| Ulcerative Colitis(UC)is a chronic non-specific intestinal inflammatory disease,and the incidence rate has increased year by year.The pathogenesis of UC is complex,as a result that there is no excellent treatment with nearly no side effects,which is a major public health problem in the world.In recent years,exosomes derived from mesenchymal stem cells(MSC-Exos),have received attention due to their anti-inflammation,regulation of immune disorders and promotion of mucosa repair similar to their source cells.In addition,MSC-Exos have good biocompantibility,low immunogenicity,strong penetration and minor malicious differentiation risk,which have been of great concern in the field of UC treatment.However,oral MSC-Exos administration is susceptible to gastrointestinal p H and enzymes,which lead the deactivation of MSC-Exos.Moreover,the current administration of MSC-Exos is mainly intravenous injection,which not only affects the bioavailability of MSC-Exos but also has certain trauma.Therefore,this project based on the biological activity of MSC-Exos and tried to solve the problem of oral administration and colon targeting,which was of great value for the location release,improvement of bioavailability,reduction of side effects,and enhancement of efficacy of drugs.The main research works are described as follows:(1)Study on the anti-inflammatory activity and mechanism of Human placental mesenchymal stem cells derived exosomes(Hpl MSC-Exos).Hpl MSC-Exos was extracted by ultracentrifugation.Transmission tracking technology(TEM),nanoparticle tracking technology(NTA)and western blot(WB)were used to characterize Hpl MSC-Exos,and MTT was used to detect the cytotoxicity of Hpl MSC-Exos.The lipopolysaccharide(LPS)-primed RAW264.7cells was used to establish an inflammatory model in vitro,and Exos were co-cultured with RAW264.7 cells.The secretion of inflammatory cytokines interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-10(IL-10)was detected by ELISA,and the m RNA level of Il-6,Tnf-α,Il-10 and inducible nitric oxide synthase(Inos)was detected by q RT-PCR.The secretion of nitric oxide(NO)was measured by Griess method.WB was used to measure the protein expression of macrophage polarization,inflammatory related signal pathways and associated apoptotic proteins.Immunofluorescence(IF)was used to evalute nuclear factor-kappa B(NF-κB)p65 nucleus translocation.The results showed that Hpl MSC-Exos were successfully extracted from Hpl MSCs conditional culture and had no cytotoxicity.Hpl MSC-Exos inhibited the secretion and transcriptional level of pro-inflammatory cytokines and NO,promoted the secretion and transcriptional level of anti-inflammatory cytokines.Hpl MSC-Exos inhibited the M1 polarization of macrophages,the nucleus translocation of NF-κB p65,the activation of toll like receptor 4(TLR4)-mediated mitogen-activated protein kinase(MAPK)/NF-κB and phosphoinositide-3 kinase(PI3K)/Akt,and suppressed apoptosis,thus exerting anti-inflammatory activity.(2)Preparation,characterization and biological properties of Lb L-Exos.Oxidized konjac glucomannan(OKGM)was performed by oxidation and was characterized via FT-IR and ~1H-NMR.Lb L-Exos were established by layer-by-layer(Lb L)technology and were characterized.MTT was used to measure the cytotoxicity of Lb L-Exos.Zeta potential was used to detect enzyme degradation of the outer membrane OKGM of Lb L-Exos.The stability and release of Lb L-Exos were measured in mimic gastric and intestinal fluid.The anti-inflammatory activity of released Exos was measured by ELISA and q RT-PCR.The uptake of Hpl MSC-Exos by RAW264.7 or HT-29 cells were observed under a confocal laser scanning microscopy.The results showed that OKGM and Lb L-Exos were successfully prepared.OKGM could be specially degraded byβ-mannanase.Lb L-Exos had no cytotoxicity,good biocompatibility,stability and sustained release.In addition,the released Exos still had anti-inflammatory activity.Furthermore,Hpl MSC-Exos were internalized into RAW264.7 and HT-29 cells.(3)Therapeutic effect of Lb L-Exos on C57BL/6J UC mice.The colitis model was induced using 3%dextran sulfate sodium(DSS).The colonic pathology was observed by H&E staining.The location of Lb L-Exos in mice was visualized by a living animal imaging system.The content of malondialdehyde(MDA)and glutathione(GSH)was measured.The secretion of inflammatory cytokines in colon and serum was measured by ELISA.The secretion of NO in colon and serum was detected by Griess method.The transcriptional level of inflammatory cytokines and i NOS was measured by q RT-PCR.The expression of myeloperoxidase(MPO)was evaluated by immunohistochemical(IHC)method.The subtypes of splenic macrophages were detected by flow cytometry,and the expression of macrophage polarization-related proteins was measured by WB.The expression of inflammation and apoptotic associated proteins was measured by WB.The expression of tight junction protein,ZO-1,was evaluated by immunofluorescence.The apoptosis of intestinal cells was detected via TUNEL.The results showed that intravenous administration of Hpl MSC-Exos and oral administration of Lb L-Exos improved the structure of colon.Lb L-Exos homed to the colon.ntravenous administration of Hpl MSC-Exos and oral administration of Lb L-Exos adjusted oxidative stress level,inhibited the secretion and m RNA expression of inflammation cytokines and NO,inhibited M1polarization in splenic and colonic macrophages and the activation of MAPK/NF-κB signaling pathway,raised the expression of ZO-1,inhibited the apoptosis of colonic tissues,thus improving DSS-induced colitis.In summary,Hpl MSC-Exos could play an anti-inflammatory role via suppressing the activation of TLR4-mediated MAPK/NF-κB and PI3K/Akt.The new oral drug delievery system,Lb L-Exos had good biocompatibility,stability,sustained release and targeting,which increased the bioavailability of Hpl MSC-Exos,and fully exerted the anti-inflammatory properties,thereby alleviating DSS-induced colitis in mice.This provided a new strategy for treatment of gastrointestinal diseases such as ulcerative colitis in future. |
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