ABSTRACT
Objective To investigate the effect of transforming growth factor β1 (TGF-β1) on the expression of matrix metalloproteinase 9 (MMP-9),tissue inhibitor of metalloproteinase 1 (TIMP-1),nuclear factor kappa B(NF-κB) and the possible signalling pathways in human amniotic cells WISH.Methods The WISH cell line was cultured.WISH cells were added with TGF-β1 of different concentrations (0,2,10 and 20 ng/ml,respectively) for 24 hours.Then,reverse transcription (RT) PCR and western blotting were used to analyze the protein and mRNA expression of TIMP-1 and MMP-9; and the expression of NF-κB was analyzed by western blot.Results (1) The profile of TIMP-1 mRNA (0.413 ±0.036,0.623 ±0.058,1.392 ±0.124,1.387 ±0.102) in WISH cells elevated when the concentration of TGF-β1 increased (0,2,10,20 ng/ml).In accordance with TIMP-1 mRNA,the expression of TIMP-1 also elevated with the increase of TGF-β1 (0.357 ± 0.031,0.596 ± 0.048,1.243 ± 0.097 and 1.359 ± 0.121,respectively).And when 2,10 or 20 ng/ml of TGF-β1 was added,the TIMP-1 mRNA and protein were significantly higher than the TIMP-1 mRNA and protein when no TGF-β1 was added(P ＜ 0.05).(2)In contrast with TIMP-1,MMP-9 mRNA (1.325 ±0.056,0.987 ±0.081,0.610 ±0.034,0.347 ±0.023) in WISH cells decreased when the concentration of TGF-β1 increased (0,2,10,20 ng/ml).The MMP-9 protein (1.119 ±0.064,1.008 ±0.052,0.578 ±0.041,0.401 ±0.015) also decreased with the increase of TGF-β1.And when 2,10 or 20 ng/ml of TGF-β1 was added,the MMP-9 mRNA and protein were significantly lower than the MMP-9 mRNA and protein when no TGF-β1 was added (P ＜ 0.05).(3) The NF-κB protein (1.423 ±0.065,1.116 ± 0.045,0.796 ± 0.041,0.359 ± 0.021) was significandy reduced with the increase of TGF-β1 (0,2,10,20 ng/ml; P ＜ 0.05).Conclusions The mRNA and protein expression of TIMP-1 decreased when TGF-β1 was low in WISH cells,whereas those of MMP-9 elevated when TGF-β1 was low.The unbalance of TIMP-1 and MMP-9 was related to the pathology of the premature rupture of membrane.And the NF-κB singalling pathway might be an important mechanism in the regulation of TIMP-1 and MMP-9 system.
ABSTRACT
Objective To investigate the effect of epidermal growth factor (EGF) on the expression of Matrix metalloproteinase-9 (MMP-9) and the signalling pathways involved in the trophoblast cell line JEG-3. Methods The JEG-3 trophoblast cell line was used in this study. (1) JEG-3 cells were cultured with various concentrations of EGF (0, 1, 10,20 ng/ml) for 24 hours and the expression of MMP-9 was tested by western blotting and reverse transcription PCR (RT-PCR). (2) Western blotting and RT-PCR were also used to investigate the expression of MMP-9 expression after incubation for 0,4,12 and 24 hours with EGF treatment (10 ng/ml) in JEG-3 cells. (3) According to the different added ingredients, JEG-3 cells were divided into some groups: control group (without EGF), EGF group (exposure to l0ng/ml EGF),EGF + inhibitors group (exposure to 10 ng/ml EGF +20 ng/ml SB203580 or exposure to 10 ng/ml EGF + 10ng/ml U0126) inhibitors group (exposure to 20 ng/ml SB203580 or exposure to 10 ng/ml U0126). Western blotting were used to investigate the expression levels of MMP-9, nuclear factor kappa B (NF-kB) ,p38MAPK, phospho-p38MAPK (p-p38MAPK) , extracellular -signal regulated kinase (ERK) and phospho-ERK (p-ERK) protein in JEG-3 cells after incubation for 24 hours. Results (1) The profiles of MMP-9mRNA were increased by various concentrations of EGF (0, 1 , 10, 20 ng/ml) in JEG-3 cells after 24hculture. The expression of MMP-9 mRNA in JEG-3 cells exposure at 1 ng/ml of EGF (0. 567 ±0. 056) , 10ng/ml of EGF (1. 392 ±0. 133) , 20 ng/ml of EGF (1. 971 ±0. 067) were significantly higher respectively (P <0. 05) , compared with 0 ng/ml of EGF treatment (0. 166 ±0. 015). Similarly, MMP-9 mRNAs were also increased with the increasing incubation time. Compared to EGF (10 ng/ml) stimulation for 0 h (0.253 ±0.044), the MMP-9 mRNA profiles were 0. 470 ±0. 026, 1.061 ±0. 115, 1. 453 ±0. 180 for 4,12 and 24 hours, respectively (P < 0. 05). (2) In accordance to the mRNA profiles, the expression of MMP-9 protein was also increased by different concentrations of EGF (0,1, 10, 20 ng/ml) in JEG-3 cells after 24 h-culture. The abundance of MMP-9 protein in the three groups was 0. 043 ±0. 012, 0. 085 ±0. 008, 0. 142 ±0. 015, with a significantly higher expression, compared with 0 ng/ml of EGF treatment (0. 004 ±0.001, P < 0.05) respectively. Similarly, MMP-9 proteins were also increased with the increasing incubation time. Compared to EGF(10 ng/ml) stimulation for 0 h (0. 030 ±0. 009) , the profiles of MMP-9 protein were 0. 137 ± 0. 010, 0. 240 ± 0. 010, 1.240 ±0.061 for 4, 12 and 24 hours, respectively (P < 0.05). (3) Both p38MAPK and ERK signalling pathways were activated by EGF in JEG-3 cells. The expression of p-p38MAPK was significantly higher (without or with 10 ng/ml EGF, 234. 1 ± 4. 1 vs.260. 9 ± 2. 5 , P < 0. 05) , however, the p38 MAPK inhibitor SB203580 markedly suppressed the increase in p-p38MAPK content induced by EGF(227. 9 ±2. 4 vs. 260. 9 ±2. 5, P<0. 05). Similarly, the expression of p-ERK was significantly higher with EGF treatment (812. 2 ±3. 5) vs. without EGF group (453.4±5.8) (P <0. 05) , while the ERK inhibitor U0126 significantly inhibited the increased p-ERK content in response to EGF treatment (71. 0 ± 1. 2 vs. 812. 2 ± 3. 5, P < 0. 05) . (4) The p38MAPK inhibitor SB203580 significantly reduced the expression of EGF-induced MMP-9 (0. 645 ± 0. 270 vs. 1. 476 ± 0. 452, P < 0. 05)and NF-kB (0.530 ± 0.026 vs. 0.959 ± 0. 017, P < 0. 05) . (5) The ERK inhibitor U0126 also significantly reduced the expression of EGF-induced MMP-9 (0. 623 ±0. 030 vs. 2. 112 ±0. 056, P <0. 05)and NF-kB (0. 325 ± 0. 082 vs. 0. 939 ± 0. 153, P < 0. 05). Conclusion EGF induced the expression of MMP-9 in a time and dose-dependant manner in JEG-3 cells. EGF enhanced MMP-9 expression through the activation of p38MAPK and ERK signalling pathways in JEG-3 cells.
ABSTRACT
Objective To study the chemical constituents from Dipentodon sinicus. Methods Isolation and purification were carried out on silica gel column and Sephadex LH-20, the structures were elucidated on the basis of chemical evidences and spectral analysis. Results Nine compounds were isolated and their structures were identified as n-1-triacontanol (Ⅰ), ?-sitosterol (Ⅱ), pyrocatechol (Ⅲ), p-dihydroxyl benzene (Ⅳ), coniferyl aldehyde (Ⅴ), vanillic acid (Ⅵ), ergosterol peroxide (Ⅶ), ?-sitosterol-3-O-?-D-glucosides-3'-O-tritriacontanate (Ⅷ), and daucosterol (Ⅸ). Conclusion All the nine compounds are isolated from the title plant for the first time, among them, compound Ⅷ is a new compound named as dipentosinin.