| Objective To explore the function of neutrophils in early endometriosis(EMs),and to reveal the role of granulocyte colony-stimulation factor(G-CSF)and interleukin-6(IL-6)on modulating neutrophil.Methods(1)To establish EMs mouse model,recruit or remove peritoneal neutrophils,record and observe the process of lesion formation and neutrophil infiltration and detect the level of G-CSF and IL-6 in serum and peritoneal fluid.(2)To establish the eukaryotic expression vector of G-CSF and/or IL-6,G-CSF receptor/IL-6 receptor/glycoprotein 130(G-CSFR/IL-6R/gp130),and transfect mouse.(3)To establish a mouse model by co-injection a mixture of neutrophils and uterine fragments.(4)To analyze the effect of G-CSF/IL-6-regulated neutrophils on lesion formation and vascular changes,and the effect of blocking this factor on neutrophil function.(5)To detect the expression of angiogenesis-related genes in neutrophils and lesions in the peritoneal cavity of co-injected mice.(6)Mice were transfected with p G-CSF and/or p IL-6 plasmids before uterine fragments injection,and the direct effects of G-CSF and/or IL-6 on neutrophils were investigated through in vivo and in vitro experiments.(7)To investigate the role of signal transducer and activator of transcription 3(STAT3)and phosphatidylinositol3-kinase(PI3K)signaling molecules in the regulation expression of matrix metalloproteinase 9(Mmp9),bombina variegata 8k Da protein(Bv8,also known as kinesin2)and Tumor necrosis factor related apoptosis inducing ligand(Trail)in neutrophils.Results(1)Adhesive fragments formed ectopic lesions on the 3rd day after uterine fragments injection;non-adherent fragments disappeared on the 5th to 6th day.Twelve hours after the uterine fragments were injected into the abdominal cavity,the number of neutrophils increased,and the number of neutrophils in the fragments also increased.Once neutrophils were depleted,the cells were reduced in the fragments.The levels of G-CSF and IL-6 in plasma and peritoneal fluid of EMs mice increased.(2)Neutrophils originated from co-injected mouse EMs group and G-CSF/IL-6 group promoted the formation of lesions.On the contrary,neutrophils from G-CSFR/IL-6R/gp130 group inhibited the formation of lesions.(3)The blood vessels in the lesions and the surrounding were dense,thickened and branches increased in the co-injected group of G-CSF/IL-6 mouse or EMs mouse.While neutrophils were removed,and the blood vessel density decreased.The m RNA expression of Mmp9 and Bv8 in neutrophils in the G-CSF/IL-6 group and the EMs group was higher than that in the control group,while the Trail was lower,and(5)the m RNA and protein expressions of the three genes in the lesions were the same.(6)In the direct effect on neutrophils,the expression of G-CSF or IL-6 alone did not affect the expression of Trail m RNA,while the co-expression of the two decreased the level of Trail and increased the levels of Mmp9 and Bv8 m RNA.This result was similar to the EMs group and was confirmed by in vitro experiments.(7)When STAT3 was inhibited,the expression of Mmp9 and Bv8 in neutrophils decreased,and Trail increased.When PI3 K was inhibited,the expression of Mmp9 and Bv8 was no different compared with the control group,but the expression of Trail was significantly reduced.The inhibitory effect of G-CSF on the expression of Trail gene in the presence of PI3 K inhibitor could be eliminated by the combined use of STAT3 inhibitor,and the expression of Trail was increased.(8)The expression of STAT3 and phosphorylated STAT3 in neutrophils of G-CSF/IL-6 group was significantly higher than single one of G-CSF group or IL-6 group,and there was no statistical difference compared with peritoneal neutrophils of EMs mice difference.Conclusions G-CSF/IL-6 regulates neutrophils through the STAT3 pathway,up-regulates the expression of Mmp9,Bv8 and down-regulates Trail genes,transforms the function of neutrophils,and promotes the formation of early ectopic lesions in mice. |
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