LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells.

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Global identification and quantitative analysis of representative components of Xin-Nao-Kang Capsule, a traditional Chinese medicinal formula, by UHPLC-Q-TOF-MS and UHPLC-TQ-MS.

Xin-Nao-Kang Capsule (XNKC), a well-known traditional Chinese medicine (TCM), is widely used for the treatment of angina pectoris, cerebral arteriosclerosis and coronary artery disease in China. However, the chemical components have not been holistically explored. In this study, a total of 173 chemical components including 48 flavonoids, 8 lactones, 35 acids, 12 phenanthraquinones, 8 monoterpene glycosides, 32 triterpenoids and 30 other compounds were identified, among which 37 were unambiguously characterized in comparison with their corresponding authentic standards using ultra-high performance liquid chromatography coupled with quadruple time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS). To further elucidate the major representative constitutes of XNKC, a high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-TQ-MS) method was used for simultaneous quantitative analysis of 34 major representative constitutes in ten batches of XNKC samples, which was validated in terms of linearity, precision, accuracy, repeatability and recovery. Meanwhile, the results of chemometric analysis indicated that puerarin and salvianolic acid B might be the most potential quantitative markers for quality control of XNKC. Taken together, the chemical constitutes of XNKC were systematically identified and a reliable quantitative method coupled with chemometric analysis was successfully employed for evaluating the holistic quality of XNKC. This study will provide a robust foundation for the holistic quality assessment of XNKC.

Monitoring the quality consistency of Fufang Danshen Pills using micellar electrokinetic chromatography fingerprint coupled with prediction of antioxidant activity and chemometrics.

A fast micellar electrokinetic chromatography fingerprint method combined with quantification was developed and validated to evaluate the quality of Fufang Danshen Pills, a traditional Chinese Medicine, which has been used in the treatment of cardiovascular system diseases, in which the tetrahedron optimization method was first used to optimize the background electrolyte solution. Subsequently, the index of the fingerprint information amount of I was performed as an excellent objective indictor to investigate the experimental conditions. In addition, a systematical quantified fingerprint method was constructed for evaluating the quality consistency of 20 batches of test samples obtained from the same drug manufacturer. The fingerprint analysis combined with quantitative determination of two components showed that the quality consistency of the test samples was quite good within the same commercial brand. Furthermore, the partial least squares model analysis was used to explore the fingerprint-efficacy relationship between active components and antioxidant activity in vitro, which can be applied for the assessment of anti-oxidant activity of Fufang Danshen pills and provide valuable medicinal information for quality control. The result illustrated that the present study provided a reliable and reasonable method for monitoring the quality consistency of Fufang Danshen pills.

Capillary electrophoresis fingerprinting coupled with chemometrics to evaluate the quality consistency and predict the antioxidant activity of Sanhuang tablet as part of its quality control.

A capillary electrophoresis fingerprint was constructed for Sanhuang tablet, a Chinese traditional patent medicine, that was commonly used in clinical practice, where the isosceles trapezoid method was first applied for the optimization of background electrolyte solution, and the resolution index was performed to assess the experimental conditions; furthermore, a novel linear quantitative fingerprint method was established for accurate qualitative and quantitative discrimination of the test samples from diverse commercial brands. The fingerprint analysis coupled with quantitative determination of two components was employed to elucidate that the quality consistency of the products was relatively good within one manufactory, but poor among different companies for the 30 batches of samples. In addition, the fingerprint-efficacy relationship between chemical components and antioxidant activity in vitro was investigated using partial least squares analysis, and the calibration and prediction of the antioxidant activity of the selected samples via fingerprint data were presented with the desired results. This work illustrates that the proposed fingerprint analysis based on linear quantitative fingerprint method can be applied for the quality evaluation of traditional Chinese medicine and herbal preparations as part of their quality control, and the constructed mathematical model is particularly suitable for depicting the fingerprint-efficacy relationship.