Discovery of a new sesquiterpenoid from Phellinus ignarius with antiviral activity against influenza virus.

Seven natural products, including one new sesquiterpenoid (eudesm-1ß, 6α, 11-triol, compound 1), one ergosta -4, 6, 8(14), 22-tetraen-3-one (compound 2), four polyphenols (compounds 3, 4, 5, 6), and one pyrone (3-hydroxy-2-methyl-4-pyrone, compound 7) were isolated from cultures of Phellinus ignarius by column chromatography. The detailed structure of compound 1 was determined using a combination of one- and two-dimensional nuclear magnetic resonance, mass spectrometry and infrared spectroscopy. The antiviral activity of these compounds against H5N1 influenza A virus was investigated using an MTT colorimetric assay system in Madin-Darby canine kidney cells. The results indicate that compound 1 possesses significant ability to inhibit influenza virus. The 50 % effective concentration was 0.14 ± 0.04 µM. Molecular modeling further suggested that the anti-influenza virus activity of this compound was partially attributed to the interactions of hydroxyl groups with an amino acid residue (Asn 170) of neuraminidase (NA) at the binding site. Moreover, the results of enzyme inhibition assays indicated that 50 % inhibition of NA was achieved by compound 1 at a concentration of 0.657 ± 0.325 mg/mL, which suggested that compound 1 is likely to interact with the NA enzyme.

[Interaction mechanism and influence between fatty acid oxidation and p38MAPK signal transduction pathway in trophoblast cells incubated with fatty acids].

OBJECTIVE: To explore the interaction mechanism and influence between fatty acids oxidation and p38MAPK signal transduction pathway in trophoblast cells stimulated by fatty acids of different chain lengths. METHODS: Serum-free trophoblast cells cultured in vitro were divided into 5 groups, i.e. incubation with DMEM/F12 medium without FFA (F-FFA), short-chain fatty acids (SC-FFA), medium-chain fatty acids (MC-FFA), long-chain fatty acids (LC-FFA) and very long-chain fatty acids (VLC-FFA). Then cells in each group were stimulated by DMEM/F12 medium, NADPH oxidase inhibitor (Apocynin) and p38MAPK inhibitor (SB203580) and were subdivided into FFA plus-DMEM group, plus-NADPH-I and plus-p38MAPK-I groups. Expressions of mRNA and protein of LCHAD in trophoblast cells were detected by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: (1) mRNA expression of LCHAD: the Δct of mRNA of LCHAD in F-FFA+DMED, SC-FFA+DMEM, MC-FFA+DMEM, LC-FFA+DMEM, LC-FFA+NADPH-I, LC-FFA+p38MAPK-I and VLC-FFA+DMEM, VLC-FFA+NADPH-I, VLC-FFA+p38MAPK-I groups were 4.57 ± 0.12, 4.36 ± 0.09, 4.55 ± 0.10, 6.84 ± 0.42, 4.45 ± 0.24, 5.08 ± 0.36, 2.23 ± 0.15, 3.90 ± 0.32, 3.81 ± 0.41. Compared with the F-FFA groups, the relative mRNA expressions of LCHAD significantly decreased in LC-FFA+DMEM/p38MAPK-I groups (P < 0.05) while increased in VLC-FFA groups (P < 0.05). Compared with the LC-FFA+DMEM groups, the relative mRNA expressions of LCHAD increased in LC-FFA+NADPH-I/p38MAPK-I groups (P < 0.05). The relative mRNA expressions of LCHAD in VLC-FFA+NADPH-I/p38MAPK-I groups significantly decreased versus VLC-FFA+DMEM group (P < 0.05). (2) Protein expression of LCHAD: The relative protein expressions of LCHAD in F-FFA+DMED, SC-FFA+DMEM, MC-FFA+DMEM, LC-FFA+DMEM, LC-FFA+NADPH-I, LC-FFA+p38MAPK-I and VLC-FFA+DMEM, VLC-FFA+NADPH-I, VLC-FFA+p38MAPK-I groups were 23.6 ± 13.0, 21.2 ± 10.2, 19.7 ± 1.9, 10.6 ± 2.6, 14.0 ± 1.8, 14.0 ± 2.8, 29.3 ± 1.9, 35.8 ± 3.2 and 35.2 ± 4.5 respectively. Compared with the F-FFA groups, the protein expressions of LCHAD significantly decreased in LC-FFA groups (P < 0.05) while increased in VLC-FFA+NADPH-I/p38MAPK-I groups (P < 0.05). CONCLUSION: Free fatty acids affect the gene and protein expressions of mitochondrial ß-oxidation enzyme of LCHAD in trophoblastic cells. Fatty acid ß-oxidation is impaired in trophoblast cells incubated with long-chain fatty acid. NADPH oxidase and p38MAPK inhibitors may alleviate such an effect. Thus p38MAPK signal transduction pathway may participate in this process. The correlation between very long chain fatty acids and fatty acid ß-oxidation is confirmed. But their interactions require further explorations.

[Interaction mechanism and influence between fatty acid oxidation in trophoblast cells and p38MAPK signal transduction pathway of severe preeclampsia].

OBJECTIVE: To investigate the effects of expression of mitochondria long-chain fatty acid oxidative enzyme (long-chain 3 hyroxyacyl CoA dehydrogenase, LCHAD) and p38 mitogen activated protein kinase (p38MAPK) signal transduction pathway in severe preeclampsia. METHODS: Serum-free trophoblast cells cultured in vitro were stimulated by early onset severe preeclampsia serum (E-PE group), late onset severe preeclampsia serum (L-PE group), HELLP syndrome serum (HELLP group), and normal pregnancy serum (NP group) respectively; each group was added DMEM/F12 medium, reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (NADPH-I) and p38MAPK inhibitor (p38-I) to stimulate cells. Expression of mRNA and protein of LCHAD in trophoblast cells were detected by real-time PCR and western blot. RESULTS: (1)The expression of mRNA of LCHAD: the level of mRNA of LCHAD in NP+DMEM, E-PE+DMEM, E-PE+NADPH-I, E-PE+p38-I, L-PE+DMEM, L-PE+NADPH-I, L-PE+p38-I and HELLP+DMEM, HELLP+NADPH-I, HELLP+p38-I groups were 1.00 ± 0.03, 0.14 ± 0.08, 0.95 ± 0.20, 1.43 ± 1.02, 0.37 ± 0.18, 1.51 ± 0.36, 1.60 ± 0.31, 0.10 ± 0.04, 0.49 ± 0.10, 0.44 ± 0.21, respectively. The relative expressions of mRNA of LCHAD were significantly reduced in E-PE+DMEM, L-PE+DMEM and HELLP+DMEM groups compared with the NP+DMEM group (P < 0.05). Compared with the NP groups, the relative expressions of mRNA of LCHAD were significantly increased in L-PE+NADPH-I and L-PE+p38-I group (P < 0.05), while reduced in HELLP groups(P < 0.05). (2) The expression of protein of LCHAD: the relative expressions of protein of LCHAD in NP+DMEM, E-PE+DMEM, E-PE+NADPH-I, E-PE+p38-I, L-PE+DMEM, L-PE+NADPH-I, L-PE+p38-I and HELLP+ DMEM, HELLP+NADPH-I, HELLP+p38-I groups were 19.4 ± 2.2, 10.7 ± 1.1, 17.9 ± 3.3, 19.1 ± 2.9, 16.4 ± 2.3, 20.3 ± 2.3, 20.9 ± 4.3, 12.4 ± 2.3, 17.6 ± 2.6, 17.7 ± 2.0 respectively. Compared with the NP groups, the protein expressions of LCHAD were significantly remarkably reduced in E-PE+DMEM, L-PE+DMEM and HELLP groups (P < 0.05). Compared with the DMEM groups, the protein expressions of LCHAD were significantly increased in NADPH-I and p38-I groups of E-PE, L-PE and HELLP groups (P < 0.05). CONCLUSIONS: These studies demonstrate that long chain fatty acid oxidation was involved in the pathogenesis and development of preeclampsia. The expressions of gene and protein of LCHAD were remarkably affected by early onset severe preeclampsia and HELLP syndrome. NADPH-I and p38-I may allay the disorder of fatty acid oxidation. p38MAPK signal transduction pathway may contributed in this process.

[Effects of expression of mitochondria long-chain fatty acid oxidative enzyme with different chain lengths of free fatty acids in trophoblast cells].

OBJECTIVE: To explore the interacting mechanisms and influences of different chain lengths of fatty acids and the expression of mitochondria long-chain 3 hydroxyacyl CoA dehydrogenase (LCHAD) in trophoblast cells. METHODS: The serum-free trophoblast cells cultured in vitro were divided into 5 groups to receive the stimulations of DMEM/F12 medium without FFA (F-FFA), short-chain fatty acids (SC-FFA), medium-chain fatty acids (MC-FFA), long-chain fatty acids (LC-FFA), very long-chain fatty acids (VLC-FFA). The expressions of mRNA and protein of LCHAD in trophoblast cells were detected by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: Compared with the F-FFA, SC-FFA and MC-FFA groups, the expressions of gene and protein of LCHAD significantly decreased (P < 0.05) in the LC-FFA group. The expression of gene of LCHAD increased significantly in the VLC-FFA group (P < 0.05). But no difference existed in protein expression between the VLC-FFA group and other three groups (P > 0.05). Gene expression of LCHAD had no difference among the F-FFA, SC-FFA, MC-FFA groups (P > 0.05). Compared with the LC-FFA group, the expression of gene of LCHAD increased significantly in the VLC-FFA group (P < 0.05). CONCLUSION: Free fatty acids may affect the expression of mitochondrial ß-oxidation enzyme of LCHAD in trophoblast cells. Long-chain fatty acid alters the LCHAD gene protein expression. The correlation between very long chain fatty acids and the gene expression of LCHAD has been detected and their interactions needs further explorations. Short or medium chain fatty acids have no significant effect on the mitochondrial metabolism of fatty acid ß-oxidation in trophoblast cells.

[Study on the oxidative stress and inflammation in trophoblast cells stimulated by different chain length fatty acids].

OBJECTIVE: To investigate the oxidative stress and inflammation in trophoblast cells stimulated by different chain length fatty acids. METHODS: Serum-free trophoblast cells cultured in vitro were divided into five groups, which were incubated with DMEM medium without free fatty acid (F-FFA), short chain fatty acids (SC-FFA), medium chain fatty acids (MC-FFA), long chain fatty acids (LC-FFA), very long chain fatty acids (VLC-FFA). Then cells in each group were stimulated by DMEM medium, reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (apocynin) and p38 mitogen-activated protein kinases (p38MAPK) inhibitor (SB203580) and were subdivided as each FFA plus-DMEM group, plus-NADPH-I and plus-p38MAPK-I groups. Expressions of mRNA and protein of p38MAPK and cyclooxygenase 2 (COX-2) in trophoblast cells were detected by real-time PCR and western blot. RESULTS: (1) The mRNA expression of p38MAPK in LC-FFA + DMEM, VLC-FFA + DMEM, LC-FFA + NADPH-I, LC-FFA + p38MAPK-I, VLC-FFA + NADPH-I, VLC-FFA + p38MAPK-I group were 4.56 ± 0.28, 22.65 ± 2.40, 0.87 ± 0.06, 1.02 ± 0.15, 19.87 ± 1.93, 10.22 ± 0.75 separately, and the protein expressions were 0.79 ± 0.02, 0.93 ± 0.10, 0.43 ± 0.06, 0.44 ± 0.19, 0.79 ± 0.10, 0.81 ± 0.14. Compared with other groups, the mRNA and protein expressions of p38MAPK in LC-FFA + DMEM, VLC-FFA + DMEM group were increased (P < 0.05). Compared with LC-FFA + DMEM group, mRNA and protein expressions of p38MAPK in LC-FFA + NADPH-I and LC-FFA + p38MAPK-I group were significantly decreased (P < 0.05). Compared with VLC-FFA + DMEM group, mRNA and protein expressions of p38MAPK had no difference in VLC-FFA + NADPH-I group (P > 0.05), mRNA expression of p38MAPK in VLC-FFA + p38MAPK-I group was significantly decreased (P < 0.05), but there was no difference in protein expression (P > 0.05). (2) The mRNA expression of COX-2 in LC-FFA + DMEM, VLC-FFA + DMEM, LC-FFA + NADPH-I, LC-FFA + p38MAPK-I, VLC-FFA + NADPH-I, VLC-FFA + p38MAPK-I group were 3.97 ± 0.03, 39.08 ± 0.63, 0.99 ± 0.13, 0.98 ± 0.18, 20.93 ± 3.70, 13.46 ± 2.31 separately, and the protein expressions were 1.32 ± 0.20, 1.33 ± 0.25, 0.59 ± 0.13, 0.58 ± 0.30, 0.88 ± 0.18, 0.91 ± 0.24. Compared with other groups, mRNA and protein expressions of COX-2 in LC-FFA + DMEM and VLC-FFA + DMEM group were significantly increased (P < 0.05). Compared with LC-FFA + DMEM group, mRNA and protein expressions of COX-2 in LC-FFA + NADPH-I and LC-FFA + p38MAPK-I group were decreased (P < 0.05). Compared with VLC-FFA + DMEM group, mRNA and protein expressions of COX-2 in VLC-FFA + NADPH-I and VLC-FFA + p38MAPK-I group were all decreased (P < 0.05). (3) The correlation analysis showed that there were significantly positive correlations between the mRNA and protein expressions of p38MAPK and COX-2 in LC-FFA group (P < 0.05). There were significantly positive correlations in protein expression (P < 0.05), but no correlation in the mRNA expression between p38MAPK and COX-2 in the F-FFA, SC-FFA, MC-FFA, VLC-FFA groups (P > 0.05). CONCLUSIONS: The oxidative stress and inflammation may exist in trophoblast cells which were stimulated by LC-FFA and VLC-FFA. p38MAPK signal transduction pathway may contributed in this process.

[Correlation between severe preeclampsia and abnormal expression of long-chain fatty acid oxidative enzyme].

OBJECTIVE: To explore the correlation between severe preeclampsia and abnormal expression of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD). METHODS: Serum-free trophoblast cells cultured in vitro were divided into 4 groups under the stimulations of normal pregnancy serum (NP group), early onset severe preeclampsia serum (E-PE group), late onset severe preeclampsia serum (L-PE group) and HELLP (hemolysis, elevated liver enzymes & low platelets) syndrome serum (HELLP group) respectively. The expressions of mRNA and protein of LCHAD in trophoblast cells were detected by real-time polymerase chain reaction (PCR) and Western blot. RESULT: (1) Expression of LCHAD mRNA: the relative expressions of mRNA of LCHAD in NP, E-PE, L-PE and HELLP groups were 1.00 ± 0.00, 3.08 ± 0.22, 1.62 ± 0.23 and 3.36 ± 0.18 respectively. The relative expressions of LCHAD mRNA were significantly reduced in the E-PE, L-PE and HELLP groups versus the NP group (P < 0.05). Compared with the L-PE group, the gene expressions of LCHAD significantly decreased in the E-PE and HELLP groups (P < 0.05) while no significant difference was found between the E-PE and HELLP groups (P > 0.05). (2) Expression of LCHAD protein: the relative expressions of LCHAD protein were 4.94 ± 0.02, 2.93 ± 0.13, 4.14 ± 0.06 and 2.80 ± 0.09 in the NP, E-PE, L-PE and HELLP groups respectively. The protein expressions of LCHAD were remarkably reduced in the E-PE, L-PE and HELLP groups versus the NP group (P < 0.05). The expressions of LCHAD protein remarkably decreased in the E-PE and HELLP groups versus the L-PE group (P < 0.05) while no significant difference was found between the E-PE and HELLP groups (P > 0.05). CONCLUSION: Long chain fatty acid oxidation is involved in the pathogenesis and development of preeclampsia. The expressions of LCHAD gene and protein are remarkably affected by early onset severe preeclampsia and HELLP syndrome. The interacting mechanism and influence between fatty acid oxidation and the development of preeclampsia are worth further exploring.

Immunomodulatory effect of polysaccharides extracted from the medicinal mushroom Antrodia camphorata (higher Basidiomycetes) in specific pathogen-free chickens.

The immunomodulatory effect of polysaccharides extracted from mycelia of the medicinal mushroom Antrodia camphorata in submerged culture was studied in 100 specific pathogen-free (SPF) chickens. The chickens were randomly divided into 2 groups (50 per group). For the treated group, each kilogram of SPF chickens was fed 5 mg of A. camphorata extract (ACE) for 35 consecutive days. Chickens were killed on days 7, 14, 21, 28, and 35, and lymphocytes were separated from the blood, spleen, thymus, bursa, kidney, and pancreas of the chickens. The results showed that, compared to the control group, the immune organ indices (except for the thymus) were higher after 14 days (P < 0.05), and the contents of globulin in blood were significantly increased on the 21st day (P < 0.05). The most of biochemical indices did not significantly changed within 35 days of treatment. Moreover, the response of proliferation and the rates of positive T lymphocytes in blood were higher than in the control group (P < 0.05).The results presented herein indicate that ACE could enhance the immune functions of the organs in SPF chickens and could be an attractive application of nutraceuticals and pharmaceuticals.