Effects Of HUC-MSCs Supernatant On Endoplasmic Reticulum Stress Induced By Rotenone In SH-SY5Y Cells

Objectives Preparation of Parkinson's Disease(PD)in vitro by rotenone(Rot)stimulating human neuroblastoma cells(SH-SY5Y),and exploring human umbilical cord mesenchymal stem cells(hUC-MSCs)supernatant The effect of endoplasmic reticulum(ER)stress on PD cell model.Methods PD cell model preparation: SH-SY5 Y cells were placed in 10%(volume fraction)inactivated fetal calf serum,1% double anti-DMEM/ F12 medium,incubated in a 37?,5%(volume fraction)CO2 incubato.Change cell culture every 2 days,and When the cells are over 80%~90% of the culture flask,they are passages according to 1:3.SH-SY5 Y cells were treated with rotenone at a concentration of 0?mol/L,0.1?mol/L,1.0?mol/L and 5.0?mol/L for 24 h.The cell viability of different experimental groups was detected by CCK-8 method.The expression of ?-synuclein in each experimental group was detected by Annexin-V/PI staining,flow cytometry detection of apoptosis and protein immunoblotting(Western blot).Strip gray analysis was performed using Image j,and the ratio of the target protein to ?-actin was used as a result.After the consent of the family members,the umbilical cord tissue of the full-term infants was harvested under sterile conditions for 15-20 cm.The cells were isolated from the umbilical cord by collagenase and trypsin digestion,cultured and passaged,and observed under an inverted phase contrast microscope.The cell morphology was identified by flow cytometry.The third generation cells were selected for starvation culture for 48 hours,and the supernatant was collected.Experimental grouping: Group Control was solvent control group,group Rot was 1.0?mol/L Rot treatment SHSY5 Y cells,group MSCs was hUC-MSCs supernatant treatment SH-SY5 Y cell group,and group MSCs+Rot was hUC-MSCs supernatant+1.0?mol/L Rot co-processes the SH-SY5 Y cell group.After 24 hours of culture in each group,the corresponding experimental studies were carried out.The cell viability,Annexin-V/PI staining of different experimental groups were detected by CCK-8 method,and apoptosis and protein immunoblotting(Western blot)of different drug groups were detected by flow cytometry.The expression levels of ?-synuclein,GRP-78,PERK,ATF6,IRE1,CHOP,Caspase12 and Caspase3 were detected in each experimental group.The results were analyzed by stripping grayscale with Image j,and the ratio of the target protein to ?-actin was used as the result.Statistical analysis: The experimental data were expressed as mean±standard deviation(x ±s).Oneway analysis of variance(ANOVA)was performed on the data using SPSS22.0 statistical software.Tukey method was used to compare the two groups.The comparison was performed using two independent sample t tests,and the difference was statistically significant at P<0.05.Results 1 After treatment of SH-SY5 Y cells with 0.1,1.0,5.0?mol/L Rot for 24 h,the cell viability decreased in a dose-dependent manner.SH-SY5 Y cell apoptosis rate is proportional to rotenone concentration(P<0.05).The expression level of ?-synuclein increases with the increase of Rot concentration(P<0.05).2 Inverted phase contrast microscopy showed that the second generation hUC-MSCs showed a uniform fusiform morphology,swirling arrangement.3 The results of hUC-MSCs flow cytometry showed that hUC-MSCs highly expressed mesenchymal stem cells labeled CD73-APC(99.9%),CD90-FITC(99.8%)and CD105-APC(98.5%);do not express hematopoietic cells markers:HLA-DR-FITC(0.1%),CD45-FITC(0.1%),CD34-FITC(0.0%),CD11b-FITC(0.0%),CD19-FITC(0.0%).4 hUC-MSCs-CM expressed GDNF,NGF and BDNF neurotrophic factors,and 48 h of GDNF and BDNF increased significantly compared with 24 h,the difference was statistically significant(P<0.05).5 hUC-MSCs-CM+Rot costimulated SH-SY5 Y increased cell viability,decreased apoptotic rate,and decreased ?-synuclein expression level(P<0.05);The hUC-MSCs-CM+Rot co-stimulation group had lower expression of GRP-78,PERK,ATF6,IRE1,CHOP,Caspase12 and Caspase3 than the Rot-stimulated group SH-SY5 Y cells(P<0.05).Conclusions 1 hUC-MSCs-CM protects rotenone-induced apoptosis in SH-SY5 Y cells.2 hUC-MSCs-CM can attenuate the endoplasmic reticulum stress in the Parkinson's disease model induced by rotenone in SH-SY5 Y cells,which may be one of the mechanisms of transplantation of hUC-MSCs for PD.Figure18;Table14;Reference 67...