Phosphorylation of RelA/p65 Ser536 inhibits the progression and metastasis of hepatocellular carcinoma by mediating cytoplasmic retention of NF-κB p65.

Background: Intrahepatic and extrahepatic metastases contribute to the high recurrence rate and mortality of hepatocellular carcinoma (HCC). Constitutive activation of nuclear factor-κB (NF-κB) is a crucial feature of HCC. NF-κB p65 (p50-p65) is the most common dimeric form. Ser536 acts as an essential phosphorylation site of RelA/p65. However, the effect of RelA/p65 Ser536 phosphorylation on progression and metastases during intermediate and advanced HCC has not been reported. Methods: Phosphorylation of RelA/p65 (p-p65 Ser536) and NF-κB p65 were detected by using immunohistochemical staining in HCC tissue samples. The biological effects of RelA/p65 Ser536 phosphorylation were evaluated by using xenograft and metastasis models. NF-κB p65 nuclear translocation was detected by using Western blotting. The binding of NF-κB p65 to the BCL2, SNAIL, and MMP9 promoters was detected by using chromatin immunoprecipitation. The biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition were assessed by using tetrazolium-based colorimetry, colony formation, EdU incorporation, flow cytometry, cell wound healing, and transwell assay. Results: NF-κB p65 is highly expressed, while p-p65 Ser536 is not well expressed in intermediate and advanced HCC tissues. In vivo experiments demonstrated that a phosphorylation-mimetic mutant of RelA/p65 Ser536 (p65/S536D) prevents tumor progression and metastasis. In vitro experiments showed that p65/S536D inhibits proliferation, migration, and invasion. Mechanistically, RelA/p65 Ser536 phosphorylation inhibits NF-κB p65 nuclear translocation and reduces NF-κB p65 binding to the BCL2, SNAIL, and MMP9 promoters. Conclusions: RelA/p65 Ser536 phosphorylation was detrimental to NF-κB p65 entry into the nucleus and inhibited HCC progression and metastasis by reducing BCL2, SNAIL, and MMP9. The phosphorylation site of RelA/p65 Ser536 has excellent potential to be a promising target for NF-κB-targeted therapy in HCC.

Distribution of Immunomodulation, Protection and Regeneration Factors in Cleft-Affected Bone and Cartilage.

BACKGROUND: Craniofacial clefts can form a significant defect within bone and cartilage, which can negatively affect tissue homeostasis and the remodeling process. Multiple proteins can affect supportive tissue growth, while also regulating local immune response and tissue protection. Some of these factors, like galectin-10 (Gal-10), nuclear factor kappa-light-chain-enhancer of activated B cells protein 65 (NF-κB p65), heat shock protein 60 (HSP60) and 70 (HSP70) and cathelicidin (LL-37), have not been well studied in cleft-affected supportive tissue, while more known tissue regeneration regulators like type I collagen (Col-I) and bone morphogenetic proteins 2 and 4 (BMP-2/4) have not been assessed jointly with immunomodulation and protective proteins. Information about the presence and interaction of these proteins in cleft-affected supportive tissue could be helpful in developing biomaterials and improving cleft treatment. METHODS: Two control groups and two cleft patient groups for bone tissue and cartilage, respectively, were organized with five patients in each group. Immunohistochemistry with the semiquantitative counting method was implemented to determine Gal-10-, NF-κB p65-, HSP60-, HSP70-, LL-37-, Col-I- and BMP-2/4-positive cells within the tissue. RESULTS: Factor-positive cells were identified in each study group. Multiple statistically significant correlations were identified. CONCLUSIONS: A significant increase in HSP70-positive chondrocytes in cleft patients could indicate that HSP70 might be reacting to stressors caused by the local tissue defect. A significant increase in Col-I-positive osteocytes in cleft patients might indicate increased bone remodeling and osteocyte activity due to the presence of a cleft. Correlations between factors indicate notable differences in molecular interactions within each group.

Biophysical characterization of RelA-p52 NF-κB dimer-A link between the canonical and the non-canonical NF-κB pathway.

The NF-κB family consists of the key transcription factors of the NF-κB signaling pathway, well known for its role in innate immune response, organogenesis, and a variety of cellular processes. The five NF-κB subunits-RelA, RelB, c-Rel, p50, and p52-are functional dimers, each of which share a conserved DNA binding domain which contains the dimerization domain (DD) as well. The NF-κB subunits can form 15 potential dimers among themselves of which, RelA-p50 is extensively studied and has largely become synonymous with NF-κB for transcription activation. While various reports have highlighted the importance of NF-κB subunit specificity in the transcription regulation of certain target genes, the dynamic nature of the NF-κB dimer composition is not well understood. In this study, we biophysically characterized six combinatorial dimers from three NF-κB subunits: RelA, p50, and p52, using NMR spectroscopy and differential scanning calorimetry. We show that the dimer composition is dynamic and can readily undergo exchange although at varied rates. Among the six dimers formed, RelA-p52 is found to be the most stable dimer with RelA-RelA being the least. Our results provide a plausible explanation as to why the RelA-p52 heterodimer is active during the later stages of the NF-κB activation and serve as a link between the canonical and non-canonical NF-κB pathway.

Silibinin Suppresses Inflammatory Responses Induced by Exposure to Asian Sand Dust.

Asian sand dust (ASD), generated from the deserts of China and Mongolia, affects Korea and Japan during spring and autumn, causing harmful effects on various bio-organs, including the respiratory system, due to its irritants such as fine dust, chemicals, and toxic materials. Here, we investigated the therapeutic effects of silibinin against ASD-induced airway inflammation using mouse macrophage-like cell line RAW264.7 and a murine model. ASD was intranasally administered to mice three times a week and silibinin was administered for 6 days by oral gavage. In ASD-stimulated RAW264.7 cells, silibinin treatment decreased tumor necrosis factor-α production and reduced the expression of p-p65NF-κB, p-p38, and cyclooxygenase (COX)-2, while increasing heme oxygenase (HO)-1 expression. In ASD-exposed mice, silibinin administration reduced inflammatory cell count and cytokines in bronchoalveolar lavage fluid and decreased inflammatory cell infiltration in lung tissue. Additionally, silibinin lowered oxidative stress, as evidenced by decreased 8-hydroxy-2'-deoxyguanosin (8-OHdG) expression and increased HO-1 expression. The expression of inflammatory-related proteins, including p-p65NF-κB, COX-2, and p-p38, was markedly reduced by silibinin administration. Overall, silibinin treatment reduced the expression of p-p65NF-κB, COX-2, and p-p38 in response to ASD exposure, while increasing HO-1 expression both in vitro and in vivo. These findings suggest that silibinin mitigates pulmonary inflammation caused by ASD exposure by reducing inflammatory signaling and oxidative stress, indicating its potential as a therapeutic agent for ASD-induced pulmonary inflammation.

Activating Transcription Factor 6 Mediates Inflammation in Experimental Varicocele-Induced Epididymal Epithelial Cells.

Introduction: Varicocele is a dilatation of the internal spermatic vein and it is generally recognized as one cause of male infertility. This study aimed to analyze the roles of activating transcription factor 6 (ATF-6) in experimental varicocele-induced epididymal epithelial cells. Methods: Experimental left varicocele was established in rats through partial left renal vein ligation. At 8 weeks after surgery, the left epididymal damage was observed using H&E and TUNEL staining. The expressions of neutral α-glucosidase (NAG), ATF-6, tumor necrosis factor (TNF)-α, and phospho-nuclear factor (p-NF)-κB p65 (S536) in the left epididymis were measured by immunohistochemistry. ATF-6 silence in rat epididymal epithelial cells was established by ATF-6 siRNA transfection. The cells were treated with hypoxia for 24 h, and cell viability was measured by CCK-8, levels of NAG, TNF-α, and interleukin (IL)-8 in cells were measured by ELISA, levels of p-NF-κB p65 (S536)/NF-κB p65 protein in cells were measured by Western blotting. Results: The results showed that the experimental left varicocele induced hypertrophy and apoptosis of epididymal epithelial cells (p<0.05), and decreased the expressions of NAG in the epididymal epithelial cells compared with the sham-operated control rats (p<0.01). Meanwhile, the expressions of ATF-6, TNF-α, and p-NF-κB p65 (S536) were increased in the epididymal epithelial cells after the experimental left varicocele compared with the sham-operated control rats (p<0.05). In the hypoxia-treated cells, ATF-6 silence increased the cell viability and decreased the levels of TNF-α, IL-8, and p-NF-κB p65 (S536) compared with the control cells (p<0.05). Discussion: The ATF-6 pathway was activated in a rat's left varicocele-induced epididymal damage. Inhibition of the ATF-6 pathway might be a possible novel therapeutic approach for left varicocele-induced epididymal damage.

Transcriptional repression by HDAC3 mediates T cell exclusion from Kras mutant lung tumors.

Histone Deacetylase 3 (HDAC3) function in vivo is nuanced and directed in a tissue-specific fashion. The importance of HDAC3 in Kras mutant lung tumors has recently been identified, but HDAC3 function in this context remains to be fully elucidated. Here, we identified HDAC3 as a lung tumor cell-intrinsic transcriptional regulator of the tumor immune microenvironment. In Kras mutant lung cancer cells, we found that HDAC3 is a direct transcriptional repressor of a cassette of secreted chemokines, including Cxcl10. Genetic and pharmacological inhibition of HDAC3 robustly up-regulated this gene set in human and mouse Kras, LKB1 (KL) and Kras, p53 (KP) mutant lung cancer cells through an NF-κB/p65-dependent mechanism. Using genetically engineered mouse models, we found that HDAC3 inactivation in vivo induced expression of this gene set selectively in lung tumors and resulted in enhanced T cell recruitment at least in part via Cxcl10. Furthermore, we found that inhibition of HDAC3 in the presence of Kras pathway inhibitors dissociated Cxcl10 expression from that of immunosuppressive chemokines and that combination treatment of entinostat with trametinib enhanced T cell recruitment into lung tumors in vivo. Finally, we showed that T cells contribute to in vivo tumor growth control in the presence of entinostat and trametinib combination treatment. Together, our findings reveal that HDAC3 is a druggable endogenous repressor of T cell recruitment into Kras mutant lung tumors.

SERPINA1 promotes the invasion, metastasis, and proliferation of pancreatic ductal adenocarcinoma via the PI3K/Akt/NF-κB pathway.

Serpin peptidase inhibitor clade A member 1 (SERPINA1) is highly expressed in a variety of solid tumors. However, its role in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, we report evidence that SERPINA1 acts as a potent oncogene to drive its extremely malignant character. We found that elevated SERPINA1 expression in primary tumors was associated with lymph node metastasis and shorter survival in PDAC patients. Mechanistic investigations revealed that overexpression of SERPINA1 induced nuclear translocation and phosphorylation of the p65 subunit through the PI3K/Akt/NF-κB pathway, thereby promoting the invasion, metastasis and proliferation of PDAC cells in vitro and in vivo. Conversely, the knockdown of SERPINA1 attenuated this signaling pathway and restored the phenotype of PDAC cells overexpressing SERPINA1. Overall, our study reveals that SERPINA1 affects the properties of PDAC through the PI3K/Akt/NF-κB pathway, and its activation confers the clinical features of epithelial-mesenchymal transition and proliferation in the disease.

Upregulation of Cullin1 neddylation promotes glycolysis and M1 polarization of macrophage via NF-κB p65 pathway in sepsis.

This study aimed to explore the underlying mechanism of neddylation in macrophage polarization during sepsis. A mouse model of sepsis was established by cecal ligation and puncture (CLP). ELISA and Flow cytometry were performed to analyze the generation of pro-inflammatory factors and M1/M2 macrophage polarization, respectively. Western blotting was applied to detect NEDD8-mediated neddylation and glycolysis-related proteins. ECAR method was used to analyze the glycolysis level. HE staining was applied to detect the lung injury. The bacterial load in peritoneal cavity and peripheral blood was determined by counting the colony-forming units. The results showed the upregulated neddylation, M1 polarization and glycolysis of macrophage in patients with sepsis and CLP-challenged mice. NEDD8-mediated Cullin1 neddylation promoted M1 polarization and glycolysis to accelerate inflammation via NF-κB p65 pathway in E.coli-treated Raw264.7 cells. MLN4924 treatment alleviated sepsis by inhibiting neddylation to prevent M1 polarization in CLP-challenged mice. In summary, this study demonstrated that upregulation of NEDD8-mediated Cullin1 neddylation promotes glycolysis and M1 polarization of macrophage via NF-κB p65 pathway, accelerating inflammation in sepsis.

Investigating the Hepatic Response to Orlistat and White Tea in Rats on a High-Fat Diet.

High-fat diets have detrimental health impacts that increase the likelihood of developing obesity and metabolic syndrome. This study aimed to examine the potential antioxidant and anti-inflammatory effects of orlistat and white tea in rats fed a high-fat diet. Thirty-two rats were randomized into four groups: control (standard diet), HFD (high-fat diet), HFD+Orlistat (high-fat diet+orlistat), and HFD+WT (high-fat diet+white tea extract). A significant increase in malondialdehyde (MDA) levels and a decrease in total thiol (TT) levels were detected in the HFD group (p < 0.001). On the other hand, a decrease in the MDA level (p < 0.001) and an increase in the TT level were observed in the orlistat and white tea groups compared with those in the HFD group (p < 0.001). Histopathological examinations revealed that, compared with the HFD alone, orlistat and white tea reduced fat accumulation, prevented degenerative changes in hepatocytes, and decreased the histopathological damage score (p = 0.001). Immunohistochemical examinations of nuclear factor-kappa B (NF-kB/p65) revealed that compared with the HFD, orlistat and white tea reduced immunopositivity (p = 0.001). White tea decreases lipid peroxidation and oxidative stress. Both white tea and orlistat decreased fat formation and inflammation in the liver and regulated inflammation by reducing Nf-kB positivity. Nevertheless, further research is needed to assess their impact on human subjects.

Micheliolide ameliorates severe acute pancreatitis in mice through potentiating Nrf2-mediated anti-inflammation and anti-oxidation effects.

Severe acute pancreatitis (SAP) is an acute inflammatory injury disease with significant mortality rate and currently without effective strategy being available. Inflammation and oxidative stress play central roles in the etiology of SAP. Micheliolide (MCL), an active monomeric component isolated from Michelia champaca, has been proved its multiple therapeutic properties including anti-inflammatory, antioxidant and anti-cancer. Nevertheless, the therapeutic effect and underlying mechanism of MCL in SAP still remain unclear. Here, we found that caerulein with lipopolysaccharide (LPS)-induced SAP murine models exhibited severe pancreatic injury, including necrosis, edema, and vacuolation of acinar cells in the pancreas, elevated serum levels of amylase and lipase, and reduced number of the exocrine cells. As expected, MCL treatment alleviated these side effects. Mechanistically, MCL triggered nuclear factor erythroid 2-related factor 2 (Nrf2) activation, thereby activating Nrf2-regulated antioxidative pathways and inhibiting nuclear factor kappa B p65 (NF-κB p65)-mediated inflammatory response, resulting in protection against pancreatic injury in SAP mice. In addition, Nrf2 gene deficiency abolished the beneficial effects of MCL on SAP-induced pancreatic inflammation and oxidative stress and blocked the ability of MCL to alleviate the pancreatic injury in SAP mice. Collectively, these findings indicated that the suppression of SAP-induced pancreatic injury by MCL was at least in part due to Nrf2-mediated anti-oxidation effect and inhibition of inflammation.

Adenanthin exhibits anti-inflammatory effects by covalently targeting the p65 subunit in the NF-κB signaling pathway.

Adenanthin is a structurally unique ent-kaurane diterpenoid isolated from Rabdosia adenantha, a traditional Chinese medicinal plant with potent anti-cancer and anti-inflammatory activities. However, its anti-inflammatory molecular mechanism remains largely elusive to date. Here, we developed an affinity-based label-free protein profiling (ALFPP) to identify potential covalent targets of electrophilic natural products with ketone or aldehyde groups. Using ALFPP, we identified 27 potential covalent targets of adenanthin, among which p65 (RelA) has been associated with its anti-inflammatory activities. Through a series of experiments, including LC-MS/MS, molecular docking, electrophoretic mobility shift assays (EMSA), and genome editing, we demonstrated that adenanthin could covalently modify the Cys38 residue of p65 to affect the binding of DNA to p65, thereby inhibiting the NF-κB signaling pathway. ALFPP will facilitate the target identification of electrophilic carbonylated natural products, especially those containing α, ß-unsaturated keto groups. Furthermore, the elucidation of the molecular mechanism of adenanthin will contribute to new drug development of adenanthin to treat inflammations and cancers, enhancing the possibility for its clinical application.

Echinatin alleviates sepsis severity through modulation of the NF-κB and MEK/ERK signaling pathways.

Sepsis, a frequently fatal condition, emerges from an exaggerated inflammatory response to infection, resulting in multi-organ dysfunction and alarmingly high mortality rates. Despite the urgent need for effective treatments, current therapeutic options remain limited to antibiotics, with no other efficacious alternatives available. Echinatin (Ecn), a potent bioactive compound extracted from the roots and rhizomes of licorice, has gained significant attention for its broad pharmacological properties, particularly its ability to combat oxidative stress. Recent research highlights the crucial role that oxidative stress plays in the onset and progression of sepsis further emphasizing the potential therapeutic value of Ecn in this context. In this study, we explored the protective effects of Ecn in a murine model of sepsis induced by cecal ligation and puncture (CLP). Ecn demonstrated a significant reduction in the levels of inflammatory cytokines and reactive oxygen species (ROS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Network pharmacology analysis identified 41 targets and top 15 pathways involved in the Ecn-mediated signaling network, revealing that Ecn might exert its effects through key targets including the NF-κB and MAPK signaling pathways. Molecular docking studies suggested a strong affinity between Ecn and MEK, with kinetic simulations and binding energy calculations confirming a stable interaction. Mechanistically, Ecn treatment inhibited NF-κB and the MEK/ERK signaling pathway, as evidenced by decreased phosphorylation of IκBα and nuclear p65, along with reduced phosphorylation of MEK and ERK in both LPS-stimulated RAW 264.7 macrophages and septic mice. Furthermore, the administration of MEK signaling agonists reversed the anti-inflammatory effects of Ecn, indicating the involvement of this signaling pathway in Ecn's protective mechanism. Notably, our investigation revealed that Ecn did not affect bacterial proliferation either in vivo or in vitro, underscoring its specific immunomodulatory effects rather than direct antimicrobial activity. In summation, our findings underscored the potential of Ecn as an innovative therapeutic remedy for sepsis-induced injury, particularly through the regulation of the NF-κB and MEK/ERK signaling pathway. This exploration unveiled a promising therapeutic approach for treating sepsis, supplementing existing interventions and addressing their constraints.

The ethanolic extract of domesticated Amauroderma rugosum alleviated DSS-induced ulcerative colitis via repairing the intestinal barrier.

Amauroderma rugosum (Blume and T. Nees) Torrend (Ganodermataceae) (A. rugosum) has been found to have anti-inflammatory ability in previous studies. The present study aimed to verify the therapeutic benefits of A. rugosum in the treatment of ulcerative colitis and to investigate its underlying mechanism of action. Acute experimental ulcerative colitis was induced by feeding the mice drinking water supplemented with dextran sodium sulfate (DSS). The findings indicated that the ethanolic extract of domesticated A. rugosum exhibited therapeutic efficacy comparable to Salazosulfapyridine (SASP) in mitigating clinical symptoms and the pathological score of the colon. Furthermore, A. rugosum exhibited the capacity to enhance the expression of tight junction (TJ) proteins, while concurrently decreasing the levels of TNF-ɑ and IL-6. A noteworthy finding is that it exhibited the capability to diminish the nuclear translocation of NF-κB p65. In conclusion, A. rugosum attenuates DSS-induced ulcerative colitis by enhancing intestinal barrier function and inhibiting mucosal inflammation. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-024-01565-5.

Dehydroevodiamine targeting IKKß to alleviate acute gastric injury via inhibiting the p65/NLRP3 axis.

BACKGROUND: Acute gastric injury, a common and recurring global digestive disorder, significantly impairs patient quality of life and overall health. Dehydroevodiamine (DHE), a bioactive natural product derived from Tetradium ruticarpum (A. Juss.) Hartley, shows potential therapeutic effects on acute gastric injury. This study investigates the underlying mechanisms of DHE's alleviating effects on acute gastric injury. METHODS: The gastric mucosal protective effect of DHE was confirmed through in vivo and in vitro acute gastric injury models. Biotin pulldown MS and molecular dynamics simulations identified DHE's target. CETSA and SPR assays validated DHE's affinity for IKKß. Protein site mutation validation and MST pinpointed the direct binding sites of DHE on IKKß. Additionally, the potential mechanism by which DHE ameliorates acute gastric injury was elucidated using WB, IHC, and IF methods, and further confirmed by rescue experiments. RESULTS: DHE effectively ameliorated IDO-induced gastric injury in GES-1 cells and rat gastric mucosa, both in vitro and in vivo. Biotin pulldown MS identified IKKß as the target of DHE in alleviating gastric injury. CETSA and SPR assays confirmed DHE's direct binding to IKKß. Molecular dynamics simulations, protein mutation experiments, and MST results pinpointed GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket as the binding sites of DHE on IKKß. Notably, DHE was found to competitively bind to IKKß with ATP. Mechanistically, DHE attenuated IDO-induced gastric injury by inhibiting the IKKß-p65/NLRP3 signaling pathway. Importantly, exogenous activation of IKKß reversed the therapeutic effect of DHE, indicating that DHE's efficacy depends on IKKß. CONCLUSION: DHE attenuated IDO-induced gastric injury by inhibiting the IKKß-p65/NLRP3 signaling pathway. Notably, DHE is a novel ATP-competitive IKKß inhibitor that prevents phosphorylation by targeting GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket. This study reveals new targets of action for DHE, providing a new molecular basis for using DHE in treating inflammation-related diseases.

Phosphorylation of Serine 536 of p65(RelA) Downregulates Inflammatory Responses.

Nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of various genes involved in inflammatory diseases and immune responses. Recently, a novel transcriptional regulatory mechanism of NF-κB involving the phosphorylation of serine 536 (534 in mice; S534) of its p65 subunit was reported; however, further research is required to elucidate the physiological role of S534 phosphorylation. Therefore, we generated S534A knock-in (KI) mice, in which the S534 of p65 was substituted with alanine. Similar to the wild-type (WT) mice, S534A KI mice developed normally. After stimulation with tumor necrosis factor α (TNFα), mouse embryonic fibroblasts (MEFs) derived from S534A KI mice exhibited increased target gene expression compared with that in the WT MEFs, which was induced by long-term binding of p65 to DNA. According to comprehensive gene expression analysis after stimulation with TNFα, the expression of genes p65ted to inflammatory and immune responses was increased, and the expression of genes p65ted to lipolysis was decreased in S534A KI MEFs. Analyses of a periodontal disease model established using WT and S534A KI mice revealed that alveolar bone resorption was enhanced in S534A KI mice owing to an increase in the number of osteoclasts, which was not attributed to the differentiation of osteoclast precursor cells but to an increased expression of interleukin-1ß and receptor activator of NF-κB ligand in the periodontal tissue. Hence, phosphorylation of S536 negatively regulates inflammatory responses in vitro and in vivo.

NF-κB Transcription Factors: Their Distribution, Family Expansion, Structural Conservation, and Evolution in Animals.

The Nuclear Factor Kappa B (NF-κB) transcription factor family consists of five members: RelA (p65), RelB, c-Rel, p50 (p105/NF-κB1), and p52 (p100/NF-κB2). This family is considered a master regulator of classical biochemical pathways such as inflammation, immunity, cell proliferation, and cell death. The proteins in this family have a conserved Rel homology domain (RHD) with the following subdomains: DNA binding domain (RHD-DBD) and dimerization domain (RHD-DD). Despite the importance of the NF-κB family in biology, there is a lack of information with respect to their distribution patterns, evolution, and structural conservation concerning domains and subdomains in animals. This study aims to address this critical gap regarding NF-κB proteins. A comprehensive analysis of NF-κB family proteins revealed their distinct distribution in animals, with differences in protein sizes, conserved domains, and subdomains (RHD-DBD and RHD-DD). For the first time, NF-κB proteins with multiple RHD-DBDs and RHD-DDs have been identified, and in some cases, this is due to subdomain duplication. The presence of RelA/p65 exclusively in vertebrates shows that innate immunity originated in fishes, followed by amphibians, reptiles, aves, and mammals. Phylogenetic analysis showed that NF-κB family proteins grouped according to animal groups, signifying structural conservation after speciation. The evolutionary analysis of RHDs suggests that NF-κB family members p50/p105 and c-Rel may have been the first to emerge in arthropod ancestors, followed by RelB, RelA, and p52/p100.

MSC-derived exosomes attenuates pulmonary hypertension via inhibiting pulmonary vascular remodeling.

BACKGROUND: Pulmonary hypertension (PH) is a serious cardiopulmonary disease with significant morbidity and mortality. Vascular obstruction leads to a continuous increase in pulmonary vascular resistance, vascular remodeling, and right ventricular hypertrophy and failure, which are the main pathological features of PH. Currently, the treatments for PH are very limited, so new methods are urgently needed. Msenchymal stem cells-derived exosomes have been shown to have significant therapeutic effects in PH, however, the mechanism still very blurry. Here, we investigated the possible mechanism by which umbilical cord mesenchymal stem cell-derived exosomes (hUC-MSC-EXO) inhibited monocrotaline (MCT)-induced pulmonary vascular remodeling in a rat model of PH by regulating the NF-κB/BMP signaling pathway. Our data revealed that hUC-MSC-EXO could significantly attenuate MCT-induced PH and right ventricular hypertrophy. Moreover, the protein expression level of BMPR2, BMP-4, BMP-9 and ID1 was significantly increased, but NF-κB p65, p-NF-κB-p65 and BMP antagonists Gremlin-1 was increased in vitro and vivo. Collectively, this study revealed that the mechanism of hUC-MSC-EXO attenuates pulmonary hypertension may be related to inhibition of NF-κB signaling to further activation of BMP signaling. The present study provided a promising therapeutic strategy for PH vascular remodeling.

Small molecule α-methylene-γ-butyrolactone, an evolutionarily conserved moiety in sesquiterpene lactones, ameliorates arthritic phenotype via interference DNA binding activity of NF-κB.

Rheumatoid arthritis (RA) is an inflammatory disease accompanied by abnormal synovial microenvironment (SM). Sesquiterpene lactones (SLs) are the main anti-inflammatory ingredients of many traditional herbs utilized in RA treatment. α-Methylene-γ-butyrolactone (α-M-γ-B) is a core moiety that widely exists in natural SLs. This study was designed to investigate the anti-arthritic potential of α-M-γ-B as an independent small molecule in vitro and in vivo. α-M-γ-B exhibited stronger electrophilicity and anti-inflammatory effects than the other six analogs. α-M-γ-B inhibited the production of pro-inflammatory mediators via repolarizing M1 macrophages into M2 macrophages. The transcriptome sequencing suggested that α-M-γ-B regulated the immune system pathway. Consistently, α-M-γ-B attenuated collagen type II-induced arthritic (CIA) phenotype, restored the balance of Tregs-macrophages and remodeled SM via repolarizing the synovial-associated macrophages in CIA mice. Mechanistically, although α-M-γ-B did not prevent the trans-nucleus of NF-κB it interfered with the DNA binding activity of NF-κB via direct interaction with the sulfhydryl in cysteine residue of NF-κB p65, which blocked the activation of NF-κB. Inhibition of NF-κB reduced the M1 polarization of macrophage and suppressed the synovial hyperplasia and angiogenesis. α-M-γ-B failed to ameliorate CIA in the presence of N-acetylcysteine or when the mice were subjected to the macrophage-specific deficiency of Rela. In conclusion, α-M-γ-B significantly attenuated the CIA phenotype by directly targeting NF-κB p65 and inhibiting its DNA binding ability. These results suggest that α-M-γ-B has the potential to serve as an alternative candidate for treating RA. The greater electrophilicity of α-M-γ-B, the basis for triggering strong anti-inflammatory activity, accounts for the reason why α-M-γ-B is evolutionarily conserved in the SLs by medical plants.

The Epstein-Barr virus small capsid protein BFRF3 disrupts the NF-кB signaling pathway by inhibiting p65 activity.

Epstein-Barr virus (EBV), a common gamma herpesvirus, establishes a life-long latent infection in the host to defend against innate immune recognition, which is closely related to a variety of malignant tumors, but its specific mechanism is unclear. BFRF3, an EBV-encoded small capsid protein, is mainly involved in the assembly of the viral capsid structure and the maintenance of its stability. Here, we showed that BFRF3 can inhibit TNF-α-mediated NF-кB promoter activation. Moreover, BFRF3 downregulates NF-кB-mediated promoter activation and transcription of inflammatory cytokines, including IL-6 and IL-8. Dual-luciferase reporter assay demonstrated that BFRF3 restrains NF-кB promoter activity at or below the p65 level, and coimmunoprecipitation analysis revealed that BFRF3 not only interacts with p65 but also binds to its critical truncated Rel homology domain (RHD) and transcriptional activation domain (TAD). However, BFRF3 does not affect the dimerization of p65-p50, but overexpression of BFRF3 reduces the nuclear accumulation of p65, and the phosphorylation of p65 (Ser536) is repressed during BFRF3 transfection and EBV lytic infection, which promotes the proliferation of EBV. Overall, our study suggested that BFRF3 may play a crucial role in antiviral immunity to defend against EBV infection by inhibiting NF-κB activity.

Silicone dressing combined with topical oxygen therapy alleviates incontinence-associated dermatitis via NF-κB p65/STAT1 signaling pathway.

BACKGROUND: Incontinence-associated dermatitis (IAD) is a tough problem in clinical settings, not only increasing the risk of complications like catheter-related urinary tract infections and pressure ulcers in elderly and critically ill patients, but also prolonging hospital stays, raising hospital costs, and possibly leading to medical disputes. This study is aimed to evaluate the therapeutic effect of silicone dressing combined with topical oxygen therapy on IAD in a rat model. METHODS: An IAD rat model induced by synthetic urine with trypsin was established. Hematoxylin & eosin staining was carried out to examine skin histology. Using immunofluorescence, the microvessel density in the affected skin tissues was determined. ELISA was performed to measure the concentrations of inflammatory cytokines and angiogenic factors in serum. The mRNA expression of EGF, PDGF, and VEGF was detected via qRT-PCR. Western blotting was employed to determine NF-κB p65/STAT1 pathway-related protein levels. RESULTS: Compared to single therapy, silicone dressing combined with topical oxygen therapy could significantly reduce the severity of IAD, improve skin histology, inhibit inflammation, and promote angiogenesis in IAD rat models. Additionally, the results showed that relatively speaking, the combined therapy suppressed the NF-κB p65/STAT1 signaling pathway more effectively. CONCLUSION: These findings indicated that silicone dressing combined with topical oxygen therapy can alleviate IAD through promoting wound healing and inhibiting inflammation via NF-κB p65/STAT1 signaling pathway in a rat model, which provided a theoretical basis for the prevention and treatment of IAD in clinic.