Exosomes Secreted By Mesenchymal Stem Cells Delay Brain Aging Through Upregulation Of SIRT1 Gene

Research background.Aging is a phenomenon that all organisms inevitably face.Globally,9.9 million people suffer from dementia each year,an indicator of an aging brain.Aging is a major risk factor for most neurodegenerative diseases,including Alzheimer’s disease（AD）and Parkinson’s disease（PD）.There are few or no effective treatments for the neurodegenerative diseases associated with aging,which often progress in an irreversible manner and are associated with significant socioeconomic and personal costs.Biological features of the aging brain include genomic instability,telomere attrition,epigenetic alterations,loss of protein homeostasis,mitochondrial dysfunction,cellular senescence,dysregulated nutrient perception,stem cell failure,and altered intercellular communication.Brain aging is associated with an accumulation of molecular damage and cellular defects that progressively reduce mental capacity and predispose to disease.Mesenchymal stem cells（MSCs）are the most widely used stem cells in regenerative medicine,MSCs are adult stem cells that reside in almost all postnatal tissues and organs,including the brain.Some evidence suggests that MSCs isolated from amniotic fluid（AF-MSCs）,dental pulp（DP-MSCs）and brain（B-MSCs）can differentiate into functional neuronal cells.In addition,MSCs act in a paracrine manner by producing immunomodulatory factors（transforming growth factor-β（TGF-β）,hepatic growth factor（HGF）,nitric oxide（NO）,indoleamine2,3-dioxygenase（IDO）,interleukin（IL）-10.IL-6,IL-1 receptor antagonist（IL-1Ra）,hemoglobinase-1（HO-1）prostaglandin E2（PGE2）,tumor necrosis factor alpha-stimulated gene/protein 6（TSG-6））and pro-angiogenic factors（basic fibroblast growth factor（b FGF））.TGF-β,platelet growth factor（PDGF）,angiopoietin-1,placental growth factor（PGF）,IL-6,monocyte chemotactic protein-1（MCP-1）,epidermal growth factor（EGF）,HGF,vascular endothelial growth factor（VEGF）)regulate the immune response,induce the production of new blood vessels,and provide nutritional support to injured neurons,resulting in enhanced repair and regeneration of neural tissue.These MSC-derived immunosuppressive and neuroprotective factors are contained in MSC-derived exosomes（MSC-Exos）,which easily penetrate neural tissue and reach target cells due to their nanoscale size and lipid envelope.Exosomes are the best defined secretory vesicles among the different extracellular vesicle types reported so far,namely microvesicles,microparticles,exosomes,exocytotic granules or apoptotic vesicles.They are 40-150 nm in diameter,have a double lipid membrane with the same orientation as the plasma membrane,and they can carry many molecules and introduce them into the recipient cell to produce various biological effects.The size of exosomes varies depending on the method used.The discovery of the broad therapeutic effect of exosome-mediated MSC eliminates many of the challenges associated with the use of live replicating cells,as it fundamentally transforms live cell-based MSC therapies into non-live MSC therapies.Sirtuin 1-7 are the seven siblings of the Sirtuin relatives（SIRT1-7）.In some cases,SIRT1 translocates from the nucleus to the cytoplasmic matrix.SIRT1 is involved in various cellular metabolic processes,including inflammation,oxidative stress,apoptosis and tumour development.s IRT1 negatively regulates p53 through deacetylation of Lys-373,Lys-382 and Lys-320,thereby antagonising premature senescence induced by H₂O₂.SIRT1 is the most evolutionarily conserved sirtuin in mammals and is essential for a variety of biological functions,including regulation of metabolism,stress response,genomic stability and ultimately ageing.It has also been suggested that SIRT1 regulates cellular energy and redox status,has NAD+-dependent protein acetylation activity,and plays a role in longevity because it defends against oxidative stress and prolongs life as we age.Therefore,in this experiment I chose Human Umbilical Cord Mesenchymal Stem Cells（HucMSC）secreted exosomes derived from HucMSC（HucMSC-EXO）as the target for analysis to investigate whether HucMSC-EXO acts through the SIRT1gene in the ageing brain.Purpose of the researchTo prepare HucMSC-EXO by ultracentrifugation using microglia（Bv2）as the experimental cell line and to establish an in vitro model of H₂O₂-induced cellular senescence;to study the restorative effects of HucMSC-EXO on the brain and senescent cells of senescent mice.Research methods.Part Ⅰ:Isolation and identification of HucMSC-EXO1.culturing human umbilical cord MSCs and collecting the culture fluid.2.gradient centrifugation（300g for 10min;2000g for 10min;10000g for 1h;100000g for 1h）to obtain exosomes of human umbilical cord MSCs.3.Transmission electron microscopy was used to capture exosome morphology,a nanoparticle tracking analyser was used to analyse exosome size and number, and Western blotting was used to confirm exosome surface markers（TSG101, CD9 and CD81）.Part Ⅱ:Establishment and treatment of microglia（Bv2）aging model in vitro1.CCK-8 assay of the toxicity of various concentrations of H₂O₂ on Bv2 cells.2.selection of Bv2 cells induced by 200μM H₂O_2.3.β-galactosidase staining to demonstrate the success of cell model establishment in vitro.4.Scratch assay to detect the migration ability of HucMSC-EXO cells.Part Ⅲ:Therapeutic effects of HucMSC-EXO on the aging brain of SAMP8 mice.According to the different culture conditions and treatment drugs,the experimental mice were divided into three groups in total:SAMR1 control group,SAMP8senescent mice group,and SAMP8-Exo treatment group1.purchasing SAMP8 rapidly senescent mice and SAMR1 as their control group.2.subjecting the mice to water maze and fear box experiments to test the learning and memory abilities of the mice.3.executing mice,taking their brain organs,performing HE staining,Nissl staining and immunohistochemical experiments.4.Preparation of histones,quantification and western blot to detect the expression of individual genes such as SIRT1,P53 and P21.5.detect the content of ROS,MDA,GSH-PX and T-SOD in the brain tissue of mice.Research findings.Part Ⅰ.1.HucMSC-EXO was successfully obtained using ultracentrifugation and its concentration was detected using the bca kit.2.HucMSC-EXO is a bilayer membrane structure as shown by transmission electron microscopy;the diameter of exosomes is approximately between 30-150 nm according to the nanoparticle tracking analysis instrument;the expression of surface markers of exosomes was confirmed by western blot.Part Ⅱ.1.CCK-8 assay showing that 200μM concentration of H₂O₂ has both inhibitory effect and allows cell growth.2.The results ofβ-galactosidase staining experiments showed that the 200u M H₂O₂-induced cell group had more senescent cells,while HucMSC-EXO treatment resulted in a significant reduction of senescent cells.3.Scratching experiments demonstrated that HucMSC-EXO could promote the migration and proliferation of senescent cells.Part Ⅲ.1.The results of water maze and fear chamber experiments showed that HucMSC-EXO treatment significantly enhanced the learning and memory abilities of the senescent mice.2.The staining of brain sections showed that the neuronal cells of SAMP8 senescent mice were solidified and reduced in number;however,HucMSC-EXO treatment regenerated and increased the number of neuronal cells,which had a significant improvement.3.The results of Western blot showed a significant improvement in apoptosis-related genes,a significant decrease in oxidative stress-related genes,and changes in senescence-related genes,demonstrating that HucMSC-EXO does have a therapeutic effect on the senescent brain.Experimental conclusion:（1）Successful extraction and validation of the HucMSC-EXO signature.（2）Successful construction of a cellular senescence model.（3）HucMSC-EXO can alleviate cellular senescence and promote cell proliferation and migration capacity.（4）HucMSC-EXO alleviates neuronal cell senescence in the brain of SAMP8senescent mice by regulating the SIRT1 gene,reducing oxidative stress and inhibiting neuronal cell apoptosis.