| Yiqi Huoxue therapy is a principle for the treatment of ischemic heart disease.Radix Astragali and Radix Salviae Miltiorrhizae are widely used as the representative of Yiqi Huoxue herbs in the treatment of clinical coronary heart disease.Astragaloside IV and Tanshinone II A are the main active ingredients of Astragalus and Salvia miltiorrhiza.Some studies have revealed that both of them have anti-oxidation,anti-inflammatory characteristics,improved microcirculation and protection of blood vessels.Our previous study showed that astragaloside and tanshinone II A can promote bone marrow mesenchymal stem cells to differentiate into endothelial cells,but the integrity and function of angiogenesis depends not only on normal vascular endothelial cells,also pericytes as an important part of the same kind of microvascular has its important role.The recruitment of pericytes by endothelial cells is a hallmark of microvascular maturation.The two interacts with each other to form a complete microvascular cavity.Pericytes and endothelial cells interact with each other through various ways to form nd maintain microvascular structure,and regulate the angiogenesis and vascular function through intercellular gap junctions.GJC is made up of intercellular Cx,allowing small molecules,ions,cell information and metabolites to pass through,enabling direct communication between adjacent cells,and plays an important role in tissue development,electrical coupling and maintaining homeostasis.Therefore,the aim of this study is to investigate whether astragaloside and tanshinone II A can promote the recruitment of pericytes to endothelial cells and promote angiogenesis,and whether their effects can be achieved by regulating pericytes Cx and GJC.It not only provides a theoretical basis for exploring the potential mechanism of Yiqi Huoxue herbs promoting angiogenesis,but also provides experimental evidence for exploring the potential targets of traditional Chinese medicine in promoting angiogenesis.Part one: Isolation,culture and identification of rat microvascular pericytes Objective:To investigate the isolation,culture and identification methods of pulmonary microvascular pericytes in the modified SD rats,and to detect the function of the pericytes obtained,so as to provide support for the next experiment.Method:1)Microvascular fragments were obtained by mechanical shear,type I collagenase digestion,microporous filtration and ultra high speed centrifugation.2)The high glucose medium(DMEM)containing 15% fetal bovine serum(FBS)was used for cell culture.3)The morphology and growth characteristics of the original PC were observed by inverted microscope.4)Detect the expression of pericyte surface marker neuron glia antigen 2(neuron-glial antigen 2,NG2),alpha smooth muscle actin(-smooth muscle alpha actin,alpha-SMA),desmin(desmin),platelet-derived growth factor receptor beta(PDGFR-beta)and CD31 related antigen.5)The growth curve of pericytes was measured by CCK-8.6)The function of pericytes was detected through co-culture of pericytes and endothelial ells,.Result:1)The improved method can obtain high purity microvascular pericytes,and the cells can be successively passaged.2)After 48 h,the pericytes were crawled out of the microvascular fragments,with irregular shapes such as long shuttle shape and triangle,which were mostly seen in the long spindle shape.A small number of endothelial cells were accompanied by the growth of the pericytes in the early stage.After 8-10 d,cells reached the request of passage,and the cells growth rapidly after the passage.3)The pericytes are mostly mononuclear,occasionally binuclear,and the nucleus is oval in the center.The cytoplasm is rich in cell.The cells were palisade or whirl like growth without contact inhibition.4)The cultured cells expressed the specific surface markers of pericytes,alpha-SMA,NG2,desmin,PDGFR-beta,and CD31 negative on the surface markers of endothelial cells.5)The pericytes were co-cultured with endothelial cells,which showed that the vascular buds and the structure of the vascular cavities were formed.Conclusion:The purified pericytes were successfully obtained by this improved method,which expressed pericytes specific markers,and possessed the characteristics and functions of mature pericytes.Part two: Regulation of Cx43 and GJC on angiogenesis by astragaloside,tanshinone II A and its combined groupObjective:To observe the effects of astragaloside IV,tanshinone II A and combination group on pericyte Cx43/GJC,and the effect of endothelial cells recruitment on pericytes and angiogenesis.The gap junction channel blocker Gap26 was further used to determine whether the regulation effect was realized by Cx43/ GJC.Method:1)Experimental grouping: P5 cells were used for experiments: blank control group,Astragaloside group(As group),Tanshinone IIA group(Ts group),combined group(As+ Ts group),Gap26 group Gap26+As group,Gap26+ Ts group,Gap26+ As+ Ts group.Astragaloside IV(0.4?g/ml),Tanshinone IIA(0.2?g/ml),and the combination group(As 0.4?g/ml+ Ts 0.2?g/ml)were given for 72h;Gap26 group was given Gap26(500?M)for 18 h.Gap26+ As group,Gap26+ Ts group and Gap26+ As+Ts group were pretreated with Gap26 for 18 hours,and then were given As,Ts,As+Ts to continue intervention for 72 hours.The working concentration of the drug was the same as before.The blank control group was given an equal volume of normal saline to intervene.2)Normal saline,astragaloside IV(0.4?g/ml),tanshinone IIA(0.2?g/ml),and compatibility group(As 0.4?g/ml+ Ts 0.2?g/ml)were used to intervene the pericytes respectively.The expression of Cx43 m RNA in PC was detected by Q-PCR method.The expression of Cx43 protein in PC was detected by Western Blot.The dyestuff scratch test was used to detect the intercellular gap junction communication function.Transwell was used to detect the migration of pericytes.PC and HUVEC were co cultured to determine the number of vascular sprouting and neovascular cavities.3)After Gap26(500?M)was used to intervene in PC for 18 h in Gap26 group,the dye scratching test was used to detect the function of GJIC,and Transwell was used to detect the migration ability of pericytes.In the double intervention group,based on the Gap26 intervention for 18 hours,the secondary interventions of As(0.4 ?g/ml),Ts(0.2 ?g/ml),and As+Ts(As 0.4 ?g/ml+Ts 0.2 ?g/ml)were continued for the next72 h.The expression of Cx43 m RNA and protein of pericytes were detected again.The PC and HUVEC co-culture tube experiments were performed again.Result:1)After the intervention of As,Ts and As+Ts,the expression level of Cx43 m RNA on pericytes was significantly higher than that in the control group(p<0.05),and the expression of Cx43 in As +Ts group,Ts group and As group decreased sequentially.The expression of Cx43 m RNA in group Gap26 was significantly lower than that in he control group(p<0.01).After Gap26 and As,Ts and As +Ts intervene pericytes,Cx43 m RNA expression in Gap26+As group,Gap26+Ts group and Gap26+As+Ts group was significantly lower than that in blank group(p<0.01),but increased compared with that in the Gap26 group.In group As+Ts,group Ts,and group As,the expression of Cx43 m RNA decreased in turn.2)After the intervention of As,Ts and As+Ts,the expression of Cx43 protein in the pericytes was up-regulated compared with the control group(p<0.01).The expression of Cx43 protein in group As,group Ts and As+Ts increased in turn.The expression of Cx43 protein in group Gap26 was lower than that in the control group(p<0.01).After Gap26 combined with As,Ts and As+Ts interference,the expression of Cx43 protein in pericytes was lower than that in the control group,which was higher than that in Gap26 group.The As+Ts group had the most significant up-regulation,followed by the Ts group,and the As group was lower than the Ts group.3)The transfer distance of the fluorescent dye was detected by the scratch test: the As+Ts group was 53.9 ± 6.1um,the Ts group was 48.4 ± 3.7um,the As group was 43.8± 2.2um,and the blank group was 28.4 ± 3.7um.Group Gap26 was 14.3 ±4.2um,and the control group was 21.7 ± 1.1um.It is suggested that the ga P junction channel blocker reduces the function of GJIC between adjacent cells.The combination grou P increased the function of the GJIC most,and the effect of tanshinone II A group and astragaloside group was weaker than that of the combination group,but there was no significant difference between the two groups.4)After the intervention of As,Ts and As+Ts groups,the migration ability of pericytes increased compared with the control group(p<0.01),of which the As+Ts group had the strongest effect and the blank group had the weakest;the gap channel blocker Gap26.After intervention,the migration ability of pericytes in the Gap26 group was significantly weaker than that in the blank group5)After intervention of As,Ts and As+Ts groups,the number of each component was significantly higher than that of the control group.There was no significant difference in the number of tube formation between the As+Ts group and the Ts group.The As roup was less than the former two groups.The Gap26 group had less control than the blank control group.6)After Gap26 combined with As,Ts,and As+Ts interfering pericytes,the number of new vascular buds and lumen like structures decreased compared with the control group,but increased compared to the Gap26 group.Conclusion:Astragaloside IV,tanshinone II A and combination group have the ability to promote pericyte migration,enhance the ability of endothelial cells to collect pericytes,and promote the formation of vascular cavity like structure.This effect may be related to astragaloside IV,tanshinone II A and combination group,which can enhance the expression of Cx43 protein on the pericytes and enhance the function of gap junctional intercellular communication. |
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