| Part 1 Replication of qi stagnation and blood stasis syndrome,the qi deficiency and blood stasis syndrome model.Objective:The rats with qi stagnation and blood stasis syndrome and qi deficiency andblood stasis syndrome were established by different time-interventions of sleep deprivation.The effects of Liqi Huoxue recipe(LQHX)and Yiqi Huoxue Recipe(YQHX)on them were established.Method:Rats were randomly divided into eight groups according to body weight:A.Normal group:normal diet,drinking water,feeding for 3 weeks;B.Qi stagnation and blood stasis group:daily sleep deprivation 16h,continuous sleep deprivation for 3 weeks;C.Qi and blood circulation high dose Group:sleep deprivation for 16h per day,continuous sleep deprivation for 3 weeks,daily administration of 5g·kg-1 per day from the second week for 2 weeks;D.Qi and blood circulation low dose group:daily sleep deprivation for 16 hours,continuous sleep deprivation 3weeks,2.5g·kg-1 was administered every day from the 2nd week for 2 weeks;E.Control group:normal diet,drinking water,feeding for 6 weeks;F.Qi deficiency and blood stasis group:sleep deprivation for 16 hours per day,Continuous sleep deprivation for 6 weeks;G.Yiqi Huoxue high-dose group:sleep deprivation for 16h every day,continuous sleep deprivation for 6 weeks,daily administration of 5.54 g·kg-1 for 2 weeks from the 5th week;H.Yiqihuoxue low-dose group:Sleep deprivation for 16 hours per day,continuous sleep deprivation for 6 weeks,and daily administration of 2.77 g·kg-1 from the 5th week for 2 weeks.After 3 weeks and 6 weeks of sleep deprivation,the general state of the rats was observed,and the body weight,grip strength,and The pulse amplitude and pain threshold were used to evaluate the qi stagnation/qi deficiency index;the R,G,and B values of the rat tongue images,blood rheology,coagulation,and echocardiography were used to evaluate the blood stasis index.Results:(1)Status observation:The normal group and the control group had good mental state and activity,and the hair was white and shiny;the rats with qi stagnation and blood stasis showed irritability,irritability,mutual biting,reduced diet and drinking,etc.Mild;qi deficiency and blood stasis group rats with apathetic,drowsiness,reduced autonomic activity,like to gather together,lethargy,slow response to external stimuli,rough hair,dull and yellow,loss of luster,body weight loss.The mental state,activity,and coat color of the rats in the high and low dose groups of LQHX and YQHX were improved.(2)Qi stagnation/qi deficiency index:The qi stagnation and blood stasis group was significantly lower than the normal group weight and pain threshold(P<0.01),pulse amplitude and grip strength were significantly increased(P<0.01);LQHX high and low dose groups were compared.The pulse amplitude and grip strength of the qi stagnation and blood stasis group were significantly decreased(P<0.01),and the pain threshold was significantly increased(P<0.01).Compared with the control group,the weight,pulse amplitude and grip strength of the Qi deficiency and blood stasis group were significantly lower(P<0.01),and the pain threshold was significantly increased(P<0.01).The YQHX high and low dose groups were more frequent than the Qi deficiency and blood stasis group.The force was significantly increased(P<0.01),and the pain threshold was significantly lower(P<0.01).(3)Blood stasis index:R,G,B values of the tongue surface:Compared with the normal group,the qi stagnation and blood stasis group had lower R,G,and B values in the color analysis of the tongue image than the control group(P<0.01),LQHX,YQHXF high,low dose compared with qi stagnation and blood stasis group,qi deficiency and blood stasis group R,G,B values were significantly increased(P<0.05-0.01).Ultrasound:Compared with the normal group,the left ventricular end-diastolic volume(Volume;d)and stroke volumewere significantly lower in the qi stagnation and blood stasis group(P<0.01).Compared with the normal control group,the qi deficiency and blood stasis group had significantly lower left ventricular end diastolic diameter(Diameter;d),left ventricular end diastolic volume(Volume;d),stroke volume,and cardiac output(P<0.05～0.01).The YQHX high-dose group significantly increased Stroke Volume,Ejection Fraction,and Cardiac Output(P<0.05-0.01)compared with Qi deficiency and blood stasis syndrome.Hemorheology:Compared with the normal group,the qi stagnation and blood stasis group showed a significant increase in whole blood viscosity(high cut,medium cut)compared with the control group(P<0.01).In the high and low dose groups of LQHX,the whole blood viscosity(high cut,middle cut)was significantly lower than that in the qi stagnation and blood stasis group(P<0.01).The high blood pressure of the high and low dose groups of YQHX was higher than that of the qi deficiency and blood stasis group(high cut,Medium cut,low cut),plasma viscosity were significantly decreased(P<0.05～P<0.01).Coagulation four:Compared with the normal group,the APTT in the Qi Deficiency and Blood Stasis group was significantly lower(P<0.05).The APTT in the high dose group of YQHX was significantly higher than that in the Qi Deficiency and Blood Stasis group(P<0.05).YQHX PT,TT and FIB were significantly lower in the high and low dose groups than in the qi deficiency and blood stasis group(P<0.05～0.01).Conclusion:Rats with qi stagnation and blood stasis syndrome were established by sleep deprivation for 3 weeks,and rats with qi stagnation and blood stasis syndrome were established for 6 weeks.The method is stable and feasible,and the establishment of this model is in line with the change process of TCM syndrome from real to virtual,which is in line with the pathogenic characteristics of modern people.Part2Qi stagnation and blood stasis syndrome,qi deficiency and blood stasis syndrome vascular endothelial homeostasisObjective:To investigate the changes of vascular endothelial homeostasis in rats with qi stagnation and blood stasis syndrome and Qi deficiency and blood stasis syndrome,and the effects of Liqi Huoxue Granules(LQHX)and Yiqi Huoxue Granules(YQHX)on them.Methods:Grouping,modeling and administration were the same as in the first part.The content of vascular endothelial cell function related indicators was detected by enzyme-linked immunosorbent assay(Elisa)and nitrate reductase method according to the kit instructions.Western blotting was performed by Western Blot.Vascular endothelial cadherin(VE-cadherin)and phosphorylated vascula r endothelial cadherin(P-VE-cadherin,Y731)were detected.The morphology of vascular tissue was observed by hematoxylin-eosin(HE)staining and scanning electron microscopy.Results:(1)Endothelial cell related functional indicators:vasoactive substance detection:qi stagnation and blood stasis group compared with normal group,qi deficiency and blood stasis group compared with control group ET-1 significantly increased(P<0.05);LQHX high dose group compared.with qi stagnation NO in the blood stasis group was significantly increased(P<0.01),and the high dose group of LQHX and YQHX was significantly lower than that of qi stagnation and blood stasis group and qi deficiency and blood stasis group(P<0.05).vascular inflammatory factor detection:qi stagnation and blood stasis group compared with the normal group,qi deficiency and blood stasis group compared with the control group vWF,IL-6,TNF-α significantly increased(P<0.05～0.01);LQHX high dose group compared with qi stagnation IL-6 and TNF-α in the blood stasis group were significantly lower(P<0.05～0.01),and IL-6 in the high dose group of YQHX was significantly lower than that in the qi deficiency and blood stasis group(P<0.01).vascular adhesion factor detection:qi stagnation and blood stasis group compared with normal group,qi deficiency and blood stasis group compared with the control group VCAM-1,P selectin significantly increased(P<0.05-0.01),LQHX,YQHX high,low dose group qi VCAM-1 and P-selectin were significantly lower in the stagnation of blood stasis group and qi deficiency and blood stasis group(P<0.05～0.01).(2)Western Blot experiment:the expression of P-VE-cadherin in the Qi deficiency and blood stasis group was significantly higher than that in the control group(P<0.01),and the YQHX high dose group was significantly lower than the Qi deficiency and blood stasis group(P<0.05).(3)Vascular HE staining:The intima of the thoracic aorta in the normal group and the control group was normal,the surface was smooth,the endothelial cells were intact without defects,shedding and cell adhesion;the qi stagnation and blood stasis group and the qi deficiency and blood stasis group were large.The intimal structure of the rat artery was disordered,the endothelium was bulged,and the defect was detached.The structure of the thoracic aorta and the integrity of the endothelial cells in the high and low dose groups of LQHX and YQHX were improved to different degrees compared with the qi stagnation and blood stasis group and the qi deficiency and blood stasis group.(4)Vascular scanning electron microscopy:the normal group and the control group had intact endothelial structure,smooth surface,neatly arranged,no cell adhesion and migration;the dysfunction of the qi stagnation and blood stasis group was abnormal,and the disorder was different in size and cell gap.Widening;the vascular endothelium surface of the qi deficiency and blood stasis group was rough and structurally disordered,and scattered platelets,white blood cells and cell debris were observed in the periphery.The LQHX and YQHX high and low dose groups showed some improvement compared with the model group.Conclusion:vascular dysregulation,endothelial dysfunction,permeability change and blood abnormality are the main pathological links of qi and blood loss,qi stagnation and blood stasis syndrome,qi deficiency and blood stasis syndrome.LQHX particles and YQHX particles play a role in regulating blood circulation and blood by regulating blood vessel endothelial function and blood biological characteristics.Part 3 The effect of glucose deprivation on endothelial cell homeostasis and energymetabolism and the protective effect of astragaloside IV and sedativeObjective:To investigate the effects of glucose oxidative deprivation on endothelial homeostasis and energy metabolism in human umbilical vein endothelial cells and the protective effects of astragaloside Ⅳ and saponin Ⅰ.Methods:(1)Toxicity and protective effect of CCK-8 on model cells:The experiment was divided into five groups:normal group,model group,and astragaloside(3.125,6.25,12.5,25,50,100,200 μmol/L).,Yangchuan azlactone group Ⅰ(3.125,6.25,12.5,25,50,100,200μmol/L),astragaloside saponin Ⅰ complex group(0.78 + 6.25,1.56 + 12.5,3.125 + 25,6.25 + 50,12.5+100,25+200,50+400μmol/L).The normal group was treated with complete medium and normal culture;the model group,the astragaloside group,the scutellarin group Ⅰ,the astragaloside saponin Ⅰ complex group Ⅰ used DMEM sugar-free medium,hypoxia for 4h,added glucose,complex Oxygen 2h,CCK-8 test drug toxicity and protective effect on model cells,and select appropriate drug concentration.(2)Western blot was used to detect the content of VE-cadherin and P-VE-cadherin protein.The total protein was extracted after modeling and administration.The content of VE-cadherin and P-VE-cadherin was detected by western blot.(3)The volume of endothelial cells was observed by Rembrand’s staining:staining was performed with Wright’s-Giemsa staining solution after modeling and administration.Dry and take a photo under the microscope.(4)Energy metabolism study:After the modeling and administration,the seahorse cell energy metabolism analysis system was used to detect the glycolysis stress test.Results:(1)The toxicity and protective effect of CCK-8 on the model cells:the absorbance of the model group was significantly lower than that of the normal group,the cell viability was decreased(P<0.01),the astragaloside group(6.25μmol/L),and the saponin Ⅰ Compared with the model group,the absorbance values of the group(50Oμmol/L,100μmol/L)and the astragaloside Ⅰ complex group(3.125+25μmol/L)were significantly increased,which had the effect of protecting cell viability.The difference was statistically significant.(P<0.01).(2)VE-cadherin,P-VE-cadherin protein expression:The expression of VE-cadherin protein in the model group was not statistically significant compared with the normal group.The P-VE-cadherin(T731)protein expression relative value model group was significantly higher than the normal group(P<0.01),and the astragaloside IV group and the astragaloside Ⅳand the lansinide Ⅰ complex group were significantly lower than the model group(P<0.05).(3)The results of Ruiji’s staining showed that the normal group had good extensibility and adhesion.After the model of sugar oxygen deprivation,the model group can cause the cell body to become rounded,the adhesion ability is weakened,the cell membrane wrinkles,the cell shrinks,and the volume is significantly reduced.Compared with the model group,the cells of the astragaloside group,the yamone azide lactone group Ⅰ,and the astragaloside sulphate groupⅠ increased the cell extensibility and adhesion,and the cells recovered to normal morphology and volume.(4)Glycolysis stress test:After modeling,the model group had significantly reduced glycolytic ability and glycolysis reserve capacity(P<0.05-0.01).C ompared with the model group,the astragaloside group could significantly increase the glycolysis storage capacity(P<0.01).The combination of the scutellarin group Ⅰ and the astragaloside saponin Ⅰ complex group could significantly increase the self-glycolytic ability and sugar.The ability of glycolysis to reserve(P<0.05～0.01).Conclusion:After glucose and oxygen deprivation,it can affect the indexes of endothelial cell homeostasis and energy metabolism.Astragaloside Ⅳ and Yangchuan ligustilide Ⅰ have protective effects. |
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