Effect And Mechanism Of N,N’-Dicyclohexyl-N-Linoleic Acylurea On Experimental Autoimmune Encephalomyelitis

Background:Multiple sclerosis(MS)is a demyelinating disease of the central nervous system caused by chronic inflammation.MS is the leading cause of disability for young people.The pathogenesis of MS is complex and not fully understood.It has been established that autoimmune processes play a key role in pathogenesis.The peripheral immune tolerance of MS patients is broken due to environmental,genetic and epigenetic factors.Autoreactive T cells may be activated by molecular simulation and bystander pathway,penetrate the into the parenchyma of the central nervous system though blood-brain barrier,and cause damage to the nervous system.MS can not be cured.At present,the main treatment methods include acute management,disease-modifying treatments(DMT)and symptomatic treatment.High dose methylprednisolone pulse therapy is the main treatment for acute MS.Disease modification therapy used to treat relapse-remitting MS is designed to reduce the symptoms and frequency of disease recurrence.More than a dozen DMT drugs have been used to treat relapse-remitting MS.However,these drugs may have serious side effects,so it is necessary to study more efficient and less toxic MS drugs.Isatidis Radix is a traditional Chinese medicine.In our previous study,a series of unsaturated fatty acids were isolated from Isatidis Radix.The structures of these unsaturated fatty acids were optimized,and then a series of urea compounds were synthesized.The toxicity of these compounds was very low.The results of acute toxicity tests showed that a single dose of 5000mg/kg of the these compounds did not cause abnormalities in mice.These compounds were found to have immunosuppressive effects in organ transplantation models.These compounds can significantly inhibit the proliferation of lymphocytes and prolong the survival of the graft.Based on the pathogenesis of MS and the action mechanism of these compounds,we speculate that these compounds may have therapeutic effects on MS.Aim:To study the efficacy and action mechanism of N,N,-dicyclohexyl-N-linoleic acylurea(DCLAA)on the animal model(experimental autoimmune encephalomyelitis,EAE)of human MS.Methods:(1)Study on safety:mice were given intraperitoneal injection of DCLAA(30mg/kg and 60mg/kg)for 20 consecutive days.The detection content is as follows:appearance,behavior and physiological changes of the animals;changes of the main organs and liver and kidney functions;apoptosis in spinal cord.(2)Study on efficacy:Mice were divided into 5 groups:control group,EAE group,EAE+DCLAA group,EAE+methylprednisolone group,and EAE+DCLAA+low-dose methylprednisolone group.The efficacy of DCLAA was determined by clinical score and demyelination of spinal cord of mice.(3)Mechanism of DCLAA on lymphocyte:experiments in vivo were conducted to explore the effect of DCLAA on the infiltration of CD4~+,CD8~+T cells,microglia and astrocytes in the central nervous system of mice;the influence of CD4~+,CD8~+T cell numbers in peripheral immune organs;effects on pro-inflammatory cytokines TNF-αand IFN-γ,anti-inflammatory cytokine TGF-βin blood of mice;effect on cell cycle and apoptosis of lymphocyte in mice.The effects of DCLAA on lymphocyte proliferation,anergy,cell cycle,apoptosis and cytokine release were further explored in vitro.(4)Mechanism of DCLAA on microglia:BV-2 cells were stimulated with LPS to establish an neuroinflammatory model of microglia in vitro.This neuroinflammation model in vitro was used to study the effects of DCLAA on the release of reactive oxygen species(ROS),NO,cytokines,and the effects of M1-activation and M2-activation markers of microglia.Results:(1)Safety:There were no obvious abnormalities in appearance,behavior and physiology in mice after 30mg/kg or 60mg/kg DCLAA treatment.HE results showed that there were no obvious abnormalities in heart,liver and kidney in mice.The liver and kidney functions were normal.No apoptosis was found in the spinal cord.(2)Efficacy:DCLAA reduced the clinical score of EAE mice and delayed the disease onset.DCLAA significantly reduced demyelination in EAE mice.The effect of DCLAA combined with methylprednisolone was not better than that of DCLAA alone.(3)Mechanism of DCLAA on lymphocyte:After DCLAA treatment,the infiltration of CD4~+,CD8~+T cells,microglia and astrocytes in the central nervous system in EAE mice reduced significantly.The number of CD4~+and CD8~+T cells in the blood and spleen,the concentration of TNF-αin the blood decreased significantly as well.The concentration of IFN-γincreased,but there was no statistical difference.However,levels of anti-inflammatory cytokine(TGF-β)elevated significantly.The results of experiment in vitro showed that the cell cycle of DCLAA-treated lymphocytes was blocked in G1 phase,cell proliferation was inhibited,mitochondrial membrane potential decreased,and cell apoptosis increased after DCLAA treatment.The concentrations of both IFN-γand TGF-βwere increased.Results of transcriptome sequencing also showed that DCLAA significantly affected the proliferation and survival of lymphocytes.Meanwhile,the expression of PI3K and P-Akt was decreased after DCLAA treatment both in vivo and in vitro.(4)Mechanism of DCLAA on microglia:After LPS stimulation,the release of ROS and NO in BV-2 cells was significantly increased.The pro-inflammatory cytokine TNF-αincreased significantly,while the anti-inflammatory cytokine IL-10 decreased significantly.The expression of i NOS increased significantly,while the expression of Arg-1 decreased significantly.However,DCLAA inhibited LPS-induced activation of BV-2 cells.The expression of the pro-inflammatory cytokine decreased when BV-2 cells were pretreated with DCLAA for 2 h and then stimulated with LPS.Meanwhile,the expression of Arg-1 increased significantly.The expression of TLR4/NF-κB signaling pathway related proteins TLR4 and P-IκBαdecreased significantly.The expression of pro-inflammatory cytokine(TNF-α、IFN-γ、IL-1β)in spinal cord and the expression of M1-activation markers(CD86 and CD16)in spinal cord and brain decreased significantly in EAE mice treated with DCLAA.Conclusion:DCLAA inhibits proliferation and promotes apoptosis of lymphocytes through PI3K-Akt signaling pathway.DCLAA reduces the number of lymphocytes,which in turn reduces peripheral lymphocyte infiltration into the central nervous system.DCLAA also inhibits the progression of EAE by reducing neuroinflammation in microglia via the TLR4/NF-κB signaling pathway.This study can provide reference for the development of MS treatment drugs.