The Protective Effects And Potential Mechanism Of HAMSCs-CM On A549 Cells Injury Induced By Paraquat

Objective: To explore the effect of human amniotic mesenchymal stem cell conditioned media on paraquat-induced A549 cell injury and its preliminary mechanism.Methods: A549 cells was cultured routinely and paraquat with different concentrations were added.CCK-8 reagent was used to detect the cell proliferation activity.Human amnion mesenchymal stem cells was obtained by a mechanical-trypsin combined method.It was conventionally cultured and the conditioned media was extracted.Different concentrations of hAMSCs-CM were added to interfere with PQ-infected cells.The cells was divided into blank control group,PQ group(model group),hAMSCs-CM intervention group(25%,50%,75% concentration according to volume fraction)and hAMSCs-CM control group(corresponding to each concentration).CCK-8 reagent was used to detect the cell proliferation activity.The cell morphology and structure of blank control group,PQ group,hAMSCs-CM intervention group(50% concentration)and hAMSCs-CM control group(50% concentration)were observed by inverted phase contrast microscope and transmission electron microscope.The distribution of reactive oxygen species(ROS)was observed by fluorescence microscope.The contents of IL-1?,IL-6 and IL-10 in cell conditioned media of each group were detected by ELISA.Flow cytometry(FCM)was used to detect apoptosis in the above groups.Western blot was used to detect the expression of Bax,Bcl-2,CHOP and GRP78 proteins in each group.Results:(1)After PQ being contaminated for 24 hours,the final concentration of 250?M,500?M,750?M,1000?M in their cell proliferation activity decreased,and the PQ concentration of IC50 was 816?M.750?M was selected as the modeling concentration of A549 cells at the later stage.(2)The cell proliferation activities of blank control group,PQ group,hAMSCs-CM intervention group(50% concentration)and hAMSCs-CM control group(50% concentration)were(99.98±4.56)%,(52.52±3.02)%,(63.18±3.97)%,(97.42±2.28)%,respectively(F = 202.919,P < 0.05).(3)After PQ being contaminated,A549 cell morphology changed,dead cells and cell debris increased.The above changes in hAMSCs-CM intervention group were less than those in PQ group.(4)Both microvilli and protuberances on cell surface decreased,endoplasmic reticulum swelled and stonechromatin aggregated and distributed abnormally.In hAMSCs-CM intervention group,microvilli can be seen on the cell surface with uniform cytoplasm,reduced organelle damage and uniform chromatin in stone.(5)Intracellular ROS increased in PQ group compared with the blank control group,and decreased in hAMSCs-CM intervention group compared with PQ group.(6)The content of IL-10 in hAMSCs-CM intervention group was higher than that in PQ group,while the content of IL-1 Beta and IL-6 in hAMSCs-CM intervention group was lower than that in PQ group(all P < 0.01).(7)The early apoptosis rates of blank control group,PQ group,hAMSCs-CM intervention group and hAMSCsCM control group were(1.96±0.50)%,(14.16±1.15)%,(7.52±2.35)% and(1.71±0.88)%(F= 44.619,P < 0.01).(8)The Bax/Bcl-2 ratio in hAMSCs-CM intervention group was lower than that in PQ group(P < 0.05).the expression of CHOP in hAMSCs-CM intervention group was lower than that in PQ group(P < 0.01).There was no difference in the expression of GRP78 between the two groups(P > 0.05).Conclusion:(1)hAMSCs-CM can reduce the production of reactive oxygen species,reduce the secretion of inflammatory factors in A549 cells caused by PQ,and reduce the damage of endoplasmic reticulum and other organelles.(2)hAMSCs-CM may reduce PQinduced A549 cell injury by reducing oxidative stress and endoplasmic reticulum stress.