Improvement Effect Of Panax Notoginseng Saponins On Ischemia-induced Endothelial Tight Junction Injury And Vascular Dysfunction

Vascular injury is the early injury during the development of ischemic cardiovascular and cerebrovascular diseases,an important risk factor in the threat to human health.Under pathological conditions,oxidative stress,inflammation and lipid metabolism,promote endothelial cell adhesion molecules increased cell gap injury,the barrier function damage,tight junctions open,lipid deposition,and edema;at the same time,endothelium-derived vasodilation factors such as the reduction of NO synthesis,biological activity decreased,causing vasodilation dysfunction,promote the formation of atherosclerosis,leading to cardiovascular and cerebrovascular diseases.Early prevention,diagnosis and treatment of vascular injury,containment has become the heart and cerebrovascular diseases developing forward-looking strategic approach.Panax notoginseng saponins(PNS)is the main active ingredient of traditional Sanqi promoting blood circulation,for the clinical prevention and treatment of stroke,coronary heart disease,angina,hypertension and other cardiovascular and cerebrovascular diseases.Previous studies have focused on the effect of PNS injection on brain and heart ischemia,vascular injury in rat evaluation also limite to the detection of apparent indicators.From the perspective of prevention and treatment of vascular injury,on the basis of evaluating the protective effect of PNS orally administered early treatment on ischemic induced cardiovascular and cerebral vascular damage,exploring the protective effect of PNS on ischemia-induced vascular endothelial cell tight junction injury and the damage to SHRSP.vasodilation dysfunction in SHRSP.ZF rats,provide a theoretical basis for clinical therapeutics.Part 1 PNS protected ischemia induced cerebralvascular and cardiovascular injury.Pre-clinical and experimental studies show that PNS has positive effect on heart and brain injury,but previous studies mainly demonstrated the positive protection via intraperitoneal injection/intravenous injection administration.In addition,research methods are mostly confined to the traditional means of molecular biology,a little lack of research expertise,intuitive sex.In this study,using the classical model of acute myocardial ischemia,acute cerebral ischemia model,the effect of PNS ultra-early treatment oral administration on ischemia induced cerebrovascular injury was evaluated;and with the microcirculation fluorescence microscope observation system dynamic visualization,investigated the improvement of PNS on cerebral ischemia/reperfusion induced microvascular leukocyte adhesion and albumin exudation;with a laser Doppler flowmeter,investigated improvement of PNS on ischemia induced cerebral blood flow decrease.Objective:To investigate whether PNS oral administration early treatment can protect acute cardiovascular and cerebrovascular ischemia and investigate whether PNS can protect cerebral ischemia-reperfusion induced microcirculation disorders and increase cerebral blood flow.Methods:Acute myocardial ischemia model:Wistar rats were randomly divided into 6 groups:the normal group,model group,Xuefuzhuyu group 480 mg/kg group,PNS 60,30,15 mg/kg group,surgery gastric administration 3d,left anterior descending coronary artery ligation copy myocardial ischemia,administrate again at 4h after operation,administered once every 12h,detect myocardial infarction,abnormal electrocardiogram,cardiac enzymes.Acute cerebral ischemia and reperfusion model:ICR mice were randomly divided into normal group,model group,Yinxingye Tablet 238.5 mg/kg group,PNS 90,45,22.5 mg/kg group.Preoperative prevention gavage 3d,2 times/d,suture method middle cerebral artery occlusion caused by cerebral ischemia,after ischemia 1.5h conducte reperfusion surgery,administrate again at 4h after operation,administered once every 12h,detect neurological score,pathological structure;using fluorescence microscopy microcirculation,leukocyte adhesion and albumin exudation were observed after 72h.Acute cerebral ischemia model:ICR mice were randomly divided into six groups,namely normal group,model group,Yinxingye Tablet 180 mg/kg group,PNS 90,45,22.5mg/kg group,preoperative oral administration 3d,the right common carotid artery ligation copy cerebral blood flow reduced model,using laser Doppler flowmetry recording before unilateral carotid artery ligation 5min and 30min after brain blood flow changes.Result:1.After ligation of the left anterior descending coronary artery,in model group,myocardial infarction,the positive rate of abnormal electrocardiogram significantly increased,serum LDH,CK,CK-MB,?-HBDH and AST were significantly increased(P<0.01).PNS 60,30,15 mg/kg significantly reduced myocardial infarction(P<0.01,P<0.05);PNS 60 mg/kg significantly inhibited abnormal electrocardiogram positive rate(P<0.01);PNS 60,30,15 mg/kg significantly decreased serum LDH,CK and CK-MB content(P<0.05,0.01).2.72 h after cerebral ischemia and reperfusion,the model group showed significantly infarction,HE staining showed ipsilateral cortex cells lose hierarchy arrangement,nerve cell swelling,degeneration,atrophy,penumbra site vacuoles;neurons lost significantly,significantly reduced the number of cells;and expanding the lumen of the blood vessel congestion.PNS 90,45mg/kg significantly reduced the infarct size,reduce the penumbra parts of the cell degeneration and loss,showed obvious proliferation of glial cells,vascular congestion occasionally expands the lumen.3.72 h after cerebral ischemia and reperfusion,in the model group,leukocyte adhesion and albumin leakage rate significantly increased(P<0.01).PNS 90,45 mg/kg significantly reduced the number of leukocytes adhesion;PNS 45 mg/kg group significantly decreased albumin leakage rate.4.After carotid artery ligation unilateral cerebral ischemia,in the model group,ipsilateral cortical blood flow decreased significantly(P<0.01).PNS 90,45,22.5 mg/kg significantly increased blood flow during 10,20,30min after ligation.Conclusion:PNS early oral treatment can protect acute cardiovascular and cerebrovascular ischemia induced injury in rats,and improve cerebral microcirculation disorders and increase cerebral blood flow.Part 2 PNS ameliorated vascular diastolic dysfunction in SHRSP.ZF rat.Vascular dysfunction is the initiating factor and carrier of "ET-blood pressure-cardiovascular events" chain.Under pathological conditions,endothelium-derived NO synthesis decreased vasodilation occurring disorders,abnormal blood pressure,accelerate the development of cardiovascular diseases.Using unilateral carotid artery ligation total cerebral ischemia mouse model,previous study demonstrated that PNS can significantly promote blood flow in ischemic ipsilateral cortex,suggesting that PNS improve vasomotor dysfunction and restore blood cycle,thereby protecting cardiovascular injury in part.SHRSP.ZF rats from the young period show a typical characteristic of the metabolic syndrome,the vasodilation in thoracic aorta and superior mesenteric artery were proved to be damaged.In this study,based on NO signal pathway,the positive effects of PNS Rgl,Rbl,Rd on vascular diastolic dysfunction were investegated with SHRSP.ZF rat thoracic aorta and superior mesenteric artery.Objective:To investigate the effects of PNS and its component Rg1,Rb1,Rd on ACh and SNP induced endothelium-dependent and endothelium-independent vasodilation disorders in thoracic aorta and mesenteric artery from SHRSP.ZF rat.Methods:Using thoracic aorta and mesenteric artery in 20-week-old WKY,SHRSP.ZF rats,observe the effect of PNS on ACh,SNP-mediated vasodilation,and examine the role of L-NAME and ODQ.In addition,we observed the effect of Rg1,Rb1 and Rd on SNP-mediated vasodilation in thoracic aorta from SHRSP.ZF rat.Using 7-week-old SHRSP.ZF rats,orally administration for 13w,observe the blood pressure,total cholesterol,triglycerides,insulin,glucose,TBARS and SOD levels in serum,detected ACh,SNP mediated thoracic artery and mesenteric artery vasodilatation and detecting the eNOS and sGC expression in thoracic aorta.Result:1.In vitro study:Compared with the normal group,ACh,SNP induced vasodilation in mesenteric artery and thoracic aorta from the model rats decreased significantly(P<0.01).PNS 30,90 mg/L group can significantly promote the SNP-mediated vasodilation in the thoracic aorta vasodilation in SHRSP.ZF rats(P<0.05),PNS 90 mg/L group can significantly promote the SNP-mediated vasodilation in the mesenteric artery in SHRSP.ZF rats(P<0.05),no significant effect on ACh-mediated thoracic aorta and mesenteric artery vasodilation in SHRSP.ZF rats.PNS had no effect on ACh and SNP-mediated thoracic aorta and mesenteric artery vasodilation in normal rats.Rb1 30.42 mg/L group significantly promoted SNP mediated vasodilation in thoracic aorta from SHRSP.ZF rats,Rg1 27.99 mg/L group,Rd 6.3 mg/L group,had no effect on the SNP-mediated vasodilation from SHRSP.ZF rats.In L-NAME intervention,PNS significantly promote SNP induced vasodilation in thoracic aorta(P<0.05);In ODQ intervention,PNS promoted SNP mediated vasodilation was completely inhibited.2.In vivo study:PNS 30 mg/kg orally administration 13w,can significantly reduce blood pressure in SHRSP.ZF model;ACh-mediated vasodilation in thoracic aorta was significantly increased,and completely abolished by L-NAME;SNP-mediated mediated vasodilation in mesenteric artery was significantly increased,and completely blocked by ODQ;protein expression of sGC in the thoracic aorta was increased,no changes of eNOS protein expression.Conclusion:PNS have a protective effect on thoracic aorta and mesenteric artery vasodilation dysfunction in SHRSP.ZF rats,associated with the NO signaling pathway;Rbl is one of the effective monomer components.Part 3 PNS ameliorated ischemia induced tight junction injuryBased on the positive effects of PNS on ischemia induced cerebrovascular injury,we further clarified the improvement of vascular diastolic dysfunction and the activation on NO signal pathway.However,NO not only through the downstream sGC/cGMP regulates vascular diastolic dysfunction,the upstream target VEGF/VEGFR2/PI3K/Akt activation involved in the regulation of vascular structures damage through mediating the apoptosis of endothelial cells,endothelial cell survival,angiogenesis and others.When cerebral ischemia-reperfusion occurs,brain capillary barrier function serious damaged,endothelial cell tight junction protein ZO-1,Claudin-5 and Occludin decreased significantly,such macromolecules penetrate into the brain tissue,causing brain edema;at the same time,the brain edema,microglial activation,proliferation of astrocytes and nerve inflammation after injury further promote the degradationof brain microvascular endothelium tight junction proteins,resulting in substantial cerebrovascular disease.Objective:To study the protective effect of PNS and its components Rg1,Rbl on ischemic induced tight junction injury and MMPs/VEGF signaling pathway.Methods:Anaerobic box were used to induce OGD 6h on HUVEC cells,after 24h reoxygenation,TEER and cell-permeablewere detected,immunofluorescence observe ZO-1,Claudin-5 protein expression;using acute cerebral ischemia and reperfusion model mice,detect ipsilateral cortex tight junction protein ZO-1/Claudin-5/Occludin and MMP-9,MMP-12 and VEGF,VEGFR2,Src,p85-PI3K,p110-PI3K,p-akt(ser 473),p-akt(thr 308),akt protein expression after 72h.Result:1.In vitro study:PNS 20,40 mg/L and ginsenoside Rbl can significantly inhibit the OGD/Reoxigen induced tight connection resistance decreases,inhibiting cell permeability increase(P<0.05).PNS 20,40 mg/L and ginsenoside Rbl can partly restored tight junctions between cells arranged around the cell membrane protein,cobblestone-like structure.2.In vivo study:PNS 45 mg/kg can significantly reduce the ultrastructure of model mice ipsilateral cortex lesions,showed perivascular edema,structure tightly packed endothelial cell tight connection is not fully open,the base film mildly wide,the base film structural integrity clear boundary.PNS 45 mg/kg significantly increased cortical ZO-1,Claudin-5 and Occludin expression in model mice and reduced MMP-9 levels,increased VEGF,VEGFR2,p85-PI3K,p-Akt(Ser473)protein expression levels(P<0.05).Conclusion:PNS ameliorate ischemia-induced vascular endothelial cell tight junction injury via MMP-9 and VEGF/VEGFR2 signaling pathway.Rbl is one of the effective monomer components.