Recruitment of neutrophils in glomeruli in early mouse sepsis is associated with E-selectin expression and activation of endothelial NF-κB and MAPK pathways.

Sepsis is a dysregulated systemic inflammatory response to an infection, which can lead to multiple organ dysfunction syndrome that includes the kidney. Leukocyte recruitment is an important process of the host immune defense in response to sepsis. Endothelial cells (EC) actively regulate leukocyte recruitment by expressing adhesion molecules following the activation of dedicated intracellular signal transduction pathways. Previous studies reported that the expression of adhesion molecules was associated with the activation of endothelial NF-κB p65 and MAPK c-Jun pathways in vitro in response to conditions that mimic processes that occur in inflammation. This study aimed to investigate the spatiotemporal patterns of leukocyte recruitment, expression of adhesion molecules, and endothelial nuclear p65 and c-Jun localization in renal microvascular beds of septic mice. Here, we used a cecal ligation and puncture (CLP) sepsis mouse model and RT-qPCR and immunohistochemical staining. We showed that neutrophils, macrophages, and T lymphocytes were all present in the kidney, yet only neutrophils accumulated in a spatiotemporally discernible pattern, mainly in glomeruli at 4 hours after CLP-sepsis initiation. E-selectin, not VCAM-1, was expressed in glomeruli at the same time point. In a subset of mice at 72 hours after CLP-sepsis started, VCAM-1 expression was prominent in glomerular EC, which was not related to changes in mmu-microRNA(miR)-126a-3p levels, a short noncoding microRNA previously shown to inhibit the translation of VCAM-1 mRNA into protein. Nuclear localization of p65 and c-Jun occurred in EC of all microvascular segments at 4 and 7 hours after CLP-sepsis initiation. In summary, sepsis-induced recruitment of neutrophils, E-selectin expression, and NF-κB p65 and MAPK c-Jun pathway activation coincided in glomeruli at the early stage of the disease. In the other microvascular beds, sepsis led to NF-κB p65 and MAPK c-Jun pathway activation with limited expression of E-selectin and no association with VCAM-1 expression or leukocyte recruitment.

Multilevel Regulation of NF-κB Signaling by NSD2 Suppresses Kras-Driven Pancreatic Tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a clinically challenging cancer with a dismal overall prognosis. NSD2 is an H3K36-specific di-methyltransferase that has been reported to play a crucial role in promoting tumorigenesis. Here, the study demonstrates that NSD2 acts as a putative tumor suppressor in Kras-driven pancreatic tumorigenesis. NSD2 restrains the mice from inflammation and Kras-induced ductal metaplasia, while NSD2 loss facilitates pancreatic tumorigenesis. Mechanistically, NSD2-mediated H3K36me2 promotes the expression of IκBα, which inhibits the phosphorylation of p65 and NF-κB nuclear translocation. More importantly, NSD2 interacts with the DNA binding domain of p65, attenuating NF-κB transcriptional activity. Furthermore, inhibition of NF-κB signaling relieves the symptoms of Nsd2-deficient mice and sensitizes Nsd2-null PDAC to gemcitabine. Clinically, NSD2 expression decreased in PDAC patients and negatively correlated to nuclear p65 expression. Together, the study reveals the important tumor suppressor role of NSD2 and multiple mechanisms by which NSD2 suppresses both p65 phosphorylation and downstream transcriptional activity during pancreatic tumorigenesis. This study opens therapeutic opportunities for PDAC patients with NSD2 low/loss by combined treatment with gemcitabine and NF-κBi.

Puerarin suppresses macrophage M1 polarization to alleviate renal inflammatory injury through antagonizing TLR4/MyD88-mediated NF-κB p65 and JNK/FoxO1 activation.

BACKGROUND: Acute kidney injury (AKI) is a clinically common and serious renal dysfunction, characterized by inflammation and damage to tubular epithelial cells. Puerarin, an isoflavone derivative isolated from Pueraria lobata, has been proven to possess exceptional effectiveness in reducing inflammation. However, the effects and underlying mechanisms of puerarin on AKI remain uncertain. PURPOSE: This study investigated the possible therapeutic effects of puerarin on AKI and explored its underlying mechanism. STUDY DESIGN AND METHODS: The effects of puerarin on AKI and macrophage polarization were investigated in lipopolysaccharide (LPS)-induced or unilateral ureteral obstruction (UUO)-induced mouse models in vivo and LPS-treated macrophages (Raw264.7) in vitro. Additionally, the effects of puerarin on inflammation-related signaling pathways were analyzed. RESULTS: Administration of puerarin effectively alleviated kidney dysfunction and reduced inflammatory response in LPS-induced and UUO-induced AKI. In vitro, puerarin treatment inhibited the polarization of M1 macrophages and the release of inflammatory factors in Raw264.7 cells stimulated by LPS. Mechanistically, puerarin downregulated the activities of NF-κB p65 and JNK/FoxO1 signaling pathways. The application of SRT1460 to activate FoxO1 or anisomycin to activate JNK eliminated puerarin-mediated inhibition of JNK/FoxO1 signaling, leading to suppression of macrophage M1 polarization and reduction of inflammatory factors. Further studies showed that puerarin bound to Toll/interleukin-1 receptor (TIR) domain of MyD88 protein, hindering its binding with TLR4, ultimately resulting in downstream NF-κB p65 and JNK/FoxO1 signaling inactivation. CONCLUSIONS: Puerarin antagonizes NF-κB p65 and JNK/FoxO1 activation via TLR4/MyD88 pathway, thereby suppressing macrophage polarization towards M1 phenotype and alleviating renal inflammatory damage.

MLKL deficiency alleviates acute alcoholic liver injury via inhibition of NLRP3 inflammasome.

Mixed lineage kinase domain-like protein (MLKL) is identified as the terminal executor of necroptosis. However, its role in acute alcoholic liver injury remains unclear. This study elucidates that MLKL can contribute to acute alcoholic liver injury independently of necroptosis. Although the expression of MLKL was upregulated, no significant increase in its phosphorylation or membrane translocation was observed in the liver tissues of mice treated with ethanol. This finding confirms that alcohol intake does not induce necroptosis in mouse liver tissue. Additionally, the deletion of Mlkl resulted in the downregulation of NLRP3 expression, which subsequently inhibited the activation of the NLRP3 inflammasome and the ensuing inflammatory response, thereby effectively mitigating liver injury induced by acute alcohol consumption. The knockout of Nlrp3 did not affect the expression of MLKL, further confirming that MLKL acts upstream of NLRP3. Mechanistically, inhibiting the nuclear translocation of MLKL reduced the nuclear entry of p65, the principal transcriptional regulator of NLRP3, thereby limiting the transcription of Nlrp3 mRNA and subsequent NLRP3 expression. Overall, this study unveils a novel mechanism of MLKL regulates the activation of NLRP3 inflammasomes in a necroptosis independent way in acute alcoholic liver injury.

CircACTR2 promotes bladder cancer progression through IKBKB-mediated NF-κB signaling pathway activation.

Background: Circular RNAs (circRNAs) have significant roles in tumor progression. The role of circRNA derived from ARP2 actin-related protein 2 homolog (circACTR2) has been reported in various human diseases. However, the functions and regulatory mechanisms of circACTR2 in Bladder Cancer (BCa) remain unknown. Objectives: This study aims to explore the biological role and regulatory mechanism of circACTR2 in BCa. Methods: We analyzed the effects of circACTR2 on BCa through bioinformatics analyses, RT-qPCR, and cell function assays. Results: We observed the upregulation of circACTR2 in BCa tissues and validated its circular structure. Loss-of-function assays demonstrated that silencing circACTR2 suppressed the proliferation, invasion, and migration of BCa cells. Mechanistic investigation revealed that circACTR2 sponges miR-219a-2-3p to elevate the expression of the inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB). This induced upregulation of IKKß protein promoted the nuclear translocation of p65, thereby activating the NF-κB signaling pathway. Conclusions: Our findings indicate that circACTR2 promotes BCa cell proliferation, migration, and invasion by activating the NF-κB signaling pathway via the miR-219a-2-3p/IKBKB axis, potentially unveiling a new therapeutic target for BCa.

Nuclear factor-κB p65 subunit determines the fate of aging epithelial cells.

Nuclear factor (NF)-κB signaling is not only important for the immune and inflammatory responses but also for the normal development of epithelial cells, such as those in the skin and tooth. Here, we generated epithelial cell-specific p65-deficient (p65Δepi-/-) mice to analyze the roles of NF-κB signaling in epithelial cell developent. Notably, p65Δepi-/- mice exhibited no abnormalities in their appearance compared to the control (p65flox/flox) littermates. Furthermore, no major changes were observed in the skin, hair growth, and shape and color of the incisors and molars. However, 65 % of p65Δepi-/- mice exhibited corneal thickening after 8 weeks of age, and 30 % of p65Δepi-/- mice exhibited hair growth from the mandibular incisors around 24 weeks of age. No hair growth was observed at 36 and 42 weeks of age. However, micro-computed tomography images revealed a large cavity below the mandibular incisors extending to the root of the incisor. Histological analysis revealed that the cavity was occupied by a connective tissue containing hair-like structures with many dark brown granules that disappeared after melanin bleaching, confirming the presence of hair. Although inflammatory cells were also observed near the eruption site of the incisor teeth of p65Δepi-/- mice, no major disturbance was observed in the arrangement of enamel epithelial cells. Overall, these results highlight the role of p65 in the maintenance of epithelial cell homeostasis during aging.

Proximity Ligation Assay to Detect the Proximity Between Host Proteins and Viral Proteins of HIV-1.

The study of host-pathogen interaction often requires interrogating the protein-protein interactions and examining post-translational modifications of the proteins. Traditional protein detection strategies are limited in their sensitivity, specificity, and multiplexing capabilities. The Proximity Ligation Assay (PLA), a versatile and powerful molecular technique, can overcome these limitations. PLA blends the specificity of antibodies, two antibodies detecting two different epitopes on the same or two different proteins, with the amplification efficiency of a polymerase to allow highly specific and sensitive detection of low-abundant proteins, protein-protein interactions, or protein modifications. In this protocol, we describe the application of PLA to detect the proximity between HIV-1 Tat with one of its cellular partners, p65, in an infected host cell. The protocol could be applied to any other context with slight modifications. Of note, PLA can only confirm the physical proximity between two epitopes or proteins; however, the proximity need not necessarily allude to the functional interaction between the two proteins.

Target NF-κB p65 for preventing posttraumatic joint contracture in rats.

RelA/p65 is as a crucial component of the nuclear factor κB (NF-κB) signaling pathway that has a significant impact on various fibrotic diseases. However, its role in the fibrosis of tissues surrounding the joint after traumatic injury remains unclear. In this study, rats were divided into three groups: non-operated control (NC) group, p65-siRNA treated (siRNA-p65) group, and negative siRNA treated (siRNA-neg) group. Then, 10 µL (10 nmol) of p65-siRNA was injected into the joint of the siRNA-p65 group. Meanwhile, 10 µL of negative siRNA was administered to the knee joint of the operated siRNA-neg group for comparison. The rats in the NC group did not receive surgery or drug intervention. After 4 weeks of right knee fixation in each group, X-ray measurements revealed significantly reduced degree of knee flexion contracture following p65-siRNA treatment (siRNA-neg: 77.73° ± 2.799°; siRNA-p65: 105.7° ± 2.629°, p < 0.0001). Histopathological examination revealed that the number of dense fibrous connective tissues decreased following p65-siRNA inhibition. Western blot analysis revealed significantly different expression levels of fibrosis-related proteins between the siRNA-p65 and siRNA-neg groups. Immunohistochemical analysis revealed a reduction in the average number of myofibroblasts in the siRNA-p65 group compared with that in the siRNA-neg group. Thus, intra-articular p65-siRNA injection could attenuate fibroblast activation and fibrosis-related protein production, suppress periarticular tissue fibrosis, and prevent joint contracture by downregulating the NF-κB p65 pathway. Statement of clinical significance: Intra-articular injection of p65-siRNA could reduce myofibroblast proliferation and fibrosis-related protein expression by downregulating the NF-κB p65 pathway, inhibit periarticular tissue fibrosis, and prevent joint adhesion, which represents a potential therapy in the prevention of joint fibrosis following traumatic injury.

The Roles of RhoA/ROCK/NF-κB Pathway in Microglia Polarization Following Ischemic Stroke.

Ischemic stroke is the leading cause of death and disability worldwide. Nevertheless, there still lacks the effective therapies for ischemic stroke. Microglia are resident macrophages of the central nervous system (CNS) and can initiate immune responses and monitor the microenvironment. Microglia are activated and polarize into proinflammatory or anti­inflammatory phenotype in response to various brain injuries, including ischemic stroke. Proinflammatory microglia could generate immunomodulatory mediators, containing cytokines and chemokines, these mediators are closely associated with secondary brain damage following ischemic stroke. On the contrary, anti-inflammatory microglia facilitate recovery following stroke. Regulating the activation and the function of microglia is crucial in exploring the novel treatments for ischemic stroke patients. Accumulating studies have revealed that RhoA/ROCK pathway and NF-κB are famous modulators in the process of microglia activation and polarization. Inhibiting these key modulators can promote the polarization of microglia to anti-inflammatory phenotype. In this review, we aimed to provide a comprehensive overview on the role of RhoA/ROCK pathway and NF-κB in the microglia activation and polarization, reveal the relationship between RhoA/ROCK pathway and NF-κB in the pathological process of ischemic stroke. In addition, we likewise discussed the drug modulators targeting microglia polarization.

Dandelion root extracts and taraxasterol inhibit LPS­induced colorectal cancer cell viability by blocking TLR4­NFκB­driven ACE2 and TMPRSS2 pathways.

Colorectal cancer is the fourth leading cause of cancer-related death worldwide. Notably, abnormalities in intestinal bacteria may contribute to the initiation or progression of colorectal cancer. Lipopolysaccharide (LPS), a bacterial endotoxin, is elevated in patients with colorectal cancer. The present study investigated the protective effects of dandelion root extracts and taraxasterol (TS; a major pharmacologically active compound in dandelion root extracts) on LPS-induced colorectal cancer cell viability, as well as the underlying mechanisms. Cell viability was assessed by MTT assay, and protein and gene expression levels were determined by western blotting and quantitative PCR. It was revealed that LPS at a low dose (0.5 µg/ml) significantly promoted the viability of human colorectal cancer cells but did not affect normal colon epithelial cells. The addition of dandelion root extracts (0.1-1 mg/ml) or TS (0.05-1 µg/ml) was able to reverse the LPS-induced increase in colorectal cancer cell viability and colony formation. Mechanistically, dandelion root extracts or TS may inhibit the LPS-promoted toll-like receptor 4 (TLR4)/NFκB-p65 pathway and transcription levels of pro-inflammatory genes (TNFα, IL4 and IL6). Compared with normal colon epithelial cells, human colorectal cancer cells had higher expression levels of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), which could be further enhanced by LPS treatment but this was reversed by co-incubation with dandelion root extracts or TS. In addition, suppression of the TLR4/NFκB-p65 pathway with CLI095 significantly reversed the stimulatory effect of LPS on the expression levels of ACE2 and TMPRSS2, whereas TNFα (10 ng/ml) markedly induced the expression levels of ACE2 and TMPRSS2. In conclusion, the present study suggested that dandelion root extracts and TS could be used as prevention strategies for reversing bacteria-driven colorectal cancer cell viability.

Electroacupuncture improves low-grade duodenal inflammation in FD rats by reshaping intestinal flora through the NF-κB p65/NLRP3 pyroptosis pathway.

Electroacupuncture (EA) is an effective alternative for the treatment of functional dyspepsia (FD). It reduces low-grade duodenal inflammation and improves the symptoms of FD by downregulating the expression of NF-κB p65 and NLRP3, but its mechanism needs to be elucidated. To examine the regulatory effect of electroacupuncture (EA) on intestinal flora and NF-κB p65/NLRP3 pyroptosis pathway in FD rats. The FD rat model was established via multi-factor stress intervention for two weeks. The rats were randomly divided into the NC group, model group, NF-kB inhibitor group (NF-κB inhibitor BAY 11-7082 was administered), EA group, and EA + NF-kB inhibitor group. After 14 days of treatment, the rats were sacrificed, and the protein and mRNA levels of NF-κB p65, IκB, and NLRP3 in the duodenum were evaluated by Western blotting assays and real-time fluorescent quantitative PCR. The Illumina MiSeq sequencing platform was used to analyze the V4 region of the 16S rRNA gene of intestinal flora and predict functional genes. The concentration of short-chain fatty acids (SCFAs) in feces was assessed by metabolomics. EA can decrease low-grade duodenal inflammation and promote gastrointestinal motility in FD rats. This effect is mediated by inhibition of the NF-κB p65/NLRP3 pyroptosis pathway, an increase in the alpha and beta diversity of gut microbiota in the duodenum, an increase in the abundance of beneficial bacteria at the phylum and genus levels, and an increase in the content of SCFAs. The protective effect of EA against FD might involve multiple hierarchy and pathways. EA may remodel intestinal flora by inhibiting the NF-κB p65/NLRP3 pyroptosis pathway, thereby improving low-grade duodenal inflammation in FD rats.

Hericenone C attenuates the second phase of formalin-induced nociceptive behavior by suppressing the accumulation of CD11c-positive cells in the paw epidermis via phosphorylated P65.

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.

MPoMA protects against lung epithelial cell injury via p65 degradation.

Numerous cases of lung injury caused by viral infection were reported during the coronavirus disease-19 pandemic. While there have been significant efforts to develop drugs that block viral infection and spread, the development of drugs to reduce or reverse lung injury has been a lower priority. This study aimed to identify compounds from a library of compounds that prevent viral infection that could reduce and prevent lung epithelial cell damage. We investigated the cytotoxicity of the compounds, their activity in inhibiting viral spike protein binding to cells, and their activity in reducing IL-8 production in lung epithelial cells damaged by amodiaquine (AQ). We identified N-(4-(4-methoxyphenoxy)-3-methylphenyl)-N-methylacetamide (MPoMA) as a non-cytotoxic inhibitor against viral infection and AQ-induced cell damage. MPoMA inhibited the expression of IL-8, IL-6, IL-1ß, and fibronectin induced by AQ and protected against AQ-induced morphological changes. However, MPoMA did not affect basal IL-8 expression in lung epithelial cells in the absence of AQ. Further mechanistic analysis confirmed that MPoMA selectively promoted the proteasomal degradation of inflammatory mediator p65, thereby reducing intracellular p65 expression and p65-mediated inflammatory responses. MPoMA exerted potent anti-inflammatory and protective functions in epithelial cells against LPS-induced acute lung injury in vivo. These findings suggest that MPoMA may have beneficial effects in suppressing viral infection and preventing lung epithelial cell damage through the degradation of p65 and inhibition of the production of inflammatory cytokines.

Prognostic Significance of Nuclear Factor Kappa B (p65) among Breast Cancer Patients in Cape Coast Teaching Hospital.

Breast cancer is the most prevalent cancer among African women, with high mortality rates in Ghana. Nuclear factor kappa B (NF-kB) has been associated with tumor progression in breast cancer. However, its clinical validation is controversial and understudied with no known published data on NF-kB (p65) among breast cancer patients in Ghana and other African countries. This study assessed the prognostic significance of NF-kB (p65) expression and its association with various clinicopathological features in breast cancer patients. Ninety formalin-fixed breast cancer tissues and 15 normal breast tissues were used to determine the expression of NF-kB (p65) using immunohistochemistry. We explored the correlation between expression of NF-kB (p65) and clinicopathological features. NF-kB (p65) was expressed in 86.7% of breast cancer tissues. There was a significant relationship between NF-kB (p65) expression and tumor grade, proliferation index (Ki67), and molecular subtype. High NF-kB (p65) expression in tumor grade 3 was about 10 times that of grade 1 (54.2% vs. 5.1%), and Ki67 > 20 was 79.7% compared to 20.3% for Ki67 ≤ 20. Patients with triple-negative breast cancer (TNBC) had 49.1% overexpression of NF-kB (p65) compared to 17%, 25.4%, and 8.5% for luminal A, luminal B, and HER2 cases, respectively. This study demonstrates that NF-kB (p65) was highly expressed among breast cancer patients at Cape Coast Teaching Hospital, Ghana, especially in TNBC. NF-kB (p65) could serve as a biomarker for cancer stage, progression, prognosis and as a therapeutic target.

Discovery of sesquiterpenoids from the roots of Chloranthus henryi Hemsl. var. hupehensis (Pamp.) K. F. Wu and their anti-inflammatory activity by IKBα/NF-κB p65 signaling pathway suppression.

Phytochemical analysis of Chloranthus henryi var. hupehensis roots led to the identification of a new eudesmane sesquiterpenoid dimer, 18 new sesquiterpenoids, and three known sesquiterpenoids. Among the isolates, 1 was a rare sesquiterpenoid dimer that is assembled by a unique oxygen bridge (C11-O-C8') of two highly rearranged eudesmane-type sesquiterpenes with the undescribed C16 carbon framework. (+)-2 and (-)-2 were a pair of new skeleton dinorsesquiterpenoids with a remarkable 6/6/5 tricyclic ring framework including one γ-lactone ring and the bicyclo[3.3.1]nonane core. Their structures were elucidated using spectroscopic data, single-crystal X-ray diffraction analysis, and quantum chemical computations. In the LPS-induced BV-2 microglial cell model, 17 suppressed IL-1ß and TNF-α expression with EC50 values of 6.81 and 2.76 µM, respectively, indicating its excellent efficacy in inhibiting inflammatory factors production in a dose dependent manner and without cytotoxicity. In subsequent mechanism studies, compounds 3, 16, and 17 could reduce IL-1ß and TNF-α production by inhibiting IKBα/p65 pathway activation.

ELMO1 ameliorates intestinal epithelial cellular senescence via SIRT1/p65 signaling in inflammatory bowel disease-related fibrosis.

Background: Intestinal fibrosis is a common complication in inflammatory bowel disease (IBD), which still lacks of reliable markers and therapeutic options. Cellular senescence has been considered an important mechanism of intestinal fibrosis, but the underlying molecular link remains elusive. Methods: Tissues were stained using α-smooth muscle actin (α-SMA), fibronectin, and collagen I as markers of myofibroblastic differentiation. Cellular senescence was confirmed through Lamin B1 staining, senescence-associated ß-galactosidase staining, and the expression of senescence-associated secretory phenotype (SASP) factors. We explored the relationship between senescence of intestinal epithelial cells (IECs) and intestinal fibrosis, as well as the molecular mechanism underlying this interaction. The effects of irisin on cellular senescence and fibrosis were determined. Results: Here, we identify engulfment and cell motility protein 1 (ELMO1) as a novel biomarker for intestinal cellular senescence and fibrosis. In fibrostrictured tissues from patients and murine models with IBD, significantly high levels of cellular senescence score and factors were noted, which positively correlated with the fibrotic regulator fibronectin. Senescent IECs, not fibroblast itself, released SASP factors to regulate fibroblast activation. Prolonging exposure to severe and persistent injurious stimuli decreased ELMO1 expression, which dampened SIRT1 deacetylase activity, enhanced NF-κB (p65) acetylation, and thereby accelerated cellular senescence. Deletion of ELMO1 led to senescent IECs accumulation and triggered premature fibrosis in murine colitis. Furthermore, irisin, inhibiting the degradation of ELMO1, could downregulate p65 acetylation, reduce IECs senescence, and prevent incipient intestinal fibrosis in murine colitis models. Conclusions: This study reveals ELMO1 downregulation is an early symbol of intestinal senescence and fibrosis, and the altered ELMO1-SIRT1-p65 pathway plays an important role in intestinal cellular senescence and IBD-related fibrosis.

TLR4 knockdown by miRNA-140-5p improves tendinopathy: an in vitro study.

Introduction: The purpose of this study was to determine whether TLR4 knockdown induced by miRNA-140-5p improves tendinopathy in an in vitro experiment. Material and methods: Extraction of tendon-derived stem cells (TDSCs) from SD rats was performed using TGF-ß1 to develop a tendinopathy cell model. In the first step, we knocked down TLR4 by si-TLR4 to investigate TLR4 in tendinopathy development, and the next we used miRNA-140-5p to investigate miRNA-140-5p in tendinopathy development. The inflammatory factors and Hyp concentration were evaluated by ELISA assay; the cell viability was measured by MTT assay; the cell apoptosis was evaluated by TUNEL and/or flow cytometry. The relative mRNA was measured by RT-qPCR assay; the relative proteins expression was evaluated by cellular immunofluorescence and/or WB assay. The correlation between miRNA-140-5p and TLR4 was analyzed by Luciferase reporter assay. Results: With miRNA-140-5p overexpression or TLR4 knockdown, the cell viability was significantly increased with cell apoptosis depressing compared with the Model group (p < 0.05, respectively). Meanwhile, the inflammatory factors TNF-α, IL-1ß and IL-6 and Hyp concentration were significantly improved (p < 0.05, respectively), whereas the TLR4, MyD88 and NF-κB(p65) protein expression levels were significantly depressed with TLR4 knockdown by si-TLR4 or miRNA-140-5p which target TLR4. Conclusions: The present results showed that TLR4 knockdown induced by miRNA-140-5p or si-TLR4 improved tendinopathy in an in vitro cell experiment.

LncRNA MTC-p65 Protein Mediates Skin Fibroblast Proliferation in Liaoning Cashmere Goat by Regulating the Nuclear Factor-Kappa B Signaling Pathway.

The aim of this study is to investigate the activation of NF-κB signaling pathway and the regulation of the expression of genes related to chorionic villus growth by the binding of LncRNA MTC (XLOC_005914) and p65 (transcription factor p65 [Capra hircus], XP_017898873.1). In addition, the regulation of LncRNA MTC and p65 binding on the proliferation of Liaoning Cashmere Goat skin fibroblasts is investigated. The upregulation of LncRNA MTC promoted the proliferation of skin fibroblasts, and the NF-κB signaling pathway played an important role in this process. Compared with the negative control (NC group), the expression of TNFα and NFKB2(NF-κB) genes was highly significantly up-regulated (P < 0.001), and NFKBIA(IκBɑ) genes were highly significantly down-regulated (P < 0.01) after LncRNA MTC overexpression (OE group). The expression levels of TNFα and NFκB-P-p65 proteins were upregulated in the OE group; NF-κB-p65 expression levels were upregulated in the nucleus, IκBα expression levels were downregulated in the cytoplasm, and P-IκBα expression levels were upregulated. LncRNA MTC and p65 proteins were co-localized in the cells. Meanwhile, LncRNA MTC and p65 protein showed significant nucleation in the OE group. RNA pull-down and LC-MS/MS verified that p65 protein was indeed an interacting protein of LncRNA MTC. LncRNA MTC binds to p65 protein, upregulates the expression of TNFα protein, nucleates p65 protein, and activates NF-κB signaling pathway to promote the proliferation of skin fibroblasts in Liaoning Cashmere Goat.

Inhibition of nucleophosmin/B23 sensitizes ovarian cancer cells to immune check-point blockade via PD-L1 in ovarian cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) that against programmed cell death protein-1 (PD-1) and its ligand PD-L1 have been approved as a promising treatment of many human cancers. However, the responses to these ICIs were limited in patients with ovarian cancer. Studies have indicated that the response to PD-1/PD-L1 blockade might be correlated with the PD-L1 expression level in cancer cells. Nucleophosmin (NPM/B23) was found to be a potential target for immunotherapy. Whether NPM/B23 plays a role in cancer-associated immunity, such as PD-1/PD-L1 axis, and its underlying mechanisms remain largely unknown in ovarian cancer. METHODS: We applied ovarian cancer cell lines as research models. The effect of modulating PD-L1 by NPM/B23 was subsequently confirmed via Western blot, flow cytometry, qRT-PCR, luciferase reporter assays, and immunoprecipitation. Protein stability and ubiquitin assay assays were used to analyze the interplay between NPM/B23 and NF-ĸB/p65 in PD-L1 regulation. The MOSEC/Luc xenograft mouse model was used to validate the role of NPM/B23-PD-L1 through tumor growth in vivo. RESULTS: Our results revealed that NPM/B23 negatively regulates PD-L1 expression via a protein complex with NF-κB/p65 and through an IFN-γ pathway. Moreover, NPM/B23 inhibitor/modulator sensitized ovarian cancer cells to the anti-PD-1 antibody by regulating PD-L1 expression in the immunocompetent mouse model. Compared to anti-PD-1 antibody alone, a combination of anti-PD-1 antibody and NPM/B23 inhibitor/modulator showed reduced tumorigenesis and increased CD8+ T-cell expansion, thus contributing to prolonged survival on MOSEC/Luc-bearing mouse model. CONCLUSION: Targeting NPM/B23 is a novel and potential therapeutic approach to sensitize ovarian cancer cells to immunotherapy.

Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway.

BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.