| BackgroundHepatic encephalopathy（HE）is a neuropsychiatric syndrome characterized by metabolic dysfunction caused by acute/chronic liver disease or portal shunt.The clinical incidence of HE is high and the prognosis is poor,which is a great burden to patients,their families and society.At present,there are still many gaps in the pathogenesis of HE and there are no effective clinical treatments.Therefore,there is an urgent need to explore the pathogenesis of HE and find potential molecular targets.Mitochondria are important organelles involved in the regulation of many cellular life processes.Healthy mitochondrial morphology and function play a critical role in systemic organ function and adaptation to pathological stressors.Mitochondria fission or fusion in response to environmental stimuli,and these dynamic changes are critical for mitochondrial function and health.In particular,mitochondrial fission affects overall cellular function by influencing changes in the morphology and functional state of the mitochondria themselves and mitochondrial respiration.Mitochondria are also a major site for the production of reactive oxygen species（ROS）,which play a critical role in determining neuronal susceptibility to oxidative stress in the brain.Previous studies have targeted mitochondrial antioxidant stress therapies for the treatment of a variety of systemic diseases（cardiovascular disease,neurological injury,etc.）and to improve health,and there is now a growing body of evidence suggesting that HE induces an increase in levels of oxidative stress and the inhibitory neurotransmitter GABA in the nucleus accumbens.For example,a recent study in Science showed that optogenetic activation of glutamic acid decarboxylase 2（GAD2）neurons in the substantia nigra pars reticulata（SNr）led to a significant improvement in locomotor termination in normal mice,suggesting that GAD2 neurons in the SNr may be important neurons in the regulation of locomotion.Previous studies from our group found that mitochondria within the SNr were abnormal in the HE.However,the relationship between oxidative stress and changes in mitochondrial morphology and function in the periphery and/or center in the HE,as well as in which neuronal cells and in which nuclei in the center of HE is mainly occurs,and the major roles and specific mechanisms involved are currently unknown.Therefore,we ask the following scientific questions:（1）What are the changes in oxidative stress within the periphery and centrally at the time of HE,and what characterizes the changes in mitochondrial morphology and functional plasticity?（2）Does targeted mitochondrial antioxidant stress therapy ameliorate HE by modulating mitochondrial morphology and functional plasticity?（3）What are the types of HE neurons within the center that are primarily involved in the response and what are their specific roles?（4）Is targeted mitochondrial therapy acting on SNr-GAD2 neurons equally effective for HE,and what is the specific mechanism of action?PurposesThe aims of this study were to（1）characterize the changes in oxidative stress and mitochondrial morphological and functional plasticity during HE;（2）investigate the mechanisms of action exerted by systemically targeted mitochondrial anti-oxidative stress therapy in HE;（3）explore which cells within which nuclei in the center respond to HE and the specific roles they play during HE;and（4）explore the means and efficacy of targeted mitochondrial therapy for HE and elucidate its mechanisms of action.Methods1.Peripheral blood was collected from normal patients（without cirrhosis or hyperammonemia）,cirrhotic patients with normal blood ammonia,and cirrhotic patients with combined hyperammonia in the clinic,and the extent of changes in oxidative stress indicators in peripheral blood was examined.2.A mouse model of acute liver injury and a choledochal ligation model were established as in vivo studies of type A and type C HE models,respectively.3.Mouse brain SNr nuclei were isolated,and the extracted proteins were used to evaluate mitochondrial fragmentation,fission protein alterations,oxidative stress and autophagy molecular changes,and to purify mitochondria to test their respiratory function.4.Biochemical tests for liver function and hematoxylin and eosin（H&E）staining were used to detect the histopathological changes in the liver of mice.5.Testing blood and brain ammonia levels.Behavioral changes in mice were detected by using Open Field and Rotating rod test.6.Mitochondrial morphology-related gene expression was observed by protein immunoblotting（Western blot,WB）,immunofluorescence staining,and Mitochondrial Network Analysis Tool（Mi NA）in combination with GAD2-Mito-GFP transgenic mice at the morphological and protein levels,respectively.7.Characterization of changes in size,number and morphology of mitochondria within SNr neurons by transmission electron microscopy.8.Reactive oxygen fluorescence probe(BBoxi Probe（?）O13,China)staining method combined with GAD2-Mito-GFP transgenic mice was used to evaluate ROS levels in SNr tissues.9.Evaluation of antioxidant stress enzyme-related expression in peripheral blood by SOD and GPx level assays.10.The effects of antioxidant stress treatment on liver function,blood ammonia,behavior,and on mitochondrial morphology and function were observed in HE mice after intraperitoneal administration of mitoquinone（Mito-Q）.11.Brain edema after HE in mice was evaluated by brain water content assay.12.Changes in neuronal number expression after HE in mice were evaluated by Nissl staining.13.Using C-FOS Cre ERT2 transgenic mice,Cre-induced viral vectors carrying a fluorescent reporter gene encoding m Cherry in combination with stereotactic brain injection of AAV viruses into bilateral SNr gave specific expression in combination with tamoxifen to capture neurons activated within the SNr upon HE or local hyperammonia stimulation.14.Using injection of viral vectors carrying designed drug（DREADDs）element（Gi or Gq）specific activation receptors into the bilateral SNr of GAD2-ire-Cre mice in combination with the artificial ligand drugs Clozapine-N-oxide（CNO）or Deschloroclozapine（DCZ）activation to precisely modulate the activity of a population of GAD2 expression-positive GABA neurons and observe the effects on behavior.15.Electrophysiological techniques were used to record spontaneous firing frequencies of SNr-GAD2 neurons following chemical activation or inhibition of h M3Dq-m Cherry and h M4Di-m Cherry,respectively.16.DIO-dependent adeno-associated virus was used in GAD2-Cre transgenic mice to overexpress or knockdown mitochondrial uncoupling protein 2（UCP2）in SNr-GAD2neurons using stereotactic brain injections,and the effects of modulating UCP2 on mitochondrial morphology and function in the HE,as well as on mouse movement,were observed.17.Changes in the respiratory metabolism of mice were assessed using a respiratory energy metabolism monitoring system（Coulumbus Oxymax/CLAMS）to monitor parameters such as O2 consumption（VO2）,CO2release（VCO2）,respiratory exchange rate（RER）,and activity level of mice.18.Purified mitochondria were incubated with Mpress Xtra probes to detect mitochondrial state III respiration,mitochondrial state IV respiration,and mitochondrial respiratory control ratio（RCR）as an assessment of the degree of coupling and structural integrity in response to mitochondrial oxidative phosphorylation.19.Mice at room temperature using a portable smart digital thermometer-monitoring anal temperature-was used to assess basal body temperature metabolism in mice.20.Measurement of ATP levels in whole SNr tissue using an enhanced ATP assay kit to assess mitochondrial synthetic function.21.The mouse mitochondrial respiratory complex III/IV Elisa kit was used to detect mitochondrial respiratory complex III/IV.ResultsⅠ.Characteristics of oxidative stress and changes in mitochondrial morphology and function in hepatic encephalopathy1.Increased oxidative stress in clinical HE patients:33 patients were recruited from normal control（no cirrhosis or high ammonia,i.e.,group A）,cirrhotic normal ammonia（i.e.,group B）,and cirrhosis combined with high ammonia（i.e.,group C）groups,Superoxide dismutase（SOD）expression was significantly lower in group C,malondialdehyde（MDA）expression was significantly higher in group C,while total antioxidant capacity（T-AOC）and glutathione peroxidase（GPP）expression were significantly lower in group C,malondialdehyde（MDA）expression was significantly higher in group C,while total antioxidant capacity（T-AOC）and glutathione peroxidase（GPP）expression were significantly higher in group C.Malondialdehyde（MDA）expression was significantly higher in group C,while there was no statistically significant difference in the expression levels of total antioxidant capacity（T-AOC）and glutathione peroxidase（GPx）,This result indicates that cirrhosis combined with high ammonia induces a decrease in the activity of antioxidant stress enzyme SOD and an increase in the synthesis of lipid peroxides MDA in the peripheral blood of the organism,which suggests that the level of oxidative stress is increased and the activity of antioxidant stress enzymes is decreased in clinical HE patients.2.Combined peripheral and central oxidative stress in the body during hepatic encephalopathy:An acute liver failure-induced HE model was prepared by intraperitoneal injection of thioacetamide（TAA）,which was found to increase the ammonia levels in the peripheral blood and central brain tissues of mice,and to decrease the total distance and average speed of movement in mice in the Open Field test,as well as to decrease the time in the Rotating rod test.,suggesting motor dysfunction of the mice.Western blot results showed that the levels of antioxidant stress enzymes SOD and GPX1 were decreased in the cerebral cortex and SNr,the levels of autophagy-related proteins PINK1/LC3B were increased,and the expression of UCP2 was increased in the nucleus accumbens of the SNr without any significant changes in the cortex during HE.3.Fragmentation-like changes of mitochondria in cerebral SNr neurons in hepatic encephalopathy:The expression of mitochondrial fission-associated proteins p DRP1Ser616/mitochondrial fission factor（MFF）/Fission 1（FIS1）was increased in cerebral SNr in HE,and it was observed by transmission electron microscopy（TEM）.The number of mitochondria in SNr neurons increased,the area of individual mitochondria decreased,and the distribution of mitochondrial grain size was more concentrated in smaller volume regions.Immediately after the application of GAD2-Mito-GFP transgenic mice,combined with Mi NA analysis,the number of mitochondria in SNr-GAD2 neurons was also increased,suggesting that the mitochondria in SNr neurons appeared to be fragmented-like altered during HE.Ⅱ.Mechanism of action of targeted mitochondrial anti-oxidative stress therapy in hepatic encephalopathy.1.Mitochondria-targeted anti-oxidative stress therapy reduces blood ammonia by alleviating acute and chronic liver injury:Mitochondria-targeted anti-oxidative stress Mito-Q therapy significantly reduced hepatocyte necrosis,ALT,AST and blood ammonia,as well as oxidative stress in peripheral blood and in the center after it was administered in various models of liver injury.Mito-Q therapy alleviates oxidative stress in vivo by reducing TAA and bile duct ligation（BDL）model-induced hepatocellular injury and ammonia levels.2.Mitochondria-targeted antioxidant stress alleviates motor dysfunction in HE mice:After Mito-Q treatment,the Cat-Walk of HE mice were clearer,the step spacing and step frequency were more stable,and the time required for exercise at the same exercise distance was reduced;the results of the Rotating rod test.showed that the mice stayed in the baton for a longer period of time;the results of the Open field trajectory showed that the spontaneous activities of the mice were significantly increased,and there was a significant increase in the trajectory in the peripheral area as well as in the central area of the open field.The Open field trajectory results showed a significant increase in spontaneous activity of the mice.These results suggest that systemic administration of Mito-Q,a mitochondrial-targeted antioxidant stress drug,can ameliorate HE-like dyskinesia.3.Targeting mitochondrial anti-oxidative stress rescues mitochondrial dysfunction and fragmentation-like changes in SNr neurons:Immunofluorescence staining results suggest that Mito-Q treatment reduces the production of ROS within the SNr.Western blot results show that Mito-Q reduces the increase of UCP2 within the SNr,restores the activity of antioxidant stress enzymes（SOD1,GPX1）,and thus reduces oxidative stress within SNr neurons.In addition,Mito-Q not only reduced the occurrence of mitochondria-associated autophagy in the SNr,but also reduced the mitochondrial fragmentation-like changes induced by the increased expression of the mitochondrial fission protein p DRPSer616/FIS1,allowing the mitochondria to function normally by maintaining mitochondrial fission-fusion homeostasis.Ⅲ.Mechanisms of GAD2 neuronal activity within the substantia nigra pars reticulata on motor modulation in hepatic encephalopathy.1.HE stimulation activates SNr GAD2 neurons:Using C-FOS Cre ERT2 transgenic mice,we found that HE induced a significant increase in the number of neurons activated by C-FOS expression in the SNr,mainly in the medial region of the SNr.In addition,local high ammonia stimulation of SNr nuclei also activated increased C-FOS neuronal expression.The activated neurons were further identified by immunofluorescence staining and were mainly GAD+neurons located mainly in the medial part of the SNr.2.SNr-GAD2 neuron activity is involved in the behavioral regulation of HE:Specific inhibition of SNr-GAD2 neuron activity using chemical genetics significantly increased the total distance traveled,average speed in Open field test,and duration of Rotating rod test of HE mice;conversely,activation of this neuron decreased the total distance traveled,average speed,and duration of Rotating rod test of HE mice.3.Abnormal mitochondrial function within the SNr may be involved in regulating the functional activity of GAD2 neurons:After chemical genetic activation,the expression of UCP2 and antioxidant stress enzymes（SOD1,GPX1）within SNr was further examined using Western blot,and it was found that chemical inhibition of GAD2 activity increased the expression of UCP2,and chemical activation of GAD2 activity decreased the expression of UCP2;while no significant effect was observed on antioxidant stress enzymes（SOD1,GPX1）and autophagy-related proteins LC3B and PINK1.It is suggested that DREADD-targeted modulation of SNr-GAD2 neuronal activity may have subsequent behavioral effects via UCP2 and thus UCP2.Ⅳ.Mechanism of action of UCP2 on mitochondrial plasticity in hepatic encephalopathy.1.UCP2 in SNr-GAD2 neurons is involved in the regulation of HE locomotion:SNr-GAD2 neurons were targeted to overexpress UCP2（Ucp2OE）using Cre-Lox P technique,and it was found that overexpression of UCP2 ameliorated the dyskinesia of HE mice by the Open-field test,whereas knockdown of UCP2（Ucp2KD）exacerbated the dyskinesia of HE mice.2.UCP2 regulates mitochondrial fragmentation within the HE-SNr by mediating p DRP1（Ser616）/FIS1:A decrease in the number and density of mitochondria within SNr-GAD2 neurons after Ucp2OE,and conversely,an increase in the number and density of mitochondria after Ucp2KD was observed by transmission electron microscopy;and by using Western blot method to detect p-DRP1（Ser616）and FIS1 expression was significantly decreased after Ucp2OE,whereas p-DRP1（Ser616）and FIS1 expression was significantly increased after Ucp2KD.It is suggested that UCP2 is involved in the regulation of mitochondrial division in HE-SNr by mediating p DRP1（Ser616）/FIS1.3.UCP2 improves mitochondrial function by reducing mitochondrial ROS generation and inhibiting excessive autophagy:Western blot assay showed that the activities of antioxidant stress enzymes（SOD1,GPX1）in HE-SNr were further reduced after Ucp2KD,whereas the activities of antioxidant stress enzymes（SOD1,GPX1）were restored after Ucp2OE;the expression of autophagy-related proteins LC3B and PINK1further increased after Ucp2KD,whereas the expression of LC3B and PINK1 decreased after Ucp2OE.The expression of autophagy-related proteins LC3B and PINK1 was further increased,whereas the expression of LC3B and PINK1 was decreased after Ucp2OE.The above results suggest that TAA-induced increase of ROS and autophagy within SNr in HE mice can inhibit oxidative stress and autophagic responses within SNr GAD2 neurons by targeting overexpression of UCP2.4.Targeted mitochondrial antioxidative stress ameliorates metabolic disorders and mitochondrial respiratory function in HE mice:The results of respiratory metabolism monitoring using metabolic cages suggest that antioxidative stress treatment improves the respiratory metabolism of mice in terms of total respiratory exchange ratio and basal body temperature of mice.The results of respiratory function assay in purified SNr mitochondria showed that targeted mitochondrial antioxidative stress（Mito-Q/Ucp2OE）alleviated mitochondrial state III respiration and respiratory control rate,and improved the activity of mitochondrial respiratory chain complexes III and IV,as well as the function of ATP synthesis.It is suggested that targeting mitochondrial antioxidant stress can maintain functional stability by promoting mitochondrial morphology and structural integrity,thereby maintaining functional stability.Conclusions1.Oxidative stress accompanies clinical HE patients and HE model mice and induces mitochondrial fragmentation and dysfunction within the central SNr.Systemic targeting of mitochondrial antioxidative stress（Mito-Q）alleviates HE by reducing peripheral and central ROS production,attenuating liver damage,and lowering ammonia levels,which in turn ameliorates mitochondrial fragmentation-like changes and dysfunction within the SNr.2.The activation response of GAD2 neurons within the SNr serves as the primary cell for HE and high ammonia stimulus messaging,and chemogenetically targeted inhibition of SNr GAD2 neuronal activity ameliorates HE by increasing UCP2 expression.3.Targeted regulation of mitochondrial UCP2 in SNr-GAD2 neurons can reduce the occurrence of abnormal mitochondrial division and excessive autophagy by reducing the production of ROS in neurons and inhibiting the activation of p DRP1（Ser616）/FIS1.4.Targeting mitochondrial antioxidative stress can improve mitochondrial respiratory metabolism,respiratory chain complexes and ATP synthesis,maintain the structural and functional integrity of mitochondria,and then promote the normal function of the cell. |
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