| Objective(s): The therapeutic effect of human umbilical cord mesenchymal stem cell-derived exosomes(HUCMSC-exos)on MPP+-induced human neuroblastoma cell(SH-SY5Y)and MPTP-induced Parkinson’s disease(PD)mouse model and whether it can inhibit the activation of NLRP3 inflammatory vesicles to alleviate neuroinflammation,so as to explore novel therapeutic methods and make up for the shortcomings of existing therapeutic methods,and provide a theoretical basis and experimental basis for the application of HUC-MSC-exos for PD in regenerative medicine.To provide theoretical basis and experimental basis for the application of HUC-MSC-exos for PD in regenerative medicine.Methods: 1.Identification of basic characteristics and specific functions of HUC-MSCs: CCK8 assay for cell viability of different generations of HUC-MSCs;flow cytometry for expression of cell surface markers CD90,CD73,CD34 and CD45;osteogenic and lipogenic induction of HUC-MSCs,and identification of their multiple differentiation potentials by oil red O,alizarin red and alkaline phosphatase(ALP)staining,respectively.2.Isolation,extraction and identification of HUC-MSC-exos: extraction of exosomes by ultracentrifugation;observation of exosomes morphology by transmission electron microscopy;detection of particle size,concentration and purity by Nanoparticle tracking analysis(NTA);identification by Western blot The expression of Exosomes surface marker proteins Alix,CD63,CD9 and CD81 were identified by Western blot.3.The effect of HUC-MSC-exos on MPP+-induced cell model and its effect: 0m M,0.1 m M,0.2 m M,0.5 m M,1 m M,2 m M,3 m M,4 m M and 5 m M were applied to SH-SY5 Y cells,and the viability of SH-SY5 Y cells was detected by CCK MPP+8after 24 h to explore the establishment of cell model The optimal concentration of MPP+;PKH67 labeled exosomes to observe the uptake of exosomes by SH-SY5 Y cells;by 0ug/m L,10 ug/m L,20 ug/m L,40 ug/m L,60 ug/m L,80 ug/m L,100 ug/m L,120 ug/m L of HUC-MSC-exos acting on SH-SY5 Y cells for 24 h SH-SY5 Y cells were treated with 1 m M of MPP+ again after 24 h.The viability of SH-SY5 Y cells was detected by CCK8 to explore the optimal concentration of HUC-MSC-exos acting on SH-SY5 Y cells;the viability and apoptosis of SH-SY5 Y cells in the three NC groups,MPP+ and MPP+/EXOs groups were detected by CCK8 and FCM again;The expression of IL-1β and IL-18 in the supernatant of the three groups of cells was detected by ELISA;the expression of NLRP3,Caspase-1 and ASC in the three groups of cells was detected by WB.4.The effect of HUC-MSC-exos on PD mouse model: 54 male C57BL/6 mice were divided into three groups according to the randomization principle: saline control group(NC),MPTP group(MPTP)and MPTP+exosome group(MPTP/EXOs).18 mice in each group were labeled with PKH67 to observe whether exosomes could cross the blood-brain barrier and reach the nigrostriatal densities of the mouse brain.PKH67-labeled exosomes were used to determine whether exosomes could cross the blood-brain barrier and be taken up by dopaminergic neurons in the nigrostriatal region of the mouse brain;firstly,mice in the MPTP and MPTP/EXOs groups were injected with MPTP(30 mg/kg)intraperitoneally for 5 consecutive days to establish a subacute PD model,while mice in the NC group were injected with saline intraperitoneally for 5 consecutive days.The mice in the MPTP/EXOs group were injected with 100 u L of exosomes in the tail vein for 7 consecutive days;behavioral changes were evaluated in the pole climbing test,hanging test,and rotating stick test before intraperitoneal injection of MPTP,after injection of MPTP,and after injection of exosomes in the tail vein;after behavioral evaluation,the mice were executed by blood sampling from the eye and brain tissue was taken and stained with hematoxylin-eosin(The hematoxylin-eosin staining(HE)was used to observe the nigrostriatal areas of each group of mice;immunohistochemistry was used to detect the positive expression levels of nigrostriatal areas and striatal tyrosine hydroxylase(TH)in each group of mice;immunofluorescence was used to detect the expression of TH and α-synuclein(α-syn)in nigrostriatal areas of each group of mice;WB was used to detect the expression of TH and α-synuclein in nigrostriatal areas of each group of mice.The expression of TH,α-synuclein,NLRP3,Caspase-1,ASC and IL-1β in the nigrostriatal region of mice was examined by WB.Results: 1.The results of the identification of basic characteristics and specific functions of HUC-MSCs: CCK8 results showed that the proliferation ability of HUCMSCs cells from P3 generation to P7 generation was vigorous;FMC results indicated that HUC-MSCs expressed high CD90 and CD73 and low CD34 and CD45;after osteogenesis induction of HUC-MSCs,alizarin red stained orange and ALP stained blue-black.After lipogenesis induction of HUC-MSCs,red lipid droplets could be observed by Oil Red O staining.2.HUC-MSC-exos isolation,extraction and identification results: transmission electron microscopy observed that the exosomes showed a teato-like bilayer structure with clear boundaries;NTA identified that the exosome concentration was 8X107-8X1010Particles/ml,the purity was 100%,and the diameter was between 30-200nm;WB results showed that the exosomes expressed surface marker proteins Alix,CD63,CD9 and CD81.3.The effect of HUC-MSC-exos on SH-SY5 Y cell model and its influence:CCK8 results showed that the cell viability was around 50% after 1 m M of MPP+treatment of SH-SY5 Y cells for 24h;HUC-MSC-exos could be taken up by SH-SY5 Y cells;80ug/m L of exosomes pretreated with SH-SY5 Y cells could well protect the viability of MPP+-induced cells and reverse apoptosis;ELISA results showed that HUC-MSC-exos treatment reduced the expression of IL-1β and IL-18 inflammatory factors in the cell supernatant of MPP+-induced cell model;WB results showed that HUC-MSC-exos treatment reduced the expression of NLRP3,Caspase-1 and ASC in MPP+ group cells,Caspase-1 and ASC expression in MPP+ group cells after HUCMSC-exos treatment.4、The effect of HUC-MSC-exos on PD mouse model: HUC-MSC-exos could reach the dense part of mouse brain substantia nigra through the blood-brain barrier and be taken up by dopaminergic neurons;the rod climbing experiment,suspension experiment and rod spinning experiment showed that HUC-MSC-exos treatment could reduce the rod climbing time,increase the suspension score and increase the adherence time and rotation speed on the rod spinning instrument in PD mice;the HE results showed that HUC-MSC-exos treatment could reduce the expression of NLRP3,Caspase-1 and ASC in MPP+ group cells.The HE results showed that HUC-MSCexos treatment reduced cell damage in the substantia nigra of PD mice;immunohistochemical results showed that HUC-MSC-exos treatment increased THpositive neurons in the substantia nigra and striatum of PD mice;immunofluorescence results and WB results showed that HUC-MSC-exos treatment increased TH expression and decreased α-synuclein expression in PD mice.The WB results also showed that HUC-MSC-exos treatment decreased the expression of NLRP3,Caspase-1,ASC and IL-1β in PD mice.Conclusion(s): In the MPP+-induced PD cell model,HUC-MSC-exos pretreatment of SH-SY5 Y cells increased cell viability and decreased apoptosis in the PD cell model,and the protective mechanism may be related to the inhibition of NLRP3 inflammatory vesicle activation in the cells.Second,in the MPTP-induced PD mouse model,HUC-MSC-exos treatment protected PD mice midbrain substantia nigra-striatal dopaminergic neurons and improved motor function in PD mice,and the mechanism may be related to inhibiting the activation of NLRP3 inflammatory vesicles in PD mice and exerting an anti-neuroinflammatory effect. |
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