Chien-Ping Chiang

Ameliorative effect of quercetin on the destruction caused by experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE The purpose of this study was to evaluate the effect of quercetin, a flavonol that exhibits anti-inflammatory properties, on experimental periodontal destruction in rats. MATERIAL AND METHODS Osteoclast formation on maxillary palatal alveolus was induced with daily lipopolysaccharide (LPS) injections (0, 1 or 5 mg/mL) for 3 d. Five days later, the osteoclasts on bony surfaces were counted after histochemical staining for tartrate-resistant acid phosphatase. The effect of intragastric quercetin on the osteoclast formation was evaluated in the following three groups: quercetin (75 mg/kg/d by oral feeding); LPS (5 mg/mL); and quercetin plus LPS. Moreover, the effect of quercetin on the ligature-induced periodontitis around maxillary second and mandibular first molars was further evaluated by microcomputerized tomography (on days 0, 4, 8 and 12) and by histometry (on day 8). RESULTS A dose-dependent increase in osteoclasts occurred after LPS injections. However, quercetin (75 mg/kg) reduced the 5 mg/mL LPS-induced osteoclasts. Using microcomputerized tomography, the bone crest levels at ligation sites were found to be significantly more apical than at the control sites on days 8 and 12; however, the apically located bone crests rebounded in rats from the quercetin-plus-ligation group. Histometry demonstrated significantly more coronal alveolar crest bone levels, less inflammatory cell-infiltrated connective tissue areas and less connective tissue attachments in the ligation-plus-quercetin group compared with those in the ligation group. CONCLUSION As the quercetin could reduce the LPS-induced osteoclast formation and the ligature-enhanced periodontal inflammation and bone loss, we suggest that it may have an ameliorative effect on periodontal destruction.

Cyclosporine-A inhibits MMP-2 and -9 activities in the presence of Porphyromonas gingivalis lipopolysaccharide: an experiment in human gingival fibroblast and U937 macrophage co-culture.

BACKGROUND AND OBJECTIVE Studies have shown that bacterial plaque and the associated gingival inflammation increase the severity of gingival overgrowth induced by cyclosporine-A (CsA). This in vitro study aimed to evaluate the effect of CsA on the activities of MMPs from the co-culture of human gingival fibroblasts and U937 macrophages in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (LPS). MATERIAL AND METHODS Activities of pro-MMP-2, MMP-2 and pro-MMP-9 in the supernatants of independent cultures and co-cultures were examined by zymography. RT-PCR was selected to evaluate the expression of mRNA for membrane type-1 (MT1) MMP in the co-cultures. RESULTS Activities of MMPs in the co-cultures were significantly greater when compared with any of the independent cultures. Lipopolysaccharide significantly increased the MMP activities in a dose-dependent manner in the co-cultures, whereas CsA inhibited these activities. In the presence of both CsA and LPS, the MMP activities inhibited by CsA could still be observed in the co-cultures. In the individual cultures, in contrast, the CsA-inhibited MMP activities, in the presence of LPS, were minimally detected. The mRNA expression of MT1-MMP was significantly enhanced after LPS treatment; however, this enhancement was inhibited by CsA. CONCLUSION This study demonstrated that, in co-cultures of human gingival fibroblasts and U937 macrophages, CsA could inhibit MMP activities in the presence of P. gingivalis LPS. It might be part of the underlying reason for the persistent overgrowth of gingiva seen when bacterial plaque and local inflammation are present during CsA therapy.

Cyclosporine A inhibits the expression of membrane type-I matrix metalloproteinase in gingiva.

BACKGROUND AND OBJECTIVE Membrane type-I matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) regulate the activation of MMP-2; however, their roles in the activation of MMP-2 in gingiva during treatment with cyclosporine A are still unknown. Therefore, the expressions of membrane type-I MMP and TIMP-2, as well as MMP-2, in gingivae upon treatment with cyclosporine A were examined in vivo and in vitro. MATERIAL AND METHODS Thirty-four rats were divided into two groups after edentulous ridges were established. The experimental group received 30 mg/kg/d of cyclosporine A and the control group received vehicle. At the end of the experimental period, the rats were killed, the gingivae were obtained and the expression of mRNA and protein of membrane type-I MMP, TIMP-2 and MMP-2 in gingiva were examined using real-time polymerase chain reaction and immunohistochemistry. In human gingival fibroblasts, the activity of MMP-2 and the expression of MMP-2, membrane type-I MMP and TIMP-2 mRNAs were examined (using zymography and reverse transcription-polymerase chain reaction, respectively) after treatment with cyclosporine A. RESULTS In gingivae of rats, cyclosporine A significantly decreased the expression of mRNA and protein of membrane type-I MMP, but not of TIMP-2. The expression of MMP-2 mRNA was unaffected but the expression of MMP-2 protein showed a significant decrease upon treatment with cyclosporine A. In fibroblast culture medium, the presence of cyclosporine A induced a decrease in MMP-2 activity in a dose-dependent manner. The expression of MMP-2, membrane type-I MMP and TIMP-2 mRNAs in fibroblasts was not significantly affected by cyclosporine A; however, in fibroblasts the ratio of mRNA expression of membrane type-I MMP to that of TIMP-2 decreased as the cyclosporine A dose was increased. CONCLUSION Cyclosporine A inhibits the expression of membrane type-I MMP in gingiva and it may further reduce the activation of MMP-2.

Human Mitochondrial NAD(P)+–Dependent Malic Enzyme Participates in Cutaneous Melanoma Progression and Invasion

Cutaneous melanoma is the most life-threatening neoplasm of the skin, accounting for most of the skin cancer deaths. Accumulating evidence suggests that targeting metabolism is an appealing strategy for melanoma therapy. Mitochondrial NAD(P)(+)-dependent malic enzyme (ME2), an oxidative decarboxylase, was evaluated for its biological significance in cutaneous melanoma progression. ME2 mRNA and protein expression significantly increased during melanoma progression, as evidenced by Gene Expression Omnibus analysis and immunohistochemistry on clinically annotated tissue microarrays, respectively. In addition, ME2 knockdown attenuated melanoma cell proliferation in vitro. ME2 ablation resulted in reduced cellular ATP levels and elevated cellular reactive oxygen species production, which activated the AMP-activated protein kinase pathway and inhibited acetyl-CoA carboxylase. Furthermore, ME2 expression was associated with cell migration and invasion. ME2 knockdown decreased anchorage-independent growth in vitro and tumor cell growth in vivo. These results suggested that ME2 might be an important factor in melanoma progression and a novel biomarker of invasion.

Angiolymphoid hyperplasia with eosinophilia affecting the scrotum: a rare case report with molecular evidence of T-cell clonality.

Angiolymphoid hyperplasia with eosinophilia (ALHE) is a rare, benign entity of unknown pathogenesis. It often presents as painful or pruritic intradermal or subcutaneous red to brown papules or nodules on the head and neck of young adults. A 38‐year‐old man had a gradually enlarging and mild pruritic plaque on the scrotum for half a year. Pathological findings showed dermal proliferation of anomalous blood vessels lined by plump endothelium with a significant perivascular inflammatory infiltrate composed of lymphocytes, histiocytes, scattered plasma cells and many eosinophils. They were consistent with the diagnosis of ALHE. In addition, the inflammatory infiltrate was analyzed by immunohistochemistry and T‐cell receptor (TCR) gene rearrangement. They were mostly CD3+ T cells and a monoclonal T‐cell population. To the best of our knowledge, this is the first case of ALHE affecting the scrotum to be reported in the published work. We present this case to expand the anatomical distribution of this rare tumor. The molecular study of our case supports that ALHE might be a low‐grade T‐cell lymphoproliferative disorder.

Effect of high glucose, Porphyromonas gingivalis lipopolysaccharide and advanced glycation end-products on production of interleukin-6/-8 by gingival fibroblasts.

BACKGROUND AND OBJECTIVE It is known that chronic periodontal infection can magnify the cytokine responses in patients with diabetes. Hyperglycemia increases the proinflammatory status, including the levels of advanced glycation end-products (AGEs), in patients with periodontitis. However, whether AGEs have additional effects on the production of those proinflammatory cytokines in diabetic patients with periodontitis is still unknown. To examine in vitro the effect of hyperglycemia and AGEs on the amounts of interleukin (IL)-6 and IL-8 produced in periodontally infected gingiva, human gingival fibroblasts (HGFs) were stimulated with glucose, AGE-modified bovine serum albumin (AGE-BSA) and Porphyromonas gingivalis LPS in the present study. MATERIAL AND METHODS Primary culture of HGFs was incubated with various concentrations of AGE-BSA (0, 50, 100 and 200 μg/mL) and LPS (0, 10, 100 or 1000 ng/mL) at two different glucose concentrations - normal glucose (5 mm) and high glucose (25 mm). The amounts of IL-6 and IL-8 produced by HGFs were evaluated using ELISA. Expression of the AGE receptor on HGFs was determined by flow cytometry. RESULTS High glucose stimulated a significant increase in the production of IL-6 and IL-8 by HGFs compared with normal glucose. This enhanced production of IL-6 and IL-8 could also be observed in the presence of LPS and/or AGE-BSA. When both LPS and AGE-BSA were present, especially at high concentrations (≥ 500 μg/mL of LPS and ≥ 25 μg/mL of AGE-BSA), a synergistic effect on IL-8 production was found in the high-glucose condition. CONCLUSIONS A synergistic effect of the production of IL-8 could be induced in HGFs with the combination of high glucose, LPS and AGEs.

Expression and bioactivities of endothelin-1 in gingiva during cyclosporine A treatment.

BACKGROUND AND OBJECTIVE This study aimed to evaluate the expression and bioactivities of endothelin-1 (ET-1) in gingiva during cyclosporine A (CsA) treatment. MATERIAL AND METHODS After establishing edentulous ridges, experimental rats were fed 30 mg/kg/day CsA while control animals received mineral oil for 4 weeks, after which a reverse transcription-polymerase chain reaction (RT-PCR) and/or immunohistochemistry was used to examine the expression of ET-1, its receptors, proliferating cell nuclear antigen (PCNA) and inducible nitric oxide synthase (iNOS) in gingivae. The roles of the endothelin receptors A and B (ET(A) and ET(B)) in CsA-enhanced expression of PCNA and iNOS were examined in cultured human gingival fibroblasts pretreated with receptor antagonists, by immunocytochemistry and RT-PCR, respectively. RESULTS The mRNA expression of ET-1, ET(A) and ET(B), as well as of PCNA and iNOS, was significantly greater in edentulous gingiva that received CsA compared with control gingiva. Immunohistochemistry revealed more cells positively stained for ET-1 and its receptors in the tissues of CsA-treated rats than in those of control rats. In fibroblast cultures, enhanced mRNA expression of ET-1, ET(A) and ET(B) was observed after CsA treatment at the concentrations of 10 and 100 ng/mL. Cyclosporine A-enhanced PCNA expression was somewhat reduced by blockade of ET(A), but not ET(B), whereas iNOS expression was somewhat reduced by blockade of ET(B). CONCLUSION Based on the present findings, we suggest that: (1) CsA upregulates the gingival expression of ET-1 and its receptors; and (2) ET(A) and ET(B) have different bioactivities, ET(A) being involved in cell proliferation and ET(B) being associated with iNOS expression.

The effects of diallyl sulfide upon Porphyromonas gingivalis lipopolysaccharide stimulated proinflammatory cytokine expressions and nuclear factor-kappa B activation in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE Diallyl sulfide (DAS), a flavor compound from garlic, has varied potential therapeutic activities. Periodontitis is a disease that develops because of host-mediated inflammation to periodontal pathogens. In this study, the effects of DAS on the common proinflammatory cytokines and nuclear factor-kappa B (NF-κB) in human gingival fibroblasts (HGFs) being stimulated with lipopolysaccharide from Porphyromonas gingivalis, a potent periodontal pathogen, were evaluated. MATERIAL AND METHODS Cytotoxicities of DAS and lipopolysaccharide on HGFs were measured with MTS assay. The mRNA and protein expressions of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, from the HGFs treated with lipopolysaccharide with and without DAS were examined with reverse transcription-polymerase chain reaction and immunocytochemistry, respectively. In addition, the activation and nuclear translocation of NF-κB with and without DAS were compared. RESULTS DAS and lipopolysaccharide treatments within 3 mm and 10 μg/mL, respectively, did not affect the survival rate of HGFs. Lipopolysaccharide (1 μg/mL) significantly increased the mRNA expressions of IL-1β, IL-6 and TNF-α; however, DAS (1 mm) inhibited these expressions. The protein expressions of TNF-α, IL-1β, as well as the NF-κB nuclear translocation were increased after lipopolysaccharide treatment, but decreased when there was a DAS pretreatment. CONCLUSION DAS diminished P. gingivalis lipopolysaccharide-stimulated cytokine expression and NF-κB activation in HGFs; we therefore suggest DAS may be beneficial on periodontal inflammation.

Erythema Ab igne after footbath with Chinese herbal remedies

Erythema ab igne (EAI) is a reticulated, telangiectatic, and hyperpigmented skin eruption resulting from chronic exposure to long-term moderate heat. The incidence has decreased substantially today because of the advent of modern central heating systems. Recently, we encountered a patient who developed EAI after 2 weeks of footbaths with Chinese herbal remedies, which she used to treat her acute ankle sprain. Alternative Chinese medicine, such as herbal footbath, is a prevalent medical practice to treat acute pains as well as many chronic musculoskeletal ailments among Chinese and Asian populations. It has also become increasingly popular in Western countries in the past decade. Herein, we would like to report an uncommon case of iatrogenic EAI caused by footbath and raise the attention of clinicians to such rare, potentially malignant-transforming, dermatosis.

Up‐regulation of retinoblastoma protein phosphorylation in gingiva after cyclosporine A treatment: an in vivo and in vitro study

BACKGROUND AND OBJECTIVE Cyclosporine A can induce gingival cell proliferation; however, the precise molecular regulation of the proliferation is uncertain. Therefore, this study was carried out to examine, in vivo and in vitro, the expression of genes and proteins associated with gingival cell proliferation after treatment with cyclosporine A. MATERIAL AND METHODS Forty Sprague Dawley rats with right maxillary posterior edentulous gingivae were assigned to a cyclosporine A group (30 mg/kg daily of cyclosporine A, administered orally) or a control group (administered mineral oil only). The animals were killed 4 wk after treatment. The edentulous gingivae were dissected out and analyzed for the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb1) mRNA and/or protein, and phosphorylated Rb1 (pRb1), by real-time RT-PCR or immunohistochemistry. In human gingival fibroblast (HGF) cultures, the expression of PCNA, CDK4, cyclin D1 and Rb1 proteins and Rb1 phosphorylation were determined by western blotting after cyclosporine A treatment (0-10(4) ng/mL). RESULTS Proliferating cell nuclear antigen and cyclin D1 mRNAs (Pcna and Ccnd1, respectively) were expressed more strongly in the gingivae of cyclosporine A-treated animals than in the gingivae of the controls. Immunohistochemical analyses showed that a greater number of gingival cells stained positive for cyclin D1, CDK4 and pRb1 in the cyclosporine A group than in the control group. Increased expression of cyclin D1, CDK4 and PCNA proteins was observed in HGFs after cyclosporine A treatment. The phosphorylation of Rb1 was enhanced in HGFs after treatment with cyclosporine A at concentrations of 10(2)-10(3) ng/mL. CONCLUSION The increases in cyclin D1, PCNA and CDK4, together with the enhanced phosphorylation of Rb1, suggest that cyclosporine A promotes cell-cycle progression through the G(1)/S transition in the gingiva.