Nimbolide ameliorates the streptozotocin-induced diabetic retinopathy in rats through the inhibition of TLR4/NF-κB signaling pathway

BackgroundDiabetic retinopathy (DR) is a common problem in the diabetic patients due to the high blood glucose level. DR affects more number of diabetic patients worldwide with irreversible vision loss.ObjectiveThe current investigation was focused to reveal the therapeutic actions of nimbolide against the streptozotocin (STZ)-provoked DR in rats through inhibition of TLR4/NF-κB pathway.MethodologyDR was provoked to the rats through administering a single dose of STZ (60 mg/kg) intraperitoneally. The DR rats were then supplemented with the 50 mg/kg of nimbolide for 60 days. The bodyweight and blood glucose level was measured using standard methods. The lipid profiles (cholesterol, TG, LDL, and HDL), inflammatory markers, and antioxidants level was detected using respective kits. The level of MCP-1, VEGF, and MMP-9 was quantified using kits. The morphometric analysis of retinal tissues were done. The mRNA expressions of target genes were studied using RT-PCR assay.ResultsNimbolide treatment effective decreased the food intake and blood glucose, and improved the bodyweight of STZ-provoked animals. The levels of pro-inflammatory mediators, cholesterol, TG, LDL, and HDL, MCP-1, VEGF, and MMP-9 was remarkably suppressed by the nimbolide treatment. Nimbolide also improved the antioxidants, retinal thickness and cell numbers. The TLR4/NF-κB pathway was appreciably inhibited by the nimbolide.ConclusionOverall, our findings demonstrated that the nimbolide attenuated the STZ-provoked DR in rats through inhibiting the TLR4/NF-κB pathway.     

Foxf1 gene increases the risk of osteoporosis in rats by inhibiting osteoblast formation and promoting osteoclast differentiation through the upregulation of NF-κB pathway

Objectives:a) To explore the expression of Foxf1 and NF-κB in bone tissue of ovariectomized rats with osteoporosis and b) to investigate the role and mechanism of NF-κB pathway regulated by Foxf1 gene in the differentiation and formation of rat osteoclasts and osteoblasts with cell experiments.Methods:Ovariectomized rat model of osteoporosis was established with 3-month-old female SD rats. The rats were divided into sham group (n=10) and osteoporosis group (n=10). Real time fluorescent quantitative PCR and Western blot were used to detect the expression levels of Foxf1 and NF-κB genes and proteins in the femur tissues of rats and analyze their correlation.Results:Both Foxf1 and NF- κB were highly expressed in the femur tissues. Upon the overexpression of Foxf1 gene in osteoblasts and osteoclasts in vitro, the gene and protein expression of NF-κB were also upregulated, significantly reducing the gene and protein expression levels of osteogenic factors, including ATF4, OCN, ALP and Runx2.Conclusions:Foxf1 gene could inhibit osteoblast formation and promote osteoclast differentiation by NF-κB pathway, which may increase the risk of osteoporosis in rats.     

Role of TLR4/NF-κB in Damage to Intestinal Mucosa Barrier Function and Bacterial Translocation in Rats Exposed to Hypoxia

The role of Toll-like receptor 4 (TLR4)/nuclear factor-kappa-B (NF-κB) in intestinal mucosal barrier damage and bacterial translocation under hypoxic exposure is unclear. Here, we investigated their role using an acute hypobaric hypoxia model. Adult Sprague-Dawley rats were divided into control (C), hypoxia (H), hypoxia+NF-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) (100 mg. kg) (HP), hypoxia+0.5 mg/kg lipopolysaccharide (HPL), and hypoxia+PDTC+LPS (HPL) group. Except control group, other four groups were placed in a hypobaric chamber set at 7000 m. Samples were collected at 72 h after pressure reduction. Damage in ultrastructure of the intestinal tract was examined by transmission electron microscopy and bacterial translocation was detected by cultivation. Kinetic turbidimetric assay was used to measure the serum LPS.ELISA was performed to detect TNF-α and IL-6 serum concentrations. Fluorescent quantitative RT-PCR was used to measure TLR4 mRNA levels was measured using quantitative RT-PCR and protein of NF-κB p65 was measured by western blotting. Different degrees of intestinal mucosa damage were observed in groups H and HL. The damage was significantly alleviated after blockage of the TLR4/NF-κB signaling pathway. PDTC- treatment also reversed hyoxia- and LPS-induced bacterial translocation rate and increased serum levels of LPS, TNF-α, and IL-6. TLR4 mRNA levels and NF-κB p65 expression were consistent with the serum factor results. This study suggested that TLR4 and NF-κB expression increased in rat intestinal tissues after acute hypoxia exposure. PDTC-treatment reversed TLR4 and NF-κB upregulation and alleviated damage to the intestinal tract and bacterial translocation. Thus, the TLR4/NF-κB signaling pathway may be critical to the mechanism underlying hypoxia-induced damage to intestinal barrier function and bacterial translocation.     

Activation of BV2 microglia by lipopolysaccharide triggers an inflammatory reaction in PC12 cell apoptosis through a toll-like receptor 4-dependent pathway

Microglia play an important role in neuronal protection and damage. However, the molecular and cellular relationship between microglia and neurons is unclear. We carried out a prospective study to detect that activation of BV2 microglia induced PC12 cell apoptosis in vitro through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. BV2 microglia were treated with different concentrations of LPS for 24 h. Western blot was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using a specific ELISA kit. The supernatant of 10 μg/ml LPS-treated BV2 cells was used as conditioned medium (CM). PC12 cells were co-culture with CM for 24 h. Cell viability was determined by MTT assay and cell apoptosis was tested by flow cytometry. BV2 microglia were treated with 10, 20, or 30 μg/ml LPS for 24 h. The expression of TLR4, MyD88, and NF-κB significantly increased. When PC12 cells were co-cultured with CM for 24 h, cell viability decreased. CM up-regulated the Bax level and down-regulated the Bcl-2 protein level in PC12 cells. PC12 cells pretreated with interleukin-1 receptor antagonist (IL-1RA) for 30 min, significantly alleviated CM-induced PC12 cell apoptosis. These results suggest that BV2 microglia activated by LPS triggered TLR4/MyD88/NF-κB signaling pathway that induced the release of IL-1β and could participate in the PC12 cells injury.     

TAK1-ECSIT-TRAF6 Complex Plays a Key Role in the TLR4 Signal to Activate NF-κB

ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) is known as a multifunctional regulator in different signals, including Toll-like receptors (TLRs), TGF-β, and BMP. Here, we report a new regulatory role of ECSIT in TLR4-mediated signal. By LPS stimulation, ECSIT formed a high molecular endogenous complex including TAK1 and TRAF6, in which ECSIT interacted with each protein and regulated TAK1 activity, leading to the activation of NF-κB. ECSIT-knockdown THP-1 (ECSIT^KD THP-1) cells exhibited severe impairments in NF-κB activity, cytokine production, and NF-κB-dependent gene expression, whereas those were dramatically restored by reintroduction of wild type (WT) ECSIT gene. Interestingly, ECSIT mutants, which lack a specific interacting domain for either TAK1 or TRAF6, could not restore these activities. Moreover, no significant changes in both NF-κB activity and cytokine production induced by TLR4 could be seen in TAK1^KD or TRAF6^KD THP-1 cells transduced by WT ECSIT, strongly suggesting the essential requirement of TAK1-ECSIT-TRAF6 complex in TLR4 signaling. Taken together, our data demonstrate that the ECSIT complex, including TAK1 and TRAF6, plays a pivotal role in TLR4-mediated signals to activate NF-κB.     

Changes in the Expression of the Toll-Like Receptor System in the Aging Rat Kidneys

Background

The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Toll-like receptors (TLRs) are one of the receptor types of the body''s innate immune system. The function of the TLR system and the mechanisms by which it functions in renal aging remain unclear. In the present study, we, for the first time, systematically investigated the role of the TLR system and the inflammation responses activated by TLRs during kidney aging.

Methods

We used western blot and immunohistochemistry to systematically analyze the changes in the expression and activation of the endogenous TLR ligands HSP70 and HMGB1, the TLRs (TLR1–TLR11), their downstream signaling pathway molecules MyD88 and Phospho-IRF-3, and the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα (NF-κB inhibition factor α), NF-κBp65, and Phospho-NF-κBp65 (activated NF-κB p65) in the kidneys of 3 months old (youth group), 12 months old (middle age group), and 24 months old (elderly group) rats. We used RT-qPCR to detect the mRNA expression changes of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b in the rat renal tissues of the various age groups.

Results

We found that during kidney aging, the HSP70 and HMGB1 expression levels were significantly increased, and the expression levels of TLR1, 2, 3, 4, 5, and 11 and their downstream signaling pathway molecules MyD88 and Phospho-IRF-3 were markedly elevated. Further studies have shown that in the aging kidneys, the expression levels of the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα, NF-κBp65, and Phospho-NF-κBp65 were obviously increased, and those of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b were significantly upregulated.

Conclusions

These results showed that the TLR system might play an important role during the kidney aging process maybe by activating the NF-κB signaling pathway and promoting the high expression of inflammation factors.     

Hydrogen Sulfide Suppresses Oxidized Low-density Lipoprotein (Ox-LDL)-stimulated Monocyte Chemoattractant Protein 1 generation from Macrophages via the Nuclear Factor κB (NF-κB) Pathway

This study was designed to examine the role of hydrogen sulfide (H₂S) in the generation of oxidized low-density lipoprotein (ox-LDL)-stimulated monocyte chemoattractant protein 1 (MCP-1) from macrophages and possible mechanisms. THP-1 cells and RAW macrophages were pretreated with sodium hydrosulfide (NaHS) and hexyl acrylate and then treated with ox-LDL. The results showed that ox-LDL treatment down-regulated the H₂S/cystathionine-β-synthase pathway, with increased MCP-1 protein and mRNA expression in both THP-1 cells and RAW macrophages. Hexyl acrylate promoted ox-LDL-induced inflammation, whereas the H₂S donor NaHS inhibited it. NaHS markedly suppressed NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter in ox-LDL-treated macrophages. Furthermore, NaHS decreased the ratio of free thiol groups in p65, whereas the thiol reductant DTT reversed the inhibiting effect of H₂S on the p65 DNA binding activity. Most importantly, site-specific mutation of cysteine 38 to serine in p65 abolished the effect of H₂S on the sulfhydration of NF-κB and ox-LDL-induced NF-κB activation. These results suggested that endogenous H₂S inhibited ox-LDL-induced macrophage inflammation by suppressing NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter. The sulfhydration of free thiol group on cysteine 38 in p65 served as a molecular mechanism by which H₂S inhibited NF-κB pathway activation in ox-LDL-induced macrophage inflammation.     

Protective effects of the notoginsenoside R1 on acute lung injury by regulating the miR-128-2-5p/Tollip signaling pathway in rats with severe acute pancreatitis

Notoginsenoside R1 (NG-R1), the extract and the main ingredient of Panax notoginseng, has anti-inflammatory effects and can be used in treating acute lung injury (ALI). In this study, we explored the pulmonary protective effect and the underlying mechanism of the NG-R1 on rats with ALI induced by severe acute pancreatitis (SAP). MiR-128-2-5p, ERK1, Tollip, HMGB1, TLR4, IκB, and NF-κB mRNA expression levels were measured using real-time qPCR, and TLR4, Tollip, HMGB1, IRAK1, MyD88, ERK1, NF-κB65, and P-IκB-α protein expression levels using Western blot. The NF-κB and the TLR4 activities were determined using immunohistochemistry, and TNF-α, IL-6, IL-1β, and ICAM-1 levels in the bronchoalveolar lavage fluid (BALF) using ELISA. Lung histopathological changes were observed in each group. NG-R1 treatment reduced miR-128-2-5p expression in the lung tissue, increased Tollip expression, inhibited HMGB1, TLR4, TRAF6, IRAK1, MyD88, NF-κB65, and p-IκB-α expression levels, suppressed NF-κB65 and the TLR4 expression levels, reduced MPO activity, reduced TNF-α, IL-1β, IL-6, and ICAM-1 levels in BALF, and alleviated SAP-induced ALI. NG-R1 can attenuate SAP-induced ALI. The mechanism of action may be due to a decreased expression of miR-128-2-5p, increased activity of the Tollip signaling pathway, decreased activity of HMGB1/TLR4 and ERK1 signaling pathways, and decreased inflammatory response to SAP-induced ALI. Tollip was the regulatory target of miR-128-2-5p.     

Chlamydophila pneumoniae induces expression of Toll-like Receptor 4 and release of TNF-α and MIP-2 via an NF-κB pathway in rat type II pneumocytes

Background

The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-κB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-α and MIP-2.

Methods

Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-κB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-κB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-α and MIP-2 release by ELISA.

Results

Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1–2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56°C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 μg/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-κB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-α and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-α and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression.

Conclusion

The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca²⁺-mediated NF-κB activity, necessary for maintenance of TLR4 expression and TNF-α and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.     

Hyperglycemia Exacerbates Burn-Induced Liver Inflammation via Noncanonical Nuclear Factor-κB Pathway Activation

Hyperglycemia and inflammation are hallmarks of burn injury. In this study, we used a rat model of hyperglycemia and burn injury to investigate the effects of hyperglycemia on inflammatory responses in the liver. Hyperglycemia was induced in male Sprague-Dawley rats with streptozotocin (STZ) (35–40 mg/kg), followed by a 60% third-degree scald burn injury. Cytokine levels (by multiplex, in cytosolic liver extracts), hormones (by enzyme-linked immunosorbent assay [ELISA], in serum), nuclear factor (NF)-κB protein deoxyribonucleic acid (DNA) binding (by ELISA, in nuclear liver extracts) and liver functional panel (using VetScan, in serum) were measured at different time points up to 7 d after burn injury. Blood glucose significantly increased after burn injury in both groups with different temporal patterns. Hyperglycemic rats were capable of endogenous insulin secretion, which was enhanced significantly versus controls 12 h after burn injury. DNA binding data of liver nuclear extracts showed a robust and significant activation of the noncanonical NF-κB pathway in the hyperglycemic versus control burn animals, including increased NF-κB–inducing kinase expression (p < 0.05). Liver acute-phase proteins and cytokine expression were increased, whereas secretion of constitutive proteins was decreased after burn injury in hyperglycemic versus control animals (p < 0.05). These results indicate that burn injury to the skin rapidly activated canonical and noncanonical NF-κB pathways in the liver. Robust activation of the NF-κB noncanonical pathway was associated with increased expression of inflammatory markers and acute-phase proteins, and impaired glucose metabolism. Hyperglycemia is detrimental to burn outcome by augmenting inflammation mediated by hepatic noncanonical NF-κB pathway activation.     

Circulating nuclear factor-kappa B mediates cancer-associated inflammation in human breast and colon cancer

BackgroundInflammation is recognized as a hallmark feature of cancer development and progression. The aim of our study was to investigate the significance of serum nuclear factor kappa-B (NF-κB) levels as a circulating marker in the monitoring of inflammation in breast and colon cancer; to show the relationship between NF-κB with inflammatory parameters as tumour necrosis factor-α (TNF-α), soluble TNF-related apoptosis-inducing ligand (sTRAIL), interleukin-6 (IL-6), pentraxin-3 (PTX-3), procalcitonin (PCT), and C-reactive protein (CRP) levels.MethodsSerum NF-κB, TNF-α, sTRAIL, IL-6, PTX-3, PCT, and serum CRP levels were measured using enzyme-linked immunosorbent assay (ELISA) in 40 patients with breast cancer, 40 patients with colon cancer and 30 healthy controls.ResultsThe serum NF-κB, TNF-α, IL-6, PTX-3, PCT, and serum CRP concentration was significantly higher, and the serum sTRAIL concentration was significantly lower in the patients with breast and colon cancer than in healthy controls. NF-κB was positively correlated with CRP and negatively correlated with sTRAIL.ConclusionsThese results suggest that increased NF-κB may decrease the clinical efficacy of sTRAIL in solid tumour cells. There is a relationship between inflammation and carcinogenesis so that the development of cancer occurs with chronic inflammation in breast and colon. The study results have shown that colon and breast cancer patients have increased systemic inflammation, as measured by increased circulating cytokines, and acute-phase proteins, or by abnormalities in circulating cells. NF-κB may combine with other markers of the systemic inflammatory response in prognostic scores in cancer. In addition to surgical resection of the tumour, and conventional radio and chemotherapy for cancer treatment, the use of sTRAIL or other agonists for cancer therapy appeared a new potential therapy.     

The Role of the Phylogenetically Conserved Cochaperone Protein Droj2/DNAJA3 in NF-κB Signaling

The NF-κB pathway is a phylogenetically conserved signaling pathway with a central role in inflammatory and immune responses. Here we demonstrate that a cochaperone protein, Droj2/DNAJA3, is involved in the activation of canonical NF-κB signaling in flies and in human cultured cells. Overexpression of Droj2 induced the expression of an antimicrobial peptide in Drosophila. Conversely, Droj2 knockdown resulted in reduced expression of antimicrobial peptides and higher susceptibility to Gram-negative bacterial infection in flies. Similarly, Toll-like receptor-stimulated IκB phosphorylation and NF-κB activation were suppressed by DNAJA3 knockdown in HEK293 cells. IκB kinase overexpression-induced NF-κB phosphorylation was also compromised in DNAJA3 knockdown cells. Our study reveals a novel conserved regulator of the NF-κB pathway acting at the level of IκB phosphorylation.     

Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

Calcific aortic valve disease (CAVD) is characterized by chronic inflammation and progressive calcification in valve leaflets. Aortic valve interstitial cells (AVICs) play a critical role in the pathogenesis of CAVD. Previous studies show that stimulation of Toll-like receptor (TLR) 2 or TLR4 in AVICs in vitro up-regulates the expression of osteogenic mediators. Double-stranded RNA (dsRNA) can activate pro-inflammatory signaling through TLR3, the NLRP3 inflammasome and RIG-I-like receptors. The objective of this study is to determine the effect of dsRNA on AVIC osteogenic activities and the mechanism of its action. Methods and results: AVICs isolated from normal human valves were exposed to polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Treatment with poly(I:C) increased the production of bone morphogenetic protein-2 (BMP-2), transforming growth factor beta-1 (TGF-β1) and alkaline phosphatase (ALP), and resulted in calcium deposit formation. Poly(I:C) induced the phosphorylation of NF-κB and ERK1/2. Knockdown of TLR3 essentially abrogated NF-κB and ERK1/2 phosphorylation, and markedly reduced the effect of poly(I:C) on the production of BMP-2, TGF-β1 and ALP. Further, inhibition of either NF-κB or ERK1/2 markedly reduced the levels of BMP-2, TGF-β1 and ALP in cells exposed to poly(I:C). Conclusion: Poly(I:C) up-regulates the production of BMP-2, TGF-β1 and ALP, and promotes calcium deposit formation in human AVICs. The pro-osteogenic effect of poly(I:C) is mediated primarily by TLR3 and the NF-κB and ERK1/2 pathways. These findings suggest that dsRNA, when present in aortic valve tissue, may promote CAVD progression through up-regulation of AVIC osteogenic activities.