An integrated fecal metabolomic based on 1 H-NMR and UPLC-QTOF-MS revealed the preventive mechanism of Gushudan on glucocorticoid-induced osteoporotic rats.

Gushudan (GSD) has the effect of strengthening bones and nourishing kidneys. However, its specific intervention mechanism still remains unclear. In this study, to investigate the pathogenesis of glucocorticoid-induced osteoporosis (GIOP) and the preventive mechanism of GSD on GIOP, fecal metabolomics based on 1 H-NMR and ultra-high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry method was established. The changes in endogenous metabolites and the relevant metabolic pathways in the control group, model group, and GSD treatment group were investigated via multivariate statistical analysis. As a result, a total of 39 differential metabolites were identified. Of these, 22 metabolites, such as L-methionine, guanine, and sphingosine, were newly discovered as differential metabolites of GIOP. Amino acid metabolism, energy metabolism, intestinal flora metabolism, and lipid metabolism were significantly changed in the fecal profiles of GIOP rats, and GSD could play an anti-osteoporosis role by regulating these metabolic pathways. Finally, compared with our previous study of the GSD to prevent kidney yang deficiency syndrome, this study suggested that there were some identical differential metabolites and metabolic pathways. It showed that there was some correlation among the metabolic profiles of the intestine, kidney, and bone in GIOP rats. Therefore, this study offered new insights into the in-depth understanding of the pathogenesis of GIOP and the intervention mechanism of GSD.

Vitamin A-modified ZIF-8 lipid nanoparticles for the therapy of liver fibrosis.

Liver fibrosis (LF) is one of the major diseases that threaten human health. Until now, no effective drugs have been approved for clinical anti-liver fibrosis treatment. In this study, zeolitic imidazolate framework-8 (ZIF-8) lipid nanoparticles loaded with pirfenidone (PFD) and modified with vitamin A (VA) were constructed (VA-PFD@ZIF-8@DMPC NPs). PFD was embedded in ZIF-8 by the "one-pot" method, and the prepared ZIF-8 had a small particle size (84.3 nm) and high drug loading (54.46%). Moreover, the inherent pH sensitivity of ZIF-8 makes it stable in a normal physiological environment and collapsed in an acidic environment, thus controlling drug release and preventing drug leakage. Besides, the phospholipid layer makes the nano-drug delivery system dispersible and improves its biocompatibility. More importantly, VA is modified on the surface of nanoparticles (NPs), which can target the highly expressed retinol-binding protein receptor (RBPR) on the surface of hepatic stellate cells (HSCs), thereby accurately increasing the local drug concentration at the site of LF. In vivo experiments showed that VA-PFD@ZIF-8@DMPC NPs can reduce liver injury, improve the degree of LF, and exert specific therapeutic effects on LF. In conclusion, this nano-delivery system may become a novel and effective anti-liver fibrosis treatment.

Integrated serum pharmacochemistry, network pharmacology and pharmacokinetics to explore bioactive components of Gushudan in the treatment of osteoporosis.

Gushudan (GSD), a compound prescription on the basis of traditional Chinese medicine (TCM) theory and clinical practice, has been used in the treatment of osteoporosis (OP) for many years. Although studies have shown that GSD can treat OP, there is a lack of systematic screening method to explore the bioactive components, which are still unclear. Therefore, this study was aimed to establish an integrated method to screen and determine bioactive ingredients of GSD in the treatment of OP by serum pharmacochemistry, network pharmacology and pharmacokinetics. Firstly, 112 components of the GSD extract and 90 serum migrating constituents were identified by the ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), most of which were derived from flavonoids, tanshinones, coumarins and organic acids. Secondly, based on the network pharmacological analysis of the serum migrating constituents, 37 core targets and 20 main pathways related to both GSD and OP were obtained. More importantly, 7 bioactive ingredients were further screened as the PK markers by the network topology parameters including icariin, icariside II, isopimpinellin, bergapten, imperatorin, osthole and tanshinone IIA. Finally, a sensitive and accurate quantitative method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established and validated for simultaneous determination of the 7 bioactive ingredients in the rat plasma after oral administration of GSD extract, which was then applied to pharmacokinetic study. Besides, the overall pharmacokinetic characteristics were further calculated: Cmax was 180.52 ± 31.18 ng/mL, Tmax was 0.46 ± 0.20 h, t1/2 was 4.09 ± 0.39 h, AUC0-∞ was 567.24 ± 65.29 ng·h/mL, which displayed quick absorption and medium elimination in rats after oral administration of GSD extract. This study provided a new and holistic insight for exploring bioactive constituents and main targets to decode the therapeutic material basis of GSD against OP.

An efficient method for identifying natural common homoisoflavonoid by 1H-NMR.

Homoisoflavone contains 16 carbon atoms in the skeleton. The homoisoflavonoid skeleton from natural products can be roughly divided into 13 kinds, among which 5 kinds of common skeletons contain a large amount of compounds and 8 kinds of abnormal skeletons comprise a small amount of compounds. In this article, the structure identification experience of homoisoflavonoids found in Caesalpinia mimosoides was used as references and an efficient 1H NMR spectroscopic method for identifying homoisoflavonoid structure has been established. Using the chemical shift differences of H-2, 3, 4 and 9, the common natural homoisoflavonoids can be quickly and conveniently determined.

The role of thyroid hormone in the renal immune microenvironment.

Thyroid hormones are essential for proper kidney growth and development. The kidney is not only the organ of thyroid hormone metabolism but also the target organ of thyroid hormone. Kidney disease is a common type of kidney damage, mainly including different types of acute kidney injury, chronic kidney disease, diabetic nephropathy, lupus nephritis, and renal cell carcinoma. The kidney is often damaged by an immune response directed against its antigens or a systemic immune response. A variety of immune cells in the innate and adaptive immune systems, including neutrophils, macrophages, dendritic cells, T lymphocytes, and B lymphocytes, is essential for maintaining immune homeostasis and preventing autoimmune kidney disease. Recent studies have found that thyroid hormone plays an indispensable role in the immune microenvironment of various kidney diseases. Thyroid hormones regulate the activity of neutrophils, and dendritic cells express triiodothyronine receptors. Compared to hypothyroidism, hyperthyroidism has a greater effect on neutrophils. Furthermore, in adaptive immune systems, thyroid hormone may activate T lymphocytes through several underlying mechanisms, such as mediating NF-κB, protein kinase C signalling pathways, and ß-adrenergic receptors, leading to increased T lymphocyte activation. The present review discusses the effects of thyroid hormone metabolism regulation in the immune microenvironment on the function of various immune cells, especially neutrophils, macrophages, dendritic cells, T lymphocytes, and B lymphocytes. Although there are not enough data at this stage to conclude the clinical relevance of these findings, thyroid hormone metabolism may influence autoimmune kidney disease by regulating the renal immune microenvironment.

Euphorfiatnoids A-I: Diterpenoids from the roots of Euphorbia fischeriana with cytotoxic effects.

Nine previously undescribed diterpenoids, euphorfiatnoids A-I, together with seven known diterpenoids, were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by the interpretation of HRESIMS, UV, and NMR data. Their configurations were determined by electronic circular dichroism (ECD) spectroscopy analysis and the structure of euphorfiatnoid A was confirmed by X-ray crystallography. To further understand the antitumor effects of E. fischeriana, we tested the cytotoxicity of these compounds against H460, HepG2, and MCF-7 cell lines in vitro using MTT assays. Euphorfiatnoid B exhibited the most promising inhibitory effect against H460 cells with an IC50 value of 9.97 µM. Euphorfiatnoid A and C also exhibited moderate cytotoxicity against HepG2 cells with IC50 values of 11.64 and 13.10 µM, respectively.

Combination of resveratrol and luteolin ameliorates α-naphthylisothiocyanate-induced cholestasis by regulating the bile acid homeostasis and suppressing oxidative stress.

Cholestasis is a common liver injury without any effective therapeutic drugs so far. Resveratrol (RES) and luteolin (LUT) are natural polyphenols that exert protective effects on multiple liver injuries. Coadministration of RES and LUT could significantly improve the bioavailability of LUT and increase the systemic exposure to RES, and the combined treatment could also benefit from their multi-component and multi-target characteristics. Our current aim is to study the protective effects of coadministration of RES and LUT on α-naphthylisothiocyanate (ANIT)-induced cholestasis. Serum biochemical indices and liver histopathology in rats indicated that coadministration of RES and LUT could improve liver function by suppressing oxidative stress. Dysregulated bile acid (BA) homeostasis is a significant pathological feature of cholestasis, which was determined to explore the potential biomarkers and to clarify the protection mechanism of coadministration of RES and LUT. The levels of cholic acid, chenodeoxycholic acid, taurine conjugates and glycine conjugates, and the ratios of taurine conjugates to their free forms could be used as diagnosis indicators for cholestasis in rats. Furthermore, the coadministration of RES and LUT could restore the BA levels and exert better protective effects than administration alone. This study suggested that the coadministration of RES and LUT could protect against ANIT-induced cholestasis and the mechanism was closely related to regulating BA homeostasis and suppressing oxidative stress.

ES-MION-Based Dual-Modality PET/MRI Probes for Acidic Tumor Microenvironment Imaging.

Among all characteristics of the tumor microenvironment (TME), which are caused by abnormal proliferation of solid tumors, extracellular acidity is an important indicator for malignancy grading. pH-low insertion peptides (pHLIPs) are adopted to discern the acidic TME. To date, different imaging agents including fluorescent, positron emission tomography (PET), single photon emission computed tomography (SPECT), and magnetic resonance (MR) contrast agents with pHLIPs to target the acidic TME have been used to image various tumor models successfully. In this article, a PET/MRI dual-modality probe, based on extremely small magnetic iron oxide nanoparticles (ES-MIONs) with pHLIPs as a targeting unit, was proposed for the first time. In the phantom study, the probe showed relatively high r 1 relaxivity (r 1 = 1.03 mM-1 s-1), indicating that it could be used as a T1-weighted MR contrast agent. The 68Ga-radiolabeled probe was further studied in vitro and in vivo to evaluate pHLIP targeting efficacy and feasibility for PET/MRI. PET with intratumoral injection and T1-weighted MRI with intravenous injection both showed pHLIP-specific delivery of the probe. Therefore, we successfully designed and developed a radiolabeled ES-MION-based dual-modality PET/MRI agent to target the acidic tumor microenvironment. Although the accumulation of the probe in tumors with intravenous injection was not high enough to exhibit signals in the PET imaging study, our study still provides further insights into the ES-MION-based PET/MRI strategy.

Complete mitogenome of the entomopathogenic fungus Orbiocrella petchii.

In this study, the complete mitogenome of an entomopathogenic fungus Orbiocrella petchii (syn. Torrubiella petchii) was assembled and annotated. This circular mitogenome was 23,794 bp in length and consisted of 2 rRNA genes (rnl and rns), 25 tRNA genes, and 14 standard protein-coding genes of the oxidative phosphorylation system. Two group I introns were identified, and they encoded ribosomal protein S3 (in rnl) or a GIY-YIG endonuclease (in nad1). Phylogenetic analysis based on mitochondrial DNA sequences confirms O. petchii in the family of Clavicipitaceae.

A natural chalcone induces apoptosis in lung cancer cells: 3D-QSAR, docking and an in vivo/vitro assay.

This study was to study the antitumor effect of lonchocarpin (34) from traditional herbal medicine Pongamia pinnata (L.) Pierre and to reveal the underlying mechanism. The cytotoxic activities of lonchocarpin were evaluated in 10 lung cancer cell lines and it exhibited 97.5% activity at a dose of 100 µM in the H292 cell line. A field-based quantitative structure-activity relationship (3D-QSAR) study of 37 flavonoids from P. pinnata was also performed, and the results obtained showed that the hydrophobic interaction could be the crucial factor for the antitumor activity of lonchocarpin. Molecular docking studies revealed that lonchocarpin bound stably to the BH3-binding groove of the Bcl-2 protein with hydrophobic interactions with ALA146. Also, lonchocarpin significantly reduced cell proliferation via modulating Bax/Caspase-9/Caspase-3 pathway. An apoptotic test using flow cytometry showed that lonchocarpin produced about 41.1% and 47.9% apoptosis after treatment for 24 h and 48 h, respectively. Moreover, lonchocarpin inhibited tumor growth in S180-bearing mice with an inhibition rate of 57.94, 63.40 and 72.51%, respectively at a dose of 25, 50 and 100 mg/kg. These results suggest that lonchocarpin is a potentially useful natural agent for cancer treatment.

Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms.

The insect pathogenic fungus Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. However, few genetic tools are currently available for studying this organism. Here we report on the conditions for the production of transformable A. placenta protoplasts using an optimized protocol based on the response surface method (RSM). Critical parameters for protoplast production were modelled by using a Box-Behnken design (BBD) involving 3 levels of 3 variables that was subsequently tested to verify its ability to predict protoplast production (R(2) = 0.9465). The optimized conditions resulted in the highest yield of protoplasts ((4.41 ± 0.02) × 10(7) cells/mL of culture, mean ± SE) when fungal cells were treated with 26.1 mg/mL of lywallzyme for 4 h of digestion, and subsequently allowed to recover for 64.6 h in 0.7 mol/L NaCl-Tris buffer. The latter was used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the nonoptimized conditions. Generated protoplasts were transformed with vector PbarGPE containing the bar gene as the selection marker. Transformation efficiency was 300 colonies/(µg DNA·10(7) protoplasts), and integration of the vector DNA was confirmed by PCR. The results show that rational design strategies (RSM and BBD methods) are useful to increase the production of fungal protoplasts for a variety of downstream applications.

Sesquiterpenoid derivatives from Ferula ferulaeoides (Steud.) Korov.

Eight sesquiterpenoids, named Ferulaeone A-H (1-8), and seven known sesquiterpenoid derivatives were isolated from the roots of Ferula ferulaeoides (Steud.) Korov. Their structures were established by comprehensive spectroscopic analysis, and biosynthetic pathways leading to these compounds were proposed. The cytotoxicity of all these isolates against HepG2, MCF-7, and C6 cancer cell lines was evaluated and compounds 6-11, 13 exihibited various degrees of cytotoxic effect. Among them, compounds 9-11 displayed the highest potency against C6 with IC(50) values 34, 36, and 31 µM, respectively.

Hepatoprotective effect of Matrine salvianolic acid B salt on Carbon Tetrachloride-Induced Hepatic Fibrosis.

The aim of this study was to investigate the hepatoprotective effect of Matrine salvianolic acid B salt on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats. Salvianolic acid B and Matrine has long been used to treat liver fibrosis. Matrine salvianolic acid B salt is a new compound containing Salvianolic acid B and Matrine. Hepatic fibrosis induced by CCl4 was studied in animal models using Wistar rats. Organ coefficient, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), hexadecenoic acid (HA), laminin (LN), hydroxyproline (Hyp), and glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) in liver tissues were measured, respectively. Histopathological changes in the livers were studied by hematoxylin-eosin (H&E) staining and Masson Trichrome (MT) examination. The expression of transforming growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) was observed by immunohistochemical analysis. A significant reduction in serum levels of AST, ALT, HA, LN and Hyp was observed in the Matrine salvianolic acid B salt treated groups, suggesting that the salt had hepatoprotective effects. The depletion of GSH and SOD, as well as MDA accumulation in liver tissues was suppressed by Matrine salvianolic acid B salt too. The expression of TGF-ß1 and α-SMA measured by immunohistology was significantly reduced by Matrine salvianolic acid B salt in a dose-dependent manner. Matrine salvianolic acid B salt treatment attenuated the necro-inflammation and fibrogenesis induced by CCl4 injection, and thus it is promising as a therapeutic anti-fibrotic agent against hepatic fibrosis.

Calcium-activated potassium channel activator down-regulated the expression of tight junction protein in brain tumor model in rats.

This study was performed to investigate the mechanism of the blood-brain tumor-barrier (BTB) permeability increase, which was induced by NS1619, a selective K(Ca) channel activator. Using a rat brain glioma (C6) model, we exam the expression of ZO-1 and occludin in mRNA and protein level at different time point after intracarotid infusion of NS1619 (30 µg/kg/min) to tumor sites via RT-PCR and Western blot analysis. The mRNA and protein expression of ZO-1 and occludin had no great change before infusion and began to decrease significantly after 2 h NS1619 infusion, which was significantly attenuated by reactive oxygen species (ROS) scavenger (N-2-mercaptopropionyl glycine, MPG). In addition, MPG also significantly inhibited the increase of BTB permeability and malonaldehyde (MDA) level induced by NS1619. This led to the conclusion that NS1619 could time-dependently increase the BTB permeability by down-regulating the expression of tight junction protein, and this effect could be reversed by ROS.

Pharmacokinetic studies of meloxicam following oral and transdermal administration in Beagle dogs.

AIM: The potential for topical delivery of meloxicam was investigated by examining its pharmacokinetic profiles in plasma and synovial fluid following oral and transdermal administration in Beagle dogs. METHODS: The experiment was a two-period, crossover design using 6 Beagle dogs. Meloxicam tablets were administered orally at a dose of 0.31 mg/kg, and meloxicam gel was administered transdermally at a dose of 1.25 mg/kg. Drug concentrations in plasma and synovial fluid were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The pharmacokinetic parameters were calculated using the Topfit 2.0 program. RESULTS: The pharmacokinetic results showed that AUC(0-t) (23.9+/-8.26 microg.h.mL(-1)) in plasma after oral administration was significantly higher than after transdermal delivery (1.00+/-0.43 microg.h.mL(-1)). In contrast, the ratio of the average concentration in synovial fluid to that in plasma following transdermal administration was higher than that for an oral delivery. The synovial fluid concentration in the treated leg was much higher than that in the untreated leg, whereas the synovial fluid concentration in the untreated leg was similar to the plasma concentration. CONCLUSION: The high concentration ratio of synovial fluid to plasma indicates direct penetration of meloxicam following topical administration to the target tissue. This finding is further supported by the differences observed in meloxicam concentrations in synovial fluid in the treated and untreated joints at the same time point. Our results suggest that transdermal delivery of meloxicam is a promising method for decreasing its adverse systemic effects.Acta Pharmacologica Sinica (2009) 30: 1060-1064; doi: 10.1038/aps.2009.73; published online 8 June 2009.

Lectin-conjugated PLGA nanoparticles loaded with thymopentin: ex vivo bioadhesion and in vivo biodistribution.

The conjugation of lectins onto PLGA nanoparticles has been demonstrated to effectively improve the intestinal absorption of thymopentin. In this study, thymopentin-loaded nanoparticles made from fluorescein isothiocyanate labeled PLGA were modified with wheat germ agglutinin (WGA). The specific bioadhesion of nanoparticles on rat intestinal mucosa was studied ex vivo. An important increase of interaction between WGA-conjugated nanoparticles and the intestinal segments was observed compared with that of the unconjugated one (p<0.05). Fluorescence photomicrographs confirmed the bioadhesion of WGA-conjugated nanoparticles on intestinal villous epithelium as well as Peyer's patches. Biodistribution of nanoparticles was evaluated using tissues obtained from rats, to which nanoparticles were orally administered. The highest amount of WGA-conjugated nanoparticles was detected in small intestine, suggesting an increase of intestinal bioadhesion and endocytosis. The systemic uptake was as high as 6.48-13.4% of dose at 1 day and 7.32-15.26% at 7 days, which representing an increase of almost 1.4-3.1 fold across the intestine compared to <4.9% of the unconjugated one. The enhanced uptake was related to the increasing of WGA density on nanoparticles. These results further revealed the promising potential of lectin-conjugated nanoparticles on the improvement of intestinal bioadhesion and absorption for oral drug delivery.

Anxiolytic effect of saponins from Panax quinquefolium in mice.

The anxiolytic effect of the saponins from Aniliaeea Panax quinquefolium L. (PQS) was studied in male mice by using a number of experimental paradigms of anxiety and compared with that of the known anxiolytic compound diazepam. Use of the elevated plus-maze test revealed that PQS (50 mg/kg, p.o.) and diazepam (2.5 mg/kg, p.o.) increased the percentage of time and entries spent in open arms. In the light/dark test, PQS (50 and 100 mg/kg, p.o.) and diazepam (2.5 mg/kg, p.o.) prolonged the time spent in the light area. In the hole-board test, PQS (50 and 100 mg/kg, p.o.) and diazepam (2.5 mg/kg, p.o.) significantly increased both head-dip counts and head-dip duration. Both PQS (50 and 100 mg/kg, p.o.) and diazepam (2.5 mg/kg, p.o.) decreased the total fighting time in the isolation-induced aggressive test. Since PQS, in contrast to diazepam, had no effect on locomotion in these tests, its side-effect profile might be considered superior to the benzodiazepines. Thus, the present findings suggest that PQS might be a potential candidate for use as an anxiolytic drug.