RNA-binding domain 2 of nucleolin is important for the autophagy induction of curcumol in nasopharyngeal carcinoma cells.

BACKGROUND & AIMS: Excessive autophagy induces cell death and is regarded as the treatment of cancer therapy. We have confirmed that the anti-cancer mechanism of curcumol is related to autophagy induction. As the main target protein of curcumol, RNA binding protein nucleolin (NCL) interacted with many tumor promoters accelerating tumor progression. However, the role of NCL in cancer autophagy and in curcumol's anti-tumor effects haven't elucidated. The purpose of the study is to identify the role of NCL in nasopharyngeal carcinoma autophagy and reveal the immanent mechanisms of NCL played in cell autophagy. METHODS & RESULTS: In the current study, we have found that NCL was markedly upregulated in nasopharyngeal carcinoma (NPC) cells. NCL overexpression effectively attenuated the level of autophagy in NPC cells, and NCL silence or curcumol treatment obviously aggravated the autophagy of NPC cells. Moreover, the attenuation of NCL by curcumol lead a significant suppression on PI3K/AKT/mTOR signaling pathway in NPC cells. Mechanistically, NCL was found to be directly interact with AKT and accelerate AKT phosphorylation, which caused the activation of the PI3K/AKT/mTOR pathway. Meanwhile, the RNA Binding Domain (RBD) 2 of NCL interacts with Akt, which was also influenced by curcumol. Notably, the RBDs of NCL delivered AKT expression was related with cell autophagy in the NPC. CONCLUSION: The results demonstrated that NCL regulated cell autophagy was related with interaction of NCL and Akt in NPC cells. The expression of NCL play an important role in autophagy induction and further found that was associated with its effect on NCL RNA-binding domain 2. This study may provide a new perspective on the target protein studies for natural medicines and confirm the effect of curcumol not only regulating the expression of its target protein, but also influencing the function domain of its target protein.

The liver metabolic features of Mogroside V compared to Siraitia grosvenorii fruit extract in allergic pneumonia mice.

BACKGROUND: For a long time, Siraitia grosvenorii fruit extract (SGFE) and its dominant compounds, mogroside V(MV) were both reported to have therapeutic effects on allergic pneumonia, while previous studies only stay on phenotype and mechanism of the two active ingredients, hardly have any studies compared the two ingredients on the effect of liver metabolic, and revealed the relationship between mechanism and liver metabolism. OBJECTIVE: Here we elucidated and compared the curative mechanisms of SGFE and MV on allergic pneumonia through liver metabolomics. METHODS: We established allergic pneumonia mice using ovalbumin, then treated the mice with SGFE, MV and positive drug of Suhuang Zhike Jiaonang. The effects of the drugs were evaluated by detecting inflammatory cytokines, pathological examination and liver oxidative stress biomarkers. We explored the metabolic features between SGFE and MV through liver metabolomics consequently. RESULTS: At phenotype, we confirmed that MV and SGFE both inhibited the expression of inflammatory cytokines including interleukins-5 (IL-5), IL-13, IL-17 and OVA-induced immunoglobulin E, which can also relieve inflammatory cells infiltration and mesenchymal thickening in lung tissue compared with positive drug. In addition, both of them can alleviate oxidative stress damage in liver, while MV showed a superior effect than SGFE. In metabolomic analysis, the two ingredients were found to ameliorate inflammatory and oxidative reaction mainly in controlling pathways of Riboflavin metabolism and Glutathione metabolism. While SGFE were found to control other metabolic pathways such as Phenylalanine metabolism, Sphingolipid metabolism, Glycerollipid metabolism, Glycine, serine and threonine metabolism and Arginine and proline metabolism. CONCLUSION: From the results we can infer that the minor ingredients except MV in SGFE contribute poor function to the treatment of allergic pneumonia and MV may be the main functional constituent that relieve allergic pneumonia in SGFE. This study will be beneficial to figuring out a systematic theory of Siraitia grosvenorii active ingredients and proposing a guidance for pharmacology development.

Curcumol simultaneously induces both apoptosis and autophagy in human nasopharyngeal carcinoma cells.

Autophagy is usually considered as a protective mechanism against cell death, and in the meantime, leads to cell injury even apoptosis. Apoptosis and autophagy are very closely connected and may cooperate, coexist, or antagonize each other on progressive occurrence of cell death triggered by natural compounds. Therefore, the interplay between the two modes of death is essential for the overall fate of cancer cells. Our previous study revealed that curcumol induced apoptosis in nasopharyngeal carcinoma (NPC) cells. Recently, curcumol was found to induce autophagy in cancer cells. However, whether curcumol can induce NPC cells autophagy and the effects of autophagy on apoptosis remain elusive. In this study, we found that curcumol induced autophagy through AMPK/mTOR pathway in CNE-2 cells. Moreover, inhibiting autophagy by autophagy inhibitor 3-methyladenine (3-MA) or apoptosis inhibitor z-VAD-fmk significantly increased proliferation while attenuated apoptosis and autophagy compared with the curcumol 212 µM group. In contrast, combining curcumol with autophagy agonist rapamycin and apoptosis inducer MG132 synergized the apoptotic and autophagic effect of curcumol. Taken together, our study demonstrates that curcumol promotes autophagy in NPC via AMPK/mTOR pathway, induces autophagy enhances the activity of curcumol in NPC cells; the combination of autophagy inducer and curcumol can be a new therapeutic strategy for NPC.

Mogroside V reduce OVA-induced pulmonary inflammation based on lung and serum metabolomics.

BACKGROUND: Mogroside V, the main ingredient of Siraitia grosvenorii, has been proved to have therapeutic effects on pulmonary diseases. The specific mechanism still remains to be clarified, which hinders the potence of its medicinal value. PURPOSE: Serum and lung metabolomics based on LC-MS analysis were applied to explore the mechanism of mogroside V against lung inflammation. METHOD: In this study, balb/c mice were divided into control, model, mogeoside V and SH groups. We evaluated the protective effects of mogroside V on lung inflammation in asthmatic mice. Suhuang Zhike Jiaonang was used as positive drug. Metabolic profiles of serum and lung samples of mice in control, model and mogroside V groups were analyzed by LC-MS. RESULTS: Administration of mogroside V effectively relieved the expression of biochemical cytokines and lung inflammatory infiltration of asthmatic mice caused by ovalbumin (OVA). And visceral index of mice treated with mogroside V was close to control group. These results indicated that mogroside V ameliorated OVA-induced lung inflammation. LC-MS based metabolomics analysis demonstrated 6 main pathways in asthmatic mice including Vitamin B6 metabolism, Taurine and hypotaurine metabolism, Ascorbate and aldarate metabolism, Histidine metabolism, Pentose and glucuronate interconversions, Citrate cycle (TCA cycle) were regulated after using mogroside V. CONCLUSION: The study firstly elucidates the metabolic pathways regulated by mogroside V on lung inflammation through metabolomics, providing a theoretical basis for more sufficient utilization and compatibility of mogroside V.

Curcumol inhibits EBV-positive Nasopharyngeal carcinoma migration and invasion by targeting nucleolin.

Metastasis is a major cause of recurrence and death in patients with EBV-positive Nasopharyngeal carcinoma (NPC). Previous reports documented that curcumol has both anti-cancer and anti-viral effects, but there is little literature systematically addressing the mechanism of curcumol in EBV-positive tumors. Previously we found that nucelolin (NCL) is a target protein of curcumol in CNE2 cells, an EBV-negative NPC, and in this experiment, we reported a critical role for NCL in promoting migration and invasion of C666-1 cells, an EBV-positive NPC, and found that the expression of NCL determined the level of curcumol's efficacy. Mechanistically, NCL interacted with Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) to activate VEGFA/VEGFR1/PI3K/AKT signaling pathway, which in turn promoted NPC cell invasion and metastasis. Moreover, further study showed that the differential expression of NCL and curcumol intervention only had a regulatory effect on the nuclear accumulation of VEGFR1, which strengthened the anti-cancer effect of curcumol mediated through NCL. Our findings indicated that curcumol exerted anti EBV-positive NPC invasion and metastasis by downregulating EBNA1 and inhibiting VEGFA/VEGFR1/PI3K/AKT signaling by targeting NCL, which provides a novel pharmacological basis for curcumol's clinical use in treating patients with EBV-positive NPC.

PAT: From Western solid dosage forms to Chinese materia medica preparations using NIR-CI.

Near-infrared chemical imaging (NIR-CI) is an emerging technology that combines traditional near-infrared spectroscopy with chemical imaging. Therefore, NIR-CI can extract spectral information from pharmaceutical products and simultaneously visualize the spatial distribution of chemical components. The rapid and non-destructive features of NIR-CI make it an attractive process analytical technology (PAT) for identifying and monitoring critical control parameters during the pharmaceutical manufacturing process. This review mainly focuses on the pharmaceutical applications of NIR-CI in each unit operation during the manufacturing processes, from the Western solid dosage forms to the Chinese materia medica preparations. Finally, future applications of chemical imaging in the pharmaceutical industry are discussed.

Evaluation of the value of near infrared (NIR) spectromicroscopy for the analysis of glycyrrizhic acid in licorice.

It has been reported that hyperspectral data could be employed to qualitatively elucidate the spatial composition of tablets of Chinese medicinal plants. To gain more insights into this technology, a quantitative profile provided by near infrared (NIR) spectromicroscopy was further studied by determining the glycyrrhizic acid content in licorice, Glycyrrhiza uralensis. Thirty-nine samples from twenty-four different origins were analyzed using NIR spectromicroscopy. Partial least squares, interval partial least square (iPLS), and least squares support vector regression (LS-SVR) methods were used to develop linear and non-linear calibration models, with optimal calibration parameters (number of interval numbers, kernel parameter, etc.) being explored. The root mean square error of prediction (RMSEP) and the coefficient of determination (R(2)) of the iPLS model were 0.717 7% and 0.936 1 in the prediction set, respectively. The RMSEP and R(2) of LS-SVR model were 0.515 5% and 0.951 4 in the prediction set, respectively. These results demonstrated that the glycyrrhizic acid content in licorice could barely be analyzed by NIR spectromicroscopy, suggesting that good quality quantitative data are difficult to obtain from microscopic NIR spectra for complicated Chinese medicinal plant materials.

[Rapid assessment of critical quality attributes of Chinese materia medica (II): strategy of NIR assignment].

The present paper firstly reviewed the research progress and main methods of NIR spectral assignment coupled with our research results. Principal component analysis was focused on characteristic signal extraction to reflect spectral differences. Partial least squares method was concerned with variable selection to discover characteristic absorption band. Two-dimensional correlation spectroscopy was mainly adopted for spectral assignment. Autocorrelation peaks were obtained from spectral changes, which were disturbed by external factors, such as concentration, temperature and pressure. Density functional theory was used to calculate energy from substance structure to establish the relationship between molecular energy and spectra change. Based on the above reviewed method, taking a NIR spectral assignment of chlorogenic acid as example, a reliable spectral assignment for critical quality attributes of Chinese materia medica (CMM) was established using deuterium technology and spectral variable selection. The result demonstrated the assignment consistency according to spectral features of different concentrations of chlorogenic acid and variable selection region of online NIR model in extract process. Although spectral assignment was initial using an active pharmaceutical ingredient, it is meaningful to look forward to the futurity of the complex components in CMM. Therefore, it provided methodology for NIR spectral assignment of critical quality attributes in CMM.

[Multivariate detection limits of baicalin in qingkailing injection based on four NIR spectrometer types].

Multivariate detection limits (MDLs) of different types of near-infrared instruments were investigated to guide the selection of device type for TCM NIR analysis. In this paper, near-infrared spectroscopy of Qingkailing injection was performed in transmission mode on four near-infrared spectrometers named a, b, c and d, respectively. High performance liquid chromatography (HPLC) was used as the reference method to determine the content of baicalin in Qingkailing injection. Partial least squares (PLS) and interval partial least squares (iPLS) quantitative models of baicalin in Qingkailing injection were established and MDLs of quantitative models based on different types of instruments were calculated. The determination coefficient of prediction (R2(pre))and the root mean square errors of prediction (SEP) of PLS models of four different near-infrared spectrometers are 0.9762 and 230.4 microg x mL(-1) (a), 0.9561 and 246.4 microg x mL(-1) (b), 0.9662 and 264.4 microg x mL*-1) (c), 0.9985 and 71.5 microg x mL(-1) (d). And the model of instrument d shows a better prediction performance than the other three types. There are no remarkable superiorities in predictability in iPLS models of instruments a and b after variable selection, since the R2(pre) and SEP values for instruments a and b are 0.9771 and 218.4 microg x mL(-1), and 0.9754 and 219.4 microg x mL(-1), respectively. Models c and d show no results of variable selection. MDLs (delta(0.05, 0.05) of different instruments are all less than 250 microg x mL(-1), and the MDLs of instruments c and d reach to 58 and 2.9 microg x mL(-1) respectively. The results reveal that the predictability of models and corresponding MDLs are different for different detection equipments. This paper innovatively used the theory of MDL to investigate the detection performance of different types of NIR instruments. The results demonstrated the feasibility of the approach. And it is expected that in actual applications, choosing the right type of instrument should be based on the characteristics of the study carrier to ensure quantitative accuracy.

Mitochondrial signaling for histamine releases in laser-irradiated RBL-2H3 mast cells.

BACKGROUND: The low power laser irradiation (LPLI) can promote the wound healing, but the mechanism is still not fully understood. We have found in our previous work that the LPLI induces mast cells to release the histamine and thus suggested that the increased histamine release is probably one of the causes for promoting the wound healing since mast cells have been found to play positive roles in the process of wound healing. This study aims to explore the mechanism of histamine release in RBL-2H3 mast cells under laser irradiations. MATERIALS AND METHODS: The wavelength effect of laser irradiations, the permeability function of mitochondrial membrane, the Bcl-2 effect, the cytosolic alkalinization and the increment of intracellular Ca(2+) ([Ca(2+)](i)), on histamine release in RBL-2H3 cells were studied, respectively, with the corresponding fluorescence probes. RESULTS: The action bands of laser irradiations were consistent with the absorption bands of cytochrome c oxidase, suggesting that cytochrome c oxidase is the photoacceptor. After laser irradiation, (1) the cytochrome c releases from mitochondrial to cytosol reflecting an increased permeability of mitochondrial membrane, (2) the cytosolic alkalinization appears, (3) [Ca(2+)](i) increases, and (4) finally the enhancement of histamine release occurs. When Bcl-2 was used to inhibit the permeability of mitochondrial membrane these cellular signaling from (1) to (4) were all suppressed obviously. CONCLUSION: As a photoacceptor, cytochrome c oxidase absorbs incident photons and initiates the mitochondrial signaling. When the signals are transferred from the mitochondrial to the cytosol, the cytosolic alkalinization appears leading to the opening of a Ca(2+) channel on the membrane, the transient receptor potential vanilloid (TRPV), and an increment of [Ca(2+)](i). The increased [Ca(2+)](i) consequently mediates an enhanced histamine release. Such a responding chain is a suggested mechanism to understand the histamine release in RBL-2H3 cells under laser irradiations.

Effects of shear stress on intracellular calcium change and histamine release in rat basophilic leukemia (RBL-2H3) cells.

Massage, one form of physical therapy, is widely used for a large number of musculoskeletal disorders, but its exact mechanism still remains to be elucidated. One hypothesis is that the shear stress caused by massage may induce cutaneous mast cells to release histamine, thereby improving the local tissue microcirculation of blood. In the present work, a mast cell line (rat basophilic leukemia cells, RBL-2H3) was used in vitro to study cellular responses to the stimulus of shear stress generated by a rotating rotor in a cell dish. The intracellular calcium ([Ca2+]c) was studied by confocal fluorescence microscopy with Fluo-3/AM staining and the released histamine was measured with a fluorescence spectrometer using o-phthalaldehyde (OPA) staining. An elevation of [Ca2+]c occurred immediately after the shear stress, followed by histamine release. However, both [Ca2+]c increase and histamine release disappeared when a Ca2+-free saline was used, indicating that the rise in the [Ca2+]c is due to a Ca2+ influx from the extracellular buffer. Furthermore, Ruthenium red, a transient receptor potential vanilloid (TRPV) inhibitor, could effectively block the shear stressinduced histamine release, suggesting that TRPV membrane proteins are the likely targets of the shear stress. Because histamine is a well-known mediator of microvascular tissue dilation, these results may have an important impact on understanding the mechanism involved in massage therapy.