The mechanism of action of Fangji Huangqi Decoction on epithelial-mesenchymal transition in breast cancer using high-throughput next-generation sequencing and network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Fangji Huangqi Decoction (FHD) is widely used in traditional Chinese medicine (TCM). FHD has been hypothesized to inhibit the epithelial-mesenchymal transition (EMT) process, which may positively impact breast cancer prevention and treatment. However, its exact mechanism of action is still unknown. AIM OF THE STUDY: This study aimed to screen potential targets of FHD for the treatment of EMT in breast cancer through network pharmacology, and to verify their therapeutic effects in vitro experiments and high-throughput second-generation sequencing. MATERIALS AND METHODS: The data sets of effective components and targets of FHD were established through the Traditional Chinese Medicine Systems Pharmacology database. The GeneCards and OMIM databases were used to establish breast cancer-related target datasets, which were then matched with the TCM target data. The interaction between key target proteins was analyzed using the STRING database; the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to identify the associated biological processes and enriched signal pathways, respectively. The active ingredient disease target network was analyzed using Cytoscape. Finally, next generation sequencing was used to verify the related pathways of FHD intervention in EMT in breast cancer. High-content screening was used to identify the genes/pathways affected by FHD. MDA-MB-231 and HCC-1937 breast cancer cell lines were used to evaluate the impact of FHD on migration, invasion, and EMT. RESULTS: Eighty possible significant targets were identified for the treatment of breast cancer EMT with FHD; GO and KEGG were used to identify 173 cell biological processes associated with breast cancer (P < 0.05), including the NF-κB and PI3K-Akt signaling pathways. The high-throughput sequencing and network pharmacology results were highly consistent. The migration and invasion ability of MDA-MB-231 cells was reduced and their EMT status could be reversed by DSHR2 knockdown. The results of morphology and scratch assays showed that FHD could improve the EMT status of HCC-1973. CONCLUSIONS: This study provides more evidence to support the clinical application of FHD, which has reliable interventional effects on breast cancer EMT. Its therapeutic effects may involve a multi-target, multi-pathway, and multi-mechanism effect.

Reduced fecundity and regulation of reproductive factors in flubendiamide-resistant strains of Plutella xylostella.

Diamondback moth (DBM), Plutella xylostella, is an important pest of crucifers worldwide. The extensive use of flubendiamide has led to the development of resistance in field populations and reports of control failures. In this study, the lab-selected (Rf) and field-collected (Rb) flubendiamide-resistant strains of P. xylostella with LC50 resistance ratios of 1890-fold and 1251-fold, respectively, were used, as well as a lab-reared flubendiamide-susceptible strain (S). The results showed that the fecundity of the Rf and Rb-resistant strains was significantly lower than that of S strain. The contents of vitellin and transcripts of P. xylostella vitellogenin (PxVg) and P. xylostella vitellogenin receptor (PxVgR) genes in the Rf and Rb strains were significantly higher than those of S strains at 0-48 h after adult eclosion. At 96 h after eclosion, the content of vitellin in the Rf and Rb strains did not differ significantly from those of S strains, whereas transcripts of the PxVg and PxVgR genes in the Rf and Rb strains were significantly lower than that of the S strain. The content of the juvenile hormone III (JH III), ß-ecdysone (20E), and the gene expression level of P. xylostella methoprene tolerant (PxMet) in the Rf and Rb strains were significantly higher than that of the S strain. The activity of trehalase was significantly higher in the Rf and Rb strains than that of the S strain in the first to the third instar larvae, whereas in the fourth instar larvae, there was no significantly difference in the three strains. At different times after adult eclosion, the differences in trehalase activity were erratic between the strains. The transcripts of P. xylostella trehalase (PxTre) gene in the Rf and Rb strains were significantly higher than that of the S strain in most developmental stages. Here, we report differences in fecundity between flubendiamide-resistant and susceptible strains of P. xylostella and discuss gene expression of several reproductive factors, which provides a possible explanation for the mechanism of fecundity reduction concurrent with flubendiamide-resistance in P. xylostella.

Resistance to Diamide Insecticides in Plutella xylostella (Lepidoptera: Plutellidae): Comparison Between Lab-Selected Strains and Field-Collected Populations.

Diamondback moth, Plutella xylostella (L.; Lepidoptera: Plutellidae), is an important pest of crucifers worldwide. The extensive use of diamide insecticides has led to P. xylostella resistance and this presents a serious threat to vegetable production. We selected chlorantraniliprole (Rf) and flubendiamide (Rh) resistance strains of P. xylostella with resistance ratios of 684.54-fold and 677.25-fold, respectively. The Rf and Rh strains underwent 46 and 36 generations of lab-selection for resistance, respectively. Low cross resistance of Rh to cyantraniliprole was found. Cross resistance to chlorfenapyr, tebufenozid, and indoxacarb was not found in Rf and Rh strains. The P. xylostella ryanodine receptor gene (PxRyR) transcripts level in the Rf and Rh strains was up-regulated. Except for Rf34 and Rh36, PxRyR expression in all generations of Rf and Rh selection gradually increased with increasing resistance. Two resistant populations were field-collected from Guangzhou Baiyun (Rb) and Zengcheng (Rz) and propagated for several generations without exposure to any pesticide had higher PxRyR expression than the susceptible strain (S). In the S strain, PxRyR expression was not related to the resistance ratio. Gene sequencing found that the RyR 4946 gene site was glycine (G) in the S, Rf, and Rh strains, and was glutamate (E) with 70% and 80% frequency in the Rb and Rz populations, respectively. The 4946 gene site was substituted by valine (V) with the frequency of 30% and 20% in Rb and Rz populations, respectively. These results increase the understanding of the mechanisms of diamide insecticide resistance in P. xylostella.

Optimizing ultrasound-assisted extraction of prodigiosin by response surface methodology.

Prodigiosin extraction from dried Serratia marcescens jx1 cells using ultrasound-assisted extraction was optimized. The experiment was carried out in accordance with a central composite design (CCD) three-level and single-variable approach. The extraction time, extraction temperature, and solute to solvent ratio with the application of ultrasonication were optimized using response surface methodology (RSM) to maximize the extraction of prodigiosin from dried S. marcescens jx1 cells. The response of prodigiosin was determined using spectrophotometry. A quadratic model was established to predict the prodigiosin extraction yield. The analysis of variance showed that the quadratic model significantly contributed to the response of prodigiosin. The optimal extraction parameters were an extraction time of 17.5 min, an extraction temperature of 23.4°C, and a solvent-to-solute ratio of 1:27.2. Under these optimum conditions, the average prodigiosin yield was 4.3 g±0.02 g from 100 g of dried cells, which matches the predicted values. The obtained optimum conditions for prodigiosin extraction provide a scientific basis for the economical large-scale production of prodigiosin.

Kinetics and computational docking studies on the inhibition of tyrosinase induced by oxymatrine.

A combination of enzymatic inhibition kinetics and computational prediction was employed to search for an effective inhibitor of tyrosinase. We found that oxymatrine significantly inhibited tyrosinase, and that this reaction was not accompanied by detectable conformational changes. Kinetic analysis showed that oxymatrine reversibly inhibited tyrosinase in a mixed-type manner. Measurements of intrinsic and ANS-binding fluorescences showed that oxymatrine did not induce any conspicuous changes in the tertiary structure. We also conducted a docking simulation between tyrosinase and oxymatrine using two docking programs, Dock6.3 and AutoDock4.2 (binding energy was -118.81 kcal/mol for Dock6 and -8.04 kcal/mol for AutoDock4). The results also suggested that oxymatrine interacts mostly with the residues of CYS83 and HIS263 in the active site of tyrosinase. This strategy of predicting tyrosinase inhibition by simulation of docking coupling with kinetics may prove useful in screening for potential tyrosinase inhibitors. Knowledge of tyrosinase inhibition can provide medical, cosmetic, and agricultural applications. Our study suggests that oxymatrine is an important agent for various applications related to pigment formation.

Mechanism-based pharmacokinetic-pharmacodynamic modeling of the estrogen-like effect of ginsenoside Rb1 on neural 5-HT in ovariectomized mice.

We sought to develop a mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) model to characterize the effects of ginsenoside Rb1 (Rb1) and estradiol (E(2)) on neural 5-hydroxytryptamine (5-HT) concentration in ovariectomized mice. PK data of Rb1 and E(2) were obtained in plasma and brain. Brain levels of 5-HT, tryptophan (TRP), 5-hydroxytryptophan (5-HTP), and 5-hydroxyindoleacetic acid (5-HIAA) were determined after a single intravenous injection of Rb1 (20mg/kg) and E(2) (0.2mg/kg) in ovariectomized mice. The activities of tryptophan hydroxylase (TPH), aromatic amino acid decarboxylase (AAAD), and monoamine oxidase (MAO) were also evaluated. Rb1 and E(2) elevated neural 5-HT levels via TPH activation and MAO inhibition, respectively. Effects were well described by the mechanism-based PK-PD model. The net effect of increased 5-HT induced by MAO inhibition is greater than TPH activation. The increased brain levels of 5-HT induced by Rb1 and E(2) were well described by the present PK-PD model, suggesting the use and further development of this mechanism-based model for the effects of ginsenoside on brain 5-HT levels.

Beneficial estrogen-like effects of ginsenoside Rb1, an active component of Panax ginseng, on neural 5-HT disposition and behavioral tasks in ovariectomized mice.

Decreased 5-hydroxytryptamine (5-HT) concentration in the brain has been linked to central nervous system dysfunctions, especially in menopausal women. Ginsenoside Rb1, a potential phytoestrogen, has been shown to improve central nervous system dysfunctions, comparable to the estrogen treatment. To investigate the estrogen-like effects of ginsenoside Rb1 on neural 5-HT disposition and behavioral tasks, we quantified the concentrations of 5-HT and other related endogenous substances in the frontal cortex and striatum of ovariectomized mice. The activities of tryptophan hydroxylase (TPH), aromatic amino acid decarboxylase (AAAD) and monoamine oxidase (MAO) were also measured to evaluate the synthesis and metabolism of neural 5-HT. Our work shows that both ginsenoside Rb1 and estradiol increased the neural 5-HT concentration. Ginsenoside Rb1 and estradiol administration resulted in elevated TPH and depressed MAO activities, indicating that modulating the synthesis and metabolism of neural 5-HT successfully elevated 5-HT concentration. Ginsenoside Rb1 and estradiol also improved object recognition and decreased immobility time in the forced swimming test. However, a pretreatment with clomiphene (an estrogen receptor antagonist) blocked the beneficial effects of ginsenoside Rb1 and estradiol, suggesting that the estrogen-like effects of ginsenoside Rb1 were estrogen receptor-dependent.