| Background: Endometriosis(EMS)refers to the occurrence,growth,infiltration and repeated bleeding of endometrial tissue outside the uterus.Neutrophil extracellular traps(NETs)is a network structure with antibacterial function formed by substances such as myeloperoxidase,citrulline histone 3,neutrophil elastase(NE),which is released by neutrophil with DNA as the skeleton.Recent studies have found that NETs is closely related to tumor proliferation and metastasis.However,the biological behavior of EMS is highly similar to that of tumor,and studies have shown that many factors that play an important role in tumors are also important pathogenic factors of EMS.Previous studies have confirmed that the content of NETs in peritoneal lavage fluid of EMS patients is increased,so we speculate that there may be a potential relationship between NETs and endometriosis.Objectives: This study aims to seek the association between NETs and EMS through vivo and vitro experiments,reveal the mechanism of action of NETs in EMS,and lay a foundation for the discovery of new biomarkers and therapeutic targets.Methods:Part ⅠThe ectopic endometrium,ectopic endometrium in EMS patients from both experimental group and control group were collected and detected.Peripheral blood of the experimental group and control group was collected;serum and neutrophil granulocyte(PMN)were separated.The formation of NETs was detected by citrullinated histone(cit H3)and myeloperoxidase(MPO)by immunofluorescence co-localization.The citrullinated histone(cit H3)was semi-quantified by Western blot assay.cit H3 and MPO were detected by immunofluorescence co-localization.The quantitative determination of MPO in peripheral blood serum was performed by enzyme-linked immunosorbent assay(ELISA).Trypan blue staining and flow cytometry were used to detect the apoptosis of peripheral neutrophils.BALB/C mice were modeled by allotransplantation,and the above experiments was repeated after modeling.The mice were dissected weekly and corresponding specimens were collected to observe the formation of NETs over time.EMS disease modeling was performed on BALB/C mice by allotransplantation,and the above experiments were repeated after modeling to further verify the results of clinical experiments.Part ⅡThe endometrium of the experimental group and control group was collected and endometrial stromal cells(HESCs)were isolated and cultured in vitro.Peripheral blood neutrophil of healthy people was collected and PMN was induced to generate NETs by phaboate(PMA)in vitro.HESC cells from different sources were treated with NETs,and cell proliferation was verified by CCK-8 and EDU live cell proliferation techniques,cell apoptosis by flow cytometry,and changes in cell migration ability by scratch assay and transwell assay.Two inhibitors,cl-amidine and DNase I,were used to reversely verify the stimulative effect of NETs and to verify the vitro effect of NETs in vivo.An animal model of mice was established,and the mice were given NETs or their inhibitors or an equivalent amount of PBS.The influence of NETs and their inhibitors on disease formation was determined by detecting the formation of extracellular snare nets in the bone marrow of mice neutrophils,the quantization of peripheral blood MPO,and the size and number of lesion tissue in mice.Part ⅢAfter separation and purification of NET-DNA and its matrix and treatment of HESC cells,CCK-8 was used to detect the proliferation of HESC cells and the migration of HESC cells of scratch assay and transwell assay.The main active components of NETs were determined according to the experimental results.Western blot assay was performed on HESC cells treated with NET-DNA and the related Toll-like(TLR)receptors were screened.The overexpression and knockdown HESC of TLR4 receptor were constructed,and the transfection rate was determined by Western blot.CCK-8 was used to detect the changes in the proliferation and invasiveness of HESC cells treated with NET-DNA after overexpression or knockdown.By reading and analyzing previous literature,possible pathway signals were screened,NETs and their inhibitors were used to treat HESC cells with overexpression of TLR4 or TLR4 knockdown respectively,and activated proteins in pathway signals were screened out by Western blot assay.Results:Part ⅠNETs increases in patients.MPO in peripheral blood of ELSIA patients was increased: the experimental group was higher than that of the control group;flow cytometry experiment PMN apoptosis rate in the experimental group was less than that in the control group.cit H3 immunofluorescence intensity analysis results: the ectopic endometrium of the experimental group was higher than that of the control group.Western blot results: The ectopic endometrium of the experimental group was higher than that of the control group.NETs increases in EMS mice.peripheral blood MPO was significantly increased in the experimental group,experimental group vs.Control group.the average fluorescence intensity of cit H3 was higher in ectopic tissue than in control group.Western blot results: The ectopic endometrium of the experimental group was higher than that of the control group.Part ⅡNETs promoted cell proliferation: OD450 value in CCK-8 proliferation assay was higher in the experimental group than in the inhibitor group and control group.EDU results: the experimental group was higher than the inhibitor DNase I and cl-amidine group and control group.NETs inhibited apoptosis.Flow cytometry showed that the experimental group was less than the inhibitor DNase I and cl-amidine group and control group,P<0.001.NETs promoted cell migration and invasion.Scratch test mobility results:experimental group was higher than inhibitor DNase I and cl-amidine group and control group.Transwell-invasion results: experimental group was higher than inhibitor DNase I and cl-amidine group and control group Results of transwell-migration: experimental group was higher than inhibitor DNase I and cl-amidine group and control group.NETs promoted the formation and metastasis of endovariant lesions in vivo: the size of small lesions in the treated mice group was larger than that in the inhibitor and cl-amidine and DNase I groups and PBS group.Part ⅢNET-DNA promoted the proliferation and metastasis of HESC cells.the results of CCK-8 proliferation experiment showed that the DNA group was higher than the matrix group.EDU results: DNA group was higher than matrix group.Scratch test results: DNA group was higher than matrix group.Transwell-invasion results: DNA groupwas higher than matrix group.Transwell-migration results: DNA group was higher than matrix group.As TLR4 was overexpression,it is demonstrated that the proliferation and metastasis of HESC improved in the results of CCK-8 and transwell compared with control group and sh-nc groups.However,the preponderance of NET-DNA group disappeared after HESC was knocked out TLR4 receptor.NET-DNA activated STAT3,ERK-MAPK signaling pathways through up-regulation of TLR4 receptor.Western blot results: After overexpression of TLR4 receptor,P-STAT3/STAT3 protein in experimental group was higher than that in control group,inhibitor group and sh-nc group;P-p38/p38 protein in experimental group was higher than that in control group inhibitor group,and sh-nc group;P-ERK/ERK protein in experimental group was higher than that in control group inhibitor group and sh-nc group,and the significant difference disappeared after TLR4 was knocked out.Conclusions:1.Abnormal activation of NETs existed in the blood and tissues of patients with endometriosis.2.NETs promoted the proliferation and invasion of HESC cells.3.NET-DNA was the main active promoter of NETs in endometriosis.4.The ability of NET-DNA that activated p38,ERK MAPK and STAT3 signaling pathways depends on TLR4 receptor. |
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