Nuclear NF-κB Expression Research Articles

New Pathways in Lipopolysaccharide-Induced Toll-Like Receptor 4-to-Nuclear Factor-κB Signaling Through the Coupled Uniform Sequential Reaction Systems Model

The coupled uniform sequential reaction systems (CUSERS) model, which allows for determining the structure of signaling pathways with incomplete information from the temporal patterns of their components, was applied to the experimental records of activities of TLR4 downstream species IKK and NF-κB in LPS-stimulated wild-type (WT), MyD88-deficient and TRIF-deficient macrophages. New signaling pathways targeting IKK were revealed in MyD88-deficient and TRIF-deficient macrophages, and shown to be described by the coupled systems formed by 3- and 5-component or 5- and 10-component pathways, respectively. By comparing the temporal pattern of IKK in WT macrophages with those in MyD88-deficient and TRIF-deficient macrophages, two new signaling pathways, which were absent in the above defective macrophages, were found and described by a system formed by coupling 9- and 10-component pathways. As a direct consequence of the above findings, a coupled system composed of six different 3-, 5-, 5-, 9-, 10- and 10-component pathways targeting IKK and describing its temporal pattern, IKK(f), in WT macrophages was constructed. This system significantly modifies the canonical NF-κB signaling by introducing novel pathways of IKK activation. The expression of nuclear NF-κB in WT macrophages was found to depend on two different signaling pathways and to be modelled by a coupled system composed of 1- and 4-component or 2- and 8-component pathways, in dependence on sampling frequencies used in different experiments. From the three-modal NF-κB(t) temporal pattern in LPS-stimulated WT fibroblasts, three 1-, 12- and 17-component signaling pathways targeting nuclear NF-κB were determined.

4-Sulfonyloxy/alkoxy benzoxazolone derivatives with high anti-inflammatory activities: Synthesis, biological evaluation, and mechanims of action via p38/ERK-NF-κB/iNOS pathway.

In an effort to discover new agents with high anti-inflammatory activity, 22 new 4-sulfonyloxy/alkoxy benzoxazolone derivatives were synthesized, characterized, and evaluated for their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production and TNF-α expression in RAW 264.7 cells in vitro. Most of these compounds displayed greater inhibitory ability against NO production than the lead compound 4-o-methyl-benzenesulfonyl benzoxazolone, and the most active compound 2h exhibited the strongest inhibitory activity against NO, IL-1β, and IL-6 production with IC50 values 17.67, 20.07, and 8.61μΜ, respectively. The effects of 2h were comparable or stronger than those of the positive control celecoxib. Compound 2h also displayed higher activity in vivo than celecoxib in a mouse model of xylene-induced ear edema, based on their inhibitory rates of 42.69% and 30.87%, respectively. Further molecular analysis revealed that compound 2h significantly reduced the iNOS levels in cell supernatant and suppressed the protein expression of iNOS, p-p38, p-ERK, and nuclear NF-κB. The results indicated that the anti-inflammatory effect of 2h might be realized through the regulation of ERK- and p38-mediated mitogen-activated protein kinase (MAPK)-NF-κB/iNOS signaling, thereby reducing the excessive release of NO, IL-1β, and IL-6. Our findings demonstrated that compound 2h, a new benzoxazolone derivative, could inhibit activation of the MAPK-NF-κB/iNOS pathway, supporting its potential as a novel anti-inflammatory agent.

TRIM9 overexpression promotes uterine leiomyoma cell proliferation and inhibits cell apoptosis via NF-κB signaling pathway

AimsUterine leiomyoma (UM) is the most common benign gynecological tumor and the leading indication for hysterectomy. Our study explored the roles of TRIM9 in leiomyoma formation and investigated the underlying molecular mechanisms. Material and methodsThe relationship between TRIM9 expression and fibroids formation was deciphered from the GEO database after bioinformatics analysis and identified by qPCR in human leiomyoma tissues. Both TRIM9 mRNA and protein expression were further detected in primary cultured uterine leiomyoma cells (UMC). The tumorigenesis potentials of TRIM9 in cell proliferation, cell cycle, cell apoptosis; cyclin D1, survivin and cleaved-caspase 3 protein expressions in primary UMC with TRIM9 overexpression (UMC-oeTRIM9); and uterine smooth muscle cells (SMC) with TRIM9 knockdown (SMC-siTRIM9) were evaluated in vitro. NF-κB p65 and its phosphorylation were further examined by western blotting, and rescue experiments on cell proliferation, cell cycle and cell apoptosis were conducted. Key findingsTRIM9 showed higher expression in UM tissue and UMC compared with normal myometrium. The overexpression of TRIM9 in UMC notably promoted UM growth via enhancement of cell proliferation, reduction of cell apoptosis, and regulation of cyclin D1, survivin, cleaved-caspase 3, and nuclear NF-κB expression, which were reversed in SMC-siTRIM9 and PDTC (an NF-κB inhibitor) intervention in UMC-oeTRIM9. SignificanceTo our knowledge, this was the first study demonstrating the roles of TRIM9 in cell growth progression of UM development. TRIM9 may be a potential therapeutic target for UM, by promoting leiomyoma cell proliferation and reducing cell apoptosis via activation of the NF-κB pathway.

Pyrrolidine dithiocarbamate reduces alloxan-induced kidney damage by decreasing nox4, inducible nitric oxide synthase, and metalloproteinase-2.

We examined the effect of the NFκB inhibitor pyrrolidine-1-carbodithioic acid (PDTC) on inducible nitric oxide synthase (iNOS), matrix metalloproteinase-2 (MMP-2) activity, and oxidative and inflammatory kidney damage in alloxan-induced diabetes. Two weeks after diabetes induction (alloxan-130mg/kg), control and diabetic rats received PDTC (100mg/kg) or vehicle for 8weeks. Body weight, glycemia, urea, and creatinine were measured. Kidney changes were measured in hematoxylin/eosin sections and ED1 by immunohistochemistry. Kidney thiobarbituric acid reactive substances (TBARS), superoxide anion (O2-), and nitrate/nitrite (NOx) levels, and catalase and superoxide dismutase (SOD) activities were analyzed. Also, kidney nox4 and iNOS expression, and NFkB nuclear translocation were measured by western blot, and MMP-2 by zymography. Glycemia and urea increased in alloxan rats, which were not modified by PDTC treatment. However, PDTC attenuated kidney structural alterations and macrophage infiltration in diabetic rats. While diabetes increased both TBARS and O2- levels, PDTC treatment reduced TBARS in diabetic and O2- in control kidneys. A decrease in NOx levels was found in diabetic kidneys, which was prevented by PDTC. Diabetes reduced catalase activity, and PDTC increased catalase and SOD activities in both control and diabetic kidneys. PDTC treatment reduced MMP-2 activity and iNOS and p65 NFκB nuclear expression found increased in diabetic kidneys. Our results show that the NFκB inhibitor PDTC reduces renal damage through reduction of Nox4, iNOS, macrophages, and MMP-2 in the alloxan-induced diabetic model. These findings suggest that PDTC inhibits alloxan kidney damage via antioxidative and anti-inflammatory mechanisms.

Abstract P127: Adverse Endothelial Cell Protein Expression is Associated With Type 1 Diabetes

Cardiovascular risk is increased in adults with type 1 diabetes (T1D), although the mechanism is poorly understood. Endothelial dysfunction may contribute to increased coronary artery calcium (CAC) in T1D, and CAC severity may be associated with reduced nitric oxide (NO) synthesis and/or increased inflammation. We tested the hypotheses that high CAC and T1D are associated with expression of endothelial NO synthase (eNOS, enzyme that produces NO), phosphorylated eNOS (p-eNOS), and NFκB (pro-inflammatory transcription factor). Participants were categorized as having high (>80 Agatston units [AU]) or low CAC (<15 AU), measured by CT scan. Protein expression was measured using quantitative immunofluorescence in endothelial cells harvested from 14 women with T1D (mean age 52±8 years; 7 high CAC); 13 men with T1D (mean age 58±7 years; 5 high CAC); 10 women without diabetes (mean age 61±4 years; 3 high CAC); and 10 men without diabetes (mean age 59±6 years; 4 high CAC). All analyses used ratios of endothelial cell protein expression to control human umbilical vein endothelial cell protein expression. Independent t-tests compared protein expression by sex and T1D. Linear regression tested the associations of high vs. low CAC and T1D with the 3 proteins of interest after adjustment for age and sex. In men, eNOS expression differed by T1D: men with T1D had reduced cytoplasmic (0.34 vs.0.52, p=0.03) and nuclear (0.44 vs. 0.60, p=0.02) eNOS expression compared with men without T1D, suggesting reduced NO synthesis. There were no differences in p-eNOS expression, indicating that eNOS levels were likely not due to differential activation. In women, NFκB expression similarly differed by T1D: women with T1D had higher cytoplasmic (0.81 vs. 0.63, p=0.01) and nuclear (0.99 vs. 0.75, p=0.01) NFκB expression compared with women without T1D, suggesting greater inflammation. After adjustment for age and sex, there was no significant association between high vs. low CAC in endothelial expression of any proteins. The data trend suggested that high vs. low CAC is associated with reduced cytoplasmic (0.36 vs. 0.47, p=0.06) and nuclear (0.48 vs. 0.55, p=0.12) eNOS expression. T1D trended in the same direction for both cytoplasmic (0.36 vs. 0.47, p=0.06) and nuclear (0.47 vs. 0.55, p=0.13) eNOS expression. High vs. low CAC also trended towards an association with greater cytoplasmic (0.72 vs. 0.66, p=0.21) and nuclear (0.85 vs. 0.80, p=0.40) NFκB expression, while T1D was significantly associated with greater cytoplasmic (0.76 vs. 0.63, p=0.006) and nuclear (0.92 vs. 0.73, p=0.003) NFκB expression. Neither CAC nor T1D was associated with p-eNOS expression in linear regression. In conclusion, eNOS and NFκB expression are potential mechanisms for increased CAC given their roles in reduced NO synthesis and increased inflammation, and may differ by sex in people with T1D. Regardless of high CAC, inflammation in T1D is increased.

Abstract TP350: Escin Attenuates the Impairment of Neurological Function by Ameliorating Systemic Inflammation in Intracerebral Hemorrhagic Mice

Background and Purpose: Escin could attenuate cerebral ischemia-induced brain injury. The present study aimed to investigate whether Escin may attenuate the impairment of neurological function by ameliorating systemic inflammation in ICH mice. Methods: The ICH model was prepared by intrastriatal injection of collagenase. At 0.5 h, 2 h, 6 h and 12 h after Escin injection, the concentration of Escin in the serum and brain were detected. The effects of Escin on the Garcia test were evaluated. Brain water content was observed via a wet/dry weight method. Evans blue extravasation in the cerebrums were also assayed. The serum IL-1β level was detected with the ELISA kit. The ICH mice were further treated with Escin plus IL-1β. At 24 h of the ICH, Garcia test, and brain water content, the Evans blue extravasation and serum IL-1β levels were evaluated. Additionally, the expression of RhoA, ROCK1, IκBα, nuclear NF-κB, cytosolic NF-κB, Occludin and Claudin-5 in mice brain tissue were investigated with the Western blot. Results: Escin was detected in the serum of the Control group and ICH group at 0.5 h, 2 h, 6 h and 12 h after Escin administration. Escin was not detected in brain tissues at any time points in the animals. At 24 h and 72 h of ICH, the Garcia test scores in the Escin groups were significantly increased ( P < 0.05 or P < 0.01). Brain water contents and Evans blue extravasation of the right basal ganglia in the Escin groups were significantly reduced ( P < 0.05 or P < 0.01). Escin abated the increase of IL-1β induced by ICH ( P < 0.05 or P < 0.01). IL-1β administration reversed the effect of Escin on Garcia test scores, the brain water contents, and the Evans blue extravasation ( P < 0.01). In the ICH group, the levels of RhoA, ROCK1, nuclear NF-κB were increased while the expression of IκBα, cytosolic NF-κB, Occludin, Claudin-5 were reduced ( P < 0.05 or P < 0.01). However, Escin abated RhoA, ROCK1, nuclear NF-κB and augmented IκBα, cytosolic NF-κB, Occludin, and Claudin-5. IL-1β administration partially blocked the Escin-mediated regulation of IL-1β/RhoA/NF-κB signaling pathway. Conclusion: Escin cannot penetrate the blood brain barrier (BBB). Escin improves neurological function and by inhibiting systemic inflammation, and then regulating IL-1β/RhoA/NF-κB signaling pathway in BBB.

Suppression of Nuclear Factor-κB by Glucocorticoid Receptor Blocks Estrogen-Induced Apoptosis in Estrogen-Deprived Breast Cancer Cells.

Our clinically relevant finding is that glucocorticoids block estrogen (E2)-induced apoptosis in long-term E2-deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E2 widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), IL6, and TNFα in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone upregulated FADS1 and IL6, but downregulated TNFα expression. Furthermore, dexamethasone was synergistic or additive with E2 in upregulating FADS1 and IL6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E2 in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, dexamethasone effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E2, but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E2 Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that dexamethasone could not reverse IRE1α-mediated degradation of PI3K/Akt-associated signal pathways activated by E2 Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E2, leading to the blockade of E2-induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E2-induced apoptosis in LTED breast cancer cells. This study provided an important rationale for restricting the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in patients with aromatase inhibitor-resistant breast cancer.

CD44v6 may influence ovarian cancer cell invasion and migration by regulating the NF-κB pathway.

Ovarian cancer (OC) has the worst prognosis among all malignancy types in females worldwide according to epidemiological studies in 2017. Although radiotherapy, chemotherapy and surgical treatment are the most common treatment methods, their curative effects are not satisfactory. The present study aimed to examine the role of cluster of differentiation 44 variant 6 (CD44v6) in the molecular mechanism of the proliferation and tumorigenicity of OC cells, and provide a novel target for the clinical treatment of OC. A total of 46 clinical samples were collected, including 24 malignant ovarian tumor tissue samples and 22 benign ovarian tissue samples. Expression of CD44v6 and nuclear factor-κB (NF-κB) in these samples was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. The A2780 OC cell line was used to establish a normal control group, a negative control group and a CD44v6-small interfering (si)RNA transfection group. The expression of CD44v6 and NF-κB mRNA was detected in each group by RT-qPCR. The proliferation, invasion and migration abilities of the cells were then assessed by Transwell and colony formation assays. Additionally, immunofluorescence was used to detect nuclear NF-κB expression. CD44v6 and NF-κB mRNA expression levels were significantly increased in malignant ovarian tumor tissues, compared with normal ovarian tissues (P<0.01), and immunohistochemistry demonstrated similar results. In the CD44v6-siRNA group, NF-κB mRNA expression was significantly reduced, compared with the control and negative control (both P<0.01) groups. Transwell and colony formation assays demonstrated that the migration, invasion and colony formation abilities of OC cells in the CD44v6-siRNA group were significantly reduced, compared with the control and negative control (both P<0.01) groups. Immunofluorescence results demonstrated that the expression of NF-κB in the cytoplasm and nucleus of the CD44v6-siRNA group was also markedly reduced, compared with the other two groups. In conclusion, CD44v6 may participate in the proliferation of OC cells through activation of the NF-κB pathway and these observations may provide a novel therapeutic target for the clinical treatment of OC.

Auricular vagus nerve stimulation protects against postoperative cognitive dysfunction by attenuating neuroinflammation and neurodegeneration in aged rats

Postoperative cognitive dysfunction (POCD) has been increasingly recognized as a significant complication after surgery, especially in senior patients. Vagus nerve stimulation (VNS) reportedly provides beneficial effects against various brain disorders, supporting a hypothesis of its protective role in POCD. However, direct stimulation of the vagus nerve is invasive, as it requires a surgical incision in the neck. Thus, we employed a non-invasive VNS method by stimulating the dermatome in the external ear, which is innervated by the vagus nerve (auricular vagus nerve stimulation; aVNS) and sought to investigate the efficacy of this method in treating surgery-induced cognitive dysfunction in an aged rat model of POCD. We observed that the treatment of aVNS alleviated postoperative memory impairment after exploratory laparotomy surgery, as demonstrated by the shorter swimming latency and distance in Morris water maze tests. Moreover, aVNS also reduced postoperative apoptosis in the hippocampus of the aged rats. Concomitant with these beneficial effects, we found that treatment with aVNS attenuated postoperative neuroinflammation (i.e., the protein level of interleukin-1β and tumor necrosis factor-α, along with the nuclear protein expression of NF-κB) and Alzheimer’s-related pathology (tau phosphorylation at AT-8 and Ser396, as well as the levels of Aβ40 and Aβ42) in the hippocampus of the aged rats. In conclusion, our study is the first to reveal the neuroprotective effect of aVNS against POCD. This effect might be attributed to the inhibition of neuroinflammation and Alzheimer’s-related pathology. This study suggests non-invasive aVNS may serve as a promising method for clinical treatment of POCD.

Rosmarinic acid attenuates inflammatory responses through inhibiting HMGB1/TLR4/NF-κB signaling pathway in a mouse model of Parkinson's disease

Inflammation contributes to the pathological processes in patients and animal models of PD. Rosmarinic acid (RA) has been demonstrated to protect neurons in PD models. The present study aimed to evaluate the anti-inflammatory effect of RA on PD and reveal possible pharmacological mechanisms. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) was injected to mice to establish PD model in vivo. BV-2 cells were exposed to 1-methyl-4-phenylpyridinium (MPP+) and α-synuclein to establish PD model in vitro. Results showed that treatment with RA dose-dependently improved motor function of PD mice, increased the number of tyrosine hydroxylase-positive cells, reduced production of pro-inflammatory cytokines, and inhibited microglia activation in ventral midbrain. In cell study, RA also decreased MPP+ or α-synuclein-induced secretion of pro-inflammatory cytokines. Furthermore, RA treatment downregulated the expression levels of HMGB1, TLR4 and Myd88 and inhibited NF-κB nuclear expression both in PD animal and cell models. These findings indicated that RA could attenuate inflammatory responses through suppressing HMGB1/TLR4/NF-κB signaling pathway, which may contribute to its anti-PD activity.

Exogenous ghrelin ameliorates acute lung injury by modulating the nuclear factor κB inhibitor kinase/nuclear factor κB inhibitor/nuclear factor κB pathway after hemorrhagic shock

Previous studies have shown that ghrelin, a peptide produced in the stomach, attenuates acute lung injury (ALI) in various animal models, and that some of these effects are associated with inhibition of the nuclear factor κB signaling pathway. This study investigated whether ghrelin exerts beneficial effects on hemorrhagic shock (HS)-induced ALI by modulating nuclear factor κB inhibitor kinase/nuclear factor κB inhibitor/nuclear factor κB (IKK/IκBα/NF-κB) pathway activity. HS was induced in male SD rats by withdrawing blood to a mean arterial pressure (MAP) of 40 mm Hg for 1 h; rats then received ghrelin (10 nmol/kg) or vehicle intravenously and were resuscitated with the shed blood and an equal volume of Ringer lactate solution followed by observation for 2 h. After resuscitation, samples were collected and analyzed for lung histopathology, wet to dry weight ratio (W/D), bronchoalveolar lavage fluid (BALF) protein, neutrophil infiltration, plasma inflammatory cytokines (TNF-α and IL-6), and cytoplasmic phosphorylated IKKβ, IκBα, phosphorylated IκBα and nuclear NF-κB expression. Compared to those in the two sham groups, lung injury, W/D, BALF protein, neutrophil infiltration, plasma TNF-α and IL-6 levels, and IKK/IκBα/NF-κB pathway activation were significantly increased in HS rats. After ghrelin administration, all parameters analyzed were decreased compared to those without ghrelin in HS rats. Moreover, ghrelin alleviated the decreased MAP after resuscitation compared to that in HS rats. Exogenous ghrelin attenuates the inflammatory response and acute lung injury after HS. These beneficial effects appear to be mediated through inhibition of IKK/IκBα/NF-κB signaling.

Anti-inflammatory effect of aged black garlic on 12-O-tetradecanoylphorbol-13-acetate-induced dermatitis in mice.

BACKGROUND/OBJECTIVESAlthough aged black garlic has various biological activities such as anti-allergy, anti-inflammation and neuroprotection, effect of aged black garlic on chemically contact dermatitis is unclarified.MATERIALS/METHODSTo evaluate anti-dermatitic activity of aged black garlic extract, we investigated effects of a fraction of aged black garlic extract (BG10) on both in vivo and in vitro.RESULTSBG10 almost inhibited formation of nitric monoxide and interleukin-6 (IL-6; IC50, 7.07 µg/mL) at 25 µg/mL, and dose-dependently reduced production of tumor necrosis factor-α (TNF-α; IC50, 52.07 µg/mL) and prostaglandin E2 (IC50, 38.46 µg/mL) in lipopolysaccharide-stimulated RAW264.7 cells. In addition, BG10 significantly inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2 and nuclear NF-κB, and improved that of cytosolic levels of NF-κB and IκBα in the cells. Consistent with in vitro studies, BG10 (0.5 mg/mL) not only reduced ear edema but also suppressed the formation of IL-6 and TNF-α induced by 12-O-tetradecanoylphorbol-13-acetate in ear tissues of mice.CONCLUSIONSThese findings suggest BG10 has anti-dermatitic activity through inhibiting activation of macrophages. Therefore, such effects of BG10 may provide information for the application of aged black garlic for prevention and therapy of contact dermatitis.

Ethyl pyruvate reduces spinal cord edema after spinal cord injury and attenuates spinal cord astrocyte swelling and aquaporin-4 expression after oxygen-glucose deprivation and reoxygenation in vitro by inhibiting high mobility group box-1 in rats

Objective To study the roles of ethyl pyruvate (EP) in spinal cord edema after spinal cord injury (SCI) and in spinal cord astrocytic swelling after oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro in rats. Methods After SCI models were established in adult Sprague-Dawley (SD) rats, an intraperitoneal injection of EP was conducted to inhibit high mobility group box-1 (HMGB1). Effects of EP on spinal cord edema, HMGB1 expression and astrocyte activation (glial fibrillary acidic protein (GFAP) expression) in SCI rats were analyzed. Spinal cord astrocytes were cultured in post-natal SD rats and incubated under OGD/R procedure. Effects of EP on cell swelling, expression of HMGB1, aquaporin-4 (AQP4) and toll-like receptor 4 (TLR4), and nuclear expression of nuclear factor-kappa B (NF-κB) in spinal cord astrocytes were observed. Results The water content in the spinal cord was increased significantly more at 1 d after SCI than at 12 h and 3 d (P<0.05). Intraperitoneal injection of EP at 50 mg/kg reduced spinal cord water content, HMGB1 expression and astrocyte activation (GFAP expression) in SCI rats significantly more than that at 25 mg/kg or 100 mg/kg (P<0.05). The volume of spinal cord astrocytes cultured in vitro after OGD 6 h/R 24 h was significantly greater than that after OGD 6 h/R 6 h or OGD 6 h/R 12 h (P<0.05). EP at 12 μmol/L reduced cell swelling, decreased expression of HMGB1, AQP4 and TLR4, and downgraded nuclear expression of NF-κB in spinal cord astrocytes after OGD/R significantly more than EP at 6 μmol/L(P<0.05). Conclusion EP may reduce early spinal cord edema after SCI, attenuate spinal cord astrocyte swelling and decrease AQP4 expression after OGD/R in vitro by inhibiting HMGB1 in rats. Key words: Spinal cord injury; Astrocyte; Ethyl pyruvate; High mobility group box-1; Aquaporin-4

Klotho Protein Protects Human Keratinocytes from UVB-Induced Damage Possibly by Reducing Expression and Nuclear Translocation of NF-κB.

BackgroundUV-related skin disease such as actinic keratosis is a major concern in public health. In view of the cell injury induced by UVB, Klotho protein it is an ideal therapy to eliminate UVB-induced cell damages and the associated signaling pathways.Material/MethodsTo gain insights into the potential role of Klotho and the underlying molecular mechanism, we constructed a Klotho-overexpress HaCaT cell line and assessed the protection against UVB insults. The effects of exposure to UVB radiation on the human keratinocyte HaCaT cells, including cell growth, apoptosis, and changes of selected biomarkers, were measured by CCK-8, flow cytometry, Quantitative real-time PCR, and Western blot analysis.ResultsWe found that enhanced NF-κB activity was accompanied by decreased expression of the anti-aging protein Klotho upon UVB stimulation, which was further confirmed with in vivo experiments. Overexpression of Klotho was able to considerably alleviate the UVB-induced damages to cells and reversed the UVB-caused biomarker changes to a great extent, which was comparable to the effects of administration of NF-κB inhibitor PDTC, suggesting the inhibition of nuclear translocation and DNA-binding activity of NF-κB. Furthermore, Klotho overexpression was proved to decrease the nuclear expression of NF-κB as much as the treatment with PDTC, which provides support for the direct regulation of NF-κB by Klotho.ConclusionsCollectively, our work provides new insight into the potential role of Klotho in the context of UVB-induced injuries in human keratinocytes, as well as providing the basis for future study of new therapies against UV-related skin disease.

Effects of Flower Buds Extract of Tussilago farfara on Focal Cerebral Ischemia in Rats and Inflammatory Response in BV2 Microglia.

To investigate the effects of the flower buds extract of Tussilago farfara Linné (Farfarae Flos; FF) on focal cerebral ischemia through regulation of inflammatory responses in activated microglia. Brain ischemia was induced in Sprague-Dawley rats by a transient middle cerebral artery occlusion (tMCAO) for 90 min and reperfusion for 24 h. Twenty rats were randomly divided into 4 groups (n=5 per group): normal, tMCAO-induced ischemic control, tMCAO plus FF extract 300 mg/kg-treated, and tMCAO plus MK-801 1 mg/kg-treated as reference drug. FF extract (300 mg/kg, p.o.) or MK-801 (1 mg/kg, i.p.) was administered after reperfusion. Brain infarction was measured by 2,3,5,-triphenyltetrazolium chloride staining. Neuronal damage was observed by haematoxylin eosin, Nissl staining and immunohistochemistry using anti-neuronal nuclei (NeuN), anti-glial fibrillary acidic protein (GFAP), and anti-CD11b/c (OX42) antibodies in ischemic brain. The expressions of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF-α), and hypoxia-inducible factor-1a (HIF-1α) were determined by Western blot. BV2 microglial cells were treated with FF extract or its main bioactive compound, tussilagone with or without lipopolysaccharide (LPS). Nitric oxide (NO) production was measured in culture medium by Griess assay. The expressions of iNOS, COX-2 and pro-inflammatory cytokines mRNA were analyzed by reverse transcription-polymerase chain reaction. The expression of iNOS, and COX-2 proteins, the phosphorylation of ERK1/2, JNK, and p38 MAPK and the nuclear expression of NF-κB p65 in BV2 cells were determined by Western blot. FF extract significantly decreased brain infarctions in ischemic rats (P<0.01). The neuronal death and the microglia/astrocytes activation in ischemic brains were inhibited by FF extract. FF extract also suppressed iNOS, TNF-α, and HIF-1α expression in ischemic brains. FF extract (0.2 and 0.5 mg/mL, P<0.01) and tussilagone 20 and 50 μmol/L, P<0.01) significantly decreased LPS-induced NO production in BV2 microglia through downregulation of iNOS mRNA and protein expression. FF extract and tussilagone significantly inhibited LPS-induced expression of TNF-α, IL-1β, and IL-6 mRNA, and also suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK and the nuclear expression of NF-κB in a dose-dependent manner. FF extract has a neuroprotective effect in ischemic stroke by the decrease of brain infarction, and the inhibition of neuronal death and microglial activation-mediated inflammatory responses.

P75 Involved in the Ubiquitination of α-synuclein in Rotenone-based Parkinson’s Disease Models

For Parkinson’s disease (PD), the regulatory mechanism of α-synuclein (α-syn) aggregation remains to be clarified. Ubiquitination modification is crucial for α-syn aggregation, with implications for Lewy body formation. Besides, ubiquitin ligase absentia homolog (siAH) is involved in the ubiquitination of α-syn. We investigated whether the p75 receptor can act as a potential regulator of α-syn accumulation through ubiquitination. Western blot, immunoprecipitation, gene transfection, and RNA interference technology were employed to detect the effect of p75 in in vivo and in vitro models. In a rotenone-based stereotactic (ST) infusion in vivo model of PD, p75 receptor and siAH expression was increased significantly compared with the control group. In cellular models of rotenone-mediated neurotoxicity, the interactions between p75 and siAH were revealed by immunoprecipitation; the colocalization of p75 with α-syn was observed in the cytoplasm; p75 promoted nuclear expression of NF-κB (p65), which might interact with the promoter of the siAH gene. Moreover, siRNA-mediated p75 depletion reduced the upregulation of α-syn and nuclear expression of p65 and protected against cell apoptosis induced by rotenone. Thus, aberrant expression of p75 may regulate the increased expression of α-syn, which is related to siAH-mediated ubiquitination and nuclear expression of p65.

Role of PI3K/Akt/NF-κB and GSK-3β pathways in the rat model of cardiopulmonary bypass-related lung injury

BackgroundApoptosis is a cellular mechanism contributing to cardiac surgery using cardiopulmonary bypass (CPB)-induced lung injury. The ubiquitous PI3K/Akt pathway regulates proliferation, apoptosis and differentiation by controlling a broad range of target proteins including NF-κB and GSK-3β. The roles of the PI3K/Akt/NF-κB and PI3K/Akt/GSK-3β pathways in CPB-related lung injury are unclear. MethodsSeventy-two male Sprague-Dawley rats were assigned into sham, CPB, Wortmannin (Wtn) and insulin-like growth factor-I (IGF-I) groups (n = 18, each). Six subjects per group were evaluated at each of three time points: Prior to CPB (T1); opening of the left hilus pulmonis (T2); and 90 min after CPB (T3). Arterial blood specimens were obtained at each time point to test respiratory and oxygenation indices. Left lung tissues were processed for H&E and TUNEL staining. Western blot was employed to evaluate protein levels and activities of Akt, phospho-Akt (p-Akt), GSK-3β, phospho-GSK-3β (p-GSK-3β) and nuclear NF-κB. ResultsLung ischemia/reperfusion and CPB caused notable lung injury, as evidenced by lung functional decline and pathological deterioration, accompanied by increases in apoptosis and expression levels of p-Akt, p-GSK-3β and nuclear NF-κB in lungs (all P < 0.05 vs. Sham). At T3, Wtn-treated CPB subjects showed worsened lung function and pathological lung structures, as well as apoptosis in lungs (all P < 0.05 vs. CPB); additionally, Wtn inhibited Akt phosphorylation and slightly, but significantly increased expression of nuclear NF-κB (both P < 0.001 vs. CPB). Conversely, treatment of subjects with IGF-I increased Akt phosphorylation (P < 0.001 vs. CPB), inhibited expression of nuclear NF-κB (P = 0.008 vs. CPB), improved lung function and tissue morphology (both P < 0.05 vs. CPB), and reduced apoptosis in lungs (P < 0.001 vs. CPB). Neither Wtn nor IGF-I did alter GSK-3β phosphorylation levels (P = 0.836 and P = 0.669 vs. CPB, respectively). ConclusionThe PI3K/Akt/NF-κB pathway played a role in CPB-related lung injury, possibly through mediating apoptosis in lungs. GSK-3β, a signaling effector that also participated in CPB-induced apoptosis in lungs, but was not regulated by the PI3K/Akt pathway in this context.

The effect of hyperuricemia on cardiac function, nuclear factor‐κB in myocardial tissue, Mn superoxide dismutases activity in mitochondria of rats

ObjectiveTo investigate the effect of hyperuricemia (HUA)on nuclear factor‐κB(NF‐κB) in myocardial tissue, Mn superoxide dismutases ( Mn‐SOD) activity in mitochondria, cardiac function of rats with high blood uric acid (HUA).MethodsSixty SD male rats, including 30 young rats and 30 aged rats, were randomly divided into four groups: the young control group, the young HUA group, the aged control group and the aged HUA group. Fifteen rats were in each group. The HUA rat models were set up by lavage method with yeast extract and ethambutol. Left ventricular systolic pressure (LVSP), left ventricular end‐diastolic pressure (LVEDP) and left ventricular pressure at the end of the rising and falling maximum velocity (+ dp/dtmax) were determinated by intubation in the left ventricular with cardiac catheter. Mn‐SOD activity in the mitochondria of rats myocardial tissue were detected by chemical colorimetry; The expression of nuclear NF‐κB in rats myocardial tissue were measured by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction(RT‐PCR). Differences between groups was analyzed with One way analysis of variance, Student‐Newman‐Keuls q tests was used to compare the mean of multiple samplesResultsHyperuricemia had no significant effect on the cardiac function of HUA groups. But the gene and protein expression of NF‐κB in myocardial tissue and Mn‐SOD activity of mitochondria were impacted by HUA. Both the gene and protein expression of NF‐κB and the activity of Mn‐SOD in HUA groups were increased (F=85.428 4, 120.683 0 and 398.228 3, P&lt;0.05). Furthermore, the increase of gene and protein expression of NF‐κB and the Mn‐SOD activity in rats myocardial tissue of young HUA group were higher than those in aged HUA group.(q= 6.818 6, 10.693 6 and 18.877 9, P &lt; 0.05).ConclusionHyperuricemia may cause inflammation and increase oxygen free radicals in myocardial tissue on one hand, and it may reduce inflammation and protect the myocardial tissue by increasing the activity of Mn‐SOD in myocardial mitochondria of HUA rats, and removing the oxygen free radicals on the other hand. The response to uric acid in young HUA group is stronger than that in the aged HUA group.Support or Funding InformationThis work supported by National Natural Science Funds of China (30600637, 81573245); Science and Technology Research Project of Shanxi Province Health and Family Planning Commission (201301068); Science and Technology Development Project of Shanxi Province (20111101); Doctor Startup Funds of Shanxi Medical University, (B03201217); The First Hospital Funds of Shanxi Medical University (YG1303); China Postdoctoral science Foundation Grant (2014M561207); The Education Reform Project of Shanxi Medical University;, The Education Reform Project of Postgraduate Students in Shanxi Province (No. 2017‐027);The Teaching Reform Program of Shanxi Higher Education (No. 2013‐033);The science and technology research projects of health department of Shanxi Province(Fund No.2016D011119).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Salvia miltiorrhiza Polysaccharides on Lipopolysaccharide-Induced Inflammatory Factor Release in RAW264.7 Cells.

This study investigated the anti-inflammatory effects and possible underlying mechanisms of Salvia miltiorrhiza polysaccharides (SMP) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The cytotoxicity of SMP was detected by the MTT method. The morphological change of RAW264.7 was observed by Diff-Quik staining. Enzyme-linked immunosorbent assay was used to evaluate the production of cytokines in LPS-induced RAW264.7 cells. The nitric oxide (NO) kit assay detected the NO release from LPS-induced RAW264.7 cells. Real-time polymerase chain reaction was used to detect the transcriptions of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), inducible NO synthase (iNOS), and cyclooxygenase (COX)-2 in LPS-induced RAW264.7 cells. The protein expression of nuclear NF-κB was measured by Western blot. The results showed that the safe medication range of SMP was less than 3 mg/mL. Compared with the LPS model group, SMP (2, 1, and 0.5 mg/mL) improved the degree of cell deformation and reduced the amount of pseudopodia, and statistically reduced the secretions of cytokines in cells induced by LPS (P < 0.01) at different time points. SMP significantly inhibited the mRNA transcriptions of TNF-α, IL-6, iNOS, and COX-2 and the protein expressions of NF-κB, p-p65, and p-IκBa. In conclusion, this study preliminarily proved the protective effect of SMP on LPS-induced RAW264.7 macrophage. Its mechanism might be related to inhibition of NF-κB signal pathway and the gene expressions and secretion of cytokines.

Inhibitory effects of Aconiti Lateralis Radix Preparata on chronic intermittent cold-induced inflammation in the mouse hypothalamus

Ethnopharmacological relevanceAconiti Lateralis Radix Preparata (AR) is the most frequently used herb to generate heat and treat symptoms associated with coldness in Asia. Aims of the studyThe hypothalamus is one of the master regulators to maintain constant core body temperature. Chronic exposure to cold stress disturbs homeostatic regulation, gradually resulting in hypothalamic inflammation. This study investigate the effects of AR, on the chronic intermittent cold (CIC)-induced release of pro-inflammatory signaling molecules in the mouse hypothalamus. Materials and methodsAconiti Lateralis Radix Preparata extract (ARE) were solubilized in distilled water and diluted with saline before administration. Male ICR mice (7 weeks old, 30–32g) were divided randomly into 6 groups: (1) control, (2) cold stress, (3) ARE 30, (4) ARE 100, (5) ARE 300, and (6) ARE 1000mg/kg groups. Groups (2)-(6) were exposed to CIC stress once a day for 14 days. CIC stress was achieved by exposing the mice to 4°C and 60 ± 10% humidity for 120min once a day. Rectal temperature was measured after terminating cold stress. Cortisol levels were measured from serum. Hypothalamus tissue was used for western blot analysis, and IL-9, IL-13, PGE1, and PGE2 levels were assessed. ResultsARE treatment prevented the CIC-induced decrease in rectal temperature and increase in serum cortisol level. ARE-treated CIC-exposed mice demonstrated decrease in nuclear c-Fos levels dose-dependently compared to CIC-exposed mice. Nuclear NF-kB expression showed significant increase in CIC-exposed mice. ARE treatment significantly blunted the increase in nuclear NF-kB expression. CIC-exposed mice had significantly increased levels of both IL-9 and IL-13. Treatment with ARE suppressed the elevated IL-9 and IL-13 levels. Between control and CIC-exposed mice PGE1 levels showed no difference. However ARE (1000mg/kg)-treated CIC-exposed mice had a significant increase in PGE1 level compared to CIC-exposed mice. PGE2 levels were significantly higher in CIC-exposed mice compared to control mice. ARE treatment significantly attenuated the increase in PGE2 levels. ConclusionsOur findings suggest CIC stress disturbs the anti-inflammatory effect of cortisol and maintenance of the body temperature. Thus AR contributes to suppress the activated proinflammatory factors, IL-9, IL-13, and PGE-2, and to increase the heat production.