Effect Of Glycolysis In Regulating Pulmonary Macrophage Polarization On Pulmonary Vascular Remodeling In Rats With Hypoxic Pulmonary Hypertension And The Protective Mechanism Of 17β-estradiol

Objective:To explore the effect of glycolysis and pulmonary macrophage polarization on pulmonary vascular remodeling in rats with hypoxic pulmonary hypertension and the pulmonary vascular protective mechanism of 17β-estradiol（E2）.Methods:1.Animal experiment:A total of 30 healthy female SD rats were randomly divided into 5 groups（n=6 each）:Ovariectomy（OVX）+normoxia group,OVX+normoxia+E2 group,OVX+hypoxia group,OVX+hypoxia+E2 group,OVX+hypoxia+dichloroacetate（DCA）group.All rats underwent oophorectomy and castration.The two groups with E2 intervention received subcutaneous injection of E2 20ug/kg daily,and the rest groups received subcutaneous injection of normal saline 0.1ml daily.The group with DCA intervention received DCA 80mg/kg daily by intragastric gavage,and the rest groups received equivalent volume of normal saline by intragastric gavage.The hypoxic groups were placed in the hypoxic chamber（24 hours per day for 8 weeks）to establish HPH model and normoxic groups were kept in the room air.Mean pulmonary artery pressure（m PAP）and right ventricular hypertrophy index（RVHI）were measured.Morphological changes of pulmonary artery remodeling were observed by HE staining.The macrophage markers NOS2 and CD206 were labeled by immunofluorescence assay.Real-time PCR and Western blot were used to detect the levels of NOS2 and CD206.The lactic acid concentrations in lung tissues were measured using a lactic acid kit.2.In vitro:Rat lung macrophages NR8383 cultured in vitro were randomly divided into 4 groups:normoxia group,hypoxia group,hypoxia+E2(10^-8mol/L)group,hypoxia+DCA（10mmol/L）groups.The macrophage markers NOS2 and CD206 were labeled by immunofluorescence assay.Real-time PCR and Western blot were used to detect the levels of NOS2 and CD206.The lactic acid concentrations from culture supernatant were measured using a lactic acid kit.Results:1.Animal experiment:Compared with OVX+normoxia group,the groups under hypoxic conditions showed thickened pulmonary artery wall,increased m PAP and RVHI,massive infiltration of macrophages,and significantly increased expression of NOS2,CD206 and lactic acid concentrations（P<0.01）.Compared with OVX+hypoxia group,morphological changes of pulmonary artery remodeling were significantly attenuated,and decreased expression of NOS2 and lactic acid concentrations,increased expression of CD206 were observed in OVX+hypoxia+E2 group and OVX+hypoxia+DCA group（P<0.01）.The difference between two normoxia groups had no statistical significance（P>0.05）.2.In vitro:Compared with normoxia group,the groups under hypoxic conditions showed obvious proliferation and significantly increased expression of NOS2,CD206 and lactic acid concentrations（P<0.01）.Compared with hypoxia group,the proliferation of macrophages were obviously reduced,and decreased expression of NOS2 and lactic acid concentrations from culture supernatant,increased expression of CD206 were observed in hypoxia+E2 group and hypoxia+DCA group（P<0.01）.Conclusions:Under environment of long-term chronic hypoxia,macrophages could regulate their phenotypes through changes in glycolysis,and participate in the pulmonary vascular remodeling in hypoxic pulmonary hypertension.E2 may promote macrophage to type M2 polarization by reducing the glycolysis level,thus improving pulmonary artery remodeling in hypoxic pulmonary hypertension.