Co-Culture of Bone Marrow Mesenchymal Stem Cell (BMSC) and Osteoclasts Regulates Cell Differentiation and Alleviates Osteoporosis by Up-Regulating miR-211

Bone marrow mesenchymal stem cells (BMSCs) can release a large amount of exosomes (EXO) during bone remodeling by osteoclasts. EXO contains miRNA-211, which has a variety of biological effects. However, little is known about whether miR-211 from BMSC-EXO affects the surrounding cells. Therefore, we aim to study the role of miRNA-211 derived from BMSC-EXO in regulating osteoclasts differentiation. Macrophage colony stimulating factor (M-CSF) and nuclear factor kappa B receptor activator (RANKL) were used to stimulate bone marrow macrophages (BMM) to obtain osteoclasts, which were treated with BMSC-EXO or LPS followed by analysis of osteoclast-related genes expression by PCR, ROS release by flow cytometry, actin ring formation by immunofluorescence, and osteoclast differentiation by anti-tartrate acid phosphatase (TRAP) staining. Finally, an in vivo experiment was conducted to verify BMSC-EXO’s effect on osteoporosis. BMSC-EXO significantly inhibited RNAKL-induced osteoclast differentiation of BMMs. During osteoclasts formation, BMSC-EXO inhibited ROS production induced by RANKL and the subsequent activation of NF-κB signaling pathway induced by ROS. In addition, BMSC-EXO significantly down-regulated the osteoclast genes including nuclear factor, cytoplasmic 1 (NFATc1), C-fos, tartrate-resistant acid phosphatase (TRAP) and osteoclast-associated immunoglobulin-like receptor (OSCAR) in activated T cells. BMSC-EXO inhibited ROS release by promoting miR-211 expression, thereby inhibiting the NF-κB signaling and ultimately participating in osteoclasts differentiation. In LPS-induced mouse osteoporosis models, BMSC-EXO inhibited LPS-induced bone loss and exerted a protective effect. In conclusion, microRNA-211 derived from BMSC-EXO can regulate osteoclasts differentiation, suggesting that it might be used as a potential approach for treating osteoporosis.

A Nitrobenzoyl Sesquiterpenoid Insulicolide A Prevents Osteoclast Formation via Suppressing c-Fos-NFATc1 Signaling Pathway

It is a viable strategy to inhibit osteoclast differentiation for the treatment of osteolytic diseases such as osteoporosis, rheumatoid arthritis and tumor bone metastases. Here we assessed the effects of insulicolide A, a natural nitrobenzoyl sesquiterpenoid derived from marine fungus, on receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and its protective effects on LPS-induced osteolysis mice model in vivo. The results demonstrated that insulicolide A inhibited osteoclastogenesis from 1 μM in vitro. Insulicolide A could prevent c-Fos and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) nuclear translocation and attenuate the expression levels of osteoclast-related genes and DC-STAMP during RANKL-stimulated osteoclastogenesis but have no effects on NF-κB and MAPKs. Insulicolide A can also protect the mice from LPS-induced osteolysis. Our research provides the first evidence that insulicolide A may inhibit osteoclastogenesis both in vitro and in vivo, and indicates that it may have potential for the treatment of osteoclast-related diseases.

IKKα plays a major role in canonical NF-kB signalling in colorectal cells

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.

Astaxanthin as a Modulator of Nrf2, NF-κB, and Their Crosstalk: Molecular Mechanisms and Possible Clinical Applications

Astaxanthin (AST) is a dietary xanthophyll predominantly found in marine organisms and seafood. Due to its unique molecular features, AST has an excellent antioxidant activity with a wide range of applications in the nutraceutical and pharmaceutical industries. In the past decade, mounting evidence has suggested a protective role for AST against a wide range of diseases where oxidative stress and inflammation participate in a self-perpetuating cycle. Here, we review the underlying molecular mechanisms by which AST regulates two relevant redox-sensitive transcription factors, such as nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor κB (NF-κB). Nrf2 is a cellular sensor of electrophilic stress that coordinates the expression of a battery of defensive genes encoding antioxidant proteins and detoxifying enzymes. Likewise, NF-κB acts as a mediator of cellular stress and induces the expression of various pro-inflammatory genes, including those encoding cytokines, chemokines, and adhesion molecules. The effects of AST on the crosstalk between these transcription factors have also been discussed. Besides this, we summarize the current clinical studies elucidating how AST may alleviate the etiopathogenesis of oxidative stress and inflammation.

Nuclear Factor κB-COX2 Pathway Activation in Non-myelinating Schwann Cells Is Necessary for the Maintenance of Neuropathic Pain in vivo

Chronic neuropathic pain leads to long-term changes in the sensitivity of both peripheral and central nociceptive neurons. Glial fibrillary acidic protein (GFAP)-positive glial cells are closely associated with the nociceptive neurons including astrocytes in the central nervous system (CNS), satellite glial cells (SGCs) in the sensory ganglia, and non-myelinating Schwann cells (NMSCs) in the peripheral nerves. Central and peripheral GFAP-positive cells are involved in the maintenance of chronic pain through a host of inflammatory cytokines, many of which are under control of the transcription factor nuclear factor κB (NFκB) and the enzyme cyclooxygenase 2 (COX2). To test the hypothesis that inhibiting GFAP-positive glial signaling alleviates chronic pain, we used (1) a conditional knockout (cKO) mouse expressing Cre recombinase under the hGFAP promoter and a floxed COX2 gene to inactivate the COX2 gene specifically in GFAP-positive cells; and (2) a tet-Off tetracycline transactivator system to suppress NFκB activation in GFAP-positive cells. We found that neuropathic pain behavior following spared nerve injury (SNI) significantly decreased in COX2 cKO mice as well as in mice with decreased glial NFκB signaling. Additionally, experiments were performed to determine whether central or peripheral glial NFκB signaling contributes to the maintenance of chronic pain behavior following nerve injury. Oxytetracycline (Oxy), a blood-brain barrier impermeable analog of doxycycline was employed to restrict transgene expression to CNS glia only, leaving peripheral glial signaling intact. Signaling inactivation in central GFAP-positive glia alone failed to exhibit the same analgesic effects as previously observed in animals with both central and peripheral glial signaling inhibition. These data suggest that the NFκB-COX2 signaling pathway in NMSCs is necessary for the maintenance of neuropathic pain in vivo.

Oral streptococci subvert the host innate immune response through hydrogen peroxide

In periodontal health, oral streptococci constitute up to 80% of the plaque biofilm. Yet, destructive inflammatory events of the periodontium are rare. This observation suggests that oral streptococci may possess mechanisms to co-exist with the host. However, the mechanisms employed by oral streptococci to modulate the innate immune response have not been well studied. One of the key virulence factors produced by oral streptococci is hydrogen peroxide (H2O2). In mammalian cells, H2O2 triggers the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key pathway mediating antioxidant defence. This study aimed to determine (1) if H2O2 producing oral streptococci activated the Nrf2 pathway in macrophages, and (2) if the activation of Nrf2 influenced the innate immune response. We found that oral streptococci downregulated the innate immune response in a H2O2 dependent manner through the activation of the Nrf2. The activation of the Nrf2 signalling pathway led to the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB), the key transcription factor regulating pro-inflammatory response. This study showed for the first time that oral streptococci are unlikely passive bystanders but could play an active role in the maintenance of periodontal health by preventing overt inflammation.

Nrf2 in the Field of Dentistry with Special Attention to NLRP3

The aim of this review article was to summarize the functional implications of the nuclear factor E2-related factor or nuclear factor (erythroid-derived 2)-like 2 (Nrf2), with special attention to the NACHT (nucleotide-binding oligomerization), LRR (leucine-rich repeat), and PYD (pyrin domain) domains-containing protein 3 (NLRP3) inflammasome in the field of dentistry. NLRP3 plays a crucial role in the progression of inflammatory and adaptive immune responses throughout the body. It is already known that this inflammasome is a key regulator of several systemic diseases. The initiation and activation of NLRP3 starts with the oral microbiome and its association with the pathogenesis and progression of several oral diseases, including periodontitis, periapical periodontitis, and oral squamous cell carcinoma (OSCC). The possible role of the inflammasome in oral disease conditions may involve the aberrant regulation of various response mechanisms, not only in the mouth but in the whole body. Understanding the cellular and molecular biology of the NLRP3 inflammasome and its relationship to Nrf2 is necessary for the rationale when suggesting it as a potential therapeutic target for treatment and prevention of oral inflammatory and immunological disorders. In this review, we highlighted the current knowledge about NLRP3, its likely role in the pathogenesis of various inflammatory oral processes, and its crosstalk with Nrf2, which might offer future possibilities for disease prevention and targeted therapy in the field of dentistry and oral health.

The transcription factor hepatocyte nuclear factor 4A acts in the intestine to promote white adipose tissue energy storage

The transcription factor hepatocyte nuclear factor 4 A (HNF4A) controls the metabolic features of several endodermal epithelia. Both HNF4A and HNF4G are redundant in the intestine and it remains unclear whether HNF4A alone controls intestinal lipid metabolism. Here we show that intestinal HNF4A is not required for intestinal lipid metabolism per se, but unexpectedly influences whole-body energy expenditure in diet-induced obesity (DIO). Deletion of intestinal HNF4A caused mice to become DIO-resistant with a preference for fat as an energy substrate and energetic changes in association with white adipose tissue (WAT) beiging. Intestinal HNF4A is crucial for the fat-induced release of glucose-dependent insulinotropic polypeptide (GIP), while the reintroduction of a stabilized GIP analog rescues the DIO resistance phenotype of the mutant mice. Our study provides evidence that intestinal HNF4A plays a non-redundant role in whole-body lipid homeostasis and points to a non-cell-autonomous regulatory circuit for body-fat management.

Prognostic Value and Immune Infiltration Analysis of Nuclear Factor Erythroid-2 Family Members in Ovarian Cancer

Ovarian cancer (OC) often presents at an advanced stage and is still one of the most frequent causes of gynecological cancer-related mortality worldwide. The nuclear factor erythroid-2 (NFE2) transcription factors include nuclear factor, erythroid 2 like 1 (NFE2L1), NFE2L2, and NFE2L3. NFE2 members bind to the antioxidant-response element (ARE) region and activate the expression of targeted genes. The distinct functions of NFE2 members in OC remain poorly elucidated. Several online bioinformatics databases were applied to determine gene expression, prognosis, mutations, and immune infiltration correlation in OC patients. NFE2L1 and NFE2L2 were decreased in OC, whereas NFE2L3 was increased. NFE2L2 and NFE2L3 were significantly correlated with the clinical stages of OC. High NFE2L1 level was significantly associated with short progression-free survival (PFS) in patients with OC ( HR = 1.18 , P = 0.021 ), while high NFE2L2 expression strongly correlated with long PFS ( HR = 0.77 , P = 0.00067 ). High NFE2L3 expression was associated with better overall survival and postprogression survival in OC. Functional analysis showed that NFE2 members mainly focused on transcription coactivator activities. Genetic alterations of NFE2 members were found in 13% of OC patients, and amplification ranked the top. The expression of NFE2 members was significantly correlated with immune infiltration of CD4+ T cells, CD8+ T cells, B cells, macrophages, and neutrophils in OC. Our study provides novel insights into the roles and prognostic potential of NFE2 family members in OC.

Role of Combination Treatment of Aspirin and Zinc in DMH-DSS Induced Colon Inflammation, Oxidative Stress and Tumor Progression in Male BALB/C Mice

Colitis-associated colorectal cancer serves as a prototype of inflammation-associated cancers which is linked with repeated cycles of inflammation and DNA repair deficits. Several preclinical and clinical data reported that aspirin has chemo preventive effect in colorectal cancer and is associated with dose dependent side effects. Further, it has been reported that zinc supplementation improves the quality of life in patients undergoing chemotherapy by alteration of colonic cancer cell gene expression. However, explication of the detailed molecular mechanisms involved in combined administration of aspirin and zinc mediated protection against the colitis associated colorectal cancer deserves further investigation. For the induction of colitis associated colorectal cancer, male BALB/c mice were administered 1, 2-dimethylhydrazine dihydrochloride (DMH) 20 mg/kg/bw thrice, before the initiation of every DSS cycle (3%w/v in drinking water). One week after the initiation of DSS treatment, aspirin (40 mg/kg; p.o.) and zinc in the form of zinc sulphate (3 mg/kg; p.o.) was administered for 8 weeks. Combination of aspirin and zinc as intervention significantly ameliorated DAI score, myeloperoxidase activity, histological score, apoptotic cells and protein expression of various inflammatory markers including nuclear factor kappa light chain enhancer of activated B cells (NFκBp65), cycloxygenase -2 (COX-2), interleukin-6 (IL-6); proliferation markers such as proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription 3 (STAT3) expression significantly decreased and antioxidant enzymes nuclear factor erythroid 2–related factor 2 (Nrf-2), metallothionein, catalase and superoxide dismutase (SOD) significantly increased as evaluated by immunohistochemistry and western blot analysis.