Study On The Expression And Role Of Adiponectin In Acute Liver Injury In Mice

Objective It is well known that adipose tissue is organ storing energy in organism. Only recently, with the discovery of a variety of adipocyte-derived cytokines, the adipose tissue has gained a role as an important endocrine organ.The adipose tissue and adipokines are potent modulators of various physiological and pathological functions, including immunity and inflammation. Adipokines include leptin ,adiponectin ,resistin and sorts of pro-inflammatory cytokines/chemokines such as, tumor necrosis factor-α(TNF-α), and interleukin-6 (IL-6), monocyte chemoattractant protein(MCP-1) and so on. However, there is only scarce knowledge on their functional roles.Adiponectin represents the most abundant adipocyte-derived protein reaching high plasma levels in both humans and rodents. Adiponectin is a 244-amino acid protein that contains a putative signal sequence and a collagen-like domain followed by a globular domain similar to compliment factor C1q. Adiponectin has been shown to elicit a broad range of biological effects such as insulin-sensitising and anti-atherogenic actions. Besides,the changes of adiponectin serum level under the conditions of inflammation make it recognized that there may be relations between adiponectin and inflammation. Apart from downregulation of the synthesis of the pro-inflammatory cytokine TNF-αand its mRNA by macrophage, adiponectin leads to an increased production of the anti-inflammatory mediators IL-10 and IL-1RA in human myeloid cells.The roudly study on whether adiponectin can relieve the inflammatory degree in virus hepatits is scarced. The pathogenesis of virus hepatitis is immunizing injury to hepacytes though TNF-αand other cytokines producing by actived T lymphocytes. Concanavalin A(ConA), a T cell mitogen ,can concentrate to liver after injection and initiate a serial of immunoreactions through activing T lymphocytes. The pathogenesis of ConA is similar to that of acute hepatitis. So this study investigated the protecting mechanism adiponectin exerted to acute liver injury using hepatitis model maked by ConA ,including the effection of adiponectin exert on several inflammatory cytokines ,such as tumor necrosis factor-α( TNF-α), interleukin-10(IL-10) and so on, and on hepatocellular apoptosis. This article also first studied the signal transduction pathway which adiponectin exert the role of anti-inflammation. Adiponectin is a physiological adipokine produced in organism. It can provide theoretical evidence for clinical treatment to hepatits using adiponectin studing its protective effect to acte liver injury. This study can also provide a new ,safe and effective route to the treatment of hepatic injuries including viral hepatitis.Methods1 The build of acute liver injury model and the expression of adiponectin Twenty-one four-week-old healthy male BALB/c mice(weight 20±3g) were purchased from the Animal Experiment Core of Hebei Medical University.After feeding with standard forage for one week ,twenty-one mice were divided into three groups randomly: ConA group(eight mice): were injected with 30 mg/kg Con A in the tail-vein in the presence or absence of 6 mg/kg adiponectin; Control group(eight mice): were injected saline with 10ml/kg in the tail-vein; adiponectin group(five mice): were injected murine adiponectin(abcam Company, England) intraperitoneally in 3mg/kg 12h and 2h before treatment with Con A intravenous injections(i.v.) and the other mice received the same volume saline intraperitoneal injections (i.p.). All reagents were fasted 12h before treatment with Con A i.v.Eight hours after ConA treatment, mice were sacrificed by losing blood from femoral vein. Blood were centrifuged immediately and serum samples were stored at -20oC refrigerator for serology detection.The livers were removed and one part was frozen in liquid nitrogen for mRNA preparation, another part were fixded in 10% buffered formalin and stained with hematoxylin-eosin for routine examination. Adiponectin mRNA is detected by reverse transcription-polymerase chain reaction(RT-PCR) and the serum level of adiponectin is detected by enzyme linked immunosorbent assay ( ELISA).The expression of adiponectin protein in liver detected in immunohistochemical stainings.2 The regulating role that adiponectin exert on cytokines in acute liver injury induced by ConALevels of circulating cytokines and expressions of cytokines messenger RNA (mRNA) in the liver were determined. Analysis of liver injury was quantified by measurement of serum enzyme activities of ALT using an automated procedure. Serum IL-10,IL-4,INF-γand TNF-αwere measured in ELISA and performed strictly according to the manufacturer's instructions . RT-PCR for TNF-α, IL-10, mRNA.3 The influence of adiponectin on hepatocellular apoptosis in acute liver injuryIn situ detection of DNA fragments was performed using a terminal deoxynucleotidy transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) test . The hepatocellular apoptotic rate and hepatocellular reproductive cycle are also determined by flow cytometry(FCM).4 The primary investigate on signal transduction route through which adiponectin exert anti-inflammationThe expression in liver of cAMP-dependent protein kinase (PKA) mRNA and phosphatidylinositol-3-kinase(PI3K) mRNA was detected by means of RT-PCR. The expression of PKA and PI3K protein in liver detected in immunohistochemical stainings. NF-κB binding activity in the liver tested by electrophoretic mobility shift assay (EMSA).Results1 The build of acute liver injury model and the expression of adiponectin1.1 Routine pathologic observation Under light microscope , there are no inflammatory cell infiltration in liver tissue of mice in control group, but lymphocytes infiltration in hepatic tissue, congestion in sinus hepaticus , degeneration and dropsy in hepatic cytoplasm ,spot and focus necrosis could be seen in ConA group. The degree of hepatic inflammatory be relieved in adiponectin group .1.2 The average serum level of adiponectin (15.4±3.0μg/ml)in mice of adiponectin group is no difference to that of control group(16.5±2.8μg/ml)(p>0.05) but higher than ConA group(11.8±2.1μg/ml)(P<0.05). The average serum level in mice of ConA group is lower than that of control group(P <0.01).1.3 Adiponectin protein is expressed mainly in central vein endothelial cells in healthy liver tissue and regulated upon injection of ConA according to immunohistochemical stainings , ConA-injection led to an increased number of adiponectin positive cells, especially the infiltrated inflammatory cells. Adiponectin-expressing cells less increased in sinus hepaticus cells and inflammatory cells in mice of adiponectin group. Statistical analysis show that the expression of adiponectin protein in mice of adiponectin group (5.39±1.72%)is higher than that of control group(1.82±0.36%)(p<0.05) but lower than ConA group(10.63±4.35%)(P<0.05). The expression of adiponectin protein in mice of ConA group is higher than that of control group(P <0.01)1.4 Adiponectin mRNA was readily detectable in healthy liver tissue and further increased in ConA-mediated acute liver failure ,adiponectin mRNA expression in mice of adiponectin intervention was also increased. The expression of adiponectin mRNA in mice of adiponectin group (0.46±0.17)is higher than that of control group(0.23±0.05)(p<0.05) but no difference to that of ConA group(0.51±0.21)(P>0.05). The expression of adiponectin mRNA in mice of ConA group is higher than that of control group(P <0.01).2 The regulating role that adiponectin exert on cytokines in acute liver injury induced by ConA2.1 The average serum level of ALT in mice of adiponectin group(192.50±45.87U/L) is higher than that of control group(44.71±21.29 U/L) (p<0.01) but lower than group (616.00±171.50 U /L)(P<0.05). The average serum level in mice of ConA group is lower than that of control group(P <0.01). The average serum level of ALT in mice of ConA group is higher than that of control group(p<0.01).2.2 The average serum level of TNF-αin control group ,adiponectin group and ConA group are 0.06±0.04 ng/ml,0.51±0.29 ng/ml,0.92±0.37 ng/ml respectively; The average serum level of IL-10 are 0.09±0.06 ng/ml,0.54±0.17 ng/ml,0.26±0.11 ng/ml respectively; The average serum level of IL-4 are 0.09±0.06 ng/ml,0.54±0.17 ng/ml,0.26±0.11 ng/ml respectively; The average serum level of INF-γare:0.71±0.28 ng/ml,2.24±0.46 ng/ml,2.97±0.61 ng/ml respectively. The serum levels of TNF--α,IL-10,IL-4,INF-γare all lowest in mice of control group and all increased after administration of Con A. Mice pretreated with adiponectin before ConA administration developed reduced the TNF-α,IL-4 and INF-γlevels,but increased IL-10 levels.2.3 Correlation analysis show that the serum level of ALT is positive correlation to the level of TNF-α,IL-4,INF-γ(r=0.738 p<0.01; r=0.693 p<0.01; r=0.637 p<0.01).2.4 TNF-αmRNA expressed mildly in liver tissue of control group(0.13±0.07), increased lightly in adiponectin group(0.51±0.19)(p<0.01) and further increased in ConA group(0.82±0.27)(p<0.01). TNF-αmRNA expressed in liver tissue of adiponectin group is lower than that of ConA group(p<0.05).2.5 IL-10 mRNA expressed mildly in liver tissue of control group(0.21±0.11), increased lightly in ConA group(0.43±0.19)(p<0.05) and further increased in adiponectin group(0.74±0.27)(p<0.01). IL-10 mRNA expressed in liver tissue of adiponectin group is higher than that of ConA group(p<0.05).3 The influence of adiponectin on hepatocellular apoptosis in acute liver injury3.1 Hepatocellular apoptotic rate detected by TUNEL and FCM show that : hepatocellular apoptotic rate of adiponectin group(5.21±0.32%) is lower than that of ConA group(5.21±0.32%)(P<0.01)but is similar to that of control group(4.96±0.23%)(P<0.01),that of ConA group is higher than control group(P<0.01).3.2 The average percent of different stage of heptocellular generation cycle in control group ,adiponectin group and ConA group are: G0/G1 atage: 36.82±3.1%,46.73±4.6%,52.70±5.9% respectively; S stage: 48.83±3.6%,37.47±4.1% , 32.15±4.6% ; G2/M stage: 15.35±1.4% , 16.38±1.8% ,15.30±1.3%. The average percent of S stage of heptocellular generation cycle is decreased ,but G0/G1 stage is increased after ConA administration ,this role of ConA can be relieved by adiponectin intervention.4 The primary investigate on signal transduction route through which adiponectin exert anti-inflammation4.1 PI3K protein ,which mainly expressed on cellular membrane, slightly expressed in murine liver of control group(10.85±5.43%)is lower than that of adiponectin group ( p<0.01)and ConA group( p<0.01).The expression of PI3K protein in murine liver of adiponectin group (53.83±20.78%)is increased than that of ConA group(32.75±14.97%)(p<0.05),which can express mainly in cytoplasm.4.2 PKA protein ,which mainly express on cellular membrane, slightly expressed in murine liver of control group (12.48±8.87%) increased markedly in ConA group(87.69±25.86%( p<0.01)) and adiponectin group(80.33±30.35%)( p<0.01), which express mainly in cytoplasm.4.3 PKA mRNA expressed markedly in murine liver tissue of control group(1.19±0.43), adiponectin group(1.04±0.11) and ConA group(1.41±0.66),there are no difference among them.4.4 PI3K mRNA expressed mildly in liver tissue of control group(0.36±0.12),is increased in adiponectin group(0.54±0.13)(p<0.05), PI3K mRNA expressed in liver tissue of control group have no difference to that of Con A group.4.5 NF-κB binding activity in the liver of mice was detected by EMSA: the activity of NF-κB in ConA group (267.30±80.65)were increased significantly than that in normal contro(l75.73±20.14)(p<0.01). The ConA-mediated hepatic NF-κB activation is significantly down-regulated by adiponectin(152.83±58.86), NF-κB binding activity of adiponectin group was higher than that of control group, (p<0.05), and lower than that of ConA group(267.30±80.65)(p<0.01)4.6 Correlation analysis show that NF-κB activation is positive correlation to the level of TNF-α(r=0.823,p<0.01),ALT(r=0.793,p<0.01).Conclusion1 Acute liver injury can be induced by Con A i.v., with decreased adiponectin serum level. A lot of inflammatory cytokines are produced after Con A i.v. and they may inhibit the secretion of adiponectin by adipose tissue. Adiponectin protein and its mRNA expression in liver is up-regulated in ConA-mediated acute liver injury because of the inflatration of inflammatory cells to liver. Application of exogenous adiponectin to BALB/c mice markedly attenuated liver inflammation induced by ConA but no influence to the expression of mRNA of adiponectin.2 The serum level of ALT,IL-4,TNF-α,IL-10 and IFN-γcan be increased by Con A i.v. . Adiponectin probably exerts its hepatoprotective effects in the model of acute liver inflammation induced by ConA by a mechanism of pro-inflammatory cytokines down-regulation, such as, IL-4,TNF-αand IFN-γ, and anti-inflammatory cytokines up-regulation,such as IL-10. The treatment with adiponectin markedly reduced serum transaminase activities .3 Hepatocellular apoptosis may be induced by ConA administration. Adiponectin treatment may protect hepatocytes through relieving the inhibited role to heptocellular generation cycle induced by ConA, decreasing hepatocellular apoptotic rate.4 The expressin of PKA and PI3K protein and NF-κB binding activity in the liver of mice were increased significantly after ConA injection. Supplying adiponectin may further raise the expression of PI3K in either protein or gene level and lower NF-κB binding activity ,but have no effect to PKA. The protective effect of adiponectin administration on liver injury may be mediated by activing PI3K/Akt/NF-κB signal transduction pathway.