Catalpol attenuates polycystic ovarian syndrome by regulating sirtuin 1 mediated NF-κB signaling pathway

Oxidative stress plays a central role in polycystic ovary syndrome (PCOS). Catalpol (CAT) is the active ingredient of Rehmannia glutinosa Libosch which has therapeutic effect on PCOS. However, little is known about the mechanism of CAT in PCOS. PCOS rats were induced by subcutaneous injection of dehydroepiandrosteronec for four weeks and then were treated with CAT (50 mg/kg) or carboxyl methyl cellulose (the solvent of CAT) or normal saline for another 4 weeks. Histopathological observation of ovarian tissues, the levels of testosterone, estradiol and progesterone in rat plasma samples, the oxidative stress related-indexes and the expressions of NF-κB pathway-related proteins were determined. KGN cell (human ovarian granulosa cell line) was used as PCOS cell model and was transfected with siSIRT1 in the presence of CAT. The viability, proliferation and apoptosis of cells and the levels of SIRT1 and NF-κB pathway-related proteins were measured. CAT lessened the anthropometric indices and improved ovarian damage in PCOS model rats, and reduced the levels of testosterone, estradiol, progesterone and MDA, increased GSH content, and elevated the activities of catalase, GSH-Px and SOD in ovarian tissues of PCOS model rats. CAT up-regulated SIRT1 level and inhibited the activation of NF-κB signaling pathway in PCOS rat model and KGN cells. Silencing SIRT1 increased the viability and proliferation, whilst decreased the apoptosis of CAT-treated KGN cells. Silencing SIRT1 counteracted the effect of CAT on the level of oxidative stress-related factors and NF-κB signaling pathway in KGN cells. CAT attenuated PCOS by regulating SIRT1 mediated NF-κB signaling pathway.     

Interaction between PKR and PACT mediated by LPS-inducible NF-κB in human gingival cells

The double‐stranded RNA‐dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double‐stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 µg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF‐κB to the nucleus after 30 min, and inhibition of NF‐κB decreased the PACT–PKR interaction induced by LPS. The level of pro‐inflammatory cytokine mRNA, including interleukin‐6 (IL‐6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro‐inflammatory cytokines was not affected by RNAi‐mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF‐κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT–PKR association in Sa3 cells. Our results also suggest that NF‐κB is involved in the PACT–PKR interaction and the production of pro‐inflammatory cytokines in periodontitis. J. Cell. Biochem. 113: 165–173, 2012. © 2011 Wiley Periodicals, Inc.     

1B/(-)IRE DMT1 expression during brain ischemia contributes to cell death mediated by NF-κB/RelA acetylation at Lys310

The molecular mechanisms responsible for increasing iron and neurodegeneration in brain ischemia are an interesting area of research which could open new therapeutic approaches. Previous evidence has shown that activation of nuclear factor kappa B (NF-κB) through RelA acetylation on Lys310 is the prerequisite for p50/RelA-mediated apoptosis in cellular and animal models of brain ischemia. We hypothesized that the increase of iron through a NF-κB-regulated 1B isoform of the divalent metal transporter-1 (1B/DMT1) might contribute to post-ischemic neuronal damage. Both in mice subjected to transient middle cerebral artery occlusion (MCAO) and in neuronally differentiated SK-N-SH cells exposed to oxygen-glucose-deprivation (OGD), 1A/DMT1 was only barely expressed while the 1B/DMT1 without iron-response-element (-IRE) protein and mRNA were early up-regulated. Either OGD or over-expression of 1B/(-)IRE DMT1 isoform significantly increased iron uptake, as detected by total reflection X-ray fluorescence, and iron-dependent cell death. Iron chelation by deferoxamine treatment or (-)IRE DMT1 RNA silencing displayed significant neuroprotection against OGD which concomitantly decreased intracellular iron levels. We found evidence that 1B/(-)IRE DMT1 was a target gene for RelA activation and acetylation on Lys310 residue during ischemia. Chromatin immunoprecipitation analysis of the 1B/DMT1 promoter showed there was increased interaction with RelA and acetylation of H3 histone during OGD exposure of cortical neurons. Over-expression of wild-type RelA increased 1B/DMT1 promoter-luciferase activity, the (-)IRE DMT1 protein, as well as neuronal death. Expression of the acetylation-resistant RelA-K310R construct, which carried a mutation from lysine 310 to arginine, but not the acetyl-mimic mutant RelA-K310Q, down-regulated the 1B/DMT1 promoter, consequently offering neuroprotection. Our data showed that 1B/(-)IRE DMT1 expression and intracellular iron influx are early downstream responses to NF-κB/RelA activation and acetylation during brain ischemia and contribute to the pathogenesis of stroke-induced neuronal damage.     

PIAS1 protects against myocardial ischemia-reperfusion injury by stimulating PPARγ SUMOylation

Background

Myocardial ischemia-reperfusion injury (IRI) has become one of the most serious complications after reperfusion therapy in patients with acute myocardial infarction. Small ubiquitin-like modification (SUMOylation) is a reversible process, including SUMO E1-, E2-, and E3-mediated SUMOylation and SUMO-specific protease-mediated deSUMOylation, with the latter having been shown to play a vital role in myocardial IRI previously. However, little is known about the function and regulation of SUMO E3 ligases in myocardial IRI.

Results

In this study, we found dramatically decreased expression of PIAS1 after ischemia/reperfusion (I/R) in mouse myocardium and H9C2 cells. PIAS1 deficiency aggravated apoptosis and inflammation of cardiomyocytes via activating the NF-κB pathway after I/R. Mechanistically, we identified PIAS1 as a specific E3 ligase for PPARγ SUMOylation. Moreover, H9C2 cells treated with hypoxia/reoxygenation (H/R) displayed reduced PPARγ SUMOylation as a result of down-regulated PIAS1, and act an anti-apoptotic and anti-inflammatory function through repressing NF-κB activity. Finally, overexpression of PIAS1 in H9C2 cells could remarkably ameliorate I/R injury.

Conclusions

Collectively, our findings demonstrate the crucial role of PIAS1-mediated PPARγ SUMOylation in protecting against myocardial IRI.

     

Emodin suppresses oxaliplatin-induced neuropathic pain by inhibiting COX2/NF-κB mediated spinal inflammation

Oxaliplatin (OXA) is a common chemotherapy drug for colorectal, gastric, and pancreatic cancers. The anticancer effect of OXA is often accompanied by neurotoxicity and acute and chronic neuropathy. The symptoms present as paresthesia and pain which adversely affect patients' quality of life. Herein, five consecutive intraperitoneal injections of OXA at a dose of 4 mg/kg were used to mimic chemotherapy. OXA administration induced mechanical allodynia, activated spinal astrocytes, and increased inflammatory response. To develop an effective therapeutic measure for OXA-induced neuropathic pain, emodin was intrathecally injected into OXA rats. Emodin developed an analgesic effect, as demonstrated by a significant increase in the paw withdrawal threshold of OXA rats. Moreover, emodin treatment reduced the pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β) which upregulated in OXA rats. Furthermore, autodock data showed four hydrogen bonds were formed between emodin and cyclooxygenase-2 (COX2), and emodin treatment decreased COX2 expression in OXA rats. Cell research further proved that emodin suppressed nuclear factor κB (NF-κB)-mediated inflammatory signal and reactive oxygen species level. Taken together, emodin reduced spinal COX2/NF-κB mediated inflammatory signal and oxidative stress in the spinal cord of OXA rats which consequently relieved OXA-induced neuropathic pain.     

Modulation of reactive oxygen species in skeletal muscle by myostatin is mediated through NF-κB

Abnormal levels of reactive oxygen species (ROS) and inflammatory cytokines have been observed in the skeletal muscle during muscle wasting including sarcopenia. However, the mechanisms that signal ROS production and prolonged maintenance of ROS levels during muscle wasting are not fully understood. Here, we show that myostatin (Mstn) is a pro-oxidant and signals the generation of ROS in muscle cells. Myostatin, a transforming growth factor-β (TGF-β) family member, has been shown to play an important role in skeletal muscle wasting by increasing protein degradation. Our results here show that Mstn induces oxidative stress by producing ROS in skeletal muscle cells through tumor necrosis factor-α (TNF-α) signaling via NF-κB and NADPH oxidase. Aged Mstn null (Mstn(-/-) ) muscles, which display reduced sarcopenia, also show an increased basal antioxidant enzyme (AOE) levels and lower NF-κB levels indicating efficient scavenging of excess ROS. Additionally, our results indicate that both TNF-α and hydrogen peroxide (H(2) O(2) ) are potent inducers of Mstn and require NF-κB signaling for Mstn induction. These results demonstrate that Mstn and TNF-α are components of a feed forward loop in which Mstn triggers the generation of second messenger ROS, mediated by TNF-α and NADPH oxidase, and the elevated TNF-α in turn stimulates Mstn expression. Higher levels of Mstn in turn induce muscle wasting by activating proteasomal-mediated catabolism of intracellular proteins. Thus, we propose that inhibition of ROS induced by Mstn could lead to reduced muscle wasting during sarcopenia.     

Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.     

NleC, a type III secretion protease, compromises NF-κB activation by targeting p65/RelA

The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC.     

Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-κB Protein p65/RelA by Meningococcal IgA Protease

Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells.     

Microbial-induced immunomodulation by targeting the NF-κB system

Virtually all eukaryotes have developed defense mechanisms to efficiently counter potential threats from prokaryotic microorganisms; an example is the conserved nuclear factor-kappaB (NF-κB) signaling system. However, bacterial pathogens and commensals have in turn evolved highly effective counter mechanisms to modulate this immune regulatory circuit. Modifications in ubiquitin, ubiquitin-like (Ubl) proteins such as neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) and other post-translational modifications (PTMs) in the NF-κB system represent attractive targets for microbial manipulation. In this review, we describe recent advances in understanding the different strategies that bacteria have evolved to interfere with PTMs in NF-κB signal transmission.