Comparative proteome analysis of Tumor necrosis factor α-stimulated human Vascular Smooth Muscle Cells in response to melittin.

BACKGROUND: Bee venom has been used to relieve pain and to treat inflammatory diseases, including rheumatoid arthritis, in humans. To better understand the mechanisms of the anti-inflammatory and anti-atherosclerosis effect of bee venom, gel electrophoresis and mass spectrometry were used to identify proteins whose expression was altered in human Vascular Smooth Muscle Cells (hVSMCs) stimulated by tumor necrosis factor alpha after 12 h in the presence of melittin. RESULTS: To obtain valuable insights into the anti-inflammatory and anti-atherosclerosis mechanisms of melittin, two-dimensional (2-D) gel electrophoresis and MALDI-TOF/TOF were used. The proteome study, we showed 33 significant proteins that were differentially expressed in the cells treated with tumor necrosis factor alpha and melittin. Thirteen proteins were significantly increased in the cells treated with tumor necrosis factor alpha, and those proteins were reduced in the cells treated with melittin. Five of the proteins that showed increased expression in the cells treated with tumor necrosis factor alpha are involved in cell migration, including calreticulin, an essential factor of development that plays a role in transcription regulation. The proteins involved in cell migration were reduced in the melittin treated cells. The observed changes in the expression of GRP75, prohibitin, and a select group of other proteins were validated with reverse transcribed-PCR. It was confirmed that the observed change in the protein levels reflected a change in the genes level. In addition, the phosphorylation of EGFR and ERK was validated by analyzing the protein pathway. CONCLUSION: Taken together, these data established that the expression of some proteins was significantly changed by melittin treatment in tumor necrosis factor alpha stimulated the cells and provided insights into the mechanism of the melittin function for its potential use as an anti-inflammatory agent.

Ascochlorin inhibits growth factor-induced HIF-1α activation and tumor-angiogenesis through the suppression of EGFR/ERK/p70S6K signaling pathway in human cervical carcinoma cells.

Ascochlorin, a non-toxic prenylphenol compound derived from the fungus Ascochyta viciae, has been shown recently to have anti-cancer effects on various human cancer cells. However, the precise molecular mechanism of this anti-cancer activity remains to be elucidated. Here, we investigated the effects of ascochlorin on hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human epidermoid cervical carcinoma CaSki cells. Ascochlorin inhibited epidermal growth factor (EGF)-induced HIF-1α and VEGF expression through multiple potential mechanisms. First, ascochlorin selectively inhibited HIF-1α expression in response to EGF stimulation, but not in response to hypoxia (1% O(2)) or treatment with a transition metal (CoCl(2)). Second, ascochlorin inhibited EGF-induced ERK-1/2 activation but not AKT activation, both of which play essential roles in EGF-induced HIF-1α protein synthesis. Targeted inhibition of epidermal growth factor receptor (EGFR) expression using an EGFR-specific small interfering RNA (siRNA) diminished HIF-1α expression, which suggested that ascochlorin inhibits HIF-1α expression through suppression of EGFR activation. Finally, we showed that ascochlorin functionally abrogates in vivo tumor angiogenesis induced by EGF in a Matrigel plug assay. Our data suggest that ascochlorin inhibits EGF-mediated induction of HIF-1α expression in CaSki cells, providing a potentially new avenue of development of anti-cancer drugs that target tumor angiogenesis.

Progesterone produces antinociceptive and neuroprotective effects in rats with microinjected lysophosphatidic acid in the trigeminal nerve root.

BACKGROUND: In our present study, we studied the role of demyelination of the trigeminal nerve root in the development of prolonged nociceptive behavior in the trigeminal territory. RESULTS: Under anesthesia, the Sprague-Dawley rats were mounted onto a stereotaxic frame and 3 µL of lysophosphatidic acid (LPA, 1 nmol) was injected into the trigeminal nerve root to produce demyelination. This treatment decreased the air-puff thresholds, persisted until postoperative day 130, and then returned to the preoperative levels 160 days after LPA injection. The LPA-treated rats also showed a significant hyper-responsiveness to pin-prick stimulation. We further investigated the antinociceptive and neuroprotective effects of progesterone in rats undergoing demyelination of the trigeminal nerve root. Progesterone (8, 16 mg/kg/day) was administered subcutaneously, beginning on the operative day, for five consecutive days in the LPA-treated rats. Treatment with progesterone produced significant early anti-allodynic effects and delayed prolonged anti-allodynic effects. The expression of protein zero (P0) and peripheral myelin protein 22 (PMP22) were significantly down-regulated in the trigeminal nerve root on postoperative day 5 following LPA injection. This down-regulation of the P0 and PMP22 levels was blocked by progesterone treatment. CONCLUSIONS: These results suggest that progesterone produces antinociceptive effects through neuroprotective action in animals with LPA-induced trigeminal neuropathic pain. Moreover, progesterone has potential utility as a novel therapy for trigeminal neuropathic pain relief at an appropriate managed dose and is therefore a possible future treatment strategy for improving the recovery from injury.

Alpha-lipoic acid attenuates atherosclerotic lesions and inhibits proliferation of vascular smooth muscle cells through targeting of the Ras/MEK/ERK signaling pathway.

An infectious burden has been suggested to be associated with atherosclerosis in humans, based on the shared and underlying inflammatory responses during infection and atherosclerosis. However, the efficacy of anti-atherogenic drugs is yet to be tested against atherosclerosis in a scenario involving an infectious burden. We have examined alpha-lipoic acid (ALA) for anti-atherogenic effects in a hypercholesterolemic diet-induced atherosclerotic mouse model with inflammatory stimulation. C57BL/6 mice were fed with a hypercholesterolemic diet for 12 weeks to induce atherosclerosis. Lipopolysaccharide was intraperitoneally injected for the 1st week of study to simulate underlying infectious burden during development of atherosclerosis. ALA treatment alleviated atherosclerotic pathologies and reduced serum cholesterol and inflammatory cytokines. Consistently, atherosclerotic markers were improved by ALA treatment. In addition, ALA attenuated the proliferation and migration of vascular smooth muscle cells upon platelet-derived growth factor stimulation through the targeting of the Ras-MEK1/2-ERK1/2 pathway. This study demonstrates the efficacy of ALA on atherosclerosis with immunological complication, by showing that ALA modulates multiple pathogenic aspects of atherosclerosis induced by a hypercholesterolemic diet with inflammatory stimulation consisting of hypercholesterolemia, inflammation and VSMC activation.

Ascochlorin suppresses TGF-ß1-induced PAI-1 expression through the inhibition of phospho-EGFR in rat kidney fibroblast cells.

Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor-ß1 (TGF-ß1), the growth factor involved in fibrosis, modulates ECM synthesis and accumulation. TGF-ß1 enhances the production of stimulators of ECM synthesis such as plasminogen activator inhibitor type 1 (PAI-1). As such, PAI-1 expression directly influences the proteolysis, invasion, and accumulation of ECM. It was shown in this study that ascochlorin, a prenylpenl antiobiotic, prevents the expression of profibrotic factors, such as PAI-1 and collagen type I, and that the TGF-ß1-induced PAI-1 promoter activity is inhibited by ascochlorin. Ascochlorin abolishes the phosphorylation of the EGFR-MEK-ERK signaling pathway to regulate the TGF-ß1-induced expression of PAI-1 without the inhibition of TßRII phosphorylation. Furthermore, the MEK inhibitor and EGFR siRNA block PAI-1 expression, and the Raf-1, MEK, and ERK signaling pathways for the regulation of PAI-1 expression. Ascochlorin suppresses the matrix metalloproteinases (MMPs) activity to activate the heparin-binding EGF-like growth factor (HB-EGF), to induce the phosphorylation of EGFR, and the MMPs inhibitor suppresses EGFR phosphorylation and the PAI-1 mRNA levels. These results suggest that ascochlorin prevents the expression of PAI-1 via the inhibition of an EGFR-dependent signal transduction pathway activated by MMPs.

Rg3-enriched red ginseng extract promotes lung cancer cell apoptosis and mitophagy by ROS production.

BACKGROUND: Red Ginseng has been used for many years to treat diseases. Ginsenoside Rg3 has documented therapeutic effects, including anticancer and anti-inflammatory activities. However, the anticancer effect of Rg3-enriched red ginseng extract (Rg3-RGE) and its underlying mechanisms have not been fully explored. We investigated whether Rg3-RGE plays an anti-tumor role in lung cancer cells. METHODS: To examine the effect of Rg3-RGE on lung cancer cells, we performed cell viability assays, flow cytometry, western blotting analysis, and immunofluorescence to monitor specific markers. RESULTS: Rg3-RGE significantly inhibited cell proliferation and induced mitochondria-dependent apoptosis. Furthermore, Rg3-RGE also increased expression of mitophagy-related proteins such as PINK1 and Parkin. In addition, treatment with Rg3-RGE and mitophagy inhibitors stimulated cell death by inducing mitochondria dysfunction. CONCLUSIONS: Rg3-RGE could be used as a therapeutic agent against lung cancer.

Protective effects of melittin on transforming growth factor-ß1 injury to hepatocytes via anti-apoptotic mechanism.

Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-ß1-induced apoptosis in hepatocytes. TGF-ß1-treated hepatocytes were exposed to low doses (0.5 and 1 µg/mL) and high dose (2 µg/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-ß1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-ß1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-ß1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-ß1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-ß1-mediated injury.

Tetracycline-dependent expression of the human erythropoietin gene in transgenic chickens.

A critical problem in the production of transgenic animals is the uncontrolled constitutive expression of the foreign gene, which occasionally results in serious physiological disorders in the transgenic animal. In this study, we report successful production of transgenic chickens that express the human erythropoietin (hEPO) gene under the control of a tetracycline-inducible promoter. A recombinant Moloney murine leukemia virus (MoMLV)-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of unincubated chicken embryos (stage X). Out of 198 injected eggs, 15 chicks hatched after 21 days of incubation and 14 hatched chicks expressed the vector-encoded hEPO gene when fed doxycycline, a tetracycline derivative, without any significant physiological dysfunctions. The expression of hEPO reverted to the pre-induction state by removing doxycycline from the diet. The biological activity of the hEPO produced in the transgenic chickens was comparable to commercially available CHO cell-derived hEPO. Successful germline transmission of the transgene was also confirmed in G1 transgenic chicks produced from crossing G0 transgenic roosters with non-transgenic hens. Tetracycline-inducible expression of the hEPO gene was also confirmed in the blood and eggs of the transgenic chickens.

Ascofuranone inhibits epidermal growth factor-induced cell migration by blocking epithelial-mesenchymal transition in lung cancer cells.

Ascofuranone, an isoprenoid antibiotic initially purified from a culture broth of Ascochyta viciae, has multiple anticancer effects. However, the impacts of ascofuranone on the epithelial-mesenchymal transition (EMT) and epidermal growth factor (EGF)-induced effects on human lung cancer cell lines have not been previously reported. Here, we show that ascofuranone exerts its anticancer effects by inhibiting the EGF-induced EMT and cell migration in human lung cancer cell lines. Ascofuranone significantly inhibited EGF-induced migration and invasion by lung cancer cells, and suppressed EGF-induced morphologic changes by regulating the expression of EMT-associated proteins. In addition, ascofuranone upregulated E-cadherin, and downregulated fibronectin, vimentin, Slug, Snail, and Twist. Inhibition of ERK/AKT/mTOR promoted EGF-induced E-cadherin downregulation and inhibited EGF-induced vimentin upregulation in response to ascofuranone, implying that inhibition of the EGF-induced EMT by ascofuranone was mediated by the ERK and AKT/mTOR pathways. Inhibition of c-Myc suppressed EGF-induced vimentin upregulation, suggesting the involvement of c-Myc. Collectively, these findings suggest that ascofuranone inhibits tumor growth by blocking the EGF-induced EMT through a regulatory mechanism involving ERK, AKT/mTOR, and c-Myc in lung cancer cells.

Bee Venom Suppresses EGF-Induced Epithelial-Mesenchymal Transition and Tumor Invasion in Lung Cancer Cells.

Bee venom of Apis mellifera is a traditional medicine in Asia. It has been used with promoting results for the treatment of pain, rheumatoid, and cancer disease. The purpose of this study was to investigate the effects of bee venom on epidermal growth factor (EGF)-induced epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) and determine possible signaling pathway affected in EGF-induced EMT in A549 cells. Bee venom inhibited EGF-induced F-actin reorganization and cell invasion, and suppressed EGF-induced EMT, processes associated with tumor metastasis in NSCLC. Bee venom enhanced the upregulation of E-cadherin and the downregulation of vimentin and inhibited EGF-induced ERK, JNK, FAK, and mTOR phosphorylation in A549 cells. However, the inhibition of JNK phosphorylation by bee venom was not related to the inhibitory effects of EMT. Furthermore, we found that bee venom suppressed the EMT-related transcription factors ZEB2 and Slug by blocking EGF-induced ERK, FAK and mTOR phosphorylation. Bee venom inhibits EGF-induced EMT by blocking the phosphorylation of ERK, FAK, and mTOR, resulting in the suppression of ZEB2 and Slug. These data suggest bee venom as a potential antimetastatic agent for NSCLC.

Generation of transgenic chickens that produce bioactive human granulocyte-colony stimulating factor.

We report here the generation of transgenic chickens that produce human granulocyte-colony stimulating factor (hG-CSF) using replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G). The recombinant retrovirus was injected beneath the blastoderm of nonincubated chicken embryos (stage X). Out of 140 injected eggs, 17 chicks hatched after 21 days of incubation and all hatched chicks were found to express vector-encoded hG-GSF gene. The biological activity of the recombinant hG-CSF was significantly higher than its commercially derived E. coli-derived counterpart. Successful germline transmission of the transgene was also confirmed in G(1) transgenic chicks produced from the cross of Go transgenic roosters with nontransgenic hens, but most of the G(1) progeny were dead within 1 month of hatching.

Disulfiram suppresses invasive ability of osteosarcoma cells via the inhibition of MMP-2 and MMP-9 expression.

Cancer cells, characterized by local invasion and distant metastasis, are very much dependent on the extracellular matrix. The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. In this study, we reported the effects of disulfiram, a clinically used anti-alcoholism drug, on tumor invasion suppression, as well as its effects on the activity of MMP-2 and MMP-9 in human osteosarcoma cells (U2OS). Disulfiram has been used for alcohol aversion therapy. However, recent reports have shown that disulfiram may have potential in the treatment of human cancers. Herewith, we showed that the anti-tumor effects of disulfiram, in an invasion assay using U2OS cells and that disulfiram has a type IV collagenase inhibitory activity that inhibits expression of genes and proteins responsible for both cell and non-cell mediated invasion on pathways. In conclusion, disulfiram inhibited expression of MMP-2 and MMP-9 and it regulated the invasion of human osteosarcoma cells. These observations raise the possibility of disulfiram being used clinical for the inhibition of cancer invasion.

Apamin suppresses biliary fibrosis and activation of hepatic stellate cells.

Cholestatic liver disease is characterized by the progressive destruction of biliary epithelial cells (BECs) followed by fibrosis, cirrhosis and liver failure. Activated hepatic stellate cells (HSCs) and portal fibroblasts are the major cellular effectors of enhanced collagen deposition in biliary fibrosis. Apamin, an 18 amino acid peptide neurotoxin found in apitoxin (bee venom), is known to block Ca2+-activated K+ channels and prevent carbon tetrachloride-induced liver fibrosis. In the present study, we aimed to ascertain whether apamin inhibits biliary fibrosis and the proliferation of HSCs. Cholestatic liver fibrosis was established in mouse models with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Cellular assays were performed on HSC-T6 cells (rat immortalized HSCs). DDC feeding led to increased hepatic damage and proinflammtory cytokine levels. Notably, apamin treatment resulted in decreased liver injury and proinflammatory cytokine levels. Moreover, apamin suppressed the deposition of collagen, proliferation of BECs and expression of fibrogenic genes in the DDC-fed mice. In HSCs, apamin suppressed activation of HSCs by inhibiting the Smad signaling pathway. These data suggest that apamin may be a potential therapeutic target in cholestatic liver disease.

Anti-fibrotic Effects of Synthetic Oligodeoxynucleotide for TGF-ß1 and Smad in an Animal Model of Liver Cirrhosis.

Liver fibrosis is characterized by changes in tissue architecture and extracellular matrix composition. Liver fibrosis affects not only hepatocytes but also the non-parenchymal cells such as hepatic stellate cells (HSCs), which are essential for maintaining an intact liver structure and function. Transforming growth factor ß1 (TGF-ß1) is a multifunctional cytokine that induces liver fibrosis through activation of Smad signaling pathways. To improve a new therapeutic approach, synthetic TGF-ß1/Smad oligodeoxynucleotide (ODN) was used to suppress both TGF-ß1 expression and Smad transcription factor using a combination of antisense ODN and decoy ODN. The aims of this study are to investigate the anti-fibrotic effects of TGF-ß1/Smad ODN on simultaneous suppressions of both Smad transcription factor and TGF-ß1 mRNA expression in the hepatic fibrosis model in vitro and in vivo. Synthetic TGF-ß1/Smad ODN effectively inhibits Smad binding activity and TGF-ß1 expression. TGF-ß1/Smad ODN attenuated the epithelial mesenchymal transition (EMT) and activation of HSCs in TGF-ß1-induced AML12 and HSC-T6 cells. TGF-ß1/Smad ODN prevented the fibrogenesis and deposition of collagen in CCl4-treated mouse model. Synthetic TGF-ß1/Smad ODN demonstrates anti-fibrotic effects that are mediated by the suppression of fibrogenic protein and inflammatory cytokines. Therefore, synthetic TGF-ß1/Smad ODN has substantial therapeutic feasibility for the treatment of liver fibrotic diseases.

Delphinidin inhibits angiogenesis through the suppression of HIF-1α and VEGF expression in A549 lung cancer cells.

Delphinidin, a polyphenol that belongs to the group of anthocyanidins and is abundant in many pigmented fruits and vegetables, possesses important antioxidant, anti­inflammatory, anti-mutagenic and anticancer properties. In the present study, we investigated the inhibitory effects of delphinidin on vascular endothelial growth factor (VEGF) expression, an important factor involved in angiogenesis and tumor progression, in A549 human lung cancer cells. Delphinidin inhibited CoCl2- and epidermal growth factor (EGF)-induced VEGF mRNA expression and VEGF protein production. Delphinidin also decreased CoCl2- and EGF-stimulated expression of hypoxia­inducible factor (HIF)­1α, which is a transcription factor of VEGF. Delphinidin suppressed CoCl2- and EGF-induced hypoxia­response element (HRE) promoter activity, suggesting that the inhibitory effects of delphinidin on VEGF expression are caused by the suppression of the binding of HIF-1 to the HRE promoter. We also found that delphinidin specifically decreased the CoCl2- and EGF-induced HIF-1α protein expression by blocking the ERK and PI3K/Akt/mTOR/p70S6K signaling pathways, whereas the p38-mediated pathways were not involved. In animal models, EGF-induced new blood vessel formation was significantly inhibited by delphinidin. Therefore, our results indicate that delphinidin has a potentially new role in anti­angiogenic action by inhibiting HIF-1α and VEGF expression.

Apamin inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration through suppressions of activated Akt and Erk signaling pathway.

The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key process in the development of atherosclerosis lesions. Platelet-derived growth factor (PDGF) initiates a multitude of biological effects that contribute to VSMC proliferation and migration. Apamin, a component of bee venom, has been known to block the Ca(2+)-activated K(+) channels. However, the effects of apamin in the regulation PDGF-BB-induced VSMC proliferation and migration has not been identified. In this study, we investigate the inhibitory effect of apamin on PDGF-BB-induced VSMC proliferation and migration. Apamin suppressed the PDGF-BB-induced VSMC proliferation and migration with no apparent cytotoxic effect. In accordance with these findings, apamin induced the arrest of cell cycle progression at G0/G1 phase. Apamin also decreased the expressions of G0/G1 specific regulatory proteins including proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinases (CDK) 4, cyclin E and CDK2, as well as increased the expression of p21(Cip1) in PDGF-BB-induced VSMC. Moreover, apamin inhibited PDGF-BB-induced phosphorylation of Akt and Erk1/2. These results suggest that apamin plays an important role in prevention of vascular proliferation and migration through the G0/G1 cell cycle arrest by PDGF signaling pathway. Thus, apamin may be a promising candidate for the therapy of atherosclerosis.

Effects of bee venom against Propionibacterium acnes-induced inflammation in human keratinocytes and monocytes.

Propionibacterium acnes (P. acnes) cause inflammatory acne and play an important role in the pathogenesis of acne by inducing inflammatory mediators. P. acnes contributes to the inflammatory responses of acne by activating inflammatory cells, keratinocytes and sebocytes to secrete pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-8. Bee venom has traditionally been used in the treatment of certain immune-related diseases. However, there has not yet been a robust trial to prove the therapeutic effect of bee venom in skin inflammation. The aim of the present study was to investigate anti-inflammatory properties of bee venom in skin inflammation induced by P. acnes using keratinocytes (HaCaT) and monocytes (THP-1). P. acnes is known to stimulate the production of pro-inflammatory cytokines such as IL-1, IL-8, IL-12 and TNF-α. In the present study, the production of interferon-γ (IFN-γ), IL-1ß, IL-8 and TNF-α was increased by P. acnes treatment in HaCaT and THP-1 cells. By contrast, bee venom effectively inhibited the secretion of IFN-γ, IL-1ß, IL-8 and TNF-α. Furthermore, P. acnes treatment activated the expression of IL-8 and toll-like receptor 2 (TLR2) in HaCaT cells. However, bee venom inhibited the expression of IL-8 and TLR2 in heat-killed P. acnes. Based on these results, it is concluded that bee venom has an effective anti-inflammatory activity against P. acnes in HaCaT and THP-1 cells. Therefore, we suggest that bee venom is an alternative treatment to antibiotic therapy of acne.

Isothiocyanates inhibit the invasion and migration of C6 glioma cells by blocking FAK/JNK-mediated MMP-9 expression.

Isothiocyanates (ITCs) derived from cruciferous vegetables, including benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit preventative effects against various types of cancers. Yet, the inhibitory effects of ITCs on C6 glioma cell invasion and migration have not been reported. Thus, we aimed to analyze ITC-regulated MMP-9 activation, a crucial enzyme of cancer metastasis that degrades the extracellular matrix, in C6 glioma cells to investigate the inhibitory effects on cancer invasion and migration by ITCs. In the present study, we found that ITCs specifically suppressed PMA-induced MMP-9 secretion and protein expression. The inhibitory effects of ITCs on PMA-induced MMP-9 expression were found to be associated with the inhibition of MMP-9 transcription levels through suppression of nuclear translocation of NF-κB and activator protein-1 (AP-1). It was also confirmed that ITCs decreased MMP-9-mediated signaling such as FAK and JNK, whereas they had no effect on the phosphorylation of ERK and p38. Moreover, wound-healing and Τranswell invasion assays showed that ITCs inhibited the migration and invasion of C6 glioma cells. These results suggest that ITCs could be potential agents for the prevention of C6 glioma cell migration and invasion by decreasing FAK/JNK-mediated MMP-9 expression.

Serum ferritin increases in hemorrhaged rats that develop acute lung injury: effect of an iron-deficient diet.

For unknown reasons, serum ferritin levels increase in patients at risk for and with acute lung injury (ALI). To improve understanding of the relationship between serum ferritin alterations and the development of ALI, we investigated the effect of iron deficiency on the serum ferritin response of rats subjected to hemorrhage. We found that rats fed an iron-deficient diet for 6 weeks had decreased hemoglobin, hematocrit, liver total iron, liver total iron-binding capacity, and liver ferritin concentrations but the same serum ferritin concentrations as rats fed a control diet. Following hemorrhage, serum ferritin concentrations increased rapidly and progressively in rats fed a control diet. Along with increases in serum ferritin concentrations, control diet rats subjected to hemorrhage also had increased lung lavage leukocyte numbers, lung myeloperoxidase activities (lung inflammation), and lung lavage protein concentrations (lung leak) compared to control diet fed rats subjected to sham treatment. By comparison, the serum ferritin concentrations, lung inflammation, and lung leak of hemorrhaged rats fed an iron-deficient diet were decreased compared to hemorrhaged rats fed a control diet. These findings indicate that serum ferritin concentrations increase and acute lung injury develops following hemorrhage in rats fed a control, but not an iron-deficient, diet. A relatively brief exposure to an iron-deficient diet reduces hemorrhage-induced ALI.

The protective effects of melittin on Propionibacterium acnes-induced inflammatory responses in vitro and in vivo.

Melittin is the main component in the venom of the honey bee (Apis mellifera). It has multiple effects including antibacterial, antiviral, and anti-inflammatory activities in various cell types. However, the anti-inflammatory mechanisms of melittin have not been elucidated in Propionibactierium acnes (P. acnes)-induced keratinocyte or inflammatory skin disease animal models. In this study, we examined the effects of melittin on the production of inflammatory cytokines in heat-killed P. acnes-induced HaCaT cells. Heat-killed P. acnes-treated keratinocytes increased the expression of pro-inflammatory cytokines and Toll-like receptor 2. However, melittin treatment significantly suppressed the expression of these cytokines through regulation of the NF-κB and MAPK signaling pathways. Subsequently, the living P. acnes (1 × 10(7) CFU) were intradermally injected into the ear of mice. Living P. acnes-injected ears showed cutaneous erythema, swelling, and granulomatous response at 24 hours after injection. However, melittin-treated ears showed markedly reduced swelling and granulomatous responses compared with ears injected with only living P. acnes. These results demonstrate the feasibility of applying melittin for the prevention of inflammatory skin diseases induced by P. acnes.