| Background:Stroke,also known as stroke in Chinese medicine,is the world's leading disability factor and the first fatal factor in my country.The incidence of ischemic stroke accounts for 86%of the total number of strokes.For diseases with such high morbidity and high mortality,the current internationally recognized treatment method is intravenous thrombolysis and/or intravascular treatment,but its treatment time window is only 3-4.5 hours,which limits the treatment of the disease.Studies have shown that the blood-brain barrier(BBB),as a barrier between brain tissue and blood,plays an important role in the treatment of ischemic stroke by maintaining the integrity of its structure and function.Shenfu Decoction,as a representative prescription for benefiting temperature and Yang Gutuo,was recorded in ancient China for the treatment of stroke with Shenfu Decoction.It is recorded in "Feng's Tips and Miscellaneous Diseases":stroke,hand-spreading,enuresis,ginseng It comes with soup.Current studies have shown that the active monomers in ginseng and aconite have a protective effect on BBB after cerebral ischemia,but whether Shenfu Decoction has a synergistic effect on the protection of BBB compared with ginseng and aconite single Chinese medicine,and whether it can be through fatty acids The Fatty acid oxidation(FAO)mechanism plays a role,and there is a certain amount of research space at present.Objective:In order to explore whether Shenfu Decoction has a synergistic effect on the protection of BBB after cerebral ischemia,this topic first verified the permeability of mouse BBB and the expression of tight junction protein through in vivo experiments;then,through in vitro experiments,an in vitro BBB model was constructed.,To further observe the effect of Shenfu Decoction-containing serum on BBB permeation after oxygen glucose deprivation and reoxygenation(OGD/R);finally,through in vivo experiments,the expression of the rate-limiting enzyme carnitine palm oil transferase IA(CPT1A)in the FAO process To study the mechanism of Shenfu Decoction.This topic is based on the basis of the clinical application of ancient prescriptions.Through experiments,it is explored whether the Yinwenyang prescription Shenfu Decoction has a synergistic effect on the protection of BBB after cerebral ischemia-reperfusion injury,and the specific mechanism of action.Method:(1)Establish a mouse middle cerebral artery occlusion ischemia-reperfusion[MCAO(I/R)]model and grouping:modeling is carried out with the aid of a microscope,after separating the mouse neck muscle,insert the thread plug from the external carotid artery,Enter the anterior cerebral artery through the internal carotid artery,block the blood supply from the anterior cerebral artery on the side of the embolization,and block the intracranial segment of the internal carotid artery that receives the blood supply of the posterior communicating artery.After 1 hour of ischemia,the thrombus is removed and reperfused.All mice were divided into 5 groups SHAM group,MCAO group,MCAO+red ginseng group(HS group),MCAO+aconite group(FZ group)and MCAO+participating group(SF group).(2)In vivo experiments to explore the effects of red ginseng and aconite single Chinese medicine on BBB permeability and tight junction protein after cerebral ischemia-reperfusion injury,as well as the compatibility of red ginseng and aconite with Shenfu Decoction and each single Whether the flavor medicines have synergistic effects:15 minutes after MCAO modeling,mice were given the corresponding drug gavage,that is,the HS group received single red ginseng gavage,FZ group received single aconite gavage,SF group Gave the stomach with Shenfu Decoction.Detect the expression of immunoglobulin(IgG),microtubule-associated protein 2(MAP2)and Claudin-5(Claudin-5)by immunofluorescence method;detect tight junction protein Claudin-5 and occlusion by Western blot(Western Blot,WB)Protein(Occludin)and closed small circle protein 1(ZO-1)expression(3)In vitro experiments further confirm the effect of Shenfu Decoction on BBB permeability:use brain microvascular endothelial cells(bEnd.3)to establish an in vitro BBB monolayer culture model.With the medicated serum of Shenfu Decoction extracted from the animal and sterilized,the model was pretreated on the third day of modeling,and the completed BBB model was placed in a hypoxic chamber for 1 hour of oxygen and glucose deprivation(OGD),after 2 hours of reoxygenation treatment to simulate cerebral ischemia reperfusion injury,during reoxygenation,use Shenfu Decoction medicated serum to intervene the injured in vitro BBB model.Fluorescein isothiocyanate-labeled dextran(FITC-dextran)was used as a fluorescent probe to detect the permeability of BBB.(4)Study the role of FAO mechanism in the protection of BBB after ischemic stroke by Shenfu Decoction:The specific modeling,grouping and administration methods are the same as in vivo experiments,and the rate-controlling enzymes of FAO are detected by immunofluorescence and western blotting techniques The expression of CPT1A was analyzed by the expression of CPT1A to analyze the expression difference between the administration group and the model group,and between the different administration groups.Judge whether Shenfu Decoction has a synergistic effect in the process of FAO.Result:(1)Establish mouse MCAO(I/R)model and grouping:After modeling,use laser speckle imaging technology and Longa scoring method to evaluate the success of modeling,and finally all MCAO models included in the experimental group meet the standards,which proves that the animals Modeling was successful.They were divided into five groups:SHAM group,MCAO group,MCAO+red ginseng group(HS group),MCAO+aconite group(FZ group),and MCAO+participating group(SF group),with 10 animals in each group.(2)In vivo experiments to explore the effects of red ginseng and aconite single Chinese medicine on BBB permeability and tight junction protein after cerebral ischemia-reperfusion injury,as well as the synergistic effect of Shenfu Decoction:Compared with the SHAM group,the rest The four groups all had obvious IgG expression,indicating that BBB permeability increased after modeling.Compared with the MCAO group,the IgG penetration of the administration group(HS group,FZ group and SF group)was significantly reduced(P<0.01),and the difference was statistically significant.However,as far as the current data is concerned,it is impossible to determine whether the BBB protective effect of Shenfu Decoction after cerebral ischemia is better than that of red ginseng or aconite alone,and further testing is needed.The following further analysis of the differences in the fluorescence expression of the tight junction protein Claudin-5,the results show that the expression level of Claudin-5 in all the groups modeled by MCAO(I/R)is lower than that of the SHAM group,indicating that BBB appears after modelling.Damage.Compared with the MCAO group,the expression levels of Claudin-5 in all the administration groups(HS group,FZ group and SF group)were greatly increased(P<0.01),and the difference was statistically significant.Further analysis of the differences between the SF group,HS group and FZ group showed that the expression of tight junction protein Claudin-5 with Shenfu Decoction was higher than that of red ginseng and Aconite alone(P<0.01),and the difference was statistically significant.The expression of compact proteins Occludin,ZO-1 and Claudin-5 was further detected by WB technology.The experimental results showed that the expression of Occludin in the HS group and the FZ group was not statistically different from that in the MCAO group,but the expression of Occludin in the SF group was significantly higher than that in the MCAO group,the effect was prominent(P<0.01),and the difference was statistically significant.In the expression of Claudin-5,the difference between HS group and FZ group and MCAO model group was not statistically significant,but the expression of SF group was stronger than that of MCAO group(P<0.05),and the difference was statistically significant.The expression of ZO-1 in the SF group was significantly higher than that in the MCAO group(P<0.01),and the difference was statistically significant.The expression of Occludin and Claudin-5 is the same,and there is no significant difference between the HS group and the FZ group and the MCAO group.(3)In vitro experiments further confirmed the influence of Shenfu Decoction on the permeability of BBB:The experiment found that the transmembrane resistance of BBB increased significantly on the third day of modeling,and reached the highest peak on day 5-6 of modeling.,Indicating that the modeling is successful.When the resistance reached the peak value,a further 4h leakage experiment found that the liquid level inside and outside the chamber dropped less than 0.1 mm after 4h,which further confirmed the establishment of the in vitro BBB model.Further detection of the leakage of FITC-dextran in the acute phase,the results showed that the intervention of Shenfu Decoction-containing serum reduced the BBB damage caused by OGD/R,suggesting the BBB protective effect of Shenfu Decoction.Further demonstration found that the FITC-dextran penetration rate of each group of Shenfutang serum intervention was lower than that of the OGD model group(P<0.05),and the difference was statistically significant.Among them,the 20%high-dose Shenfutang serum group The effect was the most significant(P<0.01),and the statistical difference was significant.Further analysis of the difference between different concentrations of Shenfu Decoction serum group,found that there is no significant difference between 5%Shenfu Decoction serum group and 10%Shenfu Decoction serum group and 20%Shenfu Decoction serum group,and the difference is not statistically significant.However,the penetration rate of 20%Shenfu Decoction serum group was lower than that of 5%Shenfu Decoction serum group and 10%Shenfu Decoction serum group(P<0.05),and the difference was statistically significant.It is judged that the 20%concentration of Shenfu Decoction has a stronger protective effect on OGD treatment in vitro BBB.However,in view of the toxic and side effects of Fuzi,the relationship between the concentration of Shenfu Decoction-containing serum and the degree of BBB protection needs further discussion.(4)Study the role of FAO mechanism in Shenfu Decoction in protecting BBB after ischemic stroke:immunofluorescence detection results showed that the expression of CPT1A in HS group,FZ group and SF group was higher than that in MCAO group(P<0.01)The difference is statistically significant.To further demonstrate whether there is a difference between the administration groups,the HS group and the FZ group were compared with the SF group,and it was found that the expression of CPT1A in the SF group was significantly higher than that of the other two groups(P<0.01).It is statistically significant.It is proved that in the process of FAO,the effect of red participating in the synergistic effect of aconite is better than its effect alone.WB results show that after the MCAO model.the level of FAO of vascular endothelial cells is reduced.Except for the SHAM group,The expression of CPT1A in each group was reduced.However,compared with the MCAO group,the expression of CPT1A in the HS group,FZ group and SF group treated with administration increased(P<0.05),and the difference was statistically significant.Conclusion:1.The study found that Shenfu Decoction has a protective effect on BBB after cerebral ischemia-reperfusion injury.Shenfu Decoction has a synergistic effect compared with each single medicine in the prescription.2.The research results of the preliminary mechanism of action showed that the SF group can increase the expression of FAO rate-limiting enzyme CPT1A,and has a synergistic effect compared with each single medicine,indicating that the protection of Shenfu Decoction on BBB may be through the FAO mechanism,but The specific upstream and downstream pathways need to be further explored. |
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