17-β Estradiol Regulates Macrophage Phenotype In Hypoxic Pulmonary Hypertension Via HIF1α/IL6/STAT3 Signaling Pathway

Objective:In this experiment,vivo studies（using hypoxia and ovariectomy rats to establish hypoxic pulmonary hypertension models）and vitro studies（in vitro alveolar macrophage culture in rats）were performed to investigate the regulatory role of E₂on macrophages in hypoxic pulmonary hypertension and whether it plays a role through the HIF1α/IL6/STAT3signaling pathway.Methods:1 Vivo studies:choose about thirty 6-8 weeks female SD rats,randomly divided into 5 groups:sham-operation normoxia group、ovariectomy normoxia+E₂ group、sham-operation hypoxia group、ovariectomy hypoxia group、ovariectomygroup.Rats of ovariectomy group were performed ovariectomized surgery.Rats of sham-operation group only opened the abdominal cavity,did not do the treatment after finding the ovary.After that,rats of hypoxia group were placed in atmospheric hypoxia chamber,24 hours per day of sustained hypoxia.Rats of E₂group was given subcutaneously with17-βestradiol 20μg/kg daily.After 8 weeks,mean pulmonary arterial pressure（m PAP）and right ventricular hypertrophy index（RVHI）of rats was measured,the morphology of pulmonary arterioles was observed by HE staining and WA%and WT%were measured.Immunofluorescence identified rat lung macrophage markers F4/80、CD206、INOS.Protein and m RNA expression levels of HIF1α、IL6 and STAT3 in rat lung tissues were determined by RT-PCR and Western blot.2 Vitro studies:NR8383 cells were cultured to randomly divided into normoxia group,hypoxia group,hypoxia+E₂(10^–8 mol/L)group.Normoxia group was incubated in a incubator with 37℃,95%O₂,5%CO₂ for 24h.The hypoxia groups were incubated in a three-gas incubator with 37℃,3%O₂,5%CO₂and 92%N2 for 24 h.Immunofluorescence identified rat lung macrophage markers CD206、INOS.m RNA expression levels of CD206、INOS、HIF1α、IL6、STAT3 in NR8383 cells were determined by RT-PCR.Protein levels of HIF1α、IL6、STAT3 in NR8383 cells were determined by Western blot.Results:1 Vivo studies:compared with sham-operation normoxia group,three groups of rats under hypoxic conditions increased their weight slowly,m PAP、RVHI、WA%and WT%were higher,local thickening of pulmonary arterioles,stenosis of lumen,and massive infiltration of macrophages,the immunofluorescence AO value of F4/80、CD206、INOS were higher;compared with ovariectomy hypoxia group,m PAP、RVHI、WA%and WT%of ovariectomygroup decreased,AO value of F4/80 and INOS decreased,AO value of CD206 increased;Protein and m RNA expression levels of HIF1α、IL6 and STAT3:compared with sham-operation normoxia group,three groups of rats under hypoxic conditions increased,compared with ovariectomy hypoxia group,protein and m RNA level of ovariectomygroup significantly decreased.2 Vitro studies:under inverted phase contrast microscope:the number of cells in hypoxia group was increased compared with that in normoxia group,most of the cells were rounded;CD206 fluorescence staining in NR8383showed that the positive level of hypoxia group was significantly higher than that of normoxia group,and that ofgroup was higher than that of hypoxia group.INOS fluorescence staining showed that the positive level of hypoxia group was significantly higher than that of normoxia group,and that ofgroup was lower than that of hypoxia group;compared with normoxia group,m RNA level of CD206、INOS、HIF1α、IL6 and STAT3 of hypoxia group increased;compared with hypoxia group,m RNA level of INOS、HIF1α、IL6 and STAT3 ofgroup decreased;m RNA level of CD206 increased;compared with normoxia group,protein level of HIF1α、IL6 and STAT3 of hypoxia group increased;compared with hypoxia group,protein level of HIF1α、IL6 and STAT3 ofgroup decreased.Conclusions:1 Long-term chronic hypoxia can induce hypoxic pulmonary arterial hypertension,and the disease of ovariectomy rats is aggravated;E₂may regulate macrophage phenotype and inhibit pulmonary vascular remodeling in hypoxic pulmonary hypertension by inhibiting the expression of HIF1α/IL6/STAT3 signaling pathway.