[Trimethoxystilbene and its effects on the proliferation and apoptosis of PASMCs]

To synthesize 3, 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).

The chemical structure of TMS was analyzed by UV- and IR- absorption spectrometry, (1)H-NMR and (13)C-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 PASMCs were divided into 8 groups. Group of A (control) was cultured without TNF-α, TMS, or Res. Group of B (TNF-α) was cultured with 100 pg/mL TNF-α. Groups of C-E (low-high concentrations of TMS) were cultured with 100 pg/mL TNF-α and 5, 10, 20 μmol/L TMS, respectively. Groups of F-H (low-high concentrations of Res) were cultured with 100 pg/mL TNF-α and 50, 100, 200 μmol/L Res, respectively. The proliferation of PASMCs after treatment was determined by MTT assay. The apoptosis of PASMCs after treatment was determined by flow cytometry.

The UV absorption map of TMS showed λmax(MeOH) at 318, 306.2, and 217.8 nm. Analysis of infrared spectrum of TMS showed IRvKBr max /cm at 2999, 2935, 2836, 1591, 1511 and 1456/cm. The (1)H-NMR map showed that the synthetic product contained three hydroxy groups, while (13)C-NMR map showed 17 carbon signals and some symmetrical structural fragments. Electospray ionization mass spectrometry of the productshowed m/z peaks corresponded to 271[M+H](+), 256[M+H-CH(3)](+) and 241[256-CH(3)](+); the implied relative molecular weight is 270 and the implied molecular formula is C17H18O3. These data confirm the product is 3,5,4' - trimethoxystilbene. The absolute bioavailability of TMS was 45.4%. TMS was well absorped in the upper small intestine; it was excreted in stool and bile and distributed into several tissues. The maximal tolerance dose (MTD) of TMS was 5.85 g/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)%, (9.35±4.26)%, (16.75±5.34)%, (23.74±7.07)%, (6.78±5.58) %, (8.81±5.16) %, and (17.81±6.03) %, respectively. Flow cytometry showed the extent of apoptosis in PASMCs (after being treated with TMS for 24 h) 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)%, (5.77±4.62)%, (11.68±5.35)%, (18.79±4.15)%, (4.11±3.59)%, (6.33±4.8) %, and (12.47±5.06)%, respectively.

We have confirmed our synthetic product as 3,5,4'-trimethoxystilbene (TMS), with the molecular formula of C17H18O3 and appropriate molecular weight and absorbption and NMR spectra. The bioavailability of TMS was to 45%. It strongly inhibits the proliferation of PASMCs in a dose-dependent manner and induces apoptosis of PASMCs.