Effects Of Cyclosporin A On Lipopolysaccharide-induced Inflammatory Cytokines Production In Genital Tract Of Female Rabbit

The genital tract of the female rabbit is more susceptible to microbial invasion than other internal organs.Infections caused by Gram-negative bacteria can cause uterine dysfunction and lead to intrauterine growth retardation,premature birth and abortion.Lipopolysaccharide(LPS),a component of Gram-negative bacteria,is recognized by Tolllike receptor 4(TLR-4)on the cell surface,and then induces the production of proinflammatory cytokines IL-1β,IL-6 and TNF-α via My D88-dependent signaling pathway to induce systemic immune response.Cyclosporin A(CsA)is a macrolide immunosuppressive agent that has been used in the treatment of spontaneous abortion and recurrent abortion due to immunoresponse.At present,there are some reports about the effect of CsA on the production and secretion of lymphocyte cytokines and its mechanism of action,however,the potential role of CsA on local infection of female genital tract has not been reported yet.In this study,LPS was infused into the uterine body of the female rabbit to simulate local infection of the genital tract,and then CsA was intramuscularly injected to investigate whether CsA could affect the expression of inflammatory cytokines induced by LPS.Twenty(3-4 months old,2.5±0.1 Kg)healthy female New Zealand white rabbits were randomly divided into four groups(n=5 each)after estrus induction,control group,LPS group,CsA group and LPS+CsA group.The rabbits in the LPS group were given an intrauterine infusion of Escherichia coli LPS(4 mg/Kg body weight(BW)).Rabbits in the CsA group were given CsA(20 mg/Kg.BW).Rabbits in the LPS+CsA group were given LPS(4 mg/Kg.BW)and CsA(20 mg/Kg.BW).The control group received only LPS and CsA carrier.The tissue specimen from the cervix,uterine body,uterotubal junction,oviductal isthmus and ampulla were collected 3 h post-injection.The gene and protein expressions of pro-inflammatory cytokines IL-1β,IL-6,IL-8,TNF-α and IFN-γ and anti-inflammatory cytokines IL-4,IL-10,IL-13 and TGF-β were observed using q RT-PCR and immunohistochemical(IHC)assay,respectively.The results shown that(1)IL-1β,IL-6,IL-8,TNF-α,IFN-γ,IL-4,IL-10,IL-13,TGF-β,TLR-2 and TLR-4 were expressed in the cervix,uterine body,uterotubal junction,oviductal isthmus and ampulla of female rabbits.(2)Compared with the control group,LPS-treatment up-regulated the expression of IL-6 and TNF-α in the uterine body and IL-1β in the uterotubal junction(P<0.05),but did not change the expression of IL-8 and IFN-γ in all of the detected tissues(P>0.05).In addition,in the cervix and oviduct,the expression levels of all detected pro-inflammatory cytokines were not different between the LPS group and the control group(P>0.05).(3)CsA-treatment alone increased the expression levels of antiinflammatory cytokines IL-4 in the uterine body,uterotubal junction and oviductal ampulla,IL-10 in the cervix,oviductal isthmus and ampulla and TGF-β in the uterotubal junction and oviductal ampulla(P<0.05),and that CsA-treatment alone significantly up-regulated the expression levels of pro-inflammatory cytokines IL-6 and IL-8 in the cervix,IL-1β in the oviductal isthmus,TNF-α in the oviductal ampulla and IFN-γ in the uterine body(P<0.05).(4)Compared with the LPS group,LPS+CsA-treatment significantly inhibited the expression of pro-inflammatory cytokines IL-6 in the uterine body,the uterotubal junction and the oviductal isthmus,TNF-α in the uterine body and IFN-γ in the uterotubal junction and the oviductal isthmus(P<0.05).(5)The results of immuno-histochemical assay(IHC)shown that IL-6 distributed in the luminal epithelium and glandular epithelium of the cervix,the luminal epithelium,glandular epithelium and stroma layer of the uterine body,the luminal epithelium and stroma layer of the uterotubal junction and the luminal epithelium,stroma layer and myometrium of the oviduct tract.IL-10 distributed in the luminal epithelium of the cervix,the uterine body and the uterotubal junction and the luminal epithelium and stroma layer of the oviduct tract.(6)Compared with the control group,LPStreatment significantly up-regulated the protein expression of pro-inflammatory cytokine IL-6 in the uterine body and the oviductal isthmus(P<0.05).(7)CsA-treatment alone did not affect the expression level of IL-6 protein in all tissues examined(P>0.05),However,CsAtreatment significantly reduced LPS-induced IL-6 protein expression in the uterine body and the oviductal isthmus(P<0.05).In addition,compared with the control group,CsA-treatment alone significantly up-regulated the protein expression of the anti-inflammatory cytokine IL-10 in the cervix,the uterine body,the oviductal ampulla and isthmus(P<0.05).However,LPS-treatment did not affect the protein expression of IL-10 in the genital tract(P>0.05).On the basis of our results,intrauterine infusion of 4 mg/Kg LPS for 3 h induced the expression of pro-inflammatory cytokines,but did not affect the expression of antiinflammatory cytokines in the genital tract of female rabbits.20 mg/Kg CsA-treatment alone for 3 h increased the expression of pro-and anti-inflammatory cytokines,LPS+CsAtreatment inhibited the increase of pro-inflammatory cytokines induced by LPS,but did not affect the expression of anti-inflammatory cytokines after LPS treatment.