YAP inhibits NF-κB signaling and ccRCC growth by opposing p65-ZHX2 cooperativity.

Hippo pathway functions as a tumor suppressor pathway by inhibiting the oncogenic potential of pathway effectors YAP/TAZ. However, YAP can also function as a context-dependent tumor suppressor in several types of cancer including clear cell renal cell carcinomas (ccRCC). Here we show that YAP blocks NF-κB signaling in ccRCC to inhibit cancer cell growth. Mechanistically, YAP inhibits the expression of ZHX2, a critical p65 co-factor in ccRCC. Furthermore, YAP competes with ZHX2 for binding to p65. Consequently, elevated nuclear YAP blocks the cooperativity between ZHX2 and p65, leading to diminished NF-κB target gene expression. Pharmacological inhibition of Hippo/MST1/2 blocked NF-κB transcriptional program and suppressed ccRCC cancer cell growth, which can be rescued by ZHX2/p65 overexpression. Our study uncovers a novel crosstalk between the Hippo and NF-κB pathways and its involvement in ccRCC growth inhibition, suggesting that targeting the Hippo pathway may provide a therapeutical opportunity for ccRCC treatment.

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.

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.

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.

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.

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.

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.

Biochanin A mitigates ulcerative colitis and intestinal inflammation in mice by inhibiting MAPK/NF-kB (p65) axis.

Ulcerative colitis (UC) is a chronic problem of the intestine and relapsing in nature. Biochanin A is a nature-derived isoflavonoid and has numerous bioactivities. However, its role against UC and intestinal inflammation remains obscure. We aimed to comprehensively explore the pharmacological effect of biochanin A in alleviating colitis and to evaluate the potential mechanisms. Initially, we explored the anti-inflammatory action of biochanin A (15, 30, and 60 µM) by employing lipopolysaccharide (LPS)-activated RAW 264.7 cells. In RAW 264.7 cells under LPS stimulation, biochanin A inhibited the elevation of reactive oxygen species (ROS) (p < 0.0001), interleukin (IL)-1ß (p < 0.0001), IL-18 (p < 0.01), and tumor necrosis factor (TNF)-α (p < 0.01) release, nitrite production (p < 0.0001), and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins. Next, we studied the effectiveness of biochanin A (20 and 40 mg/kg) in mouse colitis induced with dextran sulfate sodium (DSS) by assessing colon length, disease activity index (DAI) scoring, and performing colonoscopy and histological analysis. The pro-inflammatory cytokines were estimated using ELISA. Western blot studies were performed to assess underlying mechanisms. In mice, biochanin A treatment alleviated DAI score (p < 0.0001), restored colon length (p < 0.05) and morphology, and re-established colon histopathology. Biochanin A affects the phosphorylation of proteins associated with NF-κB (p65) and mitogen-activated protein kinase (MAPK) axis and regulates colonic inflammation by reducing the expression of inflammatory cytokines and myeloperoxidase (MPO) activity. Altogether, our findings support the idea that the anticolitis potential of biochanin A is allied with anti-inflammatory activity by inhibiting the MAPK/NF-κB (p65) axis. Hence, biochanin A may be an alternative option to alleviate the risk of colitis.

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.

PIM1 inhibitor SMI-4a attenuated concanavalin A-induced acute hepatitis through suppressing inflammatory responses.

Background: Serine/threonine kinase 1 (PIM1) plays a crucial role in cell growth, differentiation, and apoptosis. However, its role in the pathogenesis of concanavalin A (ConA)-induced acute hepatitis is not well understood. PIM1 kinase inhibitor can reduce the expression of PIM1. This study aims to investigate the effects of PIM1 kinase inhibitor and its protective mechanism in ConA-induced acute hepatitis. Methods: C57/BL six mice were injected with ConA (20, 15, and 12 mg/kg) to induce acute hepatitis, and PIM1 kinase inhibitor SMI-4a (60 mg/kg) was administered orally 24 h before ConA injection. The survival rate of the mice was observed after ConA injection. The levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Serum inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was performed on liver tissue collected at different time points. The major cytokines expression in liver tissue was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The number of macrophages, T-cell and neutrophils in liver tissue were detected by flow cytometry (FCM). PIM1 in liver tissue was detected by western blot (WB) and qRT-PCR. SMI-4a (80 µM) was pretreated for 24 h and ConA (400 µg/mL) was stimulated for 12 h in RAW264.7 cell model. Phosphorylated p65 (p-p65) and cleaved caspase-3 (c-caspase-3) in liver tissue and macrophages were detected by WB. Results: Different concentrations of ConA caused different acute hepatitis mortality, 12 mg/kg concentration within 24 h of the mortality showed a gradient increase. The levels of AST and ALT increased significantly at 12 h after ConA injection. PIM1 expression was upregulated at 12 h. SMI-4a can suppress the PIM1 expression. SMI-4a suppressed cytokines production, AST, and ALT in ConA-treated serum. SMI-4a suppressed the major cytokines in liver tissue. Tests in liver tissue showed that SMI-4a reduced the number of T cells, neutrophils, and macrophages. SMI-4a inhibited the inflammatory response by downregulating the expression of p-p65. Meanwhile, apoptosis was decreased by decreasing the expression of c-caspase-3. Conclusions: In conclusion, the protective effect of SMI-4a against acute hepatitis is by reducing the inflammatory response and apoptosis. These findings suggest that SMI-4a may have therapeutic potential in the treatment of autoimmune hepatitis.

Protective effects of dopamine against non-alcoholic steatohepatitis via inhibiting p65 pathways in vivo and in vitro.

Introduction: Currently, the role and mechanism of dopamine in non-alcoholic steatohepatitis (NASH) remains unclear. Methods: In vitro experiments utilized FFA and LPS to establish NASH cell models, while a fibrotic cell model was created using TGFß1 to investigate the impact of dopamine on cellular lipid metabolism, inflammation, and fibrosis. In vivo experiments involved the use of MCD and HFD diets to induce NASH in mouse models for observing the effects of dopamine on NASH disease progression. Results: Our study showed that dopamine significantly downregulated the expression levels of Caspase 1, IL-1ß and IL18 in the HepG2 NASH cell model. In addition, dopamine could inhibit the TGF-ß1-induced accumulation of collagen I and α-SMA in LX2 cells. In vivo experiments have shown that dopamine attenuation in mice is associated with MCD diet-induced and HFD-induced steatohepatitis. Mechanically, dopamine inhibits the p65 signaling pathway in NASH. Conclusion: In conclusion, the present study demonstrates the role of dopamine in ameliorating the symptoms of NASH and provides a direction for future research on the application of the dopaminergic system to liver disease.

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.

SLC15A3 is transcriptionally regulated by HIF1α and p65 to worsen neuroinflammation in experimental ischemic stroke.

Systemic inflammatory stimulus is a risk factor for the incidence of ischemic stroke and contributes to poorer clinical outcomes. Solute carrier 15A3 (SLC15A3) is a peptide/histidine transporter that is implicated in regulating inflammatory responses. However, whether SLC15A3 affects the progression of ischemic stroke associated with systemic inflammation is unclear. The transient middle cerebral artery occlusion (tMCAO) mice with LPS administration (LPS/tMCAO) were prepared as an in vivo model, and LPS-induced BV2 cells under oxygen-glucose deprivation (OGD) exposure were utilized as an in vitro model. We found that SLC15A3 was highly expressed in the ischemic penumbra of LPS/tMCAO mice, and its inhibition reduced infarct area, attenuated neurological deficit, recovered motor function, and mitigated apoptotic neurons. Knockdown of SLC15A3 suppressed the proinflammatory M1-type markers and promoted the levels of M2-associated genes. The in vitro results confirmed that SLC15A3 overexpression promoted microglia polarizing towards M1 subtypes, while SLC15A3 inhibition exerted an opposite effect. In addition, we demonstrated that the p65 signaling pathway and HIF1α were activated by LPS/OGD. Luciferase reporter assay showed that inhibiting p65 using its specific inhibitor BAY 11-7082 or silencing HIF1α using siRNAs reduced the transcriptional activity of SLC15A3 in LPS/OGD-induced BV2 cells. Results in NIH 3T3 cells also confirmed that p65 and HIF1α directly bound to the SLC15A3 promoter to activate SLC15A3 transcription. In conclusion, this work shows that SLC15A3, transcriptionally activated by p65 and HIF1α, contributes to poor outcomes in ischemic stroke associated with systemic inflammation by promoting microglial cells polarizing towards M1 types.

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.

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.

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.

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.