The Effects Of Sympathetic Nervous System Regulation On Acute Liver Injury By Beta-Adernergic Signaling

BackgroundLiver injury can be induced by liver surgery or trauma, viral hepatitis and adverseeffect of medicine, which would lead to liver insufficiency or liver failure. There is still noeffective therapeutic modality for this complication so far, due to the fact that themechanism of acute liver injury remains unclear.The regulation of hepatic inflammation could be categorized into two types: body fluidregulation and neural regulation. A majority of such studies were focused on body fluidregulation. Recent studies have demonstrated that, sympathetic nerve stimulation couldexacerbate hepatic injury, while removing which could alleviate injury and promoterecovery.Overseas and our previous studies demonstrated that inflammatory progression isregulated by alpha-adrenergic receptors on the Kupffer cells stimulated by norepinephrinefrom sympathetic nerve. Nevertheless, Kupffer cells also possess beta-adrenergic receptorswhich were highly expressed in the later stage of sepsis. However the mechanism is stillunclear. The current study was designed to investigate whether sympathetic nervous systemcould regulate acute liver injury by beta-adrenergic signaling and to explore its underlinemechanism.ObjectiveBeta-adrenergic receptor agonist and antagonist were applied to rats with acute liverinjury to investigate the effect on acute liver injury. Furthermore, in vivo and in vitroresearches on Kupffer cells were performed to explore the role and the mechanism ofKupffer cells on sympathetic nervous system for acute liver injury by beta-adrenergicsignaling.Methods1Beta-adrenergic receptors were activated by intraperitoneal injection of isoprenaline （ISO） to simulated sympathetic stimulation. With normal rats were used as control group,ALT, AST, Alb and TBil alteration in peripheral blood were withdrawn, combined withpathological observation from HE stain hepatic slices to determine whether ISO possesshepatotoxicity.2An acute liver injury model was made by CCl₄.Rats were then pretreated withdifferent doses of ISO （1,10and100mg/kg）, and rats without ISO injection were used ascontrol. In different time points （24,48,72and96h）, ALT, AST, Alb and TBil alteration inperipheral blood were withdrawn, combined with pathological observation from HE stainhepatic slices to investigate dose-and time-dependent effects of ISO on acute liver injury.3Propranolol （PRO） were intraperitoneally injected to block the activation ofbeta-adrenergic receptors, then on the acute liver injury model, with no PRO injection ascontrol. ALT, AST, Alb and TBil alteration in peripheral blood were withdrawn, combinedwith pathological observation from HE stain hepatic slices to observe the effect ofbeta-adrenergic receptor blockage in acute liver injury.4After Kupffer cells depletion by GdCl3, ISO were intraperinoneal injected to activatebeta-adrenergic receptors, then on the acute liver injury model, with no ISO injection ascontrol. ALT, AST, Alb and TBil alteration in peripheral blood were withdrawn, combinedwith pathological observation from HE stain hepatic slices to explore the role of Kupffercells on the regulation of sympathetic nervous system on acute liver injury bybeta-adrenergic signaling.5Kupffer cells were harvested by in situ perfusion in liver, type IV collagenase,Percoll density gradient centrifugation and selective adhesion, and then identified byphagocytosis test and ED2immunocytochemistry staining.6The in vitro cultivation of Kupffer cells were categorized into four groups: PBSgroup, LPS group, ISO+LPS group and PRO+ISO+LPS group; IL-6expression wasdetermined by ELISA kit, and the expression of TIMP-1mRNA was tested throughreal-time PCR to investigate the mechanism of the regulation of sympathetic nervoussystem on acute liver injury by beta-adrenergic signaling.Results1Exclusively intraperitoneal injection of ISO did not cause significant deviation inliver function index （P>0.05） and did not lead to liver injury. 2High ISO dose was related with severed acute liver injury （e.g. ALT: control group336.2±140.5,1mg/kg group603.7±239.7,10mg/kg group838.6±448.5,100mg/kg group1571.5±714.7, P<0.05）. From48h, all indexes of liver function were significantly differentbetween stimulus group and control group （ALT：290.8±153.9，636.0±385.2，P<0.05ï¼›AST：521.0±296.8，2289.1±1361.5，P<0.05ï¼›Alb：28.3±1.3，25.2±1.5，P<0.05ï¼›TBil：4.1±1.2，24.8±16.9，P<0.05）.3PRO prevented the worsening of liver injury via suppression of beta-adrenergicsympathetic nerve （ALT：stimulus group606.0±154.9，inhibition group210.0±176.1，P<0.05）.4After Kupffer cells depletion, liver function index were maintained withoutsignificant difference between control and stimulus group （P>0.05）.5Latex-beads phagocytosis test exhibited that95%cells were capable of phagocytosisand ED2immunocytochemistry staining showed over90%cells were positive.6Through ELISA testing of expression of IL-6: concentration of each experimentgroup were significantly raised, in contrast of PBS group （P<0.05）. Concentration of IL-6in ISO+LPS group was significantly lower than that in LPS group （P<0.05）; Concentrationof IL-6in PRO+ISO+LPS group was obviously elevated, in contrast to ISO+LPS group.Real-time PCR testing on expression of TIMP-1mRNA demonstrated that: expression ineach experiment group was significantly raised, in contrast of PBS group （P<0.05）.Expression of TIMP-1mRNA in ISO+LPS group was significantly lower than that in LPSgroup （P<0.05）; Expression of TIMP-1mRNA in PRO+ISO+LPS group was apparentlyelevated, in contrast to ISO+LPS group.Conclusions1Beta-adrenergic receptor agonist could exacerbate CCl₄-induced acute liver injury.This effect was also validated from relieved CCl₄-induced acute liver injury by blocking theactivation of beta adrenergic receptors. Therefore, sympathetic nervous system couldregulate acute liver injury by beta-adrenergic signaling.2Kupffer cells played a critical role in the regulation of sympathetic nervous systemon acute liver injury by beta-adrenergic signaling. After Kupffer cells depletion, activationof beta-adrenergic receptors was unable to exacerbate CCl₄-induced acute liver injury.3Sympathetic nervous system activated beta-adrenergic receptors on Kupffer cells, suppressed the expression and secretion of IL-6and TIMP-1, down-regulated thehepatoprotection of IL-6and TIMP-1, exacerbated CCl₄-induced acute liver injury, andfinally accomplished the regulation on acute liver injury.