Determination of protein-bound methionine oxidation in the hippocampus of adult and old rats by LC-ESI-ITMS method after microwave-assisted proteolysis.

Protein-bound methionine (Met) oxidation has been associated with normal aging and a variety of age-related diseases, including Alzheimer's disease and Parkinson's disease. Monitoring the changes of protein-bound methionine content in the brain in response to normal aging and oxidative stress is of great interest and could be used as an indicator of oxidative stress of rats in pathological conditions. We have developed a rapid analytical method for the determination of oxidized products of protein-bound methionine in rat brain. The assay involved rapid acid proteolysis with microwave irradiation and solid-phase extraction of the free amino acids followed by LC-ESI-ITMS analysis. Detection was achieved in positive ionization with an ion trap mass spectrometer operating in multiple-reaction monitoring mode. The calibration curves of the analytes were linear (r(2) > 0.99) in the range between 0.098 and 1.560 µg/mL. Intra- and inter-day relative standard deviation percentages were <9% and <8%, respectively. The assay performance was sufficient to support a rapid analytical tool for monitoring brain protein-bound methionine oxidation levels. The content of protein-bound Met and methionine sulfoxide (MetO) in the hippocampus of adult and old rats with or without H(2)O(2) treatment was determined by employing the new method. The content of protein-bound MetO was significantly increased in old rats after exposure to H(2)O(2). This result indicates increased sensitivity to Met oxidation in the hippocampus of old rats.

Fucoxanthin extracted from Laminaria Japonica inhibits metastasis and enhances the sensitivity of lung cancer to Gefitinib.

ETHNOPHARMACOLOGICAL RELEVANCE: Laminaria japonica, a brown seaweed, has been used in Traditional Chinese Medicine (TCM) to treat a variety of diseases including lung cancer. AIM OF THE STUDY: To demonstrate the effects of Fucoxanthin (FX), a major active component extracted from Laminaria japonica on metastasis and Gefitinib (Gef) sensitivity in human lung cancer cells both in vitro and in vivo. MATERIALS AND METHODS: Invasion and migration of lung cancer cells were detected using the wound healing assay and transwell assay. Epithelial-to-mesenchymal transition (EMT) factors and PI3K/AKT/NF-κB pathways were analyzed by western blotting. RNA interference (RNAi) technology was used to silence TIMP-2 gene expression in A549 cells. The anti-metastatic effect of FX was evaluated in vivo in an experimental lung metastatic tumor model. On the other hand, cell counting kit-8 assay was used to study the cell viability of human lung cancer PC9 cells and Gef resistant PC9 cells (PC9/G) after Gef, FX or FX combined with Gef treatment. PC9 xenograft model was established to explore the anti-tumor effect of FX or combined with Gef. Immunohistochemistry staining assay and immunofluorescence staining assay were used to reveal the effects of FX on lung cancer cell proliferation and apoptosis. RESULTS: FX was able to significantly inhibit lung cancer cells migration and invasion in vitro. FX suppressed the expressions of Snail, Twist, Fibronectin, N-cadherin, MMP-2, PI3K, p-AKT and NF-κB, and increased the expression of TIMP-2. Furthermore, knockdown of TIMP-2 attenuated FX-mediated invasion inhibition. Additionally, we demonstrated that FX inhibited lung cancer cells metastasis in vivo. The anti-metastatic effects of FX on lung cancer cells might be attributed to inhibition of EMT and PI3K/AKT/NF-κB pathway. We further demonstrated that the anti-tumor activity of FX was not only limited to the drug sensitive cell lines, but also prominent on lung cancer cells with Gef resistant phenotype. Furthermore, in vivo xenograft assay confirmed that FX inhibited tumor growth and enhanced the sensitivity of lung cancer cells to Gef and this effect may be due to inhibition of tumor cell proliferation and activation of apoptosis. CONCLUSION: Collectively, our findings suggested that FX suppresses metastasis of lung cancer cells and overcomes EGFR TKIs resistance. Thus, FX is worthy of further investigation as a drug candidate for the treatment of lung cancer.

HPLC and LC-MS analysis of sinomenine and its application in pharmacokinetic studies in rats.

AIM: To improve and validate analytical methods based on HPLC and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the quantitative measurement of sinomenine in rat plasma and brain tissue. METHODS: The separation of analytes and the internal standard (IS), chloramphenicol, was performed on an Agilent TC-C18 column (250×4.6 mm, 5 µm). Blood samples were measured with a Surveyor photodiode array (PDA) detector at a wavelength of 263 nm. The LCQ DECA XP(Plus) mass spectrometer was operated in the multiple reactions monitoring mode using positive electrospray ionization, and the transition from the precursor ion (m/z 279) to the product ion (m/z 224) for sinomenine was measured in brain tissue. RESULTS: Measurements were linear over the concentration range of 0.1-100 µg/mL for sinomenine in plasma and over the range of 0.01-5.00 µg/g for sinomenine in brain tissue. The intra- and inter-day variabilities were less than 10% of the relative standard deviation (RSD), and the extraction and recovery of sinomenine was 72.48%-80.26% from plasma and 73.75%-80.26% from brain tissue. The limit of quantification (LOQ) was 0.1 µg/mL for plasma, and 0.01 µg/g for brain tissue. Identification of sinomenine was reproducible at 0.5, 5, and 50 µg/mL in the plasma and at 0.05, 0.50, and 2.00 µg/g in brain tissue. The concentration of sinomenine measured in brain tissue after a single ip dose had a neuroprotective effect on H2O2-induced injury in PC12 cells in vitro. CONCLUSION: Our methods offered a sensitivity within a wide linear concentration range for sinomenine. These methods were successfully applied to evaluate sinomenine pharmacokinetics over time in rat brain tissue after a single ip dose of 30 mg/kg.

[Study the restoration technology of concentrated application-natural diffusion about amendments of acidified soil of hilly woodland].

Through concentrated application of lime, sewage sludge and lime + sewage sludge on the sloping top of the hilly woodlands, the restoration effects of the three soil amendments on the acidified soil of hilly woodland were studied. The results showed that: (1) Joint application of sewage sludge + lime can significantly (P < 0.05) decrease soil acidity, promote the rapid increase in soil organic matter and nitrogen content, increase soil cation exchange capacity, and effectively improve acidified soil. (2) Through natural diffusion mechanisms of surface and subsurface runoff, a large area of acidified soil of hilly woodlands can be restored by concentrated application of soil amendments on the sloping top of the hilly woodlands. (3) It is conducive to solve the pollution problems of the urban sewage sludge by using municipal sewage sludge to restore acidified soil, but only for the restoration of acidified soil of timber forest.