Matrine Inhibits Infiltration of the Inflammatory Gr1hi Monocyte Subset in Injured Mouse Liver through Inhibition of Monocyte Chemoattractant Protein-1

Matrine (Mat) is a major alkaloid extracted from Sophora flavescens Ait, an herb which is used in the traditional Chinese medicine for treatment of inflammation, cancer, and other diseases. The present study examined the impact of Mat on the CCl4-induced hepatic infiltration of Gr1hi monocytes to explore the possible mechanisms underlying its anti-inflammatory and antifibrotic effects. The results indicated that Mat protected mice from acute liver injury induced by single intraperitoneal injection of CCl4 and attenuated liver fibrosis induced by repeated CCl4 injection. Meanwhile, the infiltrations of Gr1hi monocytes in both acute and chronic injured livers were all inhibited, and the enhanced hepatic expression of MCP-1 was suppressed. Cellular experiments demonstrated that Mat directly inhibited MCP-1 production in both nonparenchymal cells and hepatic stellate cells derived from CCl4-injured livers. Transwell chemotaxis assays showed that Mat significantly inhibited the chemotactic activity of MCP-1. These results suggest that the anti-inflammatory and antifibrotic effects of Mat could be contributed, at least in part, to its prevention of Gr1hi monocyte infiltration into the injured livers and inhibition of MCP-1 production and activity. These findings extend our understanding of the mechanisms underlying the anti-inflammatory and antifibrotic effects of Mat.


Introduction
Chronic hepatic injury may lead to liver fibrosis [1]. After an acute liver injury, parenchymal cells regenerate and replace dead cells. This process is associated with an inflammatory response and a limited deposition of extracellular matrix (ECM). If hepatic injury persists, liver regeneration eventually fails and hepatocytes are substituted with abundant ECMs. Activated hepatic stellate cells (HSCs), portal fibroblasts and myofibroblasts, have been identified as major ECMproducing cells in injured livers [2]. Activation of liverresident macrophages, the so-called Kupffer cells (KCs), has been indicated as an initial event in the process leading to liver injury and fibrosis caused by different etiologies [3]. It is well established that activated KCs play an important role in perpetuating an inflammatory phase resulting in the massive release of proinflammatory and fibrogenic mediators as well as activation of HSCs [4][5][6][7]. However, recent studies demonstrate that these actions are only partially conducted by KCs, but they largely depend on recruitment of monocytes into the livers [8,9].
Blood monocytes are circulating precursors of tissue macrophages. Macrophages and monocytes are characterized by lack of lymphocyte markers and by expression of CD11b and CD14 in humans and of CD11b and F4/80 in mice [10][11][12]. Murine monocytes can be subdivided by their expression of Gr1 and of the chemokine receptors CCR2 and CX3CR1. Gr1 hi monocytes express high levels of C-C chemokine receptor CCR2 but lack CX3CR1, whereas Gr1 lo monocytes lack CCR2 but express high levels of CX3CR1. Their counterparts in humans are CD14 ++ CD16 − CCR2 + and CD14 + CD16 + CCR2 − monocytes, respectively. Gr1 hi monocytes actively enter inflamed tissue and are considered precursors for macrophages and dendritic cells in inflammatory conditions, whereas Gr1 lo monocytes home to noninflamed 2 Evidence-Based Complementary and Alternative Medicine tissues and may represent steady-state precursor cells for tissue macrophages [12,13]. Differential recruitment of these monocyte subsets appears to be crucially controlled by chemokine released from injured tissue. It has been suggested that CCR2 mediates entry of inflammatory Gr1 hi monocytes into inflamed tissues [14][15][16][17][18][19][20][21]. More importantly, enhanced hepatic expression of monocyte chemoattractant protein-1 (MCP-1), a specific ligand of CCR2, has been shown to contribute to the formation and maintenance of inflammatory infiltrate during chronic liver disease [22].
A more recent study further demonstrates that inflammatory Gr1 hi but not Gr1 lo monocytes are massively recruited into the carbon tetrachloride-(CCl 4 -) injured livers in a CCR2-dependent manner [18]. Furthermore, hepatic Gr1 hi monocyte-derived cells in CCl 4 -injured livers exert proinflammatory and pro-fibrogenic actions, such as promoting HSC activation, TH1 cell differentiation, and TGF-release, during liver fibrogenesis. Impaired monocyte subset recruitment in CCR2-deficient mice reduces HSCs activation and diminishes liver fibrosis. Moreover, adoptively transferred Gr1 hi monocytes traffick into the injured livers and promote fibrosis progression in wild-type and CCR2-deficient mice [18]. These experiments provide evidence for a vital role of MCP-1/CCR2-dependent Gr1 hi monocytes infiltration in the development of liver fibrosis upon hepatic injury, thus suggesting that modulation of monocyte subset recruitment into liver may represent an approach for antifibrotic strategy.
The herbal medicine Kushen consists of the dried roots of Sophora flavescens Ait. It was first described in Shen Nong Ben Cao Jing in 200 A.D. as a treatment for inflammation, solid tumors, and many other diseases. In the traditional Chinese medicine, Kushen is commonly used as decoction or powder of dried roots for treatment of a wide variety of conditions including viral hepatitis, cancer, enteritis, viral myocarditis, arrhythmia, colpitis, and eczema [23]. Kushen alkaloids are considered to be its major active components and have been widely used in China for the treatment of hepatitis and cancers. As one of the major Kushen alkaloids, Matrine (Mat) has been demonstrated to posses significant antiinflammatory and antifibrotic properties [24][25][26][27][28][29][30][31][32][33][34]. However, the underlying mechanisms have not been fully elucidated. In the present study, we investigated whether Mat could modulate the recruitment of Gr1 hi monocytes into CCl 4 -injured liver in mice. We herein demonstrated that Mat protected mice against CCl 4 -induced hepatic injury and prevented infiltration of inflammatory Gr1 hi monocytes into the injured livers, possibly through inhibiting the production and activity of MCP-1. These new findings extend our understanding of the mechanisms underlying the anti-inflammatory and antifibrotic effects of Mat. 2.3. CCl 4 -Induced Acute Hepatic Injury Model. C57BL/6 mice were injected i.p. with CCl 4 (0.6 mL/kg body weight, diluted 1 : 3 in corn oil) to induce acute hepatic injury. As normal control, animals received the same volume of corn oil intraperitoneally. To test the protective effect of Mat, mice ( = 8/group) were administrated orally either with Mat (10 mg/kg, 30 mg/kg) in 0.2 mL of PBS or with the same volume of PBS, 3 h prior to CCl 4 injection.

CCl 4 -Induced Chronic Hepatic Injury
Model. C57BL/6 mice were repeatedly injected i.p. with CCl 4 (0.6 mL/kg body weight) twice weekly for 6 weeks to induce chronic liver injury. To test the protective effect of Mat, mice ( = 8/group) were administrated orally either with Mat (10 mg/kg) in 0.2 mL of PBS or with the same volume of PBS, 5 times weekly for last three weeks. Mice were sacrificed 48 h after the the last CCl 4 injection.

Measurement of Serum Alanine Aminotransferase (ALT).
Blood samples were collected at the indicated time points after CCl 4 injection, and serum ALT levels were measured using a colorimetric assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol.
2.6. Hydroxyproline Assay. Hepatic content of hydroxyproline was determined using the Hydroxyproline Testing Kit (Jiancheng, Nanjing, China) according to the manufacturer's protocol.

Histological Analysis.
The mouse livers were removed at the indicated time points after CCl 4 challenge. Liver samples were fixed in 10% formalin solution, embedded in paraffin, and sectioned. Hematoxylin-eosin, Sirius red, and Masson staining, were performed according to standard protocols. For immunohistochemical analysis, liver specimens were fixed in 4% paraformaldehyde, and immunohistochemical staining was performed according to standard procedures using monoclonal hamster anti-mouse CCL2/MCP-1 antibody (BioLegend, San Diego, CA, USA) as the primary antibody and horseradish-peroxidase-conjugated goat antiarmenian hamster IgG (Santa Cruz Biotechnology, Inc., CA, USA) as the secondary antibodies. Sections were counterstained with hematoxylin and evaluated by light microscopy.

Isolation of Nonparenchymal Cells (NPCs) and
HSCs. NPCs were isolated as described previously [35]. Briefly, Evidence-Based Complementary and Alternative Medicine 3 under ether anesthesia, the peritoneal cavity was aseptically exposed, and the inferior vena cava was cannulated. Liver was perfused in situ first with 50 mL of 1x Hank's balanced salt solution (pH 7.4, 37 ∘ C), followed by perfusion with 1% collagenase type IV (Sigma-Aldrich) solution until the hepatic parenchyma beneath the capsule appeared liquefied (approximately for 5 min). After hepatectomy, the liver was transferred to a Petri dish containing 10 mL DMEM medium and was gently minced. This slurry was then filtered (mesh size 70 m) to remove large aggregates. Low-speed (30 g) centrifugation of the liver suspension was performed to exclude hepatocytes, followed by high-speed (500 g) centrifugation to obtain NPCs. After 3 washes, the NPCs were used for flow cytometry analysis or in vitro culture for cytokines induction examination. HSCs were enriched from NPCs by centrifugation over a 3-layer Percoll (GE Healthcare) gradient (52%, 50%, and 30%) as described previously [36]. After centrifugation, HSCs were collected from the interface, washed with Hank's balanced salt solution, and resuspended in DMEM medium supplemented with fetal bovine serum (FBS) (20%), penicillin (100 U/mL), and streptomycin (100 U/mL).

Flow Cytometry Analysis of NPCs.
Expression levels of various cell surface antigens on NPCs were analyzed by flow cytometry on a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) using combinations of fluorochrome-conjugated mAbs against CD45, CD11b, F4/80, or Gr1. All mAbs used in this study were purchased from BioLegend (San Diego, CA, USA).

In Vitro Induction of MCP-1 in NPCs and HSCs.
Freshly isolated NPCs were suspended in DMEM medium supplemented with FBS (10%), penicillin (100 U/mL) and streptomycin (100 U/mL), and wereplated onto 12-well plates at 1 × 10 6 cells/well. After 24 h incubation at 37 ∘ C, 5% CO 2 , NPCs attached to plates were stimulated with 1 g/mL of LPS for 24 h in the absence or presence of Mat at concentrations of 10, 20, 50, or 100 M. Levels of MCP-1 in supernatants were quantified with commercial mouse ELISA kit (R&D Systems, Inc., MN, USA) according to the manufacturer's instructions.
Freshly isolated HSCs were suspended in DMEM medium supplemented with FBS (20%), penicillin (100 U/ mL), and streptomycin (100 U/mL), plated onto 6-well plates at 5 × 10 6 cells/well, and was allowed to attach to plates by incubation overnight. Then, the culture medium was replaced with fresh DMEM supplemented with FBS (0.5%), penicillin (100 U/mL), and streptomycin (100 U/mL), and the cells were cultured for another 24 h. TNF-(30 ng/mL) was added to the medium in the absence or presence of different concentrations of Mat (10, 20, 50, or 100 M). After 24 h incubation, cellular mRNA levels of MCP-1 were measured by quantitative real-time PCR.

Quantitative Real-Time PCR.
Total RNA was extracted from HSCs or liver tissues with the TRIzol reagent (Invitrogen), and it was reverse transcribed with a complementary DNA reverse-transcription kit (Takara, Dalian, China) according to the manufacturer's instructions. Quantitative real-time PCR was performed on a StepOnePlus real-time PCR system (Applied Biosystems, USA) using the SYBR Premix Ex Taq PCR Kit (Takara, Dalian, China). The primers used were designed and custom synthesized at Invitrogen. The relative levels of assayed mRNAs were calculated with the comparative CT method using GAPDH expression levels as endogenous control, and they were normalized to nontreated control. The primers used were 5 -CTTCTGGGCCTGCTGTTCACAGTT-3 (MCP-1 forward), 5 -TTCTTGGGGTCAGCACAGACCTCT-3 (MCP-1 reverse) and 5 -ATCTTCTTGTGCAGTGCCAGCCTC-3 (GAPDH forward), 5 -TTTGCCACTGCAAATGGCAGCC-3 (GAPDH reverse).

Transwell Chemotaxis Assays.
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized murine blood by density separation over Ficoll-Hypaque. The blood was layered on top of the Ficoll-Hypaque at a 2 : 1 ratio in 15 mL tubes and centrifuged for 25 minutes at 2500 rpm at room temperature. PBMCs at the interface were carefully collected and washed twice in HBSS.
Transwell chemotaxis assays were performed in 24-well transwells (5 m pore size; Costar, Cambridge, MA, USA). 1 × 10 5 PBMCs in 0.1 mL of medium were added to the upper chamber of 2 compartments in the absence or presence of Mat at various concentrations. 50 ng/mL of MCP-1 in 0.6 mL of medium was added to the lower compartment. After incubating the plate at 37 ∘ C for 2 h, the nonmigrating cells were removed from the upper surface of the membrane. The cells on the lower surface of the membrane were fixed with ice-cold methanol and then stained with crystal violet. The number of migrated cells was counted with microscopy.

Statistical Analysis.
All quantitative data are expressed as the mean ± (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) test, followed by Dunnett's multiple-comparison post hoc test. < 0.05 was considered to be statistically significant.

Mat Protects Mice against CCl 4 -Induced Acute Liver Injury and Reduces
Hepatic Inflammatory Infiltratation. The present study was initiated by investigating the protective effect of Mat on CCl 4 -induced acute liver injury in mice. As shown in Figure 1, single intraperitoneal injection of CCl 4 resulted in a time-dependent increase in serum ALT levels. Significant increases in serum ALT levels were detected at 6 h after CCl 4 injection. Serum ALT levels peaked at approximately 24 h, and they then started to subside by 48 h in CCl 4 -challenged mice (Figure 1(a)). Histological examination of liver sections from CCl 4 -challenged mice showed that CCl 4 resulted in periportal necrosis with maximal damage at 24 h, and toxic damage was accompanied by a massive infiltration of leukocytes into the liver (Figure 1(b)). Mice treated with 10 mg/kg or 30 mg/kg of Mat 3 h prior to CCl 4 challenge showed significant decrease in ALT levels at 24 h after CCl 4 -injection (Figure 1(c)). Treatment with Mat also markedly attenuated pathologic changes in CCl 4 -challenged mice. It is important to note that Mat treatment resulted in a dramatic decrease in hepatic inflammatory infiltration in CCl 4 -challenged mice (Figure 1(d)).

Mat Inhibits CCl 4 -Induced Hepatic Infiltration of Gr1 hi Monocytes Subset.
To elucidate the effects of Mat on infiltration of different monocyte subsets following liver injury, we further isolated NPCs from livers of CCl 4 -challenged mice and characterized the different monocyte subset composition by flow cytometry analysis. As shown in Figure 2(a), the population of intrahepatic leukocytes was first defined as CD45 + cells. Using the myeloid marker CD11b and the macrophage marker F4/80 antigen, two distinct subsets of intrahepatic monocytes/macrophages could be identified: CD11b + F4/80 − and CD11b + F4/80 + cells. The CD11b + F4/80 + cells were found to express high level of Gr1, thereby resembling the phenotype of the peripheral Gr1 hi monocyte subpopulation. On the other hand, CD11b + F4/80 − cells expressed low levels of Gr1, corresponding to peripheral Gr1 lo monocytes (Figure 2(a)). This result is consistent with that in the previous report [18].
After isolation of NPCs from each liver, they were at first enumerated. The results in Figure 2 monocytes subset was dramatically elevated at 24 h and 48 h after CCl 4 challenge, whereas that of CD11b + F4/80 − Gr1 lo subset only mildly increased (Figure 2(b)). This result demonstrated that the infiltration of the two monocyte subsets was differentially regulated following acute liver injury, and only CD11b + F4/80 + Gr1 hi subset massively increased after CCl 4 challenge. We next investigated the effects of Mat on the recruitment of Gr1 hi monocytes following acute liver injury. As shown in Figure 3, treatment with 30 mg/kg of Mat resulted in a significant decrease in the total number of NPCs (Figure 3(a)), as well as in the percentage and the absolute number of CD45 + leukocytes in NPCs, at 24 h after CCl 4 challenge (Figures 3(b) and 3(c)). Furthermore, both the percentage and the absolute number of CD11b + F4/80 + Gr1 hi monocytes in injured livers were also significantly reduced by Mat treatment, while the infiltration of CD11b + F4/80 − Gr1 lo subset was almost not affected (Figures 3(d) and 3(e)).

Mat
Reduces the Production of MCP-1. The above finding that Mat selectively blocks the infiltration of Gr1 hi monocytes in CCl 4 -injured livers prompted us to investigate whether Mat could inhibit the production of MCP-1, a chemokine that crucially controls the recruitment of Gr1 hi monocytes. We found that serum MCP-1 was strongly upregulated following liver injury, and Mat treatment resulted in a significant decline of serum MCP-1 level in CCl 4 -challenged mice (Figure 4(a)). In line with the change in serum MCP-1 level, hepatic MCP-1 expression was strongly upregulated after damage as determined by real-time PCR and immunohistochemical analysis, which was also markedly decreased by treatment of Mat (Figures 4(b) and 4(c)). Since NPCs, especially activated HSCs, are major MCP-1-producing cells upon liver damage [21,[37][38][39], we further investigated whether Mat could directly inhibit MCP-1 production in these cells. The result showed that Mat (20-100 M) dose dependently inhibited MCP-1 secretion from LPS-stimulated NPCs isolated from CCl 4 -injured livers ( Figure 5(a)). Mat also dose dependently suppressed the TNF--stimulated upregulation of MCP-1 mRNA in HSCs isolated from CCl 4 -injured mice in the range of 50-100 M ( Figure 5(b)).

Mat Inhibits the Chemotactic Activity of MCP-1.
In addition to suppression of MCP-1 production, Mat may also act by inhibiting the chemotactic activity of MCP-1. So, we further determined this possible effect of Mat with transwell chemotaxis assays. As shown in Figure 6, MCP-1 apparently promoted the chemotactic migration of PBMCs, which can be inhibited by Mat in a dose-dependent manner in the range of 10-100 M ( Figure 6). in a liver fibrosis model. Chronic administration of CCl 4 twice weekly for 6 weeks resulted in significant collagen deposition and liver fibrosis in mice. Oral administration of Mat (10 mg/kg, 5 times weekly for the last 3 weeks) significantly reduced the accumulation of collagen and content of liver hydroxyproline in chronic CCl 4 -challenged mice (Figures 7(a) and 7(b)). Similar to the observations after acute injury, two subsets of monocyte-derived intrahepatic cells could be distinguished in the fibrotic livers. The CD11b + F4/80 + Gr1 hi subset was largely increased during fibrogenesis, while the CD11b + F4/80 − Gr1 lo subset only mildly increased. Administration of Mat significantly attenuated intrahepatic infiltration of Gr1 hi monocytes, but it did not influenced Gr1 lo subset (Figure 7(c)). Mat also inhibited intrahepatic expression of MCP-1 in chronic CCl 4challenged mice (Figure 7(d)). These results demonstrated that Mat prevented development of hepatic fibrosis and blocked recruitment of Gr1 hi monocytes into chronic injured livers through inhibiting MCP-1 production.

Discussion
Mat has been demonstrated to be effective in suppressing inflammation in various inflammatory animal models [25,28,29,31,32,40]. In particular, Mat has been shown to protect animals from acute liver injury induced by hepatotoxins and/or LPS [26,41,42]. Mat also exhibits antiinflammatory and anti-fibrotic effects in CCl 4 -induced liver fibrotic models [30]. However, the underlying mechanisms still remain elusive. Since activation of KCs and HSCs has been well established as critical initial and relevant events in the development of liver fibrosis, we have previously tested the effects of Mat on these cells in an attempt to explore the mechanisms of Mat. Indeed, Mat has been found to inhibit TNF and IL-6 production from LPS-stimulated rat KCs [33] and to suppress serum-or PDGF-induced cell proliferation as well as serum-or TGF--induced collagen synthesis in rat HSC cell line HSC-T6 in vitro [30]. However, these pharmacologic activities are quite weak, as Mat only exerts significant inhibitory effects on the aforementioned cellular functions at concentrations higher than 250 M in those in vitro studies. In the present study, we try to identify other relevant cellular events that may be targeted by Mat to further elucidate the mechanisms underlying its anti-inflammatory and antifibrotic actions. It is well known that, during liver damage caused by different etiologies, activation of local cells is always associated with leukocyte infiltration from the bloodstream. In most cases, leukocyte infiltration results in damage amplification and generation of fibrogenic stimuli via secretion of soluble mediators and oxidative stress-related products [43]. Recently, infiltration of blood-derived macrophages has been suggested to be essential for liver fibrogenesis in addition to activation of liver-resident KCs [9,44]. Furthermore, only the inflammatory Gr1 hi monocytes but not Gr1 lo monocytes are massively recruited into the injured livers following the acute and chronic challenge of CCl 4 , and they promote the progression of liver fibrosis, thus suggesting that Gr1 hi monocytes may represent an interesting target for antifibrotic strategies [18]. In the present study, we confirmed that liver injury, either acute or chronic, induced by single or repeated CCl 4 injections, was associated with selective recruitment of Gr1 hi monocytes into livers. Furthermore, in vivo administration of Mat not only alleviated the acute liver injury induced by single CCl 4 injection but also attenuated liver fibrosis in CCl 4 -induced chronic hepatic injury model. More importantly, Mat treatment significantly prevented the hepatic infiltration of the inflammatory Gr1 hi monocyte subset in livers in both acute and chronic liver injury models. Since intrahepatic Gr1 hi monocyte-derived cells have been previously demonstrated to differentiate preferentially into inducible nitric oxide synthase-producing macrophages during chronic liver damage and to exert proinflammatory and profibrogenic actions, such as promoting HSCs activation, TH1 differentiation and TGF-release [18], our present finding suggests that inhibitory effect of Mat on the infiltration of Gr1 hi monocytes may contribute, at least in part, to its antiinflammatory and antifibrotic effects.
Accumulating evidence indicates that recruitment of Gr1 hi monocytes into inflamed tissues is critically dependent on CCR2, a cognate receptor for C-C chemokine MCP-1 expressed mainly in monocytes [14-18, 45, 46]. A previous study using MCP-1 knockout mice has demonstrated that lack of MCP-1 affords protection from liver damage and development of oxidative stress in CCl 4 -induced acute liver injury model [47]. Another study using MCP-1-specific antisense phosphorothioate oligodeoxy nucleotides and specific CCR2 inhibitors has also shown that human peripheral CD14positive monocytes contribute directly to organ fibrogenesis by an MCP-1/CCR2-dependent amplification loop [48]. A more recent study has further demonstrated that toxic liver damage results in a sequence of increased hepatic MCP-1 expression, elevated serum MCP-1, and peripheral blood monocytosis in wild-type mice. By contrast, CCR2-deficient mice lack peripheral blood monocytosis after injury and subsequently harbor significantly less Gr1 hi monocyte-derived intrahepatic macrophages [18]. In the present study, upregulation of intrahepatic MCP-1 expression following liver damage in both acute and chronic liver injury models was all significantly suppressed by in vivo administration of Mat, suggesting that Mat may inhibit MCP-1 production under inflammatory conditions. Cellular experiments using in vitro cultured NPCs and HSCs derived from CCl 4 -injured livers further demonstrated that Mat directly inhibited MCP-1 production in both LPS-stimulated NPCs and TNF--stimulated HSCs in a dose-dependent manner within the range of 20-100 M. These results corroborate the inhibitory effects of Mat on MCP-1 production induced by proinflammatory stimuli. In addition to the inhibition of MCP-1 production, Mat also exhibited the inhibitory effects on the chemotactic activity of MCP-1. In our transwell chemotaxis assays, Mat significantly inhibited MCP-1-mediated chemotactic migration of PBMCs dose dependently in the range of 10-100 M. Based on these data, we attribute the inhibition of Mat on hepatic recruitment of Gr1 hi monocytes to its inhibition of both MCP-1 production and its chemotactic activity. Yet, we cannot further explore the molecular mechanisms by which Mat exerts it inhibitory effects on MCP-1 production and function in the present study. Further study on the effects of Mat on the intracellular signaling involved in MCP-1 production and activity and identification of its molecular targets will help to fully elucidate the related mechanisms. In addition, even though it has been well documented that the recruitment of Gr1 hi monocytes into inflamed tissue is critically dependent on CCR2/MCP-1, other chemokines and their receptors are also involved in monocyte trafficking in some inflammatory settings, such as IL-8, CX3CL1, CCR5, and CX3CR1 [14,20]. Therefore, the possible effects of Mat on production and function of these chemokines remain to be investigated to fully understand the mechanisms by which Mat inhibits the hepatic recruitment of Gr1 hi monocytes.

Conclusion
The present study demonstrates that in vivo administration of Mat affords protection from liver injury and development of liver fibrosis in CCl 4 -induced liver injury model, and such beneficial effects could be contributed, at least in part, to the prevention by Mat on the hepatic infiltration of the inflammatory Gr1 hi monocyte subset in injured livers, which is most possibly through its inhibition of both MCP-1 production and activity. These new findings extend our understanding on the mechanisms underlying its anti-inflammatory and antifibrotic effects.