| With the acceleration of the aging process,the proportion of chronic diseases such as cardiovascular diseases,chronic respiratory diseases,diabetes and so on in the total burden of diseases has increased year by year.It has become an important factor limiting the prolongation of life.Further research on delaying senility and reducing diseases related to aging is one of the important measures to deal with the challenges of aging.Recent studies have found that the formation mechanism of aging is complex,which can lead to age-related microvascular and macrovascular diseases,in which microcirculation disorder is one of the main mechanisms.With the aging of the body,age-related endothelial dysfunction,impaired angiogenesis,and subsequent tissue perfusion lead to microcirculation disorder,which exacerbates the decline of cardiovascular and cerebrovascular function in elderly patients.Endothelial progenitor cells(EPCs)transplantation is a breakthrough in the treatment of cardiovascular and cerebrovascular diseases.Autologous EPCs transplantation and drug mobilization of EPCs contribute to vascular repair and neovascularization,to improve microcirculation disorders.However,the number of EPCs will decrease with age,and the extraction of EPCs is also very difficult.When transplanted alone,only a few survive.It is necessary to improve the internal environment to enhance the repair function of EPCs.Traditional Chinese medicine has the characteristics of multi-target and safe and flexible medication.Therefore,optimizing the transplantation scheme and using traditional Chinese medicine combined with EPCs transplantation has clinical application value for the treatment of age-related cardiovascular and cerebrovascular diseases.Previous experiments have proved that D-galactose(D-gal)can induce Subacute Brain Aging Animal Model in a short time,and its aging mechanism and characteristics are similar to natural aging,while Yiqi Huoxue Yangyin formula can delay the aging of the heart and active pulse by protecting the function of large blood vessels.Therefore,by constructing the subacute aging mouse model induced by D-galactose,to explore the effect of Yiqi Huoxue Yangyin Recipe on cerebral microcirculation in the process of aging,and to carry out the feasibility of combined EPCs transplantation to improve cerebral microcirculation disorder,which is of great significance for exploring traditional Chinese medicine combined with stem cell transplantation,improving microcirculation disorder and preventing and treating aging-related cerebrovascular diseases.Objective:To observe the changes of cerebral microcirculation in aging mice induced by D-galactose and the intervention effect of Yiqi Huoxue Yangyin formula combined with EPCs transplantation on cerebral microcirculation disorder in aging mice.Methods:A mouse model of D-galactose-induced subacute brain aging was constructed by subcutaneous injection of D-gal solution(180 mg/kg/D)into the neck and back of 8-week-old SPF grade male C57BL/6N mice given 10 consecutive weeks.The mouse brain microcirculation was taken as the research object.The experimental animals were divided into blank control group,aging model group,traditional Chinese medicine combined with endothelial progenitor cell transplantation group,endothelial progenitor cell transplantation group,and metformin combined with endothelial progenitor cell transplantation group.The same volume of normal saline was injected subcutaneously into the back every day as the blank control group.After 2 weeks of modeling,the animals in each group were given drugs by gavage,the blank group and the model group were given distilled water,and the other animals in each group were given corresponding drugs for continuous intervention for 8 weeks.The mice in the blank group were fed normally without intervention.At the 8th week after modeling,the packaged endothelial progenitor cells(EPCs)transfected by lentivirus were transplanted by tail vein injection.EPCs were injected into the EPCs group,traditional Chinese medicine group,and metformin group,with 1%in each group × 106 EPCs suspension 0.25ml,aging model group,and blank control group were injected with PBS buffer 0.25ml.Extraction,culture,identification,transfection,and transplantation of EPCs.The success of lentivirus transfection and transplantation of EPCs was observed by fluorescence microscope and small animal in vivo optical two-dimensional imaging system;The general state of mice was observed,and the changes of spontaneous exploratory motor activity and anxiety behavior were observed by open field experiment;Laser speckle blood flow imaging system was used to measure the blood flow of mouse brain and form a visual blood flow distribution map.The pathomorphological changes of mouse brain were detected by HE staining and Nissl staining;TUNEL staining was used to observe the apoptosis of mouse brain cells;The exprsion level of the Dotelial cell adhion molecule platelet-1(P-1/CD 31)and CD 105(Doglin)The brain of the mouse were observed and detected by immunofluorkcia staining;immunohistochemical staining was used to observe andDetect the exprsion level of fibrillar acid protein(GFAP)Glial,Vascular Dotelial Protecial Factor(VEGF),Metalloproteinas-2 matrix(MMP-2)and advanced advanced glycation products(age);the exprsion of Claudin-5,OCLUDINand Zo-1 The mouse brain were detected by Wtern Blot;Nitric oxide(NO),malondialdehyde(MDA)were measured(MDA),superoxide dismutase(SOD)and Dotelfin-1(ET-1)serum.Results:1)compared with the blank control group,the aging model group was in a worse state and the weight growth rate decreased(P<0.05);In the open field experiment,the number of activities and the distance of activities in the central area decreased(P<0.05);Cerebral blood perfusion decreased;Local cell degeneration,disordered structural arrangement,pyknosis of nucleus,decrease in the number of neurons and increase in apoptosis in brain tissue;The activity of serum SOD decreased,the content of MDA increased(P<0.05),the expression levels of aging protein age and MMP-2 in brain increased significantly(P<0.01),and brain aging was obvious;The expression of neuroinflammation related protein GFAP increased significantly(P<0.01),the expression of CD 31 protein decreased,the expression of VEGF and CD 105 protein decreased significantly(P<0.01),angiogenesis decreased,micro vessel density decreased,serum NO content decreased,ET-1 content increased(P<0.05),and endothelial function was impaired;The expression of ZO-1,occludin and claudin-5(P<0.05)proteins related to blood-brain barrier in mouse brain tissue decreased,and the function of blood-brain barrier was impaired.2)Compared with the aging model group,the weight growth rate of mice in each administration combined transplantation group was better than that in the aging model group(P<0.05);The ability of autonomous activity and exploration of mice was enhanced;The average cerebral blood flow perfusion of traditional Chinese medicine combined with EPCs transplantation group increased significantly(P<0.01),and the therapeutic effect was better than that of single transplantation and metformin combined transplantation group.The cells in the mouse brain were arranged compactly and orderly,the number of neurons and Nissl bodies increased,the cell spacing decreased,and the apoptosis decreased;The activity of serum SOD increased,the content of MDA decreased(P<0.05),and the expression levels of age and MMP-2 Protein in the brain decreased(P<0.05);The content of serum no increased,the content of ET-1 decreased(P<0.05),the expression of neuroinflammation related protein GFAP increased(P<0.05),the expression of VEGF,CD 31 and CD 105 increased(P<0.01),the function of vascular endothelium improved,angiogenesis increased,micro vessel density increased,and the expression of ZO-1,occludin and claudin-5 related to the blood-brain barrier in mouse brain increased(P<0.05).Conclusion:in D-galactose-induced brain aging mice,the number of brain neurons decreased,apoptosis increased,vascular endothelial dysfunction and microvascular formation decreased,resulting in the decrease of microvessel density,the decrease of cerebral blood flow perfusion,and the impairment of blood-brain barrier function.That is,D-galactose can induce cerebral vascular aging in mice,resulting in the disturbance of cerebral microcirculation.Yiqi Huoxue Yangyin recipe can improve the living environment that is not conducive to the growth and differentiation of EPCs,cooperate with EPCs to improve the disturbance of cerebral microcirculation in mice induced by D-galactose,give full play to the role of brain protection,optimize the stem cell transplantation scheme and delay the process of vascular aging. |
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