2-methoxyestradiol Inhibit The Proliferation Of Pulmonary A Rtery Smooth Muscle Cells In Hypoxic By Estrogen Receptor

Objective: Pulmonary arterial hypertension?PAH?is prominent chara ctered with increased pulmonary artery pressure and the membrane proliferation in vascular remodeling,intimal proliferation,which can lead to gradual occlusion of a blood vessel diseases,of which the early reaction enhancement of pulmonary artery contraction and the late vascular smoo th muscle cells?VSMCs?proliferation is the main pathological changes,resulting in pulmonary vascular structure reconstruction[1].Hypoxia can p romote the occurrence of PAH,independently or as a synergistic risk fa ctors promote the occurrence and development of PAH,low oxygen pul monary hypertension?HPH?is a important type of PAH.2-methoxyestra diol?2ME?is the main metabolites of estrogen in the body,has the inh ibition of cell proliferation,the stagnation of regulation of cell cycle an d inducing apoptosis effect[2].Can reduce hypertension induced by angiote nsin II[3],the inhibition of high blood pressure,coronary arterial remodeling[4],against atherosclerosis and vascular injury[5],inhibit intimal hyperp lasia,etc[6].In recent years,many studies have shown that 2ME also play an import ant protective role in pulmonary hypertension[7-10].This experiment was to research from the cellular level of 2ME to explore the protect role of 2ME for the proliferation of human pulmonary artery smooth muscle cells?hPASMCs?under hypoxia and the estrog en receptor?ER?in mediating role of them.Methods:1 Cell culture: Used normal hPASMCs for cell culture,placed in the basal medium containing SMCM,2% fetal bovine serum,0.5% grow th factor and 0.5% of the double resistance to smooth muscle cell cultu res,every 2 day change fluid for one time,under 37 ? and 5% CO₂,saturated humidity conditions,with 0.25% trypsin digestion of subculture,cells in logarithmic phase,the experiments for 5⁸ cells.Before the intervention of 2ME,the experimental cells are kept without FBS without phenol red DMEM culture training for 24 h.2 Cell intervention: The 5⁸ generations of hPASMCs were used,t hey were given normoxia,hypoxia and different concentrations of 2ME?0.1,0.5,1,3,5 umol/l?intervention,cells were grouped into normoxia group,hypoxia group,hypoxia+2ME?0.1umol/l?group,hypoxia+2ME?0.5umol/l?group,hypoxia+2ME?1umol/l?group,hypoxia+2ME?3umol/l?grou p,hypoxia+2ME?5umol/l?group,they were observed under inverted microscope for cell proliferation,parallel determined by MTT experiments show different intervention under the condition of cell proliferation.Then ER alpha inhibitor?MPP?and ER beta inhibitor?PHTPP?were used for preprocessing normal hPASMCs respectively,given optimum concentratio n of 2ME?5 umol/l?and located under three gas train line in the hypo xia treatment,cells were grouped into hypoxia+2ME?5 umol/l?+MPP gro up,hypoxia+2ME?5 umol/l?+PHTPP group,then under inverted microsco pe to observe and determine by MTT experiment for cell proliferation again,and under the condition of different interventions were evaluated by RT-PCR method to explore the expression of ER alpha,beta and PCNA mRNA levels in hPASMCs,under the condition of different interven tions were evaluated by Western blot method to explore the expression of ER alpha,beta and PCNA protein levels in hPASMCs.Results: 1 Determined by MTT method to detect the effect of 2ME on the proliferation of hPASMCs Compared with the oxygen group,the proliferation of hPASMCs were obviously promoted under the conditi on of hypoxia,after 2ME intervented significantly inhibited the hPASMCs proliferation induced by hypoxia and the concentration was depended.After MPP intervented significantly inhibited the inhibition of cell prolif eration of 2ME,after PHTPP intervented there had no effect on the inh ibition of cell proliferation of 2ME;2 ER? ER? and PCNA mRNA expression in hPASMCs Compared with often oxygen group,hypoxic group PCNAmRNA expression level increased significantly,ER? mRNA levels significantly decreased at the same time,ER? mRNA level without obvious change;compared with the hypoxia group,after 2ME?5umol/L?intervented PCNAm RNA expression level decreased significantly,ER?mRNA level increased significantly at the same time,ER? mRNA expression has no obvious changes.After MPP intervented the role of 2ME for the inhibition of PCNAmRNA level and the promotion of ER? mRNA level was decreased significantly;after PHTPP intervented the role of2 ME for the inhibition of PCNAmRNA level had no obvious change;3 ER? ER? and PCNA protein expression in hPASMCs Compared with oxyg en group,hypoxic group PCNA protein expression level increased signifi cantly,ER? protein levels significantly decreased at the same time,ER?protein level without obvious change;compared with the hypoxia group,after 2ME?5 umol/L?intervented PCNA and protein expression level decreased significantly,ER? protein level increased significantly at the same time,ER? protein expression leveldeclined slightly.After MPP interv ented the role of 2ME for the inhibition of PCNA protein level and the promotion of ER? protein level was decreased significantly;after PHTPP intervented the role of 2ME for the inhibition of PCNA protein level and ER? protein had no obvious change.Conclusions:1 Under hypoxic conditions,2ME promote the expression of ER?.2 2ME can be regulated the expression of ER? to inhibit the prolif eration of hPASMCs,it may through this path to be play the protective role of hypoxic pulmonary hypertension and pulmonary vascular remodeling.