413

Introduction

Liver fibrosis is a scarring process that represents the liver’s response to injury. Over time, this process can result in cirrhosis of the liver in which the architectural organization of the functional units of the liver becomes so disrupted that blood flow through the liver and liver function become disrupted. Once cirrhosis has devel-oped, the serious complications of liver disease may occur including portal hypertension, liver failure and liver can-cer. The risk of liver cancer is greatly increased once cir-rhosis develops and cirrhosis should be considered to be a pre-malignant condition. Cirrhosis and liver cancer are now among the top ten causes of death worldwide, and in many developed countries liver disease is now one

of the top five causes of death in middle-age (Griffiths et al., 2005; Bosetti et al., 2007). Therefore, prevention of the fibrosis is very important in order to improve these conditions.

HSCs activation leads to retinoid acid storage, remod-eling of ECM and production of growth factors and cytok-ines (Tsukada et al., 2006). Suppression of HSC activation has been proposed as a therapeutic target against liver fibrosis (Bataller and Brenner, 2005; Lee et al., 2008). Many studies confirm that the strategy against TGF-β and its intracellular signaling pathway are of primary interest applying ligand scavengers, cytokine antagonist and a variety of large or small molecule synthetic inhibitors of Ser/Thr-kinase of Alk-5 TGF-β receptors to reverse liver

ReseaRch aRtIcle

Antifibrotic effects of protocatechuic aldehyde on experimental liver fibrosis

Chunmei Li1, Wanglin Jiang1,2, Haibo Zhu2, and Jian Hou2

1School of pharmacy, Yantai University, Yantai, P.R.China and 2State Key Laboratory of Long-acting and Targeting Drug Delivery Technologies (Luye Pharma Group Ltd.), Yantai, P.R. China

abstractBackground: Recent studies have demonstrated that transforming growth factor-β1 (TGF-β1) and connective transforming growth factor (CTGF) are associated with the pathophysiology of liver fibrosis. We isolated protocatechuic aldehyde, the major degradation of phenolic acids.

Objective: This study was carried out to investigate the potential antifibrotic effect of Protocatechuic aldehyde (PA) on experimental liver fibrosis in vitro and in vivo, and to explore its possible mechanism.

Materials and methods: Cell proliferation was determined. Type I collagen, type III collagen, transforming growth factor-β1 (TGF-β1) and connective transforming growth factor (CTGF) were measured by ELISA kits in TNF-α stimulated HSCs. In the carbon tetrachloride (CCL4)-induced rat liver fibrosis model, liver fibrosis grade and histopathological changes were evaluated, and biochemical indicators were determined. Furthermore, immunostaining and Western blot analysis were used to detect hepatic TGF-β1 and CTGF expression in liver tissue.

Results: Overall, our results indicated that PA inhibits HSCs proliferation, inhibits the levels of TGF-β1, CTGF, type I collagen and type III collagen in TNF-α stimulated HSCs. Treatment of PA causes a significant reduction in fibrosis grade, ameliorates biochemical indicators and histopathological morphology, and reduces liver TGF-β1 and CTGF expression in rat model of CCL4-induced liver fibrosis.

Conclusion: These findings suggest that PA has potentially conferring antifibrogenic effects.

Keywords: Liver fibrosis, protocatechuic aldehyde, transforming growth factor-β1, connective transforming growth factor

Address for Correspondence: Associate Prof. Wanglin Jiang, School of Pharmacy, Yantai University, 32# Qingquan Road, Laishan District, Yantai 264003, P.R. China. Tel: +86-535-3808307. Fax: +86-535-3808307. E-mail: davidjiangwl@163.com

(Received 15 April 2011; revised 06 July 2011; accepted 23 July 2011)

Pharmaceutical Biology, 2012; 50(4): 413–419© 2012 Informa Healthcare USA, Inc.ISSN 1388-0209 print/ISSN 1744-5116 onlineDOI: 10.3109/13880209.2011.608193

Pharmaceutical Biology

2012

50

4

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419

15 April 2011

06 July 2011

23 July 2011

1388-0209

1744-5116

© 2012 Informa Healthcare USA, Inc.

10.3109/13880209.2011.608193

NPHB

608193

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fibrosis (Gressner and Gressner, 2008; Gressner et al., 2002; Liu et al., 2006). However, since TGF-β has impor-tant anti-proliferative and anti-inflammatory effects in its normal expression, inhibition of TGF-β may be a double-edged sword. Therefore, alternative targets for therapeutic intervention are needed to treat this com-plication of liver fibrosis. Recent evidence supports that connective tissue growth factor (CTGF), a fibrogenic master switch in fibrotic liver diseases (Li et al., 2006; Gressner and Gressner, 2008), plays a key role in the pathogenesis of liver fibrosis (Paradis et al., 2002).

Protocatechuic aldehyde (PA), a naturally occurring compound, is extracted from the degradation of pheno-lic acids. Many bioactivities of PA have been reported previously. For example, antitumor and antioxidant activity (Kim et al., 2008), protection of vascular endo-thelial cells from ox-LDL induced injury (Han et al., 2007), suppression of TNF-α-induced ICAM-1 and VCAM-1 expression (Zhou et al., 2005). Although previ-ous studies display that PA inhibits TGF-β1 in human lens epithelial cells cultured under diabetic conditions (Kim et al., 2007), inhibits hepatitis B virus replication (Zhou et al., 2007) and may inhibit HSC growth (Lv & Yao, 2006), there are no reports about PA on TGF-β1 and CTGF expression in TNF-α stimulated HSCs or liver fibro-sis animal model. The aim of this study was to investigate the antifibrogenic effect and the possible mechanisms of PA in TNF-α stimulated HSCs and the animal model of carbon tetrachloride (CCL

4)-induced liver fibrosis, to

clarify how to protect liver fibrosis in rats.

Materials and methods

ChemicalsProtocatechuic aldehyde (PA, Formula: C

7H

6O

3, molecuar

weight: 138.12, CAS number: 139-85-5) was obtained from State Key Laboratory of Long-acting and Targeting Drug Delivery Technologies (Yantai, P.R. China). The purity of PA was 99.3% by HPLC. Dulbecco’s modified Eagle’s medium (DMEM, Grand Island, NY, US) and fetal bovine serum (FBS) were purchased from Gibco BRL (Shanghai, P.R. China). The ELISA kits of hyaluronic acid (HA), laminin (LN), collagen types I (PC I) and collagen types III (PC III) were purchased from Beixi Science (Shanghai, P.R. China). The kits of hydroxyproline were purchased from Jiancheng Science (Nanjing, P.R. China). SP kits were purchased from Maixin Biotechnology Company (Fuzhou, P.R. China). HSC-T6 was purchased from Zhongshang University (Guangzhou, P.R. China). All other chemicals were analytical grade and purchased from commercial suppliers.

AnimalsAdult male Sprague-Dawley rats were obtained from Shandong Luye Pharmaceutical Company (Yantai, P.R. China). All animals were housed individually under temperature (20 ± 2°C) and humidity (50 ± 10%) with a 12 h light/12 h dark cycle and had free access to food

and water. The experimental procedures were approved by the institutional Animal Ethics Committee of Yantai University.

HSCs proliferation assayHepatic stellate cells T6 (HSCs) were cultured according to previous methods (Mi et al., 2008). On a 24-well plate, HSCs 5 × 104 cells/mL were seeded in DMEM medium containing 20% FBS and incubated at 37°C in a humidified 5% CO

2 atmosphere. The control group and the control

+PA 27 μM group (no TNF-α 20 ng/mL stimulation) were incubated at 37°C in a humidified 5% CO

2 atmosphere.

After HSCs incubated with TNF-α 20 ng/mL, the cultures were replaced and incubated with PA 1, 3, 9, 27 and 81 μM for 24 h and 48 h, then measured by a SRB assay.

ELISA analysisHSCs were treated with 20 ng/mL of TNF-α with or with-out 1, 3, 9, 27, 81 μM of PA in 0.5% FBS medium for 48 h. Cells were washed with PBS, then collected by scraping and centrifuged at 10000 g for 5 min. The collected cells were lysed with 2 mL of ice-cold lysis buffer (50 mM HEPES, 5 mM EDTA, 100 mM NaCl, 1% Triton X-100, pH 4) for 30 min, then centrifuged at 10000 g for 5 min. The supernatant was collated and the levels of TGF-β1, CTGF, PCI and PCIII were determined by ELISA kits.

CCL4-induced liver fibrosis model and

experimental designFifty adult male Sprague-Dawley rats, aged 6 weeks and weighing 160–200 g. Except for the control rats (n = 10), all rats were subcutaneously injected with 400 g/L CCl

4

(CCl4:olive oil = 2:3) once every 3 days for continuous

6 weeks. The rats were randomly divided into two sub-groups (n = 10) according to body weight and serum alkaline phosphatase (AKP): (I) model group, (II) PA 10 mg/kg group. All animals were treated daily via intra-gastric (i.g.) of corresponding drug for a continuous 4 weeks. After 4 weeks, all rats were anesthetized with 200 g/L urethane (5 mL/kg, i.p.). Blood samples were col-lected. The serum was separated by centrifugation at 4°C and kept at −20°C for assay. Both, their body weight and liver weight were obtained. Liver tissue was stored in cold saline for pathological diagnosis.

Biochemical determinationSerum levels of albumin (ALB), globulin (GLB), ala-nine aminotransferase (ALT) and glutamate-pyruvate transaminase (AST) were measured by routine laboratory methods using AutoLAB Analyzer Medical System (Italy). Serum levels of HA, LN and PCIII were measured by ELISA kits according to the manufacturer’s instructions. The contents of hepatic hydroxyproline (HYP) were measured by the kits according to the manufacturer’s instructions.

Fibrosis grade and histopathological changesLiver condition was classified according to the standard formulated by China Medical Association (1995), and

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fibrosis was graded from 0 to 4 (0: no fibrosis; 1: portal area fibrosis; 2: fibrotic septa between portal tracts; 3: fibrosis septa and structure disturbance of hepatic lobule and 4: cirrhosis). After grading, liver tissue was fixed in a 40 g/L solution of formaldehyde containing 0.1 µM PBS (pH = 7.4), then embedded in paraffin. Five micron thick sections were prepared. All the sections were stained with hematoxylineosin (HE) and masson trichrome. The images were observed under the light microscope.

Immunohistochemical detection for liver TGF-β1 and CTGFImmunohistochemical staining of CTGF and TGF-β1 were performed according to the instructions of the SP kit. The primary antibodies were polyclonal rabbit anti-human TGF-β1 (1:100, Santa Cruze company, US) and goat anti-human CTGF C-terminal peptide anti-body (1:100, R&D company, US). The end compounds reacted with AEC reagent. The slides were counter-stained with haematoxylin and mounted. As a negative control, primary antibody was replaced with PBS.

Western blot analysis for liver TGF-β1 and CTGFThe liver samples were homogenized, and lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 µg/mL phenylmethylsul-fonyl fluoride, 1 µg/mL aprotinin, and 1% Triton X-100. After centrifugation at 1600 g, protein samples (20 μg) were resolved on 10% SDS-PAGE gels and transferred onto a polyvinylidene difluoride (PVDF) membrane in semi-dry system (Bio-Rad company, Hercules, CA). The membranes were incubated with goat anti-human CTGF C-terminal peptide antibody (1:1000, R&D com-pany, US), polyclonal rabbit anti-human TGF-β1 (1: 200, Santa Cruze company, US), and β-actin (1:500, Shanghai Xiangsheng company, P. R. China). β-Actin was used as the loading control. Optical densities of the bands were scanned and quantified with the Gel Doc 2000 (Bio-Rad company, US). Data were normalized against those of the corresponding β-actin. Results were expressed as fold increase over the control.

Statistical analysisThe difference of fibrosis grades between groups were evaluated by Nonparametric Tests. Quantitative data from experiments were expressed as mean ± SD, significance was determined by one-way analysis of AVONA fol-lowed by Tukey’s test. p < 0.05 was considered statistically significant.

Results and discussion

PA inhibited the HSCs proliferation in a concentration-dependent manner at the concentration of 3–81 μM, as shown in Figure 1. As time increases, HSCs prolifera-tion was attenuated, with IC

50 values of 24.5 μM at 24 h

and 12.9 μM at 48 h. Those data indicated that PA plays

sustained and stable inhibition of the HSCs proliferation in a time- and concentration-dependent manner.

We evaluated the effect of PA on the release of TGF-β1, CTGF, PCI and PCIII in TNF-α-stimulated HSCs, as shown in Table 1. The results indicated that TNF-α-stimulated HSCs cultured for 48 h obviously increased the levels of CTGF, PCI and PCIII. However, treatment of PA from 3 to 81 μM, the levels of CTGF, PCI and PCIII were markedly decreased in a concentration-dependent manner. Among them, TGF-β1 was decreased only from 27 to 81 μM.

Histopathologic changes of liver sections were observed by HE staining and Masson’s trichrome stain-ing, as shown in Figure 2A and 2B. Liver tissue samples from the control rats showed normal lobular architecture with central veins, radiating hepatic cords and little colla-gen deposition (Figure 2A1 and 2B1). Liver tissue samples from the model group showed more fibrous tissues and extended into the hepatic lobules to separate them com-pletely, a large number of inflammatory cells infiltrated in the intralobular and interlobular regions, the liver structure was disordered and there were more necrotic, fatty degenerated liver cells and more collagen deposi-tion compared with the controls (Figure 2A2 and 2B2). Compared with the model group, liver fibrosis grade was reduced. Hepatocyte degeneration, necrosis and infiltra-tion of inflammatory cells were all apparently amelio-rated. In addition, collagen deposition was also markedly reduced (Figure 2C and 2D) in the PA 10 mg/kg group.

Serum levels of HA, LN and PCIII were all significantly increased in the animals of liver fibrosis. After adminis-tration of PA 10 mg/kg for 4 weeks, serum levels of HA, LN and PCIII were all significantly decreased, while ALB/GLB was increased, as shown in Table 2.

The liver factor, the contents of hepatic HYP, serum levels of ALT and AST were all significantly increased in the animals of liver fibrosis. As shown in Table 3, after administration of PA 10 mg/kg for 4 weeks, the liver fac-tor, the contents of hepatic HYP, serum levels of ALT and AST were all significantly decreased.

Figure 1. Effect of PA on HSCs proliferation. Confluent HSCs in 24-well plates was incubated with PA 1, 3, 9, 27, 81 μM for 24 and 48 h and measured by a SRB assay. Data represent the means ± SD (n = 6), PA indicated protocatechuic aldehyde. **p < 0.01; **p < 0.01 vs. the control group. Significance was determined by one-way analysis of ANOVA followed by Tukey’s test.

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Figure 2. Effect of PA on pathological progress of liver morphology. (A1–A3): Representative light microscopic appearance of rat liver (hematoxylin staining; original magnification ×200) for control (A1), model (A2) and PA 10 mg/kg (A3). (B1–B3): Representative light microscopic appearance of liver (massion trichrome staining; original magnification ×200) for control (B1), model (B2) and PA 10 mg/kg (B3). (2C): Effect of PA on pathological scores in liver; (D): Effect of PA on collagen expression in liver, results of collagen expression were expressed as fold increase over control group. #p < 0.01 vs the control group; **p <0.01 vs. the model group. Significance of massion trichrome staining was determined by one-way analysis of ANOVA followed by Tukey’s test. Fibrosis scores of differences between groups were evaluated by Nonparametric Tests.

Table 1. Effect of PA on TGF-β1, CTGF, PCI and PCIII expression in TNF-α stimulated HSCs.

GroupConcentration

(μM)TGF-β1 (ng/mL)

CTGF (ng/mL)

PCI (μg/mL)

PCIII (ng/mL)

Control – 2.59 ± 0.73 0.10 ± 0.02 25.17 ± 6.01 2.67 ± 0.45Control + PA 27 2.50 ± 0.65 0.09 ± 0.02 23.98 ± 6.45 2.62 ± 0.49

TNF-α-treated – 5.59 ± 1.06 0.39 ± 0.06 105.23 ± 14.01 7.72 ± 1.03

PA + TNF-α 1 5.13 ± 0.58 0.34 ± 0.04 97.53 ± 12.73 7.15 ± 0.983 4.88 ± 0.81 0.30 ± 0.05a 83.97 ± 14.42a 6.16 ± 1.06a

9 4.50 ± 0.71 0.27 ± 0.06b 69.80 ± 9.98b 5.12 ± 0.73b

27 4.09 ± 0.69a 0.24 ± 0.02b 59.03 ± 7.80b 4.33 ± 0.57b

81 4.08 ± 0.91a 0.19 ± 0.03b 43.74 ± 11.19b 3.87 ± 0.82b

Data represent the means ± SD (n = 6). HSCs treated with or without PA (1, 3, 9, 27, 81 μM) were stimulated or unstimulated with TNF-α (20 ng/mL) for 48 h. TGF-β1, CTGF, PCI and PCIII were determined by ELISA. PA indicated protocatechuic aldehyde. ap < 0.05, bp < 0.01 vs. TNF-α-treated group. Significance was determined by one-way analysis of AVONA followed by Tukey’s test.

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Immunostaining and western blot analysis of CTGF and TGF-β1 expression were low in the control animals, while the CTGF and TGF-β1 expression were higher in the animals of liver fibrosis, as shown in Figures 3 and 4. After administration of PA 10 mg/kg for 4 weeks, the CTGF and TGF-β1 expression were significantly lower compared with the model animals, especially the levels of CTGF expression.

Our present study showed that administration of PA 10 mg/kg for 4 weeks increases ALB/GLB in hepatic injury rats caused by CCl

4. Histological examination

demonstrated that PA ameliorates liver histological change, attenuates the extent of necrosis. Moreover, PA decreased fibrosis grade. It suggested that PA significantly protects liver against fibrosis. In addition, administration of PA 10 mg/kg for 4 weeks decreased the serum levels of

Table 2. Effect of PA on fibrosis grade, serum HA, LN, PCIII and ALB/ GLB.Group Dose (mg/kg) HA (ng/mL) LN (ng/mL) PCIII ng/mL ALB/GLBNormal saline 15.8 ± 3.5 9.2 ± 2.1 15.2 ± 4.2 1.58 ± 0.13Model saline 41.5 ± 6.3 27.8 ± 6.2 40.9 ± 7.9 0.93 ± 0.12PA 10 31.6 ± 7.8b 19.4 ± 3.8b 31.9 ± 4.4b 1.15 ± 0.16b

Data represent the means ± SD (n = 10). PA indicated protocatechuic aldehyde. ap < 0.05, bp < 0.01, vs. the model group. Significance of other data was determined by one-way analysis of AVONA followed by Tukey’s test.

Table 3. Effect of PA in body weight, liver factor and content of HYP in liver tissue.Group Dose (mg/kg) Body weight (g) Liver factor HYP (mg/g) ALT (U/L) AST (U/L)Normal saline 437 ± 54 3.01 ± 0.29 0.35 ± 0.13 25 ± 4 68 ± 9Model saline 287 ± 19 3.98 ± 0.72 1.72 ± 0.50 62 ± 16 160 ± 41PA 10 10 304 ± 21 3.31 ± 0.32a 1.20 ± 0.27b 45 ± 8a 118 ± 24a

Data represent the means ± SD (n = 10). PA indicated protocatechuic aldehyde. ap < 0.05, bp < 0.01 vs. the model group. Significance was determined by one-way analysis of AVONA followed by Tukey’s test.

Figure 3. Effect of PA on liver CTGF and TGF-β1 expression by immunostaining analysis. (A1–A3): Liver immunostaining for TGF-β1 (×200) and (B1–B3): Liver immunostaining for CTGF (×200) for control (A1, B1), model (A2, B2), PA 10 mg/kg (A3, B3). (C): Effect of PA on immunostaining for CTGF and TGF-β1 expression in liver, results were expressed as fold increase over control group. PA indicated protocatechuic aldehyde. #p < 0.01 vs. the control group; **p <0.01 vs. the model group. Significance was determined by one-way analysis of ANOVA followed by Tukey’s test.

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AST and ALT. Overall, PA improves the liver function of fibrotic rats.

Serum levels of HA, LN and PCIII are the important biomarkers reflecting the degree of hepatic fibrosis, included hepatic HYP (Hayasaka & Saisho, 1998; Korner et al., 1996; Murawaki et al., 1996). After administration of PA 10 mg/kg for 4 weeks, the serum levels of HA, LN and PCIII, including the content of hepatic HYP, were reduced. It suggested that PA prevents hepatic fibrosis due to chronic liver injury, thus delaying the develop-ment of cirrhosis.

Suppression of TGF-β1 expression attenuates liver fibrosis. However, systemic application of inhibitors of TGF-β has potentially safety hazards, so it is not suit-able for human therapy (Gressner & Weiskirchen, 2003; Gressner & Gressner, 2008). For the reason described above, CTGF is a more interesting target for future anti-fibrotic therapies (Blom et al., 2002). According to present knowledge, a down-modulation of CTGF activity would shift the TGF-β/BMP-7 balance in the direction of anti-fibrosis, e.g., inhibit ECM synthe-sis, attenuate EMT and HSC-activation, and increase ECM-degradation. CTGF knockdown by gene silencing through siRNA has been shown to suppress HSC activa-tion and ECM accumulation, attenuate Type I and type III collagens, laminin and the serum level of type III procollagen, and alleviate liver fibrosis in two toxic models of rat (Li et al., 2006; George and Tsutsumi, 2007; Uchio et al., 2004). In addition, inhibiting CTGF expres-sion alleviates the severity of liver fibrosis (Li et al., 2010; Gressner & Gressner, 2008). Those studies suggest that inhibiting CTGF and TGF-β1 might be much better in liver fibrosis therapy. Based on these observations, we

evaluated the effect of PA on the expression of TGF-β1, CTGF, PC I and PC III in TNF-α stimulated HSCs in vitro, immunohistochemical staining and western blot analysis of hepatic TGF-β1 and CTGF in vivo. Our study clearly demonstrated that PA markedly inhibits CTGF expression, but suitably inhibits TGF-β1. The analogic results were also confirmed in vitro study. In addition, PA inhibited the expression of PC I and PC III in TNF-α stimulated HSCs.

conclusion

In summary, PA inhibited HSCs proliferation, and reduced TGF-β1, CTGF, PCI and PC III expression in stimulated HSCs. PA increased serum ALB/GLB, decreased serum levels of HA, LN and PCIII, reduced content of HYP in fibrotic liver, and decreased hepatic fibrosis grade. In addition, PA ameliorated liver histological morphology, reduced TGF-β1 and CTGF expression of fibrotic liver.

In conclusion, PA potentially has antifibrogenic effects, the mechanism might be due to the inhibition of CTGF expression and HSCs activation. These findings suggest PA can be a promising drug candidate for the amelio-rating liver fibrosis.

Declaration of interest

The study was financially supported by State Key Laboratory of Long-acting and Targeting Drug Delivery Technologies (Luye Pharma Group Ltd.).

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