Flavokawain B inhibits NF-κB inflammatory signaling pathway activation in inflammatory bowel disease by targeting TLR2.

Inflammatory bowel disease (IBD) is characterized by recurrent inflammatory reactions in the intestinal mucosa, including ulcerative colitis (UC) and Crohn's disease (CD). The expression of Toll-like receptor 2 (TLR2) has been observed to increase during the progression of IBD. Flavokawain B (FKB), a natural chalcone with potent anti-inflammatory activity, exerts its effects through inhibition of the NF-κB pathway. In this study, we aimed to investigate the effects and mechanisms of FKB targeting TLR2 in IBD. C57BL/6 J mice were treated with 2.5% dextran sulfate sodium (DSS) for 7 days, with administration of FKB or TLR2 inhibitor C29 starting on day 2 to establish the model of IBD. In vitro, bone marrow-derived macrophages (BMDMs) were stimulated with the TLR2 agonist Pam3CSK4 to explore the therapeutic effect of FKB and its pharmacological mechanism. Compared with the model group, the FKB-treated group showed significant reductions in colitis-related injuries in the IBD mouse model, including weight gain, increased colon length and reduced inflammation. FKB decreased the formation of TLR2-MyD88 complex by targeting TLR2, leading to suppression of downstream NF-κB signaling pathway. Similar therapeutic effects were observed in the C29-treated group. Additionally, in vitro data suggested that FKB exerted its anti-inflammatory effect by targeting TLR2 and inhibiting Pam3CSK4-induced activation of the NF-κB pathway. The anti-inflammatory effects of FKB were demonstrated through drug affinity responsive target stability assay and cellular thermal shift assay, revealing its binding affinity to TLR2. By inhibiting the activation of the TLR2/NF-κB signaling pathway, FKB effectively prevented DSS-induced IBD and exhibited promising potential as a therapeutic candidate for IBD treatment.

Deubiquitinase OTUD6A in macrophages promotes intestinal inflammation and colitis via deubiquitination of NLRP3.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract, which has been shown to increase the incidence of colorectal cancer. Recent studies have highlighted the role of ubiquitination, a post-translational modification, in the occurrence and development of colonic inflammation. Ovarian tumor deubiquitinase 6 A (OTUD6A) is a deubiquitinating enzyme, which regulates cell proliferation and tumorigenesis. In this study, we investigated the expression and role of OTUD6A in IBD. Wide-type or Otud6a-/- mice were used to develop dextran sodium sulfate (DSS)- or 2,6,4-trinitrobenzene sulfonic acid (TNBS)-induced colitis model, as well as azoxymethane (AOM)/DSS-induced colitis-associated cancer model. Bone marrow-derived macrophages (BMDMs) were isolated from wild-type and Otud6a-/- mice to dissect molecular mechanisms. Our data show that OTUD6A deficiency attenuated DSS or TNBS-induced colitis, as well as AOM/DSS-induced colitis-related colon cancer in vivo. Bone marrow transplantation experiments further revealed that OTUD6A in myeloid cells was responsible for exacerbation of DSS-induced colitis. Mechanistically, OTUD6A directly bound to NACHT domain of NLRP3 inflammasome and selectively cleaved K48-linked polyubiquitin chains from NLRP3 at K430 and K689 to enhance the stability of NLRP3, leading to increased IL-1ß level and inflammation. Taken together, our research identifies a new function of OTUD6A in the pathogenesis of colitis by promoting NLRP3 inflammasome activation, suggesting that OTUD6A could be a potential target for the treatment of IBD.

MD2 Inhibits Choroidal Neovascularization via Antagonizing TLR4/MD2 Mediated Signaling Pathway.

PURPOSE: To explore the pathological mechanism of Toll-like receptor 4 (TLR4) mediating neovascular age-related macular degeneration (nAMD) and the potential role of the TLR4 coreceptor myeloid differentiation protein 2 (MD2). METHODS: In the study, we inhibited MD2 with the chalcone derivative L2H17 and we utilized a laser-induced choroidal neovascularization (CNV) mouse model and Tert-butyl hydroperoxide (TBHP)-challenged rhesus choroid-retinal endothelial (RF/6A) cells to assess the effect of MD2 blockade on CNV. RESULTS: Inhibiting MD2 with L2H17 reduced angiogenesis in CNV mice, and significantly protected against retinal dysfunction. In retina and choroid/retinal pigment epithelium (RPE) tissues, L2H17 reduced phospho-ERK, phospho-P65 but not phospho-P38, phospho-JNK, and reduced the transcriptional levels of IL-6, TNF-α, ICAM-1 but not VCAM-1. L2H17 could protect RF/6A against TBHP-induced inflammation, oxidative stress, and apoptosis, via inhibiting the TLR4/MD2 signaling pathway and the following downstream mitogen-activated protein kinase (MAPK) and nuclear transcription factor-κB (NF-κB) activation. CONCLUSIONS: Inhibiting MD2 with L2H17 significantly reduced CNV, suppressed inflammation, and oxidative stress by antagonizing TLR4/MD2 pathway in an MD2-dependent manner. MD2 may be a potential therapeutic target and L2H17 may offer an alternative treatment strategy for nAMD.

Curcumin analog WZ26 induces ROS and cell death via inhibition of STAT3 in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive biliary epithelial tumor with limited therapeutic options and poor prognosis. Curcumin is a promising active natural compound with several anti-cancer properties, though its clinical uses remain hindered due to its poor bioavailability. We recently synthesized curcumin analogs with multifunctional pharmacological and bioactivities with enhanced bioavailability. Among these novel curcumin analogs, WZ26 is a representative molecule. However, the anti-tumor effect of WZ26 against CCA is unclear. In this study, we evaluated the anti-tumor effect of WZ26 in both CCA cells and CCA xenograft mouse model. The underlying molecular anti-cancer mechanism of WZ26 was also studied. Our results show that WZ26 significantly inhibited cell growth and induced mitochondrial apoptosis in CCA cell lines, leading to significant inhibition of tumor growth in xenograft tumor mouse model. Treatment of WZ26 increased reactive oxygen species (ROS) generation, subsequently decreased mitochondrial membrane potential and inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3), thereby inducing G2/M cell cycle arrest and cell apoptosis. Pretreatment of N-acetyl cysteine (NAC), an antioxidant agent, could fully reverse the WZ26-induced ROS-mediated changes in CCA cells. Our findings provide experimental evidence that curcumin analog WZ26 could be a potential candidate against CCA via enhancing ROS induction and inhibition of STAT3 activation.

A network-based analysis of key pharmacological pathways of Andrographis paniculata acting on Alzheimer's disease and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Andrographis paniculata (AP) is a native plant with anti-inflammatory and antioxidant properties and used as an official herbal medicine. Recently more and more researches have indicated that AP shows pharmacological effects on Alzheimer's disease (AD) but its mechanism is unclear. AIMS OF THE STUDY: Network pharmacology approach combined with experimental validation was developed to reveal the underlying molecular mechanisms of AP in treating AD. MATERIALS AND METHODS: The compounds of AP from TCM database, the AD-related targets from disease database and the targets corresponding to compounds from swissTargetPrediction were collected. Then DAVID database was used for annotation and enrichment pathways, meanwhile the compound-target, protein-protein interaction from String database and compound-target-pathway network was constructed, molecular modeling was performed using Sybyl-x. Okadaic acid (OKA)-induced cytotoxicity model in PC12 cells was established to verify the mechanism of AP and the key proteins were detected by western blotting. RESULTS: 28 AP components were identified after ADME filter analysis and 52 targets were gained via mapping predicted targets into AD-related proteins. In addition, after multiple network analysis, the 22 hub target genes were enriched onto pathways involved in AD, such as neuroactive ligand-receptor interaction, serotonergic synapse, Alzheimer's disease, PI3K-Akt and NF-kB signaling pathway. Interestingly, molecular docking simulation revealed that the targets including PTGS2, BACE1, GSK3B and IKBKB had good ability to combine with AP components. Experimental validation in an in vitro system proved that AP treatment obviously increased in levels inactive of p-GSK3ß (P < 0.05) and decreased in levels of BACE (P < 0.05), PTGS2 (namely COX2, P < 0.05) and NF-kB protein (P < 0.05) compare with OKA treated group. CONCLUSION: Our data provided convincing evidence that the neuroprotective effects of AP might be partially related to their regulation of the APP-BACE1-GSK3B signal axis and inflammation, which should be the focus of study in this field in the future.

Pseudo-allergic reaction caused by Qingkailing injection partially via the PI3K-Rac1 signaling pathway in RBL-2H3 cells.

Qingkailing injection (QKLI) is a kind of multi-component traditional Chinese medicine injection. It has been widely used in clinical practice, but in recent years, it has caused more and more adverse reactions, mainly manifested as pseudo-allergic symptoms. To explore the potential mechanism of the pseudo-allergic reaction by QKLI, basophilic leukemia cell line 2H3 (RBL-2H3) was chosen. The results showed that QKLI at doses of 5, 10 and 20 mL L-1 activated phosphoinositide 3-kinase (PI3K) activity and also increased the levels of Ras-related C3 botulinum toxin substrate 1 (Rac1), p21 protein-activated kinase 1 (Pak1), LIM kinase (Limk1) and cofilin (an actin polymerization regulator) proteins. What's more, QKLI aggravated the depolymerization of F-actin. NSC23766, a Rac1 inhibitor, reversed the previous results in QKLI-treated RBL-2H3 cells. In addition, when the Rac1 gene was knocked down using lentiviral vector-loaded shRNA in RBL-2H3 cells, the PI3K activity and depolymerization of F-actin were downregulated, hinting that the pseudo-allergic reaction was significantly reduced. In general, the pseudo-allergic reaction induced by QKLI was likely to be based on PI3K-Rac1 signaling pathways partially.

Andrographolide Protects PC12 Cells Against ß-Amyloid-Induced Autophagy-Associated Cell Death Through Activation of the Nrf2-Mediated p62 Signaling Pathway.

Recent studies mentioned that Andrographolide (Andro), the main bioactive component of traditional Chinese medicine Andrographis paniculata, may be a potential natural product for treating Alzheimer's disease, but the underlining mechanism remains to be discovered. In this study, we investigated whether Andro regulates the nuclear factor E2-related factor 2 (Nrf2)/Sequestosome 1 (p62) signaling pathway and activates autophagy to protect neuronal PC12 cells from the toxicity of the ß-amyloid (Aß) peptide. Our results revealed that Andro protected and rescued PC12 cells from Aß1⁻42-induced cell death and restored abnormal changes in nuclear morphology, lactate dehydrogenase, malondialdehyde, intracellular reactive oxygen species, and mitochondrial membrane potential. RT-PCR and Western blotting analysis demonstrated that Andro activated autophagy-related genes and proteins (Beclin-1 and LC3); meanwhile, it also augmented the Nrf2 and p62 expression in mRNA and protein levels, and reduced p-tau and p21 protein expression in Aß1⁻42-stimulated cells. Then, further study showed that the pre-transfection of cells with Nrf2 small interfering RNA (siRNA) resulted in the downregulation of p62, Beclin-1, and LC3 proteins expression, as well as the upregulation of p21. Furthermore, the pre-transfection of cells with p62 siRNA didn't block the Nrf2 protein expression, accompanying with an elevated p21. Taken together, these results showed that Andro significantly ameliorated cell death due to Aß1⁻42 insult through the activation of autophagy and the Nrf2-mediated p62 signaling pathway.

Silencing Op18/stathmin by RNA Interference Promotes the Sensitivity of Nasopharyngeal Carcinoma Cells to Taxol and High-Grade Differentiation of Xenografted Tumours in Nude Mice.

Nasopharyngeal carcinoma (NPC) is a refractory tumour, and chemotherapy is one of the primary treatment modalities. Oncoprotein 18 (Op18)/stathmin is a conserved small cytosolic phosphoprotein and highly expressed in tumours, which plays a vital role in maintaining the malignant phenotype of tumours. Taxol is a clinically widely used chemotherapeutic agent for a broad range of taxol-resistant tumours. This study showed that Op18/stathmin silencing by RNA interference (RNAi) combined taxol cooperatively improved cellular apoptosis in CNE1 cells mainly via initiating endogenous death receptor pathway, impaired the capabilities of cellular proliferation and cellular migration and down-regulated the half maximal inhibitory concentration (IC50 ) of taxol, meanwhile decreased the expression of the upstream extracellular regulated kinase 1 (ERK1) in vitro. Evidence also showed that taxol cytotoxicity was markedly augmented for Op18/stathmin RNAi in other NPC cells. In vivo animal experiments have demonstrated that early combination of Op18/stathmin silencing and taxol evidently inhibited tumourigenicity of CNE1 cells and growth of xenografted tumours in nude mice. Remarkably, silencing Op18/stathmin by RNAi still promoted transformation of late-stage CNE1 cells in NPC-xenografted tumours from moderately to highly differentiated and inhibited the pleiotropic cytokine interleukin-10 (IL-10) autocrine by transplanted tumours. These findings suggest that silencing Op18/stathmin by RNAi promotes chemosensitization of NPC to taxol and reverses malignant phenotypes of NPC, which provides a new clue for treating drug-resistant tumours.

Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps.

BACKGROUND: Neovascularization occurs through two mechanisms: angiogenesis and vasculogenesis. Therefore, there are two strategies to promote neovascularization: therapeutic angiogenesis and therapeutic vasculogenesis (endothelial progenitor cells therapy). MATERIALS AND METHODS: In this study, we examined whether or not endothelial progenitor cells combined with vascular endothelial growth factor (VEGF) gene therapy is useful for ischemia surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted endothelial progenitor cells (EPCs) transducted with VEGF165 gene and EPCs alone. EPCs were isolated from cord blood of healthy human volunteers, cultured in vitro for 7 days and identified by immunofluorescence. After transduced with VEGF165 gene in vitro, proliferative activity of EPCs was assessed using MTT assay. CM-DiI was used to trace EPCs in vivo 4 days after injection of 5 x 10(5) VEGF-transduced EPCs(VEGF-transduced EPCs group, n = 10), 5 x 10(5) EPCs (non-transduced EPCs group, n = 10) in 500 microL EBM-2 media, or 500 microL EBM-2 media (EBM-2 media group, n = 10) local, a cranially based flap was elevated on the back of nude mice. The percent flap survival, neovasculariztion and blood flow recovery of flaps was detected. RESULTS: EPCs expressed cell markers CD34, KDR, and CD133. A statistically significant increase in percent flap survival was observed in mice of VEGF-transduced EPCs group as compared with that of non-transduced EPCs group: 67.99 +/- 6.64% versus 59.43 +/- 4.69% (P < 0.01), and 41.24 +/- 2.44% in EBM-2 media group (P < 0.01). The capillary density and blood flow recovery of flaps in VEGF-transduced EPCs group were both improved. CM-DiI-labeled VEGF-transduced EPCs were observed in vivo and the numbers of cells increased. CONCLUSION: EPCs from human cord blood can increased neovascularization of ischemic flaps and augmented the survival areas, and VEGF-transduced EPCs have more powerful ability of promoting neovascularization in animal model of ischemic flaps.