The Effect Of Catalpol On Proliferation And Differentiation Of Neural Stem Cell And Study Of Neuroprotection Induced By Catalpol After Permanent Middle Cerebral Artery Occlusion In Rats

BackgroundStroke has the characteristics of high incidence, high mortality, high recurrence rate, high morbidity and high economic burden. There is less effective drug used in clinic. Neurogenesis mediated by neural stem cells become a hot treatment of stroke based on the fact that neural stem cells are present in adult animals and humans. The death of neurons in infarct center and its surrou nding areas, lack of proliferation of endogenous neural stem cells, a few neurons differentiated from neural stem cells and only 0.2% of newborn neurons survival make it hard to repair their own neurological function just based on self-repair of animals. Therefore, the key points to treat stroke are how to promote the proliferation and differentiation into neurons of endogenous neural stem cells and how to promote the survival of newborn neurons. Rehmannia Root is one of the main natural herbs that plays the most important role in treatment of stroke. Previous studies showed that Catalpol the main active ingredient from Rehmannia Root could protect neurons from ischemic injury and promote human adipose tissue-derived stem cells to differentiate into neurons. But the effect of Catalpol on neurogenesis in vivo lack researchs. In this study, we discussed the effect of Catalpol on the proliferation and differentiation of neural stem cell and explored the survival mechanisms of newborn neurons in the brains of permanent middle cerebral artery occlusion rats. This study can provide some experimental basis on the treatment of stroke.Objective1. To research the ischemia neuroprotection induced by Catalpol in male Sprague Dawley rats after 1d, 3d, 7d, 14 d, 21 d permanent middle cerebral artery occlusion.2. To investigate the proliferation and differentiation of adult neural stem cells in the SVZ and the cerebral cortex around the infarction after 7 days p-MCAO in SD rats treated with Catalpol. To observate the effect of Catalpol on the survival of neurons in the cerebral cortex around the infarction after 7 days p-MCAO in SD rats, further to investigate the survival mechanism.Methods1. Catalpol was injected intraperitoneally 6 h after p-MCAO and repeatedly each day with the dose of 5 mg/kg, 10 mg/kg. The effects of catalpol on p-MCAO rats was performed in Bederson scores, Corner Test and Ladder Rung Walking Test at different time point of 1,3,7,14,21 d after p-MCAO.2. Using immunohistochemical method to detect the number of newborn neural stem cells, newborn neurons, newborn astrocytes in the SVZ and the cerebral cortex around the infarction in 7 d p-MCAO rats. We investigated the expression of Tuj-1 and GFAP protein in the cerebral cortex around the infarction after 7 days pMCAO in each group using western blot analysis.3. Using western blot analysis to investigate the expression of Map-2 protein in the cerebral cortex around the infarction after 7, 14, 21 days p-MCAO. Investigate the expression of BDNF, Trk B, P-Trk B, Bax, Bcl-2, Caspase-9, Cleaved-Caspase3 in the cerebral cortex around the infarction after 7, 14 days p-MCAO in each group4. K252 a was used to blockade Trk B, Using Nissl staining to observe the effect of catalpol and K252 a on neurons in the cerebral cortex around the infarction in 7 days p-MCAO rats. Using western blot analysis to observe the effect of K252 a on the expression of Tuj-1, Map-2, BDNF, Trk B, P-Trk B, Bax, Bcl-2 after 7 days pMCAO.Results1. The growth of body weight of rats were inhibited when they had gone through permanent middle cerebral artery occlusion, Catalpol played a positive role in the growth of body weight of p-MCAO rats.2. The Bederson scores of each group was decreasing over cerebral ischemia time. There was no significant difference between each group in each time. Catalpol groups, at the doses of 5 mg/kg, 10 mg/kg,both could improve the behavioural performance of Corner Test and Ladder Rung Walking Test.3. There was phenomenon of division and proliferation of cells in the SVZ and the cerebral cortex of brains in each group, Catalpol groups, at the doses of 5 mg/kg,10 mg/kg,both significantly increased the number of Brdu positive cells in the ipsilateral SVZ and the cerebral cortex around the infarction after 7 days p-MCAO. In addition, Catalpol group, at the doses of 5 mg/kg, was more effective to promote division and proliferation of cells.4. After 7 day p-MCAO, Each Catalpol group could significantly increase the mean optical density of Nestin in the ipsilateral SVZ, increase the number of newborn neural stem cells in the cerebral cortex around the infarction.5. After 7 day p-MCAO, Each Catalpol group could significantly increase the mean optical density of Tuj-1 and GFAP in the ipsilateral SVZ, increase the number of newborn neurons and astrocytes and the expression of Tuj-1, GFAP protein in the cerebral cortex around the infarction.6. Each catalpol group could significantly increase the expression of Map-2 in the cerebral cortex around the infarction after 7 d, 14 d, 21 d p-MCAO. After 7 day pMCAO, in the cerebral cortex around the infarction, each Catalpol group could increase the expression of BDNF protein and the IOD ratio of Bcl-2/bax, but the expression trend of Caspase-9 protein was unstable, After 14 day of p-MCAO, Each Catalpol group could increase the expression of BDNF protein and the mean ratio of Bcl-2/bax, in addition, could decrease the expression of Caspase-9, Cleaved-Caspase3 protein. The Phosphorylation rate of Trk B protein after 7d, 14 d p-MCAO in each group had no significant difference.7. After 7 day p-MCAO, in the cerebral cortex around the infarction, K252 a group, K252 a and 5 mg / kg Catalpol combined group, 5 mg/kg Catalpol group, 10 mg/kg Catalpol group all could improve the result of Nissl staining, increase the expression of Tuj-1, Map-2, BDNF protein and the IOD ratio of Bcl-2/Bax. The Phosphorylation rate of Trk B protein had no significant difference.Conclusion1. Catalpol could promotes neurobehavioral function recovery in p-MCAO rats.2. After 7 day of p-MCAO, each Catalpol group could promote the proliferation of neural stem cells in the ipsilateral SVZ and cerebral cortex around the infarction,promote neural stem cells to differentiate into neurons, increase the number of newborn astrocytes.3. Catalpol could inhibit neuronal apoptosis in the cerebral cortex around the infarction of p-MCAO rats by increasing the IOD ratio of Bcl-2/Bax, thus reducing the expression of caspase-9 protein, ultimately suppressing the activation of Caspase-3 protein. Catalpol may contribute to neuron survival through this signaling mechanism. The activation and strength of this signaling pathways may be related to the time course of p-MCAO. Caspase-9 protein may be the key protein during regulation of this signaling pathway by Catalpol.4. Catalpol might not be through the BDNF/Trk B signaling pathway to inhibit the apoptosis of nerve cells.