Overexpression Of MiRNA-101 BMSCs Regulates Autophagy In AM After Exposure To Dust By Activating Endoplasmic Reticulum Stress

Objectives 1.To investigate the protective effects of overexpressed miRNA-101 BMSCs on ER stress of alveolar macrophages in silicotic rats.2.To explore the possible mechanism of BMSCs transfected with miRNA-101 by detecting the changes of ERS related markers in AM.Methods experimental group and model preparation: BMSCs from SD rats were cultured in vitro to the third generation by whole bone marrow attachment method.BMSCs were identified with CD90,CD106 and CD45 antibodies by flow cytometry,and the BMSCs were stably transfected with miRNA-101.CCK-8 was used to detect BMSCs,BMSCs transfected with mirna-101 and transfected empty plasmid BMSCs cell activity.Transwell coculture system was constructed with NR8383 cells stimulated by Si O2.NR8383 cells were cultured in the upper chamber,BMSCs were cultured in the lower chamber,and polycarbonate membrane with permeability was in the middle compartment with a pore diameter of 0.4 ?m.Rats were divided into five groups.Control group: NR8383 cells were cultured completely;model group: NR8383 + Si O2(50?g/ml);BMSCs +(NR8383 + Si O2)co-culture group of BMSCs intervention group;mirna-101 group: BMSCs +(NR8383 + Si O2)co-culture group of mirna-101 overexpression;IRE1 ? inhibitor group: BMSCs and NR8383 + Si O2 + STF083010(40 ?mol/L)coculture group.Nr8383 cells were cultured for 6h,12 h,24h and 48 h.The morphology of BMSCs and nr8383 cells were observed by inverted phase contrast microscope.The morphological changes and proliferation of macrophages were observed by HE staining and CCK-8 kit.The quantitative expression of IRE1?,TRAF2,JNK,LC3 and beclin-1 were detected by Western blot.The localization of IRE1 and LC3 in macrophages was detected by immunofluorescence.The data were analyzed by spss22.0 software.The data were expressed by x?±s.The measurement data were analyzed by ANOVA and t-test.The difference was statistically significant(P<0.05).Results 1 Morphological Observation: BMSCs cells became small,round or triangular,and grew in a parallel and scattered way.After being transferred to the third generation,the cells were single and fusiform in shape,rich in cytoplasm and clear in nucleus.2 under the microscope of nr8383,the cells were oval or oblate,irregular cells were occasionally seen,and some suspended cells were small.3.The percentage of positive CD106,CD90 and CD45 were 94.45%,99.26% and 17.50% respectively,which were in accordance with the characteristics of BMSCs.4.Transfection efficiency results: when MOI was 5pfu / cell,the transfection efficiency was the best.At this time,the cell growth state was good,like a honeycomb,indicating that the transfection efficiency was high and will not affect the cell growth.The peak of transfection was observed at 72 h under fluorescence microscope.5 CCK-8 showed no significant difference in the activity of the cells between BMSCs and empty plasmid cells(P>0.05),Compared with the above two groups,the activity of BMSCs transfected with mirna-101 was significantly increased(P < 0.05).6 HE staining results: NR8383 cells in the control were round,with central or partial nucleus,uniform cytoplasm color and clear cell structure.Compared with the control,the cell volume of the model group increased,the nucleus shifted,and the cytoplasm ruptured with irregular burr like changes.Compared with the model,the degree of stimulation of each intervention group was significantly lower,but still higher than control group.The cell morphology of miRNA-101 group was better than BMSCs intervention group,and the degree of stimulation of IRE1 inhibitor group was only lower than the model group and higher than other groups.7 CCK-8 cell proliferation test results: with the prolongation of culture time,the control group began to proliferate in 3-6 hours,reached the peak in 24 hours,and then slowed down into the platform stage;the proliferation trend of other groups was the same as the control group,BMSCs intervention group and miRNA-101 intervention group were significantly better than the model group(P<0.05),There was no statistical significance between the IRE1 inhibitor group and the model group,the difference between the intervention groups was not significant,but the overall level was still lower than the control group(P<0.05).8 The results of Western blot showed that LC3,beclin-1,IRE1,TRAF2 and JNK protein in the control group did not significantly change,and there was only basic expression at each time point.The expression of each protein in the model group began to increase after 6h,peaked at 24 h,and was higher than the control group at each time point(P<0.05).Compared with the model group,the protein expression of miRNA-101 intervention group decreased significantly at all time points(P<0.05),and miRNA-101 intervention group was better than BMSCs intervention group(P<0.05).The expression of IRE1,TRAF2,JNK protein were low in IRE1 inhibitor group,while the expression of LC3,beclin-1 protein were only lower than model group and higher than other groups.9 IRE1 and LC3 were only expressed in the control group at four time points.Compared with the same period,the fluorescent expression in the model group was significantly enhanced.Compared with the model group,the fluorescent expression in the BMSCs intervention group,the miRNA-101 intervention group and the IRE1 inhibitor group were decreased and showed dynamic changes over time..Conclusions BMSCs with overexpression of miRNA-101 could effectively inhibit autophagy and protect alveolar macrophages by regulating ER of macrophages,and this effect was better than that of BMSCs alone.Figure21;Table 7;Reference 117...