Study On Experimental Therapeutics Of Cyclin-dependent Kinase's Inhibitor, Indirubin-3'-monxime, In Alzheimer's Disease

SummaryAlzheimer's disease (AD), characterized by loss of memory,cognitive decline and misbehavior, is the most common form of seniledementia. The prevalence of AD is strongly associated with age. With thegrowth of the human life span, the incidence rates of AD areprogressively increased. AD has become a heaven burden for our societyand many families. Up to now, the exact pathogenesis of AD is not clear.It is urgent to elucidate the mechanism of AD and search for newtreatments for this debilitating disease.Major pathological hallmarks of AD are senile plaque (SP),neurofibrillary tangles (TNFs), neurons and synapses loss. The massiveneuronal loss in the hippocampus and cerebral cortex is associated withlearning and memory deficits. However, the pathogenesis of neuronalloss is not fully understood. One potential target that control delayedneuronal death in AD is cell cycle-dependented kinases (Cdks). Increasing evidence suggests that some postmitotic neurons attempt toreenter the cell cycle after brain various injuries, but end by neuronaldeath. The expression of cell cycle-associated proteins also often predictsthe sites of neuronal death in Alzheimer's disease. These observationssuggest that aberrant cell cycle reentry may be an important mechanismof apoptosis in neurological disorders including AD.Recently, interest in Cdks inhibitor therapy has extended beyond therealm of oncology to include neurological disease. Cdks inhibitors arebeing used as tools to determine the relationship between deregulatedCdks activities in post-mitotic neurons and neurodegeneration, and maysomeday be used as therapeutic agents for the neurology service.Although Cdks inhibitors represent potential therapeutic targets forneurological disease, there are no desired candidates with selectiveeffects on AD. Indirubin-3'-monxime (IMX) is a potent inhibitor of Cdkswith low molecular weight. Moreover, IMX exhibits good activity andminor toxicity. It is more soluble than other indirubin analogues. In thepresent study, the effects of IMX on AD were explored so as to evaluateits therapeutic value and elucidate the mechanism of AD. The studyincluding following three parts: PartⅠElevated expression of Cdk4 and cyclin B1 inAlzheimer's disease model in ratsAim: To explore the re-expression of cell cycle related proteins anddelayed neuronal death in Alzheimer's disease model and investigate therelationship between aberrant expression of cell cycle related proteinsand apoptotic cell death. Methods: Rat model of AD was established byAβ_1-40 administering intracerebroventricularly. The apoptotic cells wereassessed by TUNEL method. The expression of cell cycle relatedproteins, i.e. Cdk4 and cyclin B1, were detected byimmunohistochemical staining. The co-localization of TUNEL andmitotic markers were also preformed. Results: Aberrant expression ofCdk4 and cyclin B1 was showed at 7 d after modeling and last for a longperiod. On the other hand, TUNEL positive cells appeared as early as 14d after the surgery and peaked at 21d. Cdk4 and cyclin B1 co-localizedwith TUNEL positive cell. Conclusions: These findings indicated thatAβ_1-40 triggered neurons to reenter the cell cycle. Moreover, cell cycleproteins preceded neuronal death in this model.PartⅡStudy on pharmacokinetics of indirubin-3'-monxime in ratsAim: To investigate the pharmacokinetics of indirubin-3'-monxime(IMX) in rat by the different administrations and distributions in tissuesof IMX. Methods: A high-performance liquid chromatography (HPLC)was used to determine the plasma IMX concentration so as to study thepharmacokinetics of IMX in rat after intravenous and intraperitonealadministrations, respectively. The main pharmacokinetics parameterswere calculated. After intravenous injection of IMX, the distribution ofIMX in heart, lung, kidney, brain was assessed. Results: The resultsindicated that IMX pharmacokinetics was conformed to the twocompartment open model. By intravenous administration IMX in rat,both absorption and distribution were fast, the velocity to effect was rapid,the elimination was main process, and the retention time was long. Afterintraperitoneal administration of IMX, the peak time was at 150min.After intravenous administration (10mg/kg), killing the rats at differenttimes, 2g of each tissue in hearts, lungs, kidneys and brains wereobtained to detect the concentration of IMX by HPLC. It was found thatthere was higher concentration of IMX in heart tissues, and the peak ofits concentration was at 1h after injection; that the peak of itsconcentration in brain and lung tissues was at 0.5h after injection; that thepeak of its concentration in kidney tissues was at 3h after injection. Conclusion: It is simple and credible that applying HPLC method to testplasma concentration of IMX in rats. The pharmacokinetics of IMX byintravenous administration was conformed to two compartment openmodel and the elimination of IMX is the main process after the injectionof IMX. IMX is distributed mainly in systemic circulation and organswith abundant blood supplying. Furthermore, IMX can penetrate easilythe blood brain barrier, mainly by the passive diffusive way.PartⅢThe effects of Indirubin-3'-monxime on theAlzheimer's disease model induced by Aβ_1-40 in ratsAim: To investigate the effects of indirubin-3'-monxime on theAβ_1-40-induced Alzheimer's disease model in rats. Methods: Rat modelof AD was established by Aβ_1-40 administering intracerebroventricularly.The apoptotic cells were assessed by TUNEL method. The expression ofcell cycle related proteins, i.e. Cdk4 and cyclin B1, were detected byWestern blotting. A cyclin-dependent kinases' inhibitor, IMX, wasintraperitoneally administered at 1d before modeling. Spatial learningbehavior was assessed by the Morris water maze at 7, 14 and 21d aftermodeling. Results: (1) IMX significantly improved behavioral deficit in the Morris water maze test at 7, 14 and 21d after modeling; (2) IMXameliorated the neuronal apoptosis both in cortex and hippocampus; (3)IMX also reduced the expression of cell cycle related proteins; (4) IMXinhibited the activity of Cdc2 protein kinase and CHAT. Conclusion:IMX had a protective effect against neuronal apoptosis and thedysfunction of learning and memory induced by Aβ_1-40 through inhibitingcyclin-dependent kinases. IMX may be a potential therapeutic agent inthe neurology service.