| Objective: To explore the changes in miR-106b-5p expression in exosomes derived from macrophages infected Mtb and its effect on the survival of Mtb in macrophages and the molecular mechanism.Methods:(1)Exosomes from macrophages were extracted and identified by electron microscopy,Western blot and particle size analysis.(2)Exosomes were injected by caudal vein.The mice are divided into three groups,the normal control group(Control),the infected macrophage-derived exosome group(IM-exo),and the normal macrophage-derived exosome group(M-exo).The fluorescence intensities of living bodies and organs(lung,spleen,liver,kidney)at different injection times(12h,36 h,72h)were detected by in vivo imaging to evaluate the organ enrichment of Mtb-infected macrophage-derived exosomes.(3)HE staining to observe the pathological changes of various organs 72 hours after the tail vein injection of different exosomes.(4)Establish an exosomal co-culture macrophage model,laser confocal detection of the exosomal uptake of macrophages;infection after co-culture,colony count and acid-fast staining to detect its influence on Mtb in macrophages;CCK-8,Hoechst/PI,electron microscopy,and oil red O staining were used to detect their effects on the viability,necrosis,apoptosis,and lipid metabolism of macrophages.(5)Expression profile analysis and bioinformatics analysis,select the differentially expressed gene miR-106b-5p and its target gene Creb5 in exosomes,RT-PCR was used to detect the expression changes of miR-106b-5p in serum exosomes,alveolar lavage fluid exosomes,macrophage exosomes,macrophages and serum.(6)RT-PCR verifies the correlation between exosomes and changes in miRNA expression in macrophages.(7)Construct miR-106b-5p overexpression and inhibition model,Creb5 inhibition model,miR-106b-5p and Creb5 dual regulation model,RT-PCR and FAM-siRNA verify the transfection efficiency and miR-106b-5p and The regulatory relationship between Creb5,the effect of colony count and acid-fast staining on Mtb in macrophages;CCK-8,Hoechst/PI,Western blot,and oil red O staining on macrophage viability and macrophage apoptosis,The influence of lipid metabolism pathway.Results:(1)The results of particle size analysis showed that the diameter of exocrine body was about 100 nm,typical tray-like exocrine vesicles could be observed under electron microscope,and four transmembrane protein CD63 could be detected by Western blot.(2)In vivo imaging showed that the body surface fluorescence of IM-Exo group was stronger than that of PBS group and M-Exo group at 12 h,36H and72 h.Organ imaging showed that the lung fluorescence appeared earliest in IM-Exo group at 12 h,36H and 72 h,and was stronger than that in M-Exo group and PBS group.(3)HE staining showed that inflammatory cells gathered in the lungs of mice in IM-Exo group.(4)The exosomal co-culture macrophage model was successfully constructed.The acid-fast staining results showed that compared with the PBS group(322.67±15.50),the number of bacteria in the IM-Exo group(210.33±46.44,P<0.05)and the M-Exo group(209.00±9.64,P < 0.01)decreased,and there was no significant change in the IM-Exo group(210.33±46.44,P > 0.05)compared with the M-Exo group(209.00±9.64,P < 0.01).The colony count results showed that compared with the M-Exo group(1.57±0.02),the colony number of the IM-Exo group(1.74±0.05,P< 0.05)increased.CCK-8 results showed that after 24 h of co-culture,the activity of IM-Exo group(120.44±5.42)was higher than that of M-Exo group(86.56±15.63)(P< 0.05).After 24 h of co-culture and 24 h of infection,there was no significant change in the activity of the IM-Exo group(88.19±11.98)compared with the M-Exo group(81.12±11.37)(P > 0.05),showing no statistical difference.Hoechst/PI staining showed that the apoptosis rate of IM-Exo group(4.25±0.85)was lower than that of M-Exo group(9.64±1.55,P < 0.01).Oil red O staining showed that the IM-Exo group(53.10±2.94)compared with the M-Exo group(31.85±9.06),the average lipid droplet content per cell increased(P<0.05),and the percentage of lipid droplets in the cell area increased(P<0.01).(5)RT-PCR verification showed that the expression of miR-106b-5p in serum exosomes in the PTB group(1.10±0.30)was higher than that in the healthy control group(0.68±0.28)(P<0.001);the PTB group(2.58±1.09)Compared with the healthy control group(0.85±0.26),the expression of miR-106b-5p in alveolar lavage fluid exosomes was increased(P<0.05);the macrophage infection(0.23±0.08)group was compared with the control group(0.02±0.03)The expression level of miR-106b-5p in secreted exosomes increased(P<0.05).In clinical serum samples,the expression of miR-106b-5p was higher in the infection group(0.94±0.65)than the normal group(0.45±0.34,P<0.05);In the RAW264.7 macrophage infection model,compared with the control group(0.43±0.08),the expression of miR-106b-5p increased after infection for 24h(0.84±0.11,P<0.01);in the BMDM macrophage infection model Among them,compared with the control group(0.52±0.08),the MOI=10(1.04±0.13,P<0.01)group had higher expression levels,which was dependent on the amount of bacteria.(6)Compared with the normal macrophage group co-cultured with normal exosomes(0.61±0.08),the normal cell group co-cultured with infected macrophage-derived exosomes after infection(1.00±0.18,P<0.05)miR-106b-5p high expression.(7)The miR-106b-5p overexpression and inhibition cell model was successfully constructed.The colony count results showed that compared with the miR-106b-5p inhibitor group(1.91±0.02),the miR-106b-5p mimics group(2.15±0.04,P<0.01)increased the colony count.Hoechst/PI staining results showed that the miR-106b-5p mimics group(4.90±0.40)had a lower apoptosis rate than the miR-106b-5p inhibitor group(16.55±1.32,P<0.01).Oil red O staining showed that the miR-106b-5p mimics group(32.05±1.47)was higher than the miR-106b-5p inhibitor group(12.49±2.31)in the average lipid droplet content per cell(P<0.01)and the lipid drop area increased(P<0.01).Western blot results showed that the expression of cleaved-caspase-3 was lower in the miR-106b-5p mimics group(0.05±0.01,P<0.01)compared with the miR-106b-5p inhibitor group(0.18±0.01).(8)The PCR results of Creb5 after regulating miR-106b-5p showed that compared with miR-106b-5p inhibitor group(0.29±0.03),miR-106b-5p mimics group(0.13±0.01,P<0.01)down-regulated Creb5 expression.The dual-regulated miR-106b-5p and Creb5 models were constructed,and the Hoechst/PI staining results showed that compared with the miR-106b-5p inhibitor group(12.48±2.53),the miR-106b-5p inhibitor+Creb5-siRNA group(1.35±0.89,P<0.01)apoptosis rate decreased;Oil red O staining results showed that compared with miR-106b-5p inhibitor group(17.30±2.47),miR-106b-5p inhibitor+Creb5-siRNA group(25.45±2.84,)The average lipid droplet content per cell increased(P<0.05),and the percentage of lipid droplets in the cell area increased(P<0.05);Western blot results showed that compared with miR-106b-5p inhibitor group(0.32±0.06)(0.23±0.02),miR-106b-5p inhibitor+Creb5-siRNA group(0.17±0.02)(0.14±0.02)cleaved-caspase-9(P<0.05)and cleaved-caspase-3(P<0.05)expression decreased;compared with miR-106b-5p inhibitor group(0.95±0.14)(0.08±0.01),Compared with miR-106b-5p inhibitor group(0.95±0.14)(0.08±0.01),miR-106b-5p inhibitor+Creb5-siRNA group(1.45±0.15)(0.21±0.01)SOAT1(P<0.05)and CIDEC(P<0.001)The amount of expression increased.Conclusion:(1)The infected macrophage-derived exosomes have a smaller diameter and higher accumulation in the lungs,which can positively regulate the survival of Mtb.(2)MiR-106b-5p is up-regulated in serum exosomes,alveolar lavage fluid exosomes and macrophage exosomes after infection in patients with PTB,and the up-regulation in macrophages is time-dependent and bacterial population-dependent.(3)The positive regulation of Mtb by exosomes may be related to miR-106b-5p targeting the inhibition of Creb5 expression,thereby negatively regulating the expression of CASP9 and CASP3 and positively regulating the expression of SOAT1 and CIDEC,which caused the decrease of macrophage apoptosis and lipid droplets Accumulation increased. |
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