The Study Of Inflammationary Factor Expression Profiles On Wilson's Disease And Regulatory Mechanism Of Modified Gandou Decoction On Ceramide Signaling Pathway On The Neurons Of TX Mouse

Wilson's disease(WD)is a curable inherited disorder of Copper(Cu)metabolism neurodegenerative disease,excessive Cumainly deposited in liver and brain.When it deposited in the brain,it can lead to varying degrees of brain damage symptoms.And the neurologic symptom is still difficu Lt to reverse which will seriously impact on the patients' s study and work.However,the mechanism of tissue damage from Cuoverload is intricacy and not adequately understood.As is well-known that increased extracellu Lar Cu contributed to neuronal pathogenic process by increasing the production of dangerous radical oxygen species,but the other molecu Lar mechanisms of Cuneurotoxicity has been rarely investigated yet.It is said that the increased extracellu Lar Cu levels were strongly affect the secretions of IL-1? and IL-12.Cu was capable of shifting GN11 and primary neurons towards a pro-inflammatory state.Wilson's disease model-tx mice presents as elevated brain Cu,inflammation and neurodegeneration.Cu is essential microelement to human beings,and is indispensable to maintain the function of the central nervous system(CNS).However,impaired cellu Lar homeostasis of Cu of the CNS,high Cu can trigger nerve inflammatory injury,and aggravate pathological damage.The inflammatory response of CNS has the dual role.An acute,or short-term neuroinflammatory response is likely important for a healthy functioning brain and contributes to the repair of damaged or infected tissue.However,when the inflammatory process continues for a long period of time,damage to the surrounding brain tissue may become substantial.The release of ROS can activate Cer signaling pathway,increasing inflammatory factor,and then inducing nerve inflammatory injury.These evidence indicate that the mechanism of the high Cu induced neuronal apoptosis may be that excess Cu promotes ASM secretion and activation of Cer release by increasing ROS,Cer activates P38 MAPK,PKC and JNK,and then promote neuronal apoptosis by improving the expression of inflammation,on the other directly lead to neuronal apoptosis.Therefore,the studyaimed to investigate the mechanism and interventional pathway of neuron damage induced by high Cu,which may provide new therapeutic method for clinical Chinese medicine treatment of WD patients with neurological symptoms.The team composed Modified Gandou Decoction(MGDD)by adding mu Lti-link neuroprotective effect of traditional Chinese medicine based on Gandou Decoction,previous study found that MGDD has neuroprotective effect to WD.But it is not yet clarified the regu Latory role of MGDD on neurons inflammatory injury induced by high Cu.This study intends to expound the neuroprotective effect of MGDD by means of observing the regu Latory role of MGDD on neurons inflammatory injury induced by high Cu.The first partObjective: To detect the level of inflammatory factors expression in WD.Methods: 99 patients with WD and 24 controls were recruited for this study.Ray Biotech antibody microarray was used to detect the levels of plasma inflammatory cytokinesResults: Our resuLts showed significant increase in IL-5,IL-10,IL-13,IL-21,IL-23,TGF-?1,TNF-? and TNF-?(P<0.01),but there were no significant difference in the GM-CSF,IFN-?,IL-1?,IL-2,IL-4,IL-6,IL-12p70,IL-17,IL-17 F,IL-22,IL-28 A and MIP-3a(P>0.05)between hepatic patients and healthy controls.Higher plasma GM-CSF,IL-2,IL-5,IL-6,IL-10,IL-13,IL-21,IL-23,TGF-?1,TNF-? and TNF-?levels were found in neurological patients compared with control groups(P<0.01).There were no significant differences in the IFN-?,IL-1?,IL-4,IL-12p70,IL-17,IL-17 F,IL-22,IL-28 A and MIP-3a between neurological patients and healthy controls(P>0.05).Besides,we found IL-5,IL-6,IL-10,IL-13,IL-17,IL-23,TGF-?1,and TNF-? levels were significantly higher in hepatic and neurological patients(P<0.01).However,There were no significant differences in the IFN-?,IL-1?,IL-2,IL-4,IL-12p70,IL-17 F,IL-21,IL-22,IL-28 A and MIP-3a between hepatic and neurological patients and healthy controls(P>0.05).Conclusion: we found an alteration in plasma levels of Pro-inflammatory(IL-2,IL-5,IL-21,IL-23,TNF-? and TNF-?)and immunoregu Latory cytokines(IL-10,IL-13 and TGF-?1)in patients with WD compared to healthy people.Our resu Lts indicated that dysregu Lation of cytokines,mainly,occurred in WD patients.Cu deposition cou Ld increase expression of inflammatory factor,nerve inflammatory injury may be the important pathogenic mechanism to WD patients with neurological symptoms.The second partObjective:To detect Ceramide signaling pathway related proteins expression levels in TX suckling mouse with MGDD treatment.Explore its molecu Lar targets of regu Lating Ceramide signaling pathway.Methods: Atomic absorption method to detect the concentration of microelement in the neurons of TX suckling mouses,which were treated with different concentrations of rabbit serum with MGDD;Flow cytometry was used to analyze the experssion of ROS in the neurons of TX suckling mouses after being treated with rabbit serum with MGDD;RT-q PCR and Western blot detect the expression of related gene and protein level of Ceramide signaling pathway in the neurons of TX suckling mouses after being treated with rabbit serum with MGDD.Results:1.In the concentration range of 10%-20%,after MGDD on the neurons of TX suckling mouses for 24 h,compared with model group the concentration of Cuin cells had significant difference(P<0.01),and in dose-dependent and time-dependent manners,MGDD can significantly increase the concentration of zinc(P<0.01);MGDD decreased the concentration of iron.2.Flow cytometry resu Lts show that compared with control group the release of ROS in model group was significant raise,and MGDD can significantly reduce the release of ROS with model group(P<0.01).3.RT-q PCR analysis demonstrated that MGDD concentration-dependently decrease the expression of ASMmRNA,CermRNA,PKCmRNA,JNKmRNA,P38 mRNA,CytCmRNA,Caspase 9mRNA and Caspase 3mRNA and also increase the expression of ERK1/2mRNA and XIAPmRNA in the neurons of TX suckling mouses Compared with the model group.4.Western blot indicated that MGDD concentration-dependently decrease the expression of ASM,Cer,PKC,JNK,P38,Cyt C,Caspase 9 and Caspase 3 and also increase the expression of ERK1/2 and XIAP in the neurons of TX suckling mouses Compared with the model group(P<0.01).Conclusion: Tacking together,MGDD can reduce neurotoxic induced by Cuoverload through reducing the concentration of Cu.MGDD can reduce oxidative damage induced by Cu overload through decreasing the release of ROS.MGDD concentration-dependently decrease the expression of gene and protein level of ASM,Cer,PKC,JNK,P38,Cyt C,Caspase 9 and Caspase 3 and also increase the expression of ERK1/2 and XIAP in the neurons of TX suckling mouses.these findings suggest that MGDD reduces neurotoxic induced by Cu overload by increasing the Cu excretion which regu Lated the expression of related gene and protein level of Ceramide signaling pathway in the neurons of TX suckling mouses.