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.

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.

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.