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Background Following myocardial infarction (MI), a series of structural and functional changes evolves in the myocardium, collectively defined as cardiac remodeling. Purpose The aim of present study was to investigate the cardioprotection of salvianolicacid B (SalB) and ginsenoside Rg1 (Rg1) combination against cardiac remodeling in a rat model at the subacute phase of MI and further elucidate the underlying mechanism. Methods Rat heart was exposed via a left thoracotomy at the fourth intercostal space and MI was induced by a ligature below the left descending coronary artery. Hemodynamic assay was conducted using a Mikro-tipped SPR-320 catheter which was inserted through the right carotid artery into left ventricle.Myocardial infarct size was detected using 3,5-triphenyltetrazolium chloride (TTC) staining. Haematoxylin and eosin (HE) stain and picric sirius red stain were conducted for histopathological detection. Immunohistochemistry was used to detect the expression of α-smooth muscle actin (α-SMA) and gelatin zymography was used to evaluate the activities of matrix metalloproteinase-9 (MMP-9). Results Comparing with MI rats, 30 mg/kg SalB-Rg1 improved cardiac function verified by maximum rate of pressure development for contraction (+dp/dtmax, p Conclusion All above study indicated the potential therapeutic effects of SalB-Rg1 on heart.

Estrogen drives the development and progression of estrogen receptor (ER)-positive breast cancer. However, the detailed mechanism underlying ER-driven carcinogenesis remains unclear despite extensive studies. Previously reports indicated higher expression of long non-coding RNA (lncRNA) myocardial infarction associated transcript (MIAT) in ER-positive breast cancer tissues than in ER-negative tissues. However, the functional relevance of MIAT in ER-positive breast cancer tumorigenesis was poorly understood. Here, we investigated the role of lncRNA MIAT in ER-positive breast cancer cells. MIAT was over-expressed in ER-positive breast cancer tissues and ER-positive breast cancer cell line MCF-7. Activating estrogen signaling by diethylstilbestrol (DES) led to a dose- and time-dependent up-regulation of MIAT in MCF-7 cells that was dependent on ERα, as evidenced by ERα silencing and pharmacological inhibition using ER antagonist ICI 182780. Silencing MIAT decreased DES-induced MCF-7 cell proliferation while overexpressing MIAT increased MCF-7 cell proliferation. Further mechanistic study identified that MIAT was critical for G1 to S phase cell cycle transition. Taken together, these results suggest that lncRNA MIAT is an estrogen-inducible lncRNA and a key regulator in ER-positive breast cancer cell growth. MIAT could serve as a potential biomarker and promising therapeutic target for ER-positive breast cancer.

The fast and accurate voltage sequence component detection of unbalanced three-phase system is of major importance for the industrial continuous processing. The traditional method is to calculate the voltage in dq synchronous reference frame. This results in low order harmonics and long detection time. A novel fast voltage sequence components detector is proposed in this paper. It is to calculate the sequence component projection on dq rotating reference frame with h times the fundamental angular velocity. Simulation results demonstrate the validity of the algorithm.

Exploration of new natural compounds is of vital significance for drug discovery and development. The conventional approaches by systematic phytochemical isolation are low-efficiency and consume masses of organic solvent. This study presents an integrated strategy that combines offline comprehensive two-dimensional liquid chromatography, hybrid linear ion-trap/Orbitrap mass spectrometry, and NMR analysis (2D LC/LTQ-Orbitrap-MS/NMR), aimed to establish a green protocol for the efficient discovery of new natural molecules. A comprehensive chemical analysis of the total ginsenosides of stems and leaves of Panax ginseng (SLP), a cardiovascular disease medicine, was performed following this strategy. An offline 2D LC system was constructed with an orthogonality of 0.79 and a practical peak capacity of 11,000. The much greener UHPLC separation and LTQ-Orbitrap-MS detection by data-dependent high-energy C-trap dissociation (HCD)/dynamic exclusion were employed for separation and characterization of ginsenosides from thirteen fractionated SLP samples. Consequently, a total of 646 ginsenosides were characterized, and 427 have not been isolated from the genus of Panax L. The ginsenosides identified from SLP exhibited distinct sapogenin diversity and molecular isomerism. NMR analysis was finally employed to verify and offer complementary structural information to MS-oriented characterization. The established 2D LC/LTQ-Orbitrap-MS/NMR approach outperforms the conventional approaches in respect of significantly improved efficiency, much less use of drug materials and organic solvent. The integrated strategy enables a deep investigation on the therapeutic basis of an herbal medicine, and facilitates new compounds discovery in an efficient and environmentally friendly manner as well.

An efficient and target-oriented sample enrichment method was established to increase the content of the minor alkaloids in crude extract by using the corresponding two-phase solvent system applied in pH-zone-refining counter-current chromatography. The enrichment and separation of seven minor indole alkaloids from Uncaria rhynchophylla (Miq.) Miq. ex Havil(UR) were selected as an example to show the advantage of this method. An optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v) was used in this study, where triethylamine (TEA) as the retainer and hydrochloric acid (HCl) as the eluter were added at the equimolar of 10mM. Crude alkaloids of UR dissolved in the corresponding upper phase (containing 10mM TEA) were extracted twice with lower phase (containing 10mM TEA) and lower phase (containing 10mM HCl), respectively, the second lower phase extract was subjected to pH-zone-refining CCC separation after alkalization and desalination. Finally, from 10g of crude alkaloids, 4g of refined alkaloids was obtained and the total content of seven target indole alkaloids was increased from 4.64% to 15.78%. Seven indole alkaloids, including 54mg isocorynoxeine, 21mg corynoxeine, 46mg isorhynchophylline, 35mg rhynchophylline, 65mg hirsutine, 51mg hirsuteine and 27mg geissoschizine methylether were all simultaneously separated from 2.5g of refined alkaloids, with the purity of 86.4%, 97.5%, 90.3%, 92.1%, 98.5%, 92.3%, and 92.8%, respectively. The total content and purities of the seven minor indole alkaloids were tested by HPLC and their chemical structures were elucidated by ESI-HRMS and (1)H NMR.

Current China Pharmacopoeia (ChP) standards employ diversified and case-dependent assay methods to evaluate the quality of different Chinese patent medicines (CPMs) that contain Panax notoginseng as the monarch drug. These conventional, HPLC-based approaches, utilizing a complex sample preparation procedure, can easily result in low analytical efficiency and possible component loss. Here, a "monomethod-heterotrait matrix" (MHM) strategy is proposed, that is, developing a universal multi heart-cutting two-dimensional liquid chromatography (MHC-2D-LC) approach that facilitates the simultaneous quantitation of five P. notoginseng saponins (noto-R1, Re, Rg1, Rb1, and Rd) in eight different CPMs. The MHC-2D-LC system was constructed on a dual-gradient liquid chromatography instrument equipped with a Poroshell SB C18 column and a Zorbax SB-Aq column for respective (1)D and (2)D separation. Method validation was performed in terms of specificity, linearity (r(2) and F-test), intra-/inter-day precision (0.4-7.9%), stability (1.2-3.9%), and recovery (90.2-108.7%), and the LODs and LOQs (loaded masses) of the five analytes varied between 4.0-11.0ng and 6.0-33.0ng, respectively. The validated MHC-2D-LC approach was subsequently applied to quantify the five saponins in thirty batches of different CPMs. The method demonstrated superiority over the current ChP assay methods in respect of specificity (avoiding co-elution), resolution (Rs>1.5), sample preparation (easy-to-implement ultrasonic extraction without repeated re-extraction), and transfer rate (minimum component loss). This is the first application of an MHC-2D-LC method for the quantitative assessment of the constituents of CPMs. The MHM approach represents a new, strategically significant methodology for the quality control of CPMs that involve complex chemical matrix.

Ultra-performance liquid chromatography (UPLC) and Single Standard for Determination of Multi-Components (SSDMC) are becoming increasingly important for quality control of medicinal herbs; this approach was developed for Ganoderma lucidum. Special attention was necessary for the appropriate selection of markers, for determining the reproducibility of the relative retention times (RRT), and for the accuracy of conversion factors (F). Finally, ten components were determined, with ganoderic acid A serving as single standard. Stable system parameters were established, and with successful resolution of those issues, this analytical method could be used more broadly.

Background The herb pair of Salvia miltiorrhiza and Panax notoginseng has widely been used for improving coronary and cerebral circulation in China. However, the exact contribution of the major active components of S. miltiorrhiza and P. notoginseng to cardioprotection is far from clear. In the present study, three representative ingredients, salvianolic acid B (SalB) from S. miltiorrhiza and ginsenoside Rg1 (Rg1) and ginsenoside Rb1 (Rb1) from P. notoginseng , were selected to elucidate the mechanism of the herb pair at the ingredient level. Methods The purity of SalB, Rg1, and Rb1 was >99%, as detected by high-performance liquid chromatography. Acute myocardial infarction was introduced by ligation of the left anterior descending coronary artery near the main pulmonary artery. Cardiac contractility was detected through a Mikro-tipped catheter, and cardiac infarct size was determined using triphenyltetrazolium chloride stain. Results The combination of SalB and Rg1, and not the combination of SalB and Rb1, improved heart contractility in rats with myocardial infarction. The different contributions of Rg1 and Rb1, in combination with SalB, to cardioprotection provides further direction to optimize and modernize the herbal medicines containing S. miltiorrhiza and P. notoginseng . Conclusion The combination of SalB and Rg1 may provide potential protection against myocardial infarction.

The 2,2′-bipyridine-3,3′-dicarboxylic acid (bida) and its cobalt complex (Co-bida) were tested as corrosion inhibitors for N80 carbon steel in sulphuric acid solution by electrochemical polarization and electrochemical impedance spectroscopy method. The results indicate that the complex and ligand inhibit the corrosion of mild steel in H2SO4 solutions and the extent of inhibition increases with inhibitor concentration and decreases with temperature. The inhibition efficiency of the inhibitors follows the trend Co-bida > bida. A mixed-inhibition mechanism is proposed for the inhibitive effects of the compounds. The adsorption characteristics of the inhibitors were approximated by Temkin isotherm. Morphological study of the carbon steel electrode surface was undertaken by scanning electron microscope and the interfacial species formed on the surface in the presence of inhibitors analyzed by infrared spectroscopy.

Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.

Ethnopharmacological relevance Myocardial infarction (MI) is considered as the major inducer to the morbidity and mortality related to coronary occlusion. Salvia miltiorrhiza Bunge is widely applied in the clinic for the prevention and treatment of heart diseases. The preparation of traditional herb decoction (THD) is not only time consuming but also difficult to keep uniform for every time. New usage form of Salvia miltiorrhiza Bunge with characteristics of convenience, uniform and efficiency is needed. Aim of the study The aims of present study were to investigate the cardio-protection of ultrafine granular powder (UGP) of Salvia miltiorrhiza Bunge; and further compare the characteristics of UGP with THD. Materials and methods MI was induced by ligation of the left anterior descending coronary artery near the main pulmonary artery. Cardio-protection of UGP or THD was evaluated based on two sets of experiments, one was acute myocardial infarction (AMI) through 7 days preventive administration, and the other one was chronic cardiac remodeling through 28 days therapeutic administration. Hemodynamic measurement was conducted to evaluate heart function and histopathological detection was used to evaluate heart structure. Results No significant improvement of heart structure and function was detected for preventive administration of UGP or THD on AMI rats. While, more significant improvements on left ventricular systolic and diastolic function were detected with therapeutic treatment with 0.81 g/kg UGP than same dose of THD on rats against chronic cardiac remodeling. Both UGP and THD showed the protective effects on heart structure, especially against fibrosis with long-term therapeutic treatment. Conclusions As a new usage form of Salvia miltiorrhiza Bunge, UGP showed significant cardio-protection against myocardial remodeling with therapeutic treatment. Comparing with THD, UGP also holds the advantages of uniform, convenience and efficiency.

The present study aimed to investigate the effect of the long noncoding RNA cancer susceptibility candidate 9 (CASC9) on doxorubicin (DOX)‑resistant breast cancer and to reveal the potential underlying mechanisms. The expression of CASC9 in breast cancer tissues and cell lines, in addition to drug‑resistant breast cancer cells (MCF‑7/DOX), was detected by reverse transcription‑quantitative polymerase chain reaction. Subsequently, MCF‑7/DOX cells were transfected with the silencing vector pS‑CASC9, containing enhancer of zeste homolog 2 (EZH2), multidrug resistance protein 1 (MDR1) or control small interfering (si)RNAs. The viability, apoptosis, migration and invasion of the transfected cells were assessed via an MTT assay, flow cytometry and a Transwell assay, respectively. The expression levels of apoptosis‑associated proteins (apoptosis regulator Bcl‑2, apoptosis regulator BAX, caspase‑3 and caspase‑9) were determined by western blotting. An RNA pull‑down assay was performed to identify CASC9‑binding candidates. In addition, the expression levels of the MDR1 gene and its encoded protein, P‑glycoprotein, were detected. CASC9 expression was upregulated in breast cancer tissues and cell lines, and drug‑resistant breast cancer cells. CASC9 knockdown significantly inhibited the growth and metastasis of drug‑resistant breast cancer cells, and decreased the half‑maximal inhibitory concentration DOX in MCF‑7/DOX cells. The RNA pull‑down assay revealed that CASC9 engaged EZH2; EZH2 siRNA significantly inhibited the cell growth, metastasis and chemoresistance of MCF‑7/DOX cells. Additionally, EZH2 may regulate the MDR1 gene. The present study demonstrated the oncogenic role of CASC9 in drug‑resistant breast cancer by binding to EZH2 and regulating the MDR1 gene. Modulation of CASC9 expression may be a promising target in the therapy of breast cancer and drug‑resistant breast cancer.

This study explored the mechanism underlying long non-coding RNA ROR regulating autophagy on Tamoxifen resistance in breast cancer. Cancer tissues and adjacent normal tissues were collected from 74 breast cancer patients. Human breast cancer BT474 cells were assigned into blank, phosphate buffered saline, Tamoxifen, negative control + Tamoxifen, siROR + Tamoxifen, 3-methyladenine + Tamoxifen, and siROR + 3-methyladenine + TA groups. The expression of long non-coding RNA ROR and expressions of multi-drug resistance-associated P-glycoprotein and glutathione S-transferase-π messenger RNA were detected using quantitative real-time polymerase chain reaction. The expressions of light chain 3, Beclin 1, multi-drug resistance-associated P-glycoprotein, and glutathione S-transferase-π protein were determined using western blotting. Cell proliferation, invasion, and migration abilities were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Transwell assay, and scratch test, respectively. The long non-coding RNA ROR expression was higher in the breast cancer tissues than that in the adjacent normal tissues. Compared with the blank group, light chain 3 and Beclin 1 expressions were increased in the siROR + Tamoxifen group but decreased in the 3-methyladenine + Tamoxifen group; these data indicated that downregulated long non-coding RNA ROR promoted autophagy. In comparison with the blank group, multi-drug resistance-associated P-glycoprotein and glutathione S-transferase-π messenger RNA and protein expressions were reduced in the siROR + Tamoxifen group but elevated in the 3-methyladenine + Tamoxifen group, suggesting that downregulated long non-coding RNA ROR suppressed the drug resistance to Tamoxifen and the inhibition of autophagy reversed the effect of long non-coding RNA ROR on drug resistance. Compared with the Tamoxifen, negative control, and siROR + 3-methyladenine + Tamoxifen groups, the cell proliferation, invasion, and migration in the siROR + Tamoxifen group were much decreased; these results implied that downregulated long non-coding RNA ROR suppressed BT474 cell proliferation, invasion, and migration and reversed the effect of Tamoxifen on the BT474 cells. These results indicate that inhibition of long non-coding RNA ROR reverses resistance to Tamoxifen by inducing autophagy in breast cancer.

Aortic dissection (AD), a severe cardiovascular disease with the characteristics of high mortality, is lack of specific clinical biomarkers. In order to facilitate the diagnosis of AD, we investigated plasma amino acid profile through metabolomics approach. Total 33 human subjects were enrolled in the study: 11 coronary heart disease (CHD) patients without aortic lesion and 11 acute AD and 11 chronic AD. Amino acids were identified in plasma using liquid chromatography and mass spectrometry (LC-MS/MS), and were further subjected to multiple logistic regression analysis. The score plots of principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) showed clear discrimination of CHD patients with AD, acute AD or chronic AD patients, respectively. The contents of histidine, glycine, serine, citrate, ornithine, hydroxyproline, proline and sarcosine were significant different in acute AD patients comparing with CHD patients. The levels of citrate, GABA, glutamate and cysteine were significant different in chronic AD patients comparing with CHD patients. The contents of glutamate and phenylalanine were significant changed in acute AD patients comparing with chronic AD patients. Plasma aminograms were significantly altered in patients with AD comparing with CHD, especially in acute AD, suggesting amino acid profile is expected to exploit a novel, non-invasive, objective diagnosis for AD.

Aortic aneurysm (AA) is a life-threatening vascular disease in defect of effective pharmaceutical therapy. Matrix metalloproteinase-9 (MMP-9) is implicated in the development of chronic vascular diseases including aneurysm, but the effective MMP-9 inhibitors are far from development. To develop new candidate for AA therapy, we evaluated the efficiency of salvianolic acid A (SalA), a novel MMP-9 inhibitor, on AA progression in a mouse model and characterized the mechanism of action. SalA is a water soluble compound of the herbal drug Rhizoma Salviae miltiorrhizae (Danshen) which in China is widely used for the treatment of hypertension, coronary artery diseases and myocardial infarction. MMPs activity was evaluated by enzyme kinetic analysis in vitro and in-gel gelatin zymography in vivo. SalA showed selectivity on gelatinase (MMP-2 and MMP-9) than on collagenase (MMP-8 and MMP-13) in vitro, and specificity on MMP-9 than MMP-2 in vivo. Aortic aneurysm was induced by angiotension II (AngII) in apolipoprotein E-deficient (ApoE(-/-)) mice. Aortic structure was evaluated by hematoxylin and eosin, picrosirius red, orein stain. Macrophage infiltration was detected by immunohistochemistry in vivo and transwell in vitro. Comparing with doxycycline (Dox), a well-known MMPs inhibitor, SalA showed similar efficiency against AA progression. SalA significantly decreased aortic diameter and aneurysm severity, ameliorated integrity of vascular structure, inhibited elastin fragmentation and macrophage infiltration. Furthermore, SalA showed greater safety than Dox based on hepatotoxicity evaluation. Our results demonstrated that SalA held great potential for AA therapy.

Seven hexahydrobenzophenanthridine-type alkaloids, Ambiguanine A–G, along with eight known alkaloids, were isolated from tubers of Corydalis ambigua var. amurensis. Their structures were elucidated based on extensive spectroscopic analyses, with absolute configurations determined by CD experiments.

The quality control of Da-Fu-Fang (DFF), referring to the traditional Chinese medicine (TCM) preparations comprising more than 10 TCMs, is challenging due to their extreme chemical complexity. In this study, a strategy is proposed for the holistic quality control of DFFs based on HPLC/qTOF-MS-oriented characteristic components data set (CCDS) and chemometric analysis. Niuhuang Shangqing pill (NHSQP), composed of 19 TCMs, is used to illustrate this strategy. The fingerprint profiling of NHSQP by HPLC/qTOF-MS resulted in the characterization of 190 compounds, comprising 47 unambiguously identified by reference standard comparison. A CCDS containing 60 characteristic components was constructed by analyzing the MS spectral differentiation of the crude drugs, a laboratory-made NHSQP powder, and negative control preparations. With the established CCDS, it was possible to simultaneously monitor 16 out of the 19 drugs involved in NHSQP. Subsequently, 26 NHSQP samples from different vendors were evaluated by the qualitative and semi-quantitative analyses of their LC/MS fingerprint data. The 60 characteristic components were detected in all of the NHSQP samples, which demonstrated their authenticity. When compared with the standard sample No. 3, however, 15 of the NHSQP samples exhibited inferior quality. Samples No. 21 and No. 13 differed significantly based on a PCA score plot, and the components responsible for the differentiation were confirmed to originate from different TCMs. This strategy is a powerful and easy method to implement and provides a potential approach to establishing the holistic quality control of complex TCM preparations.

RATIONALE Licorice (Gancao) is derived from the dried roots and rhizomes of Glycyrrhiza species (Leguminosae) and appears as a component herb in about 60% of traditional Chinese medicine (TCM) prescriptions. Modern pharmacological studies have shown that flavonoids are one class of the major components responsible for the bioactivities of licorice. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/QTOF MS) has proven to be a powerful tool for rapid profiling and identification of natural products in complex herbal medicines. METHODS A UPLC/QTOF MS method was established for the first time for profiling and structural characterization of the phenolic compounds (most of them flavonoids) in licorice. The combined use of data-independent acquisition (MSE) and data-dependent acquisition (DDA) was illustrated. RESULTS Fifteen flavonoid reference compounds were used to explore the fragmentation pathways. Compound identification was based upon the exact mass, general fragmentation behaviors, retention times, UV absorption, and the related botanical biogenesis. As a result, a total of 51 compounds were characterized, three of which were reported for the first time. CONCLUSIONS The LC/MS analysis for each injection took less than 9 min. The developed method is fast, accurate and reliable due to its high resolution and high efficiency characteristics as a result of combining both UPLC separation and QTOF exact mass measurement. Copyright © 2013 John Wiley & Sons, Ltd.

Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation., In previous studies the authors discovered that miR-214 inhibits osteoblastic bone formation. Here they extend on these findings, using ovariectomized mice and samples from patients with bone fractures, to show that miR-214 is a mediator of osteoclast-osteoblast crosstalk.

Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis (RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer for a long time. However, the underlying mechanisms for RA effects are still unclear. The present study was designed to determine the cytotoxicity of agglutinin isolated from Arisema heterophyllum Blume (AHA) and explore the possible mechanisms in human non-small-cell lung cancer A549 cells. AHA with purity up to 95% was isolated and purified from Arisaema heterophyllum Blume using hydrophobic interaction chromatography. AHA dose-dependently inhibited the proliferation of A549 cells and induced G1 phase cell cycle arrest. AHA induced apoptosis by up-regulating pro-apoptotic Bax, decreasing anti-apoptotic Bcl-2, and activating caspase-9 and caspase-3. In A549 cells treated with AHA, the PI3K/Akt pathway was inhibited. Furthermore, AHA induced increase in the levels of ER stress markers such as phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), inositol-requiring enzyme 1α (IRE1α), and phosphorylated c-Jun NH2-terminal kinase (p-JNK). AHA also induced autophagy in A549 cells. Staining of acidic vesicular organelles (AVOs) and increase in the levels of LC3II and ATG7 were observed in AHA-treated cells. These findings suggested that AHA might be one of the active components with anti-cancer effects in Arisaema heterophyllum Blume. In conclusion, cytotoxicity of AHA on cancer cells might be related to its effects on apoptosis and autophagy through inhibition of PI3K/Akt pathway and induction of ER stress.

BF211 is a synthetic molecule derived from bufalin (BF). The apoptosis-inducing effect of BF211 was stronger than that of BF while the acute toxicity of BF211 was much lower than that of BF. BF211 exhibited promising concentration-dependent anti-cancer effects in nude mice inoculated with A549 cells in vivo. The growth of A549 tumor xenografts was almost totally blocked by treatment with BF211 at 6 mg/kg. Notably, BF and BF211 exhibited differences in their binding affinity and kinetics to recombinant proteins of the α subunits of Na+/K+-ATPase. Furthermore, there was a difference in the effects of BF or BF211 on inhibiting the activity of porcine cortex Na+/K+-ATPase and in their time-dependent effects on intracellular Ca2+ levels in A549 cells. The time-dependent effects of BF or BF211 on the activation of Src, which was mediated by the Na+/K+-ATPase signalosome, in A549 cells were also different. Both BF and BF211 could induce apoptosis-related cascades, such as activation of caspase-3 and the cleavage of PARP (poly ADP-ribose polymerase) in A549 cells, in a concentration-dependent manner; however, the effects of BF211 on apoptosis-related cascades was stronger than that of BF. The results of the present study supported the importance of binding to the Na+/K+-ATPase α subunits in the mechanism of cardiac steroids and also suggested the possibility of developing new cardiac steroids with a stronger anti-cancer activity and lower toxicity as new anti-cancer agents.

A rapid high-speed counter-current chromatography (HSCCC) method was used to isolate five minor compounds from rhizome of Sparganium stoloniferum namely San Leng in Chinese, including two phenylpropanoid glycosides, sparganiaside A (1) and 1-O-feruloyl-3-p-coumaroylglycerol (2), and three aromatic acids, vanillic acid (3), p-hydroxylcinnamic acid (4), and p-hydroxybenzoic acid (5), of which, compound 1 was a new one. Five compounds were preparatively enriched at top efficiency by one-step HSCCC operation in the isolation procedure. A suitable solvent system composed of chloroform-methanol-water (4:3.5:1.8, v/v/v) was used. And the operation time was less than 4 h. The purities of compounds (1-5) in the enriched fractions were determined to be 75.8%, 66.3%, 90.6%, 79.9%, and 98.2%, respectively. The mean recoveries of the five compounds were 84.8%, 87.3%, 81.8%, 90.3%, and 92.7%, respectively. Compounds 1-4 were further purified by semi-preparative high-performance liquid chromatography (HPLC). This is the first report on the use of HSCCC as a fractionation tool for preparative isolation of minor compounds from S. stoloniferum. The method was proved to be rapid, convenient, high yield, and low cost. HSCCC was shown to be a quick and effective tool in isolation of natural products even though the compounds were not abundant.

In the present study, we found that celastrol, a natural compound with well-known apoptosis-inducing effect, could also induce paraptosis-like cytoplasmic vacuolization in cancer cell lines including HeLa cells, A549 cells and PC-3 cells derived from cervix, lung and prostate, respectively. Further study using HeLa cells indicated that the vacuoles induced by celastrol might be derived from dilation of endoplasmic reticulum. And, in celastrol-treated cells, markers of autophagy such as transformation of microtubule-associated protein 1 light chain 3 (LC3)I to LC3II and LC3 punctates formation were identified. Interestingly, autophagy inhibitors could not interrupt but enhance the induction of cytoplasmic vacuolization. Furthermore, MAPK pathways were activated by celastrol and inhibitors of MEK and p38 pathways could prevent the formation of cytoplasmic vacuolization. Celastrol treatment also induced G2/M cell cycle arrest and apoptosis in HeLa cells. In conclusion, celastrol induced a kind of paraptosis accompanied by autophagy and apoptosis in cancer cells. The coincidence of apoptosis and autophagy together with paraptosis might contribute to the unique characteristics of paraptosis in celastrol-treated cells such as the dependence of paraptosis on MAPK pathways and dynamic change of LC3 proteins. Both paraptosis and apoptosis could contribute to the cell death induced by celastrol while autophagy might serve as a kind of survival mechanism. The potency of celastrol to induce paraptosis, apoptosis and autophagy at the same dose might be related to its capability to affect a variety of pathways including proteasome, ER stress and Hsp90.

To analyze the relationship between fasting plasma glucose (FPG) and body mass index (BMI) of pre-pregnancy women and the occurrence of gestational diabetes mellitus(GDM), and to explore a effective method for GDM screening.This was a retrospective clinical study including the pregnant women who underwent screening for GDM recently and also participated in national free preconception health examination at three medical centers. They were divided into normal group and GDM group base on the 2011 diagnosis criteria for GDM of our country.FPG, BMI and diastolic blood pressure before pregnancy in GDM group were significantly higher than those in normal group (P0.01). Multiple logistic-regression showed that the women with higher FPG and BMI level before pregnancy had higher risk of diagnosis of GDM. The receiver operating characteristic (ROC) curves showed that the area under the curve (AUC) were 0.600 and 0.632 for FPG and BMI, respectively, in predicting GDM (P0.01).The FPG and BMI level of women before pregnancy are valuable predictors for early diagnosis of GDM.

Our previous study indicated that the combination of salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1), the main components of Salvia miltiorrhizae and Panax notoginseng, improves myocardium structure and ventricular function in rats with ischemia/reperfusion injury. The present study aimed to determine the safety of the combined SalB and Rg1 (SalB-Rg1) in mice. The safety of SalB-Rg1 was evaluated through acute toxicity and repeated-dose toxicity. In the acute toxicity study, the up and down procedure was carried out firstly, and then, the Bliss method was applied. In the toxicity study for seven-day repeated treatment of SalB-Rg1, forty Kunming mice were randomly divided into four groups. The intravenous median lethal dose (LD50) of the SalB-Rg1 combination was 1747 mg/kg using the Bliss method. For both the acute toxicity study and the seven-day repeated toxicity study, SalB-Rg1 did not induce significant abnormality on brain, heart, kidney, liver and lung structure at any dose based on H&amp, E stain. There were no significant changes related to the SalB-Rg1 toxicity detected on biochemical parameters for two kinds of toxicity studies. The LD50 in mice was 1747 mg/kg, which was more than one hundred times higher than the effective dose. Both studies of acute toxicity and seven-day repeated dose toxicity indicated the safety of the SalB-Rg1 combination.

This study compared the sensitivity of human hepatoma Hep3B, SK-HEP-1, SMMC-7721, and BEL-7402 cells to cardiac steroids, including bufalin (BF), a bufalin derivative (BF211), ouabain (OUA), and digitoxin (DIG). Hep3B cells exhibited relatively low sensitivity to cardiac steroids. Expression levels of subunits of Na+/K+-ATPase were high in Hep3B cells. However, colocalization of Na+/K+-ATPase and caveolin was nearly undetectable in Hep3B cells. By using RNA-Seq technology, we found a total of 36 genes to be differentially expressed between Hep3B cells and SK-HEP-1 cells, which are highly sensitive to cardiac steroids. Our bioinformatics analysis determined that these genes were mostly comprised of extracellular space, protein binding, and extracellular region. Among these 36 genes, apolipoprotein E (APOE) played a critical role, as knockdown APOE expression induced colocalization of Na+/K+-ATPase and caveolin and increased sensitivity of Hep3B cells to both proliferation-inhibiting and cytotoxic effects of BF or BF211. Also, the effects of BF on PI3K/AKT/GSK3β and apoptosis signal cascades were enhanced in APOE knockdown cells. The results of our study confirmed the role of Na+/K+-ATPase signalosome in cytotoxicity of cardiac steroids and suggested that APOE regulated the sensitivity of cells to cardiac steroids by affecting formation and function of Na+/K+-ATPase signalosome. In addition, intercellular interaction with high level of Na+/K+-ATPase β1 subunit may be also a factor in the low sensitivity of Hep3B cells to cardiac steroids. Mol Cancer Ther; 15(12); 2955–65. ©2016 AACR.

Single standard to determine multi-components (SSDMC) is a novel and rational method for quality control of botanical products and traditional Chinese medicines (TCMs). However, it is restricted to wide application due to unknown fluctuation in conversion factors when it is performed in different laboratories. To evaluate the fluctuations of conversion factors, we selected Salvia miltiorrhiza as an example to determine three components of tanshinones by SSDMC method. Then ruggedness and robustness test were adopted to comprehensively investigate three kinds of factors that may influence stability of conversion factors, which were related with environmental parametric variables, operational parametric variables and peak measurement parametric variables. Nested-factorial-design was used to perform ruggedness tests. One-variable-at-a-time (OVAT) procedure and Plackett-Burman (PB) design were both used in robustness test. The results showed that stability of conversion factors was principally related with accuracy of wavelength of UV detector, peak measurement parameters and concentration of standard solution. The acceptable range of conversion factors was obtained from robustness test. Our results showed that conversion factors were inevitable to change, but when key parameters were well controlled, the range of its fluctuation was acceptable and the SSDMC method could be used widely in different laboratories.

Although doxorubicin (DXR) is an important antineoplastic agent, the serious toxicity mediated by the production of reactive oxygen species has remained a considerable clinical problem. Our hypothesis is that tanshinone II A sodium sulfonate (TSNIIA-SS), which holds significant effects against oxidative stress, protects against DXR-induced nephropathy. Firstly, the antioxidative effects of TSNIIA-SS were confirmed using oxygen radicals absorbance capacities (ORAC) assay in vitro. Then, DXR nephropathy was induced by repeated DXR treatment and verified by kidney index (20.76 ± 3.04 mg/mm versus 14.76 ± 3.04 mg/mm, p < 0.001) and histochemical stain. The mice were randomized into three groups: Control group, DXR group and DXR-TSNIIA-SS group. TSNIIA-SS treatment not only improved DXR lesion identified by histochemical stain, but also regulated the expression of several proteins related with the cytoskeleton, oxidative stress and protein synthesis or degradation detected by two-dimensional electrophoresis (2-DE). These data have provided the evidence that TSNIIA-SS is a protective agent against DXR-induced nephropathy.

Scope Aberrant vascular smooth muscle cell (VSMC) proliferation is involved in atherosclerotic plaque formation and restenosis. Mediterranean spices have been reported to confer cardioprotection, but their direct influence on VSMCs has largely not been investigated. This study aims at examining rosmarinic acid (RA) and 11 related constituents for inhibition of VSMC proliferation in vitro, and at characterizing the most promising compound for their mode of action and influence on neointima formation in vivo. Methods and results RA, rosmarinic acid methyl ester (RAME), and caffeic acid methyl ester inhibit VSMC proliferation in a resazurin conversion assay with IC50 s of 5.79, 3.12, and 6.78 µm, respectively. RAME significantly reduced neointima formation in vivo in a mouse femoral artery cuff model. Accordingly, RAME leads to an accumulation of VSMCs in the G0 /G1 cell-cycle phase, as indicated by blunted retinoblastoma protein phosphorylation upon mitogen stimulation and inhibition of cyclin-dependent kinase 2 in vitro. Conclusion RAME represses PDGF-induced VSMC proliferation in vitro and reduces neointima formation in vivo. These results recommend RAME as an interesting compound with VSMC-inhibiting potential. Future metabolism and pharmacokinetics studies might help to further evaluate the potential relevance of RAME and other spice-derived polyphenolics for vasoprotection.

Cyclophosphamide (Cy) could induce immuno-suppression such as thymus atrophy and inhibition of lymphocyte proliferation response in mice. But, the mechanism of its effect is still not clear. Polysaccharides isolated from spore of Ganoderma lucidum (GL-SP) could, at least partly, restore the immunological effects against Cy-induced immuno-suppression. In the present study, a two-dimensional gel electrophoresis (2-DE)-based comparative proteomic method was conducted to identify the possible target-related proteins of Cy in mice thymus including those could be influenced by GL-SP. Fifteen proteins differentially expressed in Cy-treated mice compared with control were identified. Among the fifteen proteins, combined use of GL-SP with Cy could not affect Cy-induced expression change of three proteins, totally prevented Cy-induced expression change of six proteins, partly prevented Cy-induced expression change of four proteins and further enhanced Cy-induced expression change of two proteins. Results of the present study shed light on the mechanism of Cy-induced immuno-suppression as well as the protective effect of GL-SP against toxicity of Cy.

Although doxorubicin (DXR) is an effective antineoplastic agent; the serious cardiotoxicity mediated by the production of reactive oxygen species has remained a considerable clinical problem. Our hypothesis is that tanshinone IIA sodium sulfonate (TSNIIA-SS), which holds significant affects on cardioprotection in clinic, protects against DXR-induced cardiotoxicity. In vitro investigation on H9c2 cell line, as well as in vivo study in animal model of DXR-induced chronic cardiomyopathy were performed. TSNIIA-SS significantly increased cell viability and ameliorated apoptosis of DXR-injured H9c2 cells using CCK-8 assay and Hoechst 33342 stain respectively. Furthermore, the cardio-protective effects of TSNIIA-SS were confirmed with decreasing ST-interval and QRS interval by electrocardiography (ECG); improving appearance of myocardium with haematoxylin and eosin (HE) stain; increasing myocardial tensile strength using tension to rupture (TTR) assay and decreasing fibrosis through picric-sirius red staining comparing with those receiving DXR alone. These data have provided the considerable evidences that TSNIIA-SS is a protective agent against DXR-induced cardiac injury.

Traditional Chinese medicine (TCM) is commonly considered to operate due to the synergistic effects of all the major and minor components in the medicines. Hence sensitive and comprehensive analytical techniques are needed to acquire a better understanding of the pharmacological basis of the herb and to enhance the product quality control. The present review mainly focuses on the phytochemical analysis of TCMs using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the two commonly used ion sources. Triple quadrupole, ion trap (IT), Fourier transform ion cyclotron resonance (FTICR) and time-of-flight (TOF) mass spectrometers are used as on-line analyzer. The relationship between structural features and fragmentation patterns should be investigated as thoroughly as possible and hence be applied in the on-line analysis to deduce the structures of detected peaks. Characteristic fragmentation behaviors of the reference standards, as well as information regarding polarity obtained from retention time data, on-line UV spectra, data from the literature and bio-sources of the compounds allowed the identification of the phytochemical constituents in the crude extracts. Although a mass spectrometer is not a universal detector, high-performance liquid chromatography coupled with multistage mass spectrometry (HPLC-MS(n)) technique was still proved to be a rapid and sensitive method to analyze the majority of the many constituents in herbal medicines, particularly for the detection of those present in minor or trace amounts. The methods established using HPLC-MS techniques facilitate the convenient and rapid quality control of traditional medicines and their pharmaceutical preparations. However, the quantitative analysis is not the topic of this review.

Salvianolic acids (SAs) extracted from Salvia miltiorrhiza could inhibit ADP-induced aggregation of rat washed platelets. To elucidate the cellular mechanism of the inhibitive effect on platelet aggregation, a proteomic method was used to find the possible target-related proteins of SA in platelets. Treatment of 150 μg/ml SA caused the regulation of platelet levels of 12 proteins, which play important roles in calcium homeostasis, antioxidant system and cytoskeleton structure. The proteomic result was also confirmed by Western blotting.

Sanqi, the root of Panax notoginseng, is a popularly used traditional Chinese medicine with cardiovascular effects. Notoginsengnosides (NG) isolated from Sanqi could inhibit ADP-induced platelet aggregation of rat washed platelets. To identify the possible target proteins of NG in platelets, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after NG treatment were identified by MALDI-TOF MS/MS. Treatment of 200 microg/ml NG caused regulation of the levels of 12 proteins, which play important roles in platelet activation, oxidative stress and cytoskeleton. In the NG-treated platelets, there were increase in the levels of growth factor receptor-bound protein 2 (Grb2), thrombospondin 1, tubulin alpha 6 and decrease in the levels of thioredoxin, Cu-Zn superoxide dismutase, DJ-1 protein, peroxiredoxin 3, thioredoxin-like protein 2, ribonuclease inhibitor, potassium channel subfamily V member 2, myosin regulatory light chain 9 and laminin receptor 1. The change in the levels of these proteins caused by NG treatment might contribute to the inhibitive effect of NG on platelet aggregation. Furthermore, analysis of the reactive oxygen species (ROS) level indicated that NG could decrease the ROS level in platelets. The regulation of ROS level might play important role in the effect of NG on platelets.

In order to substantiate the concept that cocaine behavioral effects may be influenced by histone modification, rats were trained to self-administer cocaine intravenously (0.75 mg/(kginjection)), and were systemically pretreated with sodium butyrate (NaBu), a potent histone deacetylase inhibitor, before the test session during the maintenance phase. The effect of NaBu on a control reinforcer (sucrose)-induced self-administration was also assessed. NaBu (100-200 mg/kg) was inactive in altering the cocaine (0.75 mg/(kg injection))-maintained responding and at the highest dose (400 mg/kg) it did increase cocaine-induced lever presses during the maintenance phase. On the other hand, sucrose-reinforcing potential was not altered when NaBu was given at the highest dose (400 mg/kg). These findings extend previous observations that changes in histone acetylation are relevant to cocaine-induced behavioral effects. Given that histone acetylase inhibitor enhances cocaine-induced behavioral plasticity, the therapeutic benefits of histone acetyltransferase inhibitors warrant further investigation in the experimental models of cocaine abuse.

Three new lignan glycosides (1-3) were isolated from the stems of Akebia trifoliata. Their structures were elucidated as (7R,8R,7'R,8'R)3,3',5,5'tetramethoxy-4,4'dihydroxy-7,9':7',9-diepoxylignan-4-O-beta-D-glucopyranoside (1), (7S,8S,8'R)-4,4',9-trihydroxy-3,3',5,5'-tetramethoxy-7,9'-epoxylignan-7'-one 9-O-beta-D-glucopyranoside (2), (7R,8R,8'S)-4,4',9-trihydroxy3,3',5,5'-tetramethoxy-7,9'-epoxylignan-7'-one 9-O-beta-D-glucopyranoside (3) by spectral analyses, primarily NMR, MS and CD. The NMR assignments for the compounds were carried out using 1H, 13C, DEPT, COSY, HSQC, HMBC and ROESY NMR experiments.

Lack of pharmacological strategies in clinics restricts the patient prognosis with myocardial ischemia/reperfusion (I/R) injury. The aim of this study was to evaluate the cardioprotection of combined salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) against myocardial I/R injury and further investigate the underlying mechanism. I/R injury was induced by coronary artery ligation for Wistar male rats and hypoxia/reoxygenation injury was induced on H9c2 cells. Firstly, the best ratio between SalB and Rg1was set as 2:5 based on their effects on heart function detected by hemodynamic measurement. Then SalB-Rg1 (2:5) was found to maintain mitochondrial membrane potential and resist apoptosis and necrosis in H9c2 cell with hypoxia/reoxygenation injury. Companying with same dose of SalB or Rg1 only, SalB-Rg1 showed more significant effects on down-regulation of myocardial infarct size, maintenance of myocardium structure, improvement on cardiac function, decrease of cytokine secretion including TNF-α, IL-1β, RANTES and sVCAM-1. Finally, the SalB-Rg1 improved the viability of cardiac myocytes other than cardiac fibroblasts in rats with I/R injury using flow cytometry. Our results revealed that SalB-Rg1 was a promising strategy to prevent myocardial I/R injury.

The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry (nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and pI values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology (GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING (Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins.

Isolation and structure elucidation of ten new scarce dihydrobenzophenanthridine alkaloids, ambidalmines A1 (Ia), B1 (Ib), their enantiomers ambidalmines A2 and B2, resp., ambidalmines C, D (IIa), E (IIb), and three stereoisomeric dimers, ambidimerines F1, F2 (III), and F3 are described.

Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit.

Beta-arrestin 2 plays important physiological roles in regulating the function of the mu opioid receptor (muOR) in vivo. Periaqueductal gray (PAG) is a potential structure where morphine produces its antinociception, but it is unclear whether beta-arrestin 2 plays its regulatory effect on morphine at PAG. In the present study beta-arrestin 2 overexpression was induced by adenovirus at PAG of rats. Morphine was administrated in these rats through PAG microinjection and systemic administration. The antinociceptive effects of morphine were abolished in rats received microinjection of morphine at PAG and partially attenuated in rats received systemic administration of morphine. These findings support the notion that PAG is an important site where beta-arrestin 2 plays its regulatory effects on morphine analgesia.

This study aims to explore the effect of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKIs) on the lymphangiogenesis of lung cancer with EGFR mutation, as well as to determine the function of EGFR targeted therapy in relation to the inhibition of lymphangiogenesis during lung cancer treatment.The EGFR double mutant lung cancer cell line NCI-H1975 is used to construct lung cancer xenograft models. The models are divided into two groups: the solvent control group and the EGFR-TKI treatment group. Each group includes five mice. The inhibitory effect of EGFR-TKI on the growth of transplanted tumors was observed. Immunohistochemical method and lymphatic endothelium specific antibody D2-40 were used in the experiment to observe the influence of EGFR-TKI on lymphangiogenesis in lung cancer.The weight and relative volume of tumors in the EGFR-TKI treated group were less than those in the solvent control group. The average lymphatic vessel density of EGFR-TKI-treated mice was 6.44 per case. This value was 10.70 per case in the solvent control group. Lower density of lymphangiogenesis was found in the EGFR-TKI treated group (P=0.023). The area and longest diameter of neonatal lymphatic vessel of the EGFR-TKI treated group were less than those of the solvent control group. Moreover, EGFR-TKI exhibited no significant effect on the invasion of tumor cells into the lymphatic vessel (P=0.519).EGFR-TKI can inhibit lymphangiogenesis in EGFR mutant lung cancer while suppressing vessel diameter and expansion area.背景与目的 探索表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI）对EGFR突变肺癌新生淋巴管的影响，探讨靶点治疗对新生淋巴管的抑制作用及其在肺癌治疗中所发挥的作用。方法 采用EGFR双位点突变的NCI-H1975肺癌细胞株构建小鼠移植瘤模型。设立溶剂对照组和EGFR-TKI给药组，每组5只小鼠，观察EGFR-TKI对小鼠移植瘤的生长抑制作用；运用淋巴管内皮特异性抗体D2-40，采用免疫组织化学的方法，观察新生淋巴管的密度、面积、最大径，探讨EGFR-TKI对于肺癌组织淋巴管新生的影响。结果 EGFR-TKI给药组小鼠肿瘤重量、肿瘤相对体积小于溶剂对照组。EGFR-TKI给药组小鼠平均新生淋巴管密度为6.44个/例，溶剂对照组小鼠平均新生淋巴管密度为10.70个/例，EGFR-TKI给药组小鼠平均新生淋巴管密度较低（P=0.023）。EGFR-TKI给药组小鼠新生淋巴管面积、最长径小于溶剂对照组。而EGFR-TKI对新生淋巴管的肿瘤细胞侵犯没有明显影响（P=0.519）。结论 EGFR-TKI可以抑制EGFR突变肺癌组织淋巴管的新生，抑制新生淋巴管管径及面积的扩大。

Undoubtedly, metabonomics can reveal the comprehensive efficacies of traditional Chinese medicine (TCM) formulae and its complex mechanism at the molecular biological level. In this study, an attempt was made to address the pretreatment effect of a TCM formula. In this case, as a critical point, we should first know how to really reflect the various endogenous metabolites in a disease status before a TCM formula is employed in a therapeutic procedure. Here, we explored an approach that combined high resolution LTQ-Orbitrap mass spectrometry with a spike-in method to characterize endogenous metabolites in acute myocardial ischemia (AMI) rats. As a result, 19 potential biomarkers in rat plasma were identified and 10 related disturbed pathways were perturbed in the early stages of AMI development. Subsequently, the metabonomics method was applied to investigate the pretreatment effect of the TCM formula named the Danqi Tongmai tablet (DQTM). The results revealed that the DQTM pretreatment could reduce the AMI injury and partially regulate the perturbed TCA cycle and amino and nucleotide metabolism, which were presumable related to energy metabolism and myocardial cells apoptosis/necrosis. In conclusion, UHPLC-LTQ-Orbitrap MS combined with a spike-in method were successfully applied to the metabonomics analysis of DQTM, which demonstrated that not only a comprehensive metabolic profile in the early stages of AMI development was achieved, but also that the underlying holistic efficacies were assessed and it was helpful to understand the possible mechanism of pretreatment with DQTM.

Although gamma-aminobutyric acid (GABA)C receptor rho1, rho2 and rho3 subunits are reportedly expressed in pyramidal and granule cells in the hippocampus at various developmental stages, it is not clear whether these three rho subunits are coexpressed in a single neuron. To attempt to answer this question, we performed single-cell RT-PCR for rho subunits from neurons of rat brain hippocampus. In hippocampal cultures, pyramidal cells were positive for rho1 mRNA expression in 89%, rho2 in 94% and rho3 in 94%, while granule cells were positive for rho1 mRNA in only 6%, rho2 in 36% and rho3 in 91%. Intensive amplification of the RT-PCR products by the second PCR revealed that all the three rho subunits were coexpressed in a single pyramidal and granule cells from both of the cultures and the slices. These results suggest that all the three GABAC receptor rho1, rho2 and rho3 subunits are present probably in different compositions in pyramidal and granule cells in the rat hippocampus.

The existence of Cl(-) channels in lymphocytes and neutrophils has been increasingly recognized, but the biological functions are not yet clear. We examined the effects of Cl(-) channel blockers on the cell proliferation and the cell cycle of human leukemic cell lines. The growth of leukemic cells was suppressed most efficiently by NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid), partially by 9-AC (9-anthracenecarboxylic acid) and tamoxifen, but not by stilbene compounds. NPPB increased the G0/G1 population and induced the expression of p21, one of the critical molecules for G1/S checkpoint. Antisense oligonucleotide for a NPPB-sensitive and stilbene-insensitive Cl(-) channel, ClC-2, sufficiently suppressed the ClC-2 protein synthesis, but did not affect the growth of leukemic cells. These findings suggest that NPPB-sensitive and stilbene-insensitive Cl(-) channels other than ClC-2 play important roles in cell cycles and cell proliferation of human leukemic cells.

Pearls have been widely used as a traditional medicine, in cosmetics, and as a health food supplement in China since ancient times. However, the identification and quality assessment of pearl powder have been challenging tasks because of the similar morphological features and chemical composition of its common adulterants, especially conch powders. In this study, ultra-performance liquid chromatography was combined with pre-column derivatization to rapidly quantify 14 amino acids in pearl powder and its analogues. Based upon the quantification results, a quality criterion of a total amino acid content of not less than 1.10% was proposed for pearl powder. Principal component analysis indicated that leucine and phenylalanine were the amino acids characteristic for distinguishing between pearls and nacres. The area ratio of leucine to phenylalanine was demonstrated to be an effective diagnostic marker to discriminate freshwater cultured pearls, natural seawater pearls, and nacres. The proposed method, involving both the qualitative and quantitative aspects, was subsequently applied to quality assessment of pearl powders purchased commercially in various parts of China; eight out of 18 batches were deemed authentic and unadulterated. In the future, this analytical process should play a significant role in standardizing and providing quality control to the pearl powder market.

Chinese patent medicines (CPM), generally prepared from several traditional Chinese medicines (TCMs) in accordance with specific process, are the typical delivery form of TCMs in Asia. To date, quality control of CPMs has typically focused on the evaluation of the final products using fingerprint technique and multi-components quantification, but rarely on monitoring the whole preparation process, which was considered to be more important to ensure the quality of CPMs. In this study, a novel and effective strategy labeling “retracing” way based on HPLC fingerprint and chemometric analysis was proposed with Shenkang injection (SKI) serving as an example to achieve the quality control of the whole preparation process. The chemical fingerprints were established initially and then analyzed by similarity, principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to evaluate the quality and to explore discriminatory components. As a result, the holistic inconsistencies of ninety-three batches of SKIs were identified and five discriminatory components including emodic acid, gallic acid, caffeic acid, chrysophanol-O-glucoside, and p-coumaroyl-O-galloyl-glucose were labeled as the representative targets to explain the retracing strategy. Through analysis of the targets variation in the corresponding semi-products (ninety-three batches), intermediates (thirty-three batches), and the raw materials, successively, the origins of the discriminatory components were determined and some crucial influencing factors were proposed including the raw materials, the coextraction temperature, the sterilizing conditions, and so on. Meanwhile, a reference fingerprint was established and subsequently applied to the guidance of manufacturing. It was suggested that the production process should be standardized by taking the concentration of the discriminatory components as the diagnostic marker to ensure the stable and consistent quality for multi-batches of products. It is believed that the effective and practical strategy would play a critical role in the guidance of manufacturing and help improve the safety of the final products.

Tumor-penetrating peptide, iRGD (internalizing RGD, CRGDK/RGPD/EC) with the similar affinity to αv integrins as conventional RGD cyclopeptide could enhance the tumor penetrability of drugs by binding to neuropilin-1 (NRP-1) that over-expressed on both angiogenic blood vessels and tumor cells. Comparing with our previous study, in which a RGD cyclopeptide (RGDyC) was bound to PEGylated polyamidoamine (PAMAM) dendrimer with doxorubicin (DOX) by acid-sensitive cis-aconityl linkage (PEG-PAMAM-cis-aconityl-DOX, PPCD), the present study selected iRGD instead of previous RGD to produce iRGD-PPCD conjugate. The effect of iRGD-mediated PPCD on tumor penetration was compared with the conventional RGD ones via administration of RGDs-modified PPCD (iRGD/RGDs-PPCD) and co-administration of RGDs and PPCD (iRGD/RGD + PPCD). C6 cells were selected as the cell model owing to the highest expression of αv integrins and NRP-1 among four tumor cell lines. In vitro cytotoxicity and cellular uptake showed no significant difference between RGD-PPCD and iRGD-PPCD, but glioma spheroid penetration study showed that RGD-PPCD, iRGD-PPCD and iRGD + PPCD penetrated into C6 spheroids with a depth of 115 μm, 144 μm and 150 μm, respectively, indicating that the iRGD-mediated PPCD delivery system had a stronger penetrating ability than the RGD ones. In vivo results also demonstrated the superiority of iRGD system over RGD ones. After systemic administration, iRGD-mediated PPCD increased tumor vascular permeability, decreased tumor vascular density and average vascular diameter. Correspondingly, the iRGD system exhibited stronger penetration ability, higher accumulation in brain tumor. The median survival time of iRGD + PPCD, iRGD-PPCD and RGD-PPCD treatment groups were 61, 57.5 and 43.5 days. The present findings strongly suggested that the iRGD-mediated drug delivery system could significantly improve the efficacy of tumor therapy through enhancing tumor accumulation and penetration as compared to the conventional RGD ones.