| Vascular smooth muscle cells (VSMCs) proliferation and migration are considered as the key characteristic in the development of cardiovascular diseases. VSMCs dysfunction is obverted as a prominent pathological feature of diseases such as hypertension, atherosclerosis and restenosis after angioplasty. Therefore, inhibition of VSMCs proliferation and migration provides a possible solution in the maintenance of vascular homeostasis and thus to prevent vascular disease. Many studies have been reported that natural functional components from traditional herbs, for instance, ferulic acid (FA) from angelica, oleanolic acid (OA) from ligustrum lucidum,1,8-Dihydroxyanthraqinone (DDA) from cassia seed, Diosgenin (Dio) from Chinese Yam, daidzein from soybean, were employed as Chinese medicine to the blood-stasis. In present study, we obtained a steady human umbilical VSMC line in vitro, and established a VSMC proliferation model induced by high-concentration serum. The effects of these functional components on VSMC serum-induced proliferation and migration were subsequently examined and their possible active mechanisms were explored.Firstly, human umbilical VSMC line in vitro was obtained by explant attached method. Primary culture and subculture of human umbilical VSMCs were observed and identified by inverted microscopy. The cell morphology was identified by Coomassie brilliant blue R250.Results show that human umbilical VSMCs explanted out and constituted a confluent monolayer after 3-4 weeks. Cultured cells exhibited typical "hill and valley" pattern with long spindle shape which is idetified as a typical characteristic of SMCs. There is no significant change of biological characteristics after 5 generations of subculture. These results suggest that a steady human umbilical VSMC line in vitro was successfully established by explant attached method.VSMCs proliferation was induced by high-concentration serum. Serum-induced cells showed clustered growth pattern and cell number significantly increased from 6.7×105 to 1.02×106 after incubating with 20% serum for 48 h. Cell morphology appeared changes which includes enlarged cell size, increased cell plasma/nuclear and enhanced stain contrast. Protein content increased to 10.964μg from 6.216μg of control. Incorporation of BrdU to DNA increased to 13.691μg from 7.764μg of control. Growth curve shows that proliferated cell growth enhanced and cell division phase ahead to 48 h. Proliferation index was to 77.79% with decreased G0/G1 phase cell number and increased S phase cell number. The migration ability of cells induced by serum was observed to be enhanced. The number and distance of migrated cells were respectively 11.428-fold and 4.140-fold compared with the control. Serum increased the adhesion of extracellular matrix proteins to VSMCs. The adhesion cell to osteopontin and fibronectin were respectively 2.042-fold and 1.742-fold compared to the control. Therefore, high-concentration serum increased VSMCs proliferation, migration and adhesion. Serum-induced VSMCs proliferation could be used for related researches in vitro.The effects of functional components against lipid peroxidation, serum oxidant, blood red cell (BRC) hemolysis, mitochondria swelling, and the reducing power on Fe3+ and scavenging activity on inductive free radicals were subsequently determined. It was found that FA has the scavenging activity of O2- and DPPH, in which IC50 of 119.33 and 4.57μg/mL, respectively. The reducing power of FA on Fe3+ was 2.118-fold compared with Vit C. OA could inhibit the hemolysis of BRCs in which IC50 of 277.72μg/mL, and the reducing power of OA on Fe3+ was 2-fold compared with Vit C. DDA scavenged·O2-,OH, in which IC50 of 65.91 and 48.57μg/mL, respectively. Meanwhile, DDA could inhibit serum oxidant in which IC50 of 35.93μg/mL. Dio could inhibit serum oxidant and BRCs hemolysis, in which IC50 of 197.50 and 95.37μg/mL, respectively. Dio has the scavenging activity of·OH and DPPH, in which IC50 of 19.17 and 1.84μg/mL, respectively. The results suggests that functional components inhibited lipid peroxidation, serum oxidant and BRCs hemolysis by scavenging free radicals, reducing Fe3+ and protein-binding, thus to protect vascular from peroxidation.The effects of these functional components on VSMC serum-induced proliferation, migration and adhesion were subsequently examined. Cells treated with functional components have changed cell morphology and delayed growth curve without significant cytotoxicity. At a concentration of 100μmol/L, the inhibitory rate of FA, OA, DDA and Dio on serum-induced cell energy respectively were 56.101,72.317,57.036 and 54.154%, compared to untreated control. Protein level of them were respectively inhibited to 57.519,75.924,60.559 and 54.396% compared to untreated control. Decreased BrdU incorporation to DNA of them respectively were 1.204μg,1.210μg,1.445μg and 1.275μg compared to 1.369μg of untreated control. Proliferation index of them at 100μmol/L decreased to 65.26,60.28,71.36 and 59.85%, respectively, compared to untreated control of 77.79%. Results show that functional components inhibited serum-induced VSMCs proliferation. Moreover, VSMCs migration ability was dose-dependently inhibited by functional components. At the concentration of 100μmol/L, the inhibitory effect of FA, OA, DDA and Dio on VSMCs migrated cells number were respectively 73.78,48.59,70.81and 58.96% of untreated control. Meanwhile, the cell migration distances of them were inhibited as 67.69,70.90,59.36 and 62.56%, respectively, compared with untreated control. In addition, at the concentration of 100μmol/L, their adhesion cells to osteopontin were respectively at 29.231,48.308,53.231 and 56.923%, and to fibronectin were at 51.104,54.2598,64.038 and 67.192% of untreated control, respectively. The results suggest that functional components might contribute to the prevention and treatment atherosclerosis due to their inhibitory effects on VSMCs proliferation, migration and adhesion.The absorptions and conversion of daidzein and its derivatives (DDs) in VSMCs were measured. It was found that DDs has a much better absorption by VSMCs. Treated with daidzein and DDs for 48 h, finally absorption of DD1 and DD2 respectively were 71.67 and 61.44% compared to the additive amount, they were better than that of daidzein as 54.584%. The result of conversion assay shows that derivatives were conversed to daidzein to act their functions. Subsequently, the effects of these derivatives compared with daidzein on VSMC proliferation and migration were examined. VSMCs proliferation, migration and adhere ability were dose-dependently inhibited by DDs and daidzein without significant cytotoxicity. At the concentration of 100μmol/L, the inhibitory rate of DD1, DD2 and daidzein measured against VSMCs proliferation were 84.47,68.53 and 66.35%, respectively. The inhibitory effect of migrated cell number which cells were treated with DD1, DD2 and daidzein were respectively at 58.52,54.07 and 49.63% compared with untreated control. Meanwhile, the cell migration distances of them were significantly inhibited as 81.17,71.43 and 63.64% of untreated control, respectively. Moreover, the adhesion cells to osteopontin were respectively at 48.31,47.08 and 46.77% of untreated control, while the adhesion cells to fibronectin were at 67.823,64.038 and 57.413% respectively. In addition, DDs and daidzein arrested cell cycles of serum-induced proliferation VSMCs. Proliferation index of cells treated with DD1, DD2 and daidzein of 100μmol/L decreased to 54.31,56.39 and 70.91, respectively, compared to untreated control of 77.79%. All those results from the experiment suggest that these derivatives of daidzein might contribute to the prevention of cardiovascular diseases via inhibiting VSMCs proliferation and migration.Effect of functional components on the NOS/NO regulation of VSMCs were studied. Results show that functional components promoted NOS activity and NO production. At the concentration of 100μmol/L, the levels of NO were respectively 20.749,31.797,18.624 and 16.250μmol/gprot compared to control of 10.161μmol/gprot. Meanwhile, the relative NOS activity levels of them were 1.008,1.061, 0.900 and 0.798 U/mgprot, respectively, compared with control of 0.668 U/mgprot. The effect pattern of NOS activity of functional components on VSMCs was associated with that of NO production as follows:OA>FA>DDA>Dio, suggesting that functional components probably promoted NO production via regulating NOS activity in proliferation VSMCs.To investigate the effects of functional components on serum-induced VSMCs proliferation, the mRNA expression of cell cycle factors in proliferation VSMCs was determined by real time PCR. The up-expression of CDK2 mRNA and down-expression of p27kip1 mRNA were detected in proliferation VSMCs induced by serum, while functional components down-regulated CDK2 mRNA expression and down-regulated p27kip1 mRNA expression in proliferation VSMCs. Results show that functional components probably inhibited VSMCs proliferation via modulating cell cycle with increasing p27kip1 mRNA and down-regulating CDK2 mRNA expression. However, the precise cellular mechanism(s) by which functional components regulated VSMCs proliferation were incompletely understood and required further elucidated. |
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