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Estrogen receptor-β-dependent effects of saikosaponin-d on the suppression of oxidative stress-induced rat hepatic stellate cell activation

Saikosaponin-d (SSd) is one of the major triterpenoid saponins derived from Bupleurum falcatum L., which has been reported to possess antifibrotic activity. At present, there is little information regarding the potential target of SSd in hepatic stellate cells (HSCs), which serve an important role in excessive extracellular matrix (ECM) deposition during the pathogenesis of hepatic fibrosis. Our recent study indicated that SSd may be considered a novel type of phytoestrogen with estrogen-like actions. Therefore, the present study aimed to investigate the effects of SSd on the proliferation and activation of HSCs, and the underlying mechanisms associated with estrogen receptors. In the present study, a rat HSC line (HSC-T6) was used and cultured with dimethyl sulfoxide, SSd, or estradiol (E2; positive control), in the presence or absence of three estrogen receptor (ER) antagonists [ICI-182780, methylpiperidinopyrazole (MPP) or (R,R)-tetrahydrochrysene (THC)], for 24 h as pretreatment. Oxidative stress was induced by exposure to hydrogen peroxide for 4 h. Cell proliferation was assessed by MTT growth assay. Malondialdehyde (MDA), CuZn-superoxide dismutase (CuZn-SOD), tissue inhibitor of metalloproteinases-1 (TIMP- 1), matrix metalloproteinase-1 (MMP-1), transforming growth factor-β1 (TGF-β1), hydroxyproline (Hyp) and collagen-1 (COL1) levels in cell culture supernatants were determined by ELISA. Reactive oxygen species (ROS) was detected by flow cytometry. Total and phosphorylated mitogen-activated protein kinases (MAPKs) and α-smooth muscle actin (α-SMA) were examined by western blot analysis. TGF-β1 mRNA expression was determined by RT-quantitative (q)PCR. SSd and E2 were able to significantly suppress oxidative stress-induced proliferation and activation of HSC-T6 cells. Furthermore, SSd and E2 were able to reduce ECM deposition, as demonstrated by the decrease in transforming growth factor-β1, hydroxyproline, collagen-1 and tissue inhibitor of metalloproteinases-1, and by the increase in matrix metalloproteinase-1. These results suggested that the possible molecular mechanism could involve downregulation of the reactive oxygen species/mitogen-activated protein kinases signaling pathway. Finally, the effects of SSd and E2 could be blocked by co-incubation with ICI-182780 or THC, but not MPP, thus indicating that ERβ may be the potential target of SSd in HSC-T6 cells. In conclusion, these findings suggested that SSd may suppress oxidative stress-induced activation of HSCs, which relied on modulation of ERβ.


Introduction
Hepatic fibrosis is a reversible stage of numerous chronic liver diseases, which are associated with significant morbidity and mortality (1). Hepatic stellate cells (HSCs) are in close contact with hepatocytes and sinusoidal endothelial cells within the space of Disse, and are considered the primary cells that contribute to excessive extracellular matrix (ECM) deposition during the pathogenesis of hepatic fibrosis (2). Quiescent HSCs are characterized by a lack of proliferation, and vitamin A storage. In response to a fibrogenic stimulus, HSCs undergo an activation process, which includes loss of vitamin A stores, increased proliferation rate, increased ECM protein synthesis and transformation into α-smooth muscle actin (α-SMA)-positive myofibroblast-like cells (3,4). Therefore, HSCs are considered a target for the treatment of hepatic fibrosis (4). Oxidative stress (OS) results from the increased production of reactive oxygen species (ROS), and serves a crucial role in inducing HSC activation and fibrogenic potential (5). ROS are able to stimulate expression of the critical fibrosis-associated gene, transforming growth factor (TGF)-β1, via activating the mitogen-activated protein kinases (MAPKs) signaling pathway (6).
Gender has been identified as an independent risk factor for the progression from fibrosis to cirrhosis (7), which has a male:female ratio ranging between 2.3:1 and 2.6:1. It has previously been reported that estradiol (E 2 ) can attenuate dimethylnitrosamine (DMN)-or carbon tetrachloride (CCl 4 )-induced liver fibrosis (7,8), and significantly inhibit HSC proliferation and transformation (9). Notably, E 2 suppresses hydrogen peroxide (H 2 O 2 )-induced activation of cultured rat HSCs via decreasing lipid peroxide levels (10). However, despite these benefits, the undesirable side effects of estrogen replacement therapy, including increased risk of breast and endometrial cancers, limit its clinical application; therefore, alternative drugs are required (11,12).
Traditional Chinese medicine has been used for thousands of years for the treatment of liver-related diseases. Bupleurum-containing herbal prescriptions, including sho-saiko-to (Xiao Chai Hu Tang) and Chaihu-Shugan-San, have been traditionally used in Asian countries to treat various liver diseases (13)(14)(15). Saikosaponin-d (SSd) is one of the major active pharmacological components extracted from Bupleurum falcatum L., which has been reported to alleviate CCl 4 -induced hepatocyte injury by inhibiting lipid peroxidation (16). Furthermore, it exhibits suppressive effects on hepatic fibrosis in rats, which was induced by CCl 4 injections in combination with alcohol, high fat and low protein feeding, due to its protection against inflammatory hepatocyte injury (17). It has also been reported that SSd may inhibit proliferation and activation of HSC-T6 cells (18). Notably, our previous study demonstrated that SSd can induce estrogen response elements-luciferase activity in MCF-7 cells, thus suggesting that SSd exerts estrogen-like activity (19). However, whether SSd could suppress the activation of HSCs via the estrogen receptor (ER) signaling pathway, and which ER subtype is regulated by SSd in HSC-T6 cells, remains to be elucidated. Therefore, the present study aimed to investigate the effects of SSd on OS-induced activation of HSCs, as well as the underlying mechanisms associated with ERs.

Materials and methods
Materials. SSd (batch number: 110778-201409; purity, >95%) was purchased from National Institutes for Food and Drug Control (Beijing, China). SSd is quite stable at room temperature and retains its activity following exposure to organic solvents, including dimethyl sulfoxide (DMSO). ICI-182780, DMSO, bovine serum albumin, E 2 and phenol red-free Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, uSA). Enhanced chemiluminescence (ECL) kit was purchased from EMD Millipore (Billerica, MA, uSA). TRIzol ® reagent was purchased from Invitrogen; Thermo Fisher Scientific, Inc. ReverTra Ace-α ® reverse transcription (RT) kit and SYBR ® -Green real-time polymerase chain reaction (PCR) master mix were purchased from Toyobo Life Science (Osaka, Japan were routinely cultured in DMEM supplemented with 5% FBS in an atmosphere containing 5% CO 2 at 37˚C. Cells were grown to 85% confluence and were then transferred to phenol red-free DMEM supplemented with 5% sFBS for 2 days, in order to minimize estrogenic activity of the serum. Cells were treated with SSd or E 2 . SSd, E 2 , ICI-182780, MPP and THC were all dissolved in DMSO. All were diluted in the medium immediately prior to use (final concentration of DMSO, <0.1%). DMSO (<0.1%) alone did not have any effect on the parameters measured.
Interaction with ICI-182780, MPP and THC. Some phytoestrogens, including genistein and daidzein, act as agonists and antagonists of ERs (20). In the present study, the effects of SSd alone, as well as its interaction with ICI-182780, MPP and THC, were examined. ICI-182780 is a pure ER antagonist; MPP is an antagonist specific to ERα; THC is an antagonist specific to ERβ. HSC-T6 cells were divided into 4 groups as follows: MTT growth assay. HSC-T6 cells were washed twice with PBS, counted and seeded into 96-well plates at a density of 0.8x10 4 cells/well. After 24 h, the cells completely attached to the wells. Cell proliferation was assessed after 24 h [cells in the OS groups were analyzed following 4 h induction with 0.2 mM H 2 O 2 , as previously described (21,22)]. Briefly, cells were incubated with 100 µl 0.5 mg/ml MTT solution for 4 h at 37˚C. The medium was then discarded and 200 µl DMSO was added for 24 h at room temperature. Absorbance was measured at 570 nm using an ELx800 universal microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, uSA). Cell numbers were obtained as absorbance values. The results were expressed as proliferation of the treated cells relative to the control group.
Detection of MDA, CuZn-SOD, tissue inhibitor of metalloproteinases-1 (TIMP-1), matrix metalloproteinase-1 (MMP-1), TGF-β1, Hyp and COL1 levels in cell culture supernatants. HSC-T6 cells were seeded into 24-well plates (8x10 4 cells/well) and cultured in phenol red-free DMEM containing 5% sFBS. After 24 h, SSd and E 2 were added in the presence or absence of 1 µM ICI-182780, MPP or THC. After 24 h, cell culture supernatants were collected [cells in the OS groups were analyzed following 4 h induction with 0.2 mM H 2 O 2 , as previously described (21,22)] and stored at -20˚C. Subsequently, supernatants were analyzed using ELISA kits according to the manufacturer's protocols. Western blot analysis. HSC-T6 cells were seeded into 6-well plates and cultured in phenol red-free DMEM supplemented with 5% sFBS. Whole cell extracts were prepared using the M-PER lysis buffer (cat. no. 78501; Thermo Fisher Scientific, Inc.). Lysates were then centrifuged at 13,300 x g at 4˚C for 15 min to remove insoluble substances. Protein concentration was quantified using BCA assay according to the manufacturer's protocols. Total protein extracts (50 µg) from each sample were separated by 12% SDS-PAGE followed by electrotransfer onto polyvinylidene difluoride membranes (Immobilon-P; EMD Millipore). Membranes were blocked with 5% skimmed milk, and were incubated with rabbit polyclonal anti-rat extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/p38 and phosphorylated (p)-ERK/p-JNK/p-p38 antibodies (1:1,000), mouse monoclonal anti-rat α-SMA (1:500) or mouse monoclonal anti-β-actin (1:2,000) diluted in 2% BSA at 4˚C overnight. unbound primary antibodies were washed away using Tris-buffered saline containing 0.1% Tween-20. Immune complexes were probed using HRP-conjugated anti-mouse or anti-rabbit secondary antibodies diluted in 5% skimmed milk, which were incubated at room temperature for 2 h and were detected using an ECL western blot procedure (EMD Millipore, Billerica, MA, uSA). Band density was semi-quantified following densitometric analysis of autoradiographs using a Bio-Rad GS-690 Scanner (Bio-Rad Laboratories, Inc.). Optical density values from the experimental groups were expressed as a mean percentage of control values, and differences were calculated by normalizing the density of each band to that of β-actin.

Detection of ROS in HSC-T6 cells. Intracellular
RT-quantitative (q)PCR. HSC-T6 cells were seeded onto 6-well plates and cultured in phenol red-free DMEM containing 5% sFBS. After 24 h, SSd and E 2 were added, in the presence or absence of 1 µM ICI-182780, MPP or THC. Cells in the OS groups were analyzed after 4 h induction with 0.2 mM H 2 O 2 , as previously described (21,22). Total RNA was isolated using TRIzol ® reagent, according to the manufacturer's protocol. Total RNA (3 µg) was used to generate cDNA in each sample using Superscript II reverse transcriptase with oligo(dT) primers (Toyobo Life Science, Osaka, Japan). qPCR was performed to quantify gene expression levels. Each qPCR reaction contained 2 µl diluted cDNA sample, 10 µl SyBR-Green PCR master mix, 0.5 µl 1 µM forward and reverse primers, and 7.5 µl ddH 2 O. For detection of TGF-β1 and GAPDH (housekeeping gene) expression, the following primers were used: TGF-β1, forward 5'-TTG ACT TCC GCA AGG ACC TCG G-3' , reverse 5'-GCG CCC GGG TTA TGC TGC TGG T-3'; and GAPDH, forward 5'-CCT CTA TGC CAA CAC AGT GC-3' and reverse 5'-GTA CTC CTG CTT GCT GAT CC-3' to yield 146 and 194 bp products within 35 PCR cycles, respectively. The amplification process was conducted as follows: Pre-denaturation at 95˚C for 3 min, denaturation at 95˚C for 45 sec, annealing at 50˚C for 45 sec and elongation at 72˚C for 45 sec, a final extension step at 72˚C for 10 min. qPCR was carried out using an ABI Prism 7900 HT Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The ratio of TGF-β1 to GAPDH was used to evaluate the significance of differences among the various groups. PCR results were quantified by using the 2 -∆∆Cq method (23).  Statistical analysis. Results are presented as the means + standard deviation. Data were analyzed using an analysis of variance (one-way ANOvA; for multiple groups) with SPSS version 23.0. If ANOvA revealed an overall effect, intergroup differences were analyzed using Newman-Keuls test. Two-sided P<0.05 was considered to indicate a statistically significant difference.

Effects of SSd on the proliferation and activation of HSC-T6 cells in the presence or absence of ER antagonists.
It has previously been reported that the protective effects of E 2 against hepatic fibrosis may be associated with its inhibition of HSC activation (7-9). As a phytoestrogen, SSd exerts similar effects (18). The results of an MTT assay indicated that SSd and E 2 significantly suppressed (n=4, P<0.05) the proliferation of HSC-T6 cells induced by H 2 O 2 treatment (n=4, P<0.01) (Fig. 1). Western blot analysis indicated that the expression levels of α-SMA, a fibrotic marker that is highly expressed in activated HSCs, were reduced following incubation with SSd and E 2 (Fig. 2). Conversely, these effects were suppressed by ICI-182780 and THC (n=4, P<0.05), but not by MPP.

Effects of SSd on the synthesis and degradation of ECM in
the presence or absence of ER antagonists. TGF-β1 is a key mediator that activates HSCs to produce ECM (1,24). In addition, MMP and TIMP are two important factors that regulate degradation of ECM (25). In the present study, SSd and E 2 were able to markedly decrease TGF-β1 (Fig. 3), Hyp, COL1 (Fig. 3A) and TIMP-1 expression (Fig. 4), and increase MMP-1 expression (Fig. 4), thus inhibiting H 2 O 2 -induced ECM formation.
Conversely, the effects of SSd and E 2 were suppressed by ICI-182780 and THC (n=4, P<0.05), but not MPP.

Effects of SSd on H 2 O 2 -induced OS in the presence or absence of ER antagonists.
OS is recognized as having a crucial role in HSC activation (5). Recent studies indicated that the liver-protective and antifibrotic effects of SSd may be attributed to its antioxidative capacity (26,27). Consequently, the antioxidative effects of SSd on H 2 O 2 -induced OS in HSC-T6 cells were explored. Flow cytometry revealed that the control group had low levels of ROS and that ER antagonists alone had no significant effect. Conversely, groups treated with H 2 O 2 exhibited a significant increase in ROS (n=3, P<0.01), which could be reversed by SSd and E 2 (n=3, P<0.01) (Fig. 5A). MDA, which is an end product of lipid peroxidation, and the endogenous antioxidant CuZn-SOD, which indirectly reflects OS status, were detected by ELISA. The results indicated that increased MDA content induced by H 2 O 2 (n=4, P<0.01) (Fig. 5B) was reduced by SSd and E 2 (n= 4, P<0.05), whereas CuZn-SOD content (Fig. 5B) was increased by SSd and E 2 (n=4, P<0.01). However, the suppressive effects of SSd on OS could be inhibited by ICI-182780 and THC (n=4, P<0.05), but not MPP.
Effects of SSd on the MAPK signaling pathway in the presence or absence of ER antagonists. ROS can upregulate the expression of critical fibrosis-associated genes via activation of signal transduction pathways, including MAPKs (28,29). In the present study, the phosphorylation of three MAPK proteins (p-ERK, p-JNK and p-p38) was examined by western blot analysis, in order to provide further evidence regarding the antifibrotic effects of SSd. As shown in Fig. 2, the relative expression levels of p-ERK, p-JNK and p-p38 to their respective total proteins were increased following H 2 O 2 treatment (n=4, P<0.01), whereas SSd and E 2 were able to significantly downregulate expression levels (n=4, P<0.05). Conversely, these effects were suppressed by coadministration with ICI-182780 and THC (n=4, P<0.05), but not MPP.

Discussion
Liver fibrosis and the final stage of liver fibrosis, cirrhosis, represent the final common pathway of virtually all chronic liver diseases (30)(31)(32). SSd has been reported to possess antifibrotic activity; however, there is currently little information regarding the effects of SSd on HSCs. Therefore, the present study aimed to investigate the effects of SSd on the proliferation and activation of HSCs, and the underlying mechanisms associated with ERs. The results strongly suggested that SSd may suppress OS-induced activation of HSCs in an ERβ-dependent manner. This finding may be of potential clinical interest, since previous studies have indicated that liver fibrosis is potentially reversible with the reduction of HSC activation (1,25).
Sex hormones may affect the development of hepatic fibrosis and cirrhosis (33). E 2 is an endogenous fibrosuppressant, which has been reported to attenuate liver fibrosis in DMN-or CCl 4 -induced rat models (7,8). In addition, previous studies have demonstrated that the protective effects of E 2 against hepatic fibrosis may be associated with its ability to inhibit HSC activation (7)(8)(9). E 2 inhibits intracellular pathways and activation processes stimulated by H 2 O 2 in cultured rat HSCs via its suppressive effect on lipid peroxidation (10). Previous studies have suggested that phytoestrogens, including isoflavones, resveratrol and genistein, may suppress the progression of liver fibrosis due to their weak estrogen-like activities (34)(35)(36). Our previous study demonstrated that SSd exerts estrogen-like actions via activation of the ER signaling pathway (19). The present study further verified that the suppressive effects of SSd on H 2 O 2 -induced activation of HSC-T6 cells could be reversed by coincubation with ER antagonists, thus confirming that the effects of SSd may be ER-dependent.
OS and ROS are predominant factors in HSC activation (37). Previous studies have reported that SSd possesses marked antioxidant activity (16,17,26,27). Consistently, the present study indicated that SSd could inhibit H 2 O 2 -induced OS in HSC-T6 cells, as evidenced by decreased ROS and MDA generation, and increased CuZn-SOD activity.
ROS can upregulate the expression of critical fibrosis-associated genes via activation of signal transduction pathways and transcription factors, including MAPKs, activator protein-1 and nuclear factor-κB (5). Suppression of ERK activation is associated with complete inhibition of HSC proliferation in vitro (38). In the liver, ERK1 is associated with TGF-β1-induced fibrotic signaling in HSCs (39), whereas ERK2 has a key role in hepatocyte survival (40). In addition, JNK inhibition not only prevents TGF-β-induced murine HSC activation and decreases TGF-β signaling in human HSCs in vitro, but also significantly reduces CCl 4 -induced liver fibrosis in vivo (41). p38 MAPK is also associated with ECM synthesis and degradation. It has previously been reported that phosphorylation of p38 MAPK is augmented in activated HSCs, which is involved in TGF-β1-downregulated MMP-13 expression, as well as in upregulated COL1 expression (24,29). In the present study, OS increased the expression levels of p-ERK, p-JNK and p-p38, whereas SSd and E 2 could significantly downregulate these protein levels and hence inhibit activation of the MAPK pathway. These results are in agreement with the aforementioned involvement of the MAPK pathway. In addition, TGF-β1 is a key mediator that activates HSCs to produce ECM (1,24), whereas MMPs and TIMPs regulate ECM degradation (25). In the present study, SSd and E 2 were able to decrease the expression levels of TGF-β1, Hyp, COL1 and TIMP-1, and increase MMP-1 levels. These effects may induce positive remodeling of liver fibrosis.
The physiological effects of estrogens are mediated through two receptor subtypes, ERα and ERβ. It has been reported that only ERβ, not ERα, is expressed in primary cultured rat HSCs (42). However, our previous study detected both ERα and ERβ proteins by immunofluorescence and western blot analysis in the rat HSC line, HSC-T6 (43). This discrepancy regarding the presence of ERα may be due to the use of different HSC lines. Furthermore, the effects of genistein, a phytoestrogen, on OS and mitochondria may be due, at least in part, to increased ERβ presence, and may be due to upregulation of ERβ induced by genistein (44). It has also been demonstrated that 17β-E 2 protects ARPE-19 cells from OS via an ERβ-dependent mechanism (45). These results suggested that activation of ERβ may be associated with the reduction of ROS production. Conversely, ERα activation has been revealed to inhibit high-glucose-induced proliferation of vascular smooth muscle cells by downregulating ROS-mediated ERK activation, thus suggesting that selective activation of ERα is required for reducing OS (46). In the present study, the effects of SSd together with ICI-182780 (pure ER antagonist), MPP (ERα antagonist) or THC (ERβ antagonist) were explored, in order to determine which ER subtype contributes to the suppression of OS-induced HSC-T6 activation. The results revealed that the suppressive effects of SSd on H 2 O 2 -induced activation of HSC-T6 cells could be inhibited by coincubation with ICI-182780 or THC, but not MPP. These results strongly suggested that SSd suppresses OS-induced activation of HSCs, and this effect is largely dependent on modulation of ERβ.
In conclusion, the present study is the first, to the best of our knowledge, to suggest that the suppressive effects of SSd towards OS-induced HSC activation depend on ERβ activity, and may be at least partially attributed to inhibition of the ROS-MAPK signaling pathway. These results provided novel evidence regarding the target of SSd and may establish an experimental basis for the development of novel drugs for the treatment of hepatic fibrosis.