Expression Characteristic Of Cysteinyl Leukotrienens Autocrine/Paracrine Loop In Rat Acute Hepatic Injury Model And Its Regulation By Asiatic Acid

Hepatitis is a universal disease which severely threatens People's health.Its pathogenesis is extremely complicated,with indeterminate mechanisms. Experimental models of acute hepatic injury suppy a well platform for research and development of anti-hepatitis drug.Acute hepatic injury models induced by D-Galactosamine(D-GalN) / lipopolysaccharide(LPS) or carbon tetrachloride (CC14) are well accredited and wide used animal models.Cysteinyl leukotrienes(cys-LT),as inflammatory factors,are involved in the pathogenesis of a variety of liver diseases.It was demonstrated that MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism) superfamily plays an important role on several liver inflammatory injuries.Thus, expression of LTC4 synthase(LTC4S) and microsomal glutathione-S-transferase (mGST) 2,mGST3 in MAPEG superfamily were investigated in the rat acute hepatic injury model,which will provide a better understanding between their expression and the liver pathophysiology.The mechanism of cys-LT initiating liver injuries is still unclear,which may elicit biological responses as follows via the cys-LT receptor(cysLTR):(1) vasoconstriction,result in ischemic and hypoxia and hepatocyte injury.(2) mediator network:LTs mediate the TNF,thromboxan B2,PGB2 et al.(3) Oxygen free radical impairment.It was reported that rat cysLT1R was mainly expressed in lung smooth muscle cells,eosinophils,and macrophages.However,there is no information published on its expression and regulation in D-GalN/LPS injured liver yet.Therefore,it is important to investigate the expression of cysLT1R in liver in normal or pathological conditions,which may result in cys-LT autocrine/paracrine loop or a complicated cell factors network in liver disease.On the other hand,it was reported that NF-κB and MAPK,as downstream signaling molecules of Redox pathway,were involved in liver inflammatory injuries.In our present study,it was found that LTC4S take main responsibility for hepatic cys-LT accumulation in D-GalN/LPS induced acute hepatic injury model. Therefore,the role of ROS and ERK signal transduction pathways on increased expression of LTC4S in rat HCs and KCs co-cultured system induced by D-GalN/LPS was investigated.Triterpenoid was reported to be effective as an anti-inflammatory and inhibit the PLA2,upstream protein of 5-LO pathway.In our present study,the protective effect of AA,as a triterpenoid,against D-GalN/LPS induced injuries in Hepatocytes (HCs) and Kupffer cells(KCs) co-cultured system was investigated,as well as its mechanism by inhibiting the expression of MAPEG superfamily and reversing ROS generation and GSH depletion.1 Expression of LTC4 synthesis enzymes in rat acute hepatic injury model and its regulation by ROS signal pathwayAim:To investigate the expression and activity of LTC4 synthesis enzymes and their underlying relationship with cys-LT generation in a rat acute hepatic injury model induced by D-GalN/LPS or CC14.Methods:Rats were treated with D-GalN(300 mg/kg) plus LPS(0.1 mg/kg) for 1,3,6 and 12 h or CC14 for 3,6,12 and 24 h.Enzyme immunoassay was used to determine the hepatic cys-LT content.Reverse transcription-polymerase chain reaction(RT-PCR),Western blot or Immunohistochemical assay were employed to assess the expression or location of LTC4 synthesis enzymes.Activity of LTC4 synthesis enzymes was evaluated by determination of the products of LTA4 after incubation with liver microsomes using high performance liquid chromatography (HPLC).HCs and KCs co-cultured system were exposed to a concentration of GalN (1 mmol/L)/LPS(10 ng/ml) for 1,3,6,12 h.Results:Livers were injured after treatment with D-GalN/LPS,accompanied with cys-LT accumulation at the prophase of liver injury.Both LTC4S and mGST2 were expressed in the rat liver,while the latter was specifically located in hepatocytes.Their mRNA and protein expressions were up-regulated at an earlier phase after treatment with D-GalN/LPS.Meantime,a higher activity of LTC4 synthesis enzymes was detected,although the activity of LTC4S played the main role in this case.Moreover,D-GalN/LPS treated HCs and KCs co-cultured system well simulated the injury characteristics of the in vivo model.Conclusion:The results demonstrate that 1.Cys-LT accumulation plays a role in D-GalN/LPS-induced acute hepatic injury model;2.Cys-LT accumulation is related to enhanced expression and activity of both LTC4S and mGST2;3.The increased cys-LT and LTC4 synthesis enzymes in the liver injuries induced by D-GalN/LPS need the participation of nonparenchymal liver cells;4.D-GalN/LPS treated HCs and KCs co-cultured system well simulated the injury characteristics of the in vivo model,which supply a good model for in vitro study.2 Identification of cysLT1R expression in rat liver and alteration of cysLT1R expression in D-GalN/LPS induced rat acute hepatic injury modelAim:To identify the expression of cysLT1R in rat liver and primary isolated hepatocyte;and to explore the alteration of cysLT1R expression in D-GalN/LPS induced rat acute hepatic injury model.Methods:RT-PCR,Western blot and Immunochemistry were employed to detecte the expression of cysLT1R in rat liver and primary isolated hepatocyte. Meantime,pharmacological identification was also used in primary cultured hepatocyte,which was performed to detect the cellular Ca²⁺ when in present or absent of specific cysLT1R antagonist ONO-1078.Rat acute hepatic injury model was established to investigate the expression of cysLT1R in liver pathological condition.Results:It demonstrated that mRNA and protein of cysLT1R were expressed in both liver and hepatocyte.Immunochemistry assay indicated that cysLT1R was distributed in cell membrane of endothelial cell and hepatocyte.What is more,the expression of cysLT 1R was increased at the later prophase of rat acute hepatic injury model induced by D-GalN/LPS.Conclusion:CysLT1R is expressed in rat liver and hepatocyte.Its expression in liver is increased when the rat is treated with D-GalN/LPS for 6 or 12 h. 3 Hepatoprotective effect of Asiatic acid and its mechanism against D-GalN/ LPS injured HCs and KCs co-cultured systemAim:To investigate the protective effects of Asiatic acid(AA) and the possible mechanisms on the D-galactosamine/lipopolysaccharide(D-GalN/LPS) induced hepatotoxicity in HCs and KCs co-cultured system.Methods:HCs and KCs co-cultured system was established.The time effect of ROS generation,ERK activation,NF-κB activation and LTC4S expression were detected.Subsequently,the effect on LTC4S expression was detected when the cells were pretreated with U0126 or pyrrolidine dithiocarbamate(PDTC),small molecule inhibitors specific for ERK and ROS respectively.Subsequently,the co-cultured system was pretreated by AA at concentration of 1,3,10μmol/L for 12 h,followed by D-GalN/LPS treatments.Aspartate aminotransferase(AST),lactate dehydrogenase(LDH) in the cultured medium and cell viability were measured as the indices of cell injuries.JC-1 staining was used to determine mitochondria membrane potential(△Ψm);AO-EB staining was applied to assess apoptosis or necrosis of cells;DNA fragmentation was performed to analyse the apoptosis of cells.Moreover,fluorescence assays were applied to test reactive oxygen species (ROS),reduced glutathione(GSH) and GSH/GSSG.Meantime,cys-LT content was determined by enzyme immunoassay.Protein expression of enzymes in 5-LO pathway including 5-LO,FLAP and LTC4S were detected by Western blot.Results:D-GalN/LPS induced ROS generation,subsequent ERK and NF-κB activation.The inhibition of the ERK pathway with U0126,a potent MEK protein inhibitor abolished the ERK1/2 protein phosphorylation and blunted LTC4S expression in co-cultured system induced by D-GalN/LPS.Furthermore, antioxidants PDTC inhibited the activation of ROS and NF-κB,and also blocked LTC4S expression and attenuated the injury.We found that increases in the activities of AST and LDH and decrease in cell viability induced by D-GalN/LPS were significantly inhibited by pretreatment with 1,3 or 10μmol/L of AA.Cell morphological observation further confirmed the hepatoprotective effects of AA. Apoptosis of cells was observed after treatment with D-GalN/LPS showed by JC-1 staining,AO-EB staining and DNA fragmentation,which could also be attenuated by pretreatment of AA.Furthermore,cell redox was sustained by pretreatment of AA,which was demonstrated by the results of ROS and GSH.In parallel,produced cys-LT,associated with the increased expressions of enzymes in 5-LO pathway were also reversed by the pretreatment of AA.Conclusion:Redox signaling pathway is involved in regulation of LTC4S expression in D-GalN/LPS induced rat hepatitis.AA have protective effect against D-GalN/LPS induced cell injuries,which was associated with reversion of cell redox ruin and the inhibition of 5-LO pathway.