Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy.

Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by Kelch-like ECH-associated protein 1 (Keap1) alkylation plays a central role in anti-inflammatory therapy. However, activators of Nrf2 through alkylation of Keap1-Kelch domain have not been identified. Deoxynyboquinone (DNQ) is a natural small molecule discovered from marine actinomycetes. The current study was designed to investigate the anti-inflammatory effects and molecular mechanisms of DNQ via alkylation of Keap1. DNQ exhibited significant anti-inflammatory properties both in vitro and in vivo. The pharmacophore responsible for the anti-inflammatory properties of DNQ was determined to be the α, ß-unsaturated amides moieties by a chemical reaction between DNQ and N-acetylcysteine. DNQ exerted anti-inflammatory effects through activation of Nrf2/ARE pathway. Keap1 was demonstrated to be the direct target of DNQ and bound with DNQ through conjugate addition reaction involving alkylation. The specific alkylation site of DNQ on Keap1 for Nrf2 activation was elucidated with a synthesized probe in conjunction with liquid chromatography-tandem mass spectrometry. DNQ triggered the ubiquitination and subsequent degradation of Keap1 by alkylation of the cysteine residue 489 (Cys489) on Keap1-Kelch domain, ultimately enabling the activation of Nrf2. Our findings revealed that DNQ exhibited potent anti-inflammatory capacity through α, ß-unsaturated amides moieties active group which specifically activated Nrf2 signal pathway via alkylation/ubiquitination of Keap1-Kelch domain, suggesting the potential values of targeting Cys489 on Keap1-Kelch domain by DNQ-like small molecules in inflammatory therapies.

Leocarpinolide B Attenuates Collagen Type II-Induced Arthritis by Inhibiting DNA Binding Activity of NF-κB.

Rheumatoid arthritis (RA) is a chronic autoimmune disease triggered by a cascading inflammatory response. Sigesbeckia Herba (SH) has long been utilized as a traditional remedy to alleviate symptoms associated with rheumatism. Our previous study found that leocarpinolide B (LB), a sesquiterpene lactone isolated from the whole plant of SH, possesses potent a anti-inflammatory effect on macrophages. This study was designed to evaluate the therapeutic effects of LB on RA, and further investigate the underlying mechanisms. In collagen type II-induced arthritic mice, LB was demonstrated to decrease the production of autoimmune antibodies in serum and inflammatory cytokines in the joint muscles and recover the decreased regulatory T lymphocytes in spleen. Moreover, LB significantly suppressed the inflammatory infiltration, formation of pannus and bone erosion in the paw joints. In vitro testing showed that LB inhibited the proliferation, migration, invasion, and secretion of inflammatory cytokines in IL-1ß-induced human synovial SW982 cells. Network pharmacology and molecular docking suggested NF-κB p65 could be the potential target of LB on RA treatment, subsequent experimental investigation confirmed that LB directly interacted with NF-κB p65 and reduced the DNA binding activity of NF-κB in synovial cells. In conclusion, LB significantly attenuated the collagen type II-induced arthritis, which was at least involved in the inhibition of DNA binding activity of NF-κB through a direct binding to NF-κB p65. These findings suggest that LB could be a valuable lead compound for developing anti-RA drugs.

The "whole ingredients extract" of Astragali Radix improves the symptoms of dextran sulfate sodium-induced ulcerative colitis in mice through systemic immunomodulation.

BACKGROUND: Ulcerative colitis (UC) is a common inflammatory intestinal disease. Astragali Radix (AR) is one of the traditional Chinese medicines used in clinic for UC treatment. In our previous study, the whole ingredient extract (WIE) from AR have been proved to possess better immunomodulatory effects on immunosuppressed mice compared with the conventional water extraction (WAE). In the present study, we further evaluated the therapeutic effects of WIE against dextran sodium sulfate (DSS)-induced UC in mice through systemic immune regulation. METHODS: Gradient solvent extraction has been used to prepare the WIE of AR. The HPLC-MS analysis approach has been employed to analyze and compare the chemical differences between WAE and WIE. UC model was reproduced in 3% DSS-induced C57BL/6 mice for 6 days. Flow cytometric analysis for splenic lymphocyte subset. ELISA kits were used to determine the cytokines in the serum and colon tissues. The histopathological characteristics of colon were evaluated by hematoxylin-eosin staining and immunohistochemistry. RESULTS: The chemical compositions and the contents of main active ingredients were more abundant and higher in WIE than those in WAE. The WIE treatment altered a better action on reducing colitis disease activity index (DAI) and histological scores, as well as the recovered body weight and increased colon length in mice compared to the WAE group. Additionally, WIE showed better effects in recovering the levels of peripheral white blood cells in blood and cytokines (IL-2, IL-6 and MCP-1) in serum or colon tissues, improving the percentage of CD3+ and the ratio of CD4+/CD8+ in the spleen, and inhibiting the spleen enlargement in DSS-induced UC mice. CONCLUSIONS: WIE has a more complete chemical composition than WAE. Meanwhile, WIE possesses better therapeutic effects on UC through resuming dysfunctional immunity in mice.

Anti-inflammatory/anti-oxidant properties and the UPLC-QTOF/MS-based metabolomics discrimination of three yellow camellia species.

The species of Camellia nitidissima Chi (CC) and C. euphlebia Merr. ex Sealy (CE) are two most important plant sources for commercialized herbal tea (Jinhuacha) worldwide. However, some other species of camellia genus are also sold as alternatives in market due to the great commercial value. In this study, the similarity and difference of CC and CE as well as C.insularis (CI) are comprehensively compared both in chemistry and pharmacology. Based on the ultraperformance liquid chromatography coupled with a hybrid quadrupole orthogonal time-of-flight mass spectrometer(UPLC-QTOF-MS) analysis, a sequential-optimization based new statistical model has been developed by combining the untargeted metabolomics and fingerprint analyses, and successfully applied for chemical pattern recognition and discrimination of three yellow camellias species. The results indicated that CC, CE and CI could be well discriminated with the optimized chemical combination including quercetin-3-O-rhamnoside (C2), okicamelliaside (C4), Kaempferol 7-O-rhamnoside (C6), Corymboside (C9), asiatic acid-glc-rha-xyl (C11) and 3'-methy-4'-glucoside-ellagic acid (C14). Moreover, the 30 % ethanolic extracts of yellow camellias species presented the optimal activities on anti-inflammation/anti-oxidation in LPS-stimulated Raw264.7 macrophages dose-dependently. The averaged 50 % inhibitory concentrations (IC50) on NO production were 754.68 ± 50.96, 1182.39 ± 22.10, 1527.83 ± 106.24 µg(herb)/mL, and ROS production were 311.70 ± 26.57, 332.64 ± 25.46, 917.60 ± 41.36 µg(herb)/mL for CC, CE and CI, respectively. The results indicated a certain similarity of CC and CE, as well as their significant difference from CI.

Delivery of therapeutic small interfering RNA: The current patent-based landscape.

Implementing small interfering RNA (siRNA) is a promising therapy because it silences disease-related genes theoretically. However, the efficient delivery of siRNA is challenging, which limits its therapeutic applications. Various pharmaceutical delivery systems containing key technologies have been developed and patented, which are of great concern to developers in the field. Despite numerous studies devoted to siRNA-delivery technologies, few researchers have systematically examined relevant patents. Patents, as bridges connecting academic progress with applicable innovation, encapsulate cumulative technological innovations and provide valuable information for academic research and commercial development. This study aims to analyze advances in therapeutic siRNA delivery technology from a patent perspective. A total of 11,509 patent documents from 3,309 patent families were collected, classified into 10 technological categories, and comprehensively analyzed. An overall patent landscape of siRNA delivery was presented from the temporal, spatial, organizational, and technological dimensions. This work is expected to help researchers and developers in the field of siRNA delivery form a basis for decision-making by combining our findings with supplementary data.

Deciphering the Pharmacological Mechanisms of the Huayu-Qiangshen-Tongbi Formula Through Integrating Network Pharmacology and In Vitro Pharmacological Investigation.

Rheumatoid arthritis is a chronic inflammatory autoimmune disease, causing articular and extra-articular dysfunctions among patients, and it could result in irreversible joint damages or disability if untreated. A traditional Chinese medicine formula, Huayu-Qiangshen-Tongbi (HT) formula, has been observed successful in controlling rheumatoid arthritis progression in traditional Chinese medicine clinics. In this study, we conducted a systematic analysis of the HT formula with a purpose of proposing for its potential mechanism of action using network pharmacological methods. The potential targets of the formula were collected and screened according to the topological features of their protein-protein interaction network, and we subsequently validated our prediction results through in vitro experiments. We proposed that the HT formula could interfere with the bone metabolism and the inflammatory pathways of the body. The experimental validation results indicated that HT formula could exhibit anti-inflammatory effects by regulating several signaling pathways specifically the Toll-like receptor signaling pathway, phosphoinositide-3-kinase-Akt signaling pathway, hypoxia-inducible factor 1 signaling pathway, mitogen-activated protein kinase signaling pathway and activator protein 1 signaling pathway.

Self-assembled liposome from multi-layered fibrous mucoadhesive membrane for buccal delivery of drugs having high first-pass metabolism.

A novel delivery system based on self-assembled liposome from multi-layered fibrous mucoadhesive membrane has been developed to improve the bioavailability of Carvedilol (Car). This system consisted of an electrospun layer (enable self-assembly of liposome once contacting with water), an adhesive layer (prolong the retention period in the mouth) and a backing layer. SEM, DSC and FTIR were applied to characterize the fiber. The TEM and fluorescence study demonstrated the formation of self-assembled liposome when electrospun fiber encountered water. The ratio of PC to Car and the molecular weight of PVP both had a significant impact on the drug encapsulation efficiency. In vitro and in vivo adhesive tests were conducted to evaluate the bioadhesive performance of the adhesive layer. The dialysis dissolution and permeation study through porcine buccal mucosa were carried out. The electrospun fiber showed excellent drug permeation amount compared to pure Car. The drug concentration-time curves, in rabbits, of fibrous mucoadhesive membrane and Car suspension were different, and possible reasons were analyzed. The pharmacokinetic study demonstrated 154% increase in the relative bioavailability compared to Car suspension. This drug delivery system offered a novel platform for potential buccal delivery of drugs with high first-pass effect.

Simultaneous enantioselective determination of six pesticides in aqueous environmental samples by chiral liquid chromatography with tandem mass spectrometry.

A robust and sensitive method was developed for the enantiomeric analysis of six chiral pesticides (including metalaxyl, epoxiconazole, myclobutanil, hexaconazole, napropamide, and isocarbophos) in aquatic environmental samples. The optimized chromatographic conditions for the quantification of all the 12 enantiomers were performed with Chiralcel OD-RH column using mobile phase consisting of 0.1% aqueous formic acid and acetonitrile operated under reversed-phase conditions and then analyzed using liquid chromatography with tandem mass spectrometry. Twelve enantiomers were detected in multiple reaction monitoring mode. Solid-phase extraction and dispersive liquid-liquid microextraction were employed in this study. Response surface methodology was applied to assist in the dispersive liquid-liquid microextraction optimization. Under the optimum conditions, recoveries of pesticides enantiomers varied from 83.0 to 103.2% at two spiked levels with relative standard deviation less than 11.5%. The concentration factors were up to 1000 times. Method detection and quantification limits varied from 0.11 to 0.48 ng/L and from 0.46 to 1.49 ng/L, respectively. Finally, this method was used to determination of the enantiomers composition of the six pesticides in environmental aqueous matrices, which will help better understand the behavior of individual enantiomer and make accurate risk assessment to ecosystems.