The Clinical Efficacy Of Tongluo Huatan Capsules In The Treatment Of Stroke Recovery Period And The Dynamic Changes Of NMDAR Endocytosis Mediated By Clathrin In VD Rats

Cerebral infarction is also known as ischemic stroke,refers to ischemia and hyPoxia caused by the limitation of brain tissue ischemic necrosis or softening due to the brain blood suPPly disorders,belongs to "stroke" category of traditional Chinese medicine.Tongluohuatan caPsule has the function of Promoting blood circulation and collateralization,eliminating Phlegm and dispelling wind,and is used to treat meridian Phlegm and blood stasis blocking collateralization syndrome in stroke.The team research grouP has completed the data collection of the Phase ? clinical trial of this drug in the early stage,but has not summarized the data.Now,based on the clinical data collected in the early stage,systematic summary and analysis has been conducted to further evaluate the efficacy and safety of tongluohuatan caPsule in the treatment of stroke in the recovery stage.At the same time,based on the thought of "homotheraPy for heteroPathy in traditional Chinese medicine,the syndrome of Phlegm stasis blocking collaterals is also a common syndrome of Vescular Dementia(VD)after ischemic stroke,so the hyPothesis was Put forward that tongluohuatan caPsule can treat vascular Dementia.According to the latest research,clathrin-mediated endocytosis can inhibit NMDA-induced neuronal death and thus imProve cognition,which is a marker of endocytosis.This study on VD model rats,acquired the hiPPocamPus on several different time Point--3 h/1d/3d/7d/14 d after the treatment,using Western blotting(WB)and RT-PCR detection clathrin Protein and swallowing markers--Rab5B/NMDAR1,to exPlore the dynamic change characteristics in hiPPocamPal neurons clathrin mediated Process of NMD A recePtor cell swallowed during cerebral ischemia rePerfusion,at the same time to explore the mechanism of tongluohuatan caPsule in the treatment of VD,moreover,to Prove the curative effect of treating different diseases with same traditional Chinese decoction from the microscoPic characterization level.Clinical researchobjective:To further evaluate the effect and safety of Tongluohuatan caPsule in the treatment of stroke recovery Period Phlegm and blood stasis resistance meridians collaterals syndrome,Provide reference for clinical medication.method:Using the statistical methods to analyze clinical trial data the early stage acquired.Results:(1)From January,2012 to December,2016,more than 34 hospitals Participated,and a total of 2169 cases was included in clinal research.The effective rates of Tongluohuatan caPsule was 84.09%and 81.01%,accessed by ComPrehensive effect of nerve function defect scale and TCM syndrome scale,respectively;For mRS scale,there were 817 cases(37.67%)with the total score of mRS decreased by 50%or more,1187 cases(54.73%)with the total score decreased to 0?1.(2)Age(OR=1.024,95%CI 1.009-1.039,P=0.002)Baseline hyPertension(OR=4.720,95%CI 3.513-6.341,P=0.000)Coronary heart disease(OR=2.601,95%CI 1.530-4.421,P=0.000)HyPerliPidemia(OR=2.234,95%CI 1.234-4.407,P=0.008)Baseline NIHSS score(OR=0.902,95%CI 0.849-0.957,P=0.001)affected the comPrehensive efficacy accessed by Comprehensive effect of nerve function defect scale.(3)Baseline of TCM syndrome score had an impact on the clinical efficacy of TCM syndrome(OR=62.023,95%CI 37.407-113.0265,P=0.000).(4)Baseline hypertension(OR=2.898,95%CI 2.144-3.917,P=0.000),coronary heart disease(OR=3.625,95%CI 1.516-8.673,P=0.004)had an Positive effect on the improvement of the modified Rankin scoring scale.(5)The overall incidence ofadverse reactions of tongluohuatan caPsule was 0.00%;Conclusion:(1)In the treatments of recoverable period of cerebral infarction,Tongluohuatan capsule get good effects and safety on improving nerve function defect,reducing disability and improving TCM symptoms,alone or combining with other basic treatments of western medicine.(2)In the treatment of Tongluo Huatan Capsule in the recoverable period of cerebral infarction,the small age,light baseline disease condition and no basic diseases Patients' curative effects are better.Experimental studyObjective:(1)To explore the characteristic of dynamic changes of clathrin-mediated NMDA receptor endocytosis in hippocampal neurons of rats with vascular dementia;(2)To explore the possible role of Tongluo Huatan Capsule in the early intervention of Vescular DementiaMethods:(1)After 7 days of adaptive feeding,the rats were randomly divided into sham operation group,model group,tongluohuatan capsule group(THC group),maigang group(MH group),and shenzhijiannao prescription group(SZJN group).The rats were given the corresponding drug gavage,respectively,the sham operation group and model group were given the same amount of distilled water gavage,once per day for 7 consecutive days.The VD rats model was prepared by intraperitoneal injection of nitropurna with 2VO.The rats in the sham operation group were isolated from bilateral common carotid arteries without clipping the vessels,and were not injected with sodium nitroprusside.After modeling,the rats were tested for platform jumping,new object recognition and Morris water maze to measuring the memory function.Rat hippocampal tissues were collected at 3h,1d,3d,7d and 14d later after modeling.The expression levels of clathrin,NMDAR1 and Rab5B in hippocampus at different time points after modeling were detected by RT-PCR and Western blot.Results:(1)Behavioral science?The results of platform jumping test(model evaluation)showed that there was no statistical difference in the latency and the number of errors between the sham operation group and the normal group.Compared with the normal group and the sham group,the latency period of the model group was shorter and the number of errors increased(P<0.05).? In the new object recognition experiment,compared with the sham operation group,the new object recognition index of the model group decreased(P<0.01).The discrimination index of THC group,SZJN group and MH group were higher than that of model group(P<0.01).?Water maze:The escape latency of rats in each group was shortened to different degrees(P<0.01).The effects of different groups on escape latency were different(P<0.05),and the escape latency was longer in the model group compared with the other four groups.After the removal of the platform,the model group had a shorter residence time at the target than the other four groups(P<0.01).Compared with the model group,the number of times of crossing the platform increased in the sham operation group and the SZJN group(P<0.01).(2)Pathology:HE staining showed that there was no abnormality in the structure and morphology of hippocampal CA1 area in the sham-operated group.The neuron morphology was complete and neatly arranged,and the boundary between nucleus and cytoplasm was clearly visible without necrosis.The neuron in the CA1 area of the hippocampus of VD rats has incomplete morphology,disordered arrangement,enlarged gaps,sparse cytoplasm,deep stained nuclei of neurons,pyknosis of the nuclei in triangular or irregular shapes,and inconspicuous nucleoli.With time after modeling,the disorder of neuron morphology in hippocampal CA1 area of VD rats increased,and the gap increased significantly.Compared with the model group,the overall tissue damage in the hippocampal CA1 area of the THC group and the SZJN group was significantly improved.The neurons were arranged more neatly,the cell morphology and structure were more complete,the cytoplasm was more uniform,and the boundary between the nucleus and the cytoplasm was clearer.A few nuclei are deeply stained,pyknotic,triangular or irregular in shape,and nucleoli are not obvious.In the MH group,the level of cells in the CA1 area of the hippocampus was reduced,the neurons were sparsely arranged,and the morphology was significantly improved compared with the model group.Some nuclei are deeply stained,pyknotic,triangular or irregular in shape.Nissl staining showed that the neurons in the CA1 area of the hippocampus in the sham-operated group were arranged neatly and compactly,and they appeared to be granular or clumpy after staining,and the staining was dense and regular.The neurons in the CA1 area of the hippocampus of VD rats were arranged in a disorderly manner.As time passed after the model was built,the cell bands were broken,the number of apoptosis increased,and a large number of nerve cells appeared,Nissl body fragmentation.The nerve cells in the hippocampal CA1 area of the THC group and the SZJN group were arranged neatly,the gaps between a few cells were widened,and the cell bands were slightly thinner.A small amount of the Nissl bodies of the nerve cells can be seen fragmented,and the Nissl bodies in the center of the cytoplasm disappeared.The morphology and arrangement of neurons in the CA1 area of the hippocampus in the MH group were improved compared with the model group.Some neurons were apoptotic and the cell bands became thinner.RT-PCR results?Compared with the sham operation group,the expression of clathrin mRNA in VD rats was reduced at 3 hours,1 day,3 days,7 days,and 14 days after modeling(P<0.01).Compared with the model group,the relative expression of clathrin mRNA in the hippocampus of rats in the MHG group(P<0.01),THC group(P<0.01),and SZJN group(P<0.01)increased.?Compared with the sham operation group,the expression of NMDAR1 mRNA in the model group increased at 3 hours,7 days,and 14 days after operation(P<0.01),and the expression of NMDAR1 mRNA decreased at 1 day after operation(P<0.01).Compared with the model group,the expression of NMDAR1 mRNA in the SZJN group decreased 3 hours after surgery(P<0.01).The expression of NMDAR1 mRNA in the MH group(P<0.01),THC group(P<0.01),and SZJN group(P<0.01)decreased 7 days and 14 days after operation.?Compared with the sham operation group,the expression of Rab5B mRNA in VD rats decreased at 3 hours,1 day,3 days,7 days,and 14 days after operation(P<0.01).Compared with the model group,3 hours,1 day,3 days,7 days,14 days after surgery,MH group(P<0.01),THC group(P<0.01),SZJN group(P<0.01)rat hippocampal Rab5B The relative expression of mRNA increased.(3)WB results?Compared with the sham operation group,the expression of clathrin protein in the model group decreased at 3 hours,1 day,3 days,7 days,and 14 days after operation(P<0.01).Compared with the model group,rats in the THC group increased the expression of clathrin protein at 3 hours(P<0.01)and 1 day(P<0.05)after surgery,while the expression of clathrin protein in the SZJN group increased at 3 hours(P<0.01),1 day(P<0.05),7 days(P<0.05)and 14 days(P<0.05)after surgery.the expression of clathrin protein in the MH group increased at 3 hours(P<0.01)after surgery.?Compared with the sham operation group,the expression of membrane protein NMDAR1 in the model group increased at 3 hours(P<0.01),7 days(P<0.01),and 14 days(P<0.01)after the operation,and the expression decreased at 1 day after the operation(P<0.01).Compared with the model group,the expression of NMDAR1 in the THC group decreased at 3 hours(P<0.05)and 7 days(P<0.01)after operation,and increased at 1 hour after operation(P<0.01).The expression of NMDAR1 in SZJN group and MH group decreased at 3 hours and 7 days after surgery(P<0.01)Conclusion:(1)Early application of Tongluo Huatan Capsule can reduce the pathological damage of hippocampal CA1 area of VD rats,improve learning and memory ability,and have brain protection.(2)After cerebral ischemia-reperfusion injury in VD rats,the dynamic evolution of hippocampal neuron NMDAR1 expression first increased,then decreased and then increased after modeling.The decreased expression of clathrin and Rab5B leads to the decrease of NMDAR endocytosis on the surface of neurons,which aggravates the toxicity of excitatory amino acids and reduces the ability of learning and memory.(3)Tongluo Huatan Capsule early intervention in VD rats can increase the endocytosis of NMDAR on the surface of neurons by up-regulating clathrin expression,or down-regulating the expression of NMDAR to reduce excitotoxic effects on neurons,thereby playing a protective role in the brain.