Influence of β-elemene on the secretion of angiotensin II and expression of AT1R in hepatic stellate cells

Abstract

This study aims to investigate the influence of β-elemene on the secretion of angiotensin II (ANG II) and the expression of angiotensin receptor type 1 (AT1R) in hepatic stellate cells (HSCs). In vitro, HSC-T6 were cultured for 24 hours and then treated with different doses of β-elemene (2.5, 5 and 10 mg/L). A control group was also set up. The secretion of ANG II in the supernatant was detected by radioimmunoassay. The mRNA expression of AT1R at 4, 12 and 24 h after treatment was detected by reverse transcription-polymerase chain reaction (RT-PCR), respectively. The protein expression of AT1R was detected by western blot. At the 4th h, the ANG II secretion in the supernatant was significantly inhibited by 10 mg/L β-elemene compared with the control group (P<0.05), while 5.0 mg/L and 2.5 mg/L β-elemene had no inhibitory effect on the secretion of ANG II (P>0.05). At the time point of the 12th h, the secretion of ANG II in the supernatant treated with 10 mg/L and 5.0 mg/L β-elemene was significantly lower than the control (P<0.01, P<0.05). Following the treatment with 5.0 mg/L and 2.5 mg/L β-elemene for 24 h, significant inhibition of ANG II secretion was observed (P<0.05), but 10 mg/L β-elemene had no such effect. β-elemene significantly reduced the amount of AT1R mRNA in HSCs after the treatment for 4, 12, and 24 h in a dose-dependent manner. The expression of AT1R protein also decreased after the treatment with β-elemene for 24 h. β-elemene can inhibit the secretion of ANG II and the gene and protein expression of AT1R, which may be the mechanism by which β-elemene prevents the progress of hepatic fibrosis.

Keywords

liver cirrhosis / beta-elemene / hepatic stellate cells / angiotensin II / receptor, angiotensin, type 1

Introduction

It has been widely recognized that the hepatic stellate cell (HSC) plays a central role in the process of hepatic fibrosis. The activated HSCs stimulate proliferation of HSCs and synthesize massive extracellular matrix (ECM) by secreting some active substances, such as TGF-β1, platelet derived growth factor (PDGF), and angiotensin II (ANG II), which can promote HSC activation and proliferation via positive feedback, and even accelerate the development of liver fibrosis as well as portal hypertension [1].

ANG II is the most important bioactive substance in the renin-angiotensin-aldosterone system (RAAS), and the biological response of ANG II is mediated by its specific receptors, including type 1 and type 2, and mainly by angiotensin receptor type 1 (AT1R). It has been reported that the expression of both AT1R and AT1R mRNA in human liver tissues increases significantly with the degree of liver fibrosis [2]. Therefore, down-regulating the expression of ANG II and AT1R in HSC could be useful for slowing down the process of liver fibrosis.

Zedoary rhizome, a kind of traditional Chinese drug, is commonly used to treat hepatic fibrosis clinically. Many studies show that Zedoary rhizome has an effect of inhibiting hepatic fibrosis and the production of ECM [3, 4]. In addition, in our previous experiment [5], we found that in the CCl₄-induced hepatic fibrosis model group, plasma ANG II and the expression of AT1R mRNA in rat liver tissues increased obviously. However, in the treatment group with the extract of Zedoary rhizome, plasma ANG II and the expression of TGF-β1 and AT1R mRNA in rat liver tissues decreased significantly [6]. These suggest that the anti-fibrosis effects of the Zedoary rhizome extract may be related to inhibition of the secretion of ANG II and down-regulation of the expression of AT1R and TGF-β1. β-elemene, a natural sesquiterpene extracted from Zedoary rhizome, is the active component of Zedoary rhizome. It has several activities such as anti-thrombosis, inhibiting tumor cell proliferation and inducing tumor cell apoptosis. In a previous study [7], β-elemene was found to inhibit the proliferation and induce the apoptosis of HSCs in vitro. Whether β-elemene could influence the ANG II system is still unknown. Therefore, in the current experiment, the effects of β-elemene on the secretion of ANG II and the expression of AT1R in HSC in vitro were observed.

Materials and methods

Cell line

The HSC-T6 cell line (the phenotype was activated HSCs of Sprague-Dauley rats, established by Professor S L Friedman) was gifted by Professor Lieming Xu in Shanghai University of Traditional Chinese Medicine, Shanghai, China.

Reagents

Anti-AT1R monoclonal antibodies (mAb), anti-rat β-actin mAb, anti-mouse IgG peroxidase conjugate, and anti-rabbit IgG peroxidase conjugate antibodies were purchased from Santa Cruz Biotechnology Inc, USA.

Cell culture and drug treatment

HSC-T6 were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% calf serum, 100 unit/mL penicillin, 100 μg/mL streptomycin, 2 mmol/L L-glutamine, in an incubator of 95% air and 5% CO₂ at 37°C. HSCs in logarithmic growth phase were diluted to 1×10⁵ cell/mL and inoculated into 6-well dishes. One column from each plate containing the medium alone served as a blank control, and another column containing cells without drug exposure served as an untreated control. After incubation with fresh medium for 24 h, serial dilutions of β-elemene (2.5, 5.0, and 10 mg/L) were added and the cells were incubated for an additional 4, 12 and 24 h, respectively. All experiments were repeated three times.

Detection of ANG II in the supernatant

The secretion level of ANG II in the supernatant was detected by radioimmunoassay. The ANG II kit was purchased from Beijing Beifang Institute of Biotechnology and the instrument used was a DFM-96 γ RIA counter (10-probe). The experiment was performed and reported by the Nuclear Medicine Laboratory in the Affiliated Union Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China.

RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)

Confluent HSCs in 6-well dishes were used for total RNA extraction. The TRIzol reagent (Gibco BRL, USA) was used according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg sample of total RNA by using Moloney murine leukemia virus reverse transcriptase (Promega, USA). Specific cDNA from the reverse transcriptase reaction product was amplified by using AT1R specific primer (5'-ACGTGTCTCAGCATCGACCGCTACC-3′, and 5′-AGAATGATAAGGAAAGGGAACAAGAA-3′) and β-actin primer (5'-GAAACTACCTTCAACTCCATC-3′ and 5′-CGAGGCCAGGATGGAGCCGCC-3′). Amplification was performed with Taq DNA polymerase (Promega, USA) in a DNA thermal cycler (MJ Research, America). For AT1R, the amplification was initiated by 5 min of denaturation at 94°C for 1 cycle, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. For β-actin, the amplification was initiated by 5 min of denaturation at 94°C for 1 cycle, followed by 35 cycles at 94°C for 30 s, 48°C for 30 s, and 72°C for 1 min. After the last cycle of amplification, samples were incubated for 10 min at 72°C. The amplification products were analyzed by electrophoresis on 1.2% agarose gels and the fragments visualized by ethidium bromide staining. The predicted size of the products was 278 bp for the AT1R gene and 219 bp for the β-actin gene. The gel bands were analyzed by an image-analysis system (Olympus, Tokyo, Japan).

Detection of AT1R protein by western blot analysis

Cell lysates were obtained through lyses of the cell monolayer with sodium dodecyl sulfate (SDS) lysis buffer (2% SDS, 125 mmol/L Tris-HCl, pH 6.8, and 20% glycerol). The lysates were boiled for 5 min and then clarified by a 20 min centrifugation at 4°C. The protein concentration was measured by using a BCA protein assay reagent (Pierce, USA). Equal amounts of protein samples in the SDS sample buffer (1% SDS, 62.5 mmol/L Tris-HCl, pH 6.8, 10% glycerol, 5% β-mercaptoethanol, and 0.05% bromphenol blue) were boiled for 5 min and subjected to reducing SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in Tween 20-Tris-HCl solution (T-TBS, 100 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.02% NaN₃, and 0.2% Tween 20) for 1 h at room temperature. The membranes were incubated with T-TBS containing 5% milk and with the appropriate antibodies. After three washes with T-TBS, the blot was incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The antigen was detected by using the western blot Chemiluminescence Reagent Plus (Pierce, USA), according to the manufacturer’s instruction.

Statistical analysis

Data were analyzed by Dunnett t test followed by univariate analysis of variance test using a commercially available statistics software package (SPSS for Windows, V. 12.0, Chicago, USA). Results were presented as \overline{x}\pm s. A P value less than 0.05 was considered statistically significant.

Results

β-elemene inhibited the secretion of ANG II in HSCs

We first examined the effects of β-elemene on the secretion of ANG II in HSC-T6. At the time point of the 4th h, the inhibitory effect of β-elemene (10 mg/L) on ANG II secretion in the supernatant was significant compared with the control group (P <0.05), while 5.0 mg/L and 2.5 mg/L β-elemene had no inhibitory effect on the secretion of ANG II (P >0.05). At the 12th h, the secretion of ANG II in the supernatant treated by 10 mg/L and 5.0 mg/L β-elemene was significantly lower than the control group (P<0.01, P<0.05). Following treatment with 5.0 mg/L and 2.5 mg/L β-elemene for 24 h, ANG II secretion was significantly inhibited (P<0.05), but 10 mg/L β-elemene had no such effect. These demonstrated the effect of β-elemene on inhibiting ANG II secretion in HSCs, but not in a dose-dependent manner.

β-elemene attenuated the expression of AT1R in HSCs

Most effects of ANG II on hepatic stellate cells are mediated by the ANG II type 1 receptor. To elucidate whether β-elemene inhibited the expression of AT1R of activated HSCs, the mRNA expression of AT1R was detected with semi-RT-PCR. β-elemene significantly reduced the amount of AT1R mRNA in HSCs after treatment for 4, 12, and 24 h (Fig. 1A). To further examine whether β-elemene could attenuate the protein expression of AT1R, the cell proteins were collected following treatment with β-elemene (10, 5.0, and 2.5 mg/L) for 24 h. Western blot analyses confirmed the role of β-elemene in reducing the protein level of AT1R in HSCs (P <0.01, Fig. 1B). These results demonstrated that β-elemene reduced the expression of AT1R in HSC.

Discussion

Liver fibrosis is the common consequence of chronic liver injury of any etiology. HSCs are the major effector cells of hepatic fibrosis and portal hypertension. β-elemene is believed to be the major active component of Rhizoma zedoariae, which is used widely to treat liver diseases in traditional Chinese medicine. Whether β-elemene could be effective against liver fibrosis attracts our interest. In our previous study, we found that β-elemene could attenuate development of liver fibrosis induced by carbon tetrachloride in rats [7]; however the mechanisms underlying its anti-fibrosis activity are not understood. Recent studies have shown that the ANG II systems are involved in liver fibrogenesis [8]. In the present study, we used the HSC-T6 cell line as a model for activated HSCs, and found for the first time that β-elemene significantly inhibited the secretion of ANG II, as well as the mRNA and protein expressions of AT1R in HSC-T6 cells in vitro.

It has been reported that HSCs play an important role in the pathogenesis of fibrosis and portal hypertension. HSCs are perisinusoidal cells which store vitamin A and produce some growth factors, bioactive substances, etc. They can be activated and then convert to cells with a phenotype similar to myofibroblasts [1]. Once quiescent HSCs are activated, they will present an increased capacity of proliferation, mobility, contractility and synthesizing collagen and other components of the extracellular matrix. The whole process and the effectors are associated with various cytokines and bioactive substances secreted through autocrine and paracrine mechanisms [9]. Thus, the key point of treating liver fibrosis and portal hypertension could be focused on controlling the activation of HSCs and the biological effects caused by some related factors.

The renin-angiotensin-aldosterone system (RAAS), which is an endocrine system, plays a significant role in the management of cardiovascular and renal homeostasis by regulating vascular tone, blood pressure (BP) and fluid volume. The octapeptide ANG II is a physiologically active component of the RAAS, produced through an enzymatic cascade, which begins with renin, an aspartyl protease, cleaving angiotensinogen (AGT) to form the decapeptide angiotensin I (ANG I), which is then cleaved by the angiotensin converting enzyme (ACE), a dipeptidyl carboxypeptidase to form ANG II. The biological responses of ANG II are mediated by its two distinct ANG II receptor subtypes, AT1R and AT2R, both belonging to the G-protein-coupled receptor class with seven transmembrane domains. However, most functions of ANG II are exerted by binding with AT1R. Recently, a number of studies [10-13] supported the existence of numerous organ-based or local RAAS which have diverse physiological effects via autocrine and paracrine secretion. All the components of the RAAS can be found in the liver, heart, vasculature, kidney, etc. [8]. In addition, studies have shown that the local RAAS plays an important role in contributing to the progression of hypertensive heart disease (HHD), heart failure, and renal injury [13]. Likewise, the intrahepatic RAAS also contributes to the development of liver fibrosis, and ANG II is the major active component [8]. In the course of hepatic fibrosis, the up-regulation of the expression of AT1R in HSC and the activation, proliferation, secretion, contraction and synthesis of ECM of hepatic stellate cells, the regulation on a series of cytokines and the inhibition of apoptosis of activated HSC are all correlated with ANG II and the binding of ANG II to AT1R [8,9,12]. Some studies revealed that inhibition of ANG II or angiotensin II type 1 receptor antagonist (AT1RA) could attenuate experimental hepatic fibrosis, and completely block the proliferation, contraction and collagen synthesis in HSC [14-16]. However, it was also reported that after signal transduction of ANG II was blocked in liver cirrhosis patients, a series of adverse consequences would appear, for example, hypotension and a decrease in the glomerular filtration rate (GRE) [17]. For this reason, the long-term application of AT1RA in treating hepatic fibrosis and portal hypertension should be investigated further.

The activated HSCs contain the angiotensin system. Bataller et al. [8] confirmed that ANG II and aldosterone were present in HSCs, and mRNA expression of angiotensin converting AT1R was detectable by PCR. They also found that expression of angiotensin converting AT1R was high in activated HSCs. At the same time, ANG II expression could be detected in the cytoplasm. AT1R is present in HSCs, and ANG II exerts its biological function through binding to this receptor. Additionally, ANG II could stimulate AT1R expression [2,3,8], and induce HSC proliferation, contraction and migration. Therefore, an effective therapeutic strategy for suppressing liver fibrosis should inhibit the biological activity of the ANG II system.

β-elemene is extracted from the Rhizoma zedoariae sesquiterpene derivative, which has been used in activating blood circulation to dissipate blood stasis and alleviate food retention. Modern pharmacological research has shown that β-elemene is a kind of non-cytotoxic anticancer drug and has the activities of antioxidation, regulation of the immune system, anti-thrombosis and dilatation of blood vessels, aside from inhibition of tumor cells. Further, in our previous study we observed that β-elemene can inhibit HSC proliferation and induce its apoptosis [7]. In the current experiment, at the 12th and 24th h, the secretion levels (pmol/L) of ANG II treated with β-elemene in the supernatant were lower than that in the control group (P<0.05), but the inhibitory effect was not dose-dependent on the concentration of β-elemene. When the HSCs were cultured for 4, 12 and 24 h, β-elemene at the concentrations of 2.5, 5 and 10 mg/L could significantly depress the mRNA and protein expressions of AT1R in HSC in a dose-dependent manner (P<0.05).

In summary, the activated HSC is a key component in the pathogenesis of hepatic fibrosis and portal hypertension. ANG II plays a significant role in functions of HSC. In addition, β-elemene can regulate the secretion of ANG II and down-regulate the mRNA and protein expressions of AT1R in HSC, thus influencing HSC function. Therefore, β-elemene may be a new drug for intervention in the hepatic fibrogenesis cascade.

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