[Mechanism of moistening process of Rehmanniae Radix based on kinetic process].

The moistening process of Rehmanniae Radix was characterized quantitatively by moisture phase, texture properties, and component content based on water absorption kinetics and expansion kinetics. Non-linear fitting of water absorption kinetics and expansion kinetics in the moistening process of Rehmanniae Radix was carried out. Low-field nuclear magnetic resonance and imaging(LF-NMR/MRI) technology was used to investigate the phase state and distribution changes of water during the moistening process. The Texture Analyzer was used for the determination of texture properties. The correlations between water absorption rate, expansion rate, water phase state, hardness, and compression cycle work of Rehmanniae Radix at different moistening time were analyzed. The results showed that the water absorption kinetics and expansion kinetics of Rehmanniae Radix were in accordance with the first-order kinetics. Moreover, the water absorption rate and expansion rate increased with the increase in temperature but decreased with the increase in the size of the medicinal materials.In the moistening process, the moisture was transferred from the outside to the inside, and the proportion of the moisture phase changed significantly.Within 16 hours, free water increased from 0.825% to 97.7%,while bound water decreased from 99.2% to 2.33%.Within 28 hours, the texture properties, such as hardness and compression cycle work, decreased gradually with the prolongation in moistening time.At 32 hours, water was evenly distributed throughout the whole medicinal material, and the texture properties also tended to be stable.Pearson correlation bivariate analysis showed that moistening time, water absorption rate, expansion rate, the relative content of free water and bound water, hardness, and compression cycle work were significantly correlated, suggesting that water absorption kinetics and expansion kinetics, LF-NMR/MRI,and Texture Analyzer could directly and quantitatively characterize the moistening process.This study is expected to provide a scientific basis for clarifying the scientific connotation of the moistening process of Rehmanniae Radix.

Simultaneous qualitative and quantitative analysis of eight alkaloids in Corydalis Decumbentis Rhizoma (Xiatianwu) and Corydalis Rhizoma (Yanhusuo) by high-performance liquid chromatography and high-resolution mass spectrometry combined with chemometric methods.

In this study, we established a comprehensive high-performance liquid chromatography coupled with diode array detection and high-resolution mass spectrometry method to identify 10 and quantified eight constituents in Corydalis Decumbentis Rhizoma ("Xiatianwu" in Chinese) and Corydalis Rhizoma ("Yanhusuo" in Chinese). Chemometric methods were applied to distinguish the botanical origins of the Xiatianwu and Yanhusuo samples. Chromatographic separation was achieved using an Agilent Poroshell EC-C18 column with mobile phases A (1000 ml of 0.2% acetic acid solution containing 2.8 ml of triethylamine) and B (acetonitrile) and stepwise gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, intra- and interday precision and repeatability, the limit of detection, the limit of quantitation, and recovery. Twenty-six Xiatianwu samples and 10 Yanhusuo samples were analyzed for quality evaluation. In addition, hierarchical clustering analysis and principal component analysis were used to discriminate among samples of different botanical origins. The results showed that the contents of eight alkaloids in Xiatianwu and Yanhusuo were significantly different. Moreover, it was found that chemometric methods could be applied to accurately distinguish these two often conflated Chinese medicinal materials. In conclusion, this study provides a relatively comprehensive method for botanical origin identification and Xiatianwu and Yanhusuo quality control.

Blunting TRPML1 channels protects myocardial ischemia/reperfusion injury by restoring impaired cardiomyocyte autophagy.

Accumulating evidence suggests that autophagy dysfunction plays a critical role in myocardial ischemia/reperfusion (I/R) injury. However, the underling mechanism of malfunctional autophagy in the cardiomyocytes subjected to I/R has not been well defined. As a result, there is no effective therapeutic option by targeting autophagy to prevent myocardial I/R injury. Here, we used both an in vitro and an in vivo I/R model to monitor autophagic flux in the cardiomyocytes, by exposing neonatal rat ventricular myocytes to hypoxia/reoxygenation and by subjecting mice to I/R, respectively. We observed that the autophagic flux in the cardiomyocytes subjected to I/R was blocked in both in vitro and in vivo models. Down-regulating a lysosomal cationic channel, TRPML1, markedly restored the blocked myocardial autophagic flux induced by I/R, demonstrating that TRPML1 directly contributes to the blocked autophagic flux in the cardiomyocytes subjected to I/R. Mechanistically, TRPML1 is activated secondary to ROS elevation following ischemia/reperfusion, which in turn induces the release of lysosomal zinc into the cytosol and ultimately blocks the autophagic flux in cardiomyocytes, presumably by disrupting the fusion between autophagosomes and lysosomes. As a result, the inhibited myocardial autophagic flux induced by TRPML1 disrupted mitochondria turnover and resulted in mass accumulation of damaged mitochondria and further ROS release, which directly led to cardiomyocyte death. More importantly, pharmacological and genetic inhibition of TRPML1 channels greatly reduced infarct size and rescued heart function in mice subjected to I/R in vivo by restoring impaired myocardial autophagy. In summary, our study demonstrates that secondary to ROS elevation, activation of TRPML1 results in autophagy inhibition in the cardiomyocytes subjected to I/R, which directly leads to cardiomyocyte death by disrupting mitochondria turnover. Therefore, targeting TRPML1 represents a novel therapeutic strategy to protect against myocardial I/R injury.

Evaluation of processing mechanism in Astragali Radix by low-field nuclear magnetic resonance and magnetic resonance imaging.

Astragali Radix (Huangqi) is an important herb medicine that is always processed into pieces for clinical use. Many operations need to be performed before use, among which drying of Astragali Radix (AR) pieces is a key step. Unfortunately, research on its drying mechanism is still limited. Low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) techniques were applied to study the moisture state and distribution during drying. The content of bioactive components and texture changes were measured by HPLC and texture analyzer, respectively. The moisture content of the AR pieces decreased significantly during drying, and the time to reach the drying equilibrium were different at different temperatures. The time when at 70°C, 80°C, and 90°C reach complete drying are 180 min, 150 min and 120 min, respectively. 80°C was determined as the optimum drying temperature, and it was observed that the four flavonoids and astragaloside IV have some thermal stability in AR pieces. When dried at 80°C, although the total water content decreased, the free water content decreased from 99.38% to 15.49%, in contrast to the increase in bound water content from 0.62% to 84.51%. The texture parameters such as hardness changed to some extent, with the hardness rising most significantly from 686.23 g to 2656.67 g. Correlation analysis revealed some connection between moisture content and LF-NMR and texture analyzer parameters, but the springiness did not show a clear correlation with most parameters. This study shows that HPLC, LF-NMR, MRI, and texture analyzers provide a scientific basis for elucidating the drying principles of AR pieces. The method is useful and shows potential for extension and application; therefore, it can be easily extended to other natural herb medicines.

Simultaneous qualitative and quantitative analysis of 10 bioactive flavonoids in Aurantii Fructus Immaturus (Zhishi) by ultrahigh-performance liquid chromatography and high-resolution tandem mass spectrometry combined with chemometric methods.

INTRODUCTION: Aurantii Fructus Immaturus (Zhishi in Chinese) is the dried young fruit of Citrus aurantium L. (CA) and its cultivated varieties or Citrus sinensis Osbeck (CS). The content of flavonoids in different varieties of Zhishi may be significantly different. However, there is confusion about the botanical origin of Zhishi, and there is no reliable and systematic method to control Zhishi quality. OBJECTIVES: We aimed to establish an ultrahigh-performance liquid chromatography method coupled with diode-array detection and high-resolution tandem mass spectrometry (UPLC-DAD-HRMS/MS) for the quantitative analysis of 10 flavonoids in Zhishi that could be used for quality control and botanical origin identification. METHODOLOGY: A UPLC-DAD-HRMS/MS method was established for simultaneous identification and quantification of 10 flavonoids. Separation was performed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm) with 0.1% formic acid and acetonitrile as mobile phase under gradient elution. MS was performed in positive and negative ionisation modes. The flavonoids in 41 batches were isolated and quantified. Zhishi of different botanical origins were identified by chemometrics. RESULTS: The results showed that the established method for the determination of 10 components was reliable and accurate. Chemometrics could be used to distinguish Zhishi of different botanical origins. There were significant differences in the contents of 10 flavonoids in samples of different botanical origins. CONCLUSIONS: The quantitative analysis method in this study can be used to accurately determine the content of 10 flavonoids and provide a chemical basis for quality control and botanical origin identification of Zhishi.

Autophagy inhibition mediated by MCOLN1/TRPML1 suppresses cancer metastasis via regulating a ROS-driven TP53/p53 pathway.

Compelling evidence has demonstrated that macroautophagy/autophagy plays an important role in regulating multiple steps of metastatic cascades; however, the precise role of autophagy in metastasis remains unclear. This study demonstrates that autophagy inhibition induced by MCOLN1/TRPML1 suppresses cancer metastasis by evoking the ROS-mediated TP53/p53 pathway. First, we found that MCOLN1-mediated autophagy inhibition not only profoundly inhibits both migration and invasion in malignant melanoma and glioma cell lines in vitro, but also suppresses melanoma metastasis in vivo. Second, our study reveals that autophagy inhibition induced by MCOLN1 leads to damaged mitochondria accumulation followed by large quantities of ROS release. Third, we demonstrate that the elevated ROS resulting from autophagy inhibition subsequently triggers TP53 activity, which in turn modulates expression of its downstream targets that are involved in a broad spectrum of the metastatic cascade to suppress metastasis including MMP members and TWIST. In summary, our findings have established a mechanism by which autophagy inhibition suppresses metastasis via the ROS-TP53 signaling pathway. More importantly, our study demonstrates that autophagy inhibition through stimulation of MCOLN1 could evidently be one of the therapeutic potentials for combating cancer metastasis.Abbreviations: 3-MA: 3-methyladenine; AA: amino acid; ATG5: autophagy related 5; ATG12: autophagy-related 12; Baf-A1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CQ: chloroquine; DMEM: Dulbecco's Modified Eagle Medium; EMT: epithelial-mesenchymal transition; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HEK: human embryonic kidney; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MMP: matrix metallopeptidase; NC: negative control; NRK: normal rat kidney; PBS: phosphate-buffered saline; shRNA: short hairpin RNA; siRNA: short interfering RNA; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy-activating kinase 1.

Stimulating TRPM7 suppresses cancer cell proliferation and metastasis by inhibiting autophagy.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitous cation channel possessing kinase activity. TRPM7 mediates a variety of physiological responses by conducting flow of cations such as Ca2+, Mg2+, and Zn2+. Here, we show that the activation of TRPM7 channel stimulated by chemical agonists of TRPM7, Clozapine or Naltriben, inhibited autophagy via mediating Zn2+ release to the cytosol, presumably from the intracellular Zn2+-accumulating vesicles where TRPM7 localizes. Zn2+ release following the activation of TRPM7 disrupted the fusion between autophagosomes and lysosomes by disturbing the interaction between Sxt17 and VAMP8 which determines fusion status of autophagosomes and lysosomes. Ultimately, the disrupted fusion resulting from stimulation of TRPM7 channels arrested autophagy. Functionally, we demonstrate that the autophagy inhibition mediated by TRPM7 triggered cell death and suppressed metastasis of cancer cells in vitro, more importantly, restricted tumor growth and metastasis in vivo, by evoking apoptosis, cell cycle arrest, and reactive oxygen species (ROS) elevation. These findings represent a strategy for stimulating TRPM7 to combat cancer.

Simultaneous quantitative analysis of ten bioactive flavonoids in Citri Reticulatae Pericarpium Viride (Qing Pi) by ultrahigh-performance liquid chromatography and high-resolution mass spectrometry combined with chemometric methods.

INTRODUCTION: Citri Reticulatae Pericarpium Viride (Qing Pi in Chinese) is a clinically effective Chinese herb, which contains biologically valuable flavonoids. Qing Pi is divided into two commodity specifications, Si Hua Qing Pi (SHQP) and Ge Qing Pi (GQP), based on the harvesting time. The flavonoid contents in Qing Pi from different origins and commodity specifications may vary significantly, which will affect their therapeutic functions. Thus, it is crucial to set up a reliable and comprehensive quality evaluation method for flavonoid analysis in Qing Pi. OBJECTIVES: We aimed to establish a rapid and sensitive ultrahigh-performance liquid chromatography method coupled with diode-array detection and high-resolution mass spectrometry (UPLC-DAD-HRMS) for identification and quantification of ten flavonoids in Qing Pi. Chemometric methods were further applied to distinguish Qing Pi of different origins and specifications. METHODOLOGY: An UPLC-DAD-HRMS method was developed for the simultaneous separation and quantification of ten flavonoids in 46 batches of Qing Pi samples from different sources in China. Chemometric approaches were applied to discriminate Qing Pi from different origins and commodity specifications. RESULTS: The chemometric procedures (i.e., hierarchical clustering analysis and principal component analysis) were employed to identify the differences of Qing Pi samples with different origins and commodity specifications. The results showed that the contents of ten flavonoids in Qing Pi samples of different origins were significantly different, and the same results were found out between SHQP and GQP. CONCLUSIONS: This study provides a chemical basis for quality control of Qing Pi.

MCOLN1/TRPML1 finely controls oncogenic autophagy in cancer by mediating zinc influx.

Macroautophagy/autophagy is elevated to ensure the high demand for nutrients for the growth of cancer cells. Here we demonstrated that MCOLN1/TRPML1 is a pharmaceutical target of oncogenic autophagy in cancers such as pancreatic cancer, breast cancer, gastric cancer, malignant melanoma, and glioma. First, we showed that activating MCOLN1, by increasing expression of the channel or using the MCOLN1 agonists, ML-SA5 or MK6-83, arrests autophagic flux by perturbing fusion between autophagosomes and lysosomes. Second, we demonstrated that MCOLN1 regulates autophagy by mediating the release of zinc from the lysosome to the cytosol. Third, we uncovered that zinc influx through MCOLN1 blocks the interaction between STX17 (syntaxin 17) in the autophagosome and VAMP8 in the lysosome and thereby disrupting the fusion process that is determined by the two SNARE proteins. Furthermore, we demonstrated that zinc influx originating from the extracellular fluid arrests autophagy by the same mechanism as lysosomal zinc, confirming the fundamental function of zinc as a participant in membrane trafficking. Last, we revealed that activating MCOLN1 with the agonists, ML-SA5 or MK6-83, triggers cell death of a number of cancer cells by evoking autophagic arrest and subsequent apoptotic response and cell cycle arrest, with little or no effect observed on normal cells. Consistent with the in vitro results, administration of ML-SA5 in Patu 8988 t xenograft mice profoundly suppresses tumor growth and improves survival. These results establish that a lysosomal cation channel, MCOLN1, finely controls oncogenic autophagy in cancer by mediating zinc influx into the cytosol.Abbreviation: Abbreviations: 3-MA: 3-methyladenine; AA: amino acid; ATG12: autophagy related 12; Baf-A1: bafilomycin A1; BAPTA-am: 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; co-IP: coimmunoprecipitaion; CQ: chloroquine; DMEM: Dulbecco's Modified Eagle Medium; FBS: fetal bovine serum; GAPDH: glyceraldehyde- 3-phosphate dehydrogenase; HCQ: hydroxychloroquine; HEK: human embryonic kidney; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NC: negative control; NRK: normal rat kidney epithelial cells; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RPS6KB/S6K: ribosomal protein S6 kinase B; shRNA: short hairpin RNA; siRNA: short interfering RNA; SNARE: soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TPEN: N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; TTM: tetrathiomolybdate; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; Zn2+: zinc.