| Background:Hypoxic pulmonary arterial hypertension (HPAH) is a progressive disease characterized by an elevation of pulmonary-artery pressure and pulmonary-vascular resistance, which in its late stages becomes refractory to traditional therapies, leading to right-ventricular failure and death at last. Multiple recent studies have found that abnormal proliferation of pulmonary artery smooth muscle cells and vascular remodeling induced by hypoxia have been involved and plaied a crucial role in the development of HP AH. Cells would active a series of signal pathway to adapt for hypoxic conditions, HIF-1α is recognized as the master regulator of the hypoxic response, activating the transcription of>100genes which have influence on energy metabolism, cell proliferation and vascular development. Moreover, Hypoxia activates NADPH oxidase to increase levels of ROS which has an enhancement effect on HIF-la protein’s expression. They both trigger an increase in [Ca2+] through the mitochondrial HIF-la-TRPC-Ca2+and ROS-PKCε signaling axis in pulmonary artery smooth muscle cells. The elevation of intracellular [Ca2+] results in the proliferation of PASMCs, contraction of pulmonary arteries and vascular remodeling.Yifei Huoxue Granule (YFHXG) is dialectical concocted by academician Dong Jian-hua on the basis of two classical Chinese prescriptions, Buzhongyiqi Decoction and Xuefuzhuyu Decoction, which has been used to treat PAH and COPD for many years. Xuefuzhuyu Decoction is thought to activate blood circulation, remove blood stasis and to relieve Qi stagnation and to smooth Qi circulation. It protects against pulmonary vessels injury and cardiovascular disease. Buzhongyiqi Decoctions can improve the breath and the exchange between blood and oxygen. Both of these decoctions have the ability to fight inflammation and enhance immunity. In previous in vivo experiments, we found that it has a significant effect in inhibiting the thickening of the tunica media and reducing pulmonary arterial pressure in HPH models, it may be achieves through modulating immune reaction and attenuating hypoxia-induced inflammation response. On the basis of these observations, we hypothesized that YFHXG could suppress the hypoxia-induced proliferation of rat PASMCs and has an anti-hypoxia effect by regulating the expression of HIF-la protein and the concentration of intracellular ROS and [Ca2+]. The present study was undertaken to investigate the potential mechanisms of YFHXG at cellular levels.Objective:The present study was undertaken to investigate whether Yifei Huoxue Granule has an inhibitory effect on the hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) in vitro and to understand how it decreases pulmonary arterial pressure.Methods:(1) The YFHXG crude drugs were manufactured and checked by Clinical Pharmacy Research Institute of Beijing Municipal Health Bureau. The sliced crude drug materials were extracted by decocting in decuple boiling water for3h each time, then the clear supernatant liquid was removed and condensed. Physic liquor flowed at300ml/min and was centrifuged at4000r/min, to yield YFHXG extracts. YFHXG was dissolved in a small amount of neutral saline and then diluted with the culture medium to the following final concentrations:large dose group:7.5mg/ml, medium dose group:1.5mg/ml, and low dose group:0.15mg/ml.(2) Primary cultures of PASMCs were prepared from the pulmonary artery of2-3months old male SD rats weighing150-200g by tissue block anchorage. PASMCs were identified by the morphological examination and immunocytochemical staining for β-actin. After reaching90%confluence, cells were passaged using trypsin/EDTA. Passages3 through6were used for the study. The harvested cells were randomly divided into normoxia group(20%O2,5%CO2,75%N2), hypoxia group(3%O2,5%CO2,92%N2) and hypoxia+YFHXG group(7.5、1.5、0.15mg/ml).(3) PASMCs proliferation was measured by MTT assay. Briefly, PASMCs were seeded at a density of8×103cells/10μl/well in96-well plates. The cells were incubated for24h in serum free DMEM/F12. After reaching80%confluence, the cells in the96-well plates were randomly grouped and treated or24h、48h、72h,as described above. At the end of treatment,10μl of MTT was added to the plates, and incubation for4h at37℃.100μl of formazan solution was then directly added to dissolve the formazan crystals and incubation continued for another4h. The absorbance of the solubilized product at570nm (A570) was measured with the ELIS A reader.(4) The DNA content and the cell cycle of PASMCs were determined by flow cytometry. The PASMCs were seeded at a density of8×104cells/well into a6-well plate. After reaching80%confluence, the cells were treated with hypoxia and YFHXG for24h. The harvested cells were washed twice with ice-cold PBS buffer (pH7.4), fixed with100%ice cold ethanol at4℃overnight, incubated with0.1mg/ml RNase for at least30min at37℃, and finally stained with50μg/ml propidium iodide(PI). The samples were analyzed with a flow cytometer and the proliferation index was calculated according to the following formula:Proliferation index(%)=(S+G2/M)/(G0/G1+S+G2/M)×100%.(5) HIF-1α protein expression in PASMCs was measured by immunocytochemical method. Briefly, after stimulated with hypoxia and YFHXG for24h, the cells were washed by PBS for three times and fixed with4%paraformaldehyde for30min at room temperature before the incubation in0.3%H2O2and5%normal goat serum for15min at room temperature. Finally, the samples were washed by PBS for another three times and incubated with rabbit anti-rat HIF-1α monoclonal antibody (1:200) overnight at4℃, and detected with HRP-conjugated anti-rabbit antibody with diaminobenzidine(DAB).(6) The levels of ROS in cells can be reflected by fluorescence intensity of DCF. After24h, the cover slips coated with cells were washed by PBS three times. Then, the cells were loaded with10nmol/ml DCFH-DA for20min at37℃, washed twice with serum-free DMEM/F12to wash out extracellular dye and to allow the conversion of intracellular dye into its non-ester form. DCF was excited at488nm and measured at525nm with a laser scanning confocal microscope, and the fluorescence intensity of DCF was analyzed with LASAF software.(7) The concentration of intracellular [Ca2+] was measured by laser scanning confocal microscope. As previously described, freshly isolated cells were incubated with3μM fluo-3/AM (Invitrogen, USA) at37℃for30min. The dye was excited at488nm with a TCS-SP5laser scanning confocal microscope. Fluo fluorescence at525nm was collected and analyzed with LASAF software.(8) The results were expressed as mean±standard deviation(SD), all data were processed with SPSS13.0software. Analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was used in order to compare more than two groups. Statistical significance was accepted at P<0.05.Results:MTT assay and flow cytometry showed that hypoxia could directly activate the proliferation of PASMCs, while YFHXG dose-dependently inhibited hypoxia-induced proliferation of rat PASMCs. Immunocytochemistry showed that hypoxia enhanced HIF-1α protein expression, and LSCM showed that hypoxia significantly increased intracellular ROS and [Ca2+], while YFHXG decreased the expression of HIF-1α and attenuated the hypoxia-induced increase in intracellular concentration of ROS and [Ca2+].Conclusions:The results show that YFHXG inhibits hypoxia-induced proliferation of rat PASMCs. The anti-hypoxia effect of YFHXG may be explained by a) its regulation of HIF-1α expression and of the levels of intracellular ROS and [Ca2+], and b) its inhibitory action on PASMCs proliferation that may decrease pulmonary arterial pressure and vascular remodeling. |
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