Suppressed P65 Phosphorylation Research Articles

FuKe QianJin capsule alleviates endometritis via inhibiting inflammation and pyroptosis through modulating TLR4/ NF-κB /NLRP3 pathway

Ethnopharmacological relevanceFuke Qianjin Capsule (FKC), a traditional Chinese medicine commonly employed for treating endometritis, lacks reported treatment mechanisms. Aim of the studyThe aim of the present study was to explore the role and mechanism of FKC in lipopolysaccharide (LPS)-induced endometritis. Materials and methodsThe main active ingredients of FKC were identified via high-performance liquid chromatography (HPLC) in conjunction with standard substances. Prior to endometritis induction, Sprague Dawley female rats received FKC for 7 days. The endometritis model was established through an intrauterine injection of 1 mg/kg LPS. Concurrently, an LPS-induced RAW264.7 cell inflammation model was utilized, in which the cells were treated with serum containing Fuke Qianjin Capsule. Pathological alterations in the endometrium were assessed via H&E staining and transmission electron microscopy (TEM). The contents of MPO in uterine tissues, and NO release in cells, along with the secretion of IL-18, IL-1β, IL-6, and TNF-α in both tissues and cells, were determined via assay kits. The mRNA levels of Nlrp3, Caspase-1, Gsdmd, and Il-1β in uterine tissues and cells were analyzed via qPCR. The protein levels of TLR4, p65, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β in these samples were evaluated through Western blot analysis. Immunofluorescence was used to assess the protein levels of p-P65 and NLRP3 in uterine tissues and cells. ResultsFive primary active components of FKC were identified. Treatment with FKC in vivo mitigated endometrial pathological damage and significantly decreased the levels of MPO, IL-18, IL-1β, IL-6, and TNF-α, as well as the levels of Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA in tissue samples. Treatment with FKC inhibited the expression of TLR4, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β, as well as reduced NLRP3 protein fluorescence intensity, and inhibited P65 phosphorylation. In vitro findings demonstrated that FKC-containing serum reduced IL-18, IL-1β, IL-6, and TNF-α levels, as well as reduced Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA levels. In addition, FKC-containing serum inhibited the protein expression of TLR4, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β. FKC-containing serum also reduced NLRP3 protein fluorescence intensity and suppressed P65 phosphorylation. ConclusionFKC reverses the LPS induced NLRP3 inflammasome activation, and mitigates inflammation and pyroptosis through the modulation of the TLR4/NF-κB/NLRP3 pathway, thereby alleviating endometritis.

PRELP inhibits the progression of oral squamous cell carcinoma via inactivation of the NF-κB pathway

ObjectivesThe aim of this study was to investigate the role and molecular mechanism of proline/arginine-rich end leucine-rich repeat protein (PRELP), a secreted protein in extracellular matrix, in oral squamous cell carcinoma (OSCC) progression. DesignPRELP expression in OSCC was analyzed in the Gene Set Enrichment (GSE) 138206, GSE37991, and GSE23558 datasets as well as cell lines. Also, PRELP expression and its relationship with prognosis and immune infiltration in head and neck squamous cell carcinoma (HNSCC) were confirmed by bioinformatics analysis. The proliferation, apoptosis, invasion, epithelial-to-mesenchymal transition (EMT) and NF-κB activation were detected after alteration of PRELP expression in OSCC cells using CCK-8, EdU, flow cytometry, Transwell, real-time PCR, immunofluorescence and Western blot. Additionally, an NF-κB inhibitor PDTC was used to confirm the regulation mechanism of PRELP. ResultsThe expression of PRELP in OSCC tissues, cells and in HNSCC samples was low. HNSCC patients with higher PRELP expression was associated with longer overall survival. A positive correlation between PRELP expression and immune cell infiltration was found in HNSCC. Upregulation of PRELP inhibited, whereas PRELP silencing promoted, the proliferation, invasion and EMT of OSCC cells. Also, overexpression of PRELP promoted cell apoptosis. Mechanistically, PRELP suppressed p65 phosphorylation and nuclear translocation. And PDTC treatment partially reversed the influences of PRELP knockdown on the malignant behaviors in OSCC cells. ConclusionPRELP suppressed OSCC progression via inactivation of the NF-κB pathway. Targeting PRELP may be a potential approach for OSCC treatment.

AIM2 participates in house dust mite (HDM)-induced epithelial dysfunctions and ovalbumin (OVA)-induced allergic asthma in infant mice

Objective: Allergen sensitization and high rates of concomitant allergic diseases are characteristic of severe pediatric asthma. The present study was aimed to explore the mechanism of allergic asthma via bioinformatics and experiment investigation. Methods: The GSE27011 dataset contained the expression profiles of normal and pediatric asthma white blood cells was downloaded for analyzing the different expression genes and function enrichment. The allergic asthma model in infant mice was established by ovalbumin (OVA) stimulation. The cellular model was established by house dust mite (HDM)-stimulation in human bronchial epithelial cells. The absent in melanoma 2 (AIM2) knockdown was achieved by intranasal lentivirus injection or cell infection. The bronchoalveolar lavage fluid (BALF) was collected for cell counting and ELISA assessment of cytokines. Lung tissues were collected for HE staining and immunohistochemical (IHC) staining. Real-time PCR and immunoblotting were used for the determination of key gene expressions in mouse and cell models. Results: upregulation of AIM2 gene expression was observed in pediatric asthma patients based on GSE27011 and OVA-induced infant mouse allergic asthma model. AIM2 knockdown ameliorated OVA caused elevation in airway hyper-responsiveness (AHR), elevation in cell quantities (eosinophils, neutrophils, lymphocytes), and levels of cytokines (IL-4, IL-13, TNF-α, and OVA-specific IgE) in BALF. Moreover, AIM2 knockdown relieved OVA-caused histopathological alterations in mouse lungs, up-regulation of AIM2 levels, and NOD1 and receptor-interacting protein 2 (RIP2) protein levels, as well as p65 phosphorylation. In the cell model, AIM2 knockdown partially ameliorated HDM-induced epithelial dysfunctions by promoting cell viability, down-regulating inflammatory cytokines levels, and decreasing the protein levels of AIM2, NOD1, RIP2, and phosphorylated p65. Conclusion: AIM2 participates in HDM-induced epithelial dysfunctions and OVA-induced allergic asthma progression. AIM2 could be a promising target for pediatric allergic asthma treatment regimens, which warrants further in vivo investigations.

Jietacin Derivative Inhibits TNF-α-Mediated Inflammatory Cytokines Production via Suppression of the NF-κB Pathway in Synovial Cells.

Synovial inflammation plays a central role in joint destruction and pain in osteoarthritis (OA). The NF-κB pathway plays an important role in the inflammatory process and is activated in OA. A previous study reported that a jietacin derivative (JD), (Z)-2-(8-oxodec-9-yn-1-yl)-1-vinyldiazene 1-oxide, suppressed the nuclear translocation of NF-κB in a range of cancer cell lines. However, the effect of JD in synovial cells and the exact mechanism of JD as an NF-κB inhibitor remain to be determined. We investigated the effect of JD on TNF-α-induced inflammatory reaction in a synovial cell line, SW982 and human primary synovial fibroblasts (hPSFs). Additionally, we examined phosphorylated levels of p65 and p38 and expression of importin α3 and β1 using Western blotting. RNA-Seq analysis revealed that JD suppressed TNF-α-induced differential expression: among 204 genes significantly differentially expressed between vehicle and TNF-α-stimulated SW982 (183 upregulated and 21 downregulated) (FC ≥ 2, Q < 0.05), expression of 130 upregulated genes, including inflammatory cytokines (IL1A, IL1B, IL6, IL8) and chemokines (CCL2, CCL3, CCL5, CCL20, CXCL9, 10, 11), was decreased by JD treatment and that of 14 downregulated genes was increased. KEGG pathway analysis showed that DEGs were increased in the cytokine−cytokine receptor interaction, TNF signaling pathway, NF-κB signaling pathway, and rheumatoid arthritis. JD inhibited IL1B, IL6 and IL8 mRNA expression and IL-6 and IL-8 protein production in both SW982 and hPSFs. JD also suppressed p65 phosphorylation in both SW982 and hPSFs. In contrast, JD did not alter p38 phosphorylation. JD may inhibit TNF-α-mediated inflammatory cytokine production via suppression of p65 phosphorylation in both SW982 and hPSFs. Our results suggest that JD may have therapeutic potential for OA due to its anti-inflammatory action through selective suppression of the NF-κB pathway on synovial cells.

Therapeutic Effect of Biejiaxiaozheng Pills on Carbon Tetrachloride-Induced Hepatic Fibrosis in Rats through the NF-κB/Nrf2 Pathway.

Aims To explore the effects of Biejiaxiaozheng pills on carbon tetrachloride-induced hepatic fibrosis in rats through the NF-κB/Nrf2 pathway and to explore the possible antifibrotic mechanisms of the drug. Material and Method. A rat model of hepatic fibrosis was established via CCl4 induction. Liver function and antioxidant indices were detected using commercial kits. Hematoxylin-eosin and Masson staining were used to detect pathological changes in hepatic tissues. ELISA was used to measure plasma TNF-α, IL-β, and IL-6 levels. RT-PCR was used to measure changes in TNF-α, IL-β, and IL-6 levels in hepatic tissues. Changes in p65, P-p65, Nrf2, and HO-1 protein expression were detected using western blotting. Results In rats with hepatic fibrosis, Biejiaxiaozheng pills effectively improved liver function, alleviated fibrosis in hepatic tissues, and significantly reduced collagen accumulation. The pills significantly downregulated inflammatory cytokine expression in hepatic tissues by suppressing p65 phosphorylation and reduced plasma inflammatory cytokine levels to some extent. The pills upregulated Nrf2 and HO-1 expression in hepatic tissues, enhanced antioxidant potential, and upregulated plasma antioxidant levels. Conclusion Biejiaxiaozheng pills improved hepatic fibrosis symptoms and lesions in rats, likely by inhibiting the NF-κB pathway and promoting the Nrf2 pathway.

Treatment with toll-like receptor 2 inhibitor ortho-vanillin alleviates lipopolysaccharide-induced acute kidney injury in mice.

Reducing inflammation is a promising approach for the prevention and treatment of septic acute kidney injury (AKI), since AKI is characterized by excessive inflammation in the kidney. Previous studies have demonstrated that toll-like receptor 2 (TLR2) is overstimulated, which promotes inflammation by activating the NF-κB signaling pathway, in a lipopolysaccharide (LPS)-induced model of AKI mice. For the present study, it was hypothesized that TLR2 inhibition could reduce inflammation and consequently prevent septic AKI. Therefore, the potential renal protective effects of ortho-vanillin (OV), an inhibitor of TLR2, were investigated in the present study in vitro and in vivo. In vitro treatment with OV on LPS-stimulated mouse podocyte cell line MPC5 did not affect TLR2 expression but interrupted the interaction between TLR2 and its downstream adaptor MyD88, resulting in the reduction of inflammatory cytokines IL-6 and TNF-α expression. In vivo OV treatment in an LPS-challenged mouse model effectively alleviated LPS-induced kidney injury as indicated by histology analysis and the significantly reduced blood urea nitrogen and serum creatinine levels. Additionally, inflammatory cytokines TNF-α, IL-6 and IL-1β expression were also significantly reduced in mice with OV treatment. Signaling pathway analysis further demonstrated that OV treatment did not affect the expression of TLR2 and p65 but suppressed p65 phosphorylation. Taken together, data from the present study demonstrated that OV was effective in protecting renal function against LPS-induced AKI through the inhibition of TLR2/NF-κB signaling and subsequent inflammatory cytokine production. These findings indicated that OV or targeting TLR2 signaling in general, represents a novel therapeutic approach for use in the prevention and treatment of AKI.

17β-Estradiol inhibits TNF-α-induced proliferation and migration of vascular smooth muscle cells via suppression of TRAIL

Atherosclerosis is an inflammatory disease and involves migration of vascular smooth muscle cells (VSMCs). Estrogen inhibits VSMCs migration, while the underlying mechanism remains to be revealed. Recent years, there is emerging evidence showing that TNF-related apoptosis-inducing ligand (TRAIL) increases proliferation and migration of VSMCs. In this study, we investigated the regulatory effect of estrogen on TRAIL expression in VSMCs. TNF-α greatly enhanced TRAIL protein expression and stimulated VSMCs proliferation and migration. This effect was partially inhibited by the addition of TRAIL neutralizing antibody, suggesting that TRAIL is important in TNF-α-induced migration. 17β-estradiol (E2) inhibited TRAIL expression under TNF-α stimulation in a time- and concentration-dependent manner. This effect was was mimicked by ERα agonist 4′,4″,4‴-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), but not ERβ agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN), indicating that ERα is involved in this action. TNF-α led to nuclear factor kappa B (NF-κB) p65 phosphorylation and the inhibitor pyrrolidine dithiocarbama (PDTC) inhibited TRAIL expression, suggesting that NF-κB signaling is crucial for TARIL production. E2 suppressed p65 phosphorylation in VSMCs and the overexpression of p65 subunit reversed the inhibitory effect of E2 on TRAIL expression and cell proliferation and migration. Taken together, our results indicate that E2 inhibits VSMCs proliferation and migration by downregulation of TRAIL expression via suppression of NF-κB pathway.

Suppression of casein kinase 2 sensitizes tumor cells to antitumor TRAIL therapy by regulating the phosphorylation and localization of p65 in prostate cancer.

In the United States, prostate cancer (PCa) is the most commonly diagnosed cancer in males. For PCa at the late hormone-refractory stage, substantial improvement in treatment strategies is critically needed. TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, but both intrinsic and acquired resistance to TRAIL poses a huge problem in establishing clinically effective TRAIL therapies. In the present study, we examined the role played by casein kinase2 (CK2) in the TRAIL‑induced nuclear factor κ-light-chain-enhancer of activated Bcell (NF-κB) pathway in a PCa cell line. Downregulation of CK2 combined with a sub-dose of TRAIL suppressed p65 phosphorylation at serine536. The combination treatment of TRAIL and the CK2 inhibitor decreased p65 nuclear translocation. Under the treatment of a sub-dose of TRAIL, downregulation of CK2, using both genetic and pharmacological approaches, decreased the transcriptional activity of NF-κB and the expression of NF-κB downstream anti-apoptosis genes. Therefore, we provided novel molecular mechanistic insight reporting that CK2 regulates the sensitivity of PCa cells to the antitumor effect of TRAIL. This is important for understanding how the TRAIL pathway is disrupted in PCa and may help to develop an effective combinatorial therapy for PCa.

The nexus between VEGF and NFκB orchestrates a hypoxia-independent neovasculogenesis.

Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies.

Ruscogenin Mainly Inhibits Nuclear Factor-κB but Not Akt and Mitogen-Activated Protein Kinase Signaling Pathways in Human Umbilical Vein Endothelial Cells

Our previous results suggested that ruscogenin inhibited tumor necrosis factorα (TNF-α)–induced leukocyte adhesion, which correlated with its suppression of intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. In the present studies, we further examined its effects on the main signaling pathways involved in upregulation of ICAM-1 induced by TNF-α in human umbilical vein endothelial cells (HUVECs). The results showed that ruscogenin significantly suppressed p65 phosphorylation, IκB-α phosphorylation and degradation, and inhibited IκB kinaseα (IKKα) and IKKβ activation induced by TNF-α. However, it exerted weak effects on TNF-α–induced phosphorylations of p38, JNK, ERK1/2, and Akt. Overall, our results indicated that downregulation of ICAM-1 expression by ruscogenin in HUVECs might be mediated by nuclear factor-κB (NF-κB), but not by mitogen-activated protein kinase (MAPK) and Akt signaling pathways.

Gambogic acid, a novel ligand for transferrin receptor, potentiates TNF-induced apoptosis through modulation of the nuclear factor-κB signaling pathway

AbstractGambogic acid (GA), a xanthone derived from the resin of the Garcinia hanburyi, has been recently demonstrated to bind transferrin receptor and exhibit potential anticancer effects through a signaling mechanism that is not fully understood. Because of the critical role of NF-κB signaling pathway, we investigated the effects of GA on NF-κB–mediated cellular responses and NF-κB–regulated gene products in human leukemia cancer cells. Treatment of cells with GA enhanced apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, inhibited the expression of gene products involved in antiapoptosis (IAP1 and IAP2, Bcl-2, Bcl-xL, and TRAF1), proliferation (cyclin D1 and c-Myc), invasion (COX-2 and MMP-9), and angiogenesis (VEGF), all of which are known to be regulated by NF-κB. GA suppressed NF-κB activation induced by various inflammatory agents and carcinogens and this, accompanied by the inhibition of TAK1/TAB1-mediated IKK activation, inhibited IκBα phosphorylation and degradation, suppressed p65 phosphorylation and nuclear translocation, and finally abrogated NF-κB–dependent reporter gene expression. The NF-κB activation induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKKβ was also inhibited. The effect of GA mediated through transferrin receptor as down-regulation of the receptor by RNA interference reversed its effects on NF-κB and apoptosis. Overall our results demonstrate that GA inhibits NF-κB signaling pathway and potentiates apoptosis through its interaction with the transferrin receptor.

Guggulsterone Inhibits NF-κB and IκBα Kinase Activation, Suppresses Expression of Anti-apoptotic Gene Products, and Enhances Apoptosis

Guggulsterone, derived from Commiphora mukul and used to treat obesity, diabetes, hyperlipidemia, atherosclerosis, and osteoarthritis, has been recently shown to antagonize the farnesoid X receptor and decrease the expression of bile acid-activated genes. Because activation of NF-kappaB has been closely linked with inflammatory diseases affected by guggulsterone, we postulated that it must modulate NF-kappaB activation. In the present study, we tested this hypothesis by investigating the effect of this steroid on the activation of NF-kappaB induced by inflammatory agents and carcinogens. Guggulsterone suppressed DNA binding of NF-kappaB induced by tumor necrosis factor (TNF), phorbol ester, okadaic acid, cigarette smoke condensate, hydrogen peroxide, and interleukin-1. NF-kappaB activation was not cell type-specific, because both epithelial and leukemia cells were inhibited. Guggulsterone also suppressed constitutive NF-kappaB activation expressed in most tumor cells. Through inhibition of IkappaB kinase activation, this steroid blocked IkappaBalpha phosphorylation and degradation, thus suppressing p65 phosphorylation and nuclear translocation. NF-kappaB-dependent reporter gene transcription induced by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK was also blocked by guggulsterone but without affecting p65-mediated gene transcription. In addition, guggulsterone decreased the expression of gene products involved in anti-apoptosis (IAP1, xIAP, Bfl-1/A1, Bcl-2, cFLIP, and survivin), proliferation (cyclin D1 and c-Myc), and metastasis (MMP-9, COX-2, and VEGF); this correlated with enhancement of apoptosis induced by TNF and chemotherapeutic agents. Overall, our results indicate that guggulsterone suppresses NF-kappaB and NF-kappaB-regulated gene products, which may explain its anti-inflammatory activities.

Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis.

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.