Mechanism Of HucMSCs Regulating Macrophages 15-LOX-1 To Promote The Repair Of Inflammatory Bowel Disease

Objective:The human umbilical cord mesenchymal stem cells(huc MSCs)repairing dextran sulfuric acid(DSS)induced IBD model and diphtheria toxin(DT)inducing macrophage clearance model were established in this study to discuss the role of macrophages in MSCs alleviating IBD and the potential mechanism of MSCs regulating macrophages to alleviate IBD,and then to provide theoretical basis for clinical application of MSCs to treat IBD and experimental basis.Methods:1)The homozygous CD11b-DTR transgenic mice were subjected to cage propagation,and homozygous mice were screened for subsequent experiments using common polymerase chain reaction(PCR)technique and agarose gel electrophoresis.2)6 weeks old male CD11b-DTR transgenic mice were pre-starved for 24 hours,and DT was injected intraperitoneally to construct a macrophage clearance model at a concentration of 25 ng/g.After 24 hours,mouse peritoneal macrophages were extracted and stained with F4/80+ and CD11b+ antibodies.The ratio of F4/80 and CD11 b double positive cells was detected by flow cytometry.3)Male CD11b-DTR transgenic mice of about 20 g for 6 weeks were selected and fed with 3% DSS diluted with autoclaved water to construct a DSS model.4)To explore the role of macrophages in DSS-induced IBD,mice were divided into Ctrl,DT,DSS,DT+DSS groups.DT was injected one day before DSS feeding and refilled every 4 days.Mice body weight and DAI were recorded every day.The mice were sacrificed on the 10 th day,the colon and spleen were taken for gross observation and were prepared for tissue sections and RNA samples.The tissue microstructures were analyzed by H&E staining.The expression levels of inflammatory factors in tissues were detected by qRT-PCR.5)To explore the role of macrophages in the remission of IBD from huc MSCs,mice were divided into 7 groups: Ctrl,DSS,DSS+DT,DSS+huc MSC,DSS+DT(day0)+huc MSC,DSS+DT(day3)+huc MSC and DSS+DT(day6)+huc MSC group,DT intraperitoneal injection time was 0,3,and6 days of the experiment,and refilled every 4 days.3×106 huc MSCs were intraperitoneally injected on the third day of the experiment and refilled every 3days.Body weight and DAI were recorded daily and sacrificed in about 10 days.The spleen and colon were taken to take a general view of the tissue and extract tissue sections,protein and RNA samples.H&E staining was used to analyze microscopic lesions;PCNA-positive cells were detected by immunohistochemistry;inflammatory factors were analyzed by q RT-PCR and caspase-3,PCNA and 15-LOX-1 protein expression level was detected by western blot.6)To explore whether huc MSCs alleviate IBD by regulating macrophage 15-LOX-1,mice were divided into 5 groups: Ctrl,DSS,GFP-DSS+huc MSC,15-LOX-1-DSS+huc MSC and 15-LOX-1-DSS+DT+huc MSC group,1×109 PFU of GFP or 15-LOX-1 recombinant adenovirus were injected into the tail vein two days before DSS feeding.Huc MSCs injection is the same as above.Body weight,DAI,gross view of tissue,microscopic organization,protein and RNA detection are the same as above.7)In order to screen the key molecules of huc MSCs regulating macrophage15-LOX-1,the mi RNAs predicted by the database that can target the macrophage15-LOX-1 m RNA 3'UTR was cross-aligned with the huc MSCs exosomal mi RNA sequencing results,to screen the overlapping mi RNAs.LPS-stimulated RAW 264.7 cells were co-cultured with huc MSCs for 48 h,and the expression levels of mi RNAs in RAW 264.7 cells were detected by q RT-PCR.After confirming mi R-148b-5p as a follow-up subject,mi R-148b-5p mimics,negative control and inhibitor were designed to transfect huc MSCs to overexpress or knock down mi R-148b-5p in huc MSCs respectively.After transfected huc MSCs co-cultured with LPS stimulated RAW 264.7 cells for 48 h,the expression levels of mi R-148b-5p and 15-LOX-1 m RNA in RAW 264.7 cells were detected by q RT-PCR;the expression level of 15-LOX-1 protein in RAW 264.7 cells was detected by western blot.8)In order to verify in vivo that huc MSCs down-regulate macrophage 15-LOX-1 by mi R-148b-5p to alleviate IBD,mice were divided into Ctrl,DSS,DSS+inhibitor-huc MSC,DSS+huc MSC and DSS+mimics-huc MSC groups.3×106huc MSCs transfected with mi R-148b-5p mimics,negative control and inhibitor respectively were injected intraperitoneally on the third day of the experiment and refilled every 3 days.Body weight,DAI,gross view of tissue,microscopic organization,protein and RNA detection are the same as above.Results:1)The macrophage clearance model was successfully established.Flow cytometry showed that DT treatment could effectively remove bone marrow-derived macrophages with the rate over 98.9%.2)Compared with the DSS group,after the macrophage clearance,the body weight loss was reduced but the DAI was decreased;the length of the colon was not different but the spleen was slightly reduced;although the tissues were still disordered,but to some extent reconstruction;at the same time,the expression of inflammatory factors in tissues was reduced.3)Compared with MSC group mice,huc MSCs failed to alleviate body weight loss,decrease DAI,prolong colon length or shorten spleen size in mice with macrophage clearance before or on the day of huc MSCs injection;lost the tissue remodeling ability,and failed to inhibit the level of inflammatory factors and promote tissue cell regeneration.4)Compared with the GFP-DSS+huc MSC group,the ability of huc MSCs to alleviate IBD in 15-LOX-1-DSS+MSC mice decreased,but the difference was not obvious.However,after 15-LOX-1 overexpressing macrophages in IBD mice,huc MSCs could not reduce body weight loss and DAI;failed to reduce colon and spleen size and microstructure,to promote tissue proliferation and to inhibit the level of inflammation.5)After RAW 264.7 cells were co-cultured with different transfected huc MSCs for48 h,mi R-148b-5p mimics transfected huc MSCs significantly increased the expression level of mi R-148b-5p in RAW 264.7 cells and inhibited 15-LOX-1m RNA compared with control huc MSCs.6)Compared with the control hucMSCs,the hucMSCs transfected with mi R-148b-5p inhibitor lost the functions of repairing body weight loss,DAI,colon and spleen gross and microscopic disorder in IBD mice,and failed to inhibit inflammation and 15-LOX-1 expression levels,and to increase tissue regeneration indicators.However,after overexpression of mi R-148b-5p,the function of huc MSCs to alleviate IBD and inhibit 15-LOX-1 expression was enhanced.Conclusions: Huc MSCs attenuate DSS-induced IBD by releasing mi R-148b-5p to down-regulate macrophage 15-LOX-1 expression levels.