Effects Of Substance P In Heptic Immuine Injury Induced By Con A In Mice And In Kupffer Cells

PART ⅠThe effects of NKl-receptor antagonist L-703,606on hepatic immune injury induced by concanavalin A (Con A)ObjectiveIn order to set up the mouse model of hepatic immune injury induced by concanavalin A (Con A) and investigate the role of substance P and neurogenic inflammation mediated by SP in this hepatitis model. Meanwhile, we also evaluate the protective effects of NK1-receptor antagonist L-703,606on hepatic immune injury with assessment of serological and histological. We hope to reveal the mechanism of SP in the induction and maintenance of inflammation and to provide the experimental basis for the clinical treatment or reversion of liver injury.MethodsSixty female mice were divided into three groups randomizedly:saline control group (group1)、Con A model group (group2) and L-703,606pretreated group (group3). Mice in group1and group2were injected with saline only and Con A20mg/kg via the tail vein. Mice in group3were administrated with L-703,606in dose of10mg/kg before Con A challenge.6hours later all mice were anesthetized to gain blood from eye sockets and then killed for liver. SP contents in liver homogenates of group1and2were detected by enzyme-linktimmunosorbent assay (ELISA); The expression of TNF-α、IL-6protein in hepatic tissue of each group was deteeted by radioimmunoassay. Liver damage was assessed by serum transaminase ALT、AST measurement and histological evaluation. ResultsSP contents in liver homogenates were significantly increased in model group (P<0.05)(387.23±29.36pg/mL VS138.52±13.23pg/mL); There are edema formation, hepatocellular apoptosis and granulocyte infiltration in livers of model group, and L-703,606can significantly reduce these signs; ALT、AST activity were all increased in model and L-703,606pretreated group, but the later group is dramatically reduced (P<0.01)(402.22±16.42U/L VS782.37±21.51U/L;581.45±17.51U/L VS1004.25±18.24U/L). The content of TNF-α and IL-6protein in hepatic tissue increased compared with control group (P<0.01)(49.15±9.33pg/mlVS12.21±7.15pg/ml;281.17±13.12pg/mlVS131.25±10.34pg/ml;0.83±0.11VS0.39±0.07;0.77±0.08VS0.43±0.06).ConclusionWe established hepatic immune injury in mice successfully in this study. Histological observing show that there are edema formation, hepatocellular apoptosis and granulocyte infiltration in livers of model group. SP contents in liver homogenates in model group were obviously higher than those in control group. The content of TNF-a and IL-6protein in model group were obviously higher than those in control group.These findings suggest that neurogenic inflammation induced by SP may play an important role in this hepatitis model. Neurokinin-1receptor antagonist L-703,606can significantly alleviate concanavalin A-induced liver injury by the detection of serum transaminase and histological observing. Neurokinin-1receptor antagonist L-703,606can significantly alleviate concanavalin A-induced liver injury by the detection of the content of TNF-a and IL-6protein. It demonstrated that intervention of SP and its receptor had certain therapeutic effect on liver inflammation. PART IIThe effects of substance P and NK-lreceptor antagonist L-703,606on Kupffer cell in the production of cytokines.ObjectiveTo observe the effect of substance P in mouse Kupffer cell in vitro, We cultured mouse KCs to examine the functional consequences of exposure to SP and to determine whether treatment with SP leads to pro-inflammatory signaling activities, including the production of cytokines.To investigate the mechanism of neurokinin-1receptor antagonist L-703,606and the result may give us some new ideas for clinic use.MethodsKupffer cells from mice were isolated by collagenase digestion and differential centrifugation, using Percoll density gradients.The cells were seeded onto tissue culture plates at a density of2×106/mL and cultured in RPMI1640medium containing10%heat-inactivated fetal bovine serum,100U/mL Penicillin/Streptomycin and10mmol/L HEPES at37℃with5%CO2. All adherent cells phagocytosed latex beads and stained positive for catalase, confirming that they were Kupffer cells, and the cells were cultured for24h before the experiment. KCs were normally cultured or incubated with LPS(B group) or LPS+SP(C group) or LPS+SP+L-703,606(D group),and were stimulated in3h.NK-1R was analyzed in Kupffer cells from mice based on immunohistochemical localization using fluorescein. NK1R mRNA levels were analyzed according to real-time PCR,The IL-6and TNF-a levels in the supernatant were determined using mouse-specific enzyme-linked immunosorbent assay (ELISA) kits.ResultsThe immunoreactivity was concentrated in the cell cytoplasm,which is somewhat different from the results of other studies. NK-1R mRNA levels were upregulated in Kupffer cells incubated with SP for24h. According to real-time PCR, the results further showed that SP increased NK-1R mRNA expression2.5-fold NK-1R blockade abolished the effect of SP on NK-1R mRNA expression and significantly reduced the NK-1R mRNA expression compared with the control culture medium.The cytokine levels (IL-6/TNF-a) in the supernatant resulting from the release from cultured Kupffer cells revealed a substantial increase in the SP-pretreated group compared with the control culture medium group. The IL-6/TNF-a levels of supernatant in the SP-pretreated group were significantly higher than those in the no SP-pretreated group (92.15±8.56pg/mlVS31.02±7.26pg/ml;194.12±11.09pg/ml VS71.13±9.36pg/ml). The IL-6/TNF-a levels of supernatant in the L-703,606-pretreated group were significantly lower than those in the SP-pretreated group(28.38±5.04pg/ml VS92.15±8.56pg/ml;68.16±9.51pg/mlVS194.12±11.09pg/ml).ConclusionMouse Kupffer cells express NK-1R and SP increases NK-1RmRNA expression in Kupffer cells in vitro.SP enhances IL-6and TNF-a secretion and L-703,606Inhibits SP-Induced IL-6and TNF-a release from Kupffer cells in vitro.