Crocin Attenuates Kindling Development and Associated Cognitive Impairments in Mice via Inhibiting Reactive Oxygen Species‐Mediated NF‐κB Activation

Crocin is a pharmacologically active carotenoid pigment mainly present in the stigmas of Crocus sativus L. (Iridaceae). It has been well explored in experimental animal models of cognitive impairments, depression, anxiety and epilepsy. This study was designed to understand the effect of crocin on pentylenetetrazol (PTZ)‐induced kindling development and its associated cognitive deficit in mouse. Crocin treatment at 5, 10 and 20 mg/kg p.o. doses showed a marked reduction in severity of PTZ‐induced seizures. There was an increase in novel object preference index and discrimination ratio in the crocin‐treated groups in the novel object recognition test. Its treatment also increased percentage spontaneous alternations in T‐maze test at all the tested doses. Histopathological examination by Nissl staining showed a reduction in dark neurons in the hippocampal pyramidal layer of crocin‐treated animals in contrast to vehicle control, indicating a decrease in neuronal damage. Biochemical estimations showed a significant increase in superoxide dismutase activity and reduced reactive oxygen species (ROS) in the hippocampus of crocin‐treated animals. Immunohistochemistry results revealed attenuation in the levels of nuclear factor‐κB (NF‐κB) and phosphorylated NF‐κB in the hippocampal sections of crocin‐treated animals. The results of this study concluded that crocin treatment increased seizure threshold, thus inhibiting PTZ‐induced kindling development and improving cognitive functions. The effect was found to be due to suppression of seizure‐induced ROS generation and its linked NF‐κB pathway‐associated neuronal damage.     

Puerarin Inhibits C‐Reactive Protein Expression via Suppression of Nuclear Factor κB Activation in Lipopolysaccharide‐Induced Peripheral Blood Mononuclear Cells of Patients with Stable Angina Pectoris

Abstract: Puerarin (4′‐7‐dihydroxy‐8‐β‐d ‐glucosylisoflavone), the most abundant isoflavone‐C‐glucoside extracted from the root of the plant Pueraria lobata, has demonstrated anti‐inflammatory activity in cellular models of inflammation. In this report, we examined the ability of puerarin to modulate C‐reactive protein (CRP) expression and key molecules in the nuclear factor kappa B (NF‐κB) pathway to determine its molecular target. The protein and mRNA levels of CRP were determined in lipopolysaccharide (LPS)‐induced peripheral blood mononuclear cells of patients with unstable angina pectoris. Also, we detected the I‐κBα phosphorylation and the p65NF‐κB expression in peripheral blood mononuclear cells under our experimental condition. The results indicated that puerarin inhibited the expression of the protein and mRNA levels of CRP in LPS‐induced peripheral blood mononuclear cells. Subsequently, we determined that the inhibition of CRP expression was because of a dose‐dependent inhibition of phosphorylation and degradation of inhibitor kappaB(I‐κB), which resulted in a reduction of p65NF‐κB nuclear translocation. We conclude that puerarin acts as an anti‐inflammatory agent by blocking NF‐κB signalling, and may possibly be developed as a useful agent for the chemoprevention of atherosclerosis.     

Placental Growth Factor Triggers Epithelial‐to‐Mesenchymal Transition‐like Changes in Rat Type II Alveolar Epithelial Cells: Activation of Nuclear Factor κB Signalling Pathway

Epithelial‐to‐mesenchymal transition (EMT) occurs frequently in lung epithelium in response to damage stimuli, such as inflammation and oxidative stress. Our group has already found an excessive release of placental growth factor (PLGF) in hyperoxic lung injury in newborn rodents. Nonetheless, whether this increased PLGF plays a role in mediating EMT process of alveolar epithelial cells (AECs) is unclear and requires further investigation. In this study, primary type II AECs were first isolated for neonatal Sprague Dawley (SD) rats and treated with different doses (25 or 100 ng/ml) of recombinant PLGF for 48 hr. As compared to the untreated AECs, exogenous PLGF induced a reduction in E‐cadherin (a critical epithelial maker) and increases in vimentin and fibronectin (mesenchymal markers) in a dose‐dependent manner. The higher dose was more potent in promoting the EMT‐like changes in AECs. We also found that PLGF promoted the degradation of nuclear factor κB (NFκB) inhibitor (IκBα), induced phosphorylation of NFκB p65 at Ser 536 and enhanced p65 nuclear translocation in primary AECs. In addition, the NFκB inhibitor BAY 11‐7082 partly counteracted PLGF‐induced alterations in EMT‐related proteins in rat AECs. In summary, our study demonstrates that exogenous PLGF can induce EMT‐like changes in type II AECs at least partly by activating NFκB signalling transduction in vitro. PLGF may play a role in driving fibrotic lesions and thus can be a promising target for lung diseases.     

A 5′ AMP‐Activated Protein Kinase Enzyme Activator,Compound 59, Induces Autophagy and Apoptosis in Human Oral Squamous Cell Carcinoma

5′ AMP‐activated protein kinase enzyme (AMPK), a master regulator of cellular metabolism, is recognized for its association with various metabolic diseases, inflammation and cancer. In this study, we aimed to investigate the role of compound 59, an AMPK activator, in a panel of oral squamous cell carcinoma (OSCC) cell lines. The antiproliferative effects of compound 59 were assessed by MTT assays, flow cytometry, Western blotting, confocal microscopy and transmission electron microscopy. Relative to OSCC cells, normal human oral keratinocytes were almost insensitive to compound 59 treatment. Compound 59 induced apoptosis as indicated by caspase activation and PARP cleavage. In addition, it inhibited JAK/STAT3 signalling, arrested cells in the G1 phase, increased reactive oxygen species (ROS) generation and promoted autophagy. Interestingly, pre‐treatment with a protein tyrosine phosphatase (PP2A) inhibitor, cantharidin, partially reversed compound 59‐induced down‐regulation of p‐JAK2 and p‐STAT3, thereby suggesting the involvement of a protein tyrosine phosphatase. Together, these findings substantiate the potential of compound 59 for the treatment of OSCC patients.     

Apoptosis induction and ERK/NF‐κB inactivation are associated with magnolol‐inhibited tumor progression in hepatocellular carcinoma in vivo

Although hepatitis B and/or hepatitis C virus were recognized as major risk factor for the development of hepatocellular carcinoma (HCC), certain occupational, environmental, and lifestyle factors also play key roles in HCC tumorigenesis. Moreover, in molecular signaling route, extracellular signal‐regulated kinase (ERK)/nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) signaling was found to be overexpressed and linked to poor prognosis in HCC. Thus, to identify possible nature compound that can suppress ERK/NF‐κB may be benefit to HCC patient. Magnolol, a natural compound derived from herbal plant Magnolia officinalis, has been recognized as a liver protection and antitumor reagent. However, whether magnolol‐inhibited HCC progression correlates with disruption of ERK/NF‐κB signaling is remained unclear. In this studies, we performed SK‐Hep1/luc2 HCC bearing animal model to investigate the anticancer efficacy and mechanism of magnolol on tumor progression. Tumor size and tumor growth rate were dramatically suppressed after treatment of magnolol. In addition, expression of phospho‐ERK (p‐ERK), NF‐κB p65 (Ser536), and tumor progression‐associated proteins, such as matrix metallopeptidase 9 (MMP‐9), vascular endothelial growth factor (VEGF), X‐linked inhibitor of apoptosis protein (XIAP), and CyclinD1 were all significantly decreased by magnolol. Most important, major extrinsic and intrinsic apoptosis signaling factors, including active caspase‐8 and caspase‐9 were both enhanced by magnolol. This study indicated that apoptosis induction through extrinsic/intrinsic pathways and blockage of ERK/NF‐κB activation were associated with magnolol‐inhibited tumor progression in HCC in vivo.     

Acetaminophen Induces a Caspase‐Dependent and Bcl‐xL Sensitive Apoptosis in Human Hepatoma Cells and Lymphocytes

Abstract: Acetaminophen is a widely used analgesic and antipyretic drug that exhibits toxicity at high doses to the liver and kidneys. This toxicity has been attributed to cytochrome P‐450‐generated metabolites which covalently modify target proteins. Recently, acetaminophen, in its unmetabolized form, has been shown to affect a variety of cells and tissues, for instance, testicular and lymphoid tissues and lymphocyte cell lines. The effects on cell viability of acetaminophen at a concentration comparable to that achieved in plasma during acetaminophen toxicity have now been examined with a hepatoma cell line SK‐Hep1, primary human peripheral blood lymphocytes and human Jurkat T cells. Acetaminophen reduced cell viability in a time‐dependent manner. Staining of cells with annexin‐V also revealed that acetaminophen induced, after 8 hr of treatment, a loss of the asymmetry of membrane phospholipids, which is an early event associated with apoptosis. Acetaminophen triggered the release of cytochrome c from mitochondria into the cytosol, activation of caspase‐3, 8, and 9, cleavage of poly(ADP‐ribose) polymerase, and degradation of lamin B₁ and DNA. Whereas cleavage of DNA into internucleosomal fragments was apparent in acetaminophen treated SK‐Hep1 and primary lymphocytes, DNA was only degraded to 50‐kb fragments in treated Jurkat cells. Overexpression of the antiapoptotic protein Bcl‐x_L prevented these various apoptotic events induced by acetaminophen in Jurkat cells. Caspase‐8 activation was a postmictochondrial event and occurred in a Fas‐independent manner. These results demonstrate that acetaminophen induces caspases‐dependent apoptosis with mitochondria as a primary target. These results also reiterate the potential role of apoptosis in acetaminophen hepatic and extrahepatic toxicity.     

MiR‐146a regulates PM1‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells

Exposure to particulate matter (PM) leads to kinds of cardiopulmonary diseases, such as asthma, COPD, arrhythmias, lung cancer, etc., which are related to PM‐induced inflammation. We have found that PM_2.5 (aerodynamics diameter <2.5 µm) exposure induces inflammatory response both in vivo and in vitro. Since the toxicity of PM is tightly associated with its size and components, PM₁ (aerodynamics diameter <1.0 µm) is supposed to be more toxic than PM_2.5. However, the mechanism of PM₁‐induced inflammation is not clear. Recently, emerging evidences prove that microRNAs play a vital role in regulating inflammation. Therefore, we studied the regulation of miR‐146a in PM₁‐induced inflammation in human lung bronchial epithelial BEAS‐2B cells. The results show that PM₁ induces the increase of IL‐6 and IL‐8 in BEAS‐2B cells and up‐regulates the miR‐146a expression by activating NF‐κB signaling pathway. Overexpressed miR‐146a prevents the nuclear translocation of p65 through inhibiting the IRAK1/TRAF6 expression, and downregulates the expression of IL‐6 and IL‐8. Taken together, these results demonstrate that miR‐146a can negatively feedback regulate PM₁‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells.     

NF‐κB/p53‐activated inflammatory response involves in diquat‐induced mitochondrial dysfunction and apoptosis

Inflammation generated by environmental toxicants including pesticides could be one of the factors underlying neuronal cell damage in neurodegenerative diseases. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in PC12 cells treated with diquat. We found that diquat induced apoptosis, as demonstrated by the activation of caspases and nuclear condensation, inhibition of mitochondrial complex I activity, and decreased ATP level in PC12 cells. Diquat also reduced the dopamine level, indicating that cell death induced by diquat is due to cytotoxicity of dopaminergic neuronal components in these cells. Exposure of PC12 cells to diquat led to the production of reactive oxygen species (ROS), and the antioxidant N‐acetyl‐cystein attenuated the cytotoxicity of caspase‐3 pathways. These results demonstrate that diquat‐induced apoptosis is involved in mitochondrial dysfunction through production of ROS. Furthermore, diquat increased expression of cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) via inflammatory stimulation. Diquat induced nuclear accumulation of NF‐κB and p53 proteins. Importantly, an inhibitor of NF‐κB nuclear translocation blocked the increase of p53. Both NF‐κB and p53 inhibitors also blocked the diquat‐induced inflammatory response. Pretreatment of cells with meloxicam, a COX‐2 inhibitor, also blocked apoptosis and mitochondrial dysfunction. These results represent a unique molecular characterization of diquat‐induced cytotoxicity in PC12 cells. Our results demonstrate that diquat induces cell damage in part through inflammatory responses via NF‐κB‐mediated p53 signaling. This suggests the potential to generate mitochondrial damage via inflammatory responses and inflammatory stimulation‐related neurodegenerative disease.     

4‐chloro‐1,2‐phenylenediamine Induces Apoptosis in Mardin–Darby canine kidney cells via activation of caspases

4‐Chloro‐1,2‐phenylenediamine (4‐Cl‐o‐PD) is a halogenated aromatic diamine that was used as a precursor for manufacturing permanent hair dyes. Despite its well‐documented mutagenic and carcinogenic effects in a number of in vitro and in vivo models, its cytotoxicity and mode of action have not received similar attention. Here, we investigated the effect of 4‐Cl‐o‐PD on Mardin–Darby canine kidney cells. It induced apoptosis and the evidence suggests its initiation by reactive oxygen species (ROS). The results of various assays used show a dose‐dependent (i) decrease in cell viability, (ii) increase in cells at sub‐G1 phase and the G0/G1 phase arrested in cell cycle, (iii) increase in intracellular ROS accompanied by depletion of glutathione, and (iv) that apoptotic cell death probably involves activation of both intrinsic and extrinsic pathways. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 655–664, 2014.     

Curcumin alleviates lung injury in diabetic rats by inhibiting nuclear factor‐κB pathway

Curcumin is a polyphenolic compound that is extracted from Curcuma longa. It has broad anti‐inflammation and anti‐tumor activities. Curcumin was previously reported to exert beneficial effects on diabetes. However, the effect of curcumin on diabetes‐induced lung injury is not yet clear. In this study, the effects of curcumin on lung injury induced by diabetes was explored using quantitative real time polymerase chain reaction (PCR), enzyme‐linked immunosorbent assay (ELISA), immunohistochemistry and electrophoretic mobility shift assay. The results of this study showed that curcumin reduced oxidative stress level, inhibited the synthesis of nitric oxide and prostaglandin E2, and reduced inflammatory responses in the lungs of diabetic rats, thereby alleviating diabetes‐induced lung injury. Further study of the mechanism revealed that curcumin inhibited the activation of nuclear factor (NF)‐κB which is a key player in inflammatory responses. In summary, our study demonstrated that curcumin inhibited the activation of NF‐κB in the lungs of diabetic rats, thus reducing pulmonary inflammatory responses and oxidative stress, and ultimately relieving diabetes‐induced lung injury. This study suggests that curcumin may be a promising agent to alleviate diabetic lung injury and also provides theoretical foundation for the development of diabetes therapy.     

Involvement of NF‐κB in the upregulation of cystathionine‐γ‐lyase,a hydrogen sulfide‐forming enzyme,and bladder pain accompanying cystitis in mice

Hydrogen sulfide (H₂S) is generated from l ‐cysteine by multiple enzymes including cystathionine‐γ‐lyase (CSE), and promotes nociception by targeting multiple molecules such as Ca_v3.2 T‐type Ca²⁺ channels. Bladder pain accompanying cyclophosphamide (CPA)‐induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H₂S/Ca_v3.2 pathway. Therefore, we investigated whether NF‐κB, as an upstream signal of the CSE/H₂S system, contributes to bladder pain in mice with CPA‐induced cystitis. Bladder pain‐like nociceptive behaviour was observed in CPA‐treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. β‐Cyano‐l ‐alanine, a reversible selective CSE inhibitor, prevented CPA‐induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF‐κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF‐κB inhibitor. PDTC and curcumin, which inhibits NF‐κB signals, abolished CPA‐induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA‐induced activation of NF‐κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H₂S/Ca_v3.2 signaling. Therefore, NF‐κB‐inhibiting compounds including curcumin may be useful for the treatment of cystitis‐related bladder pain.     

Sensitization of Hepatocellular Carcinoma Cells to Apo2L/TRAIL by a Novel Akt/NF‐κB Signalling Inhibitor

Hepatocellular carcinoma (HCC) cells are intrinsically resistant to tumour necrosis factor‐related apoptosis ligand (Apo2L/TRAIL), in part, due to the compensatory activation of nuclear factor‐kappaB (NF‐κB). To broaden the clinical utilization of Apo2L/TRAIL in HCC, OSU‐A9, a potent indole‐3‐carbinol‐derived Akt/NF‐κB signalling inhibitor was used to overcome the intrinsic resistance. The antitumour effects of OSU‐A9, Apo2L/TRAIL and the therapeutic combination were assessed by MTT assay, caspase activation and PARP cleavage, and the synergistic interactions were determined by Calcusyn analysis. NF‐κB reporter gene and RT‐PCR were tested for the activation of NF‐κB and the expression of death receptors (DR)4 and 5. OSU‐A9 could sensitize HCC cells to Apo2L/TRAIL with high potency through down‐regulation of Akt/NF‐κB signalling. OSU‐A9 dose‐dependently reduced Akt phosphorylation and the expression and nuclear localization of RelA/p65, accompanied by parallel decreases in the expression of NF‐κB target products, including Bcl‐xL, Mcl‐1, cIAP1, cIAP2 and survivin. Moreover, OSU‐A9 increased DR5 expression through a reactive oxygen species (ROS)‐dependent mechanism. Concertedly, these mechanisms underlie the synergistic interaction between OSU‐A9 and Apo2L/TRAIL in mediating apoptotic death in HCC cells. The ability of OSU‐A9 to accentuate Apo2L/TRAIL‐induced apoptosis by inactivating Akt/NF‐κB signalling might foster a promising therapeutic strategy for HCC.     

丹参酮ⅡA对人卵巢癌细胞株CAOV3 增殖与凋亡的影响

［摘要］目的观察丹参酮ⅡA对人卵巢癌细胞株（CAOV3）增殖与凋亡的影响。方法培养人卵巢癌细胞株(CAOV3)，四甲基偶氮唑盐（MTT）比色法测定丹参酮ⅡA对细胞增殖的抑制作用；免疫沉淀法纯化蛋白，细胞外信号调节激酶（ERK）试剂盒测定ERK活性；流式细胞术测定丹参酮ⅡA对细胞凋亡的影响；免疫印记法（Western blot）测定Bax及bcl 2表达。结果MTT实验显示丹参酮ⅡA对CAOV3细胞增殖有明显抑制作用，免疫沉淀法显示丹参酮ⅡA可抑制ERK活性，流式细胞术显示丹参酮ⅡA可诱导CAOV3细胞凋亡，免疫印记法显示丹参酮ⅡA上调Bax，同时降低bcl 2表达，使Bax/bcl 2比值增加。结论丹参酮ⅡA可通过抑制ERK通路而抑制卵巢癌细胞增殖，并诱导细胞凋亡，其途径与上调Bax表达，降低bcl 2表达有关。     

NF‐κB as the mediator of metformin's effect on ageing and ageing‐related diseases

Ageing can be defined as the progressive failure of repair and maintenance systems with a consequent accumulation of cellular damage in nucleic acids, proteins, and lipids. These various types of damage promote ageing by driving cellular senescence and apoptosis. The nuclear factor‐kappa B (NF‐kB) pathway is one of the key mediators of ageing and this pathway is activated by genotoxic, oxidative and inflammatory stress, and regulates expression of cytokines, growth factors, and genes that regulate apoptosis, cell‐cycle progression, and inflammation. Therefore, NF‐kB is increased in a variety of tissues with ageing, thus the inhibition of NF‐kB leads to delayed onset of ageing‐related symptoms and pathologies such as diabetes, atherosclerosis, and cancer. Metformin is often used as an anti‐diabetic medication in type 2 diabetes throughout the world and appears to be a potential anti‐ageing agent. Owing to its antioxidant, anticancer, cardio‐protective and anti‐inflammatory properties, metformin has become a potential candidate drug, improving in the context of ageing and ageing‐related diseases. An inappropriate NF‐kB activation is associated with diseases and pathologic conditions which can impair the activity of genes involved in cell senescence, apoptosis, immunity, and inflammation. Metformin, inhibiting the expression of NF‐kB gene, eliminates the susceptibility to common diseases. This review underlines the pleiotropic effects of metformin in ageing and different ageing‐related diseases and attributes its effects to the modulation of NF‐kB.