Matrix Remodeling Associated 7 Deficiency Alleviates Carbon Tetrachloride-Induced Acute Liver Injury in Mice

Matrix remodeling associated 7 (MXRA7) was first noted to co-express with a group of matrix remodeling related genes, and its biological functions had remained unclear. In this study, we investigated the presumed function of MXRA7 in a carbon tetrachloride (CCl4)-induced acute liver injury model in mice. Wild-type, MXRA7−/− mice, and mice that were pulsed with hydrodynamic injection of vehicle or MXRA7-harboring plasmids were challenged with a single dose of CCl4 for injury induction. The sera, spleens, and livers were harvested from mice for assay of cytokines/chemokines expression, cellular responses, or histological features. We found that MXRA7 deficiency alleviated, and MXRA7 overexpression aggravated liver damage in CCl4-challenged mice. FACS analysis showed that MXRA7 deficiency reduced the recruitment of neutrophils through downregulation the expression of CXCL1 and CXCL2 in liver, decreased the number of CD8+ T cells in liver and spleen, suppressed the release of IFNγ and TNFα from T cells, and decreased IFNγ in serum and liver. Western blot assay demonstrated that MXRA7 deficiency suppressed the activation of MAPK pathway and AKT/NF-κB pathway, respectively. Lastly, MXRA7 deficiency or overexpression regulated the expression of two matrix remodeling-related genes (fibronectin and TIMP1) in the liver. We concluded that MXRA7 was an active player in CCl4-induced liver injury, hypothetically by mediating the inflammation or immune compartments and matrix remodeling processes. Further exploration of MXRA7 as a possible new therapeutic target for management of inflammation-mediated liver injury was discussed.

Matrix remodeling associated 7 (MXRA7) was first noted to co-express with a group of matrix remodeling related genes, and its biological functions had remained unclear. In this study, we investigated the presumed function of MXRA7 in a carbon tetrachloride (CCl4)-induced acute liver injury model in mice. Wild-type, MXRA7 −/− mice, and mice that were pulsed with hydrodynamic injection of vehicle or MXRA7-harboring plasmids were challenged with a single dose of CCl4 for injury induction. The sera, spleens, and livers were harvested from mice for assay of cytokines/chemokines expression, cellular responses, or histological features. We found that MXRA7 deficiency alleviated, and MXRA7 overexpression aggravated liver damage in CCl4-challenged mice. FACS analysis showed that MXRA7 deficiency reduced the recruitment of neutrophils through downregulation the expression of CXCL1 and CXCL2 in liver, decreased the number of CD8 + T cells in liver and spleen, suppressed the release of IFNγ and TNFα from T cells, and decreased IFNγ in serum and liver. Western blot assay demonstrated that MXRA7 deficiency suppressed the activation of MAPK pathway and AKT/NF-κB pathway, respectively. Lastly, MXRA7 deficiency or overexpression regulated the expression of two matrix remodeling-related genes (fibronectin and TIMP1) in the liver. We concluded that MXRA7 was an active player in CCl4-induced liver injury, hypothetically by mediating the inflammation or immune compartments and matrix remodeling processes. Further exploration of MXRA7 as a possible new therapeutic target for management of inflammationmediated liver injury was discussed.
Keywords: matrix remodeling associated 7, acute liver injury, neutrophils, extracellular matrix, pro-inflammatory cytokines inTrODUcTiOn Liver is a vital organ which performs important functions like extensive synthesis, retinoid stor age, metabolism, detoxification, secretion of proteins, etc. (1,2). Liver diseases are one panel of the endangering problems which lead to mortality and morbidity over the world (3,4). Liver injuries or diseases can be caused by many factors, including drugs, toxins, alcohol, and virus infection (5,6). Acute liver injury (ALI) occurs within a short period and is a common pathway to many liver diseases. The pathogenesis of ALI involves oxidative stress, hepatocyte apoptosis and necrosis, immune responses, etc. (6)(7)(8). ALI also involves inflammation and may progress to chronic liver injury, hepatic fibrosis, or even hepatocellular carcinoma (9). Therefore, searching for new thera peutic options for treatment of liver injury is critical for handling liver diseases in clinical practice.
In laboratory studies concerning liver injury, carbon tetrach loride (CCl4) is commonly used as a chemical toxin to induce ALI model in mice (10). CCl4 is metabolized by cytochrome P450 (CYP2E1) in liver to form trichloromethyl and trichloromethyl peroxy radicals, which in turn induce oxidative stress, lipid per oxidation, and hepatic injury (11). Except for oxidative stress, inflammation is another important mechanism mediating CCl4 induced liver injury (12,13). In this process, proinflammatory cytokines and chemokines are crucial players that incur cell death and liver injury (14)(15)(16). Correspondingly, previous studies sug gested that the liver injury can be prevented by suppressing oxi dative stress and inflammation (17,18). Therefore, metabolism and inflammatory response are thought to be therapy targets in the treatment of liver injury. On the other side, liver injury and recovery also involve matrix remodeling, a process that underlies structural changes occurred in any tissues, including liver (19,20). Thus, dissecting the relationship between matrix remodeling and liver injury also provides new possibilities for deve loping novel therapy of liver injury. In a previous study, we sighted expression of a novel gene, matrix remodeling associated 7 (MXRA7), in liver at mRNA level (21), but its possible involvement in any physiolo gical or pathological processes of liver had not been addressed in any study. MXRA7, first named in a bioinformatics study in 2002, belongs to the MXRA family consisting of eight genes (MXRA1-MXRA8) possibly involved in cell adhesion and matrix remode ling (22). Studies have reported that some members of this family are related with a variety of critical physiological and pathological processes, such as MXRA2 in matrix degradation (23), MXRA3 in actin polymerization and cell motility (24), MXRA5 in anti inflammatory and antifibrotic responses (25), and MXRA6 in myofibroblast differentiation and extracellular matrix formation (26). However, the function of MXRA7 has been barely investi gated except for our previous study showing a dynamic change of MXRA7 mRNA in inflammatory corneal diseases models in adult mice (21).
In the present study, we used mice genetically deficient of MXRA7 (MXRA7 −/− ) or artificially overexpressing MXRA7 to investigate the hypothetical role of MXRA7 in CCl4induced ALI in mice. The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and histopathological analysis were used to evaluate liver functions. The inflammatory response and proinflammatory cytokines as well as extracellular matrix molecules were measured to elucidate the underlying mecha nism. Our findings indicated an important role of MXRA7 in liver injury in vivo.

MaTerials anD MeThODs animals
Heterozygous MXRA7deficient (MXRA7 +/− ) mice of both sexes on C57BL/6N background were purchased from the Medical Research Council (MRC, Swindon, UK). The MXRA7 −/− and wildtype (WT) breeders were generated by crossbreeding female and male MXRA7 +/− mice, and genotyped according to the pro tocol provided by MRC ( Figure S1 in Supplementary Material). The breeders were used for generating WT and MXRA7 −/− colo nies, respectively. The male mice of WT and MXRA7 −/− (aged 6-8 weeks) were used in the study. Specific pathogenfree male C57BL/6N mice (aged 6-8 weeks) were obtained from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). All mice were housed in specific pathogenfree facility in Soochow University.

MXra7-Delivery Plasmid construction and hydrodynamic gene Transfer (hgT) In Vivo
The murine MXRA7 cDNA was cloned from splenocytes of mouse and inserted into a minicircle (MC) plasmid (pMC.EF1, System Biosciences, Mountain View, CA, USA) to construct the MXRA7delivery plasmid (pMCMXRA7), while the mock pMC.EF1 plasmid was used as control plasmid in the study. Confirmation with sequencing, preparation, and purification of both plasmids were performed as previously described (27). pMC plasmids were delivered into mice by HGT technique (80 µg plas mids in 2.5 ml PBS/mouse). Three days postinjection of plasmids, the mice were subjected to ALI induction as described below.

acute liver injury
The animal experiments were divided into two sections, namely, the MXRA7 deficiency section using WT and MXRA7 −/− mice, and the MXRA7 overexpression section using mice challenged with pMC.EF1 (control) or pMCMXRA7 plasmids. CCl4 (Sino pharm, Shanghai, China) was used to induce liver injury of diffe rently severity in mice. For survival experiments, the mice were injected intraperitoneally (i.p.) with 6 ml/kg body weight CCl4 (1:1 dilution in corn oil) and the survival of mice were observed every day. Pilot experiments and previous studies had determined this lethal dose within 1 week in WT mice to be around 6 ml/kg body weight CCl4 (1:1 corn oil) (27). For shortterm ALI induc tion and acute phase assay, the mice were injected i.p. with a dose of 1 ml/kg CCl4 (1:1 dilution in corn oil). The mice were sacrificed 24 h after CCl4 administration. Blood, spleen, and liver samples were collected for further analysis as following.
analysis of serum aspartate aminotransferase and alanine aminotransferase activity The blood samples were collected and centrifuged to separate the serum. The activity of serum ALT and AST were determined by commercial reagent kit (AILEX, Shanghai, China) with a Hitachi 7600 automatic biochemical analyzer (Hitachi, Tokyo, Japan) according to the manufacturer's protocol.

histopathological analysis
The liver tissues from different groups of mice were fixed with 10% neutral buffered formalin and then embedded in paraffin. Paraffinembedded tissues were cut into 5 µm thick sections and stained with hematoxylin and eosin for histopathological examination through a light microscope (Nikon, Tokyo, Japan). The percentage of necrotic area was determined by measuring the necrotic area relative to the entire liver area using Image J software (vision1.49, NIH, Bethesda, MD, USA) as described earlier (8).

cytokine analysis
Serum cytokine levels were analyzed using BD cytometric bead array (CBA) mouse inflammation kit (BD Pharmingen, San Diego, CA, USA) on a FACS Calibur Cytometer and analyzed using BD FCAP Array software (BD Bioscience). The cytokine kit covered mouse IL6, IL10, IL12p70, TNF, IFNγ, and MCP1.
reverse Transcription-Quantitative real-Time Pcr (rT-qPcr) The total RNA was extracted from liver tissues with RNAiso plus reagent (TaKaRa, Dalian, China), and reversetranscribed into cDNA with reverse transcriptase MMLV reagent (TaKaRa, Dalian, China) according to manufacturer's instructions. Quanti tative realtime PCR (qPCR) was performed using SYBR Premix Ex Taq reagent (TaKaRa, Dalian, China) and carried out on a QuantStudio 3 realtime PCR system (Applied Biosystems, Foster City, CA, USA). The primer sequences used in the experiment were listed in supplementary Table S1 in Supplementary Material and the primers were synthesized by Genewiz Company (Suzhou, China). The βactin gene was used as an internal control and the data were analyzed using 2 −ΔΔCt method.

immunofluorescence staining
The sections of liver tissues were blocked with 3% BSA in PBS for 1 h at room temperature, and then incubated with primary antibodies overnight at 4°C. The primary antibodies were anti Ly6C/Ly6G antibody (Bio X Cell, West Lebanon, NH), antiF4/80 antibody (Abcam, Cambridge, MA, USA) and antifibronectin antibody (Abcam). After washing in PBS for three times, the sec tions were incubated with Alexa Fluor 555 antirat IgG antibody or Alexa Fluor 488 antirabbit IgG antibody (Abcam) at room temperature for 1 h in the dark. After counterstaining with DAPI (Beyotime), the sections were observed and photographed with a Leica TCS SP8 Confocal Microscope (Buffalo Grove, IL, USA).

TUnel staining
To evaluate apoptosis of hepatocytes in liver tissues, paraffin embedded sections were stained by using a One Step TUNEL Apoptosis Assay Kit (Beyotime) according to the manufacturer's instructions. Sections were counterstained with DAPI and obser ved with a Leica TCS SP8 confocal microscope.

Western Blot analysis
Total proteins were extracted from liver tissues with the strong RIPA lysate kit (Beyotime), and the protein concentration was determined by BCA protein method. Proteins were denatured, separated by SDSPAGE gel, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat powdered milk or 3% BSA in TBST for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study were against ERK, pERK, p38, pp38, JNK, pJNK, AKT, pAKT, NFκB p65, Bax, Bcl2 (all from Cell Signaling Technology, Danvers, MA, USA), MXRA7 (Sigma, St. Louis, MO, USA), GAPDH (SUNGENE, Tianjin, China), and βactin (ImmunoWay, Plano, TX, USA). Following washes with TBST three times, the membranes were incubated with HRPconjugated goat antirabbit IgG or goat antimouse IgG antibody (ImmunoWay) for 1.5 h at room tem perature. After washing off the unbound antibody three times with TBST, the protein bands were detected by an enhanced chemiluminescence kit (Beyotime) using a CLiNX Science Instrument (Shanghai, China). The intensity of the target protein bands was calculated using Image J software.

statistical analysis
All data, when appropriate, are presented as mean ± SD with sam ple size (as indicated as n=) and repeated times of experiments detailed in figure legends. Statistical analysis was performed using the Graphpad prism 5 software (Graphpad, San Diego, CA, USA). Oneway of ANOVA analysis and Student's ttest (unpaired, two tailed) were used for statistical analysis. p < 0.05 was considered to be statistically significant (*), <0.01 or 0.001 was shown as ** or ***, respectively.

MXra7 Deficiency alleviates While MXra7 Overexpression aggravates ccl 4 -induced ali in Mice
To evaluate the hypothetical role of MXRA7 in murine ALI model, WT and MXRA7 −/− mice, as well as pMCMXRA7 medi ated MXRA7overexpressing mice, were compared. The over expression of MXRA7 in the HGTpulsed liver was confirmed at both mRNA and protein levels ( Figure S2 in Supplementary Material). Compared with controls in WT mice, deficiency of MXRA7 pro longed the survival of mice after CCl4 treatment (B) C57BL/6 mice were injected with mock pMC-EF1 (control) or pMC-MXRA7 plasmids by hydrodynamic gene transfer method. Three days later, the animals were challenged with CCl4 as above. Survivals were monitored once a day for 7 days after administration. n = 30 each group. The data shown are the summary of four experiments. (c-g) WT and MXRA7 −/− mice, or pMC-EF1 and pMC-MXRA7-pulsed mice, were treated with 1 ml/kg CCl4 injection (1:1 corn oil) or control oil. Twenty four hours later, the animals (n = 6 for control group, n = 18-23) were sacrificed and serum were harvested measurement of ALT and AST (c,D), while livers (n = 3 for control group, n = 9 for CCl4) were harvested and subjected to measurement of CYP2E1 mRNAs using reverse transcription-quantitative real-time PCR (e) and structure evaluation after histological hematoxylin and eosin staining (F,g). For histological evaluation, necrotic areas were determined by microscopy. The percentage of necrotic area was determined by dividing the sum area of necrosis by the sum of the total liver area of four fields. One representative section is shown for each group (original magnification: 40×). The data shown are the summary of three experiments. *p < 0.05, **p < 0.01.  Figure 1A), while the overexpression of MXRA7 in liver slightly but statistically significantly increased the mortality of mice ( Figure 1B). To further confirm the effect of MXRA7 on ALI, the levels of ALT and AST in serum were measured, and histopathological phenotypes in liver were examined. While both transferases were significantly increased in serum in both WT and MXRA7 −/− mice upon CCl4 treatment, the deficiency of MXRA7 markedly impaired the increase of serum ALT and AST (Figure 1C), while the overexpression of MXRA7 increased the amounts of serum ALT and AST ( Figure 1D). As a marker enzyme in the process of CCl4induced liver injury, expression of CYP2E1 in livers of these mice was compared. It was found that MXRA7 deficiency decreased while MXRA7 overexpression increased the expression of CYP2E1, respectively ( Figure 1E). When the livers were subjected to histo logical analysis, the necrotic areas around the central vein and centrilobular regions in CCl4treated MXRA7 −/− mice were significantly decreased when compared with that in WT mice (Figure 1F), and MXRA7 over expression manifested an opposite effect to that of MXRA7 deficiency ( Figure 1G). These findings indicated that MXRA7 overexpression promoted ALI development in CCl4challenged mice, while MXRA7 deficiency protected mice from such injury.

MXra7 Deficiency Decreases neutrophils and Macrophages cells infiltration in liver
The inflammatory cells play important roles in the develop ment of liver injury or related liver diseases, among which neu trophils and macrophages are two classes of wellknown players (13,29,30). In order to investigate the effect of MXRA7 on the inflammatory cells during liver injury, we examined the inflammatory cells in spleen and liver by flow cytometry or immunofluorescence staining. The deficiency of MXRA7 signifi cantly decreased the percentage and cell number of Ly6C/Ly6G + neutrophils and F4/80 + macrophages in both spleens and livers (Figures 2A,B). Furthermore, the expressions of CXCL1 and CXCL2, two welldocumented neutrophils chemoattractants, were significantly decreased in MXRA7 −/− mice ( Figure 2C). However, MXRA7 overexpression had no effect on the recruit ment of neutrophils and the expression of associated chemokines ( Figure S3 in Supplementary Material). These results indicated that deficiency of MXRA7 reduced the expression of CXCL1 and CXCL2, presumably suppressed the recruitment of neutrophils.

T cells had been suggested as both targets and effector cells in
CCl4induced injury (31). To check if MXRA7 participated in liver injury processes via the immune compartment in above liver injury model, T cells in liver as well as spleen were analyzed by flow cytometry. Compared with WT group, MXRA7 deficiency reduced the numbers of CD4 + T cells and CD8 + T cells, and the effector cells (CD62L − CD44 + ) in spleens ( Figure 3A). MXRA7 deficiency also reduced CD8 + /CD4 + ratio and the number of CD8 + T cells, while decreased CD8 + T effector cells in livers ( Figure 3B). Since T or NK cellsderived cytokines were responsible for indu ction of inflammation that aggravates liver injury (32,33), we examined IFNγ and TNFα secretion by T and NK cells. While the percentages and cell numbers of CD4 + T and CD8 + T producing IFNγ were significantly decreased in spleens of MXRA7 −/− mice (Figure 4A), the cell numbers of CD4 + T and CD8 + T producing IFNγ and TNFα were decreased in livers of MXRA7 −/− mice as  well ( Figure 4B). The numbers of NK cells producing IFNγ decreased in both spleens and livers of MXRA7 −/− mice (Figures 4A,B), while the T cells and production of IFNγ and TNFα showed no difference between MXRA7 overexpression and control group ( Figures S4 and S5 in Supplementary Material). These data sug gested that MXRA7 deficiency might alleviate liver injury by reducing CD8 + T cells and suppressing the production of IFNγ and TNFα from T cells. MXRA7 in ALI Frontiers in Immunology | www.frontiersin.org April 2018 | Volume 9 | Article 773

MXra7 Deficiency reduces the expression of Pro-inflammatory cytokines in ali Model
The inflammatory cytokines of serum IFNγ, IL6, TNF, MCP1, IL12p70, and IL10 were determined by CBA mouse inflam mation kit. The deficiency of MXRA7 decreased the level of serum IFNγ (Figure 5A), and the overexpression of MXRA7 increased IFNγ level (Figure 5B). Moreover, we also measured the expression of proinflammatory cytokines in livers. The mRNA expressions of IFNγ and IL6 were significantly decreased in MXRA7 −/− group when compared to WT group (Figure 5C), while MXRA7 overexpression elevated the mRNA expression of IFNγ ( Figure 5D). None of other tested cytokines or their genes manifested significant change. These results suggested that IFNγ was the main proinflammatory cytokines mediating the differ ential responses to CCl4induced injury in mice under different MXRA7 context.

MXra7 Deficiency inhibits While MXra7 Overexpression Promotes the expression of Fibronectin and TiMP1
Liver injury induces inflammation and expression of extracellular matrix proteins, such as fibronectin and collagen I (COL1A1) (34,35). Immunofluorescence staining showed that MXRA7 deficiency decreased the expression of fibronectin in liver com pared to WT mice (Figure 6A), while MXRA7 overexpression increased fibronectin expression (Figure 6B). However, MXRA7 had no effect on the expression of COL1A1 (Figures 6C,D). Tissue inhibitor of metalloproteinase 1 was also tested since it is a welldocumented modulator of matrix remodeling (36). MXRA7 −/− mice showed lower expression of TIMP1 mRNA than WT mice, and MXRA7 overexpression increased the mRNA level of TIMP1 (Figures 6C,D). These data suggest that MXRA7 plays a role in the reconstruction of extracellular matrix.

MXra7 Deficiency suppresses the cell apoptosis Pathway and inflammatory Pathway
To clarify whether MXRA7 could affect the apoptosis of hepato cytes, the TUNEL assay was performed. MXRA7 deficiency inhibited cell apoptosis and MXRA7 overexpression promoted cell apoptosis upon CCl4 treatment (Figures 7A,B). The expres sions of Bcl2 and Bax in livers were also compared among different groups. After CCl4 treatment, the protein expressions of the proapoptotic protein Bax were decreased in livers from MXRA7 −/− mice, while the levels of the antiapoptotic factor Bcl2 was significantly increased (Figure 7C). On the contrary, MXRA7 overexpression increased the expression of Bax as compared to control group (Figure 7D).
To understand the underlying molecular mechanisms of the effects of MXRA7 on CCl4induced liver injury, we examined the MAPK and AKT/NFκB signaling pathways, which are known to regulate cell apoptosis and inflammation. Western blot analysis showed that MXRA7 deficiency reduced the phosphorylated pro tein levels of ERK and p38 when compared to WT mice (Figure 7E), while MXRA7 overexpression increased the levels of pERK and pp38 after CCl4 treatment ( Figure 7F). However, MXRA7 deficiency or overexpression did not show significant effect on phosphorylation of JNK ( Figure S6 in Supplementary Material). MXRA7 deficiency also decreased the expression of pAKT and NFκB p65 (Figure 7G), while MXRA7 overexpression increased the expression of pAKT after CCl4 treatment ( Figure 7H). Thus, the protective roles of MXRA7 deficiency against CCl4induced hepatocyte apoptosis and inflammation might be associated with suppressing MAPK and AKT/NFκB pathways.

DiscUssiOn
Since named in 2002 (22), MXRA7 had been just mentioned in some studies without any purpose investigation into its biological functions. Previous studies noted that MXRA7 was overexpressed in childhood acute lymphoblastic leukemia and in ovarian endo metriomas (37,38). In an effort to characterize the functions of MXRA7, our lab had found that MXRA7 was involved in the pathological process of ocular inflammatory models (21) and MXRA7 might play a role in tissue injury, wound healing, and cancer (manuscripts under review).
In the current study, the potential role of MXRA7 in liver disease was investigated in mice by utilizing the CCl4induced liver injury model. This model has been widely used to study both acute injury and following chronic fibrosis (39)(40)(41). We found that MXRA7 overexpression aggravated liver injury, and MXRA7 deficiency protected the liver against CCl4induced liver injury (Figure 1). Serum AST and ALT levels, which were often used as indicators of hepatic damage and functional integrity of liver (42), manifested changes in line with the animal survival or histological changes. CCl4 could induce liver damage through CYP2E1 (43), though the necrotic area would possibly not produce CYP2E1 anymore, MXRA7 might affect liver injury partly by regulating the level of CYP2E1. In summary, all these results indicated a hepatoprotective effect of MXRA7 deficiency on CCl4induced ALI. In another word, MXRA7 might be a positive modulator of CCl4induced ALI.
Though the main functions of the liver are focused on metabo lism, it is also an immunologic organ since the immune cells resided in the liver are involved in the development of inflam mation and fibrosis when responding to various insults (44). In CCl4induced liver inflammation and injury model, lymphocytes, neutrophils, and macrophages in liver tissues play important roles through secreting cytokines and chemokines (13,(45)(46)(47). Previous study reported that CD8 + T cells could induce more liver injury and fibrosis in mice treated with CCl4, and CD4 + / CD8 + ratio reduction also involved in induction of liver fibrosis in human (40,48). The data presented here demonstrated that MXRA7 deficiency not only impaired infiltration of neutrophils and marcrophages in liver induced by CCl4 (Figure 2) but also decreased the number of CD8 + T cells and CD8 + /CD4 + ratio in liver of MXRA7 −/− mice compared to WT mice (Figure 3). In line with this observation, MXRA7 deficiency suppressed the production of IFNγ and TNFα in T cells (Figure 4) and sup pressed the expression (at mRNA level) of IFNγ and IL6 in liver, hence decreased the protein level of IFNγ in serum. Meanwhile, MXRA7 overexpression increased the IFNγ mRNA in liver and  protein in serum. Collectively, these data showed that when MXRA7 was deficient in animals, CCl4induced inflammatory or immune cells activation were decreased, leading to an alleviated liver injury than in WT host receiving same challenge.
Lastly, cell apoptosis and inflammatory signaling pathways associated with CCl4induced injury (49) were measured. MXRA7 deficiency suppressed cell apoptosis, decreased Bax expression, and increased Bcl2 expression, while MXRA7 overexpression promoted cell apoptosis and upregulated the expression of Bax (Figure 7). When the hepatic apoptosis pathways, e.g., MAPK signaling pathway, were compared in context of ALI models, the phosphorylation levels of ERK and p38 regulating cell apoptosis were downregulated in MXRA7 −/− mice and upregulated in MXRA7 overexpression mice. AKT/NFκB pathway has dual fun ction in proinflammatory and cell survival, the unbalance of NFκB activation may cause increased inflammation or insuffi cient protection from cell apoptosis (50,51). However, the effect of MXRA7 deficiency or overexpression on the AKT phospho rylation or p65 expression was a little bit complicated in CCl4 induced ALI. The overall impression was that MXRA7 deficiency depressed AKT phosphorylation and NFκB p65 expression while MXRA7 overexpression increased AKT phosphorylation (Figure 7). These results suggested that in the studied ALI model, MXRA7 not only mediated liver injury via the inflammation or immune compartment but also via acting in/on hepatocytes directly or indirectly.
In summary, this study represented the first effort to explore the possibility that the seldomaddressed gene MXRA7 was involved in ALI. Using genetically MXRA7deficient mice and assisted with HGTmediated liverspecific overexpression of MXRA7, we were able to confirm that MXRA7 played a positive role in initiation of CCl4induced ALI. It will be of interest to investigate whether such a hypothesis for MXRA7 function was applicable with other acute liver injuries, or even with chronic liver injuries. For example, repeated injection of CCl4 in mice leads to chronic liver fibrosis, a process that is more closely related with matrix remodeling. This said, though the results that MXRA7 deficiency decreased and MXRA7 overexpression increased the expression of fibronectin and TIMP1 in murine livers (Figure 6) were still preliminary, they strongly implied that MXRA7 might also be involved in such chronic liver injuries as those induced by repeated CCl4 challenge, heavy alcohol consumption, drug intoxication, or consistent virus infections. Should all these hypotheses be confirmed by future studies, MXRA7 might serve a promising new therapeutic target for preventing or treating those liver injuries. However, in spite of the current data demon strating the potential significance of MXRA7 in liver injury, much more investigations are guaranteed to help fully understand the role of MXRA7 in ALI, ALIbased diseases, or in overall physiol ogy or pathology of the liver.