Study On The Function Of Mitochondria During The Regeneration Of Dugesia Japonicum

Numerous studies have shown that ROS,mitochondrial dynamics and apoptosis play an important role in regeneration.Mitochondria,which forms branchlike tubular structure network in eukaryotic cells,undergo division and fusion constantly to meet different physiological and pathological conditions.Mitochondrial morphological maintenance and changes are regulated by mitochondrial fission proteins(Drp1,Mff,Fis)and fusion related proteins(Mfn1,Mfn2,Opa1).Several studies have shown that mitochondrial fission and fusion play an irreplaceable role in the proliferation and differentiation of stem cells as well as nerve regeneration,whether the process of planaria regeneration can be influenced is not clear.Therefore,the Dugesia japonica were used to study the influence of mitochondrial fission and fusion on planaria regeneration for their strong regeneration ability.This study was to explore the Dugesia.japonica regeneration under normal,Mdivi-1 and DPI treatment conditions at different time points respectively.Using techniques such as fluorescence staining,immunofluorescence,RT-PCR and Western blot to detect ROS content,the number of active mitochondria,the expression of drpl,mfnl,bax,caspase3 respectively to discover the normal mitochondrial dynamic changes and related mechanism during regeneration.The main results of this study are as follows:1.Drp1 mRNA expression increased significantly but mfnl mRNA expression reduced in the normal regeneration of 1-2 days and 5 days,which were consistent with increased number of mitochondria.It's suggesting that mitochondrial dynamics tending to fissure to meet energy demand in planaria regeneration.2.When drpl was inhibited,head tissue regeneration and brain development were failed,indicating that the mitochondrial division which is closely related to the stage of blastema development(1-2 days)and the regeneration and differentiation of nervous system affect the development of key structures in the regeneration directly by affecting stem cell proliferation and differentation and making failure development of head structure and brain systems.3.Expressions of Bax and caspase-3 were significantly increased during normal blastema development and brain growth and differentiation.Both of them were reduced by Mdivi-1 treatment in the critical period of regeneration,indicating that inhibited division of mitochondria causes apoptosis inhibition.The number of litochondria reduction was not the result of endogenous apoptosis,but mitochondrial fusion process.4.In the normal regeneration,ROS is required for the planarian regeneration,which is related to the number of mitochondria,and affected the expression of mitochondrial divison and fusion gene and the expression of bax and caspase3 during regeneration,resulting in the abnormal regeneration of brain nerves and tissue remodeling.And its affect on planarian regeneration was not stronger than the Mdivi-1 treated planarian regeneration.5.This study found that the expression trend of drpl mRNA was consistent with Bax in planaria head regeneration.Meanwhile,mitochondrial division inhibition treated by Mdivi-1 made the expression of Bax mRNA and protein decreased.Thus affected the expression of downstream caspase3.This indicates there is a certain functional relationship between mitochondrial fission protein drpl and Bax.Conclusion:Mitochondrial division is closely related to the formation of blastema and the regeneration and development of brain nerves in the head regeneration of planaria.The mitochondrial dynamics tending to fusion treated by Mdivi-1 result in the embryo formation obstacles by affecting stem cell proliferation and differentiation.In normal regenerative tissue,regeneration can be regulated by Bax induced apoptosis and the number changes of mitochondria caused by the mitochondria dynamic changes.The changes of ROS content and the number of mitochondria forled by mitochondrial division are one of main factors in influencing planarian regeneration at the critical time point of planarian regeneration.The latter one had stronger influence on planarian regeneration.Besides,the trend of changes between Bax and Drp1 were basically synchronous in regeneration,and there may be a certain relationship between them.