| Hypoxic pulmonary hypertension(HPH)is mainly due to the increase of pulmonary vasoconstriction caused by hypoxia and the abnormal proliferation of thesmooth muscle cells of the terminal pulmonary arterioles.HPH is more common inchronic obstructive pulmonary disease and chronic plateau type lung disease.The current treatment methods are mainly to dilate blood vessels and improve heart function,However,the improvement of the quality of life and survival rate of patients is not ideal.In recent years,many studies have focused on hypoxic pulmonary hypertension,seeking targets and intervening at the genetic level in orderto find better treatments.In this research,we found that high mobility group box-1protein(HMGB1)plays an important role in HPH through early survey and research.Our research group first established a hypoxic model of cells and an animal model of hypoxic pulmonary hypertension.It was proved that HMGB1 plays a very important role in HPH;then the related roles of HMGB1 and inflammation were identified by examining relevant inflammatory markers and proteins,investigate the mechanism of action of HMGB 1.The First Part The expression and function of HMGB1 in HPASMCand HPAEC ceUs induced by hypoxia ObjectiveTo investigate the effects of hypoxia(1% oxygen)on the action of HMGB 1 and its receptors and related inflammatory factors in human pulmonary artery smooth muscle cells and human pulmonary artery endothelial cells;and to investigate the effect of exogenous HMGB1 on the proliferation and migration of HPASMC and HPAEC cells.This proves that HMGB1 induced under hypoxic conditions plays an important role in vascular injury or repair.MethodsFirstly,HPASMC and HPAEC cells were treated with hypoxia for 24 hours,Theexpression of HMGB 1 gene and receptor in cells was detected by PCR.Then cells treated with normal oxygen concentration,HPASMC and HPAEC cells to be added to HMGB 1,then ability of cell proliferation and migration was determined.The amount of related inflammatory factor genes in cells was measured by rt-PCR.ResultsAfter hypoxic treatment of cells,the expression of HMGB1 gene was increased in both cells;hypoxia significantly promoted the inflammatory response of HPASMC cells;we found that 10 μg/ml of HMGB1 significantly inhibited HPAEC by adding different concentrations of HMGB1 in The proliferation of the cells,and HMGB1 at this concentration promotes the migration of HPASMC cells,but there is no significant difference.The expression of RAGE was increased in both cells.ConclusionHypoxia promotes the secretion of HMGB1,while the secretion of HMGB1 significantly inhibits the proliferation and migration of HPAEC and no effect in the proliferation of HPASMC cells;RAGE is the receptor of HMGB1.The Second Part The role of HMGB1 in rats with hypoxic pulmonary hypertension model ObjectiveIn animals,the hypoxic pulmonary hypertension(HPH)model was induced by hypoxia,and the ftmction of the body to secrete a variety of inflammatory factors was examined in the modified condition.MethodsAfter hypoxia treatment,the rats were hypoxic(1% oxygen)for 6 days,8 hours per day for 4 weeks,and the mean pulmonary artery pressure(mPAP)and right ventricular hypertrophy index(RVHI)were measured after the completion of the model;Pathological sections were made and HE staining was used to observe the pathological changes of the lung;HMGB1 gene expression in the lungs of HPH rats was detected by PCR;HMGB1 and inflammatory factors in serum and bronchoalveolar lavage fluid of HPH rats were detected by ELISA method with macrophage-derived chemokine(MDC/CCL22)and tumor necrosis factor-a(TNF-a)expression.ResultsThe rats were treated with hypoxia for 4 weeks,the symptoms of HPH in the rats were clearly found,that is,the mean pulmonary artery pressure was significantly increased,and the pulmonary vascular lumen of the HPH rats were significantly narrowed by pathological section.And the secretion of HMGB1 by HPH rats significantly increased.In addition,the levels of inflammatory cytokines CCL22 and TNF-a in serum and bronchoalveolar lavage fluid of HPH rats were also significantly increased.However,after injection of anti-HMGBl in HPH rats,the secretion of inflammatory factors CCL22 and TNF-a was significantly inhibited in rats.ConclusionHypoxia 4w can cause pulmonary hypertension in rats,and HMGB1 plays animportant role in the development of HPH and inflammation in rats.The Third Part HMGB1 regulates HPH through RAGE ObjectiveThrough previous studies,it has been found that HMGB1 plays an important rolein the hypoxia-induced cell or tissue damage,in vivo and in vitro,but its mechanismof action is not clear.This part mainly investigates the role of HMGB1 and its receptor RAGE(receptor for advanced glycation endproducts)in the development of HPH.MethodsExogenous HMGB1 was added to HPAEC and HPASMC cells to detect theeffect of Nitric Oxide(NO)and inflammatory cytokine secretion;Transfection with plasmid,HMGB1 was used to detect the effect of HMGB1 on RAGE and eNOS promoter activity with the method of luciferase reporter gene;HPH model wasperformed on rats,And detect the role of rat serum and lung tissue on the secretion of NO.ResultsWhen HMGB1 or anti-HMGB1 was added between different groups of HPAEC cells,both hypoxia and HMGB1 inhibited the secretion of NO in HPAEC cells,whileanti-HMGB1 inhibited the damage caused by hypoxia and promoted the secretion of NO.When the concentration of HMGB1 was 4 μg/ml the activity of RAGE promoter was significantly stimulated;and when the concentration was 2 μg/ml,the activity of eNOS promoter was obviously stimulated,but μmwhen the concentration of HMGB1 was increased,it showed an inhibitory effect.After adding HMGB1 or anti-HMGB1 to different groups of HPASMC cells,hypoxia and HMGB1 were found to promote the secretion of inflammatory cytokines in HPASMC cells,while anti-HMGB1 inhibited the damage caused by hypoxia on cells and inhibited the secretion of inflammatory factors..When HMGB1 or anti-HMGB1 was added between different groups of two cells,both hypoxia and HMGB1 promoted the expression of RAGE in both cells,while anti-HMGB1 inhibited the damage caused by hypoxia and inhibited the expression of RAGE..After normoxic rat or hypoxic rat HMGB1 or anti-HMGB1 was administered,we found that hypoxia significantly inhibited NO secretion in rat serum and bronchoalveolar lavage fluid,promoted the expression of inflammatory factors,and promoted RAGE in lung tissue Expression;and anti-HMGB1 will significantly improve the damage caused by hypoxia on rats,promote NO secretion in rat serum and bronchoalveolar lavage fluid,inhibit the expression of inflammatory factors,reduce the expression of RAGE in lung tissue.ConclusionThe action receptor of HMGB 1 is RAGE,which activates RAGE to promote the secretion of inflammatory factors in vitro and in vivo,and inhibits the function of eNOS,thereby inhibiting the secretion of NO. |
