| Objective:Human umbilical cord mesenchymal stem cell-derived exosomes(hucMSC-Ex)were established to repair dextran sulfate sodium salt(DSS)-induced inflammatory bowel disease in mice.The inflammatory bowel disease(IBD)model was used to determine the role of neddylation in the alleviation of IBD in mice by hucMSC-Ex,the mechanism of hucMSC-Ex in regulating the neddylation process to repair IBD in mice was further studied,and hucMSC-Ex was further used to treat IBD.Methods:1)Isolation and identification of hucMSC-Ex: The human umbilical cord mesenchymal stem cells(hucMSC)culture supernatants were collected,and the hucMSC-Ex was extracted by the corresponding reagents and identified by projection electron microscopy,NanoSight and western blot.2)Male BABL/C mice at 6 weeks and about 20 g were selected,and they drank water containing 3% DSS to build an IBD model.To determine whether hucMSC-Ex relieved IBD in mice by regulating the neddylation process,the mice were randomly divided into 3 groups: Neg,DSS and hucMSC-Ex groups,and 1 mg hucMSC-Ex was injected into the tail vein every 3 days.The body weight of the mice was recorded at the same time point every day,the fecal characteristics of the mice and the presence of bloody stools were observed,and according to this the disease activity index(DAI)was analyzed.The mice were sacrificed at about 10 th day,and the mice’s colorectum was taken to observe the general view of the tissues.Colon tissues were collected and tissue sections were prepared,and protein and total RNA were extracted.We analyzed colonic structural disorders by H&E staining,detected neddylation-related molecules(NEDD8,cullin 1)in tissues by IHC,and uesd qRT-PCR to analyzed inflammatory factors(IL-1β,IL-6,IL-10,TNF-α)expression in colon tissues and expression of neddylation-related molecules(NAe1,Uba3,E2 M,UBC12F,DCNL1).The neddylation-related molecules(NEDD8,cullin 1,IκB,NF-κB,P-NF-κB)proteins in colon tissues were detected by western blot.The combination of NEDD8 and cullin 1 was detected by Co-IP.3)Human colorectal mucosa cells(FHC)were cultured in vitro,the inflammatory environment was simulated by lipopolysaccharide(LPS),and hucMSC-Ex was added to relieve inflammation.The cells were divided into three groups: Neg,LPS,and hucMSC-Ex groups.After 12 h of co-cultivation,the cells were collected to extract cellular proteins and total RNA.The expression of neddylation-related molecules in cells was detected by IF analysis,qRT-PCR and western blot,and the binding of NEDD8 to cullin 1 was detected by Co-IP.4)In order to determine which key molecule in hucMSC-Ex regulated the neddylation process,the miRNA predicted by the database that can target the 3’UTR of NEDD8 mRNA was cross-matched with the hucMSC-Ex miRNA sequencing results and the coincident miRNAs were screened.LPS induced the inflammatory environment of FHC.After adding hucMSC-Ex and co-culturing for 12 h,the cells were collected.QRT-PCR was used to detect the expression level of miRNAs screened in FHC.The experiment was repeated 4 times to determine miR-326 as a key molecule.miR-326 mimics,mimics NC,inhibitor and inhibitor NC were designed and transfected with FHC to overexpress or knock down miR-326 in cells.The transfected cells were cultured for 12 h under the induction of LPS,and the cells were collected to extract cellular proteins and total RNA.The expression of neddylation-related molecules in cells was detected y IF analysis,qRT-PCR and western blot,and the binding of NEDD8 to cullin 1 was detected by Co-IP.5)Transfection kits were used to transfect miR-326 mimics,mimics NC,inhibitor and inhibitor NC with hucMSC-Ex,and the expression of miR-326 in hucMSC-Ex was detected by qRT-PCR to verify the transfection effect.6)To verify that hucMSC-Ex repaired IBD in mice by inhibiting the neddylation process through miR-326,male BABL/C mice at about 6 weeks and about 20g were randomly divided into Neg,DSS,mimics,mimics NC,inhibitor and inhibitor NC groups,drinking water containing 3% DSS to construct an IBD model,and 1 mg of hucMSC-Ex transfected with miR-326 mimics,mimics NC,inhibitor and inhibitor NC were injected into the tail vein every 3 days.Body weight,DAI,gross view of colorectal tissue,tissue structure,protein and total RNA were the detected as above.Results:1)HucMSC-Ex was successfully isolated.NanoSight nanoparticle tracking analyzer and transmission electron microscope were used to detect hucMSC-Ex with a diameter of about 100 nm.Western blot detected hucMSC-Ex to express CD9,CD63 and CD81.2)The weight of the mice in the Neg group increased with time,but the weight of the mice in the DSS group decreased on the 5th day,and blood stools also appeared,and the DAI increased sharply.The mice in the hucMSC-Ex group gained weight in the first 6 days.There was almost no change,and the body weight slightly decreased after the 6th day,but it was not obvious.The colon of DSS mice was significantly shortened,while the length of colons of hucMSC-Ex mice recovered.The expression of pro-inflammatory factors was increased in the colon tissue of mice in the DSS group,but the expression was significantly reduced in the colon tissue of the mice in the hucMSC-Ex group.On the contrary, the expression of anti-inflammatory factors was reduced in the colon tissue of the mice in the DSS group and was less increased expression in mouse colon tissue.3)Compared with the Neg group,the expression of NEDD8,cullin 1,P-NF-κB in colon tissue of DSS group increased,and the expression of related molecules(NAe1,Uba3,E2 M,UBC12F,and DCNL1)increased during neddylation,the binding of NEDD8 to cullin 1 was promoted.The expression level of neddylation-related molecules in the colon tissue of mice in the hucMSC-Ex group decreased,and the binding of NEDD8 to cullin 1 was also inhibited.4)The inflammatory environment of cells were simulated in vitro.Compared with Neg group,the expression level of neddylation-related molecules in LPS group was increased.The binding of NEDD8 to cullin 1 was enhanced.After treatment with hucMSC-Ex,the expression level of neddylation-related molecules in cells were decreased,and the binding of NEDD8 to cullin 1 was inhibited.5)FHC were transfected with miR-326 mimics,mimics NC,inhibitor and inhibitor NC,then stimulated by LPS,compared with the LPS group,the expression level of neddylation-related molecules in mimics group were decreased and the binding of NEDD8 to cullin 1 was inhibited,but the expression level of neddylation-related molecules in inhibitor did not decreased or even increased,and the binding of NEDD8 to cullin 1 was promoted.6)In terms of body weight,DAI,colon length,and degree of tissue disturbance,mice in the mimics group had significantly stronger repair effects than mice in the mimics NC group.Mice in the inhibitor group had repair effects compared to the DSS group,but were significantly weaker than those in the mimics group.It was slightly weaker than inhibitor NC group.Compared with the DSS group,the expression level of neddylation-related molecules in the mouse colon was reduced in the mimics group,and the binding of NEDD8 to cullin 1 was inhibited.The inhibitor group expression level of neddylation-related molecules in the colon tissue of the mouse was enhanced compared to the inhibitor NC group,the combination of NEDD8 and cullin 1 was also enhanced.Conclusions:HucMSC-Ex relieved DSS-induced mouse IBD through miR-326 inhibiting the neddylation. |
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