Network Pharmacology and Molecular Docking Study on the Potential Mechanism of Yi-Qi-Huo-Xue-Tong-Luo Formula in Treating Diabetic Peripheral Neuropathy.

OBJECTIVE: To investigate the potential mechanism of action of Yi-Qi-Huo-Xue-Tong-Luo formula (YQHXTLF) in the treatment of diabetic peripheral neuropathy (DPN). METHODS: Network pharmacology and molecular docking techniques were used in this study. Firstly, the active ingredients and the corresponding targets of YQHXTLF were retrieved using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform; subsequently, the targets related to DPN were retrieved using GeneCards, Online Mendelian Inheritance in Man (OMIM), Pharmgkb, Therapeutic Target Database (TTD) and Drugbank databases; the common targets of YQHXTLF and DPN were obtained by Venn diagram; afterwards, the "YQHXTLF Pharmacodynamic Component-DPN Target" regulatory network was visualized using Cytoscape 3.6.1 software, and Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the potential targets using R 3.6.3 software. Finally, molecular docking of the main chemical components in the PPI network with the core targets was verified by Autodock Vina software. RESULTS: A total of 86 active ingredients and 229 targets in YQHXTLF were screened, and 81 active ingredients and 110 targets were identified to be closely related to diabetic peripheral neuropathy disease. PPI network mapping identified TP53, MAPK1, JUN, and STAT3 as possible core targets. KEGG pathway analysis showed that these targets are mostly involved in AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, and MAPK signaling pathway. The molecular docking results showed that the main chemical components of YQHXTLF have a stable binding activity to the core pivotal targets. CONCLUSION: YQHXTLF may act on TP53, MAPK1, JUN, and STAT3 to regulate inflammatory response, apoptosis, or proliferation as a molecular mechanism for the treatment of diabetic peripheral neuropathy, reflecting its multitarget and multipathway action, and providing new ideas to further uncover its pharmacological basis and mechanism of action.

The FOS/AP-1 Regulates Metabolic Changes and Cholesterol Synthesis in Human Periovulatory Granulosa Cells.

FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of FOS using primary human granulosa/lutein cells (hGLCs). Human chorionic gonadotropin (hCG) induced a biphasic increase in the expression of FOS, peaking at 1 to 3 hours and 12 hours. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Coimmunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated protein kinase A and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high-throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis, and cholesterol biosynthesis. Quantitative PCR analysis verified hCG-induced, T-5224-regulated expression of a selection of genes involved in these processes. Consistently, hCG-induced increases in metabolic activities and cholesterol levels were suppressed by T-5224. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 complex in metabolic changes and cholesterol biosynthesis in granulosa/lutein cells of human periovulatory follicles.

A Network Pharmacology-Based Strategy for Unveiling the Mechanisms of Tripterygium Wilfordii Hook F against Diabetic Kidney Disease.

BACKGROUND: Diabetic kidney disease (DKD) poses a major public-health burden globally. Tripterygium wilfordii Hook F (TwHF) is a widely employed herbal medicine in decreasing albuminuria among diabetic patients. However, a holistic network pharmacology strategy to investigate the active components and therapeutic mechanism underlying DKD is still unavailable. METHODS: We collected TwHF ingredients and their targets by traditional Chinese Medicine databases (TCMSP). Then, we obtained DKD targets from GeneCards and OMIM and collected and analyzed TwHF-DKD common targets using the STRING database. Protein-protein interaction (PPI) network was established by Cytoscape and analyzed by MCODE plugin to get clusters. In addition, the cytoHubba software was used to identify hub genes. Finally, all the targets of clusters were subjected for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses via DAVID. RESULTS: A total of 51 active ingredients in TwHF were identified and hit by 88 potential targets related to DKD. Compounds correspond to more targets include kaempferol, beta-sitosterol, stigmasterol, and Triptoditerpenic acid B, which appeared to be high-potential compounds. Genes with higher degree including VEGFA, PTGS2, JUN, MAPK8, and HSP90AA1 are hub genes of TwHF against DKD, which are involved in inflammation, insulin resistance, and lipid homeostasis. Kaempferol and VEGFA were represented as the uppermost active ingredient and core gene of TwHF in treating DKD, respectively. DAVID results indicated that TwHF may play a role in treating DKD through AGE-RAGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, insulin resistance, and calcium signaling pathway (P < 0.05). CONCLUSION: Kaempferol and VEGFA were represented as the uppermost active ingredient and core gene of TwHF in treating DKD, respectively. The key mechanisms of TwHF against DKD might be involved in the reduction of renal inflammation by downregulating VEGFA.

The diabetes drug semaglutide reduces infarct size, inflammation, and apoptosis, and normalizes neurogenesis in a rat model of stroke.

Stroke is a condition with few medical treatments available. Semaglutide, a novel Glucagon-like peptide-1 (GLP-1) analogue, has been brought to the market as a treatment for diabetes. We tested the protective effects of semaglutide against middle cerebral artery occlusion injury in rats. Animals were treated with 10 nmol/kg bw ip. starting 2 h after surgery and every second day for either 1, 7, 14 or 21 days. Semaglutide-treated animals showed significantly reduced scores of neurological impairments in several motor and grip strength tasks. The cerebral infarction size was also reduced, and the loss of neurons in the hippocampal areas CA1, CA3 and the dentate gyrus was much reduced. Chronic inflammation as seen in levels of activated microglia and in the activity of the p38 MAPK - MKK - c-Jun- NF-κB p65 inflammation signaling pathway was reduced. In addition, improved growth factor signaling as shown in levels of activated ERK1 and IRS-1, and a reduction in the apoptosis signaling pathway C-raf, ERK2, Bcl-2/BAX and Caspase-3 was observed. Neurogenesis had also been normalized by the drug treatment as seen in increased neurogenesis (DCX-positive cells) in the dentate gyrus and a normalization of biomarkers for neurogenesis. In conclusion, semaglutide is a promising candidate for re-purposing as a stroke treatment.

Curc-mPEG454, a PEGylated Curcumin Derivative, Improves Anti-inflammatory and Antioxidant Activities: a Comparative Study.

We previously demonstrated that a PEGylated curcumin (Curc-mPEG454) significantly inhibited cyclooxygenase 2 (COX-2) expression and improved the progression of liver fibrosis. The current study systematically evaluates its anti-inflammatory and antioxidant activities in vitro in a comparative study with curcumin, aspirin, NS-398, and vitamin C. RAW264.7 murine macrophages were pretreated with Curc-mPEG454, curcumin, aspirin, NS-398, or vitamin C at the indicated concentration for 2 h; then, the cells were stimulated with 1 µg/mL lipopolysaccharide (LPS) for 24 h. The levels of pro-inflammatory cytokines and mediators, including IL-6, TNF-α, PGE2, NO, and GSH, and the activities of COX-2, SOD, and CAT, and the transcription factors involved in inflammation, such as NF-κB, c-Jun, and Nrf2, were measured. Curc-mPEG454 showed lower cytotoxicity (IC50 57.8 µM) when compared with that of curcumin (IC50 32.6 µM) and inhibited the release of the inflammatory cytokines IL-6, TNF-α, IL-1ß, and MCP-1 in a concentration-dependent manner. At 16 µM, Curc-mPEG454 was most potent in the suppression of COX-2 expression at a transcriptional level rather than in the suppression of the catalytic activity of COX-2. Like curcumin, Curc-mPEG454 significantly reduced intracellular ROS production and enhanced the activities of SOD and CAT and the level of GSH to protect cells from LPS-induced oxidative injury. Further, its anti-inflammatory and antioxidation mechanisms are related to inhibition of NF-κB p65 nuclear translocation and c-Jun phosphorylation and to activation of Nrf2. Taken together, these findings indicate that PEGylation of curcumin not only improves its biological properties but also interferes with multiple targets involved in the inflammatory response. Curc-mPEG454 is a powerful and beneficial anti-inflammatory and antioxidant agent that merits further investigation. Graphical Abstract ᅟ.

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice.

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

Inhibition of BATF/JUN transcriptional activity protects against osteoarthritic cartilage destruction.

OBJECTIVE: The basic leucine zipper transcription factor, ATF-like (BATF), a member of the Activator protein-1 family, promotes transcriptional activation or repression, depending on the interacting partners (JUN-B or C-JUN). Here, we investigated whether the BATF/JUN complex exerts regulatory effects on catabolic and anabolic gene expression in chondrocytes and contributes to the pathogenesis of osteoarthritis (OA). METHODS: Primary cultured mouse chondrocytes were treated with proinflammatory cytokines (interleukin-1ß, IL-6 or tumour necrosis factor-α) or infected with adenoviruses carrying the Batf gene (Ad-Batf). Expression of BATF and JUN was examined in human and mouse experimental OA cartilage samples. Experimental OA in mice was induced by destabilisation of the medial meniscus or intra-articular injection of Ad-Batf. The chromatin immunoprecipitation assay was used to examine the binding of BATF and JUN to the promoter regions of candidate genes. RESULTS: Overexpression of BATF, which forms a heterodimeric complex with JUN-B and C-JUN, induced upregulation of matrix-degrading enzymes and downregulation of cartilage matrix molecules in chondrocytes. BATF expression in mouse joint tissues promoted OA cartilage destruction, and conversely, knockout of Batf in mice suppressed experimental OA. Pharmacological inhibition of BATF/JUN transcriptional activity reduced the expression of matrix-degrading enzymes and protected against experimental OA in mice. CONCLUSIONS: BATF/JUN-B and BATF/C-JUN complexes play important roles in OA cartilage destruction through regulating anabolic and catabolic gene expression in chondrocytes. Our findings collectively support the utility of BATF as a therapeutic target for OA.

Restoration of Corticosteroid Sensitivity in Chronic Obstructive Pulmonary Disease by Inhibition of Mammalian Target of Rapamycin.

RATIONALE: Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated. OBJECTIVES: To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD. METHODS: The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α-induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting. MEASUREMENTS AND MAIN RESULTS: mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity. CONCLUSIONS: mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.

Effects of leonurine on intracerebral haemorrhage by attenuation of perihematomal edema and neuroinflammation via the JNK pathway.

Perihematomal edema plays a critical role in secondary brain injury in intracerebral hemorrhage (ICH), which is associated with inflammation, hematoma toxicity and oxidative stress. In this work, we investigated the protective effects of leonurine, an alkaloid of Herbal Leonuri, and possible mechanisms to provide a basis for a new therapeutic approach for ICH treatment. In in vivo studies, we demonstrated for the first time that leonurine treatment substantially decreased perihematomal edema, ameliorated neurobehavioral function deficits, reduced apoptosis and protected injured cerebral tissue after ICH. These benefits appear to be ascribed to leonurine effectively attenuating bloodbrain barrier (BBB) breakdown in vivo, by inhibiting degradation of hemoglobin and alleviating inflammatory mediator release. In this study, BV-2 cells were exposed in vitro to oxyhemoglobin (OxyHb) at a concentration of 10 µM to mimic neuroinflammation after ICH. Consistent with the results of the in vivo study, leonurine significantly inhibited OxyHbinduced inflammatory proteins expression in BV-2 cells, mainly through inhibiting the c-Jun N-terminal kinase (JNK) signaling pathway. This is the first time that leonurine is proved to be capable to protect the injured cerebral tissue after ICH, based on alleviating neuroinflammation and attenuating BBB breakdown to ameliorate perihematomal edema.

Gpr177-mediated Wnt Signaling Is Required for Secondary Palate Development.

Cleft palate represents one of the major congenital birth defects in humans. Despite the essential roles of ectodermal canonical Wnt and mesenchymal Wnt signaling in the secondary palate development, the function of mesenchymal canonical Wnt activity in secondary palate development remains elusive. Here we show that Gpr177, a highly conserved transmembrane protein essential for Wnt trafficking, is required for secondary palate development. Gpr177 is expressed in both epithelium and mesenchyme of palatal shelves during mouse development. Wnt1(Cre)-mediated deletion of Gpr177 in craniofacial neural crest cells leads to a complete cleft secondary palate, which is formed mainly due to aberrant cell proliferation and increased cell death in palatal shelves. By BATGAL staining, we reveal an intense canonical Wnt activity in the anterior palate mesenchyme of E12.5 wild-type embryos but not in Gpr177(Wnt1-Cre) embryos, suggesting that mesenchymal canonical Wnt signaling activated by Gpr177-mediated mesenchymal Wnts is critical for secondary palate development. Moreover, phosphorylation of JNK and c-Jun is impaired in the Gpr177(Wnt1-Cre) palate and is restored by implantation of Wnt5a-soaked beads in the in vitro palate explants, suggesting that Gpr177 probably regulates palate development via the Wnt5a-mediated noncanonical Wnt pathway in which c-Jun and JNK are involved. Importantly, certain cellular processes and the altered gene expression in palates lacking Gpr177 are distinct from that of the Wnt5a mutant, further demonstrating involvement of other mesenchymal Wnts in the process of palate development. Together, these results suggest that mesenchymal Gpr177 is required for secondary palate development by regulating and integrating mesenchymal canonical and noncanonical Wnt signals.

Inhibition of interleukin 1ß-stimulated interleukin-6 production by cranberry components in human gingival epithelial cells: effects on nuclear factor κB and activator protein 1 activation pathways.

BACKGROUND AND OBJECTIVE: In periodontitis, gingival epithelial cells can produce interleukin (IL)-6, a regulator of osteoclastic bone resorption, in response to IL-1ß. IL-1ß regulates cytokine expression via signaling pathways, including nuclear factor (NF)-κB and mitogen activated protein kinase (MAPK)/activator protein (AP)-1. Cranberry proanthocyanidins (PACs) inhibit IL-1ß-stimulated IL-6 production, but specific mechanisms are unclear. The objectives of this study were to determine effects of cranberry PACs on NF-κB and MAPK/AP-1 activation of IL-1ß-stimulated IL-6 production in gingival epithelial cells. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), rich in PACs, was derived from cranberry juice. Human gingival epithelial cells [Smulow-Glickman (S-G)] were incubated with IL-1ß in the presence or absence of NDM or inhibitors of NF-κB, [nemo-binding domain (NBD) peptide] or AP-1 (SP600125), and IL-6 levels were measured by ELISA. Effects of NDM on IL-1ß-activated NF-κB and AP-1 and phosphorylated intermediates in both pathways were measured in cell extracts via binding to specific oligonucleotides and specific sandwich ELISAs, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß (≥ 0.1 nm) caused a time- and dose-dependent stimulation of S-G epithelial cell IL-6 production (p < 0.005). This was significantly decreased in a dose-dependent manner by NBD peptide or SP600125 [maximum inhibition ~30-40% (p < 0.02)], and together, the two inhibitors decreased IL-6 by ~80%, similar to the inhibition caused by NDM (p < 0.001). IL-1ß stimulated NF-κB and AP-1 activation (p < 0.003), which was inhibited by NDM (p < 0.0001). NDM did not significantly affect IL-1ß-stimulated levels of phosphorylated intermediates in the NF-κB pathway (IκBα) or the AP-1 pathway (c-Jun, ERK1/2). CONCLUSION: In S-G epithelial cells, IL-1ß appeared to upregulate IL-6 production via activation of both NF-κB and MAPK/AP-1 signaling pathways because cranberry NDM decreased nuclear levels of IL-1ß-activated NF-κB (p65) and AP-1 (phospho-c-Jun) and strongly inhibited IL-6 production. Lack of inhibition of phosphorylation of IκBα, c-Jun or ERK1/2 suggested that NDM might affect both pathways downstream from those points in S-G cells, such as ubiquitination and proteosomal degradation of IκBα, or inhibition of nuclear activity of c-Jun and/or ERK1/2. Defining these points of inhibition precisely may help identify molecular targets of cranberry polyphenols.

Curcumin induces cell cycle arrest and apoptosis of prostate cancer cells by regulating the expression of IkappaBalpha, c-Jun and androgen receptor.

Curcumin possesses chemopreventive properties against several types of cancer, but the molecular mechanisms by which it induces apoptosis of cancer cells and inhibits cancer cell proliferation are not clearly understood. To evaluate the antitumor activity of curcumin for prostate cancer, we used an androgen dependent LNCaP prostate cancer cell line and an androgen independent PC-3 prostate cancer cell line as experimental models. We treated these cells with curcumin and then evaluated the effects of curcumin on cell cycle profiling and apoptosis, as well as the activation of NF-kaapaB and c-jun in these cells. The results showed that the ratios of apoptosis in LNCaP and PC-3 cells were significantly elevated in a dose dependent manner after exposure to curcumin. In addition, curcumin induces the G2/M cell cycle arrest of LNCaP and PC-3 cells in a dose dependent manner. Mechanistically, we found that curcumin upregulated the protein level of NF-kappaB inhibitor IkappaBalpha and downregulated protein levels of c-Jun and AR. These data suggest that curcumin is a promising agent for the treatment of both androgen-dependent and androgen-independent prostate cancer.

Triclosan blocks MMP-13 expression in hormone-stimulated osteoblasts.

BACKGROUND: Matrix metalloproteinase-13 (MMP-13) is an important enzyme for the modulation of bone turnover and gingival recession. Elevated levels of MMP-13 are associated with alveolar bone resorption, periodontal ligament breakdown, and gingival attachment loss, which are the clinical symptoms of periodontal disease. Evidence continues to suggest that periodontal disease contributes to oral tissue breakdown and is linked to numerous systemic conditions. Triclosan (TCN) is a long-standing, proven antibacterial and anti-inflammatory agent found in the only Food and Drug Administration-approved dentifrice for the treatment of plaque and gingivitis. METHODS: This study examines the inhibitory effects of TCN on lipopolysaccharide-, parathyroid hormone (PTH)-, and prostaglandin E2 (PGE2)-induced expression of MMP-13 in UMR 106-01 cells, an osteoblastic osteosarcoma cell line. The cells were stimulated with PTH or PGE2 to induce MMP-13 mRNA expression, and real-time reverse transcription-polymerase chain reaction was performed to determine gene expression levels. Western blot analysis assessed the presence or absence of protein degradation or inhibition of protein synthesis. MMP-13 promoter reporter assay was used to explore possible direct effects of TCN on the MMP-13 promoter. RESULTS: TCN significantly reduced PTH or PGE2 elevated expression of MMP-13 in osteoblastic cells without affecting basal levels of the mRNA. Surprisingly, TCN enhanced the expression of c-fos and amphiregulin mRNA. A promoter assay indicated that TCN directly inhibits the activation of the PTH-responsive minimal promoter of MMP-13. CONCLUSION: The present study appears to have identified a nuclear mechanism of action of TCN that accounts for the ability of TCN to inhibit PTH- or PGE2-induced MMP-13 expression in osteoblastic cells.

Keratinocyte growth factor phage model peptides can promote human oral mucosal epithelial cell proliferation.

OBJECTIVE: The objective of this study was to find keratinocyte growth factor (KGF) mimic peptides by a phage display library screening and to analyze their effects on proliferation of human oral mucosal epithelial cells (HOMECs). STUDY DESIGN: A phage display library was screened by anti-KGF antibody. ELISA was performed to select monoclonal phages with higher binding activity. The promotion of the phage model peptides on HOMEC proliferation were analyzed by MTT and their cell affinities were confirmed by immunofluorescence assay. Their effect on KGFR, human beta-defensin 3, c-Fos, and c-Jun in HOMEC were analyzed by quantitative real-time PCR. RESULTS: Two model peptides with higher affinity with HOMEC were found to have promotive activity on cell proliferation, similar to that of KGF. These 2 model peptides have no KGF-like promotion effect on the expression of c-Fos and c-Jun. CONCLUSIONS: The 2 phage model peptides can promote the proliferation of HOMEC in vitro without tumorigenic effects, which suggests their possible usages in oral mucosal wound healing.

Heroin activates Bim via c-Jun N-terminal kinase/c-Jun pathway to mediate neuronal apoptosis.

Heroin is reported to cause spongiform leukoencephalopathy (SLE) in heroin addicts and the exact mechanism has not yet been identified. In the present study, we found that heroin could induce apoptosis of primary cultured cerebellar granule cells (CGCs) and Bim was upregulated both transcriptionally and post transcriptionally during CGCs apoptosis. Upregulated Bim translocated to mitochondria and Bax was activated under heroin treatment. Genetic knockdown of Bim using lentiviruses significantly prevented neuronal apoptosis induced by heroin. Meanwhile, c-Jun N-terminal kinase (JNK)/c-Jun pathway was activated in heroin-induced apoptosis. Bim was demonstrated as a downstream target of JNK/c-Jun pathway in this process because pharmacological inhibition of JNK reduced the levels of Bim mRNA and protein. These results indicate that Bim plays a critical role in the neurotoxic process by heroin and JNK/c-Jun pathway acts upstream of Bim in regulating heroin-induced neuronal death. This represents a detailed mechanism of heroin-induced neuronal apoptosis and may provide a new and effective strategy to treat heroin-induced addiction and SLE.

LPA stimulates intestinal DRA gene transcription via LPA2 receptor, PI3K/AKT, and c-Fos-dependent pathway.

DRA (downregulated in adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl(-) absorption in intestinal epithelial cells. Disturbances in DRA function and expression have been implicated in diarrheal conditions such as congenital chloride diarrhea and inflammatory bowel diseases. Previous studies have shown that DRA is subject to regulation by short-term and transcriptional mechanisms. In this regard, we have recently shown that short-term treatment by lysophosphatidic acid (LPA), an important bioactive phospholipid, stimulates Cl(-)/HCO(3)(-)(OH(-)) exchange activity via an increase in DRA surface levels in human intestinal epithelial cells. However, the long-term effects of LPA on DRA at the level of gene transcription have not been examined. The present studies were aimed at investigating the effects of LPA on DRA function and expression as well as elucidating the mechanisms underlying its transcriptional regulation. Long-term LPA treatment increased the Cl(-)/HCO(3)(-) exchange activity in Caco-2 cells. LPA treatment (50-100 µM) of Caco-2 cells significantly stimulated DRA mRNA levels and DRA promoter activity (-1183/+114). This increase in DRA promoter activity involved the LPA2 receptor and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Progressive deletions from -1183/+114 to -790/+114 abrogated the stimulatory effects of LPA, indicating that the -1183/-790 promoter region harbors LPA response elements. Utilizing EMSA and mutational studies, our results showed that LPA induced the DRA promoter activity in a c-Fos-dependent manner. LPA also increased the protein expression of c-Fos and c-Jun in Caco-2 cells. Furthermore, overexpression of c-Fos but not c-Jun enhanced the DRA promoter activity. This increase in DRA transcription in response to LPA indicates that LPA may act as an antidiarrheal agent and could be exploited for the treatment of diarrhea associated with inflammatory or infectious diseases of the gut.

Attenuation of liver ischemia/reperfusion induced apoptosis by epigallocatechin-3-gallate via down-regulation of NF-kappaB and c-Jun expression.

INTRODUCTION: Hepatic ischemia/reperfusion (I/R) activates Kupffer cells and initiates severe oxidative stress with enhanced production of reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-alpha). ROS and TNF-alpha mediate the expression of nuclear factors and kinases, activating the signal transduction pathway, and triggering apoptosis. The aim of our study was to evaluate the potential protective effect of (-)-epigallocatechin-3-gallate (EGCG) administration in inhibition of apoptosis by attenuating the expression of NF-kappaB, c-Jun, and caspase-3 in a model of severe hepatic I/R. MATERIALS AND METHODS: Thirty Wistar rats were allocated into three groups. Sham operation, I/R, and I/R-EGCG 50mg/kg. Hepatic ischemia was induced for 60min by Pringle's maneuver. Malondialdehyde (MDA), myeloperoxidase (MPO), light histology, scanning electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunocytochemistry for NF-kappaB, c-Jun, caspase-3, analysis on liver specimens and aspartate (AST), and alanine (ALT) transferases analysis in serum, were performed 120min after reperfusion. RESULTS: Apoptosis as indicated by TUNEL and caspase-3 was widely expressed in the I/R group but very limited in the EGCG treated group. Liver was stained positive for NF-kappaB and c-Jun in the I/R group but failed to be stained positive in the EGCG treated group. MDA, MPO, AST, and ALT showed marked increase in the I/R group and significant decrease in EGCG treated group. Significant alterations of liver specimens were observed by light histology and transmission electron microscopy whilst pretreatment with EGCG resulted in parenchymal preservation. CONCLUSIONS: Administration of EGCG is likely to inhibit I/R-induced apoptosis and protect liver by down-regulating NF-kappaB and c-Jun signal transduction pathways.

Induction of MMP-1 (collagenase-1) by relaxin in fibrocartilaginous cells requires both the AP-1 and PEA-3 promoter sites.

OBJECTIVES - Relaxin induces the matrix metalloproteinase MMP-1 (collagenase-1) in TMJ fibrocartilaginous cells, and this response is potentiated by beta-estradiol. We identified the MMP-1 promoter sites and transcription factors that are induced by relaxin with or without beta-estradiol in fibrocartilaginous cells. MATERIAL AND METHODS - Early passage cells were transiently transfected with the pBLCAT2 plasmid containing specific segments of the human MMP-1 promoter regulating the chloramphenicol acyl transferase (CAT) gene and co-transfected with a plasmid containing the beta-galactosidase gene. The cells were cultured in serum-free medium alone or medium containing 0.1 ng/ml relaxin, or 20 ng/ml beta-estradiol or both hormones, and lysates assayed for CAT and beta-galactosidase activity. RESULTS - Cells transfected with the -1200/-42 or -139/-42 bp MMP-1 promoter-reporter constructs showed 1.5-fold and 3-fold induction of CAT by relaxin in the absence or presence of beta-estradiol, respectively. Relaxin failed to induce CAT in the absence of the -137/-69 region of the MMP-1 promoter, which contains the AP-1-and PEA3-binding sites. Using wild type or mutated minimal AP-1 and PEA-3 promoters we found that both these promoter sites are essential for the induction of MMP-1 by relaxin. The mRNAs for transcription factors c-fos and c-jun, which together form the AP-1 heterodimer, and Ets-1 that modulates the PEA-3 site, were upregulated by relaxin or beta-estradiol plus relaxin. CONCLUSION - These studies show that both the AP-1 and PEA-3 promoter sites are necessary for the induction of MMP-1 by relaxin in fibrocartilaginous cells.

Effects of dl-praeruptorin A on cultured neonatal rat ventricular cardiomyocytes with hypertrophy induced by endothelin-1.

The present study investigated whether dl-praeruptorin (Pd-Ia) prevents endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and the potential pathways that underlie such an effect. We assessed cardiomyocyte surface area, protein synthesis, the expression of Bax/Bcl2 and Jun genes, the expression of atrial natriuretic factor (ANF) and Ca2+/calmodulin-dependent kinase II (CaMK-II) activity in cultured neonatal rat ventricular cardiomyocytes with ET-1-induced hypertrophy. It was found that Pd-Ia decreased the surface area and protein synthesis rate in cardiomyocytes exposed to ET-1. Additionally, the expression of Bcl2 and Bax was increased in both the ET-1-exposed and Pd-Ia+ET- 1-treated groups compared with the control group, although this was not significant. In cardiomyocytes incubated with ET-1, the expression of ANF (Nppa) significantly increased relative to the control and Pd-Ia groups. The expression of Jun significantly increased in cardiomyocytes incubated with ET-1, but not in the Pd-Ia group, where Jun levels were similar to those found for the control group. Moreover, it was found that Pd-Ia inhibited the ET-1-induced increase in intracellular Ca(2+) concentration. The results showed that Pd-Ia could conceivably be an effective therapeutic drug for treating the contractile defects associated with cardiac hypertrophy and failure. This activity may be associated with its Ca2+-antagonist effect and modulation of the expression of immediate-early genes that play important roles in the mitogen-activated protein (MAP) kinase pathway.

Reduction in the protein level of c-Jun and phosphorylation of Ser73-c-Jun in rat frontal cortex after repeated MK-801 treatment.

Repeated administration of NMDA antagonists can induce behavioral alterations that mimic symptoms of psychosis, as seen in schizophrenia. JNK, one of the MAPKs, and c-Jun, its downstream target molecule, play important roles in regulating apoptosis in neural cells, and have been suggested as being associated with the pathophysiology of psychosis and the mechanism of action of some antipsychotics. We investigated changes in the JNK-c-Jun pathway and other Jun family proteins in the rat frontal cortex after single and repeated administration of MK-801 to examine acute and chronic responses. Neither the protein level nor the phosphorylation of JNK changed after single or repeated doses of MK-801. However, after repeated treatments, but not a single treatment, with MK-801, a down-regulation occurred in the protein level and of Ser73 phosphorylation of c-Jun in the rat frontal cortex. Other members of the Jun family, JunB and JunD, were unchanged. Repeated exposure to MK-801 down-regulated the phosphorylation and protein level of c-Jun in the rat frontal cortex, which may be related to the long-term effects of chronic treatment with MK-801.