Antifibrotic Compounds from Liriodendron tulipifera Attenuating HSC-T6 Proliferation and TNF-α Production in RAW264.7 Cells

Abstract

The inhibition of hepatic stellate cell (HSC) proliferation has been considered as an effective therapeutic target for the treatment of liver fibrosis. The methanolic extract of Liriodendron tulipifera showed significant inhibitory activity against the proliferation of HSCs. Bioactivity-guided isolation afforded twelve compounds including (−)-sesamin (1), (−)-syringaresinol (2), (+)-dihydrodehydrodiconiferyl alcohol (3), salvinal (4), (+)-guaiacylglycerol-8-O-4′-dihydroconiferyl ether (5), (±)-guaiacylglycerol-8-O-4′-sinapyl alcohol ether (6), tanegool (7), (+)-5,5′-dimethoxy-7-oxolariciresinol (8), 3-hydroxy-4-methoxyacetophenone (9), 4-acetoxymethylphenol (10), (−)-paramicholide (11), and blumenol A (12). Among the compounds isolated, 2, 3 and 4 significantly attenuated the proliferation of the activated HSC-T6 cells. The maximal dose of these compounds, however, showed no cytotoxicity in primary cultured rat hepatocytes. Collagen deposition in the activated HSC-T6 cells was reduced by 2, 3 and 4. Also, the increased production of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α induced by lipopolysaccharide was decreased by 3 and 4 in RAW264.7 macrophage cells. Collectively, (−)-syringaresinol (2), (+)-dihydrodehydrodiconiferyl alcohol (3), and salvinal (4) isolated from L. tulipifera leaves and twigs exhibited selective antifibrotic activities toward the activated HSCs and suppressed TNF-α production in RAW264.7 macrophages. These compounds may be useful candidates for developing therapeutic agents for the prevention and treatment of hepatic fibrosis.

Hepatic stellate cells (HSCs), a non-parenchymal liver cell that resides in the space of Disse, mainly participate to regulate sinusoidal blood flow via contraction by releasing pro-inflammatory, and pro-fibrogenic cytokines.^1,2) In a normal liver, HSCs exist in a quiescent state (qHSCs) that exhibit functions including the storage of vitamin A and retinoid as well as the suppression on extracellular matrix (ECM) formation. During the wound healing response to chronic liver injury from a variety of causes including viral, autoimmune, drug induced, cholestatic and metabolic diseases, HSCs undergo a process called “activation,” by which qHSCs are trans-differentiated into myofibroblast-like activated HSCs. The activated HSCs produce the excessive ECM which leads to liver dysfunction and irreversible cirrhosis. Therefore, suppression of HSC activation and proliferation has been proposed as a therapeutic target against hepatic fibrosis.³⁾

In the course of searching for antifibrotic compounds from natural resources using HSC-T6 cells, an immortalized rat hepatic stellate cell line, as an in vitro assay system, it was found that a methanolic extract of the leaves and twigs of Liriodendron tulipifera inhibited cell proliferation in HSC-T6. L. tulipifera, also well known as the tulip tree or yellow poplar, is the family of Magnoliaceae. Sesquiterpenes and aporphine alkaloids have been isolated from this plant as bioactive constituents with anticancer, antiplasmodial and antimicrobial activities.^4–8) However, there has been no report related to the antifibrotic activity of this plant. Thus, we have attempted to isolate the antifibrotic constituents from the leaves and twigs of L. tulipifera through bioactivity-guided fractionation.

MATERIALS AND METHODS

Plant Material

The leaves and twigs of L. tulipifera were collected from the Medicinal Plant Garden, Seoul National University, Korea, in July 2008. Plant identification was authenticated by Dr. Jong Hee Park, Pusan National University, Korea. A voucher specimen (CS-243) has been deposited in the Herbarium of the Medicinal plant Garden, College of Pharmacy, Seoul National University.

Isolation of Compounds 1–12 from L. tulipifera Leaves and Twigs

The air-dried plant material (6.76 kg) was extracted four times with 80% MeOH for 3 h each in an ultrasonic apparatus. Removal of the solvent in vacuo yielded a methanolic extract (482.5 g). The methanolic extract was then suspended in distilled water and partitioned successively with n-hexane (26.7 g), CHCl₃ (34.8 g), EtOAc (21.5 g), and n-BuOH (70.0 g). The n-hexane and CHCl₃ soluble fractions which showed anti-proliferative activities on the activated HSC-T6 cells were used to elucidate bioactive compounds. The n-hexane fraction was subjected to silica gel column using mixtures of CHCl₃–MeOH of increasing polarity as eluents to give 6 fractions (HI–VI). Compound 1 (9.8 mg) was obtained from HIV by subjecting to C₁₈ RP column chromatography with a gradient elution of MeOH–water (30→100% MeOH), and further purification on Sephadex LH-20 (MeOH). The CHCl₃ fraction was subjected to silica gel column chromatography with a gradient elution of n-hexane–EtOAc–MeOH (H : E=10 : 1→E : M=1 : 1→MeOH) to give 4 subfractions (CI–IV). CII was subjected to C₁₈ RP column chromatography with a gradient elution of MeOH–water (30→100% MeOH). Compound 9 (11.2 mg) was obtained from CII-2 by additional C₁₈ RP HPLC (MeOH−H₂O 10 : 90, 2.0 mL/min, 254 nm). CIII was subjected to silica gel column chromatography with a gradient elution of CHCl₃–MeOH (30 : 1→MeOH) to give 5 subfractions (CIII-1–5). Compound 10 (6.1 mg) was obtained from CIII-5 by additional C₁₈ RP HPLC (MeOH−H₂O 25 : 75, 2.0 mL/min, 254 nm). CIV was subjected to silica gel column chromatography with a gradient elution of CHCl₃–MeOH (50 : 1→MeOH) to give 9 subfractions (CIV-1–9). Fractions CIV-7, CIV-8, CIV-9 were further purified by recrystallization with MeOH to yield compounds 3 (23.7 mg), 5 (2.2 mg), 6 (2.2 mg), 7 (4.1 mg), 8 (8.0 mg), 11 (12.1 mg) and 12 (11.5 mg), respectively. CIV-4 was further subjected to silica gel column chromatography with a gradient elution of CHCl₃–MeOH–water (50 : 4 : 1→MeOH) to give 6 subfractions. Compound 2 and 3 were obtained from CIV-4-2 and CIV-4-3 by recrystallization with MeOH.

Culture of HSC-T6 Hepatic Stellate Cells

An immortalized rat hepatic stellate cell line, HSC-T6 was kindly provided by Prof. SL Friedman (Columbia University, New York, U.S.A.). HSC-T6 cells were maintained in Dulbecco’s modified Eagle’s medinm (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin and 100 µg/mL streptomycin at 37°C in a humidified atmosphere of 95% air–5% CO₂.

Assessment of Cell Viability

For the assay, hepatocytes or HSC-T6 cells were seeded in 48-well plates at a density of 5×10⁴ cells/mL and incubated for 24 h. Cells were treated with the sample to be tested at the concentration as indicated for 24 h or 48 h. Inhibitory effect on the proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were incubated with 2 mg/mL of MTT for 30 min–2 h. Reduction of MTT to formazan was assessed in an enzyme-linked immunosorbent assay (ELISA) plate reader at 450 nm. Data were expressed as the mean of three independent experiments.

Measurement of Cell Proliferation

Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation using a colorimetric ELISA Kit (Roche Diagnostics, GmbH, Mannheim, Germany). HSC-T6 cells were plated and treated as described for MTT assay. At the end of each treatment period, the medium was discarded and the cell pellet was used for assay according to the manufacturer’s instructions.

Measurement of Intracellular Collagen Content

The collagen content was quantified by the Sirius Red-based colorimetric assay.⁹⁾ Briefly, after treatment, the cultured HSC-T6 cells were washed with phosphate buffered saline (PBS), followed by fixation with Bouin’s fluid for 1 h. After fixation, the fixation fluid was removed and the culture dishes were washed by immersion in running tap water for 15 min. The culture dishes were air dried and stained by Sirius Red dye reagent for 1 h on the rocker with the speed of about 5 rpm. Thereafter, the solution was removed and the cultures were washed with 0.01 N HCl to remove non-bound dye. The stained material was dissolved in 0.1 N NaOH and the absorbance was measured at 550 nm against 0.1 N NaOH as a blank.

Determination of Tumor Necrosis Factor (TNF)-α Production

RAW264.7 cells were plated overnight in 12 well plates at a density of 5×10⁵ cells/mL. The cells were treated with the samples to be tested for 1 h before exposure to 100 ng/mL of lipopolysaccharide (LPS). After incubation for 24 h, the supernatants were collected and used for cytokine measurement. The concentration of TNF-α in the culture medium was determined by a mouse TNF enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Jose, CA, U.S.A.).

RESULTS

Isolation of Anti-proliferative Compounds from L. tulipifera

The methanolic extract of L. tulipifera leaves and twigs (6.76 kg) was successively fractionated into n-hexane (26.7 g), chloroform (34.8 g), EtOAc (21.5 g), n-BuOH (70 g), and H₂O fractions. The n-hexane and chloroform soluble fractions showed significant antiproliferative activity, which suppressed the proliferation of HSC-T6 cells to 7.4% and 9.6% of control, respectively, at a concentration of 100 µM (Fig. 1). These two fractions were subjected to repeated column chromatography to yield twelve compounds. The isolated compounds were identified as (−)-sesamin (1), (−)-syringaresinol (2),¹⁰⁾ (+)-dihydrodehydrodiconiferyl alcohol (3),^11–13) salvinal (4),¹⁴⁾ (+)-guaiacylglycerol-8-O-4′-dihydroconiferyl ether (5),¹⁵⁾ (±)-guaiacylglycerol-8-O-4′-sinapyl alcohol ether (6),¹⁶⁾ tanegool (7),^17,18) (+)-5,5′-dimethoxy-7-oxolariciresinol (8),^17,18) 3-hydroxy-4-methoxyacetophenone (9),¹⁹⁾ 4-acetoxymethylphenol (10),^20,21) (−)-paramicholide (11),²²⁾ blumenol A (12),²³⁾ respectively, by comparison of spectroscopic data with the literature values. All compounds except for 2 are reported from L. tulipifera for the first time (Fig. 2).

Fig. 1. The Inhibitory Effects of Total Extract and Subfractions of L. tulipifera Leaves and Twigs on the Proliferation in HSC-T6 Cells

HSC-T6 cells were incubated with total methanolic extract or each fraction at the concentration of 10, 50 and 100 µg/mL for 48 h. Cell viability was measured by the MTT assay. The percent of cell viability (%) was calculated as 100×{(Absorbance of compound-treated)/(Absorbance of non-treated control)}. Results are expressed as the mean±S.D. of three independent experiments, each performed using triplicate wells. * p<0.01 compared to untreated control.

Fig. 2. The Structures of Compounds 1–12 Isolated from L. tulipifera Leaves and Twigs

Anti-proliferative Activities of 1–12 in the Activated HSC-T6

The inhibitory activities of compounds 1–12 isolated from L. tulipifera on the proliferation of the activated HSC-T6 cells were examined using MTT assays. The HSC-T6 cells were treated with each compound at a concentration of 10 and 100 µM for 48 h. The significant cellular loss was observed in compound 2, 3 or 4 treated cells from the concentration of 100 µM at both incubation times (Fig. 3). To confirm the inhibitory effect of 2, 3 and 4 on cellular proliferation, the BrdU incorporation in HSC-T6 cells was measured. The HSC-T6 cells were treated with each compound at a concentration of 10–100 µM for 24 (Fig. 4A) or 48 h (Fig. 4B). The treatment of HSC-T6 with 2 attenuated cellular proliferation from 24 h of incubation. The reduction of proliferation was more significant in 48 h of incubation by the treatment of 2, 3 or 4. The cellular growth of HSC-T6 was arrested to 51.2±0.7, 33.9±1.0 and 27.1±0.8% of untreated control by the treatment of 2, 3 and 4, respectively, at the concentration of 100 µM.

Fig. 3. The Inhibitory Effect of Compounds 1–12 Isolated from L. tulipifera Leaves and Twigs on Cell Viability in HSC-T6 Cells

HSC-T6 cells were incubated with each compound at the concentrations of 10, 100 µM for 48 h. Cell viability was measured by the MTT assay. Results are expressed as the mean±S.D. of three independent experiments, each performed using triplicate wells. * p<0.01, ** p<0.001 compared to untreated control.

Fig. 4. The Inhibitory Effects of 2, 3 and 4 on the Proliferation of HSC-T6

HSC-T6 cells were incubated with 2, 3 or 4 at the concentrations of 10, 20, 50, 100 µM for 24 h (A) or 48 h (B). Cell proliferation was measured by the BrdU incorporation assay. Results are expressed as the mean±S.D. of three independent experiments, each performed using triplicate wells.

The Effects of 2, 3 and 4 on Collagen Deposition in HSC-T6

Excessive production and deposition collagen is one of characteristic features observed in the activated HSCs. To assess the anti-fibrotic activities of 2, 3 and 4, the inhibition on collagen deposit in the activated HSC-T6 cells was measured. The production of intracellular collagen was significantly increased in the activated HSC-T6, which was reduced by the treatment of 2, 3 and 4 for 24 h or 48 h in concentration-dependent manners. The amount of collagen was reduced to 51.5±3.0, 30.7±2.2 and 31.2±1.5 over untreated control cells by the treatment of 2, 3 and 4, respectively, at the concentration of 100 µM (Fig. 5).

Fig. 5. The Effects of 2, 3 and 4 on Collagen Deposition in HSC-T6 Cells

HSC-T6 cells were incubated with 2, 3 and 4 at the concentrations of 20–100 µM for 48 h. The collagen content was measured by the Sirius Red-based colorimetric assay. Collagen content (mg/mg protein) was calibrated from a standard curve based on reference standard. Results are expressed as the mean±S.D. of three independent experiments, each performed using triplicate wells.

The Effects of 2, 3 and 4 on the Viability of Hepatocytes

To exclude the nonselective cytotoxicity of the sample, the effect of 2, 3 and 4 on the viability of primary cultured rat hepatocytes was measured. Hepatocytes were observed intact in response to the treatment with compounds 2–4 for 48 h in the concentration rage of 20–100 µg/mL (Fig. 6).

Fig. 6. The Effect 2, 3 and 4 on Cell Viability in Primary Cultured Rat Hepatocytes

Cells were incubated with each compounds at concentrations of 20–100 µM for 48 h. Cell viability was measured by MTT assay. Data are expressed as the mean±S.D. of three independent experiments, each performed using triplicate wells.

Anti-inflammatory Effects of 3 and 4 in LPS-Stimulated RAW264.7 Macrophage

Pro-inflammatory cytokine, TNF-α originated from the activated macrophages are well correlated with necrosis and apoptosis-related hepatic cell death.^24,25) To understand the possible role of 2, 3 and 4 in hepatic fibrosis, any effects on cytokine production in the activated macrophage cells, RAW264.7 were measured. The treatment of 2, 3 or 4 effectively attenuated the production of TNF-α in LPS-stimulated RAW264.7 cells (Table 1). No cytotoxicity of compounds was found in the concentration range tested.

Table 1. Inhibitory Effects of Compounds 2–4 Isolated from L. tulipifera Leaves and Twigs on TNF-α Production Induced by LPS in RAW264.7 Macrophages

Concentration	10 µM	20 µM	50 µM	100 µM
	TNF-α (ng/mL)
Control	0.25±0.02
LPS	0.68±0.03#
2	0.67±0.08	0.68±0.04	0.59±0.01*	0.56±0.01*
3	0.64±0.07	0.62±0.05	0.51±0.03*	0.47±0.05*
4	0.60±0.05	0.58±0.03*	0.44±0.02*	0.38±0.01*

RAW264.7 cells were pre-treated with each compound for 1 h before exposure to LPS for 24 h. The concentration of TNF-α in culture medium was measured as described in Materials and Methods. The values shown are mean±S.D. of data from three independent experiments. Significant compared to control ^# p<0.01, and to LPS alone * p<0.01.

DISCUSSION

Hepatic fibrosis is well characterized by the increased proliferation, and excessive production and deposition of extracellular matrix (ECM), and HSCs are considered to play pivotal roles responsible for fibrosis.^26–29) As a consequence of liver injury caused by persistent viral and helminthic infection, autoimmune, alcoholism, metabolic agents, HSCs undergo phenotypic transformation from vitamin A-storing quiescent cells into myofibroblast-like proliferative.^26,29) The activated HSCs produce a huge amount of ECM, primarily fibrillar collagens I and III,³⁰⁾ and express alpha-smooth muscle actin that subsequently lead to hepatic fibrosis.²⁶⁾ The accumulation of ECM proteins disturbs the liver architecture by forming a fibrous scar and the subsequent development of cirrhosis with nodules of regenerating hepatocytes, which often leads to progressive loss of liver function.³¹⁾ Hence, antifibrogenic therapy that suppresses the activation of HSCs has been preferentially considered as an attractive target to prevent the pathological progression to cirrhosis in chronic liver diseases.^32,33)

HSC-T6 cells, an immortalized rat hepatic stellate cell line, retains major features of activated stellate cells, including expression of desmin, α-smooth muscle actin, and glial fibrillary acidic protein, and it can esterify retinol into retinyl esters.³⁴⁾ The activation of HSCs can be promoted by addition of serum, cytokines and other factors,^32,33) and also can be induced by the specific culturing conditions in vitro. Culturing HSCs on uncoated plastic plates is known to cause spontaneous activation leading to myoblastic phenotype, mimicking the process seen in vivo. In the present study, the accelerated proliferation of HSCs by culturing on plastic plate was found to be attenuated by the treatment of 2–4 isolated from L. tulipifera leaves in MTT and BrdU incorporation assay. Among the compounds isolated, 3 and 4, dihydrobenzofuran neolignans showed the potent inhibitory effects. The proliferation of HSCs was decreased to about 30% over untreated control by the treatment with 3 or 4 at the concentration of 100 µM. The increased deposit of intracellular collagen in the activated HSCs was also decreased significantly by the treatment of 3 or 4 in dose-dependent manners.

It has been reported that cytoskeletal protein such as tubulin and lamin A plays an important role in maintaining cell structure and function, and cell proliferation including the formation of spindles.^35,36) Under the condition that the microtubule dynamics is disturbed, cell cycle arrest can be induced which lead to apoptosis. From the class of lignan and neolignan, several potent leads such as podophyllotoxin, silymarin, phyllanthin, hypophyllanthin and cleomiscosins have been obtained as hepatoprotective and/or anticancer agents.^37–41) Especially, the compounds possessing 3,4,5-trimethoxyphenyl unit in the structure has been known to be potent as antitubulins that inhibit microtubule assembly. In the present study, dihydrobenzofuran neolignans, 3 and 4 exhibited potent inhibitory activities on the proliferation of the activated HSCs. From the earlier findings that lignan and neolignan possesses the capability interacting with the tubulin, the involvement of microtubulin dynamics in hepatoprotective effects of 3 or 4 in liver fibrogenesis might be interesting study.

During liver fibrogenesis in patients with fulminant hepatic failure or chronic liver diseases, a variety of cytokines have been associated with profibrotic or antifibrotic functions.⁴²⁾ It has been known that pro-inflammatory macrophages (type M1) polarized by cytokines from Th1 cells might positively regulate hepatic fibrosis.^34,43) Upon activation, macrophage releases various growth factors, cytokines, and lipid mediators that promote inflammatory process by directing cellular migration to the site of inflammation. TNF-α is one of the important inflammatory cytokines required for the synergistic induction of nitric oxide (NO) synthesis in LPS-stimulated macrophage. TNF-α exhibits its pro-inflammatory activity by regulating several intercellular and vascular cell adhesion molecules, which results in the recruitment of leukocytes to sites of inflammation.⁴⁴⁾ TNF-α produced by macrophages activates nuclear factor-kappa B (NF-κB) signaling via the activation of the IκB-kinase complex. Several in vitro studies have proposed that HSC activation is associated with the elevation of NF-κB activity.⁴⁵⁾ The elevated hepatic level of TNF-α is observed in acute and chronic liver diseases demonstrating the crucial role of TNF-α related NF-κB signaling in HSCs activation.⁴⁶⁾ In this regard, anti-TNF strategy is believed to be beneficial for treating hepatic fibrosis. In LPS-stimulated RAW264.7 macrophase cells, the production of TNF-α in culture media was rapidly elevated, which were decreased by the pre-treatment of 3 and 4 in dose dependent manners.

On the basis of above result, the compounds isolated from L. tulipifera, especially dihydrobenzofuran neolignans, (+)-dihydrodehydrodiconiferyl alcohol (3), salvinal (4) are believed potent to inhibit the proliferation of HSCs and the activation of macrophages. These compounds might be useful candidates to develop the therapeutic agent against liver fibrosis.

Acknowledgment

This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009804) Rural Development Administration, Republic of Korea.

Conflict of Interest

The authors declare no conflict of interest.

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