MicroRNA-520c-3p suppresses vascular endothelium dysfunction by targeting RELA and regulating the AKT and NF-κB signaling pathways

Journal of Physiology and Biochemistry - Endothelial injury, which can cause endothelial inflammation and dysfunction, is an important mechanism for the development of atherosclerotic plaque. This...     

Cryptotanshinone inhibits RANKL-induced osteoclastogenesis by regulating ERK and NF-κB signaling pathways

Osteoporosis (OS) is one of the most common healthy problems characterized by low bone mass. Osteoclast, the primary bone-resorbing cell, is responsible for destructive bone diseases including osteoporosis (OS). Cryptotanshinone (CTS), an active component extracted from the root of Salvia miltiorrhiza bunge, has been shown to prevent the destruction of cartilage and the thickening of subchondral bone in mice osteoarthritis models. However, its molecular mechanism in osteoclastogenesis needs to be determined. The aim of the current study was to explore the effect of CTS on osteoclastogenesis and further evaluate the underlying mechanism. Our results showed that CTS inhibited receptor activator of NF-κB ligand (RANKL)-induced the increase in tartrate-resistant acid phosphatase (TRAP) activity in bone marrow–derived macrophages (BMMs). In addition, the expressions of osteoclastogenesis-related marker proteins and nuclear factor of activated T-cells (NFAT) activation were suppressed by CTS treatment in BMMs. Furthermore, CTS attenuated RANKL-induced ERK phosphorylation and NF-κB activation in BMMs. These findings indicated that CTS inhibited RANKL-induced osteoclastogenesis by inhibiting ERK phosphorylation and NF-κB activation in BMMs. Thus, CTS may function as an inhibitor of osteoclastogenesis and may be considered as an alternative medicine for the prevention and treatment of OS.     

Safflower yellow alleviates osteoarthritis and prevents inflammation by inhibiting PGE2 release and regulating NF-κB/SIRT1/AMPK signaling pathways

BackgroundSafflower yellow (SY) is the main active ingredient of safflower, with various pharmacological effects such as anticoagulating, antioxidant, and anti-arthritis effects.PurposeTo investigate the anti-inflammatory and chondrocyte protecting role of SY, which subsequently leads to the inhibition of cartilage degradation.MethodsRat chondrocytes were stimulated with tumor necrosis factor α (TNF-α) with or without SY treatment. Following this, CCK-8 assay was performed to detect cytotoxicity. RT-qPCR, Western blotting, and immunofluorescence staining were used to detect the gene/protein expression of typical cartilage matrix genes and related inflammatory markers. Subsequently, EdU assay was used to evaluate cell proliferation. RNA sequencing, online target prediction, and molecular docking were performed to determine the possible molecular targets and pathways.ResultsThe results showed that SY restored the TNF-α-induced up-regulation of IL-1β, PTGS2, and MMP-13 and down-regulation of COL2A1 and ACAN. Furthermore, it recovered cell proliferation by suppressing TNF-α. Gene expression profiles identified 717 differentially expressed genes (DEGs) in the cells cultured with or without SY under TNF-α stimulation. After pathway enrichment, PI3K-Akt, TNF, Cytokine-cytokine receptor interaction, NF-κB, NOD-like receptor, and Chemokine signaling pathways were notably selected to highlight NFKBIA, CCL5, CCL2, IL6, and TNF as potential targets in osteoarthritis (OA). SY inhibited TNF-α-induced activation of NF-κB and endoplasmic reticulum (ER) stress by promoting AMPK phosphorylation along with SIRT1 expression. Further, SY reduced MMP-13 expression and targeted COX-2 for decreasing PGE2 release. In addition, anterior cruciate ligament transection-induced OA was ameliorated by local administration of SY.ConclusionThese results demonstrate that SY protects chondrocytes and inhibits inflammation by regulating the NF-κB/SIRT1/AMPK pathways and ER stress, thus preventing cartilage degeneration in OA.     

p73 is required for endothelial cell differentiation,migration and the formation of vascular networks regulating VEGF and TGFβ signaling

Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity.Vascular system formation is one of the earliest events during organogenesis.¹ The original vascular plexus is established by vasculogenesis, through differentiation and assembly of mesodermal precursors.² The angiogenesis process allows the formation of new blood vessels from the existing vasculature and is perturbed in many diseases, including cancer.³ Although efforts have been made to identify factors that control vascular development, the understanding of the molecular networks remains incomplete.The formation of new capillaries and the remodeling of preexisting blood vessels is linked by signal transduction pathways.⁴ The members of the p53 family (p53, p73 and p63) coordinate cell proliferation, migration and differentiation, and could act as regulators of vascular development. TP73 function in angiogenesis is quite controversial,^{5, 6, 7} and it has never been addressed using developmental models.TP73 has a dual nature that resides in the existence of TA and DNp73 variants. TAp73 is capable of transactivating p53 targets^{8, 9, 10} whereas DNp73 can act as p53 and TAp73 repressor.^{11, 12, 13} TP73 final outcome will depend upon the differential expression of the TA/DNp73 isoforms in each cellular context, as they can execute synergic, as well as antagonist, functions.TP73 role during development is emphasized by the p73-knockout mice (Trp73−/−, p73KO from now on) multiple growth defects.¹⁴ These mice, which lack all p73 isoforms, exhibit gastrointestinal and cranial hemorrhages,¹⁴ suggestive of vascular fragility. Furthermore, TAp73 directly regulates GATA-1,⁸ which is essential for endothelial and hematopoietic differentiation.^{15, 16} This compounded information led us to hypothesize that p73 could be implicated in the regulation of vasculogenesis/angiogenesis.Regulation of these processes involves a broad range of signaling molecules essential for vascular growth and stability,¹⁷ such as vascular endothelial growth factor (VEGF)¹⁸ and transforming growth factor-β (TGF-β).¹⁹ TGF-β operates as a rheostat that controls endothelial cell (EC) differentiation, having an inhibitory effect on EC migration and proliferation by the TGF-β/TGFRI (ALK5)/Smad2/3 pathway, while the TβRII–ALK5/ALK1 complex activates Smad1/5/8, ID1 expression and a pro-angiogenic state.^{20, 21, 22}Regulation of the TGF-β and VEGF pathways by p53 family members has been documented.^{23, 24} However, p73''s function in these pathways during development remains largely unexplored. In this work, we have used mouse embryonic stem cells (mESC) and induced pluripotent stem cells (iPSCs) as models that recapitulate early vascular morphogenesis.^{25, 26, 27} ESC and iPSC form multi-cellular aggregates (embryoid bodies, EBs) that, under appropriate conditions, generate functional EC.²⁸ mESC and iPSC differentiation capacity into ECs has been fully addressed.^{29, 30} We have also performed retinal vascularization analysis to assess vascular processes in vivo.^{31, 32}We demonstrate that p73 deficiency perturbs density and stability of mouse retinal development by affecting VEGF and TGF-β signaling. Furthermore, p73 is necessary for the assembly of vascular structures under physiological conditions in mESC and iPSC. Moreover, DNp73 positively affects angiogenesis through regulation of the TGF-β pathway in human umbilical vein cells (HUVEC) and DNp73-overexpression results in enhanced angiogenic potential of B16-F10 melanoma cells.     

Analysis of p53 and NF-κB signaling in modulating the cardiomyocyte fate during hypertrophy

Cardiac hypertrophy leading to eventual heart failure is the most common cause of mortality throughout the world. The triggering mechanisms for cardiac hypertrophy are not clear but both apoptosis and cell proliferation have been reported in sections of failing hearts. In this study, we utilized both angiotensin II (AngII) treatment of cardiomyocytes and aortic ligation in rats (Rattus norvegicus, Wistar strain) for induction of hypertrophy to understand the cellular factors responsible for activation of apoptotic or anti-apoptotic pathway. Hypertrophy markers (ANF, β-MHC), apoptotic proteins (Bax, Bad, Fas, p53, caspase-3, PARP), and anti-apoptotic or cell proliferation marker proteins (Bcl2, NF-κB, Ki-67) were induced significantly during hypertrophy, both in vitro as well as in vivo. Co-localization of both active caspase-3 and Ki-67 was observed in hypertrophied myocytes. p53 and NF-κBp65 binding to co-activator p300 was also increased in AngII treated myocytes. Inhibition of p53 resulted in downregulation of apoptosis, NF-κB activation, and NF-κB-p300 binding; however, NF-κB inhibition did not inhibit apoptosis or p53-p300 binding. Blocking of either p53 or NF-κB by specific inhibitors resulted in decrease in cell proliferation and hypertrophy markers, suggesting that p53 initially binds to p300 and then this complex recruits NF-κB. Thus, these results indicate the crucial role of p53 in regulating both apoptotic and cell proliferation during hypertrophy.     

Jolkinolide B inhibits RANKL-induced osteoclastogenesis by suppressing the activation NF-κB and MAPK signaling pathways

Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. The unique function and ability of osteoclasts to resorb bone makes them critical in both normal bone homeostasis and pathologic bone diseases such as osteoporosis and rheumatoid arthritis. Thus, new compounds that may inhibit osteoclastogenesis and osteoclast function may be of great value in the treatment of osteoclast-related diseases. In the present study, we examined the effect of jolkinolide B (JB), isolated from the root of Euphorbia fischeriana Steud on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. We found that JB inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages (BMMs) without cytotoxicity. Furthermore, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), and calcitonin receptor (CTR), was significantly inhibited. JB inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκBα degradation. Moreover, JB inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases (p38, JNK, and ERK). This study thus identifies JB as an inhibitor of osteoclast formation and provides evidence that JB might be an alternative medicine for preventing and treating osteolysis.     

Notch and NF-κB signaling pathways in the biology of classical Hodgkin lymphoma

Classical Hodgkin lymphoma (cHL) is now recognized as a B-cell-derived lymphoma which is characterized by only about 1% malignant pathognomonic Hodgkin and Reed-Sternberg (HRS) cells and an abundant infiltrate of reactive bystander cells. HRS cells are unique with respect to their lost B-cell-specific gene expression pattern and recurrent genetic lesions. Aberrant activity of Notch signaling, a highly conserved developmental pathway, acts as a negative regulator of the B cell program in HRS cells and thereby contributes to their reprogramming. Another striking feature and the major pathogenetic mechanism in HRS cells is constitutive NF-κB activation. A number of aberrations that contribute to canonical NF-κB activity in HRS cells have been described such as genetic lesions, deregulated receptor signaling and Epstein-Barr virus (EBV) infection. The importance of Notch and NF-κB signaling for cHL pathogenesis, their potential cross-talk and implications for future therapeutic applications are being discussed.     

MiR-373 exacerbates renal injury and fibrosis via NF-κB/MatrixMetalloproteinase-9 signaling by targeting Sirtuin1

BackgroundRenal fibrosis is a final common pathway of chronic kidney disease. SIRT1, a NAD⁺-dependent protein deacetylase, deacetylates the p65 of NF-κB and shows protective effects in kidney disorders. miR-373 directly targets the 3′UTR of SIRT1. However, roles of miR-373 in renal fibrosis are unclear.MethodsTGF-β1, a critical regulator of fibrosis, was used to stimulate human kidney-2 cells to establish cell model for renal fibrosis. Unilateral ureteral obstruction (UUO) was performed as an in vivo model.ResultsTGF-β1 induced the level of miR-373, reduced level of SIRT1, and promoted p65 acetylation and MMP-9 expression. These effects were reversed by the miR-373 inhibitor. In the animal model, UUO caused a consistent pattern as demonstrated in vitro.ConclusionThese results indicated an undesired effect of miR-373 in the regulation of renal injury and fibrosis by targeting SIRT1-mediated NF-κB/MMP-9 signaling, which might provide a potential therapeutic strategy for renal fibrosis.     

Contribution of p38 MAPK, NF-κB and glucocorticoid signaling pathways to ER stress-induced increase in retinal endothelial permeability

Diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. Endoplasmic reticulum (ER) stress is known to play a pathogenic role in vascular impairment in DR. The present study demonstrated that the treatment of human retinal endothelial cells with ER stress inducers such as thapsigargin (Tg) and tunicamycin (Tm) significantly increased the permeability of exogenously added FITC-dextran, accompanied by a decrease of transendothelial electrical resistance (TEER). The expression of claudin-5 among tight junction proteins was significantly decreased by the treatment with Tg or Tm. A p38 MAPK inhibitor, SB203580, and an NF-κB inhibitor, dexamethasone, significantly suppressed the Tg-induced down-regulation of claudin-5, decrease of TEER and leakage of added FITC-dextran. The translocation of NF-κB p65 subunit to the nucleus was also inhibited by the addition of SB203580 or dexamethasone. The effects of dexamethasone are thought to be due to the transrepression of the above signaling and direct regulation of claudin-5 gene.     

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-κB and upregulating endothelial nitric oxide synthase

The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-κB activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP+EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-l-arginine methyl ester (l-NAME) simultaneously with EPO administration (CLP+EPO+l-NAME). A fifth group (CLP+EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP+EPO rats presented significantly higher inulin clearance than did CLP and CLP+EPO+l-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP+EPO rats; and inulin clearance was significantly higher in CLP+EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP+EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-α activation, NF-κB activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-κB downregulation.     

Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-κB and MAPK signaling pathways

The receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-RANK regulatory axis is a major regulator of osteoclast differentiation and activation. Icariin, a flavonol glycoside isolated from the Epimedium herb, has been reported to prevents bone loss in ovariectomized mice and inhibits wear particle-induced osteolysis. However, the molecular mechanism through which icariin inhibits RANKL-induced osteoclastogenesis has not been fully understood. Therefore, we aimed to investigate the effects of icariin on RANKL-induced osteoclastogenesis and to elucidate the mechanism underlying this effect. Our results showed that RANKL-induced osteoclastogenesis was inhibited by icariin in bone marrow macrophages (BMMs) and RAW264.7?cells, and that this effect was due to suppression of NF-κB and mitogen-activated protein kinase (MAPK) activation. In addition, icariin inhibited F-actin ring formation and attenuated the bone resorption ability of mature osteoclasts. Collectively, our results indicate that icariin may be a promising potential candidate for the treatment of osteolytic diseases such as osteoporosis. Moreover, our findings lay the foundation for understanding and intervening in osteoclast-related diseases at the molecular level.     

Amelioration of experimental autoimmune encephalomyelitis by plumbagin through down-regulation of JAK-STAT and NF-κB signaling pathways

Plumbagin (PL), a herbal compound derived from roots of the medicinal plant Plumbago zeylanica, has been shown to have immunosuppressive properties. Present report describes that PL is a potent novel agent in control of encephalitogenic T cell responses and amelioration of mouse experimental autoimmune encephalomyelitis (EAE), through down-regulation of JAK-STAT pathway. PL was found to selectively inhibit IFN-γ and IL-17 production by CD4(+) T cells, which was mediated through abrogated phosphorylation of JAK1 and JAK2. Consistent with IFN-γ and IL-17 reduction was suppressed STAT1/STAT4/T-bet pathway which is critical for Th1 differentiation, as well as STAT3/ROR pathway which is essential for Th17 differentiation. In addition, PL suppressed pro-inflammatory molecules such as iNOS, IFN-γ and IL-6, accompanied by inhibition of IκB degradation as well as NF-κB phosphorylation. These data give new insight into the novel immune regulatory mechanism of PL and highlight the great value of this kind of herb compounds in probing the complex cytokine signaling network and novel therapeutic targets for autoimmune diseases.