Potent Inhibition of Human Cytochrome P450 3A4 by Biflavone Components from Ginkgo Biloba and Selaginella Tamariscina.

CYP3A4-mediated Phase I biotransformation is the rate-limiting step of elimination for many commonly used clinically agents. The modulatory effects of herbal medicines on CYP3A4 activity are one of the risk factors affecting the safe use of drug and herbal medicine. In the present study, the inhibitory effects of nearly hundred kinds of herbal medicines against CYP3A4 were evaluated based on a visual high-throughput screening method. Furthermore, biflavone components including bilobetin (7-demethylginkgetin, DGK), ginkgetin (GK), isoginkgetin (IGK), and amentoflavone (AMF) were identified as the main inhibitory components of Ginkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST), which displayed very strong inhibitory effects toward CYP3A4. The inhibitory effects of these biflavones on clinical drugs that mainly undergo CYP3A4-dependent metabolism were evaluated. The IC 50 of GK toward tamoxifen, gefitinib and ticagrelor were found to be of 0.478 ± 0.003, 0.869 ± 0.001, and 1.61 ± 0.039 µM, respectively. These results suggest the potential pharmacokinetic interactions between the identified biflavones and clinical drugs undergoing CYP3A4-mediated biotransformation. The obtained information is important for guiding the rational use of herbal medicine in combination with synthetic pharmaceuticals.

Electroacupuncture improves metabolic and ovarian function in a rat model of polycystic ovary syndrome by decreasing white adipose tissue, increasing brown adipose tissue, and modulating the gut microbiota.

BACKGROUND: Polycystic ovary syndrome (PCOS) affects 8%-15% of reproductive-age women and is associated with reproductive disorders, abdominal obesity, hyperinsulinemia, insulin resistance, type 2 diabetes, and cardiovascular diseases. Acupuncture, as a traditional physical therapy method, could affect various metabolic disorders such as obesity, hyperplasia, gout, and cardiovascular and cerebrovascular diseases in clinical practice. Moreover, electroacupuncture (EA) has been shown to decrease body weight in rats with PCOS; however, the mechanism of weight loss and the relationship between adipose tissue and gut microbiota remain unclear. OBJECTIVE: To explore the effect and mechanism of EA on white and brown adipose tissues and gut microbiota, and its follow-up effect on reproductive function, in a rat model of PCOS. METHODS: Daily EA treatment was administered at ST29 and SP6 in a dihydrotestosterone (DHT)-induced PCOS-like rat model (PCOS + EA group). Effects of EA on in vivo and in vitro adipose volume and weight, organ weight coefficients, body weight, hormonal profiles, and estrous cyclicity were measured, and compared with untreated PCOS model rats (PCOS group) and healthy rats (control group). Microbial DNA was extracted from the fecal samples to analyze group abundance and diversity. RESULTS: EA improved estrous cyclicity, decreased body weight, decreased visceral and subcutaneous fat content, and increased brown adipose tissue weight. EA also normalized serum DHT and progesterone levels and improved glucose tolerance. There were few significant differences in the composition or diversity of the gut microbiota between control, PCOS, and PCOS + EA groups, except for the relative abundances of Tenericutes at the phylum level and Prevotella_9 at the genus level, which were significantly different in the PCOS group before and after EA treatment. Both are important microflora, strongly related to body weight. CONCLUSION: EA regulated the metabolic disorders and improved reproductive function in this PCOS-like rat model by adjusting visceral fat and brown fat, as well as intestinal flora.

Decoding neuronal composition and ontogeny of individual hypothalamic nuclei.

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

Mindfulness-Based Cognitive Therapy for Treating Chronic Pain A Systematic Review and Meta-analysis.

Chronic pain is a significant public health problem with emotional and disabling factors, which may not completely respond to current medical treatments such as opioids. The systematic review and meta-analysis aimed to examine the effectiveness and safety of MBCT for patients with chronic pain. Database searches of PubMed, Medline, EMBASE, the Cochrane Library, PsycINFO, Web of Science, Scopus and CINAHL up to 15 October 2019. Included studies assessed with the Cochrane risk-of-bias tool. Eight RCTs involved 433 patients, including chronic low back pain, fibromyalgia, migraine, rheumatoid arthritis and mix etiology. MBCT intervention demonstrated a short-term improvement on depression mood [standardized mean difference -0.72; 95% confidence interval = -1.22 to -0.22, p = 0.005] compared with usual care and was associated with short-term improvement in mindfulness compared with non-MBCT [SMD 0.51; 95% CI = 0.01 to 1.01, p = 0.04]. Between-group differences in pain intensity, pain inference and pain acceptance were not significant at short- or long-term follow-up. Compared to active treatments, MBCT intervention not found significant differences in either short- or long-term outcomes. MBCT showed short-term efficacious on depressed mood and mindfulness of chronic pain patients. Longer follow-ups, large sample and rigorous RCTs that can be best understand remaining uncertainties needed.

Ovarian Innervation Coupling With Vascularity: The Role of Electro-Acupuncture in Follicular Maturation in a Rat Model of Polycystic Ovary Syndrome.

Low-frequency electro-acupuncture (EA) has been shown to restore ovulation in patients with polycystic ovary syndrome (PCOS), and previous animal experiments showed that EA improves ovarian blood flow and angiogenesis. We performed EA for 4 weeks in dihydrotestosterone (DHT)-induced PCOS-like rats and investigated the three-dimensional (3D) ovarian innervation to determine the role of innervation in folliculogenesis and vascularity. Ovarian tissues were made transparent following the CUBIC 3D tissue-clearing protocol and were immunostained using antibodies against platelet endothelial cell adhesion molecule-1 and tyrosine hydroxylase to visualize the ovarian vasculature and innervation, respectively. This was followed by 3D imaging using lightsheet microscopy and analysis using the Imaris software. In control rats, ovarian innervation increased with age, and the neuronal branching started from the ovarian hilum and reached the individual follicles at different follicle stages. At the individual follicle level, each follicle was mainly innervated by one neuronal fiber. Compared with control rats, ovaries from DHT-treated PCOS-like rats had more antral follicles and fewer preovulatory follicles and corpora lutea. Furthermore, PCOS ovaries showed decreased innervation of blood vessels near the hilum and the surrounding individual antral follicles. EA in PCOS-like rats led to increased numbers of preovulatory follicles and corpora lutea together with increased innervation of blood vessels near the hilum. To determine the role of ovarian innervation, we further performed unilateral sectioning of the superior ovarian nerve (SON) in PCOS + EA rats and found that the left sectioned ovary had fewer preovulatory follicles and corpora lutea compared with those in the right non-sectioned ovary. In conclusion, ovarian innervation likely played an important role in folliculogenesis, and EA might restore PCOS pathophysiology by regulating ovarian innervation, at least partially mediated through the SON.

Endogenous Ovarian Angiogenesis in Polycystic Ovary Syndrome-Like Rats Induced by Low-Frequency Electro-Acupuncture: The CLARITY Three-Dimensional Approach.

We sought to determine the role of ovarian vascularity and neo-angiogenesis in the development of mature follicles in polycystic ovary syndrome (PCOS) and to identify any changes induced by low-frequency electro-acupuncture (EA). Twenty-eight 21-day-old female Wistar rats were randomly divided into four groups-Control, Obesity, PCOS-like, and PCOS-like-EA (n = 7/group). Rats in the Obesity group were fed a high-fat diet throughout the experiment. Rats in the PCOS-like and PCOS-like-EA groups were implanted with a sustained-release tube containing 5α-dihydrotestosterone (DHT) beneath the skin of the neck. Rats in the PCOS-like-EA group received low-frequency EA treatment starting at 70 days for 30 min five times a week for four weeks. At the end of the experiment, all rats were euthanized and perfused with hydrogel. The ovaries were collected for clarification and imaging, and ovarian vascularity and neo-angiogenesis were analyzed. Compared with Control and Obesity rats, the ovaries in DHT-induced PCOS-like rats were smaller in size and had fewer mature follicles and corpora lutea. EA increased angiogenesis in the antral follicles of PCOS-like rats, which in turn promoted follicle maturation, ovulation, and CL formation. Therefore, endogenous ovarian angiogenesis plays a very important role in follicular maturation and might be one of the peripheral and direct mechanisms of EA on PCOS.

Hypothalamic DNA methylation in rats with dihydrotestosterone-induced polycystic ovary syndrome: effects of low-frequency electro-acupuncture.

NEW FINDINGS: What is the central question of this study? What is the role of hypothalamic DNA methylation in the development of polycystic ovary syndrome (PCOS) and the response to electro-acupuncture treatment. What is the main finding and its importance? Global DNA methylation and expression of DNA methyltransferases (DNMTs) were increased in PCOS-like rats, and electro-acupuncture (EA) decreased global DNA methylation and DNMT3b expression. Pyrosequencing showed that the DNA methylation of some PCOS candidate genes was changed in the PCOS and PCOS+EA groups, suggesting that hypothalamic DNA methylation plays an important role in the development of PCOS and in mediating the effects of electro-acupuncture treatment. ABSTRACT: Polycystic ovary syndrome (PCOS) is a common reproductive and endocrine disease of unknown aetiology. Recently, epigenetic studies focusing on DNA methylation in PCOS have received much attention, but the mechanisms are still unclear. In the present study, we used the 5α-dihydrotestosterone-induced PCOS-like rat model and treated the rats with electro-acupuncture (EA). Rats were randomly divided into four groups - controls, diet-induced obesity, PCOS and PCOS+EA. We examined the reproductive, metabolic and behavioural phenotypes, validated the effect of EA, and explored the role of hypothalamic DNA methylation by analysing the methylation of global DNA and selected candidate genes. The PCOS rats presented with reproductive dysfunctions such as lack of regular oestrous cyclicity, metabolic disorders such as increased body weight and insulin resistance, and depression and anxiety-like behaviours. EA improved the reproductive functions, decreased body weight and improved experimental depressive behaviour. Furthermore, global DNA methylation and the expression of DNA methyltransferases (DNMTs) were increased in PCOS rats compared to the control group, and EA decreased the global DNA methylation and the expression of DNMT3b. In addition, pyrosequencing showed that the DNA methylation of certain CpG sites in targeted genes (Plcg1, Camk2b, Esr2 and Pgr) was increased in the PCOS group, but the DNA methylation of Camk2b and Ar was decreased after EA treatment. These results indicate that hypothalamic DNA methylation might be correlated with the development of PCOS and that EA has an effect on hypothalamic DNA methylation in PCOS rats.

Tubeimoside-1 induces oxidative stress-mediated apoptosis and G0/G1 phase arrest in human prostate carcinoma cells in vitro.

AIM: Tubeimoside-1 (TBMS1), a triterpenoid saponin extracted from the Chinese herbal medicine Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), has shown anticancer activities in various cancer cell lines. The aim of this study was to investigate the anticancer activity and molecular targets of TBMS1 in human prostate cancer cells in vitro. METHODS: DU145 and P3 human prostate cancer cells were treated with TBMS1. Cell viability and apoptosis were detected. ROS generation, mitochondrial membrane potential and cell cycle profile were examined. Western blotting was used to measure the expression of relevant proteins in the cells. RESULTS: TBMS1 (5-100 µmol/L) significantly suppressed the viability of DU145 and P3 cells with IC50 values of approximately 10 and 20 µmol/L, respectively. Furthermore, TBMS1 dose-dependently induced apoptosis and cell cycle arrest at G0/G1 phase in DU145 and P3 cells. In DU145 cells, TBMS1 induced mitochondrial apoptosis, evidenced by ROS generation, mitochondrial dysfunction, endoplasmic reticulum stress, modulated Bcl-2 family protein and cleaved caspase-3, and activated ASK-1 and its downstream targets p38 and JNK. The G0/G1 phase arrest was linked to increased expression of p53 and p21 and decreased expression of cyclin E and cdk2. Co-treatment with Z-VAD-FMK (pan-caspase inhibitor) could attenuate TBMS1-induced apoptosis but did not prevent G0/G1 arrest. Moreover, co-treatment with NAC (ROS scavenger), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor) or salubrinal (ER stress inhibitor) significantly attenuated TBMS1-induced apoptosis. CONCLUSION: TBMS1 induces oxidative stress-mediated apoptosis in DU145 human prostate cancer cells in vitro via the mitochondrial pathway.

CFTR chloride channel as a molecular target of anthraquinone compounds in herbal laxatives.

AIM: To clarify whether CFTR is a molecular target of intestinal fluid secretion caused by the anthraquinone compounds from laxative herbal plants. METHODS: A cell-based fluorescent assay to measure I(-) influx through CFTR chloride channel. A short-circuit current assay to measure transcellular Cl(-) current across single layer FRT cells and freshly isolated colon mucosa. A closed loop experiment to measure colon fluid secretion in vivo. RESULTS: Anthraquinone compounds rhein, aloe-emodin and 1,8-dihydroxyanthraquinone (DHAN) stimulated I(-) influx through CFTR chloride channel in a dose-dependent manner in the presence of physiological concentration of cAMP. In the short-circuit current assay, the three compound enhanced Cl(-) currents in epithelia formed by CFTR-expressing FRT cells with EC(50) values of 73 ± 1.4, 56 ± 1.7, and 50 ± 0.5 µmol/L, respectively, and Rhein also enhanced Cl(-) current in freshly isolated rat colonic mucosa with a similar potency. These effects were completely reversed by the CFTR selective blocker CFTR(inh)-172. In in vivo closed loop experiments, rhein 2 mmol/L stimulated colonic fluid accumulation that was largely blocked by CFTR(inh)-172. The anthraquinone compounds did not elevate cAMP level in cultured FRT cells and rat colonic mucosa, suggesting a direct effect on CFTR activity. CONCLUSION: Natural anthraquinone compounds in vegetable laxative drugs are CFTR potentiators that stimulated colonic chloride and fluid secretion. Thus CFTR chloride channel is a molecular target of vegetable laxative drugs.

Identification of natural coumarin compounds that rescue defective DeltaF508-CFTR chloride channel gating.

1. Deletion of phenylalanine at position 508 (DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis (CF). Effective pharmacological therapy of CF caused by the DeltaF508-CFTR mutation requires the rescue of both intracellular processing and channel gating defects. 2. We identified a class of natural coumarin compounds that can correct the defective DeltaF508-CFTR chloride channel gating by screening a collection of 386 single natural compounds from Chinese medicinal herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells coexpressing DeltaF508-CFTR and an iodide-sensitive fluorescent indicator (YFP-H148Q/I152L). 3. Dose-dependent potentiation of defective DeltaF508-CFTR chloride channel gating by five coumarin compounds was demonstrated by the fluorescent iodide influx assay and confirmed by an Ussing chamber short-circuit current assay. Activation was fully abolished by the specific CFTR inhibitor CFTR(inh)-172. Two potent compounds, namely imperatorin and osthole, have activation K(d) values of approximately 10 micromol/L, as determined by the short-circuit current assay. The active coumarin compounds do not elevate intracellular cAMP levels. Activation of DeltaF508-CFTR by the coumarin compounds requires cAMP agonist, suggesting direct interaction with the mutant CFTR molecule. Kinetics analysis indicated rapid activation of DeltaF508-CFTR by the coumarin compounds, with half-maximal activation of < 5 min. The activating effect was fully reversed for all five active compounds 45 min after washout. 4. In conclusion, the natural coumarin DeltaF508-CFTR activators may represent a new class of natural lead compounds for the development of pharmacological therapies for CF caused by the DeltaF508 mutation.