| OBJECTIVE:To synthesize3,5,4’-trimethoxystilbene (TMS) by methylation of resveratrol (Res), a natural compound extracted from polygonum cuspidatum, to identify the chemical structure of TMS, to test its pharmacokinetics, and to determine the effects of TMS on the growth inhibition and apoptosis in pulmonary artery smooth muscle cells (PASMCs).METHODS:The chemical structure of TMS was analyzed by UV-and IR-absorption spectrometry,1H-NMR and13C-NMR spectroscopy and mass spectrometry. We measured the bioavailability, the characteristics of intestinal absorption, and the distribution of TMS in body and excretions of SD rats after oral administration of TMS. The acute toxicity of TMS in mice was tested. PASMCs were prepared from pulmonary artery of SD rats. The proliferation of PASMCs after treatment was determined by MTT assay. The apoptosis of PASMCs after treatment was determined by flow cytometry. The intervention of TMS on protein expression of Bax and Bcl-2in PASMCs was detected immunohistochemically. The intervention of TMS on in mRNA expression of bax and bcl-2in PASMCs after treatment was analyzed by in situ hybridization assay.RESULTS:The UV absorption map of TMS showed Xmax(MeOH) at318,306.2, and217.8nm. Analysis of infrared spectrum of TMS showed IRvmaxKBr/cm at2999,2935,2836,1591,1511and1456/cm. The1H-NMR map showed that the synthetic product contained three hydroxy groups, while13C-NMR map showed17carbon signals and some symmetrical structural fragments. Electospray ionization mass spectrometry of the product showed m/z peaks corresponded to271[M+H]+,256[M+H-CH3]+and241[256-CH3]+; the implied relative molecular weight is270and the implied molecular formula is C17H18O3. These data confirm the product is3,5,4’-trimethoxystilbene. The absolute bioavailability of TMS was45.4%. TMS was well absorbed in the upper small intestine; it was excreted in stool and bile and distributed into several tissues. The maximal tolerance dose (MTD) of TMS was5.85g/kg. MTT assay showed TMS inhibited the proliferation of PASMCs in a dose-dependent manner. The extent of growth inhibition in A-H groups were (4.07±2.12)%,(6.54±4.78)%,(23.74±7.07)%,(16.75±5.34)%,(9.35±4.26)%,(17.81±6.03)%,(8.81±5.16)%and (6.78±5.58)%respectively. Flow cytometry showed the extent of apoptosis in PASMCs (after being treated with TMS for24h) was significantly higher than that in PASMCs treated only with TNF-α. The apoptosis rates of A-H groups were (2.63±0.74)%,(3.54±0.81)%,(18.79±4.15)%,(11.68±5.35)%,(5.77±4.62)%,(12.47±5.06)%,(6.33±4.8)%and (4.11±3.59)%respectively. Immunohistochemistry assay showed the protein expression of Baxin PASMCs (after being treated with TMS for24h) increased while the protien expression of Bcl-2decreased. The effect stimulated by TMS was10times stronger than that by Res. The ratio ofBcl-2/Bax of A-H group were(0.99±0.24)%,(1.07±0.22)%,(0.74±0.37)%,(0.84±0.41)%,(0.92±0.32)%,(0.86±0.45)%,(1.02±0.42)%and (1.12±0.51)%respectively. In situ hybridization technic showed the mRNA expression of bax in PASMCs (after being treated with TMS for24h) increased while the mRNA expression of bcl-2mRNA decreased in a dose-dependent manner. Compared with Res, the effect stimulated by TMS increased10times. The ratio of bcl-2/bax of A-H group were (1.00±0.14)%,(1.09±0.11)%,(0.90±0.43)%,(0.97±0.44)%,(1.05±0.50)%,(0.95±0.42)%5(1.03±0.56)%and (1.09±0.53)%respectivelyCONCLUSION:We have confirmed our synthetic product as3,5,4’-trimethoxystilbene (TMS),with the molecular formula of C17H18O3and appropriate molecular weight and absorption and NMR spectra. The bioavailability of TMS was to45%. TMS strongly inhibited the proliferation of PASMCs in a dose-dependent manner and the effect of inhibition of TMS was10times stronger than that of Res. Apoptosis of PASMCs could be induced by TMS, extent of which was10-fold of that by Res. TMS induced apoptosis of PASMCs by down-regulation of Bcl-2expression and up-regulation of Bax expression. |
PDF Full Text Request