Yoon-Yub Park

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.

Bee venom suppresses PMA-mediated MMP-9 gene activation via JNK/p38 and NF-κB-dependent mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE Bee venom has been used for the treatment of inflammatory diseases such as rheumatoid arthritis and for the relief of pain in traditional oriental medicine. AIM OF THE STUDY The purpose of this study is to elucidate the effects of bee venom on MMP-9 expression and determine possible mechanisms by which bee venom relieves or prevents the expression of MMP-9 during invasion and metastasis of breast cancer cells. We examined the expression and activity of MMP-9 and possible signaling pathway affected in PMA-induced MCF-7 cells. MATERIAL AND METHODS Bee venom was obtained from the National Institute of Agricultural Science and Technology of Korea. Matrigel invasion assay, wound-healing assay, zymography assay, western blot assay, electrophoretic mobility shift assay and luciferase gene assay were used for assessment. RESULTS Bee venom inhibited cell invasion and migration, and also suppressed MMP-9 activity and expression, processes related to tumor invasion and metastasis, in PMA-induced MCF-7 cells. Bee venom specifically suppressed the phosphorylation of p38/JNK and at the same time, suppressed the protein expression, DNA binding and promoter activity of NF-kappaB. The levels of phosphorylated ERK1/2 and c-Jun did not change. We also investigated MMP-9 inhibition by melittin, apamin and PLA(2), representative single component of bee venom. We confirmed that PMA-induced MMP-9 activity was significantly decreased by melittin, but not by apamin and phospholipase A(2). These data demonstrated that the expression of MMP-9 was abolished by melittin, the main component of bee venom. CONCLUSION Bee venom inhibits PMA-induced MMP-9 expression and activity by inhibition of NF-kappaB via p38 MAPK and JNK signaling pathways in MCF-7 cells. These results indicate that bee venom can be a potential anti-metastatic and anti-invasive agent. This useful effect may lead to future clinical research on the anti-cancer properties of bee venom.

Protective effects of melittin on transforming growth factor-β1 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)-β1-induced apoptosis in hepatocytes. TGF-β1-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-β1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-β1-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-β1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-β1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-β1-mediated injury.

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 G1 transgenic chicks produced from the cross of Go transgenic roosters with nontransgenic hens, but most of the G1 progeny were dead within 1 month of hatching. Mol. Reprod. Dev. 75: 1120–1126, 2008.

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.

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% O2) or treatment with a transition metal (CoCl2). 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. J. Cell. Biochem. 113: 1302–1313, 2012.

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)-1β 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-1β, 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-1β, 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.

Moringa Fruit Inhibits LPS-Induced NO/iNOS Expression through Suppressing the NF-κB Activation in RAW264.7 Cells

In this study, we evaluated the anti-inflammatory effects of moringa (Moringa oleifera Lam.), a natural biologically active substance, by determining its inhibitory effects on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophage RAW264.7 cells. Extracts from different parts of moringa (root, leaf, and fruit) reduced LPS-induced nitric oxide (NO) release in a dose-dependent manner. The moringa fruit extract most effectively inhibited LPS-induced NO production and levels of inducible nitric oxide synthase (iNOS). The moringa fruit extract also was shown to suppress the production of inflammatory cytokines including IL-1β, TNF-α, and IL-6. Furthermore, moringa fruit extract inhibited the cytoplasmic degradation of I κ B -α and the nuclear translocation of p65 proteins, resulting in lower levels of NF -κ B transactivation. Collectively, the results of this study demonstrate that moringa fruit extract reduces the levels of pro-inflammatory mediators including NO , IL-1β, TNF-α, and IL-6 via the inhibition of NF -κ B activation in RAW264.7 cells. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of moringa fruit extract.

Ischemia/reperfusion Lung Injury Increases Serum Ferritin and Heme Oxygenase-1 in Rats

Intestinal ischemia/reperfusion (I/R) is one of common causes of acute lung injury (ALI). Early and accurate diagnosis of patients who are like to develop serious acute respiratory distress syndrome (ARDS) would give a therapeutic advantage. Ferritin and heme oxygenase-1 (HO-1) are increased by oxidative stress and are potential candidates as a predictive biomarker of ARDS. However, the mechanisms responsible for the increases of ferritin and HO-1, and their relationship to ALI, are unclear. In order to elucidate the interactions between ferritin and HO-1, we studied the changes in ferritin and HO-1 levels in serum and bronchoalveolar lavage (BAL) fluid after intestinal I/R injury in rats. Leukocyte number and protein contents in BAL fluid were elevated following I/R, and the increases were attenuated by mepacrine pretreatment. Both serum ferritin and HO-1 concentrations were progressively elevated throughout the 3 h observation period. Mepacrine pretreatment attenuated the increase of serum and BAL fluid ferritin concentrations, but did not suppress the increase of serum HO-1. Moreover, BAL fluid HO-1 levels did not change after I/R or after mepacrine pretreated I/R compared with sham rats. Unlike ferritin, HO-1 levels are not exactly matched with the ALI. Therefore, there might be a different mechanism between the changes of ferritin and HO-1 in intestinal I/R-induced ALI model.

Melittin inhibits TGF-β-induced pro-fibrotic gene expression through the suppression of the TGFβRII-Smad, ERK1/2 and JNK-mediated signaling pathway.

Renal fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins such as type I collagen, fibronectin, and by the increased expression of PAI-1. This study evaluated the anti-fibrotic effect of bee venom and its major compounds (melittin and apamin) on TGF-β-induced pro-fibrotic gene expression. Bee venom and melittin significantly suppressed type I collagen, fibronectin, and PAI-1 protein expression in the TGF-β-treated kidney fibroblast. However, apamin only inhibited the expression of fibronectin and type I collagen. These results indicated that the inhibitory effects of bee venom on TGF-β-induced pro-fibrotic gene expression are caused by melittin. Moreover, we attempted to elucidate mechanisms underlying the anti-fibrotic effect of melittin. Melittin dramatically inhibited the phosphorylation of TGFβRII and Smad2/3. Also, melittin inhibited the phosphorylation of ERK1/2 and JNK, but not the phosphorylation of PI3K, Akt, and p38. These results suggested that melittin inhibits TGF-β-induced pro-fibrotic genes expression through the suppression of TGFβR-Smad2/3, ERK1/2, and JNK phosphorylation, and melittin can be used as a clinical drug for the treatment of fibrosis associated with renal diseases.