Unconjugated
Phloretin (Ph) existing in apples, pears and various vegetables is known to have antitumor activities in several cancer cell lines. However, little is known about its effect on human lung cancer cells. The aim of the present study was to see whether Ph could induce apoptosis of non-small cell lung cancer (NSCLC) cells, and explore the possible underlying mechanism of action. We found that Ph markedly induced cell apoptosis of NSCLC cell line A549, and inhibited the migration of A549 cells in a dose-dependent manner. The expression level of BAX, cleaved caspase-3 and -9, and degraded form of PARP was increased and Bcl-2 was decreased after Ph treatment. In addition, the phosphorylation of P38 MAPK, ERK1/2 and JNK1/2 was increased in a dose‑dependent manner in parallel with Ph treatment. Inhibition of P38 MAPK and JNK1/2 by specific inhibitors significantly abolished the Ph-induced activation of the caspase-3 and -9. In vivo tumor-suppression assay further indicated that Ph (20 mg/kg) displayed a more significant inhibitory effect on A549 xenografts in tumor growth. All these findings indicate that Ph is able to inhibit NSCLC A549 cell growth by inducing apoptosis through P38 MAPK and JNK1/2 pathways, and therefore may prove to be an adjuvant to the treatment of NSCLC.
Interleukin-17 (IL-17 or IL-17A), a pleiotropic cytokine produced by T helper (Th) 17 cells, is involved in the pathogenesis of various autoimmune and inflammatory disorders, including periodontitis. Although the ability of pro-inflammation in periodontitis have been widely investigated, the other biological functions of IL-17, including its role in bone remodeling and the underlying molecular mechanism, have not been well clarified. In the present study, IL-17 could significantly enhance the expression of receptor activator for nuclear factor-κB ligand (RANKL) and inhibit the expression of osteoprotegerin (OPG) in human periodontal ligament cells (hPDLCs), the two critical indicators for osteoclastogenesis, suggesting IL-17 may play a destructive role in the pathogenesis of periodontal bone remodeling. Pharmaceutical signal inhibitors targeted at MAPKs, Akt or NF-κB signals, inhibited IL-17-induced RANKL and OPG regulation. Notably, the enhancement of RANKL was significantly blocked by the inhibitors of JNK and NF-κB signals. The upstream signals were further investigated with the small interfering RNA (siRNA). Both TRAF6 and TBK1 were found to be the critically signal molecules for IL-17-dependent RANKL regulation in hPDLCs. These findings may provide comprehensive understanding of the role of IL-17 in the pathogenesis of periodontitis and might also provide a reasonable way for periodontitis therapy. This article is protected by copyright. All rights reserved.
Cathepsin B (CB), an important proteinase that participates in joint destruction in rheumatoid arthritis (RA), exhibits higher expression in fibroblast-like synoviocyte (FLS) of abnormal proliferative synovial tissues. Whether and how it affects the biological behaviours of RA-FLS, such as migration and invasion, are poorly understood. In the present study, CB expression in synovial tissues of patients with RA and ostearthritis (OA) were measured by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC), respectively. Stable depletion of endogenous CB was achieved by small interfering RNA (siRNA) transfection, and decrease of CB activity was acquired by using its specific inhibitor (CA074Me). The effects of CA074Me and RNA interference (RNAi) treatments on proliferation, migration, invasion, matrix metalloproteinase (MMP)-2/-9 expression, focal adhesion kinase (FAK) activation, and mitogen-activated protein kinases (MAPKs) phosphorylation of FLS were analysed. In RA synovial tissues, CB was expressed at elevated levels compared with OA synovial tissues. CA074Me could inhibit invasion of FLS obtained from RA patients in an ex-vivo invasion model. CA074Me and siRNA treatments suppressed the migration and invasion of FLS, reduced the activity, expression and mRNA level of MMP-2, restrained the activation of FAK and reduced the expression of F-actin. Moreover, CA074Me decreased the phosphorylation of P38 MAPK and c-Jun N-terminal kinase (JNK) in FLS, while siCB treatment reduced the phosphorylation of P38 but not JNK. CB substantially contributes to the invasive phenotype of FLS that leads to joint destruction in RA. This proteinase may show promise as a therapeutic target in inflammatory arthritis.
It has been documented in in vitro studies that zinc oxide nanoparticles (ZnO NPs) are capable of inducing oxidative stress, which plays a crucial role in ZnO NP-mediated apoptosis. However, the underlying molecular mechanism of apoptosis in neurocytes induced by ZnO NP exposure was not fully elucidated. In this study, we investigated the potential mechanisms of apoptosis provoked by ZnO NPs in cultured primary astrocytes by exploring the molecular signaling pathways triggered after ZnO NP exposure. ZnO NP exposure was found to reduce cell viability in MTT assays, increase lactate dehydrogenase (LDH) release, stimulate intracellular reactive oxygen species (ROS) generation, and elicit caspase-3 activation in a dose- and time-dependent manner. Apoptosis occurred after ZnO NP exposure as evidenced by nuclear condensation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. A decrease in mitochondrial membrane potential (MMP) with a concomitant increase in the expression of Bax/Bcl-2 ratio suggested that the mitochondria also mediated the pathway involved in ZnO NP-induced apoptosis. In addition, exposure of the cultured cells to ZnO NPs led to phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Moreover, JNK inhibitor (SP600125) significantly reduced ZnO NP-induced cleaved PARP and cleaved caspase-3 expression, but not ERK inhibitor (U0126) or p38 MAPK inhibitor (SB203580), indicating that JNK signaling pathway is involved in ZnO NP-induced apoptosis in primary astrocytes.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer, because it preferentially induces apoptosis in numerous cancer cells with little or no effect on normal cells. 5,7-Dihydroxyflavone is a dietary flavonoid commonly found in many plants. Here we show that the combined treatment with 5,7-dihydroxyflavone and TRAIL at subtoxic concentrations induced strong apoptotic response in human hepatocarcinoma HepG2 cells, acute leukemia Jurkat T cells, and cervical carcinoma HeLa cells. We further investigated the mechanisms by which 5,7-dihydroxyflavone augments TRAIL-induced apoptosis in HepG2 cells. 5,7-Dihydroxyflavone up-regulated the expression of pro-apoptotic protein Bax, attenuated the expression of anti-apoptotic proteins Bcl-2, Mcl-1, and IAPs, and reduced the phosphorylation levels of Akt and STAT3, weakening the anti-apoptotic signals thus facilitating the process of apoptosis. Moreover, 5,7-dihydroxyflavone and TRAIL were well tolerated in mice, and the combination of 5,7-dihydroxyflavone and TRAIL reduced tumor burden in vivo in a HepG2 tumor xenograft model. Interestingly, 5,7-dihydroxyflavone-mediated sensitization to TRAIL-induced cell death was not observed in normal human hepatocytes L-O2. These results suggest that the 5,7-dihydroxyflavone in combination with TRAIL might be used for cancer prevention and/or therapy.
BACKGROUND:
The dipeptidyl peptidase-4 inhibitor sitagliptin, a new anti-diabetic medicine, is effective in treating type 2 diabetes mellitus by increasing the activation and duration of action of glucagon-like peptide-1. Since atherosclerosis is the main pathological feature of diabetic cardiovascular complications, it is important to investigate the anti-atherosclerotic effect of sitagliptin and explore the relevant mechanisms.
METHODS:
Male apolipoprotein-E-knockout mice were randomly divided into two groups and fed either high-fat diet (HFD) or HFD plus sitagliptin at a concentration of 0.3% for 16 weeks. Body weight, food intake, blood glucose, serum lipids and adhesion molecules were measured. The atherosclerotic plaque area and its histological composition were analyzed using Sudan staining and immunohistochemistry. The expression of inflammatory cytokines (monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6) and the activation of AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK) in the aortas were determined using quantitative polymerase chain reaction and western blot, respectively.
RESULTS:
Mice treated with sitagliptin developed fewer atherosclerotic plaques than the control group (7.64 ± 1.98% vs 12.91 ± 1.15%, p < 0.001), particularly in the aortic arch and abdominal aorta, where plaques were decreased 1.92- and 2.74-fold, respectively (p < 0.05 and p < 0.01). Sitagliptin significantly reduced the content of collagen fiber in plaques 1.2-fold (p < 0.05). Moreover, sitagliptin significantly reduced the expression of monocyte chemoattractant protein-1 and interleukin-6 in the aorta (p < 0.01 and p < 0.05), as well as the serum levels of soluble vascular cell adhesion molecule-1 and P-selectin (both p < 0.05). In addition, Sitagliptin induced phosphorylation of AMPK and Akt (p < 0.05 and p < 0.01), while suppressed phosphorylation of p38 and extracellular signal-regulated kinase (Erk) 1/2 (p < 0.05 and p < 0.01) in aortas.
CONCLUSIONS:
Our present study indicates that sitagliptin can reduce the area of the atherosclerotic lesion, possibly by regulating the AMPK and MAPK pathways and then reducing leukocyte -endothelial cell interaction and inflammation reactions. These actions are independent of weight loss and glucose-reducing effects.
The anti-cancer effects of dioscin have been widely reported. However, its effect on laryngeal cancer remains unknown. In the present paper, our results showed that dioscin markedly caused cell apoptosis and DNA damage, increased reactive oxygen species (ROS) level, induced S-phase arrest, and inhibited invasion of human laryngeal cancer HEp-2 and TU212 cells. Mechanism investigation showed that dioscin markedly up-regulated p53 level, and down-regulated cyclin-dependent kinase 2 (CDK2) and Cyclin A levels. In addition, dioscin significantly down-regulated the levels of p-ERK, Bcl-2, up-regulated the levels of p-JNK, p-p38, Bax, cleaved caspase-3/-9, and caused Cytochrome c release. Furthermore, U0126, an ERK1/2 inhibitor, markedly down-regulated Bcl-2 level, up-regulated the levels of Bax, cleaved caspase-3/9, and enhanced Cytochrome c release inducted by dioscin. While, SP600125 (one JNK inhibitor) and SB203580 (one p38 inhibitor) markedly up-regulated Bcl-2 level, down-regulated the levels of Bax, cleaved caspase-3/9, and obviously boosted Cytochrome c release induced by dioscin. Interestingly, dioscin also markedly down-regulated the levels of MMP2 and MMP9 associated with tumor invasion. Taken together, our study indicated that dioscin suppressed laryngeal cancer cells growth via inducting cell-cycle arrest, MAPK-mediated mitochondrial- derived apoptosis and inhibiting tumor invasion, which could be used as one potential candidate for the treatment of laryngeal cancer in the future.
Gastric ulcers (GUs) are a common type of peptic ulcer. Alcohol overdose is one of the main causes of GU, which is difficult to prevent. Although the protective effect of butyrate on inflammation-related diseases is well understood, its effect on GUs has not been reported. We investigated the protective effects of butyrate against ethanol-induced lesions to the gastric mucosa in mice and the underlying mechanisms. BALB/c mice were orally pretreated with butyrate for 30min prior to the establishment of the GU model by challenge with absolute ethanol. Ethanol administration produced apparent mucosal injuries with morphological and histological damage, whereas butyrate pretreatment reduced the gastric mucosal injuries in a dose-dependent manner. Butyrate pretreatment also significantly ameliorated contents of malondialdehyde (MDA) and carbonyl proteins, and decreased levels of IL-1β, TNF-α and IL-6. The Western blot results consistently demonstrated that butyrate pretreatment attenuated the phosphorylation of NF-κB p65, p38 MAPK and ERKs in the gastric tissues. Additionally, gastric wall mucus (GWM), a parameter reflecting mucosal defense, was clearly increased by butyrate pretreatment. Butyrate pretreatment protects the gastric mucosa against ethanol-induced lesions by strengthening the mucosal defense and anti-oxidant and anti-inflammatory activities. As a necessary substance for the body, butyrate may be applied to the prevention and treatment of GUs.
Endotoxin can stimulate inflammatory cytokine release from monocytes/macrophages and result in septic shock. Glycyrrhetinic acid (GA), the main bioactive component of licorice, possesses substantial anti-inflammatory activity. Here, we explored effect of 11-deoxy-18α-glycyrrhetinic acid-30-ethyl ester (DGAEE), a newly synthesized derivative of GA, on septic shock. DGAEE and its main metabolite 11-deoxy-18α-glycyrrhetinic acid (DGA) significantly alleviated septic shock as evidenced by improvements of survival rates, lung histopathological changes and wet/dry ratio in lipopolysaccharide (LPS)/D-galactosamine-stimulated mice, and decreased blood pressure in LPS/D-galactosamine-stimulated rats. The two compounds decreased serum levels of NO, TNF-α, IL-6, IL-1β, and increased the level of IL-10 more potently in mice. In LPS-stimulated RAW 264.7 cells, DGA but not DGAEE showed marked regulation of NO, TNF-α, IL-6 and IL-10 levels, suggesting that DGAEE display anti-shock effect by DGA rather than itself. Moreover, the neutralizing antibody against IL-10 markedly prohibited the inhibitory effect of DGA on the production of cytokines from RAW 264.7 cells, and AS101 (an inhibitor of IL-10 biosynthesis) almost completely reversed the anti-shock effect of DGA in mice. In addition, DGA did not affect activation of NF-κB-p65 and p38 MAPK as well as IκBα degradation, but moderately reduced activation of ERK and JNK, and markedly increased phosphorylation of GSK3β in LPS-stimulated RAW 264.7 cells. LY294002 (an inhibitor of GSK3β phosphorylation) and LiCl (an inhibitor of GSK3β activity) diminished and potentiated increase of IL-10 levels by DGA, respectively. In conclusion, DGAEE alleviates septic shock through DGA in an IL-10-dependent manner, and the mechanism is related to inactivation of GSK3β.
Norisoboldine (NOR), the main active constituent of Radix Linderae, was previously demonstrated to ameliorate collagen-induced arthritis in rats through regulating the imbalance of T cells in intestines, which implied its therapeutic potential in inflammatory bowel disease. Here, we investigated the effect of NOR on ulcerative colitis (UC) induced by dextran sulfate sodium (DSS) in mice. Results showed that NOR (20, 40mg/kg) markedly reduced the symptoms of colitis, the levels of IL-1β and TNF-α, and the activation of ERK, p38 MAPK and NF-κB-p65. NOR only slightly decreased the levels of IFN-γ and IL-17A in mouse colons, but it dramatically increased the level of IL-10 at both protein and mRNA grades. Consistently, NOR increased the number of CD4(+)CD25(+)Foxp3(+) Treg cells more obviously than it decreased that of CD4(+)IL-17(+) Th17 cells in mesenteric lymph nodes (MLNs) and colonic lamina proprias (LPs) of colitis mice, and promoted the expression of Foxp3 mRNA in colon tissues. It could facilitate the in vitro differentiation of Treg cells from naive T cells and promote the phosphorylations of Smad2/3 in colon tissues of colitis mice. On the other hand, NOR did not affect the expressions of homing receptors CCR9 and α4β7 in SPs, and homing ligands CCL25 and Madcam-1 in MLNs and colonic LPs, suggesting that the increase of Treg cells in colons by NOR was not due to gut homing. In conclusion, NOR can ameliorate DSS-induced UC in mice, and the mechanisms involve reduction of pro-inflammatory cytokines and selective induction of Treg cells in colons.
Ruscogenin, a natural steroidal sapogenin, presents in both food and medicinal plants. It has been found to exert significant anti-inflammatory activities. Considering that activation of neutrophil is a key feature of inflammatory diseases, this study was performed to investigate the inhibitory effect of ruscogenin and its underlying mechanisms responsible for neutrophil activation. Ruscogenin displayed potent antioxidative effects against Formyl-Met-Leu-Phe (FMLP)-induced extra- and intracellular superoxide generation in mouse bone marrow neutrophils, with IC50 values of 1.07±0.32 μM and 1.77±0.46 μM, respectively. Phorbol myristate acetate (PMA)-elicited extra- and intracellular superoxide generation were also suppressed by ruscogenin, with IC50 values of 1.56±0.46 μM and 1.29±0.49 μM, respectively. However, ruscogenin showed weak inhibition in NaF-induced response. Inhibition of superoxide generation was mediated neither by a superoxide-scavenging ability nor by a cytotoxic effect. Furthermore, ruscogenin inhibited the membrane translocation of p47phox and p67phox. It reduced FMLP-induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and p21-activated kinase (PAK). The cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were increased by ruscogenin. Moreover, ruscogenin inhibited phosphorylation of protein kinase B (Akt), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). In addition, the inhibitory effects of ruscogenin on superoxide production and the phosphorylation of Akt, p38MAPK, and ERK1/2 were reversed by PKA inhibitor (H89), suggesting a PKA-dependent mechanism. In summary, our data suggest that ruscogenin inhibits activation of neutrophil through cPLA2, PAK, Akt, MAPKs, cAMP, and PKA signaling pathways. Increased PKA activity is associated with suppression of the phosphorylation of Akt, p38MAPK, and ERK1/2 pathways.
The aim of this study was to explore the intracellular mechanisms underlying the cardiovascular toxicity of air particulate matter (PM) with an aerodynamic diameter of less than 2.5 µm (PM2.5) in a human umbilical vein cell line, EA.hy926. We found that PM2.5 exposure triggered reactive oxygen species (ROS) generation, resulting in a significant decrease in cell viability. Data from Western blots showed that PM2.5 induced phosphorylation of Jun N-terminal kinase (JNK), extracellular signal regulatory kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), and activation of nuclear factor kappa B (NF-κB). We further observed a significant increase in expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Moreover, the adhesion of monocytic THP-1 cells to EA.hy926 cells was greatly enhanced in the presence of PM2.5 . However, N-acetylcysteine (NAC), a scavenger of ROS, prevented the increase of ROS generation, attenuated the phosphorylation of the above kinases, and decreased the NF-κB activation as well as the expression of ICAM-1 and VCAM-1. Furthermore, ERK inhibitor (U0126), AKT inhibitor (LY294002) and NF-κB inhibitor (BAY11-7082) significantly down-regulated PM2.5 -induced ICAM-1 and VCAM-1 expression as well as adhesion of THP-1 cells, but not JNK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580), indicating that ERK/AKT/NF-κB is involved in the signaling pathway that leads to PM2.5 -induced ICAM-1 and VCAM-1 expression. These findings suggest PM2.5 -induced ROS may function as signaling molecules triggering ICAM-1 and VCAM-1 expressions through activating the ERK/AKT/NF-κB-dependent pathway, and further promoting monocyte adhesion to endothelial cells.
The study aims to investigate the effects of protocatechuic acid (PCA) separated from Chinese herbs, on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. The mouse model was induced by intraperitoneal injection of LPS at the dose of 5mg/kg body weight. Three doses of PCA (30, 15, 5 mg/kg) were administered to mice with intraperitoneal injection one hour prior to LPS exposure. Six hours later after LPS administration, the effect of PCA on ALI mice was assessed via histopathological examination by HE staining, inflammatory cytokine production by ELISA assay and RT-PCR, p38MAPK and NF-κB activation by Western blot analysis. We found that PCA administration significantly ameliorated lung histopathological changes and decreased protein concentration in the bronchoalveolar lavage fluid. Furthermore, the overproduction of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was reduced by PCA. Additionally, PCA at the dose of 30 mg/kg could block the activation of p38MAPK and NF-κB signal pathways induced by LPS. In conclusion, our findings demonstrate that PCA possesses a protective effect on LPS-induced ALI in mice via suppression of p38MAPK and NF-κB signal pathways. Therefore, PCA may be useful in the therapy of lung inflammatory diseases, especially for ALI.
Green tea (Camellia sinensis) is an extremely popular beverage worldwide. Epigallocatechin-3-gallate (EGCG) is one of the major catechins isolated from green tea and contributes to its beneficial therapeutic functions including antioxidant, anti-inflammatory and anti-cancer effects. However, the effect of EGCG on mastitis is not yet known. This study was to investigate the protective potential of EGCG against mastitis in rats. The rat mastitis model was induced by injecting lipopolysaccharide (LPS) into the duct of mammary gland. The mammary gland was collected after the experimental period. The levels of mammary oxidative stress and inflammatory responses were assessed by measuring the local activities of antioxidant enzymes and the levels of inflammatory cytokines. The mammary expressions of mitogen-activated protein kinases (MAPKs), nuclear factor κB-p65 (NFκB-p65) and hypoxia-inducible factor-1α (HIF-1α) were evaluated by western blot analysis. It was found that EGCG obviously normalized LPS-induced low activities of antioxidant enzymes as well as decreased the high levels of inflammatory cytokines. Additionally, EGCG inhibited the mammary over-expression of MAPKs, NFκB-p65 and HIF-1α. These results indicated that EGCG was able to attenuate LPS-induced mastitis in rats by suppressing MAPK related oxidative stress and inflammatory responses.
Urocortin (UCN) has exhibited antiinflammatory and neuroprotective effects on intracerebral hemorrhage (ICH). However, the underlying mechanisms are still not clear. Therefore, this study was aimed to investigate effects of UCN1 on ICH in vitro and in vivo and further explore the possible mechanism. ICH was induced by an infusion of autologous blood into the unilateral striatum of anesthetized male Sprague-Dawley rats. The rats were randomly divided into three groups (8 rats per group): sham ICH control group, ICH saline group and ICH UCN1 group. UCN1 was infused into the lateral ventricle after 1h post-ICH. Neurological deficits were evaluated by modified neurological severity score (mNSS). Brain edema was assessed using the dry/wet method. The neurological cell metabolic activity of N2a and SH-SY5Y was detected by CCK-8. The level of VEGF, JNK and p38 were determined by enzyme-linked immunosorbent assay and western blot. Post-treatment with UCN1 could improve neurological deficits and reduce brain edema. Moreover, UCN1 could increase the metabolic activity of neuron cells dose-dependently and these effects could be abolished by corticotropin-releasing factor receptor 2 (CRFR2) antagonist anti-Svg-30. Furthermore, the level of VEGF, JNK and p38 were up-regulated by post-treatment with UCN1 via CRFR2. The protective effects of UCN1 against ICH are possibly mediated by activating the phosphorylation of JNK and p38 and further increasing the level of VEGF via CRFR2.
The aim of the present paper was to investigate the effects and possible mechanisms of the total saponins from Dioscorea nipponica Makino (TSDN) against type 2 diabetes mellitus. Streptozotocin (STZ) with high-fat diet induced type 2 diabetes mellitus (T2DM) rats were treated with TSDN. Some biochemical parameters, target proteins and genes were investigated. The results showed that TSDN decreased the levels of food/water intake, fasting blood glucose and serum lipid parameters, ameliorated oral glucose and insulin tolerance test levels, markedly increased body weight and serum insulin, reduced excess free radicals and affected ossification and renal protection. Histopathological examination indicated that TSDN increased liver glycogen, decreased the production of lipid vacuoles and lightened liver damage. Further investigation showed that TSDN down-regulated the protein expressions of NF-κB, GRP78, ATF6, eIF2 and the levels of MAPK phosphorylation and up-regulated the protein expressions of IRS-1, GLUT-4, p-Akt and p-AMPK. In addition, TSDN obviously decreased the gene expressions of TNF-a, IL-6, PEPCK, G6Pase, GSK-3β and GSK-3β activity, and increased the gene expressions of PFK, PK and GK activity. These findings show the anti-diabetic activity of total saponins from D. nipponica Makino, which should be developed as a new potent drug for treatment of diabetes mellitus in future.
The crude powder of the fruit of Arctium lappa L. (ALF) has previously been reported to attenuate experimental colitis in mice. But, its main effective ingredient and underlying mechanisms remain to be identified. In this study, ALF was extracted with ethanol, and then successively fractionated into petroleum ether, ethyl acetate, n-butanol and water fraction. Experimental colitis was induced by dextran sulfate sodium (DSS) in mice. Among the four fractions of ALF, the ethyl acetate fraction showed the most significant inhibition of DSS-induced colitis in mice. The comparative studies of arctigenin and arctiin (the two main ingredients of ethyl acetate fraction) indicated that arctigenin rather than arctiin could reduce the loss of body weight, disease activity index and histological damage in the colon. Arctigenin markedly recovered the loss of intestinal epithelial cells (E-cadherin-positive cells) and decreased the infiltration of neutrophils (MPO-positive cells) and macrophages (CD68-positive cells). Arctigenin could down-regulate the expressions of TNF-α, IL-6, MIP-2, MCP-1, MAdCAM-1, ICAM-1 and VCAM-1 at both protein and mRNA levels in colonic tissues. Also, it markedly decreased the MDA level, but increased SOD activity and the GSH level. Of note, the efficacy of arctigenin was comparable or even superior to that of the positive control mesalazine. Moreover, it significantly suppressed the phosphorylation of MAPKs and the activation of NF-κB, including phosphorylation of IκBα and p65, p65 translocation and DNA binding activity. In conclusion, arctigenin but not arctiin is the main active ingredient of ALF for attenuating colitis via down-regulating the activation of MAPK and NF-κB pathways.
Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC50 values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.
The effect of the total saponins from Rosa laevigata Michx fruit (RLTS) against acetaminophen (APAP)-induced liver damage in mice was evaluated in the present paper. The results showed that RLTS markedly improved the levels of liver SOD, CAT, GSH, GSH-Px, MDA, NO and iNOS, and the activities of serum ALT and AST caused by APAP. Further research confirmed that RLTS prevented fragmentation of DNA and mitochondrial ultrastructural alterations based on TdT-mediated dUTP nick end labeling (TUNEL) and transmission electron microscopy (TEM) assays. In addition, RLTS decreased the gene or protein expressions of cytochrome P450 (CYP2E1), pro-inflammatory mediators (IL-1β, IL-4, IL-6, TNF-α, iNOS, Bax, HMGB-1 and COX-2), pro-inflammatory transcription factors (NF-κB and AP-1), pro-apoptotic proteins (cytochrome C, p53, caspase-3, caspase-9, p-JNK, p-p38 and p-ERK), and increased the protein expressions of Bcl-2 and Bcl-xL. Moreover, the gene expression of IL-10, and the proteins including LC3, Beclin-1 and Atg5 induced by APAP were even more augmented by the extract. These results demonstrate that RLTS has hepatoprotective effects through antioxidative action, induction of autophagy, and suppression of inflammation and apoptosis, and could be developed as a potential candidate to treat APAP-induced liver damage in the future.
Dentin, the predominant mineralized tissue of the tooth, comprises an extracellular matrix of collagen and a heterogeneous mixture of non-collagenous components, many of which have cellular signaling properties. These properties may be important in signaling stem cell involvement in tissue regeneration following injury and the present study investigates their morphogenic effects on differentiation of Bone Marrow Stromal Stem Cells (BMMSCs) in vitro. Non-collagenous dentin matrix proteins (DMPs) were isolated from healthy human teeth and their effects on BMMSCs behavior examined during in vitro culture. In vitro, DMPs enhanced alkaline phosphatase activity and mineralization in BMMSCs cultures as well as increasing the expression of dentinogenic and osteogenic differentiation markers (including runt-related transcription factor 2, osterix, bone sialoprotein, dentin sialophosphoprotein and osteocalcin) at both transcript and protein levels, with 10 μg/mL DMPs being the optimal stimulatory concentration. Expression of phosphor-ERK/phosphor-P38 in BMMSCs was up-regulated by DMPs and, in the presence of the ERK1/2- and p38-specific inhibitors, the differentiation of BMMSCs was inhibited. These data indicate that DMPs promote the dentinogenic/osteogenic differentiation of BMMSCs via the ERK/p38 MAPK pathways.
The present study was to investigate the effects and possible mechanisms of the total saponins from Dioscorea nipponica Makino (TSDN) against CCl₄-induced hepato-toxicity in mice. The mice were orally administrated with TSDN for seven days and then given CCl₄ (0.3%, 10 ml/kg i.p.). The results showed that TSDN significantly attenuated the activities of ALT and AST, consistent with hematoxylin-eosin staining. The ALP levels and relative liver weight were significantly decreased by TSDN compared with model group. Moreover, TSDN dramatically decreased MDA, iNOS and NO levels, while the levels of GSH, GSH-Px and SOD were increased. Further investigations showed that TSDN inhibited CCl₄-induced metabolic activation and CYP2E1 expression, down-regulated the levels of MAPKs phosphorylation, NF-κB, HMGB1, COX-2 as well as effectively suppressed the expressions of Caspase-3, Caspase-9, PARP and Bak. Quantitative real-time PCR assay demonstrated that TSDN obviously decreased the gene expressions of TNF-a, IL-1β, IL-6, IL-10, Fas, FasL, Bax as well as modulated Bcl-2 mRNA level. This is the first time to report the protective actions of the TSDN against CCl₄-induced liver damage in mice through suppression of inflammation and apoptosis. This natural product should be developed as a new drug for treatment of liver injury in future.
Apoptosis of microvascular endothelial cells plays a crucial role in the progression of various lung diseases and triggers microcirculatory disorder and organ dysfunction. LPS, an outer membrane component of Gram-negative bacteria, is one of the major virulence factors for lung diseases. Recent studies have shown that the Rho/Rho kinase (ROCK) pathway plays an important role in the regulation of apoptosis, inflammatory cell migration and chemokine production in various cell types and animal models. We therefore undertake this study to investigate the inhibitory effect of fasudil, a potent and selective inhibitor of ROCK, on LPS-induced apoptosis of rat pulmonary microvascular endothelial cells (PMVECs). The results suggested that fasudil effectively prevented LPS-induced injury of rat PMVECs, as determined by MTT assay, LDH activity assay, apoptosis and western blot analysis of apoptosis-related proteins Bcl-2 and Bax. Furthermore, the mechanisms underlying the protective effect were evaluated. We found that LPS-induced MYPT-1 phosphorylation was markedly suppressed by fasudil. Moreover, fasudil pretreatment obviously inhibited the activation of JNK and p38 MAPKs induced by LPS, whereas that of ERK1/2 was not affected by fasudil. In addition, inhibiting the JNK and p38 pathways by SP600125 and SB203580 respectively attenuated the LPS-induced apoptosis and regulated the expression of apoptosis-related proteins Bcl-2 and Bax. Taken together, these results demonstrate that fasudil exerts an anti-apoptotic effect in rat PMVECs, which is mediated by the inhibition of Rho/ROCK and its downstream JNK and p38 MAPKs.
In this study, the anticancer effect of LW-214, a newly synthesized flavonoid, against MCF-7 human breast cancer cells and the underlying mechanisms were investigated. LW-214 triggered the mitochondrial apoptotic pathway by increasing Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (ΔΨm) and caspase-9 activation, degradation of poly (ADP-ribose) polymerase (PARP), cytochrome c (Cyt c) release and apoptosis-inducing factor (AIF) transposition. Further research revealed that both the reactive oxygen species (ROS) generation and the apoptosis signal regulating kinase 1 (ASK1) activation by LW-214 were induced by down-regulating the thioredoxin-1 (Trx-1) expression. The ROS elevation and ASK1 activation induced a sustained phosphorylation of c-Jun N-terminal kinase (JNK), while SP600125, as known as JNK inhibitor, almost reversed LW-214-induced apoptosis in MCF-7 cells. Overexpression of Trx-1 in MCF-7 cells attenuated LW-214-mediated apoptosis as well as the JNK activation and reversed the expression of mitochondrial apoptosis-related protein. Accordingly, the in vivo study showed that LW-214 exhibited a potential antitumor effect in BALB/c species mice inoculated MCF-7 tumor with low systemic toxicity, and the mechanism was the same as in vitro study. Taken together, these findings indicated that LW-214 may down-regulated Trx-1 function, causing intracellular ROS generation and releasing the ASK1, and lead to JNK activation, which consequently induced the mitochondrial apoptosis in vitro and in vivo.
Multiple studies have indicated that selective cyclooxygenase-2 (COX-2) inhibitors possess clinically chemopreventive and preclinically anticancer activities. Their long-term use, however, may be limited by the cardiovascular toxicity. This study tried to investigate whether an apple oligogalactan (AOG) could enhance the growth inhibitory effect of celecoxib on colorectal cancer. Caco-2 and HT-29 cell lines were exposed to different concentrations of AOG (0-1 g/L), celecoxib (0-25 μmol/L), and their combination. COX-2 levels were assessed by reverse transcription PCR and Western blot. COX-2 activity was evaluated by measuring prostaglandin E2 concentration. A colitis-associated colorectal cancer (CACC) mouse model was used to determine the effect of the combination in vivo. AOG (0.1-0.5 g/L) could potentiate the inhibitory effect of physiologic doses of celecoxib (5 μmol/L) on cell growth and decrease COX-2 expressions both at RNA and protein levels. In vivo, the combination (2.5% AOG plus 0.04% celecoxib, w/w) prevented against CACC in mice effectively. Our data indicate that AOG could potentiate the growth inhibitory effect of celecoxib on colorectal cancer both in vitro and in vivo through influencing the expression and function of COX-2 and phosphorylation of MAPKs, which suggests a new possible combinatorial strategy in colorectal cancer therapy.
Our previous studies have shown that dioscin has protective effect against liver injury. However, the action of the compound against ethanol-induced liver injury is still unknown. In the present paper, ethanol-induced acute and chronic liver damage rat models were used, and the results showed that dioscin significantly alleviated liver steatosis, reduced the levels of alanine aminotransferase, aspartate aminotransferase, total triglyceride (TG), total cholesterol and malondialdehyde, and increased the levels of high-density lipoprotein, superoxide dismutase, glutathione and glutathione peroxidase. Transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays showed that dioscin prevented mitochondrial ultrastructural alterations and apoptosis caused by ethanol. In addition, dioscin significantly inhibited ethanol-induced cytochrome P450 2E1 activation, down-regulated the levels of mitogen-activated protein kinases phosphorylation, inhibited the expressions of nuclear factor kappa B, glucose regulated protein 78, activating transcription factor 6 and alpha subunit of translation initiation factor 2 to attenuate oxidative damage, decreased the expressions of tumor necrosis factor alpha and interleukin-6, and down-regulated the expressions of apoptosis-related proteins including p53, caspase-3, caspase-9, poly (ADP-ribose)-polymerase and cytokeratin-18. Further investigation indicated that dioscin markedly increased the expressions of peroxisome proliferators-activated receptor α and its target genes including medium-chain acyl-CoA dehydrogenase, carnitine palmitoyl-CoA transferase I and acyl-CoA oxidase to advance fatty acid β-oxidation, up-regulated the expressions of acyl-CoA synthetase long-chain family member 1, acyl-CoA synthetase long-chain family member 5, alpha-aminoadipic semialdehyde dehydrogenase and acyl-CoA dehydrogenase to promote fatty acid metabolism, and down-regulated the expressions of glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase 1 and diacylglycerol acyltransferase 2 to accelerate TG synthesis. However, dioscin had no effects on the expressions of sterol regulatory element-binding protein-1c, fatty acid synthase, acetyl-CoA carboxylase 1 and stearoyl-CoA desaturase 1 associated with fatty acid synthesis. In conclusion, dioscin shows excellent protective effect against ethanol-induced liver injury through ameliorating ethanol-induced oxidative stress, mitochondrial function, inflammatory cytokine production, apoptosis and liver steatosis, which should be developed as a new drug for the treatment of ethanol-induced liver injury in the future.
The RNA-binding protein Musashi2 (Msi2) has been identified as a master regulator within a variety of stem cell populations via the regulation of translational gene expression. A recent study has suggested that Msi2 is strongly expressed in leukemic cells of acute myeloid leukemia patients, and elevated Msi2 is associated with poor prognosis. However, the potential role of Msi2 in leukemogenesis is still not well understood. Here, we investigated the effect of Msi2 knockdown on the biological properties of leukemic cells. High expression of Msi2 was found in K562 and KG-1a leukemic cell lines, and low expression was observed in the U937 cell line. We transduced K562 cells with two independent adenoviral shRNA vectors targeting Msi2 and confirmed knockdown of Msi2 at the mRNA and protein levels. Msi2 silencing inhibited cell growth and caused cell cycle arrest by increasing the expression of p21 and decreasing the expression of cyclin D1 and cdk2. In addition, knockdown of Msi2 promoted cellular apoptosis via the upregulation of Bax and downregulation of Bcl-2 expression. Furthermore, Msi2 knockdown resulted in the inactivation of the ERK/MAPK and p38/MAPK pathways, but no remarkable change in p-AKT was observed. These data provide evidence that Msi2 plays an important role in leukemogenesis involving the MAPK signaling pathway, which indicates that Msi2 may be a novel target for leukemia treatment.
Alfa-calendic acid and β-calendic acid, geometric and positional isomers of linolenic acid were previously shown to possess potent anticancer properties. In this study, we found that α-calendic acid and β-calendic acid could induce apoptosis and suppress invasion of human choriocarcinoma JEG-3 cells in vitro. Treatment with α-calendic acid and β-calendic acid significantly increased oxidative stress in human choriocarcinoma JEG-3 cells detected by the level of reactive oxygen species (ROS), lipid peroxidation production malondialdehyde (MDA), glutathione (GSH) and the effects of antioxidants NAC and α-tocopherol. Furthermore, oxidative stress activated the phosphorylation of p38MAPK. SB203580, a selective p38MAPK inhibitor, blocked the apoptosis induced by α-calendic acid and β-calendic acid by upregulating Bcl-2/Bax ratio and inhibition of the activation of Caspase-3 and Caspase-9. SB20350 also partially abrogated the cell invasion effects of α-calendic acid and β-calendic acid. These results suggested that α-calendic acid and β-calendic acid induced apoptosis and inhibited invasion in JEG-3 cells by activation of oxidative stress pathways and subsequent activation of P38MAPK.
Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer-related mortality in developed countries. Many ingredients of apples have been proven to have anti-inflammatory and anti-carcinogenic characteristics, and show benefits for CRC prevention. The aim of this study, therefore, was to evaluate inhibitory effect of an apple oligogalactan (AOG) on pro-inflammatory endotoxin lipopolysaccharide (LPS)-activated human colon carcinoma cells HT-29 and SW-620 and investigate the possible mechanisms. The two cell lines were pretreated with AOG (0.1-1 g/L) for 30 min and then treated with 10 μg/mL LPS. Real time PCR, Western blot, electrophoretic mobility shift assay (EMSA), and ELISA were used to detect the expression and activity of cyclooxygenase-2 (COX-2), NF-κB and MAPKs pathways. AOG significantly inhibited the expression and activity of COX-2 in LPS-activated human colon carcinoma cells HT-29 and SW-620. The mechanisms of AOG-suppressed COX-2 expression may be through inhibiting the phosphorylation of MAPKs and the activation of NF-κB and AP-1. These data may provide another molecular basis for understanding how apples act to prevent CRC and indicate that AOG may be useful for treatment of colitis and prevention of carcinogenesis.
Diosgenin (Dio), a major active component of steroidal sapogenin of the traditional Chinese herb Dioscorea zingiberensis C.H.Wright, shows various activities including anti-inflammatory, anti-thrombotic activities, anti-cancer properties etc. In the present study, we found that diosgenin significantly suppressed the phosphorylation of lung NF-κB p50/p65 and MAPK/p38 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice, when given orally at doses of 0.1, 1.0 and 10mg/kg 1h prior to LPS challenge (30 mg/kg, intravenous injection). Moreover, diosgenin attenuated the lung histopathological changes such as pulmonary edema, coagulation and infiltration of inflammatory cells. In addition, diosgenin significantly decreased the lung wet to dry weight (W/D) ratio and nitrite/nitrate content at three doses, and also markedly inhibited LPS-induced body temperature decrease and nitrite/nitrate elevation in plasma. Besides, diosgenin could significantly suppress activation of NF-κB p65/p50, p38 and expression of inducible nitric oxide synthase (iNOS) in LPS-induced THP-1 cells. Our findings indicate the potential application of diosgenin for ALI treatment.
The adhesion of monocytes to activated vascular endothelial cells is a critical event in the initiation of atherosclerosis. Adhesion is mediated by oxidized low-density lipoprotein (ox-LDL) which up-regulates inflammatory markers on endothelial cells. Here we report that (±) 7, 8-dihydroxy-3-methyl-isochromanone-4 (XJP-1), an inhibitor of ox-LDL-induced adhesion of monocytes to endothelial cells blocks cellular functions which are associated with adhesion. We show that XJP-1 down-regulates ox-LDL-induced over-expression of adhesion molecules (ICAM-1 and VCAM-1) in a dose-dependent manner in human umbilical vein endothelial cells (HUVECs), attenuates ox-LDL-induced up-regulation of low-density lipoprotein receptor (LOX)-1, decreases generation of reactive oxygen species (ROS), blocks translocation of nuclear factor-kappa B (NF-κB) activity, and prevents activation of c-Jun N-terminal kinase (JNK)/p38 pathways in endothelial cells. These findings suggest that XJP-1 may attenuate ox-LDL-induced endothelial adhesion of monocytes by blocking expression of adhesion molecules through suppressing ROS/NF-κB, JNK and p38 pathways.
Cardiac hypertrophy is a response of the myocardium to increased workload and is characterised by an increase of myocardial mass and an accumulation of extracellular matrix (ECM). As an ECM protein, an integrin ligand, and an angiogenesis inhibitor, all of which are key players in cardiac hypertrophy, mindin is an attractive target for therapeutic intervention to treat or prevent cardiac hypertrophy and heart failure. In this study, we investigated the role of mindin in cardiac hypertrophy using littermate Mindin knockout (Mindin ( -/- )) and wild-type (WT) mice. Cardiac hypertrophy was induced by aortic banding (AB) or angiotensin II (Ang II) infusion in Mindin ( -/- ) and WT mice. The extent of cardiac hypertrophy was quantitated by echocardiography and by pathological and molecular analyses of heart samples. Mindin ( -/- ) mice were more susceptible to cardiac hypertrophy and fibrosis in response to AB or Ang II stimulation than wild type. Cardiac function was also markedly exacerbated during both systole and diastole in Mindin ( -/- ) mice in response to hypertrophic stimuli. Western blot assays further showed that the activation of AKT/glycogen synthase kinase 3β (GSK3β) signalling in response to hypertrophic stimuli was significantly increased in Mindin ( -/- ) mice. Moreover, blocking AKT/GSK3β signalling with a pharmacological AKT inhibitor reversed cardiac abnormalities in Mindin ( -/- ) mice. Our data show that mindin, as an intrinsic cardioprotective factor, prevents maladaptive remodelling and the transition to heart failure by blocking AKT/GSK3β signalling.
Keloid is a specific skin scar that expands beyond the boundaries of the original injury as it heals. The invasive nature of keloid and notable migratory activity of fibroblasts are a hallmark, which distinguishes keloids from other common scars. Madecassoside, a triterpenoid saponin occurring in Centella asiatica herbs, possesses unique pharmacological properties to enhance wound-healing and diminish keloid formation. However, the effects of madecassoside on the formation of keloid scars have been poorly understood. Here, we focused on the potential of madecassoside on the migration of keloid-derived fibroblasts (KFs) and its mechanism. Primary KF, originating from human earlobe keloids, were purified and cultured, and then treated with madecassoside (10, 30, and 100μM). In both transwell migration assays and scratch-wound-closure assays, KF migration was considerably suppressed by madecassoside pretreatment. Furthermore, KFs treated with madecassoside showed decreased F-actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. By Western blot analysis, madecassoside was shown to remarkably attenuate the phosphorylation of cofilin, p38 MAPK and phosphatidylinositol-3-kinase (PI3K)/AKT signaling, but only exhibited a minor effect on MMP-13 and little effect on ERK1/2 phosphorylation. It was concluded that madecassoside could be of great use in the treatment and/or prevention of hypertrophic scars and keloids.
Insulin-like growth factor 1 (IGF-1) is a potent mitogenic protein which can enhance the osteogenic differentiation of periodontal ligament (PDL) fibroblasts. However, it remains unclear whether IGF-1 can stimulate the osteogenic differentiation and osteogenesis of human periodontal ligament stem cells (PDLSCs). In this study, STRO-1(+) PDLSCs were isolated from human PDL tissues, treated with IGF-1, and their osteogenic capacity was investigated in vitro and in vivo. Dimethyl-thiazol-diphenyl tetrazolium bromide assay and flow cytometry results demonstrated that 10-200 ng/mL IGF-1 can stimulate the proliferation ability of PDLSCs and 100 ng/mL is the optimal concentration. Exogenous IGF-1 can modify the ultrastructure, enhance the alkaline phosphatase activity, the mineralization ability of PDLSCs, and increase the expression of osteogenic markers (runt-related transcription factor 2, osterix, and osteocalcin) at mRNA and protein levels. In vivo transplantation illustrated that IGF-1 treated implants generated more mineralized tissues, and presented stronger expression of RUNX2, OSX, and OCN than control group. Moreover, the expression of phosphor-ERK and phosphor-JNK in these stem cells was upregulated by IGF-1, indicating that MAPK signaling pathway was activated during the osteogenic differentiation of PDLSCs mediated by IGF-1. Together, the results showed that IGF-1 can promote the osteogenic differentiation and osteogenesis of STRO-1(+) PDLSCs via ERK and JNK MAPK pathway, suggesting that IGF-1 is a potent agent for stem cell-based periodontal tissue regeneration.
In the past years, free fatty acids (FFAs) and obesity have been reported to play an important role in cancer development. Palmitic acid (PA) is the most prevalent saturated FFA in circulation. However, the mechanism underlying the effect of PA on cell proliferation is still to be elucidated. In this report, we, for the first time, investigate the signaling pathway in human normal hepatocytes (QZG) responsible for PA-induced proliferation. The results demonstrate that PA promotes cell cycle progression, accelerates cell proliferation, and induces a transient and sequential activation of a series of kinases. The employment of several inhibitors and antioxidants indicates that a ROS-induced stress-sensitive p38 MAPK/ERK-Akt cascade plays a critical role in the regulation of PA on cell cycle and cell proliferation. Moreover, PA dose and time dependently activates Nrf2 and this activation relies on ROS-induced stimulation of p38 MAPK/ERK-Akt signaling, demonstrating that Nrf2 activation may be associated with the regulation of PA on cell cycle transition and proliferation. In conclusion, our study elucidates the importance of PA metabolism on cell proliferation, and suggests that PA stimulates hepatocyte proliferation through activating the ROS-p38 MAPK/ERK-Akt cascade which is intersected with the activation of Nrf2 and that the effect of ROS on signal transduction is in a dose- and time-dependent manner. All the above noted provide a new clue for the central role of ROS in cell proliferation and tumorigenesis.
Chronic hyperglycemia is the hallmark of diabetes and its complication. High glucose-induced excessive reactive oxygen species (ROS) production has been considered to play an important role in the development of diabetes. However, the influence of high glucose on the liver remains to be clarified. Rosiglitazone (RSG) is a member of thiazolidinediones (TDZs) family, which is the ligand of the of nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPARγ), being used clinically for the treatment of type 2 diabetic patients through their insulin-sensitizing effect. In the present study, we investigated the cytotoxicity of high glucose in QZG hepatocytes and evaluated the protective effect of RSG. The results showed that high glucose significantly reduced cell viability through generation of ROS via activation of PKC, which was inhibited by RSG. On the one hand, RSG notably inhibited the activation of PKC induced by high glucose independent of PPARγ, leading to the decrease of ROS generation. On the other hand, RSG notably increased the expression of key antioxidant transcription factor Nrf2 and antioxidant enzyme HO-1 in a PPARγ-dependent manner, leading to the elimination of excessive ROS. In addition, RSG also inhibited the decrease of COX-2 expression induced by high glucose through activating PPARγ. Furthermore, the activation of Akt and MAPKs was involved in the effect of RSG on Nrf2, HO-1 and COX-2. In summary, our study supports the hypothesis that RSG protect hepatocytes from high glucose-induced toxicity through PPARγ-dependent and PPARγ-independent pathways.
Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia. Although the clear mechanisms of DM and insulin resistance are still to be cleared, it has been well documented that reactive oxygen species (ROS) play a pivotal role in DM and multiple types of insulin resistance. For the past few years, natural substances have been shown to have the potential to treatment DM. Attention has been especially focused on plants rich in triterpenoids, which generally show antioxidant and antiglycation effect. In our previous studies, it was shown that oleanolic acid (OA), a natural triterpenoid and an aglycone of many saponins, is a potent antioxidant acting as not only a free radical-scavenger through direct chemical reactions but also as a biological molecule, which may enhance the antioxidant defenses. The present study aimed to investigate the potential antidiabetic effect of OA. Oleanolic acid showed a significant blood glucose-lowering and weight-losing effect in diabetic animals induced by streptozotocin (STZ). In the insulin resistant model, it was also shown that OA may promote insulin signal transduction and inhibit oxidative stress-induced hepatic insulin resistance and gluconeogenesis, in which process the phosphorylation of ERK and the protective effect on mitochondrial function may be involved. These findings may significantly better the understanding of the pharmacological actions of OA and advance therapeutic approaches to DM.
AIMS:
The radiation-induced bystander effect (RIBE) has potential implications in cancer risks from space particle radiation; however, the mechanisms underlying RIBE are unclear. The role of the MAPK pathway in the RIBEs of different linear energy transfer (LET) was investigated.
MAIN METHODS:
Human macrophage U937 cells were irradiated with γ-rays or carbon ions and then co-cultured with nonirradiated HMy2.CIR (HMy) lymphocytes for different periods. The activation of MAPK proteins and the generation of intracellular nitric oxide (NO) and reactive oxygen species (ROS) in the irradiated U937 cells were measured. Micronuclei (MN) formation in the HMy cells was applied to evaluate the bystander damage. Some U937 cells were pretreated with different MAPK inhibitors before irradiation.
KEY FINDINGS:
Additional MN formation was induced in the HMy cells after co-culturing with irradiated U937 cells, and the yield of this bystander MN formation was dependent on the co-culture period with γ-ray irradiation but remained high after 1h of co-culture with carbon irradiation. Further investigations disclosed that the time response of the RIBEs had a relationship with LET, where ERK played a different role from JNK and p38 in regulating RIBEs by regulating the generation of the bystander signaling factors NO and ROS.
SIGNIFICANCE:
The finding that the RIBE of high-LET radiation could persist for a much longer period than that of γ-rays implies that particle radiation during space flight could have a high risk of long-term harmful effects. An appropriate intervention targeting the MAPK pathway may have significant implications in reducing this risk.
Copyright © 2015 Elsevier Inc. All rights reserved.
SCOPE:
Flavonoids have well-known antioxidant, anti-inflammatory, and anti-cancer activities. Isoflavone genistein is considered a potent antioxidant agent against oxidative stress. Although several mechanisms have been proposed, a clear antioxidant mechanism of genistein is still remained to be answered.
METHODS AND RESULTS:
In this study, we focused on the concerted effects on expression of Nrf2 and phase II enzyme pathway components. Transient transfection assays, RT-PCR and immunoblot analysis were performed to study its molecular mechanisms of action. In Caco-2 cells, treatment with genistein markedly attenuated H(2)O(2) -induced peroxide formation; this amelioration was reversed by buthionine sulfoximine(GCLC inhibitor) and zinc protoporphyrin(HO-1 inhibitor). Genistein increased HO-1 and GCLC mRNA and protein expression. Genistein treatment activated the ERK1/2 and PKC signaling pathway; therefore increased Nrf2 mRNA and protein expression. The roles of the ERK1/2 and PKC signaling pathway were determined using PD98059 (ERK1/2 inhibitor) and GF109203X (PKC inhibitor) and RNA interference directed against Nrf2. Both inhibitors and siNrf2 abolished genistein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK1/2, PKC, and Nrf2 in inducing HO-1 and GCLC by genistein.
CONCLUSION:
Our studies show that genistein up-regulated HO-1 and GCLC expression through the EKR1/2 and PKC /Nrf2 pathways during oxidative stress.
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
AIM:
We investigated whether 8-dihydroxy-3-methyl-isochromanone (XJP-1), a novel angiotensin-converting enzyme inhibitor (ACEI), exhibited inhibitory activity to lipopolysaccharide (LPS)-accelerated vascular inflammation.
METHODS:
Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords and cultured. The direct effect of XJP-1 on the activation of endothelial cells was measured using MTT assay. Nitric oxide (NO) in the culture medium was measured using Griess method. The expression of cell adhesion molecules (ICAM-1 and VCAM-1) was determined by flow cytometry and RT-PCR. The protein expression levels of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein (MCP)-1, and endothelin-1 (ET-1) secretion were measured using ELISA. Quantitative analysis of eNOS, iNOS, inhibitory factor NF-κB (IκB) and MAPKs were determined using Western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined using immunofluorescence.
RESULTS:
XJP-1 significantly inhibited LPS-mediated endothelial cell dysfunction, as measured by NO production, iNOS expression, adhesion molecule (ICAM-1, VCAM-1) expression, and chemokine (TNF-α, MCP-1) production in vitro. It up-regulated eNOS expression in the same experimental setting. XJP-1 alone was found non-cytotoxic at the concentration up to 1000μM. The mechanistic investigations of XJP-1 suppression LPS-induced inflammation in HUVECs revealed that XJP-1 blocked NF-κB nuclear entry in an IκB-dependent manner, as well as inhibited MAPK activation induced by LPS. XJP-1 reduced endothelin-1 secretion and increased nitric oxide metabolite production by HUVECs. However, the effect of XJP-1 on nitric oxide and endothelin-1 metabolite production is mediated by the activation of bradykinin B(2) receptor being counteracted, at least in part, by a specific antagonist.
CONCLUSION:
XJP-1 inhibited LPS-induced cytotoxicity and inflammatory response. The mechanism underlying this protective effect might be related to the inhibition of MAPK and NF-κB signaling pathway activation, suggesting the potential inhibition of the atherosclerotic process by suppressing the expression of chemoattractant molecules and monocyte adhesion. XJP-1 also has an effect in improving endothelin-1 through activating bradykinin B(2) receptor. These findings indicated that XJP-1 is potentially a novel therapeutic candidate for the treatment of atherosclerosis.
Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
OBJECTIVE AND DESIGN:
The anti-inflammatory effect of methyl-1-hydroxy-2-naphthoate (MHNA), a novel naphthol derivative, was evaluated in the lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages.
MATERIALS AND METHODS:
The release of nitric oxide (NO), interleukin-1beta (IL-1β) and interleukin-6 (IL-6) were detected by the Griess reagent and ELISA methods. The protein expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were examined by Western blotting. The mRNA expressions of IL-1β, IL-6, iNOS and COX-2 were determined by real-time PCR. Activation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) pathways were detected by Western blotting, reporter gene assay and electrophoretic mobility shift assay.
RESULTS:
MHNA significantly inhibited the release of NO, IL-1β and IL-6 as well as the protein expression of iNOS and COX-2 in LPS-stimulated macrophages. It also inhibited the mRNA expression of iNOS, COX-2, IL-1β and IL-6. Further studies indicated that MHNA inhibited LPS-induced increases in NF-κB DNA-binding activity and NF-κB transcriptional activity as well as IκB-α degradation and NF-κB translocation in a dose-dependent manner. Meanwhile, the activation of p38 MAPK and c-Jun N-terminal kinases (JNK) induced by LPS were decreased by MHNA.
CONCLUSIONS:
MHNA inhibits the LPS-induced inflammatory response in murine macrophages via suppression of NF-κB and MAPKs signaling pathways activation.
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