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.

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.

Sigesbeckia orientalis L. Derived Active Fraction Ameliorates Perioperative Neurocognitive Disorders Through Alleviating Hippocampal Neuroinflammation.

Neuroinflammation is closely related to the pathogenesis of perioperative neurocognitive disorders (PNDs), which is characterized by the activation of microglia, inflammatory pathways and the release of inflammatory mediators. Sigesbeckia orientalis L. (SO) is a traditional Chinese medicine which demonstrates anti-inflammatory activities in different models. In this study, we aim to isolate the active fraction from the extract of SO with higher anti-inflammatory potential and confirm if the selected fraction exerts neuroprotection against the development of PND in an animal model. Moreover, the components in the selected fraction would be determined by UPLC-PDA analysis. Three fractions were prepared by column chromatography packed with three different macroporous resins. Anti-inflammatory activities of prepared fractions were accessed in microglial BV2 cultures by nitric oxide release, gene expression of inflammatory cytokines and activation of inflammatory JNK and NF-kB pathway molecules. Our results demonstrated that the fraction prepared from D101 macroporous resin (D101 fraction) exhibited a more potent anti-neuroinflammatory effect. The neuroprotective effect of D101 fraction was further examined in postoperative mice. Our results showed that surgery-induced cognitive dysfunction was attenuated by the D101 fraction treatment. This fraction also reduced microglial activation, inflammatory cytokines and inhibiting JNK and NF-kB pathway molecules in the hippocampus. In addition, surgery induced dendritic spine loss while D101 fraction ameliorated the spine loss in the hippocampus. For safety concerns, anti-thrombotic effect was examined by tail bleeding assay and no significant change of the bleeding pattern was found. UPLC-PDA analysis indicated that flavonoids (rutin, isochlorogenic acid A, isochlorogenic acid C) and terpenoid (darutoside) were the most important components in the D101 fraction. Our results support a therapeutic, as well as the translational potential for D101 fraction in ameliorating postoperative neuroinflammation and subsequent PND in the clinical setting without increasing bleeding tendencies.

Brij-functionalized chitosan nanocarrier system enhances the intestinal permeability of P-glycoprotein substrate-like drugs.

The highly expressed P-glycoprotein (Pgp) in the intestine plays a key role in preventing drugs across the intestinal epithelium, which linked by tight junctions (TJs). Thus increasing the oral bioavailability of Pgp substrate-like drugs (PSLDs) remains a great challenge. Herein, we construct a nanocarrier system derived from Brij-grafted-chitosan (BC) to enhance the oral bioavailability and therapeutic effect of berberine (BBR, a typical PLSD) against diabetic kidney disease. The developed BC nanoparticles (BC-NPs) are demonstrated to improve the intestinal permeability of BBR via transiently and reversibly modulating the intercellular TJs (paracellular pathway) and Pgp-mediated drug efflux (transcellular pathway). As compared to free BBR and chitosan nanoparticles, the BC-NPs enhanced the relative oral bioavailability of BBR in rats (4.4- and 2.7-fold, respectively), and the therapeutic potency of BBR in renal function and histopathology. In summary, such strategy may provide an effective nanocarrier system for oral delivery of BBR and PSLDs.

Sigesbeckia orientalis L. Extract Alleviated the Collagen Type II-Induced Arthritis Through Inhibiting Multi-Target-Mediated Synovial Hyperplasia and Inflammation.

Excessive proliferation and inflammation of synovial fibroblasts accelerate and decorate the pathological process of rheumatoid arthritis (RA). Sigesbeckia orientalis L. (SO) is one of the main plant sources for Sigesbeckiae Herba (SH) which has been used traditionally in treating various forms of arthritis and rheumatic pain. However, the anti-arthritic mechanisms of SO are still not clearly understood. In this study, we investigated the therapeutic effects and the underlying mechanisms of SO against collagen type II (C II)-induced RA in rats as well as the interleukin (IL)-1ß-induced human synovial SW982 and MH7A cells. For the in vivo studies, thirty-six Wistar male rats were randomly arranged to six groups based on the body weight, and then C II-induced to RA model for 15 days, followed by treatment with the 50% ethanolic extract of SO (SOE, 0.16, 0.78, and 1.56 g/kg) for 35 days. The results suggested that SOE significantly inhibited the formation of pannus (synovial hyperplasia to the articular cavity) and attenuated the cartilage damaging and bone erosion in the CIA-induced rats' hind paw joints. Moreover, SOE decreased the production of C-reactive protein (CRP) in the serum and the expression of IL-6 and IL-1ß in the joint muscles, as well as recovered the decreased regulatory T lymphocytes. The results obtained from the in vitro studies showed that SOE (50, 100, and 200 µg/ml) not only inhibited the proliferation, migration, and invasion of human synovial SW982 cells but also decreased the IL-1ß-induced expression of IL-6 and IL-8 both in SW982 and MH7A cells. Besides, SOE reduced the expression of COX-2, NLRP3, and MMP9, and increased the expression of MMP2 in the IL-1ß-induced SW982 cells. Furthermore, SOE blocked the activation of NF-κB and reduced the phosphorylation of MAPKs and the expression of AP-1. In conclusion, SOE attenuated the C II-induced RA through inhibiting of MAPKs/NF-κB/AP-1-mediated synovial hyperplasia and inflammation.

Sigesbeckia glabrescens Makino extract attenuated the collagen-induced arthritis through inhibiting the synovial hyperplasia and inflammation.

BACKGROUND: Sigesbeckia glabrescens Makino (SG) has been traditionally used for rheumatism and joint protection. However, the anti-arthritic effects and underling mechanisms of SG have not been demonstrated. In this study, we investigated the anti-arthritic effects and mechanisms of SG extract (SGE) on collagen-induced arthritic rats and interleukin (IL)-1ß-stimulated human synovial SW982 cells. METHODS: Rats were induced to arthritis by collagen for 15 days and then received SGE treatment for 35 days. The body weight and arthritis severity score of the rats were monitored weekly. At the end of the experiment, the radiographic and histological changes of rats' hind paw were obtained; the serum C-reactive protein was detected by enzyme-linked immunosorbent assay (ELISA); the expression levels of interleukin (IL)- 1ß, IL6 and IL-10 in the joint muscles were determined by ELISA and immunohistochemical staining; and the level of regulatory T cells (Tregs) in the spleen was detected using flow cytometry. In addition, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and scratch wound healing assay were used to evaluate the proliferation of SW982 synovial cells. ELISA, western blot and immunofluorescence staining were used to investigate the anti-inflammatory mechanisms of SGE on IL-1ß-induced SW982 cells and joint muscles of CIA rats. RESULTS: SGE attenuated the collagen-induced hind paw swelling, cartilage damage and bone erosion. SGE inhibited the synovial hyperplasia to the articular cavity in the toe joint and ankle. Moreover, SGE decreased the production of C-reactive protein in serum and the expression of IL-6, IL-1ß, cyclooxygenase-2 (COX-2) and phosphorylation of NF-κB p65 in the joint muscles. SGE also recovered the decreased Tregs. Results from the in vitro experiments showed that SGE not only inhibited the proliferation and migration of human synovial cell but also inhibited the IL-1ß-induced expression of IL-6 and IL-8. Similarly, SGE inhibited the activation of NF-κB and the expression of COX-2. CONCLUSIONS: SGE attenuated the collagen-induced arthritis through inhibiting the synovial hyperplasia and inflammation.

Bu-Shen-Fang-Chuan formula attenuates cigarette smoke-induced inflammation by modulating the PI3K/Akt-Nrf2 and NF-κB signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic obstructive pulmonary disease (COPD) is a respiratory inflammatory disease. Unlike asthma, COPD is insensitive to glucocorticoid treatment; thus, it is of great importance to find alternative medications, including Chinese medicine, to suppress inflammation. Bu-Shen-Fang-Chuan formula (BSFCF) is commonly used for the treatment of COPD in China. However, the mechanisms of BSFCF in COPD treatment are still unclear. AIM OF THE STUDY: To verify the anti-inflammatory efficacy of BSFCF in COPD and to explore the possible mechanisms underlying its anti-inflammatory efficacy based on the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)-Nuclear factor erythroid 2-related factor 2 (Nrf2) and Nuclear factor (NF)-κB signalling pathways. MATERIALS AND METHODS: A rat model of COPD was established by chronic exposure to cigarette smoke (CS) for 6 months. Bronchoalveolar lavage fluid (BALF) and blood were obtained to detect inflammatory cytokines. Lung samples were harvested, and part of each sample was fixed for subsequent H&E staining and immunohistochemical (IHC) analysis. The remaining lung tissues were used for RNA sequencing analysis and western blotting. RESULTS: BSFCF significantly reduced inflammatory infiltration in the lungs of CS-exposed rats and decreased the concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in both the BALF and serum. Additionally, BSFCF evidently attenuated NF-κB activation and downregulation of glucocorticoid receptor (GR) caused by CS. Furthermore, BSFCF increased the activation of PI3K/Akt-Nrf2 signalling in response to CS. CONCLUSIONS: BSFCF attenuated CS-induced inflammation in COPD, which was partially achieved through the PI3K/Akt-Nrf2 and NF-κB signalling pathways.

Comprehensive comparison on the anti-inflammatory effects of three species of Sigesbeckia plants based on NF-κB and MAPKs signal pathways in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Sigesbeckiae Herba (SH), a traditional anti-inflammatory Chinese herbal medicine, is originated from the plants of Sigesbeckia pubescens Makino (SP), S. orientalis L. (SO) and S. glabrescens Makino (SG). The current studies reported that the chemical constituents in the three species of plants were different. AIM OF THE STUDY: The aim of this study is to provide a systemic comparison on the anti-inflammatory effects and the underlying molecular mechanisms among the three plants based on their effects on nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signal pathways in vitro. MATERIAL AND METHODS: Twenty-four batches of three Sigesbeckia herbs were collected from different regions of China and extracted with 50% ethanol. The distribution of 6 compounds in the 24 batches of SH extracts were characterized by UPLC analysis. The cytotoxicity of all extracts to RAW264.7 cells in the absence or presence of lipopolysaccharide (LPS) were examined by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The anti-inflammatory effects of the extracts were investigated using Griess reagent and enzyme-linked immunosorbent assay. The underlying mechanisms of the representative samples (SP007, SO005 and SG003) for individual species were examined by western blotting and immunofluorescence staining. RESULTS: The estimated average sub-lethal dose (LD15) of SP, SO and SG on RAW264.7 cells were 181.7 ± 15.7, 291.5 ± 33.9 and 317.1 ± 16.3 µg/mL, respectively. In LPS-stimulated RAW264.7 cells, the inhibitory effects of SH species were determined to be SP > SO > SG on NO release, while SP ~ SO > SG on secretion of post-inflammatory cytokines (TNF-α, IL-6 and MCP-1). Moreover, suppression on LPS-induced excessive expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as the activation of NF-κB and phosphorylation of MAPKs were investigated to be associated to the anti-inflammatory effects for all SH species. CONCLUSIONS: We firstly reported a systemic comparison on the anti-inflammatory properties for the three main plant origins of SH. Although SG showed lower toxicity and less anti-inflammatory effects compared with SP and SO in LPS-induced RAW264.7 cells, comparable inhibitory effects on NF-κB and MAPKs pathways and the reduction of LPS-induced iNOS and COX-2 were observed in the anti-inflammatory process for all Sigesbeckia plants.

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.