The Inhibitory Effect Of3,4-dihydroxyacetophenone On Human Pulmonary Artery Smooth Muscle Cell Proliferation Induced By Hypoxia

ObjectiveThis study was designed to administrate3,4-dihydroxyacetophenone （3,4-DHAP, or DHAP） into human pulmonary artery smooth muscle cells （HPASMCs）and to explore its inhibitory effect and mechanism in suppressing the proliferation ofHPASMCs.MethodsCultured HPASMCs were divided into five groups under these conditions:①normoxia group;②hypoxia group, and the hypoxia group treated with either;③0.6×10^-4mol/L DHAP, or④3.3×10^-4mol/L DHAP, or⑤6.0×10^-4mol/L DHAP. Foreach of these five groups, cell cycle was analyzed by flow cytometry. MTTcolorimetric was applicated by calulating OD value under different conditions. theamount of PCNA OD value was detected by Fluorescence staining. the protein inexpression location of proliferating cell nuclear antigen （PCNA） were detected byimmunohistochemistry, and the expression of PCNA in HPASMCs was analyzed byWestern-blot. The intracellular Ca2+concentration （[Ca2+]i） in a single HPASMC weremeasured using laser scanning confocal microscopy.ResultsWhen compared with the normoxia group with the cell distributions of92.46,4.20%in G0/G1and S. respectively, these distributions changed into77.77,14.39%ofhypoxia group, respectively. Both these decrease in cell proportion at G0/G1phaseand increase in S phase were statistically significant with P <0.01or0.05,respectively. Compared with hypoxia group, DHAP1（hypoxia group treated with the low concentration of DHAP） was increased in cell proportions in G0/G1（85.88%）and reduced cells in S phase （9.63%）. With the increase of DHAP concentration（DHAP2and DHAP3）, these differences had the tendency of increase with statisticalsignificant difference （P <0.01or0.05） between two groups（Table4）. Theseobservations clearly demonstrated that DHAP reduced HPASMCs proliferationinduced by hypoxia.The results of the absorbance value （OD） by MTT assay at570nm showedhypoxia group0.250±0.028, DHAP1group0.190±0.009, DHAP20.170±0.015,DHAP3group0.160±0.023, respectively. Which demonstrated that OD value of thehypoxia group was significantly higher than other group administrating with DHAP（P <0.05）. With concentration increasing of DHAP in a certain range，the OD valuehad a downward trend, the difference between the groups was statistically significant（P <0.05）.Immunohistochemical techniques showed the PCNA staining mainly located inthe nucleus; Western-blot showed ratio of PCNA/Actin in hypoxia group was （0.322±0.021）, higher than normal oxygen group （0.025±0.002）（P <0.05） and DHAP1group （0.063±0.004）, DHAP2group （0.055±0.004）, DHAP3group（0.037±0.002）（P <0.05）, with increasing in the dose of DHAP, the ratio of PCNA/Actindeclined,the difference between the groups was statistically significant （P <0.05）.The [Ca2+]i of single cell by double wavelength fluorescence intensity ratiomethod was determined with the average values25.0±8.9,64.0±10.2,30.0±9.5,23.0±8.4, and17.0±3.5from Normoxia group, Hypoxia group,DHAP1group, DHAP2group, and DHAP3group, respectively. The results showed [Ca2+]i of Hypoxia,was significantly higher than that of Normoxia group,（P <0.05）. However,[Ca2+]iof DHAP1group was lowered than Hypoxia group （P <0.05）, and [Ca2+]i wasdecreasing as the increase of DHAP concentration. All of these group differenceswere statistically significant （P<0.05） by neighboring comparative analysis. Theseobservations demonstrated that the administrated DHAP inhibited hypoxia-inducingrising of [Ca2+]i in HPASMCs, thus also inhibited the proliferation of HPASMCs. ConclusionsHypoxia could induce the cell proliferation of HPASMCs. DHAP could suppressthe cell proliferation,which mechanism might be related to the suppression ofintracellular free calcium concentration. The mechanism of DHAP improve humanpulmonary circulation may be relevant of it.