Mechanism Of A Novel Therapeutic Approach Targetting Telomere To The Oxidative Damage Of Nervous System

Objective:Cardiovascular and cerebrovascular accident,sepsis and acute lung injury of acute and critical disease,produce a large amount of active oxygen plasma(ROS),which reduce the first organ resuscitation success rate.The accumulation of ROS and oxidative stress,hypoxia cause irreversible oxidative damage to the nonrenewable nerve cells.Therefore,the recovery of the brain is the bottleneck in the field of emergency treatment.The latest study found that DNA damage is an important pathophysiological consequence of oxidation and anti-oxidation and affect the normal cell metabolism and tissue function.Telomeres in end of linear chromosome of the eukaryotic cells is a DNA protein complex.Telomere and telomere binding protein together constitute the special chromosome ends "hat" structure that maintain genome integrity and inhibition of DNA damage.Therefore,to explore the inhibition of DNA oxidative stress injury targeted telomere may be a new way to improve the recovery of the brain.In this paper,we first prove Bo to vancomycin,ICRF-193 that mediated oxidative stress can directly or indirectly disrupt telomere specific protein TRF2,activation of ?H2AX and DNA damage signal.Protection of telomeres is proposed to reduce the toxic effects of DNA damage and oxidative damage.And in oxidative nerve injury in mice model,TRF2 can maintain telomere structure and strengthen the anti-oxidation of the telomere in neural tissue,which indicates an important role in the protection of nervous system.To clarify the mechanism between inhibition of DNA damage and eliminate amyloid beta protein,phosphorylation of tau,which mediated oxidative damage to nerve related factor.Methods:DNA damage are induced by gene toxic drugs ICRF-193 in HT1080 cells.The expression of TRF2 was knocked down by cell transfection technique,and the changes of DNA damage in cells were detected by immunofluorescence.The protein,DNA and RNA of brain tissue were extracted from wild type mouse and APPSwe/PS1dE9C57BL/6 mouse model,and the contents of TRF2 protein in mouse brain and other tissues were detected by Western Blot and other techniques.The expression of TRF2 in brain tissue and other tissues was detected by RT-PCR.Analysis of the changes of telomere length in brain tissue of mice with PNA FISH technique.Extract intact brain tissue by left heart perfusion.The expression of TRF2 in mouse brain tissue was detected by immunofluorescence staining.The primary neurons were extracted from the wild type and APPSwe/PS1dE9C57BL/6 model mouse brain tissues.The expression of TRF2 and ?H2AX in the neurons are detected after seven days.The morphological changes of the neurons are evaluated.over expression of Tau,Analysis of P-tau and ? H2 AX using immunofluorescence,Blot Western and other techniques in SHSHY5 Y that overexpressed Tau.TRF2 ‘s expression in SHSY5 Y cells was inhibited by siRNA,and the expression of TAU protein was detected.A beta oligomers are constructed in vitro and put in cells culture medium,and the expression of ?H2AX protein and TRF2 was detected in cells with different conditions.Results:1)Inhibition of TRF2 alleviates the specific telomere damage caused by ICRF-193.2)The expression of TRF2 is higher in TG and WT mice brain than other tissues.3)TRF2 is much higher in transgenic mice than wild type mice,and the phenomenon is much sever in older mice brain.4)Exogenous oligomeric A? can insult damage in SHSY5 Y,especially after interfering the translation of TRF2 by siRNA.5)SHSY5Y overexpressing TAU has a higher expression of P-TAU,TAU and ?H2AX.6)Knocking down the TRF2 level in SHSY5 Y over-expressing TAU cause a much higher DNA damage and the expression of phosphorylated TAU is also elevated.7)The neurons extracting from mice that are born into 24 hours have a higher expression of TRF2 and ?H2AX.Conclusions:TRF2 can alleviate telomere specific damage incicating a crucial role in DNA damage.The higher expression of TRF2 in mice brain indicates a crucial role of TRF2 in nervous system.Transgenic mouse has a relative higher expression of TRF2 compared with wild type mouse,and the phenomenon is more evident in 12 months old mice.We guess that TRF2 may affect the neurons in AD.By building up a cell line overexpressing MAPT and synthetic oligomeric A ?,we find that the cell has a higher DNA damage and p-TAU level.When we knocking down the TRF2 expression,the DNA damage is much sever.All of those may imply that the TRF2 may regulate the nerve cells in AD.To find out the mechanism we have much to be done in the future.