The Mechanism Of Bone Marrow Mesenchymal Stem Cells-Derived Exosomes On The HMGB1 In The LPS-induced RAW264.7 Cells

Objective(s):RAW264.7 cells were stimulated by lipopolysaccharide(LPS)to make an inflammatory model.The current study aims to investigate the effect of Bone Marrow Mesenchymal Stem Cells-Derived Exosomes(BMSC-exo)on high mobility group protein B1(HMGB1)in RAW264.7 macrophages.Methods:1.The whole bone marrow adherence was used to make mouse BMSCs,and flow cytometry was applied to identify specific markers on BMSCs.The BMSC-exo was extracted by differential centrifugation.The morphology of BMSC-exo was observed by transmission electron microscope.The diameter distribution range of BMSC-exo was detected by nanoparticle tracking analysis(NTA),and the specific markers of mouse BMSC-exo(CD9,CD63,CD81 and Calnexin)were detected by flow cytometry and Western Blot.2.RAW264.7 cells were stimulated by LPS to make an inflammatory model.Cell Counting Kit-8(CCK8)was used to evaluate the cell viability.The level of inflammatory factors tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the cell supernatant was detected by ELISA to determine the optimal concentration of LPS on RAW264.7 CCK8 and ELISA were used to further determine the optimal concentration of BMSC-exo on RAW264.7 cells;3.The experiment was divided into 4 groups:Ⅰ.sham group(Normal RAW264.7 cell);Ⅱ.LPS group[RAW264.7+LPS(10 ng/ml)];Ⅲ.EXD group(RAW264.7+LPS+deleted cell exosomes supernatant group);Ⅳ.EXO group[RAW264.7+LPS+BMSC-exo(4 μg/ml)].After 3 hours of LPS stimulation,the EXD group and EXO group were treated with the same volume deleted exosomes cell supernatant and BMSC-exo solutionboth.CCK8 was used to detect the cell viability;The apoptosis level was detected by flow cytometer;The levels of TNF-α and IL-1β in each group were examed by Elisa;The mRNA and protein expression of HMGB1 and NF-κB were tested by quantitative PCR detecting system(qPCR)and western blot.Results:1.The results of flow cytometry showed:the surface markers CD34,CD45,CD73,and CD105 of BMSCs accounted for 1.39%,22.1%,81.1%,and 89.4%,respectively.The identification of BMSC-exo on transmission electron microscopy showed that the typical cup-shaped,round or elliptical vesicles,with uneven sizes;NTA showed that the diameter of vesicles ranges from 123 nm to 130 um,and the particle concentration in the sample is 1.57×1010 particles/ml;western blot showed that the expression levels of CD9,CD63,and CD81 in BMSC-exo were significantly higher than those in BMSCs(P<0.05),and the expression level of Calnexin was significantly lower than that in BMSCs(P<0.05).The flow cytometry indicated CD9,CD63,CD81 and Calnexin labeled cells accounted for 87.87%,81.93%,85.71%and 8.04%respectively.2.Successfully established RAW264.7 cell inflammatory model:CCK8 cell proliferation experiment and ELISA were used to explore the effect of LPS(0.1 ng/ml,1.0 ng/ml,10 ng/ml,100 ng/ml,1 μg/ml,10 μg/ml)on RAW264.7 cell viability and inflammatory factors.The results showed that the optimal concentration of LPS are 10 ng/ml.Similarly,CCK8 and ELISA were applied tothe optimal concentration of BMSC-exo(4 μg/ml)on RAW264.7 cell inflammatory model.3.CCK8 proliferation experiment and apoptosis results:compared with sham group,the cell viability of LPS group,EXD group and EXO group was reduced,the apoptosis rate was increased(P<0.05).Compared with the LPS group,the cell viability in EXD group and EXO group increased,the apoptosis rate was decreased(P<0.05).Compared with the EXD group,the cell viability in EXO group increased,the apoptosis rate was decreased(P<0.05) ELISA results:compared with sham group,the expression levels of TNF-α and IL-1βin LPS group,EXD group and EXO group increased(P<0.05).However,the levels of TNF-α and IL-1β in EXD and EXO group decreased compared with LPS group,Compared with EXD group,the expression levels of TNF-α and IL-1β in the EXO group decreased(P<0.05).qPCR and Western blot results:Compared with sham group,the mRNA and protein expression levels of HMGB1 and NF-κB in the LPS group,EXD group and EXO group were increased(P<0.05);compared with the LPS group,the mRNA and protein expression levels of HMGB1 and NF-κB in the EXD group and EXO group decreased(P<0.05);compared with the EXD group,the HMGB1,NF-κB mRNA and protein expression levels decreased in EXO group(P<0.05).Conclusion(s):(1)The whole bone marrow adherence method can successfully culture and amplify mouse BMSCs in vitro.Differential centrifugation,transmission electron microscopy,NTA,flow cytometry and Western blot can successfully extract and identify BMSC-exo.(2)The inflammatory model of RAW264.7 cells could be constructed successfully by LPS stimulation.(3)Our results demonstrated that both BMSC-exo and BMSC-EXD can increase the cell proliferation in RAW264.7 cell inflammatory model and reduce the rate of apoptosis,inhibited inflammation,down-regulated the level of HMGB1 and NF-κB,therefore reduced LPS-induced inflammation.The effect of BMSC-exo is better.