The Study On The Effect And Mechanism Of Aspirin Promotes Oligodendrogenesis And Myelination

Chronic cerebral hypoperfusion induced cerebral white matter injury (WML) is one of theimportant factors leading to cognitive impairment in elderly patients, which seriouslyaffecting the quality of life of patients, whereas there is no clear and effective treatment. Ithad shown that, oligodendrocyte (OLs) degeneration resulting in abnormal structure andfunction of myelin sheath, neuronal axons fiber degeneration necrosis in the cerebral whitematter is an important cause of cognitie decline in older rats, and promoting remyelinationplays a curical role in ameliorating cognitive function induced by WML. The mature OLsin situ and oligodendrocyte precursor cells (OPC) in subentricular zone represent twoimportant endogenous source of remyelination cell, mature OLs can be directly involvedin the synthesis of myelin, OPCs in the site of damage and migrate from the SVZ are alsoinvolved in myelin sheath after differentiation. In the previous study, we have found thatthe traditional anti-platelet aggregation drug ASA can promote neonatal OPC proliferationin SVZ, and migrate to the white matter lesion then differentiated into new OLs. For thefurther study of the effect of ASA on OPC proliferation and OLs myelin forming in situ of white matter damage, as well as the role of ASA on the oligodendroglial cell lines in vitroenvironment, and the behavioral verification of ASA effect on cognitive function in WMLrat model, then finally to explore its possible mechanism of the role on theoligodendroglial cell lines. In a word, provide experimental basis for ASA using forclinical treatment of WML, and ideas of new drugs development for WML treatment.Experiment1The effect of aspirin on oligodendrogenesis incorpus callosum of WML model ratsObjective: To study the effect of ASA on OPC proliferation, maturation and OLs myelinregeneration in situ of the corpus callosum in WML rat model; Methods: WML rat modelwere induced by CCAO method, then treated with different doses (25,50,100,200mg/kg)of ASA or the same proportion of Laigan solution.4weeks later, the expression ofNG2/BrdU, PLP/BrdU positive cells in CC was observed by immunofluorescence doublestaining, and the protein levels of PDGF α R, CNPase, MBP were analyzed by Westernblot detection; Addtionally, the structure of myelin sheath in CC were detected byUltrastructure electron microscope and G-ratio were calculated; Results:1) In thetreatment of100and25mg/kg aspirin, the number of PLP+/BrdU+double-stainednew-differentiated oligodendrocytes and that of NG2+/BrdU+double-labeled proliferatingOPCs reached the peak, respectively, the variance has statistical difference (p<0.05);2)Immunoblotting results showed that the expression of PDGFaR was upregulated aftercerebral ischemia and further enhanced by25mg/kg aspirin treatment, which, however,was decreased by the treatment with100and200mg/kg aspirin. By contrast, ischemiacaused downregulation of MBP expression whereas25–200mg/kg aspirin amelioratedischemia-induced decreased MBP expression. The MBP expression levels in100mg/kgaspirin-treated rats were higher than that in normal group, The discrepancy has statisticaldifference (p<0.05). Unexpectedly, aspirin did not affect CNPase expression, thedifference has no statistical significance (p>0.05);3) Compared with the normal, demyelination and reduction of myelin thickness were evident in the control rats subjectedto ischemia. Four-or six-week treatment of aspirin attenuated ischemia-induceddemyelination and reduction of myelin thickness and an attenuated G-ratio inaspirin-treated rats, also; Conclusion: Depending on its dosage, aspirin may protectagainst ischemia-induced WML by affecting OPC fate and oligodendrocyte myelination.Experiment2The influence of aspirin on the development of NSCand oligodendrocytes cultured in vitroObjective: To study the influence of ASA on the oligodendrocyte lineage in vitroconditions; Methods: Cultured NSCs and OPCs were treated with different concentrations(0.1,0.5,1,5,10μmol/L) of ASA or the same volume of ethanol, then the expression ofO4+, CNPase+and MBP+positive cells were detected by immunofluorescence staining forobserve the efficiency of NSC proliferation and differentiation to oligodendroglia, andA2B5+/Ki67+, CNPase+positive cells were calculated to observe the effect of ASA onOPC proliferation and differentiation; Results:1) In cultured NSCs, aspirin with differentconcentrations (0.1–10μmol/L) increased the number of O4+cells compared with ethanolcontrol; Differently, only relatively high concentrations of aspirin (5.0–10μmol/L)increased the number of CNPase+or MBP+cells, as compared to the controls, thedifferences has statistical significance（p<0.05）；Tuj1+neurons were increased in differentconcentrations (0.1-10μmol/L) of ASA treatment groups compared with the control,accompanied by Tuj1-/MBP-astrocytes reduced, while the expression of MBP+OLswas obviously increased only in5-10μmol/L ASA treatment group.2) In cultured OPCs,more A2B5+/Ki67+cells were present with the treatment of0.1μmol/L aspirin whereasfewer were observed with the treatment of10μmol/L compared with the controls, thedifference has statistical significance（p<0.05）; more CNPase+cells were observed in thepresence of1.0–10μmol/L aspirin than the controls, while0.1–0.5μmol/L did not have thiseffect; Conclusion: ASA could promote the differentiation of NSC to OPC, and promote OPC proliferation, differentiation and myelination.Experiment3The effect of aspirin on the cognitive function ofWML model ratObjective: To verify whether ASA has the effect of ameliorate the cognitive impairmentin WML rats; Methods: Open field test and Elevated plus maze test were used to excludethe influence of anxiety on the detection results cognitive function, and then Morris watermaze test was selected to testify the ability of learning and memory changes of the rats;Results: After four days of navigation training, ASA treatment group showed shortenescape latency compared with the control group, especially the fourth day of training,the difference is statistically significant (p<0.05); while the swimming speed of rats hasnot significant difference (p>0.05); In addition, it has no significant difference in theanxiety state between the ASA treatment group and the control group (p>0.05);Conclusion: ASA could ameliorate the learning and memory ability of the WML ratsindependently, which has nothing to do with the anxiety and depression status of rats.Experiment4The study on the mechanism of aspirin promotesoligodendrogenesis and myelinationObjective: It have shown that, ERK and Rho signaling pathway play an important role inoligodendroglia development and myelination, this study mean to investigate whetherASA promoting oligodendroglia development and myelin formation through the twopathways; Methods: Cultured OPCs were processed with10μmol/L ASA alone orsupplement with ERK inhibitor (PD98059) also, the expression level of phosphorylationERK (p-ERK), MBP and the activity of RhoA were tested by Western blot; Results: Theexpression of MBP and p-ERK increased significantly in10μmol/L ASA treatment group compared with the control group, the differences have statistical significance (p<0.05);After a ERK inhibitor (PD98059) was added, the expression of which were decreased tocontrol levels, while the expression of total ERK had no obvious changes; In contrast tothe ERK pathway, RhoA activity decreased about80%in10μmol/L ASA treatment groupcompared with the control group, it has significant difference (p<0.05); Conclusion: ASAprobably promotes oligodendroglia development and myelination through increasing thelevel of ERK phosphorylation, and reduce the activity of RhoA pathway.