| Objective:To observe the anti-inflammatory effect of iMSC-Exo on LPS-induced AM inflammatory response,and to explore the molecular mechanism of iMSC-Exo exerting anti-inflammatory effect.Method:iMSC was cultured in vitro,and iMSC-Exo in cell supernatant was extracted by spin ultrafiltration.The cells were identified by transmission electron microscopy(TEM),high-resolution adjustable resistance pulse(TRPS)and Western blotting.AM was cultured and treated with PBS,iMSC-Exo,LPS,LPS+iMSC-Exo;differential miRNA expression profiles between LPS group and LPS+iMSC-Exo group were compared by second-generation high-throughput sequencing technology;miR-125b-5p mimic or inhibitor was transfected into LPS+iMSC-Exo-induced AM;ELISA was used to detect the contents of TNF-?,IL-1? and IL-6 in the supernatant of each group,and each group was detected by qRT-PCR.TNF-?,IL-1?,IL-6 were used to verify the expression of miR-125b-5p in AM and iMSC-Exo.Western Blot was used to detect TRAF-6 and NF-?B(p65)in AM.The protein expression of p-p65 was observed by immunofluorescence to observe the co-localization of iMSC-Exo and AM and the fluorescent expression of TRAF-6 in cells.Result:1.The observed iMSC-Exo was a circular or elliptical membranous vesicle with a diameter of about 40-100 nm and expressed exogenous protein markers CD9 and CD63.2.After iMSC-Exo was co-cultured with LPS pre-stimulated AM for 24 h,the protein and mRNA levels of TNF-?,IL-1? and IL-6 in the supernatant and cells were significantly lower than those in the LPS group;It can be seen that the fluorescently labeled iMSC-Exo wraps around the nucleus of AM,suggesting that iMSC-Exo canbe engulfed by AM.3.The second-generation high-throughput sequencing results showed that the expression ratio of LPS+iMSC-Exo group and LPS group was ?2 times as differentially expressed miRNAs,and LPS+iMSC-Exos stimulated AM 6h and 24 h,respectively,and up-regulated by more than 2 times.There were 90 and 30 miRNAs,including 12 miRNAs including miR-125b-5p,miR-9b-3p and miR-135a-5p.The expression levels of 12 miRNAs were significantly up-regulated at two time points,and the inflammatory response was selected.The miRNAs were verified in vitro and found that miR-125b-5p was up-regulated most,and miR-125b-5p was also highly expressed in iPSC-MSC-Exo.4.By transfecting miR-125b-5p mimic overexpressing miR-125b-5p by LPS+iMSC-Exo-induced AM,it can significantly enhance the anti-inflammatory effect of iMSC-Exo on LPS-induced AM inflammatory response;Silencing miR-125b-5p by 125b-5p inhibitor can attenuate the anti-inflammatory effect of iMSC-Exo on LPS-induced AM inflammatory response.5.Bioinformatics analysis predicted that there is a complementary pairing sequence between the 3'-UTR of TRAF-6 gene and the miR-125b-5p seed region;the dual luciferase reporter gene verified that transfection of miR-125b-5p can make wild-type TRAF6 The luciferase activity of the-3' UTR vector was reduced by about30%,while the luciferase activity of the mutant TRAF6-3'UTR vector was not significantly affected.6.After iMSC-Exo was co-cultured with LPS pre-stimulated AM for 24 h,the expression levels of TRAF-6 and P-P65 in AM were significantly lower than those in LPS group,while the protein level of NF-?B was not significantly changed.7.LPS+iMSC-Exo-induced AM transfection of miR-125b-5p mimic overexpressing miR-125b-5p,the expression levels of TRAF-6 and P-P65 protein were decreased compared with the negative control group;and transfected miR-125 bSilencing of miR-125b-5p by 5p inhibitor resulted in up-regulation of TRAF6 protein expression and increased nuclear translocation of NF-?B in AM.Conclusion:iMSC-Exo targets TRAF-6 via miR-125b-5p,inhibits the activation of NF-?B inflammatory signaling pathway in AM,and attenuates LPS-induced inflammatory responses. |
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