Expression of integrin in hepatic fibrosis and intervention of resveratrol

Abstract

The aim of this study was to explore the expression of integrin-β1 in different stages of hepatic fibrosis and intervention of resveratrol as well as the way by which integrin-β1 promoted hepatic fibrosis. Hepatic fibrosis models of male Sprague Dawley (SD) rats were created and intragastric administration of resveratrol was given in low (40 mg/kg), middle (120 mg/kg) and high (200 mg/kg) dose groups. The expression of integrin-β1, tumor growth factor-β (TGF-β) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in different stages of hepatic fibrosis was detected by using RT-PCR. The expression of hexadecenoic acid (HA) and precollagen III (pc III) was assayed by radioimmunoassay. The expression of integrin-β1, TGF-β and TIMP-1 was determined in each group. Liver function and pathological sections of each group in different stages of hepatic fibrosis was tested to judge the therapeutic efficacy of resveratrol at different doses. The expression of integrin-β1 in normal control group was low and steady and was not increased as the development of hepatic fibrosis, but it is increased in other groups. The expression levels of integrin-β1 in the model control group (0.878±0.03, P<0.01) and low dose group (0.855±0.04, P<0.01) were higher than other groups, but there was no difference between model control group and low dose group (P>0.05). The expression levels of integrin-β1 and TGF-β in middle dose group and high dose group were higher than other groups (P<0.01). The expression levels of integrin-β1 and TGF-β in model control group and low dose group were lower than the normal control group (P<0.01). The expression levels of TIMP-1 in the model control and low dose groups were higher than the other groups (P<0.01). The expression levels of TIMP-1 in the middle dose group and the high dose group were lower than the normal control group (P<0.01). The expression of integrin-β1 existed in all stages of hepatic fibrosis of SD rats, and it was increased as the development of hepatic fibrosis. The expression of TGF-β and TIMP-1 was consistent with that of integrin-β1 in different stages of hepatic fibrosis. Resveratrol could improve the degree of hepatic fibrosis of SD rats and decrease the expression of integrin-β1 markedly at a dose of 120 mg/kg.

Keywords

liver fibrosis / integrin-β1 / resveratrol / tumor growth factor-β / tissue inhibitor of metalloproteinase-1

Introduction

Integrin-β1 is one of members of cellular adhesion molecule (CAM) family, the most important receptor of cells through which cells can communicate with the extracellular matrix (ECM) [1]. It plays a very important role in the migration, identification, growth and survival of cells. Recent research indicated that integrin-β1 was highly related with hepatic fibrosis [2-4]. Its functions in hepatic fibrosis appear in two aspects: improving activation of hepatic stellate cell (HSC) and synthesis of ECM, inhibiting the degradation of ECM [5,6]. In this experiment, the expression of integrin-β1 [7], tumor growth factor-β (TGF-β) and tissue inhibitor of metalloproteinase (TIMP-1) in different stages of hepatic fibrosis was observed, and their correlation and change of the expression by intervention of resveratrol were explored.

Materials and methods

Materials

Animals

Sprague Dawley (SD) male rats, weighing (250±50) g, were provided by Shanghai Experimental Animals Center of Chinese Academy of Sciences (China).

Reagents

Resveratrol was purchased from the Shanghai Nabei Biotechnology Co., Ltd (China). Trizol was from Intivigen (USA) and was used to extract total RNA. The two-step RT-PCR kit [RNA PCR KIT(AMV)Ver3.0] was procured from TAKARA (Japan). Integrin β1 mRNA hybridization in situ kit (MK1209), TGF-β mRNA hybridization in situ kit and TIMP-1(r) ELISA kit (EK0583) were all from the Boshide Biotechnology Company (Wuhan, China). Serum pc III radioimmunoassay kit was from Yusheng Company (China).

Methods

Grouping of Rats

One hundred and twenty five SD male rats were divided randomly into 5 groups (25 rats per group): normal control, model control, low dose (resveratrol: 40 mg/kg), middle dose (120 mg/kg) and high dose (200 mg/kg) groups.

Establishment of hepatic fibrosis model

CCl₄ was diluted with liquid paraffin to the concentration of 40%. Hypodermic injection of CCl₄ (0.75 mL/100 g) was performed in the rats for the hepatic fibrosis model, 3 times a week, for a total of 8 weeks.

Intragastric administration of resveratrol

Resveratrol was diluted with sterile distilled water to the concentrations of 10, 30 and 50 mg/mL. Intragastric administration was performed in the low dose group (concentration of resveratrol: 10 mg/mL, dose of intragastric administration: 40 mg/kg), middle dose group (30 mg/mL, 120 mg/kg) and high dose group (50 mg/mL, 200 mg/kg), once a day starting just from the beginning of preparation of hepatic fibrosis model, for a total of 14 weeks.

Extraction of the materials from rat liver

Rats were executed at day 10, 30, 50, 70 and 90 after the beginning of preparation of hepatic fibrosis model, 5 rats each time per group, 1 cm×1 cm×1 cm hepatic tissues were taken and fixed in 4% neutral buffer formalin, and surplus hepatic tissue was frozen at -70°C for reserve.

Liver function test

Alanine aminotransferase (ALT), albumin (ALB), globulin (GLB) and A/G (ALB/GLB) in serum were determined by using the automatic biochemistry analyzer.

Radioimmunoassay of hexadecenoic acid (HA) in serum

Standard liquid or sample product (50 μL) was added into 10 mL test tube and 125I-HA (iodinated 125 hexadecanoic acid) (100 μL) was added into each test tube. Then, the test tubes were put into 37°C water bath for 60 min after misce bene. First antibody (100 μL) was added into the test tubes at 37°C water bath for 30 min after misce bene. Then, the second antibody (100 μL) was added, at 37°C in a water bath for 30 min, centrifuged at 3500 r/min for 15 min. The supernatant was discarded, and the radioactivity (B) of sediments was determined. Standard density was the abscissa and the corresponding B/B0×100 Y-axis, and a standard curve was drawn. The density of HA sample product was on the standard curve.

Radioimmunoassay of pc III in serum

Standard liquid or sample product (100 μL) was added into 10 mL test tube and first antibody (100 μL) was added into the test tubes overnight at room temperature after misce bene. Then, the second antibody (1000 μL) was added, placed at room temperature for 30 min, placed at 4°C for 30 min after shaking and centrifuged at 3500 r/min for 15 min. The supernatant was discarded and the radioactivity (B) of the sediments was determined. Standard density was abscissa, corresponding B/B0×100 was Y-axis, and the standard curve was drawn.

Semi-quantitative RT-PCR

Two-step RT-PCR kit was used. Amplification primer of rat Integrin β1 was as follows: sense, 5'-agtagaggtggtccttcagtt-3'; antisense, 5'-cagtgtagttgggatagcatt-3', which was designed by Primer Premier 5.0. Length of amplification was 401 bp. Amplification primer of rat TIMP-1 was as follows: sense, 5'-atattctgtctggatcggc-3'; antisense, 5'-gcttcgtcatactcctgttt-3'. The amplification primer of rat TGF-β1 was as follows: sense, 5'-TAA TGG TGG ACC GCA ACA ACG-3'; antisense, 5'-CTT GCT GTA CTG TGT GTC CAG-3'. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the reference, and the amplification primer was as follows: sense, 5′-tccctcaagattgtcagcaa-3′; antisense, 5′-agatccacaacggatacatt-3′. The length of amplification was 309 bp. The conditions of the reaction were as follows: the initial steps were 95°C for 10 s, followed by a denaturing step for 5 s at 95°C, and an annealing/extension step at 60°C for 30 s performed in 40 cycles. The products of RT-PCR in every group were analyzed by agarose gel electrophoresis, and then photographed by using the Tianneng GIS-1000 digital jelly photograph system. Absorbance (A) values of all straps were determined, and the specific value of A values of every strap were compared, with that of the corresponding GAPDH being used as the last value so as to avoid error by the difference of A values of sample RNA.

Statistical analysis

All data were presented as \overline{x}\pm s. Statistical comparison among groups was made by one-way analysis of variance (ANOVA) by using SPSS12.0 statistical software (Lead Technologies, Inc, USA). A P value less than 0.05 was considered statistically significant.

Results

Pathology

Hematoxylin-Eosin (HE) staining

The staining shows the structure of the liver in normal control group was integrated, while wide degeneration, necrosis of hepatic cells and disappearance of normal structure of the folial were observed in the model control group (Fig. 1). Collagen staining shows that there were different degrees of hepatic fibrosis in resveratrol-treated groups, among which the most serious hepatic fibrosis was found in low dose group, indicating the success of model establishment and intragastric administration (Fig. 2).

Van Giesons (VG) collagen staining

he collagen fibril was red and matrix was stramineous after VG collagen staining. There was almost no red collagen except the vessel wall in the normal control group under light microscope. In the model control group, there was an increase of collagen fibrils in increase liver portal canal and liver parenchyma, and the enlarged red fiber bundle was composed, and arranged in a crisscross pattern. In the high dose group, there was a trifle of red collagen fibrils, which were thin and unshaped reticulate conjunction. In the low dose group, there were a lot of red collagen fibrils except on the vessel wall and they were thinner than those of the model control group. The increase of collagen fibrils in middle dose group was less than that of low dose group but more than that of high dose group (Fig. 2).

Effect of resveratrol on rat liver function

The levels of ALT in the model control group at the 6th and 12th week were higher than those in normal controls. The levels of ALT in the high dose group and the middle dose group at the 6th and 12th week were significantly lower than those in the model control group (P<0.01). The levels of ALB in the model control group at the 6th and 12th week were lower than those in the normal control group. The levels of ALB in the high dose group and middle dose group at 6th and 12th week were significantly higher than those in model control group (P<0.01) (Tables 1-3).

Effect of resveratrol on rat HA, precollagen III (pc III) in serum

The expression levels of HA and pc III in the model control group were significantly higher than those in the normal control group (P<0.01). The contents of HA and pc III in the three resveratrol-treated groups were significantly lower than in the model control group (P<0.01, Tables 4, 5).

RT-PCR

As shown in tables, the expression of integrin-β1 during the whole process of hepatic fibrosis was steady and at a low level, but it was increased in other groups with the development of hepatic fibrosis, and the expression levels of integrin-β1 in model control and low dose groups were significantly higher than the other groups (P<0.01). The expression levels of integrin-β1 in the middle dose group and high dose group were higher than in the normal control group (P<0.01, Table 6). The level of TGF-β in the model control group was higher than in the normal control group (P<0.01, Table 7). The level of TIMP-1 in the model control group was higher than that in the normal control group at the 6th week. The level of TIMP-1 in the high dose group was significantly lower than that in the model control group (P<0.01, Table 8).

The expression of integrin-β1 was increased with the development of hepatic fibrosis, which was consistent with the expression of TIMP-1 and TGF-β (Fig. 3).

The expression of integrin-β1 existed in all stages of hepatic fibrosis of the SD rats, and increased with the development of hepatic fibrosis. Resveratrol could improve the hepatic fibrosis in SD rats and decrease the expression of integrin-β1 markedly at the dose of 120 mg/kg.

Discussion

Hepatic fibrosis is still a tough problem to resolve through medical science. It is now accepted that the process of hepatic fibrosis actually refers to the activation of HSC by the continual stimulation of all impairing factors and a great deal of synthesis of ECM leading to structural change of hepatic lobules [8-10]. Integrin plays a very important role in the signal communications between cells and the ECM which correlates to migration, identification, growth and the survival of cells [11-13]. Integrin is an important cytokine in the process of hepatic fibrosis. Recent research indicated that integrin had an intimate relationship with activation of HSC and degradation of ECM [14,15].

The function of integrin in the process of hepatic fibrosis includes two parts: to promote activation of HSC and synthesis of ECM, and to inhibit degradation of ECM. HSC is the main cell which can produce ECM. Interaction between HSC and ECM is very important during the damage and repair process of hepatic tissues, which is mediated by integrin (receptor of ECM). In the process of hepatic fibrosis, fibronectin is increased firstly, and then activated HSC assembles at the place where the fibronectin deposits and conducts information into the cell through integrin (α4β1, α5β1) which is located on the surface of HSC. That causes the changes in the cytoskeleton of HSC [16,17]. Thus, HSC turns into collagenoblast and myofibroblast. The ability of HSC synthesizing collagen is enhanced and the process of hepatic fibrosis is switched on [18].

Integrin inhibits degradation of EMC. The degradation of EMC depends mainly on matrix metalloproteinases (MMPs) [19]. A key mechanism of hepatic fibrosis is the increased expression of TIMP, which can inhibit the expression of MMPs and lead to the decrease of the degradation of EMC depositing in liver. Some components of EMC can increase the expression of TIMP while decreasing the expression of MMPs during hepatic fibrosis through the mediation of integrin. In addition, EMC as a ligand signaling molecule can bind to integrin located on the surface of the collagenoblast [20,21], affecting the quantity and activity of TGF-β receptor and on the physiological metabolism of collagen through a series of signal transduction [22,23]. While in hepatic fibrosis this adjustment path is blocked. Collagenoblasts produce large quantities of EMC which leads to hepatic fibrosis under continual stimulation of TGF-β [24,25].

The chemical name of resveratrol is 3,4,5-trihydroxysitlbene, molecular formula C₁₄H₁₂O₃, molecular weight 228.25, achromatic color, needle crystal, including four forms, cis form resveratrol, trans form resveratrol, cis form resveratrol glycoside and trans form resveratrol glycoside. The last two forms can be decomposed by glucosidase in the intestinal tract and be turned into resveratrol which reduces its pharmacologic function. The physiological activity of trans-isomer is stronger than cis-isomer and metamer. The trans form resveratrol can be converted to cis form resveratrol under ultraviolet radiation. Resveratrol resides widely in plants, including grapes, peanuts and giant knotweed rhizomes, especially in the cutex of grapes with about 50-100 µg/g, mainly in the trans form of resveratrol.

Recent research shows resveratrol has widely biological pharmacologic activities, including anti-tumor, anti-cardiovascular diseases, anti-mutation, anti-oxygen, anti-inflammation, liver protectin, anti-hepatic fibrosis, inducing apoptosis and hormone-like effects.

The mechanism of action by which resveratrol exerts its anti-hepatic fibrosis effects include: (1) Resveratrol and its glycoside have strong effects in restraining lipid peroxidation, cutting down the lipoids in serum and liver, reducing lipid peroxidate in the body and protecting the liver from damage; (2) Resveratrol has hormone-like effects that can protect the cellular membrane of the liver from damage. Huang [26] studied the protective effect of resveratrol on primary culture liver cells in rats under damage from phenixin and found it could efficiently protect cells from being damaged. At concentrations of 1×10^-7-1×10^-4 mmol/L, it could elevate cell survival notably and at a rate of 88.7% at a concentration of 1×10^-5 mmol/L that is close to level of normal cultured cells. (3) Resveratrol can induce change of liver collagenoblast phenotype, reducing significantly the reduplication of liver collagenoblast and at the same time reducing the expression of collagen protein type-I and metalloprotease 9. It makes significant sense in the treatment of hepatic fibrosis and liver cancer [27]. Besides, resveratrol can make hepatic stellate cells remain in the G1 stage [28] by selective down-regulation of the expression of cyclinD1, which restrains the formation of hepatic fibrosis. Lu [29] studied the effect of resveratrol on rat hepatic fibrosis induced by phenixin through experiments and discovered that the level of hydroxyproline in the liver, the degree of collagenous fibrosis in the liver and the level of ALT in the serum were significantly decreased as compared with the model control group after treatment of resveratrol. It indicated resveratrol had obvious therapeutic effect on hepatic fibrosis caused by phenixin.

Under the intervention of resveratrol, the expression of integrin-β1 was reduced, leading to the decrease of the expressions of both TGF-β1 and TIMP1. Thus, progression of hepatic fibrosis was relieved or inhibited, which may be another important mechanism by which resveratrol exerts its effect on anti-hepatic fibrosis.

To accompany the deep research on the mechanism of hepatic fibrosis, more and more attention is paid to the function of integrin in hepatic fibrosis. Integrin may be the new target site in the therapy of hepatic fibrosis. Resveratrol monomer is thought highly of because of its redundant pharmacologic action and is hoped to be a new drug used to treat hepatic fibrosis [30].

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