Selective α1B- and α1D-adrenoceptor antagonists suppress noradrenaline-induced activation, proliferation and ECM secretion of rat hepatic stellate cells in vitro.

AIM
To explore the effects of noradrenaline (NA) on hepatic stellate cells (HSCs) in vitro and to determine the adrenoceptor (AR) subtypes and underlying mechanisms.


METHODS
The distribution and expressions of α1A-, α1B-, and α1D-ARs in HSC-T6 cells were analyzed using immunocytochemistry and RT-PCR. Cell proliferation was evaluated with MTT assay. The expression of HSC activation factors [transforming factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA)], extracellular matrix (ECM) secretion factors [tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagen-Ι (ColΙ)] and PKC-PI3K-AKT signaling components (PKC, PI3K, and AKT) in the cells were detected by Western blotting and RT-PCR.


RESULTS
Both α1B- and α1D-AR were expressed in the membrane of HSC-T6 cells, whereas α1A-AR was not detected. Treatment of the cells with NA concentration-dependently increased cell proliferation (EC50=277 nmol/L), which was suppressed by the α1B-AR antagonist CEC or by the α1D-AR antagonist BMY7378. Furthermore, NA (0.001, 0.1, and 10 μmol/L) concentration-dependently increased the expression of TGF-β1, α-SMA, TIMP-1 and ColΙ, PKC and PI3K, and phosphorylation of AKT in HSC-T6 cells, which were suppressed by CEC or BMY7378, or by pertussis toxin (PT), RO-32-0432 (PKC antagonist), LY294002 (PI3K antagonist) or GSK690693 (AKT antagonist).


CONCLUSION
NA promotes HSC-T6 cell activation, proliferation and secretion of ECM in vitro via activation of Gα-coupled α1B-AR and α1D-AR and the PKC-PI3K-AKT signaling pathway.


Introduction
Liver fibrosis is a chronic liver damage caused by a variety of pathogenic factors [1] . It is a common pathology and a necessary step on the way to liver dysfunction and cirrhosis [2] . The mechanisms of pathogenesis are the excessive production and low degradation of extracellular matrix (ECM), which leads to ECM deposits in liver and the formation of liver fibrosis [3] . The HSC is the most important cell type involved in liver fibrosis [4] . The activation of hepatic stellate cells (HSCs) is central to liver fibrosis, whereas the apoptosis of HSCs can reverse liver fibrosis [5] .
The sympathetic nervous system (SNS) is widely distributed in the body and participates in the regulation of body functions [6] . Liver tissue is rich in autonomic nervous system (ANS) tissue, which is distributed around the Disse cavity [7] . Stoyanova [8] showed that there are many autonomic nerve fibers in the hepatic portal area and hepatic lobule. HSCs express catecholamine biosynthetic enzymes and can synthesize and release noradrenaline (NA), dopamine, 5-hydroxytryptamine (5-HT) and other neurotransmitters [9] . Studies have shown that liver fibrogenesis requires sympathetic neurotransmitters [10] and that SNS blockers significantly reduce liver fibrosis [11] . Numerous evidence is available showing that excessive activation of the SNS is related to the occurrence and development of liver fibrosis [12] .

Cell culture
The HSC-T6 cell line was obtained from the Institute of Clinical Pharmacology, Anhui Medical University. The cells were cultured at 37 °C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) from Gibco BRL Life Technologies Inc (Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO, USA).

Immunocytochemistry
The cells were cultured in 6-well plates containing cover slips. The cover slips were washed twice with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min. Immunohistochemical staining for α 1A -AR, α 1B -AR, and α 1D -AR were performed according to the standard SABC protocol described in the SABC Reagents Kit (Boster, Wuhan, China). Positive expression was detected as brown or yellow staining. PBS was used as a negative control to replace the primary antibodies.
Cell proliferation assay Plated HSC-T6 cells were cultured in the presence or absence of α 1B -AR antagonist CEC (10 -5 mol/L) or α 1D -AR antagonist BMY7378 (10 -5 mol/L) for 12 h. Various concentrations of NA were then added [18,19] . Proliferation was measured with MTT assay (Sigma, St Louis, MO, USA) to determine the effects of NA with or without α 1B -AR and α 1D -AR antagonists on cell proliferation. After treatment, MTT solution (5.0 mg/mL in PBS) was added (20.0 μL/well), and the plates were incubated for another 4 h at 37 °C. The purple formazan crystals were dissolved in 150.0 μL of dimethyl sulfoxide (DMSO) per well. After 10 min, the plates were read on a microplate reader (Biotech Instruments, NY, USA) at an absorbance of 490 nm. Untreated cells were used as a control. The assays were performed in three independent experiments. The cell proliferation was calculated using the following formula: cell proliferation (%)=[(OD of the experimental samples/OD of the control)-1] ×100% (n=3, mean±SD).

Western blot analysis
Proteins were extracted from cells treated with NA in the presence or absence of α 1B -AR or α 1D -AR antagonists and signaling pathway molecule inhibitors using RIPA lysis buffer (Beyotime, Haimen, China) and phenylmethylsulfonyl fluoride (PMSF) (99:1). Each protein sample was mixed with 5× sample  buffer (4:1) (Bio-Rad, Hercules, CA, USA) and heated in boiling water for 10 min. The proteins were separated by 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA), and incubated overnight at 4 °C with various primary antibodies at a 1:500 dilution. The secondary antibody [peroxidase (HRP)-labeled goat anti-mouse or goat anti-rabbit] was added and incubated at 1:30 000 dilution. Immunodetection was performed with an enhanced chemiluminescence system (ECL, Pierce, Rockford, IL, USA) using hydrogen peroxide and luminol as a substrate.

RT-PCR
Total RNA was extracted from HSC-T6 cells using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's protocol. The integrity and concentration of RNA were determined by measuring absorbance at 260 nm and total RNA was stored at -80 °C. RNA was separated for RT-PCR by agarose gel electrophoresis (Thermo, MA, USA). Total RNA from each sample (2 µg) was re-suspended in a final volume of 25 µL of reaction buffer. PCR for GAPDH was performed on each individual sample as an internal control. The PCR conditions were 95 °C for 5 min, followed by 35 cycles of 95 °C for 5 s, 40-60 °C for 30 s and 72 °C for 30 s. After the last cycle of amplification, samples were incubated for 10 min at 72 °C. The amplified RT-PCR products were subjected to electrophoresis at 85 V on 2% agarose gels (Sigma, St Louis, MO, USA) for 30 min. A DNA ladder was used as a molecular marker. Agarose gels were stained with 0.5 mg/mL ethidium bromide (Sigma, St Louis, MO, USA) in TAE buffer. The gel bands were examined using the gel image system (Tanon, Shanghai, China).

Statistical analysis
Statistical analyses were performed using the SPSS 11.0 software program (SPSS Software Products, Chicago, IL, USA). All data are presented as number or mean±SD. Statistical analysis among groups was performed by one-way analysis of variance (ANOVA). Statistical analysis between two groups was performed using Dunnett′s method. The Bliss [20] method was used to determine the linear regression equation to calculate EC 50 . The threshold for statistical significance of differences was P<0.05.
NA promotes HSC-T6 cell proliferation and adrenoceptor antagonists reduce HSC-T6 cell proliferation We plated HSC-T6 cells in the presence and absence of α 1B -AR antagonist CEC (10 μmol/L) or α 1D -AR antagonist BMY7378 (10 μmol/L) for 12 h and then added various concentrations of NA. Using the MTT assay to measure the proliferation of HSC-T6 cells, we found that NA significantly induced HSC-T6 cell proliferation in a concentration-dependent manner compared with the control group (P<0.05) (Figure 2A). The EC 50 was 277 nmol/L ( Figure 2B). The α 1B -AR antagonist CEC and the α 1D -AR antagonist BMY7378 both reduced HSC-T6 cell proliferation compared with the equivalent concentration of NA only (P<0.01) (Figure 2A). The α 1B -AR antagonist may be a competitive antagonist and the α 1D -AR antagonist may be a noncompetitive antagonist, because the HSC proliferation curve moved to the right when the cells were cultured with the α 1B -AR antagonist. With an increased concentration of NA, the curve did not reach the original E max . However, the HSC cell proliferation curve moved in parallel to the right when the cells were cultured with the α 1D -AR antagonist. With an increased concentration of NA, the curve reached the original E max ( Figure 2C).
NA promotes the expression of TGF-β 1 , α-SMA, TIMP-1, PKC, and PI3K protein and the phosphorylation of AKT but α 1 -AR antagonists inhibit their expressions in HSC-T6 cells To explore the effects and possible mechanisms of action of NA and α 1 -AR antagonists on HSC-T6 cells, and to explore the function of the PKC-PI3K-AKT signaling pathway, we measured the expression of HSC activation factors (TGF-β 1 and α-SMA), ECM secretion factors (TIMP-1) and PKC-PI3K-AKT signaling pathway molecules (PKC, PI3K, and AKT) in HSC-T6 cells after treatment with NA with or without α 1 -AR antagonists. The results showed that NA significantly promoted the expression of TGF-β 1 , α-SMA, TIMP-1, PKC, PI3K protein and the phosphorylation of AKT in HSC-T6 cells (P<0.05). The α 1B -AR and α 1D -AR antagonists significantly inhibited their expression (P<0.05) (Figure 3 and 4).

Discussion
Studies have shown that the SNS is involved in regulation in the liver and plays an important role in the development of liver fibrosis. The HSC could be the target of SNS regulation [21] . Oben et al [11] suggested that leptin deficient ob/ob mice have the characteristic of fibrosis resistance in chronic liver injury, because the expression of NA is low and the activation of the SNS is suppressed in these mice. Drugs that have effects on the SNS may provide new strategies for the clinical treatment of liver fibrosis. We are interested in understanding the effects and mechanisms of SNS action on HSC cells and determining the AR subtypes that play a role in this process. We are interested in finding alternative therapeutic targets to increase drug effectiveness and reduce adverse reactions. Studies have suggested that sympathetic nerve neurotransmitters promote the repair of liver injuries. They also promote the activation of HSCs by coupling with ARs [22] . Sancho-Bru et al [23] confirmed that liver tissue expressed α 1A -AR, α 1B -AR, α 2A -AR, α 2B -AR, β 1 -AR, and β 2 -AR. HSCs also express a variety of adrenoceptor subtypes such as α 1A -AR, α 2B -AR and β 2 -AR. However, Oben et al [18] showed that HSCs express α 1B -AR, α 1D -AR, β 1 -AR, and β 2 -AR. Currently, the distribution and function of adrenoceptor subtypes in liver tissue and HSCs are controversial and need further research. Our study examined this issue further, and we observed the expression of three α 1 -AR subtypes (α 1A -AR, α 1B -AR, and α 1D -AR) in HSCs. We found that α 1B -AR and α 1D -AR are expressed in cell membranes but α 1A -AR not. Previous studies have shown that NA promotes HSC proliferation and inhibits apoptosis in vitro, mainly through α-AR and β 2 -AR [13] . Other results suggested that α 1 -AR and β 2 -AR expression increased in the liver tissue of rats with liver fibrosis [24] . Duan et al [25] also suggested that NA, α 1 -AR, and β 2 -AR were more highly expressed in rat liver tissue with liver fibrosis. α 1 -AR plays important roles in many physiological processes [26] . We studied the various subtypes of α 1 -AR to further define the mechanism of action of the SNS in the development of liver fibrosis. The results showed that blocking either α 1B -AR or α 1D -AR down-regulated the activation, proliferation and secretion of NA treated HSC cells.
The SNS acts through neurotransmitters interacting with different adrenoceptor subtypes, and then activating downstream signaling pathways. α-AR can activate multiple signaling pathways including the phosphoinositide-calcium signal-  ing system, and the PKC signaling system. β-AR can activate the G protein-cAMP-PKA signaling system. Various receptor subtypes also have different characteristics in coupling with G protein. α 1 -AR couples with G αq protein and α 2 -AR couples with G αi protein. β 1 -AR only couples with G αs protein but β 2 -AR couples with G αs and G αi proteins [27] .
Studies of heart failure have found that SNS regulates the apoptosis of myocardial cells through β-AR coupling with G protein [28] . β 1 -AR promoted apoptosis through the mitogen activated protein kinase (MAPK) signaling pathway and β 2 -AR inhibited apoptosis through the PI3K signaling pathway [29] . The PI3K signaling pathway is important in cell proliferation [30] . Studies of this pathway are important for elucidating the mechanisms of action of the SNS in the development of liver fibrosis. We would like to identify new methods for the effective treatment of liver fibrosis.
The PKC-PI3K-AKT signaling pathway regulates platelet derivation growth factor (PDGF) to promote HSC proliferation and secretion [31] . Blocking this pathway can inhibit HSC proliferation and ECM expression, leading to an improvement in patients with liver fibrosis [32] . Marra et al [33] showed that the activation of the PKC-PI3K-AKT signaling pathways promoted the mitosis and chemotaxis of HSC cells. Our  Previous experiments have shown that NA promotes HSC proliferation [34] . We demonstrated this action by MTT and performed further experiments. We found that NA promoted the proliferation of HSCs in a concentration-dependence manner. The EC 50 was 277 nmol/L. Understanding the EC 50 of drugs is important in guiding clinical use of the drug. And 277 nmol/L is approximately 10 -7 mol/L. In our experiments, we used 10 -7 mol/L of NA in co-culture with antagonists or inhibitors to study the effect of NA on HSCs after blocking receptors or signaling pathways. Based on the concentrationeffect curves, we demonstrated that the α 1D -AR antagonist is competitive and the α 1B -AR antagonist is noncompetitive in HSCs. In addition to HSC proliferation, our experiments studied the activation and secretion of HSC-T6 cells. The results showed that NA promoted the activation and secretion of HSC-T6 cells and that the selective α 1B -AR and α 1D -AR antagonists and the inhibitors of PKC-PI3K-AKT signaling pathways  In conclusion, our study demonstrated that, through α 1B -AR and α 1D -AR, NA activates G α protein coupling with the PKC-PI3K-AKT signaling pathway, which might be one of the mechanisms to promote HSC-T6 activation, proliferation and secretion of ECM.