| Objective In this study,the effects of manassantin A(MA)derivative LXY6099,a specific inhibitor of hypoxia-inducible factor(HIF-1α),on the abnormal proliferation,migration,apoptosis resistance and interstitial transformation of pulmonary artery endothelial cells by using in vitro cell experiments and in vivo animal experiments were used to investigate the effects of hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension in two ways.In this experiment,we used cell experiments in vitro and animal experiments in vivo to explore the effects of the specific inhibitor of hypoxia inducible factor(HIF-1 α),the derivative of the Manassantin A(MA),LXY6099,on the abnormal proliferation,migration,apoptosis resistance of pulmonary artery smooth muscle cells and the interstitial transformation of pulmonary artery endothelial cells were investigated to determine whether LXY6099 can inhibit hypoxic-induced pulmonary vascular remodeling and the mechanism of pulmonary hypertension in two ways.Methods一、 Animal experiment Rat models were established in normoxic(21% O2)group,hypoxic(10% ±0.5% O2)group,hypoxic solvent(Hypoxia+0.5% CMC-Na)group and hypoxic drug(Hypoxia +LXY6099)group.Normal oxygen group: in normal environment containing oxygen;Hypoxia group: kept under hypoxia for 4 weeks;Hypoxic solvent group: hypoxia and 0.5% sodium carboxymethylcellulose(CMC-Na)were given by gavage for 4 weeks;Hypoxia drug group: LXY6099(50 mg · kg-1)was given by gavage under hypoxia for 4 weeks.Four weeks later,the right ventricular systolic pressure(RVSP)of rats was measured,and then the rats were killed.The lung tissue was fixed,dehydrated,and embedded in OCT.After sectioning,it was stained with hematoxylin-eosin(HE)and photographed under the microscope.Calculate the percentage of medial wall thickness(WT%)and the percentage of medial wall area(WA%)of pulmonary arterioles.Comparison of HIF-1 in each group by Western Blot α Protein,PCNA,BCL-2,Cleared-Case3 and other proteins were expressed;Endothelial cell marker(VE-cadherin)and interstitial cell marker(Slug,Vimentin α-SMA).二、Cell experiment The models of rat pulmonary artery smooth muscle cells(PASMCs)were established in normoxic(control,21% O2)group,hypoxic(Hy+48h,3% O2)group,hypoxic solvent(Hy48h+DMSO)group,hypoxic low concentration administration(Hy48h+LXY6099 0.1nmol/L)group,hypoxic high concentration administration(Hy48h+LXY6099 1nmol/L)group.Control group: cells were cultured under normal oxygen;Hy+48h group: cells were cultured under hypoxia for 48h;Hy48h+DMSO group: cells were cultured with DMSO under hypoxia for 48h;Hy48h+LXY6099(0.1nmol/L)group: treated with LXY60990.1nmol/L and cultured under hypoxia for 48h;Hy48h+LXY6099 1nmol/L group:treated with LXY6099(1nmol/L)and cultured under hypoxia for 48 h.The migration ability of cells in each group was tested by scratch test and Transwell test.Compare the HIF-1 of each group through Western Blot experiment αExpression of protein,PCNA,BCL-2,Cleared-Case3 and other proteins in PASMCs.Rat pulmonary artery endothelial cells(PAECs)normoxic(21% O2)group,hypoxic(Hy+6d)group,hypoxic solvent(Hy6d+DMSO,3% O2)group,hypoxic low concentration administration(Hy6d+LXY6099 0.1nmol/L)group,hypoxic high concentration administration(Hy6d+LXY6099 1nmol/L)group models were established.Control group: cells were cultured under normal oxygen;Hy+6d group: cells were cultured under hypoxia for 6d;Hy6d+DMSO group:cells were cultured with DMSO under hypoxia for 6d;Hy6d+LXY6099(0.1nmol/L)group: treated with LXY6099 0.1nmol/L and cultured under hypoxia for 6d;Hy6d+LXY6099 1nmol/L group: treated with LXY6099 1nmol/L and cultured under hypoxia for 6d.Comparison of HIF-1 in each group by Western Blot α Protein,endothelial cell marker(VE-cadherin)and interstitial cell marker(Slug,Vimentin α-SMA).Results一、 LXY6099 reduces pulmonary artery pressure and vascular remodeling in rats after hypoxia In rats,compared with the normoxia group,the RVSP in the hypoxic group increased;In hypoxic group,the lumen was also significantly narrowed and occluded,and WT% and WA% were significantly increased;Hypoxia group HIF-1 α The expression of protein,cell proliferation marker PCNA protein,apoptosis resistance marker Bcl-2 protein increased significantly,and the expression of apoptotic protein Cleared-Case3 decreased;In hypoxia group,the expression of VE-cadherin protein,a marker of endothelial cells,decreased,and interstitial cell markers(Slug,Vimentin α-The expression of SMA)and other proteins increased.After administration of LXY6099,compared with the hypoxic group,the lumen of the hypoxic group was significantly widened,WT(%)and WA(%)were significantly decreased,the expression of PCNA protein,apoptosis resistance marker Bcl-2 protein,and the expression of apoptotic protein Cleared-Case3 were significantly decreased in the hypoxic group;In hypoxic group,the expression of VE-cadherin protein increased,and the expression of interstitial cell markers(Slug,Vimentin α-SMA)and other proteins decreased.二 、 LXY6099 reduces the proliferation,migration and apoptosis resistance of PASMCs after hypoxia,and promotes apoptosis In PASMCs,compared with normoxic group,HIF-1 cells in hypoxic group αThe protein expression was significantly increased;Cell proliferation increased;Apoptosis decreased and apoptosis resistance increased;Increased migration capability.LXY6099 can inhibit HIF-1 of PASMCs under hypoxia α Compared with the hypoxic group,the cell proliferation ability,apoptosis resistance,apoptosis and migration ability of the hypoxic low concentration and high concentration groups were significantly decreased,and were positively correlated with the concentration of LXY6099.三 、 LXY6099 reduces the interstitial transformation of PAECs after hypoxia In PAECs,compared with normoxic group,HIF-1 cells in hypoxic group αThe protein expression was significantly increased;The expression of endothelial cell marker(VE-cadherin)protein decreased,and interstitial cell marker(Slug;Vimentin;α-SMA)protein expression increased and End MT occurred.LXY6099 can inhibit HIF-1 of PAECs under hypoxia α Compared with the hypoxic group,the expression of protein in both the hypoxic lowconcentration and high-concentration groups can reduce End MT,and is positively correlated with the concentration of LXY6099.Conclusions To sum up,this study found that:(1)LXY6099 inhibits HIF-1 by α Protein alleviates hypoxic-induced pulmonary hypertension and vascular remodeling in rats;(2)LXY6099 inhibits HIF-1 α Protein to inhibit the abnormal proliferation,migration,apoptosis resistance and interstitial transformation of pulmonary artery endothelial cells induced by hypoxia,thus improving hypoxic pulmonary hypertension. |
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