Study On The Neuroprotective Effect And Its Possible Mechanisms Of Cyclin-dependent Kinase's Inhibitor, Indirubin-3'-monxime, Against A?-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 A?-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:Part?The neuroprotective effect of IMX against A?25-35-induced apoptosis in cultured neuroblastoma SH-SY5Y cellsAim: To investigate the neuroprotective effect of IMX against A?25-35-induced apoptosis in cultured neuroblastoma SH-SY5Y cells. Methods: Alzheimer's disease cellular model was established by cultured neuroblastoma SH-SY5Y cells with A?25-35-treatment. Cell Counting Kit 8(CCK-8) assay was used to measure the cell viability of A?25-35 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 3.0?M IMX resulted in a significant reduction in cell viability, which is compared with the control group (P<0.01 at 3.0 and 6.0?M; P<0.001 at 12.0?M).(2)Treatment with a dose of 10-50?M of aged A?25–35 for 48 hr and 72 hr or 30-50?M A?25–35 for 24 hr resulted in a significant reduction in cell viability, which is compared with that in the control group (P<0.01 at 10-30?M; P<0.001 at 40-50?M).(3) Pretreatment with IMX (0.25–1.0?M) concentration-dependently reversed A?25–35-induced SH-SY5Y cell death (P<0.05 at 0.25?M; P<0.01 at 0.5 and 1.0?M).(4) A?25–35-treated cells exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis. After 48 hr exposure of 40?M of aged A?25–35, 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 1.0?M 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 A?25–35-treated-alone group were markedly increased to 20.33±2.02% (P<0.01) with 20?M A?25-35 treatment. This increase, however, was prevented by the addition of 1.0?M 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 40?M of aged A?25–35 (n=5, P <0.001). However, pretreatment with 0.5 and 1.0?M of IMX concentration-dependently suppressed the apoptosis induced by 40?M of aged A?25–35 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 A?25–35-induced neurotoxicity, indicating the neuroprotective activity of IMX. Part?The effect and its possible mechanisms of IMX on A?25-35-induced tau phosphorylation in SH-SY5Y cellsAim: To investigate the effect and its possible mechanisms of IMX on A?25-35-induced tau phosphorylation in SH-SY5Y cells. Methods: (1) SH-SY5Y was incubated with 0, 0.5 and 1.0?M IMX. Following 24 hr treatment, 40?M of aged A?25–35 was added. 48 hr later, caspase-3 activity was measured with the caspase-3 assay kit. (2) 0.5?M and 1.0?M IMX were added in the serum-deprived media of SH-SY5Y cells for 1 hr prior to the 6 hr A?25-35 (20?M) exposure. Western blotting analysis was performed to detect tau phosphorylation at the site of pS396, pS199 and pT205 and p-GSK3?(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 A?25-35 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 A?25-35-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 A?25–35 treatment, the expression of p-GSK3?(Ser9) was decreased, which meaned that GSK3?phosphorylation was increased. However, the expression of p-GSK3?(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 A?25-35-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 A?25–35-induced neurotoxicity. (2) IMX attenuates A?25–35-induced neurotoxicity possibly by inhibiting tau phosphorylation at the site of pS396, pS199 and pT205. (3) IMX decreases tau phosphorylation by inhibiting GSK3?phosphorylation. (4) The expression of p-JNK and p-ERK were not involved the effect of IMX.