The Mechanism Of Astragaloside Inducing Bone Marrow Mesenchymal Stem Cells Differentiating Into Pericytes

The pathological features of ischemic heart disease is vascular obstruction and stenosis,which is harmful to our healthe.It has been proposed that rapid establishment of effective circulation is important for the ischemic heart and to reduce the incidence of mortality.Therapeutic angiogenesis has become the direction of the treatment for the ischemic heart disease currently.Pericytes,residing at the interface between the endothelium and the surrounding tissue,which can regulate the proliferation and apoptosis of endothelial cells,readjust the local hemodynamics and stabilize the vascular structure,play an important role in angiogenesis and maturation.NG2,?-SMA are important surface markers,which are used in the identification of pericytes.In the process of angiogenesis,mesenchymal stem cells,actig as the precursors of the pericytes,which is recruited by the endothelial cells,are essential for the remodeling of blood vessels.TGF-?₁plays an important role in the differentiation of mesenchymal stem cells into pericytes.In the area of traditional medicine,ischemic heart disease has been equal to obstruction of qi in the chest.Blood stasis due to qi deficiency is the main syndromes of thoracic obsrtuction.Based on the thought of“treating disease from the root”and“deficiency of genuine qi and excess of pathogenic factor”,yiqi method is particularly important for treating ischemic heart disease.Other treatments such as enriching qi and activating blood,eliminating phlegm and freeing channels are also supplemented by yiqi drugs.Salvia miltiorriza,as one of the major qi-tonics,is widely applied for the treament of ischemic heart disease.Astragaloside is the main effective components of salvia miltiorrhiza,which is frequently used as a substitute for astragalus in the basic research.It has been proved that astragaloside can stimulate angiogenesis via activation of the vascular endothelial growth factor?VEGF?and hypoxia inducible factor-1??HIF-1??in vitro.However,the role of astragaloside on pericytes is not clear.In this study,we use the bone marrow mesenchymal stem cells?BMSCs?of rats as the precursors of the pericytes to explore the fuction of astragaloside,in the process of precursors differentiating into pericytes.It may help us for a better understanding on the role of qi-tonics in the treatment of ischemic heart disease.Part 1 The promotion of Astragaloside on bone marrow mesenchymal stem cells differentiating into pericytes1.ObjectiveObserve the effect of astragaloside on the differentiation of BMSCs into pericytes.2.Method?1?The MSCs were isolated from bone marrow of SD rats?5d-7d?by whole bone marrow primary culture method.?2?The primary mesenchymal stem cells were purified by amplification in vitro.?3?The cell growth were observed by inverted microscope.?4?Flow cytometry was used to detect the expression of surface antigens CD29,CD44,CD45,and CD90 of the third generation cell.?5?The P₃-P₅ generation were divided into three groups:blank control group,incubate with normal saline 2?L intervention for 3 day;positive control group?TGF-?₁group?,cultured with TGF-?₁ 10 ng for 3 day,the concentration of which was adjusted to 5ng/mL;Ast group,cultured with astragaloside 8?g for 1 day,2 day,3 day,the density of which was adjusted to 4?g/mL,while the samples were marked as Ast 1d group,Ast 2d group and Ast 3d group.?6?Expression levels of NG2,?-SMA were examined by immunocytochemistry,which were the specific marker for the authentication of pericytes.?7?Expression levels of NG2,?-SMA were examined by western blot,which were the specific marker for the authentication of pericytes.?8?Expression levels of NG2,?-SMA were examined by RT-PCR,which were the specific marker for the authentication of pericytes.3.Result?1?The MSCs were in fusiform shape,arranged in bundles or whorls,under the inverted microscope.?2?The expression of surface antigens CD29 and CD90 were positive,while the CD34 and CD45 were negative on the other hand.?3?Immunofluorescence assay showed NG2,?-SMA positive in the treated cells,in the mean time,the blank control group showed negetive.?4?The protein expression level of NG2,?-SMA were higher in all Ast group,after the stimulation of astragaloside.The protein expression level of NG2,?-SMA were higher in Ast 3d group,compared with the negative control group and the positive control group?p<0.05?.?5?The mRNA expression level of NG2,?-SMA were higher in all Ast group after the stimulation of astragaloside.The mRNA expression level of NG2,?-SMA were higher in Ast 3d group,compared with the negative control group and the positive control group?p<0.05?.4.Conclusion?1?The extracted cells from the rats were in fusiform shape,arranged in whorls under the inverted microscope and they expressed mesenchymal stem cell-related antigen CD29and CD90,but not expressed hematopoietic cell antigen CD44 and CD45,which was ident-ical to bone marrow-derived mesenchymal stem cells and met the experiment requirment.?2?Immunofluorescence assay showed NG2,?-SMA positive in the treated cells.West-ern blot and RT-PCR showed significant increase in protein and mRNA expression levels of NG2 and?-SMA,after the stimulation of astragaloside on MSCs.The results showed that astragaloside can stimulate MSCs into pericytes,compared with the blank control group,and the effect of differentiation was better than the TGF-?_1.Part 2 The mechanism of astragaloside relate to promoting the differentiation of bone marrow mesenchymal stem cells into pericytes1.ObjectiveExplore the mechanism of astragaloside ralate to promoting the differentiation of mesenchymal stem cells into pericytes.2.Method?1?The third generation cells were divided into four groups:the blank control group,the TGF-?₁ group,the Ast group and the blocker group.The blank control group was treated with normal saline 2?L,while the samples were collected at day 0 and day 3.The TGF-?₁ group was cultured with TGF-?₁ 10 ng,the concentration of which was adjusted to 5 ng/mL,while the sample were collected at day 3.The Ast group was cultured with astragaloside 8?g,the density of which was adjusted to 4?g/mL,while the samples were collected at day 1,day 2,day 3,day 5,day 7,which were markerd as Ast 1d group,Ast 2d group,Ast 3d group,Ast 5d group and Ast 7d group.The blocker group was administered with TGF-?₁ receptor sb431542 10?M,prior to Ast exposure,while the samples were collec-ted at day 1,day 2,day 3,marked as sb431542+Ast 1d group,sb431542+Ast 2d group,and sb431542+Ast 3d group.?2?Expression levels of NG2,?-SMA were examined by immunocytochemistry,which were the specific marker for the authentication of pericytes.?3?Expression levels of NG2,?-SMA,p-Smad2,Smad3 were examined by western blot.?4?Expression levels of NG2,?-SMA were examined by RT-PCR,which were the specific marker for the authentication of pericytes.3.Result?1?The p-Smad2 protein level increased by day 1,reached a maximum at day 3,and then decreased,after the stimulation of the astragaloside.The protein expression level of p-Smad2 was higher in Ast 3d group,compared with the Ast 0d group?p<0.01?.?2?The effect of the astragaloside is blocked by the inhibitor of the TGF-?₁ receptor sb431542 exposure,leading to the down-regulation of the protein level of p-smad2.Compar-ed with the Ast group at the same time point,the protein expression level of p-Smad2reduced significantly in the blocker group at day 3?p<0.001?.?3?Immunofluorescence assay showed NG2,?-SMA negetive in the sb431542+Ast3d group,compared with the Ast group.?4?The effect of the astragaloside is blocked by the inhibitor of the TGF-?₁ sb431542exposure,leading to the down-regulation of the protein and mRNA level of NG2 and?-SMA.Compared with the Ast group at the same time point,the protein and mRNA expression level of NG2 and?-SMA reduced significantly in the blocker group at day3?p<0.001?.4.Conclusion?1?The expression of p-smad2 protein was increased,after the stimulation of astragaloside on BMSCs,suggesting that astragaloside can activate TGF-?₁/Smad2signaling pathway.?2?For the BMSCs,the negetive staining results,the down-regulation related to the protein and mRNA expression level of NG2 and?-SMA,after the delivery of sb431542prior to astragaloside,suggesting that the effect of the astragaloside is blocked by the inhibitor of the TGF-?₁ receptor sb431542.?3?The down-regulation of the p-smad2 protein,combined with the fact that the down-regulation of the NG2 and?-SMA,after the pro-exposure of sb431542,suggesting that Astragaloside can induce bone marrow mesenchymal stem cells differentiating into pericytes through the TGF-?₁/Smad2 signaling pathway.