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The Mitochondrial Apoptosis Pathway Pattern and the Protective Effect of Curcumin in Experimental Au

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Tutor: QinXinYue
School: Chongqing Medical University
Course: Neurology
Keywords: multiple sclerosis,experimental autoimmuneencephalomyelitis,apoptosis,mitochondr
CLC: R744.3
Type: PhD thesis
Year:  2013
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BackgroundMultiple sclerosis (MS) is a central nervous system (CNS) autoimmunedisease, with the pathological characteristic of demyelination in whitematter, inflammatory infiltration and axonal injuries. The pathological basisof new formed lesions in MS is not very clear, which generally consideredas demyelinating events caused by T-cell-mediated immune response.Recently studies on autopsy specimen found that the initial pathologicalfindings when new lesion formed were different from the traditional MSpathological findings. Extensive oligodendrocytes apoptosis was the earliestpathological changes in the new formed lesions. The area ofoligodendrocytes apoptosis were gathered with activated microglia whilethe morphological of myelin was normal. The infiltrating of T, Blymphocytes and myelin phagocyte were found after a few days. But thereason for oligodendrocytes apoptosis is still not very clear. Experimentalautoimmune encephalomyelitis (EAE) model is a classic animal model for MS. But the relationship between the apoptosis and the typical pathologicalchanges in the EAE model is still unknown. This study was performedaimed to clarify the early pathological events of MS/EAE in order tofurther understand the pathological mechanism and to help looking foreffective intervention from the early stage of MS.Recently studies showed that mitochondria injury as an importantnon-immune mechanism in the pathology of MS. Mitochondrialdysfunction and a higher demand of energe have been found in MS.Whether mitochondria injury is involes in the early pathology of MS/EAEis still not very clear.Curcumin is a kind of plant polyphenol with the role ofanti-inflamation and anti-oxidant. It was also been showed neuro-protectionroles in EAE. But it is uncertain that whether curcumin can protectmitochondria from injury and suppress the apoptosis in the early pathologyof MS/EAE.ObjectiveIn this study, we explored whether and when apoptosis andmitochondria damage happened in C57BL/6EAE mice at different timepoint post-immunization. We aim to explicit the initial event in EAE/MSwhich occurred before the traditional pathological destruction such asdemyelination and to explore the underlying mechanism on it. Methods1. C57BL/6mice EAE model was successful conducted. The micewere divided into the3d,7d,16d (acute phase), and30d (chronic phase)groups according the post immunized days. Also the normal group wasadded. By using TUNEL in suit apoptosis detection, transmission electronmicroscopy (TEM), the ATP level detection, and double-labelimmunofluorescence, we explore the cell apoptosis and the damage ofmitochondria in different time point post-immunization. The relationshipbetween apoptosis/mitochondrial and demyelination and axonal injury alsobeen detected.2. C57BL/6mice were divided into the normal group, the EAE group,the control group and the curcumin groups. The last three groups weredivided into the16d and30d groups again according the post immunizeddays. The mice in the curcumin group were intraperitoneal injection withcurcumin (200mg/kg/d) every day from the day of immunization.Neurological function scores were performed to evaluate the neurologicalfunction of EAE mice. H-E stain was used to observe theinflammatory infiltration in the spinal cord. Luxol fast blue (LFB) was usedto observe the extent of demyelination. Bielschowsky sliver stain was usedto evaluate the axonal injury. Immunohistochemistry was performed to testthe activation of astrocytes and caspase-3. TUNEL in suit apoptosisdetection was used to evaluate the extent of apoptosis in each group. 3. C57BL/6mice were divided into the normal group, the EAE group,the control group and the curcumin group. The last three groups were thendivided into the3d,7d,16d and30d groups according the post immunizeddays. The mice in curcumin group were intraperitoneal injection withcurcumin (200mg/kg/d) every day from the day of immunization. Westernbolt was performed to observe the expression of caspase-3,caspase-9,Cytochrome c (cyt-c),MBP and NRF2in each group. The ATP level wastested by using ATP level detection kits. The study of the mechanism thatcurcumin inhibits apoptosis through mitochondria protection wasperformed.Results1. The apoptosis was observed in the spinal cord only3days afterEAE model was conducted. The highest expression of apoptosis cells wasobserved in the16d group (acute phase)(P<0.05). The initial apoptosiscells in the3d group were proved to be oligodendrocytes and neuron cells.The ATP level was start to descending3days post immunization comparedto the normal group (P<0.01). The lowest level of ATP was found in the16d group (P<0.05). Mitochondrial morphology abnormal such asmitochondrial swelling, mitochondrial crista squeezed to one side can beobserved only3days post immunization by TEM. But the morphology ofmyelin and axonal was still normal in the same time point. At16days postimmunization, the mitochondria was found to be swelling, vacuolation or even dissolved accompanying myelin sheath destruction and axoplasmicatrophy.2. The neurological function in the curcumin group was significantlyimproved compared to the EAE group (P<0.05) and the onset time of thedisease was significantly delayed (P<0.05). The inflammatory infiltrationin the curcumin group was significantly alleviated compared to the EAEgroup at the same time point (P<0.05) by H-E stain. The area ofdemyelination was significantly reduced in the curcumin group (P<0.05)detected by LFB stain. The extent of axonal injury in both acute andchronic phase in the curcumin group was reduced at the same time pointcompared to the EAE group by bielschowsky sliver stain. A less activatedastrocytes can be seen in the curcuim group at16days post immunization(P<0.01) but not at30days post immunization (P>0.05) at the same timepoint compared to the EAE group. The number of apoptosis cells weresignificantly reduced in the curcumin group both at16days and30dayspost immunization tested by TUNEL and the expression ofcaspase-3(P<0.05).3. Western blot showed the decreasing expression of MBP protein wasobserved at16days post immunization. Curcumin significantly suppressthe decreasing of MBP protein (P<0.05) so as to protect myelin from injury.Compared to the EAE group, curcumin significantly suppress theexpression of caspase-3, a apoptosis marker protein, and the decreasing of ATP level both at7days,16days and30days post immunization(P<0.05).4. The expression of cyt-c and caspase-9, which are the key proteins ofmitochondria induced apoptosis, was significant increased at7days postimmunization (P<0.01). And the expression keep in a higher levelcompared with the normal group in the whole course of EAE. Curcumincan significantly suppress the expression of cyt-c and caspase-9comparedwith the EAE group (P<0.05). The increased expression of NRF2in EAEgroup was found at7days,16days and30days post immunization and amore higher expression of NRF2was seen in the curcumin group comparedto the EAE group (P<0.01).Conclusions1. The initial apoptosis cells in the CNS occur at3days after EAEmice was immunized. The apoptosis cells are oligodendrocytes and neuroncells. Apoptosis happened before the demyelination and axonal injury,which were regard as the classic pathological characteristic of MS/EAE.And the extent of apoptosis is in accordance with the severity of EAE.2. Curcumin significantly inhibits the infiltration of inflammatory cells,the injuries of axonal and mitochondria, which comprise its protectioneffects on EAE.3. Curcumin suppress the apoptosis induced by mitochondria injury byinhibits the release of cyt-c to the cytoplasm. This probably relate to theincreasing expression of NRF2. The results will provide us useful clues in finding effective methods aimed at early intervention and relapseprevention in the MS treatment.
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