Targeted metabolomics combined with network pharmacology to reveal the protective role of luteolin in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease that significantly endangers human health, where metabolism may drive pathogenesis: a shift from mitochondrial oxidation to glycolysis occurs in diseased pulmonary vessels and the right ventricle. An increase in pulmonary vascular resistance in patients with heart failure with a preserved ejection fraction portends a poor prognosis. Luteolin exists in numerous foods and is marketed as a dietary supplement assisting in many disease treatments. However, little is known about the protective effect of luteolin on metabolism disorders in diseased pulmonary vessels. In this study, we found that luteolin apparently reversed the pulmonary vascular remodeling of PAH rats by inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). Moreover, network pharmacology and metabolomics results revealed that the arachidonic acid pathway, amino acid pathway and TCA cycle were dysregulated in PAH. A total of 14 differential metabolites were significantly changed during the PAH, including DHA, PGE2, PGD2, LTB4, 12-HETE, 15-HETE, PGF2α, and 8-iso-PGF2α metabolites in the arachidonic acid pathway, and L-asparagine, oxaloacetate, N-acetyl-L-ornithine, butane diacid, ornithine, glutamic acid metabolites in amino acid and TCA pathways. However, treatment with luteolin recovered the LTB4, PGE2, PGD2, 12-HETE, 15-HETE, PGF2α and 8-iso-PGF2α levels close to normal. Meanwhile, we showed that luteolin also downregulated the gene and protein levels of COX 1, 5-LOX, 12-LOX, and 15-LOX in the arachidonic acid pathway. Collectively, this work highlighted the metabolic mechanism of luteolin-protected PAH and showed that luteolin would hold great potential in PAH prevention.

Cyhalofop-butyl and Glyphosate Multiple-Herbicide Resistance Evolved in an Eleusine indica Population Collected in Chinese Direct-Seeding Rice.

Eleusine indica is a typical xerophytic weed species with a cosmopolitan distribution. It is invasive and highly adaptable to diverse habitats and crops. Due to rice cropping-pattern changes, E indica has become one of the main dominant grass weeds infecting direct-seeding paddy fields. A Chinese E. indica population has evolved multiple-herbicide resistance to cyhalofop-butyl and glyphosate. In this study, the multiple-resistance profile of E. indica to these two different types of herbicides and their resistance mechanisms were investigated. Whole-plant dose-response assays indicated that the multiple-herbicide-resistant (MHR) population exhibited 10.8-fold resistance to cyhalofop-butyl and 3.1-fold resistance to glyphosate compared with the susceptible (S) population. ACCase sequencing revealed that the Asp-2078-Gly mutation was strongly associated with E. indica resistance to cyhalofop-butyl. The MHR plants accumulated less shikimic acid than S plants at 4, 6, and 8 days after glyphosate treatment. In addition, no amino acid substitution in the EPSPS gene was found in MHR plants. Further analysis revealed that the relative expression level of EPSPS in MHR plants was 6-10-fold higher than that in S plants following glyphosate treatment, indicating that EPSPS overexpression may contribute to the glyphosate resistance. Furthermore, the effectiveness of nine post-emergence herbicides against E. indica were evaluated, and one PPO inhibitor pyraclonil was identified as highly effective in controlling the S and MHR E. indica populations.

Molecular basis of resistance to ACCase-inhibiting herbicide cyhalofop-butyl in Chinese sprangletop (Leptochloa chinensis (L.) Nees) from China.

Chinese sprangletop (Leptochloa chinensis (L.) Nees) is one of the most troublesome grass weeds in rice in China. Seven suspected cyhalofop-butyl-resistant L. chinensis populations were collected from different rice fields with a history of cyhalofop-butyl use. The level of resistance and resistance mechanisms in seven populations were studied. Dose-response tests indicated that five populations (JS3, JS4, JS6, JS7 and JS8) had evolved high-level resistance (26.9 to 123.0-fold) to cyhalofop-butyl compared with the susceptible (S) population, and other two populations (JS2 and JS5) were still sensitive to the herbicide. Two acetyl-coenzyme A carboxylase (ACCase) genes were cloned from each population, and three different ACCase mutations (Ile-1781-Leu, Trp-1999-Cys, and Trp-2027-Cys) in ACCase2 gene were determined in different resistant (R) populations. In addition, no resistance-conferring mutations was detected in the R population (JS7), and ACCase gene expression was similar between the S and R populations. Thus, non-target-site resistance mechanisms may be involved in the JS7 population. Moreover, the patterns of cross-resistance of JS6 (Ile-1781-Leu), JS4 (Trp-1999-Cys), JS8 (Trp-2027-Cys), and JS7 (unknown resistance mechanisms) populations to other ACCase-inhibiting herbicides were determined. The JS6 and JS8 populations showed resistance to fenoxaprop-P-ethyl, metamifop, clethodim and pinoxaden, the JS4 population was resistant to fenoxaprop-P-ethyl, metamifop and pinoxaden, and the JS7 population had resistance only to fenoxaprop-P-ethyl and metamifop. These results indicated the diversity of the target-site mutations in ACCase gene of L. chinensis, and provide a better understanding of cross-resistance in L. chinensis, which would be helpful for the management of cyhalofop-butyl-resistant L. chinensis.

Isobutylhydroxyamides from Sichuan Pepper and Their Protective Activity on PC12 Cells Damaged by Corticosterone.

The pericarp of Zanthoxylum bungeanum Maxim., commonly known as Sichuan pepper, is a widely used spice to remove fishy odor and add palatable taste. A phytochemical investigation of the 95% ethanol extract of Sichuan pepper resulted in the isolation of 21 isobutylhydroxyamides, including 8 new ones named ZP-amides G-N, among which the chiral resolution of racemic ZP-amide A and ZP-amide B was successfully accomplished. The protective activity on corticosterone-treated PC12 cells of the isolated isobutylhydroxyamides was also evaluated. The new compounds 3-5 and the known compounds 1, 1a, 2, 2a, 11, and 15 improved the survival rate of PC12 cells. The bioactivity studies disclosed the potential of Sichuan pepper to be developed as new neuroprotective functional food.

Phenolic constituents of the Bangladeshi medicinal plant Pothos scandens and their anti-estrogenic, hyaluronidase inhibition, and histamine release inhibitory activities.

Extracts from the stem and roots of the Bangladeshi medicinal plant Pothos scandens L. (Araceae) were isolated, and three hemiterpene glucoside aromatic esters, pothobanosides A (1), B (2), and C (3), and a phenylisobutanoid, pothobanol (4), along with 14 known compounds, were characterized. The isolates were tested for their estrogenic/anti-estrogenic activity using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D, and syringoyl derivatives (2, 3, and canthoside B) showed strong inhibitory activity against both cell lines. Their less oxygenated analogs (1, and markhamioside F) were almost inactive. The isolates were also evaluated for hyaluronidase and histamine release inhibitory activities, and pothobanoside A (1) showed significant hyaluronidase inhibitory activity among the isolated compounds, which was similar to that of the positive control rosmarinic acid. Because hyaluronidase produces an angiogenic response that has been implicated in tumor invasiveness and metastasis, 1 could be valuable as an anti-tumor compound with a different mechanism of action from related compounds (2, 3). Pothobanoside C (3) and pothobanol (4) were also found to inhibit histamine release to a similar degree to the positive control epigallocatechin 3-O-(3"-O-methyl)-gallate. The histamine release inhibitory potency of these isolates may support the traditional uses of this plant in folk medicine.

Magnetic resonance spectroscopy for detection of choline kinase inhibition in the treatment of brain tumors.

Abnormal choline metabolism is a hallmark of cancer and is associated with oncogenesis and tumor progression. Increased choline is consistently observed in both preclinical tumor models and in human brain tumors by proton magnetic resonance spectroscopy (MRS). Thus, inhibition of choline metabolism using specific choline kinase inhibitors such as MN58b may be a promising new strategy for treatment of brain tumors. We demonstrate the efficacy of MN58b in suppressing phosphocholine production in three brain tumor cell lines. In vivo MRS studies of rats with intracranial F98-derived brain tumors showed a significant decrease in tumor total choline concentration after treatment with MN58b. High-resolution MRS of tissue extracts confirmed that this decrease was due to a significant reduction in phosphocholine. Concomitantly, a significant increase in poly-unsaturated lipid resonances was also observed in treated tumors, indicating apoptotic cell death. MRI-based volume measurements demonstrated a significant growth arrest in the MN58b-treated tumors in comparison with saline-treated controls. Histologically, MN58b-treated tumors showed decreased cell density, as well as increased apoptotic cells. These results suggest that inhibition of choline kinase can be used as an adjuvant to chemotherapy in the treatment of brain tumors and that decreases in total choline observed by MRS can be used as an effective pharmacodynamic biomarker of treatment response.

Somatostatin 4 receptor activation modulates TRPV1[correction of TPRV1] currents in dorsal root ganglion neurons.

Somatostatin (sst) is a cyclic neuropeptide known to have inhibitory roles in the central nervous system. It exerts its biological effects via the activation of the 5 sst receptor subtypes, which belong to the family of G-protein coupled receptors (GPCR). This peptide has analgesic properties, specifically via the activation of the sst4 receptor subtype. Although this is established, the precise molecular mechanisms causing this have not yet been fully elucidated. This research aimed to identify a possible anti-nociceptive mechanism, showing functional links to the transient receptor potential vanilloid type 1 (TRPV1) within the pain processing pathway. Calcium imaging and whole cell voltage clamp experiments were conducted on DRG neurons prepared from adult rats, utilizing capsaicin stimulations and the sst4 receptor specific agonist J-2156. The complete Freund's adjuvant (CFA) inflammatory pain model was used to examine if effects are augmented in pain conditions. The sst4 receptor agonist J-2156 was able significantly to inhibit capsaicin induced calcium and sodium influx, where the effect was more potent after CFA treatment. This inhibition identifies a contributory molecular mechanism to the analgesic properties of sst4 receptor activation.

Harzianolide, a novel plant growth regulator and systemic resistance elicitor from Trichoderma harzianum.

A detailed understanding of the effect of natural products on plant growth and protection will underpin new product development for plant production. The isolation and characterization of a known secondary metabolite named harzianolide from Trichoderma harzianum strain SQR-T037 were described, and the bioactivity of the purified compound as well as the crude metabolite extract in plant growth promotion and systemic resistance induction was investigated in this study. The results showed that harzianolide significantly promoted tomato seedling growth by up to 2.5-fold (dry weight) at a concentration of 0.1 ppm compared with the control. The result of root scan suggested that Trichoderma secondary metabolites may influence the early stages of plant growth through better root development for the enhancement of root length and tips. Both of the purified harzianolide and crude metabolite extract increased the activity of some defense-related enzymes to response to oxidative stress. Examination of six defense-related gene expression by real-time reverse transcription-PCR analysis revealed that harzianolide induces the expression of genes involved in the salicylic acid (PR1 and GLU) and jasmonate/ethylene (JERF3) signaling pathways while crude metabolite extract inhibited some gene expression (CHI-II and PGIP) related to basal defense in tomato plants. Further experiment showed that a subsequent challenge of harzianolide-pretreated plants with the pathogen Sclerotinia sclerotiorum resulted in higher systemic resistance by the reduction of lesion size. These results indicate that secondary metabolites of Trichoderma spp., like harzianolide, may play a novel role in both plant growth regulation and plant defense responses.

Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

BACKGROUND: Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. METHODOLOGY/PRINCIPAL FINDINGS: ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. CONCLUSION/SIGNIFICANCE: Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

Diaminobutane (DAB) dendrimers are potent binders of oral phosphate.

Reduction of blood phosphorus is a critical component in the management of secondary hyperparathyroidism in chronic kidney disease patients. In addition to dialysis treatment and dietary phosphorus restriction, oral phosphate binders are often consumed with meals to reduce the availability of food phosphorus. Several oral phosphate binders are approved for use in chronic kidney disease patients, but all have practical limitations because of toxicity, poor efficacy, or high cost. Using an in vivo method to measure intestinal phosphate absorption in rats using radiolabeled phosphate, we found that first-, second-, third-, and fifth-generation diaminobutane dendrimer compounds, DAB-4-Cl, DAB-8-Cl, DAB-16-Cl, and DAB-64-Cl, respectively, drastically reduce the absorption of inorganic phosphate in a dose-dependent manner. To avoid complications of metabolic acidosis caused by hydrochloride salts, an acetate salt, DAB-9-AcOH, was prepared and shown to be equally effective at binding radiolabeled phosphate as DAB-8-Cl. DAB-8-AcOH was further shown to increase fecal phosphorus and decrease serum phosphorus in a dose-dependent manner when fed to rats. These data suggest that dendrimer compounds are of great potential use in the binding of food phosphate for the management of hyperparathyroidism secondary to chronic kidney disease.

A comparison of reactivating efficacy of newly developed oximes (K074, K075) and currently available oximes (obidoxime, HI-6) in cyclosarin-and tabun-poisoned rats.

The potency of newly developed oximes (K074, K075) and commonly used oximes (obidoxime, HI-6) to reactivate nerve agent-inhibited acetylcholinesterase was evaluated in rats poisoned with tabun or cyclosarin at a lethal dose corresponding to the LD50 value. In vivo determined percentage of reactivation of tabun-inhibited blood and brain acetylcholinesterase showed that obidoxime is the most efficacious reactivator of tabun-inhibited acetylcholinesterase among studied oximes in the peripheral compartment (blood) although the differences between obidoxime and newly developed oximes were not significant. On the other hand, one of the newly developed oximes (K074) seems to be a significantly more efficacious reactivator of tabun-inhibited acetylcholinesterase in the central compartment (brain) than the other studied oximes. In addition, the oxime HI-6 is unable to sufficiently reactivate tabun-inhibited acetylcholinesterase in rats. In vivo determined percentage of reactivation of cyclosarin-inhibited blood and brain acetylcholinesterase in poisoned rats showed that HI-6 is the most efficacious reactivator of cyclosarin-inhibited acetylcholinesterase among the studied oximes in the peripheral (blood) as well as central (brain) compartment although the differences between the oxime HI-6 and other tested oximes in the brain were not significant. Due to their reactivating effects, both newly developed K-oximes can be considered to be promising oximes for the antidotal treatment of acute tabun poisoning while the oximes HI-6 is still the most promising oxime for the treatment of acute cyclosarin poisonings due to its high potency in reactivating cyclosarin-inhibited acetylcholinesterase in the peripheral as well as central compartment.

Effects of the somatostatin receptor subtype 4 selective agonist J-2156 on sensory neuropeptide release and inflammatory reactions in rodents.

BACKGROUND AND PURPOSE: Substance P (SP) and calcitonin gene-related peptide (CGRP) released from capsaicin-sensitive sensory nerves induce local neurogenic inflammation; somatostatin exerts systemic anti-inflammatory actions presumably via sst4/sst1 receptors. This study investigates the effects of a high affinity, sst4-selective, synthetic agonist, J-2156, on sensory neuropeptide release in vitro and inflammatory processes in vivo. EXPERIMENTAL APPROACH: Electrically-induced SP, CGRP and somatostatin release from isolated rat tracheae was measured with radioimmunoassay. Mustard oil-induced neurogenic inflammation in rat hindpaw skin was determined by Evans blue leakage and in the mouse ear with micrometry. Dextran-, carrageenan- or bradykinin-induced non-neurogenic inflammation was examined with plethysmometry or Evans blue, respectively. Adjuvant-induced chronic arthritis was assessed by plethysmometry and histological scoring. Granulocyte accumulation was determined with myeloperoxidase assay and IL-1beta with ELISA. KEY RESULTS: J-2156 (10-2000 nM) diminished electrically-evoked neuropeptide release in a concentration-dependent manner. EC50 for the inhibition of substance P, CGRP and somatostatin release were 11.6 nM, 14.3 nM and 110.7 nM, respectively. J-2156 (1-100 microg kg(-1) i.p.) significantly, but not dose-dependently, inhibited neurogenic and non-neurogenic acute inflammatory processes and adjuvant-induced chronic oedema and arthritic changes. Endotoxin-evoked myeloperoxidase activity and IL-1beta production in the lung, but not IL-1beta- or zymosan-induced leukocyte accumulation in the skin were significantly diminished by J-2156. CONCLUSIONS AND IMPLICATIONS: J-2156 acting on sst4 receptors inhibits neuropeptide release, vascular components of acute inflammatory processes, endotoxin-induced granulocyte accumulation and IL-1beta synthesis in the lung and synovial and inflammatory cells in chronic arthritis. Therefore it might be a promising lead for the development of novel anti-inflammatory drugs.

Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of the choline kinase inhibitor MN58b in human carcinoma models.

MN58b is a novel anticancer drug that inhibits choline kinase, resulting in inhibition of phosphocholine synthesis. The aim of this work was to develop a noninvasive and robust pharmacodynamic biomarker for target inhibition and, potentially, tumor response following MN58b treatment. Human HT29 (colon) and MDA-MB-231 (breast) carcinoma cells were examined by proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment with MN58b both in culture and in xenografts. An in vitro time course study of MN58b treatment was also carried out in MDA-MB-231 cells. In addition, enzymatic assays of choline kinase activity in cells were done. A decrease in phosphocholine and total choline levels (P < 0.05) was observed in vitro in both cell lines after MN58b treatment, whereas the inactive analogue ACG20b had no effect. In MDA-MB-231 cells, phosphocholine fell significantly as early as 4 hours following MN58b treatment, whereas a drop in cell number was observed at 48 hours. Significant correlation was also found between phosphocholine levels (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008) following MN58b treatment. Phosphomonoesters also decreased significantly (P < 0.05) in both HT29 and MDA-MB-231 xenografts with no significant changes in controls. 31P-MRS and 1H-MRS of tumor extracts showed a significant decrease in phosphocholine (P < or = 0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors.

Hemodynamic effects of di-sec-butyl phenol, an anesthetic substituted phenol.

Dose- and age-related hemodynamic effects were determined for an anesthetic substituted phenol, 2,6-di-sec-butyl phenol (DSB). DSB, 7.5 mg/kg, induced hypnosis in young rabbits and increased mean blood pressure to 170 +/- 14% and heart rate to 150 +/- 21% of control values. In elderly rabbits, 7.5 mg/kg DSB induced hypnosis, had no effect on blood pressure, but increased the heart rate to 130 +/- 2% of control. After ganglionic blockade with hexamethonium, 7.5 mg/kg DSB caused a decline in mean blood pressure (71 +/- 5% of control) without change in heart rate. DSB increased norepinephrine release from SH-SY5Y cells, a human neuroblastoma cell line (5.4 +/- 1.7% vs. 3.5 +/- 0.3%). DSB produced age-dependent elevation of mean blood pressure in rabbits, probably by causing release of catecholamines from the sympathetic nervous system.

Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 4: synthesis and structure-activity relationships for 1-[N-(methyl)-N-(phenylsulfonyl)amino]-2-(phenyl)-4-(4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidin-1-yl)butanes.

(2S)-2-(3-Chlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2,3-dihydrobenzthiophene-3,4'-piperidin-1'-yl)]butane S-oxide (1b) has been identified as a potent CCR5 antagonist having an IC50=10 nM. Herein, structure-activity relationship studies of non-spiro piperidines are described, which led to the discovery of 4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidine derivatives (3-5) as potent CCR5 antagonists.

CPI-1189 prevents apoptosis and reduces glial fibrillary acidic protein immunostaining in a TNF-alpha infusion model for AIDS dementia complex.

AIDS dementia complex (ADC) is characterized by increased apoptosis, gliosis, and oxidative stress in the CNS, as well as a compromised blood-brain barrier. TNF-alpha has been shown to be elevated in AIDS dementia complex brains and may contribute to AIDS dementia complex. To model elevated TNF-alpha in AIDS dementia complex, TNF-alpha was infused ICV bilaterally into rats for 3 days. TNF-alpha treatment increased apoptosis around the infusion site and selectively in the septum and corpus callosum. Co-administration of the synthetic antioxidant CPI-1189 prevented TNF-alpha induced apoptosis. Both TNF-alpha and CPI-1189 treatment suppressed glial fibrillary acidic protein (GFAP) staining at the infusion site. TNF-alpha did not significantly affect the integrity of the blood-brain barrier, but CPI-1189 treatment increased blood-brain barrier integrity at the infusion site. No effect of TNF-alpha or CPI-1189 treatment was found on measures of oxidative stress. These results support TNF-alpha as a key agent for increasing apoptosis in AIDS dementia complex. Additionally, CPI-1189 treatment may protect against TNF-alpha induced apoptosis and astrogliosis in AIDS dementia complex. Lastly, the toxic effect of TNF-alpha and the protective effect of CPI-1189 may not be mediated primarily through manipulation of classic reactive oxygen species.