[Effect of transforming growth factor ß1 on the expression of matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1 and nuclear factor kappa B signalling pathway in the human amniotic cells WISH].

OBJECTIVE: To investigate the effect of transforming growth factor ß1 (TGF-ß1) 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-ß1 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-ß1 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-ß1 (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-ß1 was added, the TIMP-1 mRNA and protein were significantly higher than the TIMP-1 mRNA and protein when no TGF-ß1 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-ß1 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-ß1. And when 2, 10 or 20 ng/ml of TGF-ß1 was added, the MMP-9 mRNA and protein were significantly lower than the MMP-9 mRNA and protein when no TGF-ß1 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 significantly reduced with the increase of TGF-ß1 (0, 2, 10, 20 ng/ml;P < 0.05). CONCLUSIONS: The mRNA and protein expression of TIMP-1 decreased when TGF-ß1 was low in WISH cells, whereas those of MMP-9 elevated when TGF-ß1 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.

Anti-diabetic effects of TongGuanWan, a Chinese traditional herbal formula, in C57BL/KsJ-db/db mice.

In the present study, the anti-diabetic effects of a traditional Chinese medicinal formula extract, TongGuanWan, were investigated in type 2 diabetic animals. It was orally administered to C57BL/KsJ-db/db mice once a day for 4 weeks at the doses of 62, 125, and 250 mg/kg body weight. TongGuanWan significantly lowered the blood glucose and glycosylated haemoglobin levels as well as improved the glucose tolerance in db/db mice. The serum triglyceride levels in the db/db mice were significantly decreased, whereas the high-density lipoprotein cholesterol levels were significantly increased, after treatment with this herbal formula. TongGuanWan also markedly decreased the animals' body weights compared to those of the control db/db group but did not alter food intake. The effects of TongGuanWan were compared to those of the drug rosiglitazone. In addition, five main constituents of TongGuanWan, mangiferin, berberine, cinnamic aldehyde, timosaponin BII, and timosaponin AIII, were quantified using high performance liquid chromatography coupled with a diode array and an evaporative light scattering detector (HPLC-DAD-ELSD). These results suggest that TongGuanWan may be useful for the treatment of type 2 diabetes.

[Effect of epidermal growth factor on the expression of matrix metalloproteinase-9 and the signalling pathways involved in the trophoblast cell line JEG-3].

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 10 ng/ml EGF), EGF+ inhibitors group (exposure to 10 ng/ml EGF+ 20 ng/ml SB203580 or exposure to 10 ng/ml EGF+ 10 ng/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-κB), 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-9 mRNA were increased by various concentrations of EGF (0, 1, 10, 20 ng/ml) in JEG-3 cells after 24 h-culture. The expression of MMP-9 mRNA in JEG-3 cells exposure at 1 ng/ml of EGF (0.567±0.056), 10 ng/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 p38MAPK 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-κB (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-κB (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.

Complete NMR spectral assignments of two new iridoid diastereoisomers from the flowers of Plumeria rubra L. cv. acutifolia.

Two new iridoid diastereoisomers (1, 2), together with five known compounds, were isolated from the flowers of Plumerian rubra L. cv. acutifolia. Their structures were elucidated by the means of in-depth spectroscopic and mass-spectrometric analyses, particularly 1D and 2D NMR spectroscopy.

Cytotoxic triterpenoid saponins from Vaccaria segetalis.

By the guidance of bioassay, one new cytotoxic triterpenoid saponin, 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] quillaic acid 28-O-beta-D-glucopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-fucopyranosyl-(1-->4)]-beta-D-fucopyranoside (1), and five known cytotoxic triterpenoid saponins, vaccaroside E (2), vaccaroside G (3), vaccaroside B (4), segetoside H (5) and segetoside I (6), were isolated from Vaccaria segetalis. Their structures were established on the basis of ESI-MS, IR, extensive NMR ((1)H NMR, (13)C NMR, TOCSY, (1)H-(1)H COSY, DEPT, HMQC, HMBC and ROESY) analyses, chemical degradation, and by comparing with previously reported data. Compounds 1-6 showed moderate cytotoxic activities against LNcap, P-388 and A-549 cell lines with IC(50) values in the range 0.1-12.9 microM.

Identification and determination of four metabolites of mangiferin in rat urine.

Four metabolites of mangiferin were firstly isolated and identified from rat urine. The structures of the four metabolites were determined to be 1,3,7-trihydroxyxanthone (M-1), 1,3,6,7-tetrahydroxyxanthone (M-2), 1,3,6-trihydroxy-7-methoxyxanthone (M-3) and 1,7-dihydroxyxanthone (M-4), respectively. A simple and specific analytical method for determination of the four metabolites in rat urine was developed by high performance liquid chromatography (HPLC). Quercetin was employed as an internal standard. The correlation coefficients of the calibration curves were higher than 0.997, both intra- and inter-day precision of four metabolites were determined and their R.S.D. did not exceed 10%. The accuracy and linear range had been investigated in detail. The cumulative urinary excretions of the four metabolites were measured and the possible metabolic pathway of the metabolites was discussed.

LC-MS characterization of efficacy substances in serum of experimental animals treated with Sophora flavescens extracts.

Anti-DHBV (duck hepatitis B virus) activity was found in the aqueous extracts of Sophora flavescens Ait. in vivo. Liquid chromatography/electrospray ionization ion trap mass spectrometry was applied to characterize the components in duck serum after oral administration of S. flavescens extract. Oxymatrine (1), sophoranol (2), sophoridine (3) and matrine (4) were identified in the serum. Further research on the four compounds was evaluated for their antiviral activity against HBV (hepatitis B virus) in cell culture. The results suggested that oxymatrine, sophoranol and matrine were the efficacy substances for anti-HBV activity in aqueous extracts of S. flavescens Ait.

Structure elucidation and complete NMR spectral assignments of a novel sesquiterpene glycoside from Ixeris sonchifolia.

A novel sesquiterpene glycoside was isolated from the whole plant of Ixeris sonchifolia. The structure was established as 1(10)E-4Z-3alpha-hydroxy-germacra-1(10),4,11(13)-trien-6, 12-olide-14-O-beta-D-glucopyranoside (1) on the basis of spectroscopic techniques and chemical analysis.