The Role And Mechanism Of High Mobility Group Box 1 In Experimental Pulmonary Hypertension

Objective:To explore the role and mechanism of high mobility group box 1?HMGB1?in experimental pulmonary hypertension?PH?.Methods:?1?Chronic hypoxia?CH?exposure and monocrotaline?MCT?,glycyrrhizin and anti-HMGB1 neutralizing antibody treatment.?2?The hemodynamic measurements were performed at the end of the animal experiments by powerlab system;right ventricular/left ventricular+septal ratio?RV/LV+S?were performed to evaluate right heart hypertrophy.?3?Hematoxylin-eosin and in the lung of control,hypoxia and MCT rats with or without Glycyrrhizin?GLY?and anti-HMGB1 neutralizing antibody treatment respectively to evaluate pulmonaryvascularwallthicknessandHMGB1compartmentdistribution;immunohistochemical staining were also performed to detect HMGB1compartment distribution in the lung of control,hypoxia and MCT rats with or without GLY.?4?HMGB1expression level were detected by quantity real time PCR and western blotting;and PI3K/Akt expression level was also detected by western blotting.?5?PASMCs cytosolic Ca²⁺([Ca²⁺]_i)was measured by calcium fluorescence probe fura-2AM and CCD imaging system.?6?Immunofluorescence staining of HMGB1 in PASMCs was performed to observe its cell compartment location.?7?Cell Counting Kit-8?CCK8?and wound healing migration assay were used to assess the proliferation and migration of PASMCs incubated with HMGB1 or exposed to hypoxia condition respectively.?8?HMGB1 levels in the culture medium of PASMCs exposed to normoxia and hypoxia?1%O₂ for the indicated time?and in the blood plasma of hypoxia and MCT-treated rats were measured by Enzyme-Linked ImmunoSorbent Assay?ELISA?.Results:?1?Hypoxia and MCT-induced PH model successfully established by the increased pressure of pulmonary artery?Ppa?,right ventricular systolic pressure?RVSP?,pulmonary vascular resistance?PVR?,?right ventricular?/?left ventricular+septal?[RV/?LV+S?]ratio and pulmonary vascular remodeling.?2?HMGB1 mRNA and monomer protein level increased in lung tissue of hypoxia rats,however,the monomer protein level decreased in MCT rats;HMGB1 tetramer expression level consistently upregulated in hypoxia and MCT rats and the ratio of tetramer to monomer was also consistently increased in these two models.?3?The exposure of PASMCs either to H₂O₂ or to 1%O₂ for 24h induced the translocation of nuclear HMGB1 to cytosol with a concomitant increased HMGB1 staining in cytoplasm;pretreatment with catalase inhibited the HMGB1 translocation in PASMCs;cytoplasm positive HMGB1cells increased in the lung tissue of rats after hypoxia exposure and MCT treatment.?4?HMGB1 induced PASMCs[Ca²⁺]_i mobilization via TLR4 activation of TRPC-dependent calcium entry and this effect was blocked by TLR4 inhibotor TAK-242 and TRPC suppression with 2-APB and SKF-96365.?5?HMGB1 promoted the migration of PASMCs via TLR4-dependent PI3K/Akt signal pathways activation,however,it had no effect on PASMCs proliferation.?6?Gly treatment attenuated the hemodynamic change,vascular remodeling and right ventricular hypertrophy in the above rat models of PH;The inflammation of lung tissue and particularly the perivascular tissue were alleviated in Gly-treated rats.?7?anti-HMGB1neutralizing antibody attenuated lung inflammation and PH establishment in rats after hypoxia exposure and monocrotaline treatment.Conclusion:This study demonstrates that HMGB1 is critical in experimental PH and identifies HMGB1 as a potential diagnosis biomarker and therapeutic targets for the treatment of this incurable disease.