| ObjectiveGuillain-Barre syndrome (GBS) is a common acute peripheral nerve autoimmune disease, characterized with the demyelination of acute peripheral nerve and inflammatory cells infiltration, clinical flaccid limb symmetry paralysis as the main feature. For the treatment of GBS, both intravenous gamma globulin and plasma exchange, are able to shorten the course of the disease and speed up the recovery of the disease, but there are still approximately3%to10%fatality rate and20%of patients are left with varying degrees of disability. It is necessary to find new therapeutic modalities for GBS.Prostaglandin E2(PGE2), an important substances of arachidonic acid metabolites, has a strong immune activity. PGE2also regulates immune cell development, function and survival. Prostaglandin E2receptor (EPs) is divided into four subtypes, namely EP1, EP2, EP3, and EP4, EP4receptors on T lymphocytes and dendritic cells promote the differentiation of Thl cells and Th17cells amplification. In recent years, studies showed that PGE2-EP4signaling play an increasingly important role in some autoimmune diseases.Experimental autoimmune neuritis (EAN), a CD4+T-cell-mediated autoimmune demyelination disease of acute peripheral nerve, is publicly known as a classic animal model for studying GBS. EAN is peripheral nerve demyelination and inflammatory cell infiltration as the main lesion and its proinflammatory cytokines, such as IFN-γ, IL-17, IL-12, play a key role. Based on previous in vitro and in vivo studies of PGE2-EP4receptor antagonists on immunologic system, PGE2-EP4receptor antagonist L161982was used in treatment of EAN on the immunization phase and onset phase in this study. The therapeutical effect of L161982on EAN rats and the possible immunologic mechanisms were evaluated by examining the changes in clinical, histopathological and immune indicators.Methods1. Antigen extract:Bovine peripheral nerve myelin (BPM) was prepared from intra-and extradural spinal nerve roots by ultracentrifugation.2. Experimental animals:Twenty-one female Lewis rats,7-8weeks old, weighing150-170g were used in the present study. All of the rats were maintained in specific-pathogen free condition.3. Induction and clinical evaluation of EAN:10%chloral hydrate was used for the animal anesthesia (300mg/kg) by intraperitoneal (i.p) injection. EAN was induced by subcutaneous (s.c) injection into both hind footpads with200μL of antigen emulsion including5mg of BPM, and2mg of mycobacterium tuberculosis and100μL of incomplete Freund’s adjuvant and100μL of saline). Clinical score were assessed one day before immunization and thereafter daily until day16post immunization. Clinical score:0=no illness;1=flaccid tail;2=dragging both hind linbs;3=paralysis of both hind limbs;4=paralysis of four limbs or death; intermediate scores of0.5increment were given to rats with intermediate signs.4. L161982treatment:Twenty-one rats were randomly divided into treatment group A, treatment group B and control group (n=7). Treatment group A were intraperitoneally injected daily with L161982(5mg/kg) one day before immunization to eight days after immunization (immunization phase). Treatment group B were intraperitoneally injected daily with L161982(5mg/kg) from days5to16after immunization (onset phase) and control group was intraperitoneally injected with the same volume L161982dissolvent.5. Histopathological examination:The EAN rats were sacrificed on day16post immunization, at a time when the clinical signs of EAN peaked. Sciatic nerves were excised near the lumbar spinal cord segment, fixed in10%phosphate-buffered formalin, and embedded in paraffin. Multiple longitudinal sections (4μm slices) of sciatic nerves were stained with hematoxylin and eosin to observe the histopathology.6. Immunohistochemistry:Paraffin tissue sections (4μm) were deparaffinized, hydrated, repairing antigen and treated for blocking endogenous peroxidase activity. The sections were incubated with polyclonal rabbit anti-rat IFN-y, IL-17antibody and then stained according to the avidin-biotin technique. The numbers of positive cells were counted at×40magnification in the entire section area. The average results were expressed as cells per mm2tissue section.7. CCK8determination of lymphocyte proliferation in vitro:The draining lymph nodes were removed under aseptic conditions on day16post immunization. The lymphocyte suspension density of2×106/mL, was inoculated into96-well plates.200μL/holes were added to10μL BPM, ConA and RPMI1640, and cultured for72h, and then join CCK810μL/hole, and incubated for4h, its optical density (OD) was measured in a microplate reader.8. Statistical analysis:The SPSS18.0computer program was used for all calculations and statistical evaluations. Results were expressed as means±tandard deviation (SD). Differences between groups were evaluated by one-factor analysis of variance (ANOVA) or rank-sum test. The level of significance was set to P<0.05.Results1. L161982treatment delayed disease onset and decreased disease peak clinical scores of EAN:Treatment group A and treatment group B displayed a significant delay in onset than control group (P<0.05). Compared to treatment group B, treatment group A also displayed disease onset (P<0.05). Treatment group A disease peak of clinical scores were significantly lower than treatment group B and control group (P<0.05). Treatment group B showed lower mean peak clinical scores than control group, but there was no statistically significant difference.2. L161982treatment inhibited peripheral nerve inflammatory cell infiltration: The sciatic nerves of EAN rats were examined histologically at the height of the clinical course of EAN. Treatment group A and treatment group B significantly reduced the infiltrating cells in sciatic nerves sections compared with the control group (P<0.05). Treatment group A significantly reduced the infiltrating cells than treatment group B (P<0.05).3. L161982treatment suppressed the cytokine expression in sciatic nerves: Immunohistochemical studies showed that the numbers of IFN-γ and IL-17expressing cells were strongly decreased in the sciatic nerves of L161982treatment group A and treatment group B compared to control group (P<0.05). Treatment group A had lower sciatic nerve IFN-y, IL-17-positive cells than treatment group B (P <0.05).4. L161982inhibited in vitro reaction:The draining lymph nodes were tested on day16post immunization for lymphocyte proliferation. When compared to control group, treatment group A and treatment group B could significantly suppress BPM and ConA-induced lymphocyte proliferation (P<0.05).Conclusions1. In this study, L161982was administered to EAN rats on the immunization phase and onset phase. Rats treated with L161982at the both phases displayed significant delay in onset, lower peak clinical score comparison with control group. These data suggest that L161982has therapeutic potential for alleviating EAN.2. Rats treated with L161982at the both phases significant decreased inflammatory cell infiltration and numbers of IFN-y, IL-17expression cells in the sciatic nerve, BPM-stimulated lymphocyte proliferation. Our findings suggest that PGE2-EP4receptor antagonist L161982could inhibit cellular immunological response by suppressing the autoreactive T lymphocyte proliferation and over-expression of pro-inflammatory cytokines.3. L161982treatment group reduced sciatic inflammatory cytokines IFN-y, IL-17expression, indicating that the PGE2-EP4receptor antagonist, non-specific effects on T-cell subsets.4. Our data showed that immunization phase treatment group significantly delayed the time of onset of EAN, reduced the peak period of the disease clinical scores compared with the onset phase of the treatment group. The results suggest that the immunization phase application of PGE2-EP4receptor antagonist L161982treatment effect is superior to the onset phase. |
PDF Full Text Request