Study On The Neuroprotective Effect And Its Possible Mechanisms Of Cyclin-dependent Kinase’s Inhibitor, Indirubin-3’-monxime, Against Aβ-induced Neuronal Toxicity

Alzheimer’s disease is a progressive neurodegenerative disease, which is the most common form of dementia. There are as many as 24 million patients in the world and about 4 million people living with Alzheimer’s disease in China. With the progress of the disease, it can cause problems with memory, thinking and behavior severe enough to affect work, daily or social life. AD has become a heaven burden for our society and 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 new treatments for this debilitating disease.Some new recognitions of pathologic feature of AD patients have provided new important clues about the mechanisms of neuronal apoptosis which make us to see new desire of AD therapy. Accumulating data have shown that neuronal death is accompanied by the induction of cell cycle proteins in a variety of experimental neuronal models. It is indicated that post-mitotic neurons attempt to reenter the cell cycle in respond to various neuronal stressors. But due to their post-mitotic state, these neurons enter into an adverse condition which would create a conflict of signals and result in cell death by apoptosis.These results suggest that aberrant cell cycle reentry may be an important mechanism of neuron loss in neurological disorders. Not surprisingly, treatment with pharmacological inhibitors of Cdks may therefore potentiate the role for treatment of AD. However, the development of highly selective clinical therapies targeting Cdks activity is still in its infancy. Some relative reports used in vitro and acute cerebral ischemia models. At present, it is destitute to develop effective, hypotoxic, selective Cdks inhibitors which can pass the blood brain barrier. Indirubin was identified as the active component of Danggui Luhui Wan, a mixture pill of 11 herbal medicines traditionally utilized against certain types of leukemias by the Chinese Academy of Medicine. It is a selective Cdks inhibitor, which can efficiently inhibit the activities of Cdk1, Cdk2 and Cdk5. But the water-solubility and liposolubility are poor. Indirubin-3’-monoxime (IMX), a synthetic derivative of indirubin, is a potent inhibitor of Cdks with low molecular weight, with better solubility characteristics than indirubins. It has hypotoxicity and membrane permeability and acts by competition with ATP at the catalytic site of Cdks. The aim of the present study is to evaluate the neuroprotective effect of IMX against Aβ-induced apoptosis in cultured neuroblastoma SH-SY5Y cells. In addition, we have explored the effect on tau protein hyperphosphorylation and its possible mechanisms.The study included the following two parts:PartⅠThe neuroprotective effect of IMX against Aβ25-35-induced apoptosis in cultured neuroblastoma SH-SY5Y cellsAim: To investigate the neuroprotective effect of IMX against Aβ25-35-induced apoptosis in cultured neuroblastoma SH-SY5Y cells. Methods: Alzheimer’s disease cellular model was established by cultured neuroblastoma SH-SY5Y cells with Aβ25-35-treatment. Cell Counting Kit 8(CCK-8) assay was used to measure the cell viability of Aβ25-35 and(or) IMX at different time and concentrations.The morphology of SH-SY5Y cell apoptosis of different groups was observed by phase-contrast microscopy and Hoechst 33258 staining. In addition, neuronal apoptosis was measured by a flow cytometric assay. Results: (1) Treatment with a dose of more than 3.0μM IMX resulted in a significant reduction in cell viability, which is compared with the control group (P<0.01 at 3.0 and 6.0μM; P<0.001 at 12.0μM).(2)Treatment with a dose of 10-50μM of aged Aβ25–35 for 48 hr and 72 hr or 30-50μM Aβ25–35 for 24 hr resulted in a significant reduction in cell viability, which is compared with that in the control group (P<0.01 at 10-30μM; P<0.001 at 40-50μM).(3) Pretreatment with IMX (0.25–1.0μM) concentration-dependently reversed Aβ25–35-induced SH-SY5Y cell death (P<0.05 at 0.25μM; P<0.01 at 0.5 and 1.0μM).(4) Aβ25–35-treated cells exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis. After 48 hr exposure of 40μM of aged Aβ25–35, the number of apoptotic cells was markedly increased to 42.4±3.86% (P< 0.001), which, however, was prevented by the addition of 0.5 and 1.0μM IMX. The percentage of apoptotic cells was 27.4±3.09% and 19.2±2.02%, respectively (n=5, P<0.001). (5) Apoptotic cells of Aβ25–35-treated-alone group were markedly increased to 20.33±2.02% (P<0.01) with 20μM Aβ25-35 treatment. This increase, however, was prevented by the addition of 1.0μM IMX. The percentage of apoptotic cells was 12.4±1.82% (P<0.01). Basal apoptotic cell death was 8.1±1.42% and rose to 50.2±4.77% in cells treated for 48 hr with 40μM of aged Aβ25–35 (n=5, P <0.001). However, pretreatment with 0.5 and 1.0μM of IMX concentration-dependently suppressed the apoptosis induced by 40μM of aged Aβ25–35 for 48 hr, and the percentage of apoptotic cells was 33.9±3.09% and 24.5±3.47%, respectively (n=5, P<0.001). Conclusion: IMX markedly reversed Aβ25–35-induced neurotoxicity, indicating the neuroprotective activity of IMX. PartⅡThe effect and its possible mechanisms of IMX on Aβ25-35-induced tau phosphorylation in SH-SY5Y cellsAim: To investigate the effect and its possible mechanisms of IMX on Aβ25-35-induced tau phosphorylation in SH-SY5Y cells. Methods: (1) SH-SY5Y was incubated with 0, 0.5 and 1.0μM IMX. Following 24 hr treatment, 40μM of aged Aβ25–35 was added. 48 hr later, caspase-3 activity was measured with the caspase-3 assay kit. (2) 0.5μM and 1.0μM IMX were added in the serum-deprived media of SH-SY5Y cells for 1 hr prior to the 6 hr Aβ25-35 (20μM) exposure. Western blotting analysis was performed to detect tau phosphorylation at the site of pS396, pS199 and pT205 and p-GSK3β(Ser9)expression. (3) The cultures were treated as described above, 24 hr and 2 hr later, western blotting analysis was performed to detect p-JNK and p-ERK expression. Results: (1) Caspase-3 activity was significantly increased in the Aβ25-35 treated group when compared to the control group (P<0.01). These changes were partially prevented by pretreatment with IMX (P<0.05). (2)In the Aβ25-35-treated group, protein levels of phosphorylated tau at the site of pS396, pS199 and pT205 detected by western blotting were significantly increased when compared to the control group (P<0.05). However, these changes were partially or completely prevented by pretreatment with IMX (P<0.05). (3) After Aβ25–35 treatment, the expression of p-GSK3β(Ser9) was decreased, which meaned that GSK3βphosphorylation was increased. However, the expression of p-GSK3β(Ser9) was significantly increased when pretreatment with IMX (P<0.05). (4) The expression of p-JNK and p-ERK were significantly increased in the Aβ25-35-treated group compared to the control group (P<0.05). But there were no significant changes after IMX treatment. Conclusion: (1) IMX can inhibit caspase-3 activity efficiently to ameliorate Aβ25–35-induced neurotoxicity. (2) IMX attenuates Aβ25–35-induced neurotoxicity possibly by inhibiting tau phosphorylation at the site of pS396, pS199 and pT205. (3) IMX decreases tau phosphorylation by inhibiting GSK3βphosphorylation. (4) The expression of p-JNK and p-ERK were not involved the effect of IMX.