Prevention of TGF-β-induced early liver fibrosis by a maleic acid derivative anti-oxidant through suppression of ROS, inflammation and hepatic stellate cells activation

Current anti-fibrotic effect of antioxidants in vivo is disappointing due probably to the fact that once liver fibrogenesis is established it is too advanced to be reversed by anti-oxidation mechanism. We consider antioxidant may only act on the early phase of fibrogenesis. Thus, we had previously established an early liver fibrosis animal model using an inducible expression vector (pPK9a), which contains TGF-β gene and was hydro-dynamically transferred into mice to induce a transient liver fibrosis. TGF-β1 has been well documented to up-regulate the expression of α2(1) collagen (Col 1A2) gene in the liver via the reactive oxygen species (ROS); the process triggers inflammation, leading to hepatic stellate cells (HSC) activation and liver fibrogenesis. Using our animal model and ROS, cyclooxygenase-2 (Cox-2) and Col 1A2 promoter assays as screening targets, we report here that a maleic acid derivative isolated from the Antrodia camphorata mycelium strongly decreases ROS production, promoter activity of Cox-2 and Col 1A2, intracellular calcium, expression of alpha-smooth muscle actin (α-SMA), Smad4-p-Smad2/3 co-localization in cell nucleus and the DNA binding activity of Sp1. Our results suggest that the maleic acid derivative prevents liver fibrosis at an early phase both in vitro and in vivo through the inhibition of ROS, inflammation and the activation of HSC.


Introduction
Advanced liver fibrosis represents a significant and severe healthcare problem and there are no effective drugs for therapy so far.Hence, prevention of progression of fibrogenesis and revival of endogenous repair activities at an early stage is an important matter for both current and future therapies.Accordingly, it becomes important to have a suitable fibrosis model for studying the initiating stage of fibrosis and finding therapeutic molecules for targeting in early fibrogenesis.
TGF-β1 is one of the powerful and widely distributed profibrogenic mediators and plays predominant role during organ fibrogenesis.In the liver, TGF-β1 transforms hepatic stellate cells (HSC) into myofibroblasts which are the main source of liver extracellular matrix (ECM).In response to TGF-β stimulation HSC induces up-regulated expressions of Col 1A2 and αsmooth muscle actin (α-SMA), the two indicators of fibrosis; the process is mediated by phosphorylation of Smad2 and Smad3 and subsequent nuclear translocation of a Smad containing complex.Moreover, it has also been documented that TGF-β1 up-regulates the expression of Col 1A2 gene in the liver via ROS generation and calcium influx [1,2].The up-regulated Ca 2+ influx is associated with activation of HSC and cell contraction [3].The cascade process triggers inflammation, leading to HSC damages and liver fibrogenesis [4].
Hydrodynamics-based gene delivery is an efficient, simple and convenient transfection procedure for laboratory animals where a large-volume naked plasmid DNA is intravenously injected into the animal tail vein at a high-speed.The method allows the achievement of a high expression level of exogenous gene in the liver [5,6].Using this approach we have recently established a transient and early fibrosis animal model by using a pPk9a plasmid that contains TGF-β1 gene and was transferred via tail vein injection into a testing animal.TGF-β1 expression in this transgenic model was high and transient [5,6].
Various molecules designed to affect the TGF-β system have been successfully used for anti-fibrogenesis studies [7].Blockage of ROS generation by HSC in response to TGF-β and alleviation of the downstream proteins is a strategy to inhibit liver fibrosis.We hence set out to screen small molecules isolated from some herbal medicines with the hope that they may antagonize fibrosis.In the process we used ROS, Col 1A2 and Cox-2 promoter assays as screening targets.Out of a panel of approximately 96 chemical compounds a small maleic acid derivative molecule isolated from the mycelia of Antrodia camphorata (designated here as compound 2) (Fig 1) was found to be a promising candidate molecule for inhibiting liver fibrogenesis.The fruiting body of A. camphorata is a well-known traditional herbal medicine in Taiwan, it has been shown to have antioxidant and anti-inflammatory effects [8][9][10][11].The maleic acid derivative is attributed to be the main component in A. camphorata for the antioxidant and anti-inflammatory activities.Our finding may have interesting clinical implication.

Experimental procedures
Cell line.HSC-T6 cells are immortalized and closely resembling primary stellate cells of rat liver.This cell line is a generous gift from Professor S. L. Friedman of the Mount Sinai School of Medicine (NY, USA) [12].The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37˚C with 5% CO 2 in a humidified incubator and medium was replaced every two days.
Mice.Mice of BALB/c strain were used as a transient fibrosis animal model for this study.All procedures of animal handling were approved by the Institutional Animal Care and Use Committee of the National Cheng Kung University.The mice (8 weeks old) were divided into seven groups (n = 21), fed ad libitum standard laboratory chow and water with or without 25 mM of ZnSO 4 plus 5% sucrose.Compound 2 was dissolved in DMSO and given intraperitoneally (i.p.) at 30, 150 or 300 μg/kg.For hydrodynamic transfer of pPK9a vectors containing TGF-β1 gene into mice, the vectors were injected through tail vein simultaneously with ZnSO 4 and compound 2 [13].
Chemical.Compound 2 was isolated and purified from A. camphorata mycelium by the Simpson Biotech Co. Ltd., Taiwan [14].The compound is a maleic acid derivative with a formula shown in Fig 1 .cDNA constructs.A pPK9a vector containing TGF-β1 cDNA is a gift from Professor Paturu Kondaiah.TGF-β1 gene is triggered by the promoter of metallothionein gene, which is expressed in liver.As indicated by reference [15], both Cys 223 and Cys 225 in the TGF-β propeptide were converted into serine, resulting in the dissociation of the pro-peptide and de novo secretion of bioactive TGF-β1 (25 KD).The bioactive TGF-β1 significantly stimulates the activation of a 353-bp fragment of the Col 1A2 gene promoter [16].The -353/-1 fragment constructed into the pGL2-basic vector is a generous gift of Professor Maria Trojanowa.The promoter-reporter construct pxc918 containing -918/+49 COX-2 promoter fragment is also a gift from Dr. Wen-Chang Chang [17].
Measurement of [Ca 2+ ]i in HSC-T6 cells.Observation of [Ca 2+ ]i under the treatment of TGF-β and compound 2. Cells were first pretreated with compound 2 (30, 150 or 300 nM) for 8 h then loaded with the fluorescent calcium indicator fura-3/AM before the addition of TGF-β1 (2 ng/ml).Cell images were taken using a florescence microscope.
Measurement of ROS production from HSC-T6 cell.To analyze the effects of compound 2 on ROS production after TGF-β treatment, ROS was measured by chemiluminescence (CL) count.CL are highly sensitive and specific to the different kinds of ROS simultaneously, such as HO Á , O 2 Á-and H 2 O 2 .The luminol-dependent CL have been used frequently for the detection of superoxide radical anions in various biological systems.HSC-T6 cells (1 × 10 6 cells) were incubated at 37˚C with different concentrations of compound 2 (0-300 ng/ml) and 2 ng/ml TGF-β for 4 hours.After treatment, HSC-T6 cells were collected and suspended in 0.2 ml PBS.
The CL count was measured in a completely dark chamber of the Chemiluminescence Analyzing System.After determination of a 60-s background level, 0.5 ml of 25 mM luminol in PBS (pH 7.4) was injected into the sample.The CL was monitored continuously for an additional 240 s.Dual-luciferase assay.HSC-T6 cells (1.5 × 10 5 ) were cultured in each well of six-well plates.The calcium phosphate precipitation method was used for transient transfection [18].The plasmids pRL-TK and p353 Lux (Col 1A2-luciferase) or pxc918 Lux (Cox-2-luciferase) were co-transfected into cells.The pRL-TK vector was used to normalize the transfection efficiency.After transfection for 12 h, the medium was changed and the cells were treated with various concentrations of compound 2 (30, 150, 300 nM) and TGF-β (2 ng/ml) for additional 24 h.The cells were washed in PBS for recovering cell lysate by scraping the cells in the presence of 1 × passive lysis buffer (Promega, Madison, WI).Firefly and Renilla luciferase activities were analyzed using the Dual-luciferase assay system (Promega) and measured by a Sirius luminometer (Berthold Detection System, Pforzheim, Germany).
Cellular immunofluorescence imaging.HSC-T6 (1×10 5 cells/well) were seeded into sixwell Lab-Tek II chamber glass slides (Nalge Nunc International, Naperville, IL) with complete medium.After 24-h culture, the medium was changed with fresh medium containing 0.1% FBS.Following treatment with 2 ng/ml of TGF-β1 and various concentrations of compound 2 (30, 150, 300 nM) for 8 h, cells were incubated on ice for 5 min, washed with ice-cold PBS, fixed with 100% methanol at -20˚C, and washed twice at room temperature with PBS.The fixed cells were subjected to immunostaining and observed using a fluorescent or confocal microscopy [19,20].
Hydrodynamics-based gene transfer.Ten mg of pPK9a plasmid was dissolved in 3.0 ml Ringer's solution and injected in bolus into the mouse tail vein in a short duration of 5-7 s according to the hydrodynamics-based transfection protocol as described [5,21,22].Drinking water contains 25 mM of ZnSO 4 for gene-transferred animals to activate the metallothionein promoter and express TGF-β [13].
Histology and immunohistochemistry. Liver tissues of TGF-β gene-transferred mice were embedded in an optimal cutting temperature compound (Miles Inc., Elkhart, IN) and frozen in liquid nitrogen for following experiments.Five mm of cryosections were performed by using cryostats (Leica CM 1800, Nussloch, Germany).Histology and immunohistochemistry were performed as described previously [5].For the detection of α-SMA, a mouse monoclonal antibody (Santa Cruz) was used.Signals were visualized by anti-mouse IgG, horseradish peroxidase labeled secondary antibody, and 3, 3'-diaminobenzidine substrate (Vector Laboratories, Burlingame, CA).All sections were viewed under a microscope (Leica Mikrosysteme Vertrieb GmbH, Bensheim, Germany).
Masson's trichrome staining.Liver specimens from TGF-β gene-transferred mice were treated with 4% paraformaldehyde in PBS and dehydrated in a graded alcohol series.Following xylene treatment, the specimens were embedded in paraffin blocks and cut into 5 μm-thick sections that were stained with Masson's trichrome as described [5,23].
Gel electrophoretic mobility shift assay (EMSA).The preparation of liver nuclear extracts and EMSA was based on the method described previously with minor modifications [20,25].
Statistics. Results were expressed as mean ± SE.Statistical analysis was performed by unpaired Student's t test for pairwise comparisons.A p value <0.05 was regarded as statistically significant.

Effects of compound 2 on intracellular ROS production
The intracellular oxidation state of the cell was analyzed by flow cytometry, when ROS were induced by TGF-β1 (2 ng/ml) in the presence or absence of compound 2. Pretreatment of the cells with 30-300 nM of compound 2 eliminated the ROS generation in a dose-dependent manner (Fig 2A).This result indicates that compound 2 may act as an antioxidant to block TGF-β-induced ROS.

Effects of compound 2 on the promoter activities of Col 1A2 and Cox-2 genes
Transcription factors Sp1, AP-1, and Smad binding sites on the promoter region (-334 to -184) of Col 1A2 have been implicated in TGF-β1 induced transcription in fibroblasts [26].We

Compound 2 directly decreased the level of intracellular calcium ([Ca 2+ ]i) in HSC-T6 cells
Generation of ROS has been reported to be a potential mechanism for alteration of intracellular calcium and up-regulation of ECM protein accumulation [2].Therefore, we examined whether compound 2 could change the concentration of [Ca 2+ ] i in TGF-β treated HSC-T6 cells.After incubation with compound 2 at 30, 150, or 300 nM for 4 h, [Ca 2+ ] i was measured immediately upon addition of TGF-β1 to the cells (Fig 3).[Ca 2+ ] i compared with positive control were significantly reduced.We also used another calcium indicator fura-2/AM to measure real-time intracellular calcium concentration and found the same results (data not shown).The results imply that a trace amount of compound 2 is sufficient enough to block both [Ca 2+ ] i .

Effects of compound 2 on Smad4 and p-Smad2/3 in HSC-T6 cells
Activation of HSC by TGF-β plays a key role in liver fibrosis at the early phase and the activated cells are accompanied with high accumulation of p-Smad2/3 and Smad4 in cell nucleus [2].As shown in Fig 4, compound 2 alleviates both p-Smad2/3 and Smad4 accumulation in the nuclei in a dose-dependent manner and compound 2 also decreased levels of the p-Smad2/3 and Smad4 in cells.The result reveals that compound 2 also affects the activation of fibrosisassociated transcription factors.

Effects of compound 2 on collagen expression in hydrodynamics-based fibrosis mice
We next assessed the effects of compound 2 in vivo using TGF-β transgenic mice.The degree of fibrosis was assessed by using the histopathological analysis of liver section under light Western blot analysis for p-Smad2/3, α-SMA and Cox-2 TGF-β plays a major role in fibrogenesis via the activation of HSC and accumulation of p-Smad2/ 3 in cell nucleus [26,27].Our in vitro study also showed the same finding (Fig 4).Therefore, we subsequently examined the levels of p-Smads and Cox-2 protein as well as HSC activation in vivo.We used immunohistostaining to measure the levels of p-Smads, Cox-2 and α-SMA in liver tissues of transgenic mice.α-SMA was used to detect the HSC activation.We found that compound 2 reduced the levels of p-Smad2/3, α-SMA and Cox-2 proteins in the liver of pPK9a-transferred mice in a dose-dependent manner (Fig 6A & 6B).The results indicate that compound 2 prevent fibrogenic effects of liver by decreasing p-Smad2/3, Cox-2 and HSC activation.

Effects of compound 2 on Sp1 binding activity
TGF-β1 acts as a strong activator of ECM accumulation to induce the Col 1A2 gene expression by increasing the binding activity of a Sp1 and p-Smad2/3-Smad4 complex to Col 1A2 promoter element [27].Since Sp1 is a critical mediator of Col 1A2 expression, we examined if Sp1 was induced in pPK9a-transferred mice treated with ZnSO 4 and reversed by compound 2. The binding activity of Sp1 was performed with EMSA.Fig 6C shows that compound 2 indeed reduced the binding activity of Sp1 in liver in a dose-dependent manner.This result reveals that compound 2 not only affects the transcriptional activity of Smads but also Sp1 to regulate the expression of Col 1A2.

Discussion
ROS intermediates act as signaling molecules of TGF-β in regulation of inflammatory and fibrotic responses [1,2].Therefore, first selective platform is used to ROS screen whether small molecules could act as an antioxidant to block TGF-β-induced ROS in HSC-T6 cells.The second and third selective platforms are the Col1A2 and Cox-2 promoter activities assays.We checked the molecules of TGF-β downstream signal pathway, such as Smads and Sp1, and we also checked the levels of collagen in vivo.In this study, we have demonstrated that compound   6).We hence conclude that compound 2 acting as an oxygen free radical scavenger has antifibrotic effects and can suppress early phase of fibrosis.
The signaling relationship between TGF-β1 and ROS or Ca 2+ has not been well elucidated in detail, although both ROS and Ca 2+ have been documented to play important roles in the early phase of TGF-β1 stimulation [1].It also has been reported that generation of ROS increases [Ca 2+ ] i and up-regulates ECM protein accumulation [2].In this connection three points can be considered from our current work for possible blocking targets of TGF-β signaling pathways in liver fibrosis (Fig 7).First, our results demonstrate that a trace amount of compound 2, acting as an antioxidant, is sufficient enough to impede the generation of ROS (Fig 2 ) and the accumulation of [Ca 2+ ] i (Fig 3).The decrease of [Ca 2+ ] i interferes both the activation of HSC, in which α-SMA expression was reduced (Figs 3, 5 and 6), and the phosphorylation of Smad 2/3 (Fig 6).Secondly, the activation of protein kinase C is also able to induce Cox-2, which although is not addressed in our study but its downstream effecter Cox-2 was observed to be reduced in our study (Fig 6).And lastly, EMSA showed that compound 2 reduced the binding of Sp1 in the liver tissue in a dose-dependent manner, providing direct evidence that compound 2 down regulates the expression of Col 1A2 gene both in vivo and in vitro and therefore retards liver fibrosis induced by TGF-β1.
Our hydrodynamically transgenic mice procedure provides an animal model for studying early and initial fibrosis and the effects of antioxidants which may reverse fibrosis [5,22].Using this three selective platform and early liver fibrosis mice model, we had found two small molecules which are maleic acid derivative and retinoid acid derivative can inhibit fibrogenesis through the antioxidant and anti-inflammatory effects [22] And we had found two small molecules presents closely data in vitro and in vivo, it may cause by two small molecule through the same of tree selective platform.The phenomenon may seem to non-steroid anti-immflamentory drugs althought the drug different but function also similar.Our findings are important in that we present an interesting chemical and demonstrate its effects on the cascade molecular fibrogenetic events following ROS scavenging by compound 2 both in vitro and in vivo.Taken

Fig 2 .
Fig 2. Reduction of ROS generation and promoter activity by compound 2 in TGF-β1-treated HSC-T6 cells.(A) Measurement of ROS under the treatment of TGF-β1 and compound 2. Cells incubated for 4 h in the presence of TGF-β1 and compound 2 were harvested for analyzing the oxidation state by adding 0.5 ml of 25 mM luminol.(B) & (C) Effects of compound 2 at various concentrations on the promoter activity of Col 1A2 (B) and Cox-2 (C) in the presence of TGF-β.The relative luciferase activity was expressed as ratio of firefly to Renilla luciferase activity.Values are represented as mean ± SD.; *, p<0.05, and **, p<0.01, unpaired t test, n = 4, compared with TGF-β only.https://doi.org/10.1371/journal.pone.0174008.g002

Fig 6 .
Fig 6.Effects of compound 2 on TGF-β1 downstream molecules in the livers of pPK9a-transferred mice.The total and nuclear proteins were extracted from the livers of pPK9a-transferred mice administrated with ZnSO 4 water for 2 days.(A) & (B) Detection of protein levels of α-SMA, Cox-2, p-Smad, and GAPDH.Thirty μg of total proteins were subjected to Western blot and GAPDH is used as an internal control.https://doi.org/10.1371/journal.pone.0174008.g006