| BackgroundTraditonal Chinese Medicine is attractive for the better clinical effects on cerebral stroke,and with a long history. Rehmannia Root is the main natural herb medicine that plays the most important role in treatment of stroke. Recently, Catalpol, a main active component of Rehmannia Root,were endued with an extensive ischemic neuroprotection.But researcheres focused on neuronal protection, ignoring the neurovascular unit protection,which is immportant in stroke especially.So intensive researches should be done to further elucidate the effects of catalpol on the neurovascular protection and the neurovascular repairment after stroke.OBJECTIVE(1)To investigate the effects on HUVECs adhesion,proliferation migration,and expresstion of EPO and VEGF treatment with Catalpol isolated from Dihuang;then to investigate the role of Catalpol on survival activity and neurite outgrowth from cerebral cortical neurons in culture.(2)To research the ischemia neuroprotection and its mechanism induced by Catalpol in male Sprague Dawley rats after permanent occlusion of the distal right middle cerebral artery (p-MCAO).(3)The signal transducers and activators of transcription (STAT) proteins are a group of transcriptional factors.Among them,STAT3 initiates a pro-survival signaling cascade and VEGF induced angiogenesis . So far,little has been known about the role of STAT3 in neovascularization in cerebral ischemia.This study examines the phosphorylation status of STAT3 in the cerebral cortex surrounding the ischemic core in the subacute stage after permanent occlusion of the distal right middle cerebral artery (pMCAO) in rats and to investigate the role of Catalpol in regulating angiogenesis at the transcription and translation level.METHODS1.At the cellular level,HUVECs and primary cerebral cortical neurons were cultured,we observed the catalpol effects on HUVECs adhesion,prolifersion,migration and survival activity and outgrouth of primary cortical neurons in culture,using cell counting,MTT, micrometter,respectively.In addition,the expression of EPO and VEGF on HUVECs induced by catalpol were observed with immunohistochemical method.2.The neuroprotection of catalpol was evaluated in unilateral ischaemia in male Sprague Dawley rats induced by pMCAO.(1) To investigate neuroprotection treatment with catalpol on pMCAO rats,five groups were designed as sham-operated,model, ischemia-treated,with catalpol,Citicoline and saline respectively.Catalpol was injected intraperitoneally 6h and 24h after pMCAO and repeatedly each day for 7d with the dose of 1,5.0,10 mg?kg-1.The neuroprotection was estimated by the indexes of behavior:the effects of catalpol on pMCAO rats was performed in a battery behavioral testing(Bederson scores,muscle strength scores,Beam walking scores and affected forelimb skilled reaching test)at different time point of 1,4,7,14,21d after pMCAO.(2) To evaluate the possibility of Catalpol crossing BBB with HPLC assay.3.Evaluating the possible neuroprotection mechanism of catalpol(1) Uhrastructural observation of nerve cells and synapse and brain capillary endothelial cells were investigated at 15d after pMCAO.(2) we investigated the expression of GAP-43,EPO,VEGF and KDR protein on pMCAO rats brain in each group using immunohistochemical method and western blot analysis.4.Evaluating the possible role of STAT3 on angogenesis and the mechanism of catalpol's angogenesis on pMCAO rats(1) AG490 was used to blockade phosphorylation of STAT3,six groups were designed as sham-operated,model,of AG490 and catalpol.Catalpol was injected intraperitoneally 24h after p-MCAO and repeatedly each day for 7d with the dose of 5.0 mg?kg-1.At the time point of 15d after pMCAO,catalpol's angogenesis effects was indentfied by double staining ofⅧfactor (vWF),a marker of endothelial cells,and proliferating cell nuclear antigen (PCNA),a marker of cell proliferation.(2) In addition,Stat3 actived by ischemia and the possible role of Stat3 on angogenesis was also examined by using immunohistochemical method and western blot analysis.Stat3 protein levels and the levels of the activated form of Stat3 (pStat3 ) at 15d were determined by western blot analysis,pStat3 were also demonstrated by immunohistochemical method.(3) At the same time, we investigated the expression of VEGF on pMCAO rats brain in each group using immunohistochemical method and western blot analysis.RESULTS一,Results in vitro1. Catalpol enhanced HUVECs adhesion,proliferation and migrationCompared with blank group at each time point,catalpol in each concentrtion enhanced HUVECs adhesion significantly(p<0.05);At the dose of 0.25mg?ml-1,the adhesion of HUVECs was increased in time-dependent way.Compared with negtive group, 0.5～1mg?ml-1 of catalpol improved HUVECs proliferation,with the peak occurring at 1mg?ml-1 of catalpol(p <0.05).But it inhibits HUVECs adhesion and proliferation at dose of 2.5mg?ml-1.Compared with blank group and normal saline group, catalpol stimulates HUVECs migration at the dose of 0.5~5 mg?ml-1, with the peak occurring at 1mg?ml-1 of catalpol,migration length was 102±27.6μm and 142±35.5μm at the time point of 24h and 48h,respectivly ( p <0.01).Migration length of HUVECs begins to decline at the concentrtion 5mg?ml-1 of catalpol.2. Upregulating EPO and VEGF expression on HUVECs induced by catalpolCompared with blank and normal saline group,at the dose of 0.5,1,2.5mg?ml-1,a statistically significant dose-dependent increase in the EPO expression on HUVECs was observed (p<0.05),with the peak occurring at 2.5mg?ml-1 of catalpol, IOD (integral optic density,IOD) of each group was 120.65士0.014,138.39±0.015,142.19±0.026, respectively.VEGF expression on HUVECs was like as EPO,but no dose-dependent relation,IOD of catalpol group at concentration of 0.5,1,2.5,5mg?ml-1 was 117.68士0.022,210.63士0.045,184.46士0.044 and 174.46士0.024,respectivly,it was higher than that of blank and normal saline group significantly(p<0.05).3. Catalpol doesn't affect survival activity on primary cortical neurons, but promotes its axon growth Primary cortical neurons was cultured successfully and identified by NF-200 antibody. Its purity was more than 95%.Catalpol doesn't affect survival activity on primary cortical neurons by MTT assay. Compared with blank group and Citicoline group, each dose group on neurons survival activity has no remarkably difference(P>0.05).At the dose of 1~5mg?ml-1, a statistically significant dose-dependent increase in the axon growth of primary cortical neurons was observed, with the peak occurring at 2.5mg?ml-1 of catalpol, there was significant difference(P<0.05).But cortical neurons axon growth began to decline at dose of 5mg?ml-1.二,Results in vivo1. Catalpol improved SD rats neurobehavioral consequences after pMCAO evaluated by a battery of behavioral testsTo explore the effects of catalpol on the functional recovery of nerves(NFR) in rats with pMCAO,Bederson score,balance function,muscle strength and percent success on forelimb skilled reaching were assessed at 1 d,4 d,7 d,15 d and 21 d after the operation. The percent success on forelimb skilled reaching in catalpol group was 48.7%±5.4% and 47.3%±4.8% at dose of 5 and 10 mg?kg-1 respectivly, it was increased significantly than that of control group and model group (P<0.05). It shows that catalpol can contribute to functional recovery of nerves after pMCAO.2. Catalpol crosses the BBB and detected in CSF by HPLC The ability of Catalpol to cross the blood-brain barrier (BBB) was investigated, Intravenous (i.v.) Catalpol was rapidly distributed into brain. After 40min, Catalpol in CSF could be detected by HPLC.3. Catalpol enhanced brain angiogenesis in p-MCAO rats(1) The vascular pattern in cerebral cortex surfaceAfter pMCAO 15d, it was thus clear that liquefactive necrosis of the brain was observed in model group,with scarce and derangement microvascular,However, focus of cerebral ischemia was near heal in Catalpol group, with more branch vessels crossing and gathering to focus surface of cerebral ischemia in radiat way. All these vessels arborize to form a continuous network of small blood vessels .(2) Angiogenesis surrounding ischemic cortex core showing by LSCM(laser scanning confocal microscope)Catalpol's angogenesis effects was indicated by double staining of vWF,a marker of endothelial cells,and proliferating cell nuclear antigen (PCNA),a marker of cell proliferation. This study demonstrates that Stroke induces the mother vessels from preexisting microvessels and the mother vessels,a transient structure,generate smaller daughter vessels over the course of weeks in ischemic brain,the microvessels formed connections with the surrounding proliferating vessels.There was sparseness vessel double staining of vWF and PCNA in sham-operation rats. But signifycant remodeling of the microvessel network occurs in the infarcted hemisphere.The number of vessels with small diameter and short segment increased at 15 days after stroke,more vessels double staining of vWF and PCNA could be seen in group treatment with catalpol and Citicoline; Microvessel double staining density with catalpol was more than that of Citicoline, normal saline group and model group, there was significant difference (P<0.05). Neurological functional recovery in each group is strongly correlated with an increase in angiogenesis in the cortex surrounding the ischemic lesion after p-MCAO.Compared with sham operation group,number of co-localization point in model group (40.67±2.30) increased significantly (P<0.05).The number of co-localization point treatment with catalpol is up to 233.67±89.51, which is more than that of model and Citicoline group obviously,there was remarkably difference (P<0.05).The rerults as above is conformity with the results of IOD analysis in co-localization area.It shows that Catalpol plays a important role in cerebral ischemia angiogenesis.(3) Effects of uhrastructural observation on brain capillary endothelial cells(BCECs)The changes of BCECs microstructure were observed by transmission electron microscopy.Compared with normal saline group and model group,Catalpol reduced the BCECs edema significantly.The number of chondriosome in Catalpol group is more than that of in model group, and near normal levels.4. Effects on uhrastructural structure of nerve cells synapse and axon growth(1) Catalpol upregulates GAP-43 expression,the marker of axon growthThere was no difference between the group of model and normal saline and sham oprationed group, although GAP-43 expression in model and normal saline group are higher than that of in sham oprationed group. Compared with model and Citicoline group, Catalpol at the dose of 5 mg?ml-1 dramaticaly upregulated GAP-43 expression, IOD was 359.24±12.38, there was significant difference (P<0.05). western blot analysis results is also shown as same as Immunofluorescence results.(2) Ultrastructure changes of neurons treatment with catalpolCatalpol play a certain role in protection of the neurons following pMCAO injury.In model group, the changes of neuron ultrastructure include:mitochondria swelling,loss of mitochondria crest and matrix, decreased nuclear size,scattered nucleoplasm,and reduced synaptic number. The ultrastructure of the neurons in the Catalpol group was near normal.5. Catalpol upregulates EPO expression in pMCAO rats brainImmunofluorescence results as below:IOD of the normal saline group, model group,and Citicoline group was 4.80±0.41,6.88±0.60,5.02±0.45, respectively.Compared with sham operation group, EPO expression is increased significantly (P<0.05);Catalpol at the dose of 5 mg?ml-1 dramaticaly upregulated EPO expression,IOD was 9.43±1.27,Compared with normal saline group,model group,and Citicoline group,there was significant difference (P <0.01).The same results were obtained by western blot analysis.6. Catalpol upregulates VEGF expression in pMCAO rats brainImmunofluorescence results as below:Compared with sham operation group,model group dramaticaly upregulated VEGF expression(P<0.05), IOD was 325.10±26.82;Catalpol at the dose of 5 mg?ml-1 upregulated VEGF expression,IOD was 628.56±36.63,there was significant difference between catalpol group and other groups (P<0.05).The same results were obtained by western blot analysis.7. Catalpol upregulates KDR expression in pMCAO rats brainImmunofluorescence results demonstreated that IOD of catalpol group was 116.3±10.23,Compared with normal saline group,model group and Citicoline group,catalpol at the dose of 5 mg?ml-1 dramaticaly upregulated KDR expression,there was significant difference(P <0.05);IOD in normal saline,model and Citicoline group was 69.99±6.59,73.43±7.85,88.18±7.36 respectively.The same results were obtained by western blot analysis. 8. Angiogenesis mechanism treatment with catalpol in pMCAO rats(1) The level of tyrosine phosphorylation of Stat3 (pStat3) pStat3 immunoreactivity was detected in p-MCAO brain,Ischemia induced by pMCAO activates the JAK/STAT3 pathway in infarcted hemisphere in rats brain.Ctalpol can activate cytokine-mediated signal transduction from the membrane to the nucleus,it can reverse inhibiting transduction effects of AG490,but this pheminon was not visible in DMSO group.Western blot analysis also revealed that a significant increase in the pStat3 protein in combination of AG490 and catalpol group or catalpol group,there was significant difference,compared with AG490 group (P<0.05).(2) Ratio of pStat3/ tStat3The level of tyrosine phosphorylation of Stat3 was demonstrated by p-Stat3/ t-Stat3.AG490 reduced the ratio of pStat3/tStat3 obviously(P<0.05),it was 7%,but combination of AG490 and catalpol reverses the inhibiting effects of AG490 significantly (P < 0.05),the ratio of pStat3/tStat3 is 16%;the ratio of pStat3/tStat3 is up to 23% treatment with catalpol only.(3) VEGF expressionImmunofluorescence shows that VEGF expression were high in pMCAO brain after pMCAO 15d.IOD of VEGF expression in catalpol group was 628.56±36.63,It revealed a significant increase in the VEGF protein treatment with catalpol,there was significant difference (P<0.05);VEGF expression was downregulated by AG490 significantly,IOD of AG490 group was 534.46±24.45,catalpol could reverses the AG490 effects on VEGF protein expression.Western blot analysis results as same as immunofluorescence analysis results.(4) Interrelated parameters of brain angiogenesisMicrovessel double staining density was dramaticly increased in catalpol group,compared with Citicoline normal saline group and model group,there was significant difference (P < 0.05);Microvessel double staining density was downregulated by AG490 significantly and reversed by the catalpol.It revealed a significant increase in the microvessel double staining density treatment with combination with catalpol and AG490,there was significant difference (P<0.05).CONCLUSION1. The amelioration of behavioral after ischemia was observerd in pMCAO rats received treatment with Catalpol at the dose of 1,5,10 mg?kg-1,intraperitoneally immediately after pMCAO at 6 h or 24 h and repeatedly once a day for 7 days.it was the best effects of nueroprotection at the dose of 5 mg?kg-1.2. Protective effects and its mechanism of Catalpol isolated from dihuang on cerebral ischemia is pleiotropic functions and multiple pathways.In peri-infarct cortex,Catalpol may mediate neurovascular remodeling.(1) Catalpol can crosses the BBB and be detected in CSF by HPLC.(2) Catalpol stimulates neurite outgrowth from cerebral cortical neurons in culture and increase the synapse number in pMCAO rats via upregulating expression of GAP-43,Vascular endothelial growth factor and EPO.(3) Treatment with Catalpol increased angiogenesis,as indicated by double staining of vWF,a marker of endothelial cells,and proliferating cell nuclear antigen (PCNA),a marker of cell proliferation.The angiogenesis stimulated by Catalpol was accompanied by an increase protein expression of VEGF and EPO.3. To evaluate the significance of JAK2/STAT3 signaling in brain,we inhibited JAK2 with AG490 and inhibits STAT3 activation,Inhibition of JAK2 down-regulated pSTAT3.The blockade of JAK2/STAT3 signaling significantly decreased pMCAO brain angiogenesis through down-regulation of VEGF.(1) Signal transducer and activator of transcription 3 is required for brain capillary growth after pMCAO in adult rats.(2) Catalpol enhences endothelial cell proliferation and adhesion, stimulates endothelial cell migration accompanied by upregulating EPO and VEGF protein.(3) Catalpol given after pMCAO in adult rats provides significant neuroprotection and angiogenesis,mediated partly by activation of the Janus kinase-signal transducer-3 and activator of transcription VEGF signaling pathways.
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