Woon-Hae Kim

Anti-fibrotic Effects of Synthetic Oligodeoxynucleotide for TGF-β1 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 β1 (TGF-β1) is a multifunctional cytokine that induces liver fibrosis through activation of Smad signaling pathways. To improve a new therapeutic approach, synthetic TGF-β1/Smad oligodeoxynucleotide (ODN) was used to suppress both TGF-β1 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-β1/Smad ODN on simultaneous suppressions of both Smad transcription factor and TGF-β1 mRNA expression in the hepatic fibrosis model in vitro and in vivo. Synthetic TGF-β1/Smad ODN effectively inhibits Smad binding activity and TGF-β1 expression. TGF-β1/Smad ODN attenuated the epithelial mesenchymal transition (EMT) and activation of HSCs in TGF-β1-induced AML12 and HSC-T6 cells. TGF-β1/Smad ODN prevented the fibrogenesis and deposition of collagen in CCl4-treated mouse model. Synthetic TGF-β1/Smad ODN demonstrates anti-fibrotic effects that are mediated by the suppression of fibrogenic protein and inflammatory cytokines. Therefore, synthetic TGF-β1/Smad ODN has substantial therapeutic feasibility for the treatment of liver fibrotic diseases.

The Protective Effect of Melittin on Renal Fibrosis in an Animal Model of Unilateral Ureteral Obstruction

Renal fibrosis is the principal pathological process underlying the progression of chronic kidney disease that leads to end-stage renal disease. Melittin is a major component of bee venom, and it has anti-bacterial, anti-viral, and anti-inflammatory properties in various cell types. Thus, this study examined the therapeutic effects of melittin on the progression of renal fibrosis using the unilateral ureteral obstruction (UUO) model. In addition, the effects of melittin on inflammation and fibrosis in renal fibroblast cells were explored using transforming growth factor-β1 (TGF-β1). Histological observation revealed that UUO induced a considerable increase in the number of infiltrated inflammatory cells. However, melittin treatment markedly reduced these reactions compared with untreated UUO mice. The expression levels of inflammatory cytokines and pro-fibrotic genes were significantly reduced in melittin-treated mice compared with UUO mice. Melittin also effectively inhibited fibrosis-related gene expression in renal fibroblasts NRK-49F cells. These findings suggest that melittin attenuates renal fibrosis and reduces inflammatory responses by the suppression of multiple growth factor-mediated pro-fibrotic genes. In conclusion, melittin may be a useful therapeutic agent for the prevention of fibrosis that characterizes the progression of chronic kidney disease.

Bee Venom Inhibits Porphyromonas gingivalis Lipopolysaccharides-Induced Pro-Inflammatory Cytokines through Suppression of NF-κB and AP-1 Signaling Pathways

Periodontitis is a chronic inflammatory disease that leads to destruction of tooth supporting tissues. Porphyromonas gingivalis (P. gingivalis), especially its lipopolysaccharides (LPS), is one of major pathogens that cause periodontitis. Bee venom (BV) has been widely used as a traditional medicine for various diseases. Previous studies have demonstrated the anti-inflammatory, anti-bacterial effects of BV. However, a direct role and cellular mechanism of BV on periodontitis-like human keratinocytes have not been explored. Therefore, we investigated the anti-inflammatory mechanism of BV against P. gingivalis LPS (PgLPS)-induced HaCaT human keratinocyte cell line. The anti-inflammatory effect of BV was demonstrated by various molecular biological methods. The results showed that PgLPS increased the expression of Toll-like receptor (TLR)-4 and pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, and interferon (IFN)-γ. In addition, PgLPS induced activation of the signaling pathways of inflammatory cytokines-related transcription factors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1). BV effectively inhibited those pro-inflammatory cytokines through suppression of NF-κB and AP-1 signaling pathways. These results suggest that administration of BV attenuates PgLPS-induced inflammatory responses. Furthermore, BV may be a useful treatment to anti-inflammatory therapy for periodontitis.

Apamin inhibits TNF-α- and IFN-γ-induced inflammatory cytokines and chemokines via suppressions of NF-κB signaling pathway and STAT in human keratinocytes

BACKGROUND Atopic dermatitis (AD) is identified by an increase in infiltrations of several inflammatory cells including type 2 helper (Th2) lymphocytes. Th2-related chemokines such as thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), and pro-inflammatory cytokines including interleukin (IL)-1β and IL-6 are considered to play a crucial role in AD. Tumor necrosis factor (TNF)-α- and interferon (IFN)-γ induce the inflammatory condition through production of TARC, MDC, IL-1β and IL-6, and activations of related transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT) in keratinocytes. Apamin, a peptide component of bee venom, has been reported its beneficial activities in various diseases. However, anti-inflammatory effects of apamin on inflammatory condition in keratinocytes have not been explored. Therefore, the present study aimed to demonstrate the anti-inflammatory effect of apamin on TNF-α- and IFN-γ-induced inflammatory condition in keratinocytes. METHODS HaCaT was used as human keratinocytes cell line. Cell Counting Kit-8 was performed to measure a cytotoxicity of apamin. The effects of apamin on TNF-α-/IFN-γ-induced inflammatory condition were determined by real-time PCR and Western blot analysis. Further, NF-κB signaling pathways, STAT1, and STAT3 were analyzed by Western blot and immunofluorescence. RESULTS Apamin ameliorated the inflammatory condition through suppression of Th2-related chemokines and pro-inflammatory cytokines. Further, apamin down-regulated the activations of NF-κB signaling pathways and STATs in HaCaT cells. CONCLUSIONS These results suggest that apamin has therapeutic effect on AD through improvement of inflammatory condition.

Anti-Inflammatory Effect of Melittin on Porphyromonas Gingivalis LPS-Stimulated Human Keratinocytes

Periodontitis is a chronic inflammatory disease that contributes to the destruction of the gingiva. Porphyromonas gingivalis (P. gingivalis) can cause periodontitis via its pathogenic lipopolysaccharides (LPS). Melittin, a major component of bee venom, is known to have anti-inflammatory and antibacterial effects. However, the role of melittin in the inflammatory response has not been elucidated in periodontitis-like human keratinocytes. Therefore, we investigated the anti-inflammatory effects of melittin on a P. gingivalis LPS (PgLPS)-treated HaCaT human keratinocyte cell line. The cytotoxicity of melittin was measured using a human keratinocyte cell line, HaCaT, and a Cell Counting Kit-8. The effect of melittin on PgLPS-induced inflammation was determined with Western blot, real-time quantitative PCT, and immunofluorescence. PgLPS increased the expression of toll-like receptor (TLR) 4 and proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and interferon-γ (IFN-γ). Moreover, PgLPS induced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), extracellular signal-regulated kinase (ERK), and protein kinase B/Akt. Melittin also inhibited the expression of proinflammatory cytokines by suppressing the activation of the NF-κB signaling pathway, ERK, and Akt. Melittin attenuates the PgLPS-induced inflammatory response and could therefore be applied in the treatment of periodontitis for anti-inflammatory effects.

Beneficial effects of melittin on ovalbumin-induced atopic dermatitis in mouse

Atopic dermatitis (AD) is an inflammatory skin disease characterized by intense pruritus and relapsable eczematous lesions. The hallmarks of AD are defects in the epidermal barrier and immunoglobulin E (IgE)-mediated sensitization to several environmental allergens, as well as an immune disorder mediated by an imbalance toward T-helper-2 response. Melittin, a major component of bee venom, has been studied in various inflammatory diseases. However, the beneficial effects of melittin on mouse with AD-like symptoms have not been explored. Therefore, we investigated the anti-allergic effects of melittin. AD was induced by ovalbumin (OVA) patch. After agent treatment, skin tissues and sera were extracted from the sacrificed mice were used to demonstrate the effects of melittin through various molecular biological methods. The results showed that OVA-induced skin thickening and inflammatory infiltration were decreased in the melittin-treated group. Melittin prevented OVA-induced filaggrin deficiency and imbalanced inflammatory mediators. Furthermore, melittin inhibited IL-4/IL-13-induced filaggrin downregulation through the blockade of STAT3 activation in human keratinocytes. In summary, this study has shown that melittin ameliorated OVA-induced AD-like symptoms from various perspectives. The findings of this study may be the first evidence of the anti-inflammatory effects of melittin on OVA-induced AD.

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.

Antifibrotic Effect of Smad Decoy Oligodeoxynucleotide in a CCl4-Induced Hepatic Fibrosis Animal Model

Hepatic fibrosis is the wound-healing process of chronic hepatic disease that leads to the end-stage of hepatocellular carcinoma and demolition of hepatic structures. Epithelial–mesenchymal transition (EMT) has been identified to phenotypic conversion of the epithelium to mesenchymal phenotype that occurred during fibrosis. Smad decoy oligodeoxynucleotide (ODN) is a synthetic DNA fragment containing a complementary sequence of Smad transcription factor. Thus, this study evaluated the antifibrotic effects of Smad decoy ODN on carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice. As shown in histological results, CCl4 treatment triggered hepatic fibrosis and increased Smad expression. On the contrary, Smad decoy ODN administration suppressed fibrogenesis and EMT process. The expression of Smad signaling and EMT-associated protein was markedly decreased in Smad decoy ODN-treated mice compared with CCl4-injured mice. In conclusion, these data indicate the practicability of Smad decoy ODN administration for preventing hepatic fibrosis and EMT processes.

Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro

Atopic dermatitis (AD) is a multifactorial skin condition with complex interactions of innate and adaptive immune responses. There are several existing therapies for AD, including topical glucocorticosteroids, emollients, phototherapies, calcineurin inhibitors and immunosuppressants, such as cyclosporine A. Although these therapies reduce inflammation, they also cause serious side effects. Therefore, it is necessary to develop new therapeutic approaches for AD treatment without side effects. There are several studies on natural materials or toxins, such as herbs, ginseng extract and snake venom, for AD treatment. However, treatment of AD with bee venom and its major component, melittin has rarely been studied.