| Objective: This search is to explore the effect of Bushen Tiaochong Decoction on the expression of mi RNA in BMSCs exosomes,and then to explore the possible mechanism of treatment of thin endometrium.Methods:(1)Through the digital random method,42 SD female rats aged 7-8 weeks were randomly divided into 6 groups: normal group,model group,western medicine group and low-dose,medium or high-dose Chinese medicine group.Except for the normal group,the rats in the remaining 5groups were established as thin endometrial rat models during estrus and identified by HE staining.Low,medium,and high-dose traditional Chinese medicine groups were given Bu Shen Tiao Chong decoction which crude drug amounts of 0.8525 g/ml,1.705 g/ml,and 3.410 g/ml respectively by gavage.The western medicine group was given estradiol valerate which concentration of 0.01mg/100 g by gavage.The normal group and the model group were given 1ml/100 g normal saline by gavage.Once a day for 7 consecutive days.Then blood was collected from the abdominal aorta to prepare medicated serum.(2)The whole bone marrow adherence method was used to extract BMSCs from 4-week-old rats.And pass them down to the third generation.Observe the cell morphology under a microscope,and detect CD34、CD44、CD90 by flow cytometry for identification.(3)The P3 generation BMSCs were randomly divided into 7 groups: control group,normal group,model group,western medicine group and low,medium or high dose Chinese medicine group.To culture for 24 h,48h and 72 h by the corresponding medium,and to detect the proliferation of BSMCs by CCK-8.The best dose of Bushen Tiaochong Decoction was determined.(4)The BMSCs exosomes were extracted by ultra-high-speed centrifugation.The morphology was detected by electron microscopy,particle size was detected by NTA and TSG101 and Calnexin were detected by Western Blot for identification.(5)High-throughput sequencing technology was used to screen the differentially expressed mi RNAs of BMSCs exosomes among each group.Through GO function and KEGG pathway enrichment analyswasof target genes,in order to explore the biological processes involved.Results:(1)Compared with the normal group,the rat’s endometrium of the model group became thinner,the interstation was loosened,and the blood vessels and glands were reduced.(2)Microscopic showed that BSMCs grew adherently,long fusiform,vortex-like or radially trending,and tightly connected.Flow cytometry showed that BSMCs highly expressed CD44 and CD90,but hardly expressed CD34.(3)The CCK-8 method test results showed:(1)Compared with the control group in the same time period,the OD values of the western medicine group and the low,medium and high dose Chinese medicine group increased at 24 h,48h and 72h(P<0.05);(2)Compared with the western medicine group and the low-dose Chinese medicine group,the OD value of the high-dose Chinese medicine group increased at 24 h,48h,72h(P<0.05);(3)Compared with the middle-dose Chinese medicine group at the same time period,the high-dose Chinese medicine group.The OD value of the group at 48 h and 72 h increased(P<0.05);(4)The OD value of the low,medium and high dose Chinese medicine group increased with the increase of time,24h<48h<72h(P<0.05).(4)Observed under the electron microscope,the exosomes were oval or round vesicle-like structures,with a complete double-layer membrane,resembling a "saucer".The average particle size of exosomes detected by NTA was 73.09 nm.Western Blot detects that exosomes express TSG101 but not Calnexin.(5)The differentially expressed mi RNAs between each group were screened by high-throughput sequencing technology:(1)Between normal serogroup and model serogroup: mi R-141-3p was an up-regulated gene,mi R-351-5p and mi RNA-451-5p were down-regulated genes;(2)Between model serogroup and traditional Chinese medicine serogroup: mi R-21-5p,mi R-31a-5p,mi R-181a-5p,mi R-195-5p,mi R-503-5p,mi R-132-3p were up-regulated genes,let-7bc-5p,let-7b-5p,mi R-223-3p,mi R-122-5p were down-regulated genes;(3)Between model serogroup and western medicine serogroup: mi R-103-3p,mi R-31a-5p,mi R-351-5p was an up-regulated gene,mi R-223-3p,mi R-126a-3p,mi R-122-5p,mi R-141-3p were down-regulated genes;(4)Between traditional Chinese medicine serogroup and western medicine serogroup: mi R-103-3p was Up-regulated genes,mi R-150-5 and mi R-503-5p were down-regulated genes.(6)Enrichment analyzed by GO function and KEGG pathway:(1)Between normal serogroup and model serogroup,there were 110 cytological components,including: synapses,synaptic vesicles,et al;there were 609 biological processes,including: potassium ion transport across membranes,cellular potassium ion transport,et al;there were 80 molecular biological functions,including: potassium channel activity,voltage-gated potassium channel Activity,et al;there were 39 signal pathways,including: hippo signal pathway,mucin-type O-glycan biosynthesis,et al.(2)Between the model serogroup and the traditional Chinese medicine serogroup,there were 130 cytological components,including: neuron projection,synapse,et al;there were 488 biological processes,including: neuron projection Extension,localization regulations,et al;there were 103 molecular biological functions,including: transferase activity,transfer of phosphorus-containing groups,et al;there were mainly 50 KEGG pathways,including: axons Guidance,AMPK signaling pathway,et al.(3)Between the model serogroup and the western medicine serogroup,there were 93 cytological components,including:dendrites,neuron projections,et al;there were 611 biological processes,including: stress-activated MAPK the regulation of cascade reaction,the regulation of JUN kinase activity,etc.;there were 81 molecular biological functions,including: metal ion transmembrane transport protein activity,transferase activity,et al;there were 43 KEGG pathway,including: Wnt signaling pathway,hippo signaling pathway,et al.(4)Between the traditional Chinese medicine serogroup and the western medicine serogroup,there were75 cytological components,including: potassium channel activity,voltage-gated potassium channel activity,et al;there were 483 biological processes 483,including: negative regulation of protein depolymerization,glycoprotein biosynthesis process,et al;there were 86 molecular biological functions,including: peptide binding,transcription factor activity,et al;there were 46 KEGG pathways,including: hippo signaling pathway,insulin resistance,et al.Conclusion:(1)Different doses of Bushen Tiaochong Decoction can promote the growth of BMSCs.High-dose Bushen Tiaochong Decoction has the best proliferation-promoting ability of BMSCs.(2)Bushen Tiaochong Decoction interferes with the differential expression of mi RNAs in BSMCs exosomes,possibly by regulating the Wnt signaling pathway to induce the proliferation of endometrial glandular epithelial and stromal cells,promote endometrial blood vessels and glands,and reduce endometrial fibrosis;by regulate the HIF-1 signal pathway and its downstream factor VEGF to induce endometrial vascular regeneration;by regulate the Hedgehog signal pathway to induce endometrial angiogenesis,reduce blood flow resistance,restore endometrial blood supply,and then participate in the thin endometrial membrane Organization’s repair process. |
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