OBJECTIVES: To observe the reparative effects of human umbilical cord mesenchymal stem cell (hUC-MSC) transplantation on white matter injury (WMI) in neonatal rats and explore its mechanism through the nuclear factor-kappa B (NF-κB) signaling pathway mediated by microglial cells. METHODS: Sprague-Dawley rats, aged 2 days, were randomly divided into three groups: sham-operation,WMI, and hUC-MSC (<i>ni>=18 each). Fourteen days after modeling, hematoxylin-eosin staining was used to observe pathological changes in the white matter, and immunofluorescence staining was used to measure the expression level of ionized calcium-binding adapter molecule 1 (Iba1). Western blotting was used to measure the protein expression levels of inhibitory subunit of nuclear factor-kappa B alpha (IκBα), phosphorylated IκBα (p-IκBα), phosphorylated NF-κB p65 (p-NF-κB p65), myelin basic protein (MBP), and neuron-specific nuclear protein (NeuN). Quantitative real-time PCR was used to assess the mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), MBP, and NeuN. Immunohistochemistry was used to measure the protein expression levels of MBP and NeuN. On day 28, the Morris water maze test was used to evaluate spatial cognitive ability. RESULTS: Fourteen days after modeling, the sham-operation group exhibited intact white matter structure with normal cell morphology and orderly nerve fiber arrangement. In the WMI group, large-scale cell degeneration and necrosis were observed, and nerve fiber arrangement was disordered. The hUC-MSC group showed relatively normal cell morphology and more orderly nerve fibers. Compared with the sham-operation group, the WMI group had significantly higher proportions of Iba1-positive cells, increased protein levels of p-IκBα and p-NF-κB p65, and higher mRNA levels of TNF-α and IL-1β. The protein expression of IκBα and the positive expression of MBP and NeuN, as well as their protein and mRNA levels, were significantly reduced in the WMI group (<i>Pi><0.05). Compared with the WMI group, the hUC-MSC group showed reduced proportions of Iba1-positive cells, decreased protein levels of p-IκBα and p-NF-κB p65, and lower mRNA levels of TNF-α and IL-1β. Furthermore, IκBα protein expression and MBP and NeuN expression (both at the protein and mRNA levels) were significantly increased in the hUC-MSC group (<i>Pi><0.05). On day 28, the Morris water maze results showed that compared with the sham-operation group, the WMI group had significantly longer escape latency and fewer platform crossings (<i>Pi><0.05). In contrast, the hUC-MSC group had significantly shorter escape latency and more platform crossings than the WMI group (<i>Pi><0.05). CONCLUSIONS hUC-MSC transplantation can repair WMI in neonatal rats, promote the maturation of oligodendrocytes, and support neuronal survival, likely by inhibiting activation of the NF-κB signaling pathway mediated by microglial cells.
Animals ; Rats, Sprague-Dawley ; White Matter/metabolism* ; Rats ; Signal Transduction ; Mesenchymal Stem Cell Transplantation ; Humans ; NF-kappa B/metabolism* ; Animals, Newborn ; Umbilical Cord/cytology* ; Male ; NF-KappaB Inhibitor alpha/metabolism* ; I-kappa B Proteins/genetics* ; Microfilament Proteins/analysis* ; Calcium-Binding Proteins/metabolism* ; Female

This study aimed to further explore the relevant mechanism of action by network pharmacology integrated with animal experimental verification based on previous proven effective treatment of vertebral artery type of cervical spondylosis(CSA) by Panlongqi Tablets. Bionetwork analysis was performed to establish drug-disease interaction network, and it was found that the key candidate targets of Panlongqi Tablets were enriched in multiple signaling pathways related to CSA pathological links, among which phosphatidylinositol 3-kinase(PI3 K)/serine-threonine kinase(AKT/PKB) signaling pathway was the most significant. Further, mixed modeling method was used to build the CSA rat model, and the rats were divided into normal, model, Panlongqi Tablets low-, medium-and high-dose(0.16, 0.32, 0.64 g·kg~(-1)) and Jingfukang Granules(positive drug, 1.35 g·kg~(-1)) groups. After successful modeling, the rats were administered for 8 consecutive weeks. Pathological changes of rat cervical muscle tissues were detected by hematoxylin-eosin(HE) staining, and the content of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), vascular endothelial cell growth factor(VEGF) and chemokine(C-C motif) ligand 2(CCL2) in rat serum and/or cervical tissues was determined by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to detect the protein expression levels of chemokine(C-C motif) receptor 2(CCR2), PI3 K, AKT, phosphorylated AKT(p-AKT), I-kappa-B-kinase beta(IKK-beta/IKKβ), nuclear factor kappa B(NF-κB P65) and phosphorylated nuclear factor kappa B(NF-κB p-P65) in rat cervical tissues, and positive expression of p-NF-κB P65 in rat cervical muscle tissues was detected by immunofluorescence. The results showed that Panlongqi Tablets at different doses improved the degree of muscle fibrosis and inflammation in cervical muscle tissues of CSA rats, and reduced the content of inflammatory factors IL-1β, TNF-α, VEGF, CCL2 and CCR2 in serum and/or cervical tissues. The protein expression levels of PI3 K, p-AKT, IKKβ and p-NF-κB P65 as well as the nuclear entry of p-NF-κB P65 in cervical tissues were down-regulated. These findings suggest that Panlongqi Tablets can significantly inhibit the inflammatory response of CSA rats, and the mechanism of action may be related to the down-regulation of the activation of PI3 K/AKT signaling pathway.
Animals ; Drugs, Chinese Herbal ; I-kappa B Kinase/pharmacology* ; NF-kappa B/metabolism* ; Network Pharmacology ; Phosphatidylinositol 3-Kinases/metabolism* ; Protein Serine-Threonine Kinases ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Signal Transduction ; Spondylosis/drug therapy* ; Tumor Necrosis Factor-alpha/metabolism* ; Vascular Endothelial Growth Factor A/genetics* ; Vertebral Artery/metabolism*

Berberine (BBR) is an isoquinoline alkaloid extracted from Rhizoma coptidis and has been used for treating type 2 diabetes mellitus (T2DM) in China. The development of T2DM is often associated with insulin resistance and impaired glucose uptake in peripheral tissues. In this study, we examined whether BBR attenuated glucose uptake dysfunction through the cholinergic anti-inflammatory pathway in HepG2 cells. Cellular glucose uptake, quantified by the 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-deoxy-D-glucose (2-NBDG), was inhibited by 21% after HepG2 cells were incubated with insulin (10(-6) mol/L) for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor (α7nAChR) protein was reduced without the change of acetylcholinesterase (AChE) activity. The level of interleukin-6 (IL-6) in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-β (IK&kappa;β) Ser181/IKKβ and the expression of nuclear factor-kappa B (NF-&kappa;B) p65 protein were also increased. However, the treatment with BBR enhanced the glucose uptake, increased the expression of α7nAChR protein and inhibited AChE activity. These changes were also accompanied with the decrease of the ratio of pIKKβ Ser181/IKKβ, NF-&kappa;B p65 expression and IL-6 level. Taken together, these results suggest that BBR could enhance glucose uptake, and relieve insulin resistance and inflammation in HepG2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of AChE activity.
Berberine ; pharmacology ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; pharmacology ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; Insulin ; metabolism ; Insulin Resistance ; Interleukin-6 ; metabolism ; Transcription Factor RelA ; metabolism ; alpha7 Nicotinic Acetylcholine Receptor ; genetics ; metabolism

<b>OBJECTIVEb>This study observed attenuating effect of hydroxysafflor yellow A (HSYA), an effective ingredient of aqueous extract of Carthamus tinctorius L, on lipopolysaccharide (LPS)-induced endothelium inflammatory injury.

<b>METHODSb>Eahy926 human endothelium cell (EC) line was used; thiazolyl blue tetrazolium bromide (MTT) test was assayed to observe the viability of EC; Luciferase reporter gene assay was applied to measure nuclear factor-&kappa;B (NF-&kappa;B) p65 subunit nuclear binding activity in EC; Western blot technology was used to monitor mitogen activated protein kinase (MAPKs) and NF-&kappa;B activation. Reverse transcription polymerase chain reaction (RT-PCR) method was applied to observe intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin mRNA level; EC surface ICAM-1 expression was measured with flow cytometry and leukocyte adhesion to EC was assayed with Rose Bengal spectrophotometry technology.

<b>RESULTSb>HSYA protected EC viability against LPS-induced injury (P <0.05). LPS-induced NF-&kappa;B p65 subunit DNA binding (P <0.01) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (I&kappa;Bα) phosphorylation was inhibited by HSYA. HSYA attenuated LPS triggered ICAM-1 and E-selectin mRNA levels elevation and phosphorylation of p38 MAPK or c-Jun N-terminal kinase MAPK. HSYA also inhibited LPS-induced cell surface ICAM-1 protein expression P <0.01) and leukocyte adhesion to EC (P <0.05).

<b>CONCLUSIONb>HSYA is effective to protect LPS-induced high expression of endothelium adhesive molecule and inflammatory signal transduction.

Cell Adhesion ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Cell Survival ; drug effects ; Chalcone ; analogs & derivatives ; chemistry ; pharmacology ; therapeutic use ; E-Selectin ; genetics ; metabolism ; Endothelium, Vascular ; drug effects ; pathology ; Gene Expression Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; pathology ; Humans ; I-kappa B Proteins ; metabolism ; Inflammation ; drug therapy ; pathology ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Leukocytes ; cytology ; drug effects ; Lipopolysaccharides ; MAP Kinase Signaling System ; drug effects ; NF-KappaB Inhibitor alpha ; Phosphorylation ; drug effects ; Protective Agents ; pharmacology ; Protein Binding ; drug effects ; Quinones ; chemistry ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism

The host takes use of pattern recognition receptors (PRRs) to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.
DNA, Bacterial ; immunology ; metabolism ; DNA, Viral ; immunology ; metabolism ; Gene Expression Regulation ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Interferon Regulatory Factor-3 ; genetics ; immunology ; Interferon Type I ; biosynthesis ; immunology ; Membrane Proteins ; genetics ; immunology ; Models, Molecular ; NF-kappa B ; genetics ; immunology ; Nucleotides, Cyclic ; biosynthesis ; immunology ; Nucleotidyltransferases ; genetics ; immunology ; Protein Binding ; Protein-Serine-Threonine Kinases ; genetics ; immunology ; Signal Transduction

<b>OBJECTIVEb>The study was aimed to investigate the inhibitory effect and mechanism of astragaloside IV (ASI) on the activation of microglial cells.

<b>METHODb>After pre-incubated with ASI for 2 h, microglial cells BV-2 were stimulated with interferon-γ (IFN-γ) for 1. 5 h and 24 h, respectively. Secretion of nitric oxide (NO) in the medium was measured by Griess method. Production of tumor necrosis factor alpha (TNF-α) was detected by ELISA approach. Cellular gene expressions of CD11b, TNF-α, interleukin 1β (IL-1β) and induced nitric oxide synthase (iNOS) were examined by quantitative-PCR analysis. Total and phosphorylation of STAT1, I&kappa;B and NF-&kappa;B was analyzed by Western blot method.

<b>RESULTb>ASI could significantly inhibit the increased secretion of TNF-α and NO from BV-2 cells upon IFN-γ stimulation (P < 0.001). Further study showed that ASI significantly down-regulated gene expression of IL-1β and TNF-α (P < 0.01, P < 0.05) and exhibited a trend to reduce that of iNOS. IFN-γ and ASI have no obvious effect on gene expression of CD11b. Moreover, ASI inhibited the phosphorylation of STAT1, I&kappa;B and NF-&kappa;B elicited by IFN-γ stimulation.

<b>CONCLUSIONb>ASI could restrain microglial activation through interfering STAT1/I&kappa;B/NF-&kappa;B signaling pathway, reducing gene expres- sion of IL-1β and TNF-α, and thus inhibiting the production of proinflammatory mediators such as NO and TNF-α.

Animals ; Astragalus Plant ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; I-kappa B Proteins ; genetics ; metabolism ; Interferon-gamma ; genetics ; metabolism ; Mice ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Triterpenes ; pharmacology

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction

<b>OBJECTIVEb>To study the effect of E3 ubiquitin ligase RNF31 knockdown on nuclear factor-&kappa;B (NF-&kappa;B) pathway activation and cell apoptosis.

<b>METHODSb>Human RNF31 siRNA sequences were cloned into the lentiviral vector pGreenPuro and transiently transfected in HEK293T cells to screen the most effective fragments, which were co-transfected along with the packaging plasmids PMD and SPA in 293T cells. The cell supernatant was collected at 24 h and 48 h after the transfection and the viral titers were determined with flow cytometry. Real-time PCR and Western blotting were used to evaluate the effect of RNF31 knockdown on the expression of NF-&kappa;B downstream target genes and I&kappa;Bα activity; the changes of NF-&kappa;B pathway transcriptional activity were assessed with dual luciferase reporter gene. Hochest dying was used to examine the influence of RNF31 down-regulation on cell apoptosis.

<b>RESULTSb>RNF31 knockdown mediated by the lentiviral vector pGreenPuro-RNF31 suppressed the transcriptional activity of NF-&kappa;B and the downstream target genes in HEK293 cells stimulated with TNF-α. RNF31 knockdown also resulted in suppression of NF-&kappa;B-stimulated expression of pI&kappa;Bα and in increased apoptosis of cells stimulated with TNF-α for 24 h.

<b>CONCLUSIONb>RNF31 down-regulation inhibits NF-&kappa;B pathway activation induced by TNF-α.

Apoptosis ; Down-Regulation ; Gene Expression Regulation ; Genetic Vectors ; HEK293 Cells ; Humans ; I-kappa B Proteins ; metabolism ; Lentivirus ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transfection ; Tumor Necrosis Factor-alpha ; pharmacology ; Ubiquitin-Protein Ligases ; genetics ; metabolism

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination

The effects of black rice anthocyanidins (BRACs) on retinal damage induced by photochemical stress are not well known. In the present study, Sprague-Dawley rats were fed AIN-93M for 1 week, after which 80 rats were randomly divided into two groups and treated with (n = 40) or without BRACs (n = 40) for 15 days, respectively. After treatment, both groups were exposed to fluorescent light (3,000 +/- 200 lux; 25degrees C), and the protective effect of dietary BRACs were evaluated afterwards. Our results showed that dietary BRACs effectively prevented retinal photochemical damage and inhibited the retinal cells apoptosis induced by fluorescent light (p < 0.05). Moreover, dietary BRACs inhibited expression of AP-1 (c-fos/c-jun subunits), up-regulated NF-kappaB (p65) expression and phosphorylation of IkappaB-alpha, and decreased Caspase-1 expression (p < 0.05). These results suggest that BRACs improve retinal damage produced by photochemical stress in rats via AP-1/NF-kappaB/Caspase-1 apoptotic mechanisms.
Animal Feed/analysis ; Animals ; Anthocyanins/administration & dosage/*pharmacology ; Antioxidants/administration & dosage/*physiology ; Blotting, Western ; Caspase 1/*genetics/metabolism ; Diet ; Dietary Supplements/analysis ; I-kappa B Proteins/genetics/metabolism ; NF-kappa B/*genetics/metabolism ; Neoplasm Proteins/genetics/metabolism ; Nucleocytoplasmic Transport Proteins/genetics/metabolism ; Oryza sativa/chemistry ; Proto-Oncogene Proteins c-fos/genetics/metabolism ; Proto-Oncogene Proteins c-jun/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Retinal Diseases/etiology/*prevention & control ; Signal Transduction/*drug effects/radiation effects ; Transcription Factor AP-1/*genetics/metabolism