (PBS), the total RNA was prepared by the TriPure isolation reagent (Roche, Mannheim, Germany) following the manufacturer's directions. After that, cDNA preparation was carried out and a RevertAid™ First Strand cDNA synthesis kit (Fermentas, USA) was used for PCR reaction according to the manufacturer's protocol. For real-time PCR, the amplification was carried out in a total volume of 20 μL containing 0.5 μM of each primer, 4 mM MgCl2, 10 μL SYBR Green Master (Roche), and 10 μL of 1:10 diluted cDNA. Real-time PCR was carried out on a LightCyclers480 (Roche). PCR reactions were performed in duplicate and heated to 95 1C for 5 min, followed by 45 cycles of denaturation at 95 1C for 10 s, and then annealed at 55 1C for 10 s, and extended at 72 1C for 12 s. The sequences of primers for real-time PCR are listed in Supplementary Table S1. Data are represented as the fold induction of fibrosis-related genes (VEGF, VEGFR, MCP-1 and TGF-β1) by the LPS-stimulated group compared to the spontaneous group (Lee et al., 2011). 2.5. Monocyte chemotactic protein 1 (MCP-1) assay rLSECs were cultured on a 24-well culture plates at the density of 1.2  105 cells/well. After 12 h incubation, cells were washed with PBS and treated with either extract or pure compound (25 μg/mL or 10 μg/mL, respectively) for 1 h, and then stimulated with LPS (100 ng/mL) for another 18 h. The culture supernatants were collected for MCP-1 assay. Analysis of MCP-1 was performed by a rat MCP-1 enzyme-linked immunosorbent assay (ELISA) kit (Life Technologies) according to the manufacturer's protocol.

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24-well culture plates at the density of 1.2  105 cells/well. After 24 h serum starvation, a monolayer of cells was scratched by 200-μL tip and washed with PBS. Cells were treated with isolated pure compound (25 μg/mL for baicalin, or 10 μg/mL for baicalein, wogonin, and oroxylin-A) directly or with a changed culture medium composed of an isolated pure compound treated rLSEC conditioned medium, respectively. rHSC migration was quantified by the number of cells that had migrated to the scratched area after 6 h. Bindarit (300 μM), an MCP-1 inhibitor, was used as a positive control (Maddaluno et al., 2012). 2.8. Transwell chemoattraction assay To confirm the effects of SBE on rHSC migration, transwell assay was performed according to a previously reported method (Kuo et al., 2012). Serum-starved rHSCs with or without a test sample (25 μg/mL for baicalin, or 10 μg/mL for baicalein, wogonin, and oroxylin-A) were seeded in the upper chamber (8 mm pores Transwell; Corning Costar, Cambridge, MA, USA). LPS (100 ng/mL) or the conditioned medium was added into the lower chamber as a chemoattractant agent. rHSCs migrated to the other side of the membrane were detected after 6 h incubation. Non-migrating cells were wiped off with a cotton swab, and the membrane was fixed and stained with hematoxylin (Sigma) to define the cell nuclei. Cells that had migrated through the membrane were assessed by counting the number of migrating cells in five random microscopic fields per well (at 400  magnification). All experiments were repeated at least 3 times. Bindarit (300 μM) was used as a positive control to inhibit cell migration. The proportion of migrating cells, which we refer to as the migration index, was calculated as follows: Migrationindex ð%Þ ¼ ðnumber of cells on the lower surface of the membrane=number of cells on the upper and lower surfaces of the membraneÞ  100:

2.9. Cell viability Cell viability was assessed by MTT assay and LDH leakage assay to evaluate the cytotoxicity of crude extract, subfractions and isolated pure compounds. Mitochondrial dehydrogenase activity was used as an index of cell viability and was assessed using the MTT test. The extent of reduction of MTT to formazan within cells was quantified by measurement of OD540 nm. LDH activity can be used as an indicator of relative cell viability as well as a function of membrane integrity. LDH leakage was determined by a colorimetric LDH assay kit (Sigma Chemical Co., St. Louis, MO). 2.10. Statistical analysis

2.6. Preparation of rLSEC conditioned medium rLSECs were seeded onto 24-well culture plates at the density of 1.2  105 cells/well and incubated overnight at 37 1C. The attached cells were washed with PBS and the medium was replaced with serum-free DMEM for 24 h. Cells were treated with extract or isolated pure compound (25 μg/mL or 10 μg/mL, respectively) for 1 h, and then stimulated with LPS (100 ng/mL) for another 18 h. The medium was collected and the cells were removed by centrifugation.

Results are expressed as the mean 7standard deviation (SD) for three experiments. Differences between specific means were analyzed by one-way analysis of variance (ANOVA) using the SPSS system, vers. 11.0 (SPSS, Chicago, IL, USA). Group means were compared using one-way ANOVA followed by an S–N–K test. p-values less than 0.05 were considered significant.

3. Results

2.7. Wound-healing assay

3.1. LPS induced fibrogenic gene expression in rLSECs

To investigate the migration of HSCs, the wound-healing assay was performed according to a previously reported method (Lee et al., 2011). rHSCs were seeded on collagen-coated (2 mg/mL)

Rat liver sinusoidal endothelial cells (rLSECs) was isolated by hepatic artery perfusion and Percoll density gradient centrifugation (Friedman and Roll, 1987). The purity of cells ( 490%) were

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Fig. 2. LPS-induced fibrogenic gene expressions in rat liver sinusoidal endothelial cells (rLSECs). rLSECs were stimulated with LPS (100 ng/mL) for 1.5, 3, 6, 12 or 24 h. mRNA levels of VEGF, VEGFR, MCP-1 and TGF-β1 were quantified by real-time PCR. GAPDH was used as an internal control. Data shown are mean 7S.D. from three independent experiments. np o0.05, compared with the non-stimulated control.

components, the subfractions of SBE-EtOAc, -BuOH, and -H2O were further investigated. Both SBE-EtOAc (25 μg/mL) and SBEBuOH (25 μg/mL) significantly reduced the mRNA expression of MCP-1 without cytotoxicity, as determined by MTT assay and LDH leakage assay at the working concentration (Supplementary Fig. S2). This shows that the major active components might exist in these subfractions. Salvianolic acid B (Sal B), an anti-fibrotic polyphenols from Salvia miltiorrhiza, was used as a positive control (Tsai et al., 2010). Under activity-guided fractionation, six compounds were isolated from SBE-EtOAc and -BuOH including baicalin, baicalein, wogonin, oroxylin-A, scutellarein, and 5,7,2′-trihydroxy-6-methoxyflavone (6) (Fig. 1). Fig. 4 shows the effect of these compounds on the mRNA expression of MCP-1 in LPS-induced rLSECs. Baicalin showed no effect on the expression of MCP-1 mRNA (Fig. 4A), and oroxylin-A had an inhibitory effect that was not significant (Fig. 4D). Each of the other four compounds, baicalein (an aglycone of baicalin), wogonin, scutellarein, and compound 6, were reduced in the expression of MCP-1 mRNA in a concentration-dependent manner (Fig. 4B, C, E, and F). However, scutellarein and compound 6 revealed significant effects at higher concentrations (25 μg/mL¼ 35, 33 μM, respectively) only (Fig. 4E and F). In addition, both our isolation and Li et al. (2004) found that baicalin (14.40%), baicalein (3.08%), wogonin (0.97%) and oroxylin-A are the major components in Scutellariae radix. Therefore, these four major components were selected for further investigation in the expression of MCP-1 protein in rLSECs.

3.3. Baicalein and wogonin inhibited the secretion of MCP-1

Fig. 3. Scutellariae radix suppressed the gene expression of MCP-1 in LPS-stimulated rLSECs. rLSECs were pre-treated with extracts of Scutellariae radix for 1 h, and then stimulated with LPS (100 ng/mL) for another 6 h. mRNA levels of MCP-1 were quantified by real-time PCR and normalized to GAPDH mRNA expression. Data shown are mean 7 S.D. from three independent experiments. #po 0.05, compared with the non-stimulated control. np o0.05, compared with the LPS-stimulated group.

determined by immunostaining with the von Willebrand factor (Supplementary Fig. S1). To explore the major fibrogenic marker from liver endothelial cells, we evaluated the expression of fibrosis-related genes (VEGF, VEGFR, MCP-1 and TGF-β1) in rLSECs induced by LPS, a permanent endotoxin of peripheral circulation. As shown in Fig. 2, rLSECs were stimulated with LPS (100 ng/mL), and the expressions of fibrogenic genes were measured in different time courses. The mRNA expression of MCP-1 was significantly enhanced after LPSstimulation for 6 h in rLSECs (6 folds). Therefore, the expression of MCP-1 was selected as a biomarker in the screening platform to find the herbs with potential in the inhibition of LPS-induced rLSEC activation.

3.2. Extracts and isolated pure compounds of Scutellariae radix suppressed gene expression of MCP-1 SBE (25 μg/mL) significantly decreased the mRNA expression of MCP-1 by 93% in LPS-induced rLSEC (Fig. 3). To explore the active

Monocyte chemoattractic protein-1 (MCP-1) has been identified as a chemoattractant for activated HSCs and has been shown to play a crucial role in the development of hepatic fibrosis in the perpetuation phase during HSC activation (Seki et al., 2009). According to evidence, the secretion of MCP-1 is more directly involved in refining the anti-fibrotic effect of Scutellariae radix. To measure MCP-1 secretion, the consistent cell number was seeded into a 24-well plate (Supplementary Fig. S3) and a rat ELISA kit was used in an LPS-induced rLSEC model. As shown in Fig. 5, the MCP-1 level was significantly higher in LPS-induced rLSECs cultured supernatant than in the spontaneous control (4.8 ng/mL vs 2.6 ng/mL, po0.05). HSC migration was also markedly enhanced at the concentration of 5 ng/mL of MCP-1 (Supplementary Fig. S4). Both baicalein- (10 μg/mL, 37 μM) and wogonin- (10 μg/mL, 35 μM) treated groups had significantly reduced MCP-1 expression (2.8 and 3.4 ng/mL vs 4.8 ng/mL, respectively; both po0.05). Of the four major components of Scutellariae radix, baicalein (10 μg/mL, 37 μM) suppressed MCP-1 level by 43%, and the MCP-1 level of the baicalein treated group was almost equal to that of the spontaneous control (p¼0.597). 3.4. Baicalein suppressed rHSC migration The wound-healing assay is used to mimic cell migration during chronic injury, and is suitable for studies on the effects of cell–matrices and cell–cell interactions in cell migration (Rodriguez et al., 2005). With a fresh medium, LPS significantly enhanced rHSC migration (Fig. 6A, open bar). Only the baicalein treated group (10 μg/mL, 37 μM) markedly suppressed rHSC migration, which shows that baicalein directly inhibited the migration of rHSC induced by LPS. To investigate the effect of rLSECs on rHSC migration, rLSEC conditioned medium was used for the rHSC culture medium. The resulting changes of rHSC migration were compared with the culture in the fresh medium. rHSCs treated with LPS-induced rLSEC conditioned medium

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Fig. 4. Effects of pure compounds isolated from Scutellariae radix on the gene expression of MCP-1 in LPS-stimulated rLSECs. rLSECs were pre-treated with (A) baicalin, (B) baicalein, (C) wogonin, (D) oroxylin-A, (E) scutellarein, or (F) compound 6 for 1 h, then stimulated with LPS (100 ng/mL) for another 6 h. mRNA levels of MCP-1 were quantified by real-time PCR and normalized to GAPDH mRNA expression. Data shown are mean 7S.D. from three independent experiments. #p o 0.05, compared with the non-stimulated control. *p o 0.05, compared with the LPS-stimulated group.

markedly increased rHSC migration in comparison to those in the fresh medium (Fig. 6A, 285% vs 210%). As expected, baicalein (37 μM) effectively attenuated rHSCs migration (p o0.05) with a similar level of Sal B (200 μM). However, baicalin (25 μg/mL, 56 μM), oroxylin-A (35 μM), and wogonin (35 μM) were without significant effect. Transwell chemoattraction assay was further used to confirm the suppressed effect of baicalein on rHSC migration (Fig. 6B). For both the fresh medium and conditioned medium, the rHSC

migration significantly decreased in only the baicalein treated group. The MCP-1 synthesis inhibitor, Bindarit, was used as a positive control in both assays.

4. Discussion The present study established a platform for screening the potential effect of herb in the MCP-1 gene expression in

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Fig. 5. Major compounds isolated from Scutellariae radix inhibited the secretion of MCP-1 from LPS-stimulated rLSECs. rLSECs were pre-treated with baicalin, baicalein, wogonin, or oroxylin-A for 1 h, then stimulated with LPS (100 ng/mL) for another 18 h. The medium samples were collected and measured by a rat MCP-1 ELISA kit. Data shown are mean 7S.D. from three independent experiments. # p o 0.05, compared with the non-stimulated control. np o0.05, compared with the LPS-stimulated group.

LPS-stimulated rLSECs. Under the activity-guided fractionation, the active components in Scutellariae radix were explored and the effect of the rLSEC conditioned medium on rHSC migration was examined as well. We demonstrated that the gene and protein levels of MCP-1 in the rLSEC cultured medium were significantly enhanced by LPS (Figs. 2 and 5). Baicalein was identified as a major active component in Scutellariae radix, which effectively inhibited rHSC migration by suppressing the expression of MCP-1 mRNA and protein in rLSECs. LSEC is an important scavenger for the removal of pathogenic materials in blood derived from the gastrointestinal tract via the portal vein (Braet and Wisse, 2002). LSECs also regulate vascular homeostasis and sinusoidal structural changes that occur in response to liver injury and chronic liver diseases (Deleve et al., 2008). In normal physiologic situations, LPS tolerance in LSECs contributes to local hepatic control of inflammation (Uhrig et al., 2005). However, when the intestinal barrier function is disrupted due to liver injury, the increased intestinal permeability leads to the translocation of gut microflora-derived LPS into the liver through the portal vein (Lumsden et al., 1988; Dangi et al., 2012), and these HSCs can also interact directly with cells of the immune system and stimulate it to synthesize many mediators (Seki and Schnabl, 2012). Both LSECs and HSCs are both associated with Toll-like receptor four regulated liver fibrosis signaling under LPS-stimulation (Paik et al., 2003; Jagavelu et al., 2010). In a time course study, we found that gene expression of MCP-1 (but not VEGF, VEGFR, or TGF-β1) was potentially enhanced in LPS-induced rLSECs, and its protein level was significantly increased after 18 h stimulation as well (Figs. 2 and 5). MCP-1, also called CC chemokine ligands 2 (CCL2), has been implicated in HSC activation and migration (Marra et al., 1999; Friedman, 2008). Liver-derived chemokines can therefore function in both paracrine and autocrine fashions, further expanding their role in liver diseases (Saiman and Friedman, 2012). Recruitment of activated HSCs to the injured sites and production of ECM such as collagen I are believed to lead to postnecrotic fibrosis in the liver (Lumsden et al., 1988; Lee et al., 2007). HSCs regulate leukocyte trafficking and activation through secretion of chemokines such as MCP-1. MCP-1 expression is elevated in patients with hepatitis (Leifeld et al., 2003) and in experimental animal liver injury models (Paik et al., 2003). Our study indicated that the concentration of MCP-1 in a medium conditioned by LPS-induced rLSECs is 4.8 ng/mL after 18 h culturing.

Fig. 6. Baicalein suppressed rHSC migration. rHSC was cultured for 6 h in either fresh medium or rLSEC conditioned medium, and treated with baicalin, baicalein, wogonin, or oroxylin-A. Cell migration was evaluated by wound-healing assay (A) and transwell chemoattraction assay (B). Sal B and Bindarit were used as positive controls. Data shown are mean 7 S.D. from three independent experiments. #p o 0.05, compared with the non-stimulated control. np o 0.05, compared with the LPS-stimulated group. Φ p o 0.05, compared with the fresh medium.

A wound-healing assay revealed that rHSC migration significantly increased at 5 ng/mL of MCP-1 in human liver stellate cell line LX-2 cells (Supplementary Fig. S3). Sal B, a positive control, has been reported to inhibit smooth muscle cell migration (Sun et al., 2010) and HSC activation (Yang et al., 2008; Tsai et al., 2010). Scutellariae radix is widely used in TCM for treating liver diseases. Previous reports have demonstrated that baicalin, a major flavone glucuronide from Scutellariae radix, induces HSCs apoptosis to suppress liver fibrosis (Pan et al., 2012). The present study demonstrates that Scutellariae radix inhibits LPS-induced rLSEC activation and attenuates rHSC migration through suppressing MCP-1 expression. It is noteworthy that baicalein exhibited better activity than other isolated compounds. A previous in vivo study reported that oral administration of baicalein showed an anti-fibrotic effect through suppressing the PDGFR expression of HSCs in CCl4 liver fibrotic rats (Sun et al., 2010). Moreover, baicalin suppressed LPS-mediated inflammatory responses and thereby ameliorated ischemia/reperfusion-induced liver injury (Kim and Lee, 2012). As is known, baicalin, which is a glucurolide of baicalein, is degraded to baicalein in the GI

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tract, indicating an anti-fibrotic effect (Pan et al., 2012). In our study, baicalin had no significant effect on mRNA and protein expression of MCP-1, even at the concentration of 25 μg/mL (56 μM) (Fig. 4A). On the other hand, baicalin could have an anti-fibrotic effect by a mechanism other than MCP-1. Yang et al. (2012) recently reported that baicalin, one of the active components in Yang-Gan Wan (a Chinese herbal prescription used for preventing liver fibrosis), prevents liver fibrosis by stimulating PPARγ activity in HSCs. A series of flavones including baicalein, oroxylin-A, wogonin, and others isolated from Scutellariae radix can protect the liver from CYPIA2induced metabolic activation of protoxicants (Guo et al., 2007). It has also been reported that wogonin has potent anti-HBV activity, both in vitro and in vivo studies (Kim et al., 2002). In the present study, wogonin suppressed mRNA and protein expressions of MCP-1 in rLSECs, but without significant effect in the inhibition of rHSC migration (Figs. 5 and 6). Moreover, oroxylin-A, an isomer of wogonin, had no significant effect on either of these two activities. Under normal conditions, differentiated LSECs prevent HSCs activation and promote reversion of activated HSCs to quiescence, but capillarization of LSECs promotes HSCs activation (Deleve et al., 2008). HSCs, which are located in the space of Disse, probably influence hepatic sinusoidal blood flow and play a major role in mediating fibrosis and inflammation in an injured liver. HSCs further regulate leukocyte trafficking and activation through secretion of chemokines. LSEC conditioned medium has been reported to contain several growth and pro-inflammation factors, such as TNF-α, IL-6 and MCP-1 (Adrian et al., 2007). In our study, rHSCs were activated by LPS directly and caused cell migration (210% compared with control). However, conditioned medium resulted in more potent rHSC migration than in the directly LPS-induced group (285% vs 210%, po0.05), because the conditioned medium contained 5 ng/mL MCP-1 secreted from LPS-induced LSECs. In addition, baicalein treatment, both in the fresh medium and conditioned medium, suppressed rHSC migration. Baicalein can directly inhibit rHSC migration and decrease MCP-1 secretion from rLSECs. This result shows that baicalein may play multiple functions in liver protective activity. However, the action mechanism remains to be further investigated.

5. Conclusion This is the first investigation on the effects of Scutellariae radix on LPS-induced rLSEC attenuating MCP-1 and the effects of the isolated active components on rHSC migration in an LPS-induced rLSEC conditioned medium. In this study, we found six major flavones that were isolated from the active fractions of Scutellariae radix. Among them, four compounds, baicalein, wogonin, scutellarein and compound 6, inhibited both MCP-1 mRNA and protein expressions. Only baicalein had a significant effect on rHSC migration. However, baicalin, a major component in SBE-BuOH, had no significant inhibitory effects on either rLSEC activation or rHSC migration. This study demonstrates that Scutellariae radix attenuated LPS-induced rLSEC activation and rHSC migration with downregulation of MCP-1 expression. The results provide supporting evidence that Scutellariae radix may be beneficial for ameliorating liver fibrosis.

Acknowledgments This work was supported by the National Science Council (99-2628-B-077-001-MY3) and the National Research Institute of Chinese Medicine (NRICM100-DMC-02) in Taiwan.

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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2013.08.049.

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