Sesamin induces cell cycle arrest and apoptosis through p38/C-Jun N-terminal kinase mitogen-activated protein kinase pathways in human colorectal cancer cells.

Sesamin, a lignan compound, exhibits a variety of biological activities and possesses potent anticancer properties on some human cancers. However, its effect on human colorectal cancer (CRC) remains to be elucidated. To investigate the effects of sesamin on CRC cells and further to explore the mechanisms, cell viability, cell cycle and apoptosis assays were performed in this study. We found that sesamin had a selective antiproliferation of CRC cell line HCT116 in a dose- and time-dependent manner, but no obvious effect on human normal colorectal mucosa epithelial cell FHC. Further study showed that sesamin-induced cell cycle arrest and decreased the expression of Cyclin D1 significantly and dose-dependently in HCT116 cells. Moreover, sesamin dose-dependently triggered apoptosis of HCT116 but not FHC, and promoted the expression levels of proapoptotic biomarkers Bax, cleaved caspase-3 and cleaved PARP-1 and inhibited the expression of antiapoptotic biomarker Bcl-2. Western blot analysis was used to reveal the possible signaling pathways, and we found that sesamin upregulated the phosphorylation expression levels of C-Jun N-terminal kinase (JNK) and p38 except ERK1/2 in a dose-dependent way in both HCT116 and another CRC cell line SW480. Moreover, we found that the apoptosis effect induced by sesamin was partially eliminated by inhibiting JNK or p38 activation. Finally, we showed that sesamin effectively reduced the growth of xenograft tumors derived from cell lines with limited toxicity. Taken together, the potential ability of sesamin to induce cell cycle arrest and apoptosis was shown to be via the p38 and JNK mitogen-activated protein kinase signaling pathways, which may be one of the mechanisms of the anticancer activity of this low-toxic agent.

Apoptotic effects of dehydrocrenatidine via JNK and ERK pathway regulation in oral squamous cell carcinoma.

Dehydrocrenatidine, a ß-carboline alkaloid isolated from Picrasma quassioides, has been demonstrated to exert analgesic effects and play essential roles in janus kinase inhibition and exert analgesic effects through the suppression of neuronal excitability. Alkaloids such as paclitaxel and vincristine had been well explored to be chemotherapeutic agents. However, the anticancer effects of dehydrocrenatidine remain unclear. In the present study, we found that dehydrocrenatidine induced apoptosis in human oral cancer cells through both extrinsic and intrinsic pathways involving proteins such as caspase-3, caspase-8, caspase-9, poly (adenosine diphosphate-ribose) polymerase, and members of the Bcl-2 family. Cotreatment with dehydrocrenatidine and mitogen-activated protein kinase (MAPK) inhibitors indicated that dehydrocrenatidine induced apoptosis through the activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). The findings provide insight into the potential of dehydrocrenatidine for a new perspective on molecular regulation.

Dual functional liposomes carrying antioxidants against tau hyperphosphorylation and apoptosis of neurons.

Quercetin (QU) and rosmarinic acid (RA) were loaded in phosphatidic acid-liposomes (QU/RA-PA-liposomes) with surface apolipoprotein E (ApoE) using a process of thin-film hydration, followed by covalent crosslinking to activate biological pathways for penetrating the blood-brain barrier (BBB) and redeeming the neuronal apoptosis from attack of ß-amyloid 1-42 (Aß1-42) and neurofibrillary tangles. The conjugation of liposomes with PA improved the activity of QU and RA against neurotoxicity of Aß1-42. The fluorescent images of brain capillaries revealed that surface modification with ApoE improved the permeation ability of QU/RA-PA-ApoE-liposomes across the BBB. In addition, the highest therapeutic efficacy was obtained in the case of QU/RA-PA-ApoE-liposomes, compared to other QU/RA formulations studied using in vivo Aß1-42-insulted rats mimicking Alzheimer's disease (AD). The cellular and molecular evidence from AD rats included the decrease in Aß1-42 plaque formation and interleukin-6 secretion, increase in the neuronal count in Nissl staining, and reduction in the expression of phosphorylated extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, p38 kinase and tau protein at serine 202 as well as caspase-3. The use of PA-ApoE-liposomes as a dual targeting formulation enhances the QU and RA ability to infiltrate the BBB, docks Aß1-42 plaques and can be a potent approach to rescue degenerated neurons from AD.

Dexmedetomidine Protects SK-N-SH Nerve Cells from Oxidative Injury by Maintaining Iron Homeostasis.

Ferroptosis is characterized by the accumulation of iron-derived reactive oxygen species (ROS). Ferroptosis causes neuronal death in multiple neurological disorders. Dexmedetomidine (Dex), an extensively used anesthetic, has neuroprotective effects against ROS, but its effect on iron metabolism remains unknown. In this study, SK-N-SH cells were treated with Dex for 24 h before treatment with 100 µM tert-butyl hydroperoxide (t-BHP; an ROS inducer) for 1 h. Afterward, intracellular ROS and labile ferrous iron [Fe(II)] levels were assessed. Dex hindered the increase in cellular ROS and labile Fe(II) levels caused by t-BHP, although Dex alone had no effect on labile Fe(II) level. t-BHP increased the expression of iron importers, transferrin receptor-1 and divalent metal transporter-1, and iron regulatory protein 1 and 2. These effects were abrogated by Dex treatment and SP-1 knockdown. t-BHP increased the phosphorylation of c-Jun N-terminal kinase (JNK) and signal transducer and activator of transcription 4 (STAT4), the primary up-stream activators of SP-1, but Dex decreased this. This study, for the first time, revealed that the antioxidative effect of Dex is partly associated to the inhibition of intracellular iron accumulation induced by t-BHP. Dex regulates iron metabolism by regulating iron importers and exporters through JNK/Sp1 and Stat4/Sp1 signaling. It is worth investigating whether Dex can protect neurons from ferroptosis.

Sanguinarine inhibits the proliferation of BGC-823 gastric cancer cells via regulating miR-96-5p/miR-29c-3p and the MAPK/JNK signaling pathway.

Sanguinarine (SAN), a quaternary benzophenanthridine alkaloid extracted from the root of Papaveraceae plants, has shown antitumour effects in multiple cancer cells. However, the therapeutic effects and the underlying mechanisms of SAN in gastric cancer (GC) remain elusive. In this study, the in vitro proliferation inhibition effect of SAN in GC cells was determined using CCK-8 assay, the in vivo antitumor effect of SAN was evaluated in mice with xenotransplanted tumor. The mechanism underlying the antitumor activity of SAN was explored by gene microarray assay and bioinformatics analysis. The levels of differentially expressed miRNAs and target genes were verified by real-time RT-PCR and immunohistochemistry. SAN inhibited the proliferation of BGC-823 cells in a concentration-dependent manner in vitro and in vivo. The miR-96-5p and miR-29c-3p were significantly upregulated in untreated BGC-823 cells and significantly downregulated in SAN treated cells. The mRNA and protein expression of their target gene MAP4K4 were upregulated in SAN treated xenotransplanted tumors, and pMEK4 and pJNK1 proteins in the MAPK/JNK signaling pathway were also upregulated by SAN. These indicate that SAN may inhibit the proliferation of BGC-823 cells through the inhibition of miR-96-5p and miR-29c-3p expression, and subsequent activation of the MAPK/JNK signaling pathway.

Effects of pomegranate aril juice and its punicalagin on some key regulators of insulin resistance and oxidative liver injury in streptozotocin-nicotinamide type 2 diabetic rats.

Nowadays, medicinal plants have been widely used everywhere to provide essential care for many disorders including diabetes. Recent reports assumed that the antidiabetic activities of pomegranate aril juice (PAJ) may be ascribed to its punicalagin (PCG). Therefore, the present study evaluated and compared the antidiabetic activities of PAJ and its PCG, and monitored some mechanisms of their actions in streptozotocin-nicotinamide (STZ-NA) type 2 diabetic rats. STZ-NA diabetic rats were given, orally/daily, PAJ (100 or 300 mg/kg body weight, containing 2.6 and 7.8 mg of PCG/kg body weight, respectively), pure PCG (2.6 or 7.8 mg/kg body weight), or distilled water (vehicle) for 6 weeks. PAJ (especially at the high dose) alleviated significantly (P < 0.05-0.001) most signs of type 2 diabetes including body-weight loss, insulin resistance (IR) and hyperglycemia through decreasing serum tumor necrosis factor-α concentration and the expression of hepatic c-Jun N-terminal kinase, and increasing the skeletal muscle weight and the expression of hepatic insulin receptor substrate-1 in STZ-NA diabetic rats. Also, it decreased significantly (P < 0.001) the oxidative liver injury in STZ-NA diabetic rats through decreasing the hepatic lipid peroxidation and nitric oxide production, and improving the hepatic antioxidant defense system. Although the low dose of PCG induced some modulation in STZ-NA diabetic rats, the high dose of PCG did not show any valuable antidiabetic activity, but induced many side effects. In conclusion, PAJ was safer and more effective than pure PCG in alleviating IR and oxidative liver injury in STZ-NA diabetic rats.

Astragalus polysaccharides decrease proliferation, migration, and invasion but increase apoptosis of human osteosarcoma cells by up-regulation of microRNA-133a.

Osteosarcoma (OS) has a high incidence, malignity, and frequency of recurrence and metastasis. In this study, we aimed to explore the potential anti-cancer effects of Astragalus polysaccharides (APS) on human OS MG63 cells as well as underlying mechanisms. Viability of MG63 cells was assessed by CCK-8 assay to determine the adequate concentration of APS. Then, effects of APS on MG63 cell proliferation, cell cycle distribution, apoptosis, and migration and invasion were analyzed by BrdU incorporation, PI staining, flow cytometry, and transwell assays, respectively. The expression levels of proteins involved in these physiological processes were assessed by western blot analysis. Afterwards, miR-133a level in APS-treated cells was determined by qRT-PCR, and whether APS affected MG63 cells through regulation of miR-133a was determined. Finally, the activation of c-Jun N-terminal protein kinase (JNK) pathway was detected. We found that APS treatment suppressed the viability, proliferation, migration, and invasion of MG63 cells, as well as induced cell apoptosis. Moreover, APS enhanced the expression of miR-133a in MG63 cells. Knockdown of miR-133a reversed the APS treatment-induced MG63 cell proliferation, migration and invasion inhibition, as well as cell apoptosis. Furthermore, APS inactivated JNK pathway in MG63 cells. Knockdown of miR-133a reversed the APS treatment-induced inactivation of JNK pathway in MG63 cells. To conclude, APS repressed proliferation, migration, and invasion while induced apoptosis of OS MG63 cells by up-regulating miR-133a and then inactivating JNK pathway.

Chinese Herbal Medicine Effects on Muscle Atrophy Induced by Simulated Microgravity.

BACKGROUND: Skeletal muscle atrophy is a striking example of the multiple changes in the physiological state of humans and animals induced by microgravity. Previous studies have shown that a blood circulation disorder may be a cause of this atrophy, and traditional Chinese medicine has been regarded as a potential countermeasure to reverse the atrophy in China. This study was carried out to test the effects of Xuefuzhuyu capsules (XFZY) on the skeletal muscle atrophy induced by simulated microgravity. METHODS: The mass and cross-sectional area of the soleus muscle were compared in rats treated with XFZY that were hindlimb unloaded for 30 d (XFZY-TS group), untreated rats that were hindlimb unloaded for 30 d (TS group), and control rats (CON group). The expression and phosphorylation levels of key proteins of the sarcoplasmic reticulum stress system were also measured. RESULTS: Treatment with XFZY attenuated the loss of muscle mass and cross-sectional area induced by hindlimb unloading. Western blot analysis showed that the phosphatidyl-inositol-3-kinase/phospho-Akt (PI3K/p-Akt) pathways were down-regulated after 30 d in the TS group compared with the CON group. This effect was partly reversed by XFZY. Hindlimb unloading increased the expression of glucose-regulated protein 78 (GRP78), cytosine-cytosine-adenosine-adenosine-thymidine/enhancer-binding protein homologous protein (CHOP), C-Jun N-terminal kinase (JNK), and Caspase 12. Treatment with XFZY alleviated this increased protein expression. DISCUSSION: Our results suggest that XFZY could partially reverse the effects of hindlimb unloading on muscle atrophy and perhaps target the sarcoplasmic reticulum stress system, possibly through the GRP78-CHOP-JNK-Caspase 12 pathway.Zhang S, Yuan M, Cheng C, Xia D, Wu S. Chinese herbal medicine effects on muscle atrophy induced by simulated microgravity. Aerosp Med Hum Perform. 2018; 89(10):883-888.

Zafirlukast and vincamine ameliorate tamoxifen-induced oxidative stress and inflammation: Role of the JNK/ERK pathway.

AIMS: This study investigated the hepatoprotective effects of both zafirlukast and vincamine and their possible role in the treatment of tamoxifen-induced liver injury in rats. MATERIALS AND METHODS: Female Wistar rats were divided into five groups (10 rats each). Groups I and II received 1% Tween 80 and served as normal and tamoxifen controls, respectively. Groups III, IV and V were treated with zafirlukast (80 mg/kg), vincamine (10 mg/kg) and a combination of zafirlukast (80 mg/kg) and vincamine (10 mg/kg), respectively for 10 successive days. Tamoxifen was given orally to all groups, except for 1st group, in the dose of 45 mg/kg for 10 days to induce liver injury. Subsequently, rats were sacrificed for biochemical, histopathological, Immunohistochemistry, PCR and western blot assessment. KEY FINDINGS: Tamoxifen-induced liver injury was reflected by alterations in estimated biochemical parameters, activation of JNK/ERK pathway, increased expression of NF-κB, liver oxidative stress and inflammatory markers parallel to histopathological changes in liver tissue. Treatment of rats with zafirlukast and vincamine ameliorated tamoxifen induced hepatic cell injury via suppressing oxidative stress, inflammatory markers, caspases-3, p-JNK/p-ERK and NF-κB pathways. SIGNIFICANCE: Zafirlukast and vincamine may be regarded as potential therapeutic strategies with antioxidant and anti-inflammatory activities against tamoxifen-induced oxidative damage in rat liver.

The Red Algae Compound 3-Bromo-4,5-dihydroxybenzaldehyde Protects Human Keratinocytes on Oxidative Stress-Related Molecules and Pathways Activated by UVB Irradiation.

Skin exposure to ultraviolet B (UVB) irradiation leads to the generation of reactive oxygen species (ROS). Excessive ROS cause aging of the skin via basement membrane/extracellular matrix degradation by matrix metalloproteinases (MMPs). We recently demonstrated that 3-bromo-4,5-dihydroxybenzaldehyde (BDB), a natural compound of red algae, had a photo-protective effect against UVB-induced oxidative stress in human keratinocytes. The present study focused on the effect of BDB on UVB-irradiated photo-aging in HaCaT keratinocytes and the underlying mechanism. BDB significantly impeded MMP-1 activation and expression, and abrogated the activation of mitogen-activated protein kinases and intracellular Ca2+ level in UVB-irradiated HaCaT cells. Moreover, BDB decreased the expression levels of c-Fos and phospho-c-Jun and the binding of activator protein-1 to the MMP-1 promoter induced by UVB irradiation. These results offer evidence that BDB is potentially useful for the prevention of UVB-irradiated skin damage.

The bradykinin-cGMP-PKG pathway augments insulin sensitivity via upregulation of MAPK phosphatase-5 and inhibition of JNK.

Bradykinin (BK) promotes insulin sensitivity and glucose uptake in adipocytes and other cell types. We demonstrated that in rat adipocytes BK enhances insulin-stimulated glucose transport via endothelial nitric oxide synthase, nitric oxide (NO) generation, and decreased activity of the mitogen-activated protein kinase (MAPK) JNK (c-Jun NH2-terminal kinase). In endothelial cells, NO increases soluble guanylate cyclase (sGC) activity, which, in turn, activates protein kinase G (PKG) by increasing cGMP levels. In this study, we investigated whether BK acts via the sGC-cGMP-PKG pathway to inhibit the negative effects of JNK on insulin signaling and glucose uptake in rat adipocytes. BK augmented cGMP concentrations. The BK-induced enhancement of insulin-stimulated glucose uptake was mimicked by the sGC activator YC-1 and a cell-permeable cGMP analog, CPT-cGMP, and inhibited by the sGC inhibitor ODQ and the PKG inhibitor KT 5823. Transfection of dominant-negative PKG reduced the BK augmentation of insulin-induced Akt phosphorylation. The activation of JNK and ERK1/2 by insulin was attenuated by BK, which was mediated by the sGC-cGMP-PKG pathway. Whereas insulin-stimulated phosphorylation of upstream activators of JNK and ERK, i.e., MKK4 and MEK1/2, was unaffected, BK augmented insulin-mediated induction of MKP-5 mRNA and protein levels. Furthermore, zaprinast, a phosphodiesterase inhibitor, enhanced cGMP and MKP-5 and prolonged the action of BK. These data indicate that BK enhances insulin action by inhibition of negative feedback by JNK and ERK via upregulation of MKP-5, mediated by the sGC-cGMP-PKG signaling pathway.

The Novel HDAC8 Inhibitor WK2-16 Attenuates Lipopolysaccharide-Activated Matrix Metalloproteinase-9 Expression in Human Monocytic Cells and Improves Hypercytokinemia In Vivo.

Dysregulated human monocytes/macrophages can synthesize and secrete matrix metalloproteinases (MMPs), which play important roles in the progression of sepsis. In this study, we investigated the effects and mechanism of a novel histone deacetylase (HDAC8) inhibitor, (E)-N-hydroxy-4-methoxy-2-(biphenyl-4-yl)cinnamide (WK2-16), on MMP-9 production and activation in stimulated human monocytic THP-1 cells. Our results demonstrated that the acetylation level of structural maintenance of chromosomes 3 (SMC3) was up-regulated by WK2-16 in THP-1 cells. Consistently, an in vitro enzyme study demonstrated that WK2-16 selectively inhibited HDAC8 activity. Moreover, the WK2-16 concentration dependently suppressed MMP-9-mediated gelatinolysis induced by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS). Additionally, WK2-16 significantly inhibited both MMP-9 protein and mRNA expression without cellular toxicity. Nevertheless, WK2-16 suppressed the extracellular levels of interleukin (IL)-6 from LPS-stimulated THP-1 cells. For the signaling studies, WK2-16 had no effect on LPS/TLR4 downstream signaling pathways, such as the NF-κB and ERK/JNK/P38 MAPK pathways. On the other hand, WK2-16 enhanced the recruitment of acetylated Yin Yang 1 (YY1) with HDAC1. Finally, in vivo studies indicated that WK2-16 could reduce the serum levels of TNF-α and IL-6 in endotoxemic mice. These results suggested that HDAC8 inhibition might provide a novel therapeutic strategy of hypercytokinemia in sepsis.

Adenovirus-Mediated Small Interfering RNA Targeting TAK1 Ameliorates Joint Inflammation with Collagen-Induced Arthritis in Mice.

Transforming growth factor ß-activated kinase-1 (TAK1) is a key upstream kinase in cell signaling during inflammation, which regulates the expression of inflammatory mediators. Small interfering RNA (siRNA) against TAK1 offers promise as a potential therapeutic strategy in immune-mediated inflammatory disorder including rheumatoid arthritis. Here, we are to evaluate the therapeutic effects of intra-articular administration of adenoviral-mediated siRNA against TAK1 (ad-siRNA-TAK1) on collagen-induced arthritis (CIA) in mice. Ad-siRNA-TAK1 was constructed. The murine RAW 264.7 macrophages were infected with ad-siRNA-TAK1, and the silencing specificity of TAK1 was assessed by quantitative polymerase chain reaction (PCR) and western blot. DBA/1 mice were injected intra-articularly with ad-siRNA-TAK1. Development and severity of arthritis was assessed histologically. Synovial inflammation and bone destruction were determined by hematoxylin and eosin (HE) staining. Articular and serum concentrations of tumor necrosis factor-α, interleukin-1, and interleukin-6 were determined using enzyme-linked immunosorbent assay. Levels of phosphorylated p38, c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase (ERK) were detected by western blot. In vitro, ad--siRNA-TAK1 efficiently inhibited the expression of TAK1 at both mRNA and protein levels. In vivo, intra-articular injection of ad-siRNA-TAK1 efficiently alleviated joint inflammation, decreased the expression of pro-inflammatory mediators, and suppressed JNK pathways. Our results demonstrate the efficiency of ad--siRNA-TAK1 in controlling joint inflammation of CIA, which is associated with the suppression of the expression of pro-inflammatory cytokines and JNK activation.

α-1 Antitrypsin Enhances Islet Engraftment by Suppression of Instant Blood-Mediated Inflammatory Reaction.

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic ß-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in ß-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

Rapid Communication: Prolactin and hydrocortisone impact TNFα-mediated mitogen-activated protein kinase signaling and inflammation of bovine mammary epithelial (MAC-T) cells.

The objective of this study was to evaluate the effects of the hormones prolactin (PRL) and hydrocortisone (HC) on bovine mammary alveolar (MAC-T) cells mitogen-activated protein kinase (MAPK) inflammatory signaling and inflammatory gene expression. MAC-T cells were cultured in the presence (+PRL +HC; Dulbecco's modified Eagle's medium [DMEM] 10% fetal bovine serum, 10 µg/mL of insulin, 100 IU/mL penicillin, 100 µg/mL streptomycin, 1 µg/mL ovine PRL, 0.5 µg/mL HC, and 10 m sodium acetate) or the absence (-PRL -HC; DMEM 10% fetal bovine serum, 10 µg/mL insulin, 100 IU/mL penicillin , and 100 µg/mL streptomycin) of PRL and HC, and MAPK (extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38) phosphorylation and inflammatory gene expression were examined in response to tumor necrosis factor α (TNFα). Statistical analysis was assessed using 1-way ANOVA, and Tukey's post hoc analysis was used to assess statistical significance when ≤ 0.05. MAC-T cells cultured in +PRL +HC and -PRL -HC were co-stimulated with increasing concentrations of TNFα (0, 10, 30, 100, 300, and 1,000 p). Cell lysates were harvested 15 min after TNFα stimulation and assessed for MAPK phosphorylation using immunoblotting. c-Jun N-terminal kinase and p38 phosphorylation increased in a dose-dependent manner and was greater in cells cultured in -PRL -HC. MAC-T cells cultured in +PRL +HC and -PRL -HC were next stimulated with TNFα (300 p), and lysates were harvested over time (0, 15, 30, 60, 120, and 180 min) after TNFα stimulation. c-Jun N-terminal kinase and p38 phosphorylation was transiently increased in MAC-T cells stimulated with TNFα; however, JNK and p38 signaling was greater in MAC-T cells cultured in -PRL -HC. We next examined inflammatory gene expression in MAC-T cells cultured in +PRL +HC and -PRL -HC. Cells were co-stimulated with (300 p) or without TNFα. Ribonucleic acid was isolated 1 h after TNFα stimulation, and a PCR array was performed to examine the expression of 83 inflammatory genes. Gene expression was increased in MAC-T cells in response to TNFα. Consistent with enhanced MAPK signaling, inflammatory gene expression was increased in MAC-T cells cultured in -PRL -HC. Real-time quantitative PCR of 6 target genes was used to validate the PCR array findings. Collectively, our data demonstrate that -PRL -HC MAC-T cells are more responsive to TNFα stimuli. These findings suggest that cell culture conditions (e.g., treatment with hormones) greatly impact cellular response and should be considered prior to experimental design and hypothesis testing.

Effect of Alkaloids from Nelumbinis Plumula against Insulin Resistance of High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease in Mice.

This study aimed to investigate the effects of total alkaloids from Nelumbinis Plumula (NPA) on insulin resistance (IR) of high-fat diet- (HFD-) induced nonalcoholic fatty liver disease (NAFLD). Rats were fed with HFD for 8 weeks to induce NAFLD. Then, the effect of NPA on ameliorating IR in HFD-induced NAFLD was evaluated. Fasting serum insulin was determined using an enzyme-linked immunosorbent assay (ELISA) kit for insulin following the manufacturer's protocol. Some inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were determined using ELISA kits to assess the inflammatory burden in rats. The results showed that HFD could induce a significant increase in blood glucose and IR in rats. However, rats treated with NPA (400 or 600 mg/kg) showed improved IR and reduction in serum inflammatory cytokines TNF-α and IL-6. Further investigation indicated that NPA could inhibit IR by restoring the insulin receptor substrate-1 (IRS-1) and suppressing the expression of c-Jun N-terminal kinase (JNK) phosphorylation. The present results supported the view that the pathogenesis of NAFLD was complex with inflammation, together with increasing serum glucose and IR. Also, JNK and IRS phosphorylation were suggested for their involvement in the modulating of IR during NAFLD progression. Therefore, NPA may serve as a potential natural remedy against IR in NAFLD.

Low Dose Cadmium Inhibits Proliferation of Human Renal Mesangial Cells via Activation of the JNK Pathway.

Cadmium (Cd) is a heavy metal and environmental pollutant. The kidney is the principal target organ of Cd exposure. Previously, we found that low concentration of Cd damages the integrity of the glomerular filtration barrier. However, little is known about the effects of Cd on renal mesangial cells, which provide structural support for the glomerular capillary loops and regulate intraglomerular blood flow. In this study, human renal mesangial cells (HRMCs) were cultured in the presence of serum and treated with 4 µM Cd. We found that Cd activates the c-Jun N-terminal kinase (JNK) pathway, and increases the protein levels of c-Jun and c-Fos. Cd treatment also induces a decrease in proliferation and an increase in apoptosis of HRMCs, but only the decrease in HRMC proliferation was reversed by pretreatment with SP600125, an inhibitor of the JNK pathway. In addition, Cd does not change the expression of α-smooth muscle actin and platelet-derived growth factor receptor-ß, the markers of mesangial cells, or the alignment of the filamentous actin (F-actin) cytoskeleton of HRMCs. Our data indicate that the JNK pathway mediates the inhibitory effects of Cd on HRMC proliferation.

Xanthoangelol and 4-hydroxyderrcin suppress obesity-induced inflammatory responses.

OBJECTIVE: Obesity-induced inflammation plays a pivotal role in the pathogenesis of insulin resistance and type 2 diabetes. Xanthoangelol (XA) and 4-hydroxyderrcin (4-HD), phytochemicals extracted from Angelica keiskei, have been reported to possess various biological properties. Whether XA and 4-HD alleviate obesity-induced inflammation and inflammation-induced adipocyte dysfunction was investigated. METHODS: For the in vitro study, a co-culture system composed of macrophages and adipocytes and macrophages stimulated with conditioned medium derived from fully differentiated adipocytes was conducted. For the in vivo study, mice were fed a high-fat diet supplemented with XA for 14 weeks. RESULTS: XA and 4-HD suppressed inflammatory factors in co-culture system. Moreover, treatment of RAW macrophages with XA and 4-HD moderated the suppression of uncoupling protein 1 promoter activity and gene expression in C3H10T1/2 adipocytes, which was induced by conditioned medium derived from LPS-stimulated RAW macrophages. Also, XA and 4-HD inhibited c-Jun N-terminal kinase phosphorylation, nuclear factor-κB, and activator protein 1, the last two being transcription activators in activated macrophages. Furthermore, in mice fed the high-fat diet, XA reduced inflammatory factors within the white adipose tissue. CONCLUSIONS: These results suggest that XA and 4-HD might be promising phytochemicals to suppress obesity-induced inflammation and inflammation-induced adipocyte dysfunction.

Green tea (-)-epigallocatechin gallate induced growth inhibition of human placental choriocarcinoma cells.

This study investigated the pathways involved in the effect of green tea epigallocatechin gallate (EGCG) on mitogenesis in BeWo, JEG-3, and JAR placental choriocarcinoma cells. EGCG inhibited cell proliferation in dose-dependent and time-dependent manners, as indicated by the number of cells and incorporation of bromodeoxyuridine (BrdU). A catechin-specific effect of green tea was evident; EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in suppressing cell growth. When all three of the mitogen-activated protein kinase (MAPK) subfamilies, i.e., ERK, p38, and JNK, were examined, EGCG significantly increased levels of phospho-ERK1/2 (pERK1/2) and phospho-p38 (pp38) and did not alter the total protein levels of ERK1/2, p38 MAPK, JNK, and phospho-JNK. EGCG-induced increases in the levels of pERK1/2 and pp38 proteins were prevented by pre-treatment with specific inhibitors of ERK1/2 MAPK and p38 MAPK, respectively. These inhibitors also suppressed EGCG-induced decreases in both cell number and BrdU incorporation. Moreover, pre-treatment with an AMP-activated protein kinase (AMPK) inhibitor prevented the actions of EGCG on proliferation and AMPK phosphorylation. These data suggest that EGCG mediates choriocarcinoma cell growth via the AMPK, ERK, and p38 pathways, but not JNK pathway.

The anacardic 6-pentadecyl salicylic acid induces macrophage activation via the phosphorylation of ERK1/2, JNK, P38 kinases and NF-κB.

Amphipterygium adstringens is a plant traditionally used to treat gingivitis, gastric ulcer and even gastric cancer but the mechanism involved in the regulation of the immune response is not elucidated yet. The 6-pentadecylsalicylic acid (6SA) is the main anacardic acid found in A. adstringens. In order to evaluate the immune-modulatory abilities of 6SA, we used mouse splenocytes and determined the phosphorylation of the transcription factor NF-κB and MAP kinases ERK1/2, JNK and p38 in helper and cytotoxic T cells, natural killer (NK) cells and F4/80(+) macrophages. Treatment with 6SA was not cytotoxic as measured by both trypan blue exclusion and tetrazolium salts (MTT) tests. Additionally, 6SA did not alter the proportion of helper and cytotoxic T lymphocytes, NK cells or macrophages. Moreover, 6SA treatment significantly increased the phosphorylation of ERK1/2, JNK, P38 and NF-κB mainly in macrophages. In this cells (peritoneal macrophages), treatment with 6SA increased the secretion of nitric oxide (NO), interleukin (IL)-6 and tumour necrosis factor (TNF)-α and decreased the secretion of IL-4 and IL-10 depending on MAPK and NF-κB phosphorylation. In addition, 6SA increased the migration and phagocytic activity of macrophages also depending on the phosphorylation of different kinases. These data suggest that 6SA induces the classical activation pathway in macrophages via the phosphorylation of MAP kinases and NF-κB thus activating the adaptive immune system.