NF-κB P65 Protein Levels Research Articles

Potential protective effects of rhEGF against ultraviolet A irradiation-induced damages on human fibroblasts

BackgroundUltraviolet A (UVA) rays reach the dermal skin layer and generate oxidative stress, DNA damage, and cell inflammation, which in turn lead to photo-aging and photo-carcinogenesis. While there have been many studies about the beneficial effects of topical epidermal growth factor (EGF) treatment in the healing of wounds, the effect of EGF on UVA-induced skin irritation remains unknown. To clarify the effects of EGF on UVA-induced skin damage, it was investigated whether EGF signaling can affect intracellular reactive oxygen species (ROS) and DNA damages in UVA-irradiated human dermal fibroblasts.Materials and methodsFibroblasts cultured with or without rhEGF were UVA-irradiated at 40 mJ/cm2 twice per day for 5 days. After the irradiation, the intracellular ROS levels and expression of catalase and superoxide dismutase-1 (SOD-1) in the fibroblasts were ascertained. Further investigation to determine the effects of EGF on UVA-induced DNA damage, including a single cell gel electrophoresis assay and an enzyme-linked immunosorbent assay (ELISA), was carried out. Moreover, the NF-κB activity was ascertained in order to investigate the effects of EGF on UVA-irradiated fibroblasts.ResultsAs a result, it was revealed that recombinant human EGF (rhEGF) inhibited UVA- increased intracellular ROS in the fibroblasts and increased the expression of catalase and SOD-1. Moreover, in UVA-irradiated fibroblasts, the longest DNA-damaged tails were observed, but this phenomenon was not detected in cells cotreated with both UVA and rhEGF. Also, it was observed that DNA damage induction, including that of cyclobutene pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts, and 8-hydroxy-2-deoxyguanosine, was caused by UVA irradiation. Similar to previous results, it was downregulated by rhEGF. Furthermore, rhEGF also inhibited NF-κB gene expression and the NF-κB p65 protein level in the nucleus induced by UVA irradiation.ConclusionThese results suggest that EGF might be a useful material for preventing or improving photo-aging.

Therapeutic Effect of Penta-acetyl Geniposide on Adjuvant-Induced Arthritis in Rats: Involvement of Inducing Synovial Apoptosis and Inhibiting NF-κB Signal Pathway

Previous studies demonstrated that penta-acetyl geniposide ((Ac)5GP, an acetylated derivative of geniposide) exhibited better pharmacological functions than geniposide. This study was aimed to observe the potential effect of (Ac)5GP on adjuvant-induced arthritis (AIA) in rat and explore the involved mechanisms. Rat AIA was induced by complete Freund's adjuvant. (Ac)5GP (30, 60, 120mg/kg) was given to AIA rats by intragastric administration. Paw swelling, polyarthritis index, serum pro-inflammatory cytokines levels, histological assessments of ankle joint, and proteoglycan expression were respectively measured to evaluate the therapeutic effect of (Ac)5GP on rat AIA. Immunohistochemistry for Ki67 and TUNEL assay were performed to reveal the anti-proliferative and pro-apoptotic effects of (Ac)5GP on AIA synoviocytes in vivo. Protein levels of Bcl-2, Bax, caspase 3, IκBα, p-IκBα, and NF-κB p65 in synovial tissues were detected by Western blot. We found that (Ac)5GP treatment could suppress secondary hind paw swelling, reduce polyarthritis index, decrease TNF-α and IL-1β serum levels, attenuate pathological damage of ankle joint, and promote proteoglycans expression. (Ac)5GP treatment also could reduce Ki67 positive expression rate and raise the synovial apoptosis index in synovial tissues. Additionally, (Ac)5GP (120mg/kg) could significantly decrease Bcl-2 protein level, increase Bax and cleaved caspase 3 protein levels, and normalize the ratio of Bcl-2 to Bax. Moreover, (Ac)5GP (120mg/kg) could inhibit the degradation and phosphorylation of IκBα and reduce NF-κB p65 protein level in nuclear extracts. In conclusion, (Ac)5GP showed a potent anti-arthritic effect on AIA rats via inducing synovial apoptosis and inhibiting NF-κB activation in synovial tissues.

Acetazolamide protects rat articular chondrocytes from IL-1β-induced apoptosis by inhibiting the activation of NF-κB signal pathway.

Because the excessive apoptosis of articular chondrocytes contributes to extracellular matrix (ECM) loss and cartilage damage in rheumatoid arthritis (RA), inhibiting chondrocyte apoptosis might be a promising strategy for RA. Aquaporin1 (AQP1) is overexpressed in RA cartilage and synovial tissues, and play a vital pathogenic role in RA development. Particularly, we previously reported that acetazolamide (AZ) as an AQP1 inhibitor suppressed secondary inflammation and promoted ECM production in cartilage of adjuvant-induced arthritis rats. Here, we investigated the antiapoptotic effect of AZ on interleukin-1β (IL-1β)-induced apoptosis, a classic in vitro model of chondrocyte apoptosis. AZ treatment could inhibit IL-1β-induced apoptosis, evidenced by increasing cell viability, relieving apoptotic nuclear morphology, decreasing apoptosis rates, and restoring mitochondrial membrane potential. Additionally, AZ reversed IL-1β-induced decrease of Bcl-2 protein and reduced IL-1β-induced increases of Bax and caspase 3 protein, accompanied by inhibiting IκBα degradation and phosphorylation in cytoplasm, reducing NF-κB p65 protein level in nucleus and preventing NF-κB p65 translocation from cytoplasm to nucleus. In conclusion, our findings indicated that AZ could effectively attenuate IL-1β-induced chondrocyte apoptosis mediated by regulating the protein levels of apoptosis-related genes and inhibiting the activation of NF-κB signal pathway, suggesting that AZ might be of potential clinical interest in RA treatment.

Hepatoprotective Effects of Lactobacillus on Carbon Tetrachloride-Induced Acute Liver Injury in Mice.

The aim of this study was to investigate and compare the effects of heat-killed and live Lactobacillus on carbon tetrachloride (CCl4)-induced acute liver injury mice. The indexes evaluated included liver pathological changes, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the serum, related gene expression (IL-1β, TNF-α, Bcl-2, and Bax), and related proteins levels (Bax, Bcl-2, Caspase 3, and NF-κB p65). Compared with the model group, the results indicated that the levels of ALT, AST, and MDA in the serum, the expression levels of IL-1β, TNF-α, and Bax, and the protein levels of Bax, Caspase 3, and NF-κB p65 significantly decreased, and the pathologic damage degree all significantly reduced after live Lactobacillus fermentum (L-LF) and live Lactobacillus plantarum (L-LP) treatment. Additionally, the levels of SOD and GSH in the serum, the gene expression of Bcl-2, and the protein level of Bcl-2 significantly increased after L-LF and L-LP treatment. Although HK-LF and HK-LP could also have obvious regulating effects on some of the evaluated indexes (ALT, AST, the expression levels of TNF-α and Bax, and the protein level of Bcl-2) and play an important role in weakening liver damage, the regulating effects of L-LF or L-LP on these indexes were all better compared with the corresponding heat-killed Lactobacillus fermentum (HK-LF) and heat-killed Lactobacillus plantarum (HK-LP). Therefore, these results suggested that LF and LP have an important role in liver disease.

The levels of NF-κB p50 and NF-κB p65 play a role in thyroid carcinoma malignancy in vivo.

BackgroundTo investigate the relationship between the levels of nuclear factor (NF)-κB p50 and NF-κB p65 and tumour characteristics in patients with thyroid carcinoma.MethodsThis prospective study enrolled consecutive patients with thyroid carcinoma. Tumour samples were collected and the levels of NF-κB p50 and NF-κB p65 protein and mRNA were measured using immunohistochemistry and quantitative real-time reverse transcription–polymerase chain reaction (qRT–PCR).ResultsA total of 73 patients with thyroid carcinoma were included in the study (20 males; 53 females; mean ± SD age, 44.8 ± 12.7 years, range, 18–76 years). There were no significant differences in sex, age and pathological type between the NF-κB p50 positive group and the NF-κB p50 negative group, but tumour diameter and lymph node metastasis were significantly higher in the NF-κB p50 positive group compared with the NF-κB p50 negative group. Similar findings were observed for NF-κB p65. The levels of NF-κB p50 were positively correlated with NF-κB p65 in samples of thyroid carcinoma (rs = 0.653).ConclusionThe levels of NF-κB p50 and NF-κB p65 in samples of thyroid carcinoma were positively associated with tumour diameter and the presence of lymph node metastasis.

Protective Effects of Astragaloside IV on Delayed Cerebral Vasospasm in an Experimental Rat Model of Subarachnoid Hemorrhage

Delayed cerebral vasospasm is an important cause of morbidity and mortality in patients with subarachnoid hemorrhage (SAH). This study aimed to assess the effects of Astragaloside IV (AS-IV) on delayed cerebral vasospasm after SAH. A rat model of SAH was established by puncturing one side of the internal carotid artery. Then, rats received daily intraperitoneal injections of AS-IV (20 mg/kg; SAH-AS-IV group), 0.1% dimethyl sulfoxide (DMSO) (SAH-DMSO group), or saline (SAH group) for 5 days; an additional control group consisted of rats receiving sham surgery and saline injections. Morphologic characteristics of the basilar artery (BA) were measured from histologic sections stained with hematoxylin and eosin and used as indicators of cerebral vasospasm. Immunohistochemistry was used to detect Toll-like receptor-4 (TLR4) and nuclear factor kappa B (NF-κB) p65 protein levels in the BA. Enzyme-linked immunosorbent assay was used to measure the plasma concentrations of tumor necrosis factor-alpha and interleukin-6. Compared with controls, the SAH-DMSO and SAH groups showed increased wall thickness and reduced luminal cross-sectional area (indicative of vasospasm) and increased TLR4 expression and enhanced NF-κB activation in the BA, as well as elevated plasma levels of tumor necrosis factor-alpha and interleukin-6. Administration of AS-IV was associated with significant attenuation of all the aforementioned changes induced by SAH (P < 0.05). AS-IV may attenuate delayed cerebral vasospasm after SAH through inhibition of TLR4/NF-κB-mediated inflammatory signaling pathways.

Preventive effects of guanosine on intestinal inflammation in 2, 4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rats.

Guanosine, a guanine-based purine, is an extracellular signaling molecule exerting anti-inflammatory and antioxidative effects in several in vivo and in vitro injury models. We aimed to investigate its protective effects on 2, 4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rat. Rats were divided into five groups and colitis was induced by intracolonic instillation of DNBS (15mg/rat). Guanosine (4 or 8mg/kg) was administered for 6days i.p. starting the day of the colitis induction. Body weight loss, stool consistency, colon weight/length, histological analysis, myeloperoxidase activity (MPO) and pro-inflammatory cytokine levels were assessed. Immunoblotting of nuclear factor-κB (NF-κB) p65 protein levels and detection of oxidative and nitrosative stress markers were also performed. Guanosine, in a dose-dependent manner, significantly ameliorated the severity of DNBS-induced colitis, reducing body weight loss and diarrhea incidence, preventing the DNBS-induced macroscopic and microscopic damage to the colonic mucosa, and the MPO increase. Guanosine treatment also lowered interleukin-1β, interleukin-6, and tumor necrosis factor-α mRNA levels. Importantly, guanosine in DNBS rats down-regulated the expression of NF-κB p65 and the levels of reactive oxygen species and nitrite. In conclusion, guanosine exerts beneficial effects in DNBS-induced colitis in rats, through modulation of colonic inflammation, downregulating of NFκB-mediated signaling.

The effect of NF-κB on NLRP3 of lung tissue in rats with acute lung injury induced by phosgene

Objective To observe the effect of signal transduction pathway of nuclear factor-kappa B (NF-κB) on Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and pyroptosis in rats with acute lung injury induced (ALI) by phosgene. Methods The rats were randomly(random number) divided into 3 groups: air exposure control group, phosgene exposure group and PDTC group with phosgene exposure after 100 mg/kg pyrrolidine dithiocarbamate (PDTC) administration. The specimens of serum, bronchoalveolar lavage fluid (BALF) and lung tissue were collected 6 h after exposure. Morphological changes were observed by HE staining. The expression of NLRP3 in the lung of three groups was detected by immunohistochemistry. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expressions of NF-κB p65,NLRP3, ASC and caspase-1 mRNA in the lung tissue. NF-κB p65, NLRP3, ASC and caspase-1 protein levels in the lung tissue were quantified by Western blot. The concentrations of IL-1β, IL-18 and IL-33 in the serum and BALF were measured by enzyme-linked immunosorbent assay (ELISA). Pyroptosis was observed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). Results The model of phosgene-induced ALI was successfully established in rats. Morphological changes with inflammatory cell infiltration were observed in the lung tissues of phosgene group, in which NLRP3 positive cells also could be observed by immunohistochemical staining. The mRNA expressions and protein levels of NF-κB p65, NLRP3 and caspase-1 in lung tissues were significantly increased (P<0.05) in phosgene group, compared with air control group. The mRNA expressions and protein levels of NF-κB p65, NLRP3 and caspase-1 in lung tissues were significantly decreased (P<0.05) in PDTC group, compared with phosgene group. The IL-1β, IL-18 and IL-33 protein levels in serum and BALF were significantly increased (P<0.05) in phosgene group, compared with air control group. The IL-1β, IL-18 and IL-33 protein levels in serum and BALF were significantly decreased (P<0.05) in PDTC group, compared with phosgene group. TUNEL results showed that pyroptosis in the lung tissue obviously increased in phosgene group, while decreased in PDTC group. Conclusions NLRP3 inflammasome and lung cell pyroptosis were induced through NF-κB signal transduction pathway in rats with acute lung injury caused by phosgene inhalation. Blockade of NF-κB can alleviate acute lung injury by down-regulating the expression of NLRP3 inflammasome to inhibit pyroptosis. Key words: Nuclear factor-kappa B (NF-κB); Phosgene; acute lung injury; Pyrin domain-containing 3 (NLRP3) innammasome; Inhalation; pyroptosis; Inflammation; Pyrrolidinedithiocarbamic acid (PDTC)

Salvianolic Acid B Suppresses Inflammatory Mediator Levels by Downregulating NF-κB in a Rat Model of Rheumatoid Arthritis.

BackgroundSalvianolic acid B (SB) is a major active phyto-component of the plant Radix Salvia miltiorrhiza, which is traditionally used to treat joint pain and arthritis. The present study examined the anti-rheumatoid arthritis efficacy of SB on collagen-induced rheumatoid arthritis (CIA) in a rat model.Material/MethodsForty-eight rats were divided into 4 groups: Control rats treated with saline (Group I), rats subjected to CIA induction by intradermal injection of bovine collagen II type at the tail (Group II), and rats subjected to CIA and supplemented with either 20 or 40 mg/kg of SB for 28 days (group III or IV).ResultsPaw swelling, edema, arthritis score, thymus and spleen indexes, and neutrophil infiltration were significantly decreased (p<0.01) by treatment with 20 or 40 mg/kg of SB. The levels of inflammatory cytokines (interleukin-1β, -6, and -17, and TNF-α) and anti-collagen II-specific immunoglobulins (IgG1 and IgG2a) were markedly decreased (p<0.01), and those of antioxidant enzymes (SOD, CAT, and GSH) were significantly increased (p<0.01) in SB-treated rats. Administration with SB (20 or 40 mg/kg) resulted in lower phosphorylated IκB-α and NF-κB p65 protein levels and markedly downregulated IκB-α expression. Furthermore, CIA rats revealed the presence of highly diffused polymorphonuclear cells (PMNs) infiltration with eroded cartilage; however, these phenomena were considerably ameliorated by SB.ConclusionsSB alleviates oxidative stress and inflammation in CIA rats, thus verifying its anti-rheumatoid arthritis property.

The anti‐inflammatory effect of 10‐oxo‐trans‐11‐octadecenoic acid (KetoC) on RAW 264.7 cells stimulated with Porphyromonas gingivalis lipopolysaccharide

There is rapidly developing interest into the role of several anti-inflammatory agents to resolve inflammation in periodontal disease. A bioactive polyunsaturated fatty acid, 10-oxo-trans-11-octadecenoic acid (KetoC), is known to have various beneficial physiological effects; however, the effect of KetoC on inflammation remains unclear. Here, we investigated the effect of KetoC on RAW 264.7 cells stimulated with Porphyromonas gingivalis lipopolysaccharide, and explored the intracellular mechanism responsible for its anti-inflammatory effects. RAW 264.7 cells were pre-treated with or without KetoC, and then stimulated with or without P.gingivalis lipopolysaccharide. Levels of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1β were determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Specific antagonists for G protein-coupled receptor (GPR)40 and GPR120 were used to clarify the receptor for KetoC. The intracellular mechanism was investigated using western blotting analysis to separate nuclear and cytosolic NF-κB p65 protein. KetoC (5μmol/L) was not toxic to RAW 264.7 cells, and significantly reduced the expression of TNFα and IL-6 mRNA and protein, and IL-1β mRNA. No protein production of IL-1β was observed. Additionally, when bound to GPR120, KetoC trended to downregulate nuclear NF-κB p65 protein levels. However, the antagonist for GPR40 failed to diminish the action of KetoC. KetoC suppressed the proinflammatory cytokines TNFα, IL-6 and IL-1β via NF-κB p65, by binding to its receptor GPR120. KetoC is a promising candidate in future studies as a bioactive anti-inflammatory agent in treating periodontal disease.

HMGB1-RAGE signaling pathway in pPROM

ObjectiveIncreased inflammation of the placenta is considered as a risk factor and a promoter of preterm premature rupture of the membranes (pPROM). High-mobility group box 1 (HMGB1) is a recently identified inflammatory cytokine, and HMGB1-RAGE signaling pathway has been associated with many pathophysiological processes. This study aims to reveal the mechanisms of HMGB1-RAGE signaling pathway in pPROM. Materials and methodsThe mRNA levels of relative gene of HMGB1 pathway, HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2, were analyzed by real-time PCR in placentas collected from 60 normal term women, 60 women with PROM and 60 women with pPROM. Additionally, levels of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 protein were detected in frozen placental specimens by western blot, and the locations of HMGB1, RAGE and NF-κBp65 were evaluated in the well-characterized tissue microarray (TMA) by immunohistochemistry. ELISA was further used to detect HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 level in maternal and cord serum. ResultsCompared with normal term and PROM women, we found that (1) The mRNA expressions of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 in HMGB1-RAGE pathway of pPROM placentas were higher. (2) The protein levels of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 in pPROM placentas were higher. (3) HMGB1 and RAGE immunoreactivity in pPROM placenta TMA were increased in the cytoplasm of syncytiotrophoblast (STB), extravillous trophoblast (EVT) and mesenchymal cells, while NF-κBp65 was enhanced in the nucleus of STB and EVT. (4) Maternal serum concentrations of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 in pPROM group were greater. (5) Cord serum concentrations of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 among the 3 groups had no significant differences. ConclusionHMGB1 nuclear-cytoplasmic translocation in pPROM placenta may lead to the binding of HMGB1 to its receptor RAGE, resulting in provoking NF-κBp65 activity, and then inducing the release of MMP-9 and MMP-2, which all above activities contributed to the process of pPROM. Consequently, HMGB1-RAGE signaling pathway may be involved in the pathogenesis of pPROM.

Intermittent hypoxia promotes melanoma lung metastasis via oxidative stress and inflammation responses in a mouse model of obstructive sleep apnea

BackgroundRecently, increased tumor incidence and cancer-related mortality have been reported among patients with obstructive sleep apnea (OSA). Intermittent hypoxia (IH), the hallmark feature of OSA, contributes to the metastasis of tumors. However, the molecular mechanisms by which tumor metastasis is accelerated by OSA-like IH remain to be elucidated.MethodsC57BL/6 J male mice were subjected to intravenous injection of B16F10 melanoma cells before receiving IH treatment. Then, the animals were randomly distributed into three groups (n = 8 each): normoxia (N) group, IH group, and antioxidant tempol group (IHT, exposed to IH after treatment with tempol). After the mice were sacrificed, the number and weight of lung metastatic colonies were assessed. The lung tissues with tumor metastasis were analyzed for markers of oxidative stress and inflammation and for HIF-1α using western blotting and real-time PCR (qRT-PCR). The level of reactive oxygen species (ROS) in B16F10 cell was also assessed after N, IH and IH with tempol treatments.ResultsCompared with normoxia, IH significantly increased the number and weight of mouse lung metastatic colonies. Treatment of B16F10 cells with IH significantly enhanced ROS generation. Lung tissues with tumor metastasis provided evidence of increased oxidative stress, as assessed by p22phox and SOD mRNA levels and the NRF2 protein level, as well as increased inflammation, as assessed by TNF-α and IL-6 mRNA levels and the NF-κB P65 protein level. HIF-1α protein levels were increased in response to IH treatment. Tempol, an important antioxidant, ameliorated IH-induced melanoma lung metastasis in mice and reduced oxidative stress and inflammation responses.ConclusionsThese results support the hypothesis that oxidative stress and inflammation responses play an important role in the pathogenesis of OSA-like IH-induced melanoma lung metastasis in mice. Antioxidant intervention provides a novel strategy for the prevention and treatment of cancer in OSA populations.

Ingenol-3-Angelate Suppresses Growth of Melanoma Cells and Skin Tumor Development by Downregulation of NF-κB-Cox2 Signaling.

BackgroundA recent focus in skin cancer prevention intervenes though modulating molecular links between inflammation and cell growth signaling, such as NF-κB. This study elucidates the effect of a non-tumor promoting phorbol ester, ingenol-3-angelate (I3A), on the growth of human melanoma cells and on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation and 7,12-Dimethylbenz(a)anthracene (DMBA)-induced skin carcinoma in mice.Material/MethodsCell viability was assessed by MTT assay, cell proliferation by clonogenic assay, apoptosis and cell cycle arrest was analyzed by flow cytometry, protein expression was studied by IHC and Western blotting, and gene expression by qPCR.ResultsI3A suppressed the survival and proliferation of human melanoma cells with estimated IC50 values around 38 and 46 μM for A2058 and HT144 cell, respectively. I3A activated the protein levels of PKCδ and PKCɛ, which induced apoptosis by activating caspase-9 and caspace-3 followed by lowering of mitochondrial membrane potential and enhancing DNA fragmentation. I3A induced G1 phase cell cycle arrest as well as G2/M phase arrest in both cell lines. I3A inhibited the levels of NF-κB p65 protein as well as phosphorylation of p65 and its nuclear translocation. I3A suppressed the gene expression of NF-κB, COX-2 and iNOS. I3A inhibited TPA-induced inflammation and epidermal hyperplasia in female ICR mice by downregulating NF-κB and iNOS. I3A suppressed the growth of skin tumor in DMBA-induced mice in dose-dependent manner.ConclusionsThe mechanism of I3A induces apoptosis in human melanoma cells and suppresses skin inflammation and carcinoma via downregulation of NF-κB-iNOS-COX-2 signaling.

Therapeutic effects of polysaccharides extracted from Porphyra yezoensis in rats with cerebral ischemia/reperfusion injury

The polysaccharides of Porphyra yezoensis (PPY), porphyrans, are recognized as the major active components as they have several biological properties. This study was performed to assess the effect of PPY administration against cerebral ischemia-reperfusion injury (IRI). Thirty-two adult Sprague-Dawley (SD) rats were chosen and divided into 4 groups as follows: group I – rats received only saline; group II – subjected to middle cerebral artery occlusion (MCAO) for 60 min followed by 24 h of reperfusion (IRI-induced); group III – pretreated with PPY (100 mg/kg) for 7 days, followed by IRI induction; group IV – treated with PPY (100 mg/kg) for 7 days without IRI induction. All the data were analyzed by Dunnett’s (multiple comparisons) test using SPSS software. Pretreatment with PPY significantly ( p <0.01) lowered the neurological deficit and cerebral infarct volume in comparison with IRI-induced rats. A pronounced ( p <0.01) increase in the levels of antioxidant components (superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH)) was observed in PPY-supplemented rats. Also, the proinflammatory markers: interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and the nuclear factor-kappa B protein, subunit p65 (NF-κB p65) were substantially ( p <0.01) suppressed in PPY-administered rats. Moreover, the protein levels of TNF-α and NF-κB p65 were considerably ( p <0.01) downregulated upon pretreatment with PPY. Our data suggest that PPY could exhibit neuroprotective activity by attenuating oxidative stress and the inflammatory response. https://doi.org/10.2298/ABS170621039S Received: June 21, 2017; Revised: October 8, 2017; Accepted: October 8, 2017; Published online: October 16, 2017 How to cite this article: Sun C, Wu F, Chen D, Ge J. Therapeutic effects of polysaccharides extracted from Porphyra yezoensis in rats with cerebral ischemia/reperfusion injury. Arch Biol Sci. 2018;70(2):233-9.

Effect of NF-κB signaling pathway mediated by miR-711 on the apoptosis of H9c2 cardiomyocytes in myocardial ischemia reperfusion.

The aim of this study was to explore the change of the expression of miR-711 in myocardial ischemia-reperfusion (I/R) injury and the possible mechanism. The cardiomyocyte model of I/R injury was constructed. Real-time quantitative fluorescence polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of miR-711 as well as the mRNA and protein levels of NF-κB (p65). Flow cytometry, CCK-8 kit, and enzyme-linked immunosorbent assay (ELISA) were used to detect the apoptosis, cell viability, and the content of LDH and MDA, respectively. Compared to control cells, the expression levels of miR-711, the mRNA, and protein levels of NF-κB were higher in H9c2 cardiomyocytes of I/R, the apoptosis rate of H9c2 cardiomyocytes of I/R was higher, the levels of LDH and MDA were higher in the supernatant of cell culture, and the cell viability was lower. In comparison to the cells of I/R, the apoptosis rate of H9c2 cardiomyocytes of I/R plus miR-711 inhibitors was lower, the levels of LDH and MDA were lower in the supernatant of cell culture, and the cell viability was higher. In comparison to control cells, the expression level of Bcl-2 was lower, and the expression levels of Bax and Caspase-3 were higher in the cells of I/R. In comparison to the cells of I/R, the expression level of Bcl-2 was lower, and the expression levels of Bax and Caspase-3 were higher in the cells of I/R plus miR-711 inhibitors. Overexpression of miR-711 could promote the expression of NF-κB (p65) in the cardiomyocytes of I/R and accelerate the transportation of NF-κB (p65) into the nucleus, thus promoting the apoptosis of cardiomyocytes.

The Study on Mechanism of Nf-Κb Signal Molecule In Kashin-Beck Disease

Kashin-Beck disease (KBD) is an endemic osteoarthropathy.This study is to observe the change of NF-κB signaling in KBD patients and expression of NF-κB p65 in human C28/I2 chondrocyte for analyzing the effect of NF-κB p65 on chondrocyte apoptosis. 161 Patients with KBD and 312 healthy controls were randomly selected from Shaanxi, China matched by age and sex. Venous blood was collected from patients and healthy controls. The model of C28/I2 chondrocyte oxidative damage was established by using tert-butyl hydroperoxide (tBHP), and cell apoptosis and reactive oxygen species (ROS) was detected by Hoechst 33342 and dichlorofluorescein methods, respectively. The proteins of whole blood and cells was extracted by Trizol method, expression level of NF-κB p65 molecule in whole blood were detected by Western blotting. Compared with age and sex, differences were not statistically significant between KBD group and control group (t=0.336，P=0.737；χ2=0.407，P=0.523). The protein expression level of NF-κB p65 in KBD group was 1.833 times as high as that of control group (P<0.05). In C28/I2 chondrocyte oxidative damage model, the number of cell apoptosis increased in tBHP group and level of ROS and protein expression level of NF-κB p65 were higher than that of control group (P<0.05), however, levels of ROS were lower than that of control group (P<0.05), protein levels of NF-κB p65 were down-regulated in low and middle selenium pre-protection group compared to tBHP group (P>0.05). The NF-κB signaling pathway is up-regulated in KBD patients, moreover, chondrocyte experiments showed cell apoptosis was mediated via up-regulation of NF-κB p65, and the expression of p65 is down-regulated by Na2SeO3intervention, which suggest NF-κB signaling pathway play an important role in pathogenesis of KBD (This research was supported by National Natural Science Foundation in China No.81573104,81172610, 81673117).

Tumor necrosis factor-alpha inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated nuclear factor-kappaB signaling pathway.

The majority of experiments show that tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of mesenchymal stem cells and pre-osteoblasts by activated nuclear factor-kappaB (NF-κB) signaling. However, the underlying mechanisms by which NF-κB signaling inhibits osteogenic differentiation are not fully understood. The aim of the present study was to investigate whether EphB4 signaling inhibition mediates the effects of TNF-α-activated NF-κB signaling on osteogenic differentiation of pre-osteoblasts. Murine MC3T3-E1 pre-osteoblasts were treated with 10ng/mL of TNF-α. NF-κB inhibitor, pyrrolidine dithiocarbamate, was used to achieve NF-κB signaling inhibition. EphB4 signaling was activated using ephrinB2-fc. The mRNA expressions of runt related transcription factor 2 (Runx2), bone sialoprotein (BSP) and EphB4 were determined using reverse transcription-polymerase chain reaction. The protein levels of Runx2, BSP, Col Ia1, osteopontin, EphB4, p-NF-κB p65 and NF-κB p65 were evaluated using western blot assays. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells was evaluated by ALP activity kit, and mineral nodule formation was evaluated by Alizarin Red S staining. TNF-α inhibited EphB4 expression, while it suppressed Runx2, BSP expression from gene and protein levels as well as ALP activity and mineral nodule formation in MC3T3-E1 cells. Activation of EphB4 signaling by ephrinB2-fc promoted osteogenic differentiation of MC3T3-E1 cells, whereas TNF-α impaired the osteogenic differentiation enhanced by ephrinB2-fc. Pyrrolidine dithiocarbamate blocked the activation of NF-κB signaling induced by TNF-α, while it prevented the downregulation of Runx2, BSP and EphB4, induced by TNF-α. TNF-α inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated NF-κB signaling pathway.

Baicalin may alleviate inflammatory infiltration in dextran sodium sulfate-induced chronic ulcerative colitis via inhibiting IL-33 expression

AimsTo investigate the therapeutic effect of baicalin treatment in chronic ulcerative colitis (UC), and explore the potential anti-inflammation mechanism(s) via IL-33 pathway. Main methodsUC model were established by giving three cycles of 5-day 2% dextran sodium sulfate (DSS) with two intervals of 14-day recovery in mice, totaling 43days. At the 13th day of the UC modeling, mice received baicalin at doses of 50, 100, or 150mg/kg, respectively. Disease activity index (DAI) assessment as well as HE and PAS staining were performed. Serum levels of TNF-α, IL-1β and IL-6 were determined by ELISA. Myeloperoxidase (MPO) activity and nitric oxide (NO) contents in colon were measured. The expressions of IL-33 and Ly6/G were examined by immunochemistry. And contents of IL-33 protein and NF-κB-related proteins were tested by Western blot. Key findingsMorphological and histological analyses revealed that baicalin administration had a significant effect on reducing the severity of DSS-induced UC in mice. Besides, baicalin treatment significantly reduced the levels of MPO and NO. Moreover, increased levels of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, have been identified in damaged colon tissue, which was noticeably reduced by baicalin treatment. Our data demonstrated that protein levels of IL-33 and NF-κB p65 were elevated in colon tissues of chronic UC mice. Baicalin treatment significantly suppressed levels of IL-33 and NF-κB p65, whereas levels of IκB-α were increased. SignificanceBaicalin treatment effectively alleviated DSS-induced chronic UC, and the protective mechanisms may involve inhibition of IL-33 expression and subsequent NF-κB activation.

Protective Role of Apocynin via Suppression of Neuronal Autophagy and TLR4/NF-κB Signaling Pathway in a Rat Model of Traumatic Brain Injury.

Neuronal autophagy and inflammatory responses are important in the pathogenesis of traumatic brain injury (TBI), and toll-like receptor 4 (TLR4) may play an important role in the related molecular cascade. The present study investigated the protective effect of apocynin, an inhibitor of NADPH oxidase, in a TBI rat model and further examined neuronal autophagy and the TLR4-mediated pathway. Adult male Sprague-Dawley rats were subjected to controlled cortical impact injury and intraperitoneally injected with apocynin (50mg/kg) immediately after the trauma. In addition to motor and behavioral studies, brain water content and histology analyses were performed. Expression of autophagy-related proteins as well as TLR4/NF-κB signaling and inflammatory mediators was analyzed. The apocynin treatment significantly attenuated TBI-induced motor and behavioral impairment, brain edema and neuronal damage in rats. Immunohistochemical and Western blot analyses revealed that apocynin treatment significantly reduced the expression of NOX2, LC3 and Beclin1 in the hippocampus at 12-48h after injury. Double immunolabeling demonstrated that apocynin decreased the co-localization of LC3 or TLR4-positive cells with hippocampal neurons at 24h following TBI. In addition, CD11b (microglial marker) and GFAP (astrocyte marker)-immunopositive cells were also clearly decreased in hippocampal tissues. Meanwhile, protein levels of TLR4, NF-κB p65, TNF-α and IL-1β were found to be significantly downregulated by Western blot analysis. In conclusion, our findings indicate that the protective effects of apocynin may be related to modulation of neuronal autophagy and the TLR4/NF-κB signaling pathway.

The sinomenine enteric-coated microspheres suppressed the TLR/NF-κB signaling in DSS-induced experimental colitis

Sinomenine is a pure alkaloid with immunosuppressive effects that is extracted from the Chinese medicinal plant Sinomenium acutum. We studied the therapeutic effects of sinomenine on inflammatory bowel disease. In this study, we randomly divided mice into the following ten groups: Control group; DSS-induced colitis group; Salicylazosulfapyridine (SASP)-treated group; Chitosan-treated group; low-, medium-, and high-dose sinomenine-treated and sinomenine enteric-coated microspheres-treated groups. We recorded changes in colon length, disease activity index (DAI), and colon pathology, measured TLR4, MyD88, SIGIRR, NF-κB p65 protein levels and inflammatory serum cytokine levels.Except for the Control group, the weight of mice in each group decreased, the DAI of the DSS-induced colitis group was significantly higher than the other groups, and the DAIs of the sinomenine- and sinomenine enteric-coated microspheres-treated groups were significantly lower than that of the SASP-treated group. TLR4, MyD88, NF-κB p65 and proinflammatory cytokine expressions decreased dose dependently in the sinomenine and sinomenine enteric-coated microspheres-treated groups and were generally lower in the sinomenine enteric-coated microspheres groups. However, SIGIRR and anti-inflammatory IL-10 expressions exhibited the opposite pattern. Based on the superior therapeutic effect, sinomenine enteric-coated microspheres might regulate TLR/NF-κB signaling and would be beneficial for an effective and safe therapy of inflammatory bowel disease.