| BackgroundAlzheimer's disease(Alzheimer's disease,AD)is a progressive disability disease.At present,there is no effective treatment to alleviate the disease.With the increasing aging of the global population,the prevalence of AD is increasing year by year,so it is extremely urgent to develop effective prevention strategies and treatment methods.The pathogenesis of AD is complex.Some theories have been confirmed,such as Amyloid ?(A?)deposition,astroglial degeneration,hyperphosphorylation and accumulation of tau protein,neuronal dystrophy,oxidative stress,mitochondrial dysfunction,biological metal dynamic balance disorder,decreased level of acetylcholine(Acetylcholine,ACh)and so on.Extracellular A? deposition to form senile plaque and intracellular hyperphosphorylated tau protein to form neurofibrillary tangles(Neurofibrillary tangles,NFTs)are the main pathological features of Alzheimer's disease.At present,the drug research and development for the pathological processes such as A? aggregation,NFTs formation after tau protein hyperphosphorylation and neuroinflammation all ended in failure.Therefore,it is confirmed that AD is caused by the parallel participation of a series of pathological events,rather than the cascade amplification caused by an event(such as ?-amyloid or chronic inflammation).The best direction of AD therapeutic drug research and development or therapeutic intervention should be "one point multi-target" or "single intervention,multiple effects".Neural stem cells((Neural stem cells,NSCs)have the commonness of self-renewal stem cells and the characteristics of oriented neural differentiation.NSCs transplantation is a promising method for the treatment of central nervous system degenerative diseases such as AD.It plays a protective role in many ways:inhibiting the accumulation of ?-amyloid protein,inhibiting the activation of intracranial microglia,promoting the proliferation of endogenous neural stem cells and neuronal differentiation,promoting endogenous angiogenesis and so on.However,the acquisition of NSCs is difficult,and there are ethical problems in clinical application.At the same time,the low survival rate of exogenous transplanted cells in vivo due to host immune rejection and the destruction of pathological microenvironment greatly limits the clinical application of NSCs transplantation.The repair mechanism of neural stem cell transplantation involves the replacement of primitive neural tissue,that is,cell substitution,immune regulation,neuronutritional support of paracrine products,paracrine effect of neural stem cells and so on.Studies have shown that neural stem cell conditioned medium(NSC-conditioned medium,NSC-CDM)has the same organ protective effect as neural stem cells,and the main mechanism of stem cell transplantation is not cell substitution,but depends on the pathological microenvironment,namely stem cell paracrine.Therefore,the effective use of NSC-CDM(including a variety of neural stem cell paracrine substances)to replace neural stem cell transplantation has become a new treatment strategy,and can effectively avoid many limitations of stem cell therapy.Mitochondrial dysfunction is an early event of AD.A? locates and accumulates in mitochondria,which directly or indirectly destroys the structure and function of mitochondria and induces oxidative stress.These processes can promote the production of A?,further aggravate the damage of mitochondria and lead to a vicious circle,and finally neuronal degeneration and apoptosis.Another important aspect of mitochondria in cells is the regulation of programmed cell death.In mitochondrial-mediated apoptosis,intrinsic signal-triggering proteins are released from the intermembrane space of mitochondria to the cytoplasm,the most significant of which is cytochrome c(Cytochrome c(cyto c).Cyto c is a key component of mitochondrial apoptosis.,Cyto c passes through the mitochondrial membrane,participates in the initiation of Caspase 9,and further activates Caspase 3,which leads to apoptosis.As part of the main mechanism of amplifying apoptosis signals,mitochondrial protection may be an appropriate target for therapeutic agents.In the age-related degenerative disease AD,there must be cell senescence,especially the degradation of macromolecular structure and function such as DNA and protein.In various reactions that cause protein damage,the abnormal formation of iso-aspartic acid(isoAsp)peptide bond is very common,which changes the three-dimensional structure of protein and leads to cell dysfunction.Protein L isoaspartate methyltransferase 1(protein L isoaspartate O-methyltransferase 1)is a product of the conserved gene PCMT1,which can selectively methylate the carboxyl group of L-isoaspartic acid residues,demethylate methyl iso-aspartic acid into succinimide,and restore normal a-connection,so as to repair or degrade damaged proteins.PCMT1 is expressed in all mammalian tissues and cells studied so far,especially in brain tissue.PCMT1 is highly active and polytropic,and it has been found to have more than 30 major substrates in the brain,so even small differences in PCMT1 activity may have a profound impact on the long-term function of the central nervous system((Central Nervous System,CNS).In recent years,it has been confirmed that PCMT1 plays a key role in multiple regulation in many physiological processes,such as longevity,apoptosis,heat shock response,different kinds of oxidative stress and so on.Mammalian male sterile line 20-like protein kinase 1((Mammalian Sterile 20-like protein kinase-1)is a pre-apoptotic protein,which is the core protein of classical Hippo signaling pathway.Its active form,MST1,can up-regulate Bax,inhibit Bcl-2 and activate Caspase3,to induce apoptosis.Studies have shown that PCMT1 may have the effect of regulating MST1,that is,PCMT1 can inhibit the activation of MST1 and inhibit cell apoptosis.To sum up,neural stem cell transplantation is one of the most promising treatments for Alzheimer's disease,but there are many limitations in its clinical application.The conditioned medium of neural stem cells has many protective effects similar to that of neural stem cells,and the effective use of conditioned medium of neural stem cells can effectively avoid many limitations faced by neural stem cell transplantation.Therefore,we will explore the protective effect of neural stem cell conditioned medium on AD model,and provide theoretical support for the clinical transformation of neural stem cell conditioned medium.Mitochondrial dysfunction runs through the occurrence and development of Alzheimer's disease and is the target of therapeutic agent research.We will first explore the protective effect of neural stem cell conditioned medium on mitochondrial function.PCMT1 is a conserved gene expressed in mammalian cells,which participates in the basic life activities of cells,and its activity is significantly decreased in the brain of patients with Alzheimer's disease.We speculate that neural stem cell conditioned medium can interfere with neuronal apoptosis by up-regulating the expression of conservative gene PCMT1 and regulating Hippo apoptosis pathway with MST1 as the core protein,and regulate cell survival from the "source" of nerve cells.Furthermore,we will explore whether neural stem cell conditioned medium regulates apoptosis in AD model through PCMT1/MST1 pathway,in order to confirm the multi-pathway protective mechanism of neural stem cell conditioned medium.Part one:Neural stem cell conditioned medium attenuates apoptosis of SH-SY5Y cells induced by A?25-35.Purpose of the study:Neural stem cells are the precursor cells(source)of neuronal differentiation,with strong self-renewal ability and significant paracrine effect.The conditioned medium of neural stem cells rich in paracrine products has a function similar to that of neural stem cells,and can avoid many limitations of stem cell therapy.We use A? 25-35 to induce SH-SY5Y cell injury to establish an in vitro AD cell model,and give neural stem cell conditioned medium intervention to explore whether it can reduce AD cell injury.Research methods:1.To establish an in vitro model of Alzheimer's disease:SH-SY5Y cells were treated with A ?25-35,and the drug concentration and action time of AD cell model were determined by CCK-8 assay.2.The experiment was divided into four groups:control(Control)group,AP 25-35(40 ? M)group,A?25-35(40 ?M)+neural stem cell complete medium(NSC-complete medium,NSC-CPM)group,A? 25-35(40 ?M)+neural stem cell conditioned medium(NSC-CDM)group.3.The four groups were treated with different media for 24 hours,cell viability and apoptosis were detected by 1CCK-8 assay,apoptosis was detected by TUNEL staining,and apoptosis was detected by flow cytometry.Results:1.When SH-SY5Y cells were treated with A?25-35 30 ? M at different time(12h,24h,36h,48h),the cell survival rate decreased with time.2.SH-SY5Y cells were treated with Ap 25-35 at different concentrations(10?M,20?M,30?M,40?M,50?M)for 24 h.With the increase of A?25-35 concentration,the cell survival rate decreased gradually.Compared with the control group,the cell viability of SH-SY5Y cells treated with 40?M A?25-35 for 24 hours decreased to(56.62±1.26)%.3.CCK-8 results:compared with Control group,the cell viability of A?25-35 group decreased significantly(***p<0.001).Both NSC-CPM and NSC-CDM reversed the effect of A?25-35 on SH-SY5Y cell viability(***P<0.001).NSC-CDM group had higher cell viability than NSC-CPM group.4.TUNEL staining results:the characteristic nuclear fragments of apoptotic cells were clearly observed in A?25-35 induced SH-SY5Y cells,in addition,concentrated nuclei and nuclear fragments were also found.The number of TUNEL positive nuclei in NSC-CPM or NSC-CDM group was significantly lower than that in A?25-35 group,and the number of TUNEL positive nuclei in NSC-CDM group was lower than that in A?25-35 group.In addition,the results of apoptosis detected by flow cytometry showed the same trend.Summary of this chapter:1.SH-SY5Y cells were treated with 40?M A?25-35 for 24 hours to establish AD cell model.2.After AD cell model was intervened by conditioned medium of neural stem cells,the expression of apoptotic proteins(Cytochrome c,Caspase-9,Caspase-3,Bax)in mitochondrial pathway decreased,the content of ROS and MDA decreased,and the content of MMP increased,and the effect was better than that of complete culture medium of neural stem cells(NSC-CPM,).The conditioned medium of neural stem cells NSC-CDM(some components of paracrine of NSCs)attenuated the injury of SH-SY5Y cells induced by A?25-35 through mitochondrial protection.Part two:Neural stem cell conditioned medium improved the damage of SH-SY5Y cells induced by A?25-35 by protecting the function of mitochondria.Purpose of the study:Mitochondrial dysfunction is one of the pathogenesis of Alzheimer's disease and is considered to be an early event of Alzheimer's disease.A? is deposited in mitochondria,which affects the function of mitochondrial complex and leads to mitochondrial toxicity and neuronal apoptosis.The function of mitochondria is abnormal,which produces high level of reactive oxygen species((Reactive oxygen species,ROS),which damages the specific components of mitochondria and aggravates the dysfunction of mitochondria.Another important function of mitochondria in cells is to regulate programmed cell death.In mitochond rial-mediated apoptosis,intrinsic signal-triggering proteins are released from the intermembrane space of mitochondria to the cytoplasm,the most significant of which is cytochrome c(Cytochrome c(cyto c).Cyto c is a key component of mitochondrial apoptosis.,Cyto c passes through the mitochondrial membrane,participates in the initiation of Caspase 9,and further activates Caspase 3,which leads to apoptosis.The first part of the experiment has confirmed the protective effect of neural stem cell conditioned medium on AD cell model.In this part,we will continue to explore its regulatory effect on mitochondrial function and oxidative stress,and determine whether the conditioned medium of neural stem cells can protect the integrity of mitochondria and maintain the normal function of mitochondria.Research methods:Research methods:1.To determine the drug concentration and action time of AD cell model.2.The experiment was divided into four groups:control group,A?25-35 group,A ?25-35+NSC-CPM group and Ap 25-35+NSC-CDM group.3.Experimental methods:? apoptotic proteins of mitochondrial pathway were detected by Western blotting(WB),?mitochondrial function tests:mitochondrial membrane potential(MMP)detection,reactive oxygen species(ROS)detection,malondialdehyde,MDA)content detection,and?structural changes of mitochondria were observed by projection electron microscope.Results:1.The modeling condition is that the concentration of Ap 25-35 is 40 ? M and the action time is 24 hours.2.The results of WB assay showed that compared with Control,the expression levels of Cytochrome c,Caspase 9,Caspase 3 and Bax in A?25-35 group increased(**P<0.01),while the expression of Bcl 2 decreased in NSC-CPM and NSC-CDM group,and NSC-CDM group was better than NSC-CPM group(*P<0.05).3.ROS determination showed that the content of ROS in A?25-35 group was significantly higher than that in Control group.The content of ROS in NSC-CPM or NSC-CDM+A?25-35 group was significantly lower than that in A?25-35 group(*P<0.05),and the effect of NSC-CDM was better than that of NSC-CPM(*P<0.05).The result of MDA content determination was similar to that of ROS.Compared with Control group,MMP in A?25-35 group decreased significantly(**P<0.01).Similarly,NSC-CPM or NSC-CDM could reverse this effect,and the effect of NSC-CDM was better(*P<0.05).4.The results of electron microscope showed that the mitochondria of SH-SY5Y cells treated with AP 25-35 were swollen,especially the cristae of mitochondria almost disappeared or disintegrated.In NSC-CPM and NSC-CDM+A?25-35 groups,although most of the mitochondria were swollen,we observed some normal mitochondrial cristae.The mitochondrial swelling in NSC-CDM+AP 25-35 group was lighter than that in NSC-CPM+A?25-35 group,and the number of normal mitochondria was more.Summary of this chapter:1.SH-SY5Y cells were treated with 40?M A?2s-35 for 24 hours to establish AD cell model.2.After AD cell model was intervened by conditioned medium of neural stem cells,the expression of apoptotic proteins(Cytochrome c,Caspase-9,Caspase-3,Bax)in mitochondrial pathway decreased,the content of ROS and MDA decreased,and the content of MMP increased,and the effect was better than that of complete culture medium of neural stem cells(NSC-CPM,).The conditioned medium of neural stem cells NSC-CDM(some components of paracrine of NSCs)attenuated the injury of SH-SY5Y cells induced by A?25-35 through mitochondrial protection.Part three:Neural stem cell conditioned medium attenuates SH-SY5Y cell injury induced by A?25-35 through PCMT1/MST1 pathway.Purpose of the study:Neural stem cells are the precursor cells(source)of neurons,and PCMT1 is a conservative gene expressed in mammals.Its basic function is to repair senescence proteins,which has many substrates and participates in the basic life activities of cells,so PCMT1 may play an important role in the process of neuronal differentiation and repair.Studies have confirmed that PCMT1 and MST1 co-locate in cells,PCMT1 may be the upstream regulatory factor of MST1,and MST1 is the core protein of classical Hippo apoptosis pathway.At the same time,MST1 can inhibit autophagy and induce apoptosis through multiple pathways.Therefore,we speculate that neural stem cell conditioned medium may regulate neuronal apoptosis by acting on PCMT1/MST1 pathway.In this part of the experiment,we will explore whether NSC-CDM plays a cytoprotective role through this pathway.Research methods:Section 1.Bioinformatics analysis was used to analyze the difference of PCMT1 expression in the brain of patients with AD and normal elderly.?download the dataset GSE5281;?from the gene expression database GEO and analyze the differentially expressed genes with Limma package.Section 2.1.Four groups(1 control group,2 A? 25-35 group,3 A?25-35+NSC-CPM group,4 A?25-35+NSC-CDM group)were cultured for 24 hours,and the expression of PCMT1 was detected by WB and real-time quantitative PCR respectively.2.The overexpression of PCMT1 and the evaluation and screening of silencing efficiency,and the expression of PCMT1 in each group was detected by WB method and qRT-PCR method.3.The experiment is divided into two groups:The first group:1 normal control group(Control check,CC group),2 negative control group(Negative control,NC group),3 silencing expression of PCMT1 group(sh-PCMT1 group),4 silencing expression of PCMT1+NCS-CDM group(sh-PCMT1+NSC-CDM group).The second group:1 unloaded plasmid group(Vector group),2 overexpressed PCMT1 group(PCMT1-OV group),3 AP 25-35(40?M for 24 h)+empty plasmid group(A?25-35),4 overexpression of PCMT1+A?25-35(40?M for 24 h)group(A? 25-35+PCMT1-OV group).4.The cell viability was detected by CCK-8 assay,apoptosis was detected by TUNNEL staining,apoptosis-related protein content was detected by WB method,and the expression of PCMT1,total MST1(T-MST1)and phosphorylated MST1(p-MST1)was detected by WB method.Results:Section 1.Using Limma to analyze the differential genes of GSE5281 dataset downloaded from GEO database,it was found that the expression of PCMT1 decreased significantly in hippocampus,medial temporal gyrus and posterior cingulate brain regions of patients with AD and normal elderly.Section 21.The results of WB showed that compared with Control group,the expression of PCMT1 in A?25-35 group decreased(**-P<0.001),and the expression of PCMT1 in NSC-CDM group increased significantly compared with A?25-35 group(***p<0.001),which was higher than that in NSC-CPM group.The results of qRT-PCR method showed the same trend.2.Evaluation and screening of plasmid transfection efficiency:compared with NC group,the expression of mRNA and protein in PCMT1 in sh-PCMT1 group was significantly lower than that in Vector group(***P<0.001),and the expression of mRNA and protein in PCMT1 in PCMT1-OV group was significantly higher than that in PCMT1-OV group(***p<0.001).The effect of constructing transfection plasmid is good.3.The results of CCK-8 showed that the viability of SH-SY5Y cells in sh-PCMT1 group was lower than that in NC group,and that in PCMT1-OV group was higher than that in Vector group,and that in AP 25-35+Vector group was significantly higher than that in A?25-35+PCMT1-OV group(**P<0.01),indicating that overexpression of PCMT1 reversed the inhibitory effect of A?25-35 on cell viability.TUNEL staining showed that the number of apoptotic cells in sh-PCMTl group was higher than that in NC group,and the overexpression of PCMT1 reversed the apoptosis of SH-SY5Y cells induced by A?25-35(Ap 25-35+Vector group,vs.A?25-35+PCMT1-OV group,**P<0.01).4.The results of WB showed that compared with NC,Bcl 2/BAX decreased significantly and Cleaved caspase 3/Caspase3 increased significantly in sh-PCMT1 group(*P<0.05).After injury induced by A?25-35/Bcl 2/BAX decreased significantly(A?25-35+Vector group,vs.Vector group,**P<0.01),Cleaved caspase 3/Caspase3 increased significantly(A?25-35+Vector group,vs.Vector group,*P<0.05).After overexpression of PCMT1,the decreasing trend of Bcl 2/BAX was reversed(A?25-35+Vector group,vs.A?25-35+PCMT1-OV group,**P<0.01,the increasing trend of),Cleaved caspase 3/Caspase3 was reversed(Ap25-35+Vector group,vs.A?25-35+PCMT1-OV group,**P<0.01).5.The results of WB showed that the expression level of silent PCMT1,PCMT1 was significantly decreased(NC group vs.sh-PCMT1 group,**P<0.01),p-MST1/T-MST1 was increased(NC group vs.sh-PCMT1 group,**p<0.01),overexpression PCMT1,PCMT1 expression was increased(PCMT1-OV group vs.Vector group,**P<0.01),p-MST1/T-MST1 was decreased(PCMT1-OV group,vs.Vector group,**P<0.01).After injury induced by A?25-35,the expression of PCMT1 decreased significantly(A?25-35+Vector group vs.Vector group,**P<0.01),p-MST1/T-MST1 increased(A?25-35+Vector group vs.Vector group,**P<0.01),and the rising trend of p-MST1/T-MST1 was reversed after PCMT1 treatment(AP 25-35+Vector group vs.A? 25-35+PCMT1-OV group,*P<0.05).Summary of this chapter:1.The results of bioinformatics analysis showed that compared with the normal elderly,the PCMT1 gene in hippocampus,medial temporal lobe and posterior cingulate gyrus of AD patients was significantly down-regulated,2.Compared with the control,the expression of PCMT1 in AD cell model decreased significantly.3.After the intervention of NSC-CDM in AD cell model,the decreasing trend of PCMT1 was reversed,which confirmed that NSC-CDM could up-regulate the expression of PCMT1 in AD cell model.4.?compared with the negative control,after silencing PCMT1,the cell viability decreased,the apoptosis increased,and the expression of apoptosis-related protein increased.?compared with the plasmid no-load group,the cell viability of overexpressed PCMT1 group increased,and the expression of apoptosis-related protein decreased.?compared with the plasmid no-load group,A?25-35 injured cells decreased cell viability,increased apoptosis and up-regulated the expression of apoptosis-related protein.?compared with A?25-35 injury group,after overexpression of PCMT1,cell viability increased,apoptosis decreased and apoptosis-related protein expression decreased,The above results suggest that PCMT1 regulates apoptosis and up-regulates PCMT1 can reduce A ? 25 35-induced apoptosis.5.? compared with negative control,MST1 phosphorylation was up-regulated after silencing PCMT1.?compared with plasmid no-load group,MST1 phosphorylation was decreased after the overexpression of PCMT1.?MST1 phosphorylation was up-regulated after A?25-35 injury compared with plasmid no-load group,and?MST1 phosphorylation was decreased after overexpression of PCMT1 compared with A?25-35 injury group.These results suggest that PCMT1 regulates MST1 phosphorylation and up-regulates PCMT1 can inhibit Ap25-35-induced MST1 phosphorylation.To sum up,neural stem cell conditioned medium up-regulated the expression of PCMT1 and reduced the apoptosis of SH-SY5Y cells induced by A?25-35 by inhibiting the activation of MST1 and acting on the classical Hippo apoptosis pathway.Conclusion:Combined with the research background,we designed and carried out the corresponding experiments on whether the conditioned medium of neural stem cells has multiple neuroprotective effects,and achieved the following results:1.We found that the injury of SH-SY5Y cells induced by A?25-35 was time-and dose-dependent.The model was established under the condition of 40?M A? 25-35 for 24 hours.Under this condition,the cell viability decreased to(56.62±1.26)%.2.We confirmed that conditioned medium of neural stem cells could reduce the apoptosis of SH-SY5Y cells induced by A?25-35 by CCK8 detection,TUNEL staining and flow cytometry.3.We used WB to detect the apoptotic protein of mitochondrial pathway,electron microscope to observe the structure of mitochondria and flow cytometry to detect the membrane potential of mitochondria.We confirmed that the conditioned medium of neural stem cells maintained the integrity of the structure and function of mitochondria,and alleviated the damage of SH-SY5Y cells induced by A?25-35 through the protective effect of mitochondria.4.We used Limma package to analyze the gene differential expression of neurons in the same brain region between Alzheimer's disease patients and normal elderly people,and found that the expression of PCMT1 gene was significantly down-regulated in the three brain regions of AD patients.5.We silenced and overexpressed PCMT1,by plasmid transfection and found that PCMT1 could regulate the survival of SH-SY5Y cells.The viability of silenced PCMT1 cells decreased and apoptosis increased,while the viability of overexpressed PCMT1 cells increased.PCMT1 can regulate the phosphorylation(activation)of MST1.Silenced PCMT1,the phosphorylation of MST1 increased,while overexpressed PCMT1,the phosphorylation of MST1 decreased,6.In vitro experiment,we found that the injury induced by A?25-35 led to the down-regulation of PCMT1 expression in SH-SY5Y cells,while the conditioned medium of neural stem cells could reverse this change.MST1 phosphorylation was enhanced by A?25-35 induction,and overexpression of PCMT1 could reverse this change.It is proved that the conditioned medium of neural stem cells can reduce the apoptosis of SH-SY5Y cells induced by A?25-35 by up-regulating the expression of PCMT1,inhibiting MST1 phosphorylation and acting on Hippo pathway.To sum up,the experiment confirmed that the conditioned medium of neural stem cells alleviated the damage of SH-SY5Y cells induced by A?25-35 through multi-pathway("one point and multi-target")protection mechanism.This provides theoretical support for the clinical transformation of neural stem cell conditioned medium. |
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