| Objective:Under the guidance of collateral disease theory,we study medicine senile dementia (AD) pathogenesis of cerebral microvascular injury in Neuronal damage, which will provide reliable experimental data to clarify the compound-Cardio Contact (TXL) basised of collateral disease theory on brain microvascular injury and its protection of Neuron by the way of protecting cerebral microvascular.1.The cognitive and memory dysfunction of AD model and the intervention of TXLIn this study, mice were divided into six groups, SAMR1 control group, SAMP8 group, TXL three dose groups (high, medium, low), Huperzine A (SSJJ) group, Each group included 20 mice which were Fedded of 10m1/kg basis of body weight, once a day for 90 days, the same volume of solvent of SAMR1. in the End of the experiment, mice were characterized by changes observed in each group, cognitive and memory dysfunction,neuronal loss, cortical amyloid (Aβ) deposition conditions.2.The cerebral microvascular injury of AD model and and the intervention of TXLIn this study, SAMP8 mice continue to be used as the research object, SAMR1 still as control, following the first part of the experiment proved that Aβdeposition in brain cortex after immunohistochemical method to observe the changes in the number of mice brain microvascular endothelial function damage, the expression in perivascular inflammatory changes.3. The injury of brain microvascular endothelial cells (HBMEC) induced by Aβ, and the intervention of TXL In this study, cell experiment, divided into six groups,control group: HBMEC with 10% FBS in DMEM medium for 30h after the test; model group: HBMEC with 10% FBS in DMEM culture medium after 6h, adding a final concentration of 20μmol/L the role of Aβ1-42 induced cell damage 24 h; TXL Group:HBMEC DMEM with 10% FBS in the culture medium and the final concentration was added 100,200,400 ug/ml of the TXL preconditioning 6h, adding a final concentration of 20μmol/L Aβ-42,24h, induced cell damage. SSJJ Group:HBMEC with 10% FBS in DMEM culture.And add a final concentration of 2μmol/L of SSJJ pretreatment 6h, adding a final concentration of 20μmol/l Aβ1-42 effect 24 h induced cell damage. In the End of the experiment, cells were observed under the inverted microscope, morphological changes, SRB detection of cell activity, nitric acid were detected by enzyme NO reduction levels, various groups in the supernatant VEGF, IL-lβ, IL-6, TNF-a expression by ELISA detection; eNOS, HIF-la, VEGF protein expression by westernblot detect; the expression of VEGFmRNA by RealtimePCR detect.4. The effect of cerebral microvascular endothelial cells injuried by Aβto neurons and the intervention of TXLIn this study, the method of primary cultured neurons were used, neurons were divided into normal group (control group), normal HBMEC supernatants+neurons group (normal group), Aβinjury HBMEC supernatants+neurons (model group), Aβgroup of damaged neurons (Aβinjury group), TXL intervention HBMEC supernatants+Aβ+neurons group (TXL group), SSJJ intervention HBMEC supernatants+Aβ+neurons group (SSJJ group), a total of six experimental group, control group adding 100% serum-free DMEM with nutrient solution, and the remaining 50% of each group were on 50% of endothelial cells in serum-free supernatant and DMEM medium, cells were placed in temperature of 37℃,5%CO2, saturated humidity The incubator 12h. After the end of the experiment the cells were observed under inverted microscope morphological changes, HE staining cells morphological changes, immune cell necrosis was observed under fluorescence microscope, the activity of neurons in each group by MTT test, the apoptosis rate by flow cytometry, apoptotic protease Caspase-3, apoptotic protein bax, bcl-2 expression by Western Blot detection, the bax mRNA, bcl-2 mRNA expression realtime PCR detection.1.1 The characterization of SAMR1 mice showed changes in white coat color, lively, responsive; SAMP8 mouse pelage is dull, lassitude, mental Weidun, limbs curled up, move slowly, squinting jitter, reduced shoulder Arch aging demeanor. And SAMP8 compared, TXL each dose group are characterized by different degrees of improvement, and high-dose group improved significantly, SSJJ mice compared with SAMP8 mice had improved, compared with TXL, middle dose group little worse.1.2 The water maze latency of mice experiments showing:in the reference memory detection, and SAMR1 control group, SAMP8 group gradually extend the spatial reference memory latency, P<0.01, TXL group latency, and SAMP8 group, P<0.05 or P<0.01, especially with high-dose group; SSJJ group latency, and SAMP8 group was not statistically significant, P>0.05. Detection in spatial working memory, and SAMR1 normal control group, SAMP8 group of spatial working memory latency gradually extended, P<0.05 or P<0.01, TXL group in 1,2,3 days latency, and SAMP8 control group, P<0.05 or P<0.01, set in 1,2 days SSJJ latency, and SAMP8 control group, P<0.05.1.3 The cerebral cortex of mice neuronal damage showing that compared with SAMR1 normal control group, the neurons serious of SAMP8 group, P<0.01, TXL dose of neurons to reduce, and SAMP8 group, P<0.05 or P<0.01, SSJJ group of neurons to reduce, and SAMP8 group, P<0.01, two-drug treatment showed no statistical significance, P>0.05.1.4 The cortex of mice showing that Aβdeposition in Congo red amyloid deposition was painted brick red or cherry, purple weak positive is into. And SAMR1 normal control group, SAMP8 group of more Aβdeposition, P<0.05, TXL of three dose groups and SSJJ cortical cells mostly colored purple, heavier than the SAMR1 group of cortical staining, and SAMP8 group, the deposition Aβdecreased significantly, P<0.01, no significant difference between the two drugs, P>0.05.2.1 The changes of NO in plasma among groups showed that:compared with SAMR1 mice, the levels of NO in SAMP8 group plasma significantly decreased, P<0.01, the levels of NO in each dose TXL group was significantly higher, and SAMP8 group, P<0.05 or P<0.01. TXL higher dose group which is most obvious, SSJJ group had no obvious improvement, compared with SAMP8 group, P>0.05.2.2 The number of microvascular changes in mice brain cortex showed that:compared with SAMR1 control group, the number of cortical microvessels reduced significantly in SAMP8 group, P<0.01, each TXL dose group were increased significantly, compared with SAMP8 group, P<0.05 or P<0.01, SSJJ number of obvious improvement in brain microvessels, compared with SAMP8 group, P> 0.05.2.3 The expression of ET-1 in mice cerebral cortex showed that: compared with SAMR1 group, the expression of ET-1 in SAMP8 group was increased significantly, P<0.01. Each TXL dose group was reduced, compared with the SAMP8 group, P<0.05 or P<0.01, the expression of ET-1 in SSJJ group was not improved obviously, compared with SAMP8 group, P>0.05.2.4 The expression of IL-6 in mice cerebral cortex showed that: compared with SAMR1 group, the expression of IL-6 in SAMP8 around microvessels was increased, P<0.01, TXL dose groups decreased, compared with SAMP8, P<0.01 or P<0.05, the expression of IL-6 in SSJJ group was not changed significantly, compared with SAMP8 group, P>0.05.2.5 The expression changes of IL-1βin brain cortex showed that: compared with SAMR1 control group, that in SAMP8 group increased, P<0.01, the expression of IL-lβin TXL group was significant lower, compared with SAMP8 group, P<0.01, the IL-1βaround capillaries decreased, compared with SAMP8 group, P<0.05.2.6 The expression of TNF-a in mice cerebral cortex showed that: compared with SAMR1 control group, the TNF-a around the capillaries in SAMP8 group brain was increased significantly, P<0.01, the microvascular expression in TXL dose group was decreased significantly, compared with the model group P<0.05, SSJJ group of capillaries around the TNF-a expression was not significantly changed, compared with SAMP8 group, P>0.05.3.1 The morphological changes in each group showed visible microscope, normal HBMEC fusiform cell bodies, the typical "cobblestone" shape, cell quantity, while the number of cells in model group decreased significantly, a large number of cell damage, but also cell death can be seen floating in the culture medium. TXL cells compared with the model number of larger number of good adherent cells, morphologically normal, a rare injury and death of cells, compared with model group, SSJJ cells form was little improvement.3.2 The change of cells in each group showed that:compared with the control group, model group HBMEC cytotoxicity, P<0.05, the activity TXL group and SSJJ group HBMEC improved than the model group, P<0.05 or P<0.01, compared with the TXL and SSJJ group, P<0.05.3.3 The change of NO in HBMEC in each group showed that:compared with control group, model group, NO levels in HBMEC decreased, P<0.01, while the TXL Group NO improvement compared with model group, P<0.05 or P<0.01, SSJJ Group NO levels had no obvious improvement, compared with model group, P>0.05.3.4 The changes of eNOS in the HBMEC group showed that:compared with the control group, the expression of eNOS in model group HBMEC decreased, P<0.01, compared with model group, the eNOS in TXL group improved, P<0.05 or P<0.01, compared with the model group, the eNOS level of in SSJJ group was not obvious, P>0.05, compared with the TXL group, P<0.05.3.5 The expression of VEGF change in cells supernatant showed that: compared with the control group, VEGF levels decreased in model group, P<0.01, compared with model group, VEGF expression TXL group improved P<0.05 or P<0.01. compared with model group, the VEGF level SSJJ Group increased, P<0.05.3.6 The expression of VEGF change in HBMEC showed that:compared with the control group, the VEGF expression in model group was decreased, P<0.01, compared with model group, the VEGF expression in TXL group improved, P<0.01 the VEGF expression in SSJJ Group improved, compared with the model group, P<0.05.3.7 The expression of VEGFMRNA in each HBMEC group showed that: compared with the control group, the VEGFMRNA expression model group reduced, P<0.01, compared with model the model group, the VEGFMRNA expression in TXL group, P<0.05 or P<0.01. SSJJ group had no obvious improvement, P>0.05.3.8 The changes of HIF-1a in HBMEC group cells showed that: compared with control group, the HIF-1a protein expression in model group was increased, P<0.01, compared with the model group, the expression of TXL high dose group was enhanced, P<0.05, the expression changes of HIF-la protein in SSJJ group was not obvious, P>0.05, compared with two drugs, TXL group was better than SSJJ group, P<0.05.3.9 The expression of IL-1β, IL-6, TNF-a in HBMEC group showed that: compared with the control group, the IL-1β, IL-6, TNF-a in model group were higher, P<0.05 or P<0.01, the IL-1β, IL-6, TNF-a expression in TXL group was reduced, P<0.05 or P<0.01, the IL-1βin SSJJ group decreased, P<0.05, the expression of IL-6, TNF-a was no obvious improvement, compared with model group, P>0.05.3.10 The results of the expression of NF-κB signaling pathway in HBMEC:compared with the control group, the expression of NF-κB protein in model group increased, P<0.01, the expression in TXL three doses was reduced, Compared with model group, P<0.01, the NF-kB protein expression of SSJJ group decreased was not obvious, compared with the model group, P>0.05.4.1 Cultured neurons showed morphological changes in cerebral cortex of the experimental portion of selected primary culture, grown on plates in the cortical neurons were round, transparent, uniform, smooth membrane, refractive good, clear halo. After 24h cells fully adherent, some short and small cells can be seen protruding.2-3 days, with significantly increased cell processes, the processes have become the trend of long, large cell body bes spindle, spherical and triangular cells with three-dimensional. The role of adding cytosine arabinoside, the flat polygons almost eliminate glial cells, neuronal cells showed morphological characteristics.4-5 days, the rapid differentiation of neuronal cells, cell shape is very typical cell dimension a strong, full cell body, cytoplasm-rich, clear nucleolus, bipolar and multipolar neurons everywhere, protruding over the rough, at end of branches, the formation of neurites extended network.4.2 The showing of morphological changes of neurons in each group4.2.1 Under inverted microscope:the control group and normal group of spindle cell bodies of cortical neurons, or triangle, poly heap growth, interwoven into the mesh, good refraction. Aβinjury group and model group cell injury seriously, cell morphology, cell processes became shorter, or fewer, refraction difference, and there are dead cells floating, and Model group, the most significant performance, compared with the model group, TXL group and SSJJ group improved, and TXL group showed the most significant.4.2.2 HE staining:the group of neurons showed morphological changes in the control group, normal control group, neuronal cell bodies large and bright, clearly visible nucleus and nucleolus, neurite length, and more, forming a typical nerve terminal branch network; model group and Aβinjury group reduction in the number of neurons, cell bodies fuzzy, sparse neuronal processes significantly, fewer shorter, or even disappear, and Model group, the most serious injury. compared with the model, TXL and SSJJ group was improved, and the TXL group improved significantly.4.3 In each group cell, necrosis and apoptosis of neurons to change PI staining showed the blank group and control group showed less cortical neurons in the red-stained cells, indicating that very few necrotic cells, while Aβinjury group and model group of Red dye cells more, the number of dead cells increased and the model group of cells changed significantly, TXL and SSJJ group had improved to the TXL group. Hoechst33342 staining the blank group and control group showed low cortical neurons stained blue nucleus and membrane integrity, and few apoptotic cells, Aβgroup and model group injury serious cell damage, cells were stained bright blue, some cells were typical morphology of apoptosis, in cells surrounding the formation of apoptotic bodies. And changes to the model group significantly, TXL and SSJJ group were improved, especially TXL group.4.4 The group of neuronal activity changes in results with the control group and normal group, Aβinjury group and model group neuron activity decreased, especially the model group, P<0.05, TXL group of neurons in the energy than the model group, P<0.05. SSJJ group activity of neurons compared with model group, but not statistically significant, P>0.05.4.5 Apoptosis rate of neurons in each group showed a change with the control group and normal group, Aβinjury group and model group increased rate of neuronal apoptosis, P<0.01, between two groups, the model group increased significantly, P<0.05, compared with model group, the apoptotic rate of TXL and SSJJ group were decline, especially the model group, P<0.05, between two groups, P<0.05.4.6 The group of neuronal apoptosis protease caspase-3 showed no change with the control group and normal group, Aβinjury group and model group, neuronal apoptosis protease caspase-3 expression, especially the model group, P<0.05, the caspase-3 expression of TXL group was decreased, compared with the model group, P<0.05, SSJJ group reducing was not obvious, compared with the model group, P>0.05.4.7 The group of neuronal apoptosis gene bax, bcl-2 showed changes in Aβinjury group and model group, neuronal expression of apoptosis protein bax, bcl-2 expression was decreased, and the model group was most significant, P<0.05, compared with the model group,TXL and SSJJ group was improved, P<0.05,.4.8 The group of neuronal apoptosis gene bax, bcl-2mRNA showed changes in Aβinjury group and model group, the expression of neuronal cells in baxmRNA increased, bcl-2mRNA expression decreased, and the model group was most obvious, P<0.05 or p<0.01, compared with the model group, TXL Group baxmRNA lower, P<0.05, bcl-2mRNA increase, p<0.01. The SSJJ group baxmRNA not changed significantly, P<0.05, bcl-2mRNA expression was not obvious improvement, P>0.05.1. Firstly, using the theory of Traditional Chinese Medicine collateral disease to investigate pathogenesis and treatment of AD. Cerebral collaterals divide into gas collaterals that transport gas and blood collaterals that transport blood, which is the material basis to maintain the brain activity of God, based on blood-related Collateral Theory characteristics. Although AD is the disease on the gas collaterals, but the abnormal structure and function of blood-related Collateral can not be ignored. Based on the transforming relationship among god, gas, precision air, we find that deficiency of vital energy, the injury of gas collaterals is an important pathogenesis of AD,because that the weakness of gas and blood, Vein stasis, the disorders about the blood supply, Tianjin blood exchange, nutrition, metabolic, in the end of vein can also increase the air contact injury, which will cause loss of brain with god, as the pathological basis of AD pathogenesis, yiqihuoxuetongluo is an effective therapies to AD.2. Based on the Correlation between Traditional Chinese Medicine context and Western medicine about the small blood vessels and microcirculation anatomy relevant, Learning Western medicine on the pathogenesis of AD microvessels latest break through in the last year on AD simply understood as the concept of neurodegenerative diseases, by the microvascular injury mechanism, we explore the pathogenesis of AD. The results show that brain structure and function of microvascular damage occurs on rapid aging model mice:cortical ET-1 increased expression, NO secretion was decreased, the number of capillaries reduced significantly, perivascular inflammatory factors increased significantly; somatic cell studies Show that Aβcan not only damage the structure and function of HBMEC, but also Aβincubated with the supernatant of endothelial cells also induced significant neuronal apoptosis, which can not only provide an experimental basis for Western medicine microvascular hypothesis of AD pathogenesis, but also Enrich the scientific content of the Context theory of gas-blood related.3. The results show that TXL could improve the rapid aging of memory and cognitive function in type mice and protect the brain microvascular structure and function significantly; by reducing the microvascular endothelial cell injury Aβ-induced to reduce brain neuronal apoptosis, which reveale Initially that the clinical treatment of TXL is by the protection of AD microvascular mechanisms, which will provide the experimental basis for Clinical study of AD, but also prove the guiding value to AD of the collateral disease theory.
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