The Effect And Mechanism Of Mitochondrial Uncoupling Protein 2 (UCP2) On Dyskinesia Of Hepatic Encephalopathy

Hepatic encephalopathy(HE)is a broad spectrum of neurological abnormalities that can only be diagnosed early by specific neuropsychological tests due to impaired liver function or portacaval shunts.In fact,about 40% of cirrhotic patients developed to HE and subsequently die.In clinic,patients with HE accompanied by decreased motor function,behavioral disorders or reduced activity,which seriously affected the living conditions of patients with HE,but the current pathogenesis of HE and dyskinesia remained unclear.In recent years,studies have found that oxidative stress(OS)plays an important role in the development and progression of HE,and revealed that overexpression of Uncoupling Protein 2(UCP2)could reduce neuronal damage in other neurological diseases such as Parkinson's disease(PD)and Alzheimer's disease(AD)thus protecting neuro and promoting motility.However,there is no related research that explores the relationship between the expression and function of UCP2 in Substantia Nigra reticular part(SNr)and HE.Therefore,this paper investigate the effects of UCP2 on dyskinesia of HE through monitoring the changes of UCP2 expression in SNr of HE mice and regulating the expression of UCP2 in SNr,providing potential mechanism for clinical treatment.Objective: 1.In order to observe the change of mitochondrial morphology in the substantia nigra(SN)of HE mice,thioacetamide(TAA)was utilized to construct the model of hepatic encephalopathy(Paper 1)2.To explore the possible mechanisms of improvement the dyskinesia in mice withHE providing the potential target for future clinical treatment of HE and the basis of theory,this paper monitored the expression of UCP2 in SN at different time points and the movement of HE mice.Furthermore,the UCP2 AAV virus of overexpression or downregulation was injected into the SNr to study the changes of the motility of mice with HE.(Paper 2)Method: The model of hepatic encephalopathy was constructed by continuous intraperitoneal injection of TAA 300 mg/kg for three days.After the successful preparation of the models,the liver tissues and blood samples were collected on day 1,day 4 and day 7 to perform the H&E staining and detect liver function(ALT,AST)and blood ammonia.The substantia nigra was separated from the brain tissue by brain matrix and total protein and mitochondrial protein were extracted to detect the expression of mitochondrial fission protein(DRP1),and autophagy protein(PINK1 and PARKIN).The morphology of mitochondria in substantia nigra of hepatic encephalopathy was observed by the electron microscope.(Paper 1)Next,UCP2-null(overexpression or interference)AAV virus was injected into SNr.TAA was injected intraperitoneally to prepare the model of hepatic encephalopathy on the 14 th day when the virus expressed in the vivo.After 4 days,the expression of UCP2 and the specificity of virus were determined by immunofluorescence,Western Blot and RT-q PCR.The effect of UCP2 on the motor of HE mice was detected by behaviour test.In order to clarify the possible mechanisms,the level of oxidative stress and ATP were detected by the Elisa in SNr of the different group(the model group,UCP2 KD and UCP2 OE group).Next,we further explored the relationship between the UCP2 and GAD67(GFAP/IBA-1)in SNr by immunofluorescence.We also collected the liver tissues and blood samples to perform H&E staining and detect blood ammonia to evaluate the function of UCP2.Finally,we used the Western Blot and RT-q PCR to detect the expression of KCC2 in SNr of hepatic encephalopathy.(Paper 2)Result:First of all,the model of acute liver failure was successfully induced byintraperitoneal injected TAA and the enhanced levels of blood ammonia signified that the model of hepatic encephalopathy was constructed.The mitochondrial dynamics significantly changed in SN of HE mice,which manifested that the mitochondrial fission proteinin(DRP1)was increased in SN accompanied with the increased mitochondrial autophagy protein(PINK1 and PARKIN).The results of electron microscopy further confirmed the significant changes of mitochondrial morphology that the number of mitochondria was increased and the total area reduced in substantia nigra of hepatic encephalopathy.(Paper 1)Next,targeted interference of the UCP2 in SNr was carried out to observe the effect of UCP2 on the motility in HE mice.Firstly,it was found that UCP2 was increased in protein and m RNA level in SNr of HE mice,but UCP4 had no significantly change.Then the rotarod,open field and elevated-plus maze were employed to measure the behaviour of HE mice and found that mice with HE were accompanied by dyskinesia.We found that the antioxidant enzyme(GSH,SOD)and the level of ATP were decreased in SNr of HE mice.Next,the empty vector virus of UCP2(over-expression or interference)was injected in SNr by stereotaxic localization of brain and the results of immunofluorescence indicated that the virus injection site,effect of expression and specificity were very well,and protein and m RNA levels also verified this results.Results of behavioristics showed that up-regulation of UCP2 in SNr significantly improved the dyskinesia of hepatic encephalopathy mice,whereas downregulation of UCP2 aggravated motor and balance dysfunction.To declare possible mechanisms,we examined the effect of UCP2 on oxidative stress and ATP expression within SNr.Results revealed that overexpression of UCP2 could reduce the level of oxidative stress in SNr,restore part of the antioxidant enzymes and increase the level of ATP in SNr,while interferring UCP2 obtained opposite results.To further clarify the effect of UCP2,immunofluorescence in SNr of HE was performed and found that UCP2 was mainly expressed in the GABAergic neurons but not in glial cells(GFAP,IBA-1).Finally,we observed the expression of KCC2 protein and found that UCP2 could significantly restore the expression of KCC2 in GABAergic neurons of SNr.(Paper 2)Conclusion: 1.Mitochondrial fission and mitophagy could illustrate a new mitochondria strategy to explain the changes within SNr of HE in neuropathology(Paper 1)2.Increasing UCP2 in the SNr of mice with HE could restore the expression of KCC2 by decreasing the level of oxidative stress in GABAergic neurons,which reconstruted the function of GABAergic neurons in SNr and further improve the motor function of HE mice.This study elucidated a potential mechanism of dyskinesia in mice with HE and provided a possible therapeutic target for new drug development and clinical treatment of HE.(Paper 2)...