Expression Of MAPEG Family And Its Products In Concanavalin A-induced Mice Hepatitis And Hepatoprotect By Cyclosporin A

In present study, we employed Concanavalin A(ConA)-induced mice hepatitis model to investigate the metabolism of arachidonic acid and the role of MAPEG family, during the immune liver injury; we also observate the protect effect of (Cyclosporin A)CsA on ConA-induced hepatitis, and investigate whether leukotriene pathway involved in this protection, as assessed by the both protein expression and its product.The thesis was divided into two parts.Part 1. Alternation of metabolism of Arachidonic Acid and expression of MAPEG family in ConA-induced mice hepatitisObjective: To investigate the change of the two important metabolites of arachidonic acid, PGE2 and cysLTs, and expression of MAPEG family.Methods: Male Balb/c mice were randomly divided into 5 groups, 6 mice/group. ConA (20 mg/kg) was administratered through the tail vein and mice were sacrificed at 1 h, 2 h, 6 h and 8 h, respectively. Saline in the corresponding volume served as control group. Liver injury were evaluated by the determination of ALT and AST and histological examination of HE staining. The serum TNF-αand IFN-γand hepatic cysLTs content were assayed by the corresponding ELISA Kit, whereas the hepatic PGE2 content by RIA method. The expression of LTC₄S, mGST₂ and mGST₃ mRNA levels, the mGST₂ protein expression, and protein expression of other MAPEG members and MAPK were examined by RT-PCR, Immunohistochemistry and Western blot, respectively.Results: when mice were intravenously injected with ConA, the animals suffered from acute liver failure, with dramatical elevatation of ALT and AST and histopathological changes within 8 h. There are differential expression of two metabolites of arachidonic acid, the cysLTs increased at the early of liver injury and peaked between 1 h～2 h after ConA administraion, whereas PGE₂ changed insiginificantly. As compared with control, little mPGES expression was found in both mRNA and protein, while accumulative increment of both mGST₃ and LTC₄S, as well as mGST₃ expression found by immunohistochemistry. Besides, the dramatical increase of cysLTs might be also attributed to its upstream catalysis related proteins such as 5-lipoxygenase (5-LO) or 5-lipoxygenase -activating protein (FLAP), as well as cysteinyl leukotriene receptor type 1 (cysLT₁R). The serum TNF-αand IFN-γand MAPK pathway were also investigated and both peaked in the early phage of ConA-induced mice liver injury.Part 2. Alternation of MAPEG and its product cysLTs involved in the protect effect of CsA on ConA-induced hepatitisObjective: To explore the alternation of MAPEG and its product cysLTs whether involved in the protect effect of CsA on ConA-induced hepatitis.Methods: Male Balb/c mouse liver injuried-model were developed by i.v. injection of ConA (20 mg/kg) and protected by CsA pretreatment (150 mg/kg) before ConA administration and sacrifice at 2 h and 8 h. Saline and CsA treatement alone were served as control. Liver injury were evaluated by the determination of ALT and AST and histological examination of HE staining. The hepatic cysLTs content were assayed by ELISA Kit, whereas expression of MAPEG family were assessed by Western blot. Results: Pretreatment with CsA prevented mouse liver from ConA injury, accompanied with decrease of hepatic cysLTs. Besides,CsA abrogated an ConA-induced up-regulation of mGST₃, but failed in LTC4S regulation. CsA significantly reduced the expression of 5-LO, whereas little effect on cysLT₁R and FLAP, all of which were upregulated by ConA administration.Conclusions:1.Balb/c mice developed severe liver injury after ConA administration, with thedramatical increase of cysLTs, and up-regulate both levels of mRNA and protein ofFLAP,LTC₄S,mGST₂ and mGST₃, while little effect on mPGES expression; CsApretreatment protect liver injuried by ConA and inhibit the increase of cysLTs,withdown-regulate mGST₃ and little effect on FLAP and LTC₄S;2.ConA enhanced the expression of 5-LO and cysLT₁R, and CsA pretreatmentreduced the elevation of 5-LO, but failed to inhibit the up-regulation of cysLT₁R;3.The dramatical increase of serum TNF-αand IFN-γ, and activation of MAPK wereboth found early, which maybe initialed the disorder of arachidonic metabolism andliver injury.