京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20236号 / 医博第4195号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 松原 和夫, 教授 川村 孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In dieser Dissertation wurden am Ferkelmodell (8-15 kg, drei Gruppen: „Kontrolle“ n=7, „Herz-Lungen-Maschine (HLM)“ n=10, „(-)-Epigallocatechin-3-Gallat (EGCG)“ n=6, die Kontrollgruppe wurde thorakotomiert, die HLM- und die EGCG-Gruppe wurden thorakotomiert und für 90 Minuten an eine HLM angeschlossen, die EGCG-Gruppe erhielt vor und nach der HLM-Zeit EGCG) drei Fragestellungen behandelt: Erstens wurde untersucht, ob die Verwendung einer HLM während eines kardiochirurgischen Eingriffes unter hypothermen Bedingungen mit nicht-pulsatilem Blutfluss und Kardioplegie die Niere schädigte. Dafür wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe mit Hämatoxylin-Eosin (HE) angefärbt und unterschiedliche Strukturen betrachtet, wobei histopathologische Veränderungen in der HLM-Gruppe auffielen. Paraklinisch fanden sich erhöhte nierenspezifische Blutwerte (Serumkreatinin und -harnstoff) in der HLM-Gruppe. Diese Ergebnisse waren hinweisend für eine funktionell relevante Schädigung der Niere durch die HLM. Unterstützend kam ein Absinken des Gesamteiweißes im Serum der HLM-Gruppe hinzu, was auf eine generelle Schädigung des Organismus durch die HLM hindeutete. Zweitens wurde betrachtet, ob die gesetzten Schäden die Merkmale eines Ischämie-Reperfusionsschadens aufwiesen. Hierzu wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe immunhistochemisch (Hypoxie-induzierter-Faktor-1-alpha-Tyramide- Signal-Amplification (HIF-1-alpha-TSA)-, Nitrotyrosin-3-Amino-9-Ethylcarbazol (Nitrotyrosin-AEC)- und Apoptose-induzierender-Faktor-Tyramide-Signal-Amplification (AIF-TSA)-Färbung) angefärbt. Dabei zeigte sich, dass sich die HLM-Gruppe in einer hypoxischen Situation befand (HIF-1-alpha Akkumulation in den Zellkernen), nitrosativem Stress ausgesetzt war (Nitrotyrosin in den Tubuli) und dass sie teilweise so stark geschädigt wurde, dass Apoptose induziert wurde (AIF in Zellkernen) – alle drei Färbungsergebnisse waren hinweisend für einen ischämischen Zustand, in dem sich die HLM-Gruppe befunden hat. Auch die Ergebnisse der durchgeführten renalen Reversed Phase High Performance Liquid Chromatography (RP-HPLC) deuteten auf ebendies hin. Unterstützend wirkten die Ergebnisse des arteriellen Laktats – die HLM-Gruppe zeigte eine Hyperlaktämie – und die Tatsache, dass einige der histologischen Merkmale für eine frühe Schockniere (welche ischämischen Ursprungs sein kann) in der HLM-Gruppe gefunden wurden. Dies alles zeigte, dass der HLM-assoziierte Nierenschaden vorrangig die Natur eines Ischämie-Reperfusionsschadens aufwies. Drittens wurde untersucht, ob EGCG diese HLM-assoziierte Schädigung abmildern konnte. Dafür wurden bei der EGCG-Gruppe alle oben genannten Untersuchungen durchgeführt. Die Ergebnisse zeigten, dass EGCG in der Dosierung 10 mg/kg eine renoprotektive Wirkung gegen die HLM-assoziierten Schäden hatte, und diese abmildern bzw. ihnen entgegenwirken konnte. Diese Ergebnisse sind für die pädiatrische Kardiochirurgie interessant, welche zum Beispiel bei der Korrektur angeborener Herzdefekte auf die Verwendung der HLM angewiesen ist. Komplikationen wie eine Nierenschädigung post operationem sind nicht selten und verkomplizieren den Verlauf. Die vorliegende Dissertation zeigt das renoprotektive Potential des in grünem Tee vorkommenden Katechins EGCG im Umfeld eines kardiochirurgischen Eingriffes mit Verwendung einer HLM. Die Wirksamkeit dieser Substanz ist wahrscheinlich darin begründet, dass sie mehr als ein Antioxidans ist. Neben seiner Radikalfänger- und Stickstoffmonoxidscavenger-Fähigkeiten ist EGCG außerdem antiapoptotisch wirksam. Derzeit wird die Kardiochirurgie mit Verwendung einer HLM in der Veterinärmedizin nur in wenigen Zentren angewendet. Es besteht für die Zukunft jedoch die Hoffnung, dass gerade für Kleintierbesitzer, die ihre Tiere als Familienmitglied betrachten, und auch für zoologische Einrichtungen bei der Diagnose eines Herzfehlers die Kardiochirurgie mit Verwendung einer HLM als Therapiemöglichkeit eine interessante und realistische Alternative zur bislang angewandten palliativen medikamentösen Therapie darstellen kann. / In this dissertation a piglet model (8-15 kg, three groups: “control” n=7, “extracorporeal circulation (EC)” n=10, “EGCG” n=6, the control-group was thoracotomized, the EC- and the EGCG-group were thoracotomized and underwent cardiopulmonary bypass (CPB) for 90 minutes, and the EGCG-group received EGCG before and after the CPB) is presented. Three questions were raised and answered: Firstly, it was investigated if the use of a CPB during cardiac surgery with hypothermia, non-pulsatile blood flow and cardioplegia caused damage to the kidney. In order to answer this question, paraffin slices of the kidney of the control- and the EC-group were stained with hematoxylin-eosin (HE), and different structures were evaluated – this staining showed histopathological changes in the EC-group. Paraclinical, the EC-group showed elevated kidney-specific blood parameters (serumcreatinine and -urea). These findings indicated a functionally relevant impairment of the kidney caused by the CPB. Supporting this, the EC-group also showed a decline of the total amount of proteins in the serum, which was suggestive of a generalized injury of the body by the CPB. Secondly, it was investigated whether the injury of the kidney might have been caused by an ischemia/reperfusion injury. Therefore, paraffin slices of the kidney of the control- and the EC-group were immunhistochemically stained (hypoxia-induced-factor-1-alpha-tyramidesignal-amplification (HIF-1-alpha-TSA)-, nitrotyrosine-3-amino-9-ethylcarbazole (nitrotyrosine-AEC)- and apoptosis-inducing-factor-tyramide-signal-amplification (AIF-TSA)-staining). These stainings revealed, that the EC-group had suffered from a hypoxemic situation (accumulation of HIF-1-alpha in the nuclei), from nitrosative stress (presence of nitrotyrosine in the tubuli), and that the kidney was partly damaged to the point of an induction of apoptosis (presence of AIF in the nuclei) – all three of these findings indicated, that the kidneys of the EC-group were put into an ischemic situation. The findings of the renal reversed phase high performance liquid chromatography (RP-HPLC) indicated the same thing. This was also supported by the blood parameter of lactate – the EC-group showed a hyperlactemia – and by some histological findings in the EC-group, which were characteristical for an early shock-kidney (which may be caused by ischemia). Taken together, these findings showed that the CPB-associated kidney injury was primarily caused by an ischemia/reperfusion injury. Thirdly, it was investigated, whether EGCG might attenuate the CPB-associated kidney injury. For that purpose, all of the investigation methods mentioned above were carried out with the samples of the EGCG-group. The findings showed that EGCG (dose: 10 mg/kg) had a protective effect on the kidney, protecting it against the damage caused by the CPB, and was able to partly attenuate this damage and partly even fully counteract it. These findings are of interest for pediatric cardiac surgery, which for example for the correction of innate heart defects depends on the use of CPB. Complications – like acute renal injury post operationem – occur frequently and complicate the recovery. This dissertation demonstrates the renoprotective potential of the natural compound EGCG in the setting of cardiac surgery with the use of CPB. The reason for the effectiveness of EGCG in this situation probably is that EGCG is more than an antioxidant. EGCG not only works as a radical- and nitric-oxide-scavenger, but also is antiapoptotic. In veterinary medicine cardiac surgery with CPB is done by few centers only. However for the future there is hope that people – especially pet owners who view their companion animals as family members, and zoos – become more and more willing to and interested in having an animal diagnosed with a heart defect treated with cardiac surgery including the use of an CPB, instead of – like its usually done nowadays – only giving palliative medication to the animal.

O chá verde vem sendo utilizado na prevenção e tratamento de variadas doenças infecciosas e imunoinflamatórias, por apresentar efeitos benéficos decorrentes da presença de Epigalocatequina-3-galato (EGCG). Esta catequina apresenta papel antiinflamatório, anti-oxidante, anti-microbiano e mineralizador e poderia ser utilizado no tratamento da doença periapical. O objetivo do presente trabalho foi avaliar o efeito uma pasta à base de EGCG utilizada como curativo de demora em lesões periapicais experimentalmente induzidas em cães. Um total de 80 raízes de pré-molares de cão com rizogênese completa e lesões periapicais experimentalmente induzidas, foram aleatoriamente divididos em 4 grupos submetidos à diferentes protocolos de tratamento endodôntico: 1) curativo de demora com pasta à base de EGCG; 2) curativo de demora com EGCG em veículo aquoso; 3) curativo de demora com pasta à base hidróxido de cálcio (Pasta Calen); 4) tratamento endodôntico em sessão única. Para avaliação da resposta tecidual, os dentes foram avaliados radiograficamente e histopatologicamente. As imagens radiográficas obtidas antes e 120 dias após o tratamento, foram fotografadas e digitalizadas para o programa Image J 1.28 para mensuração das áreas (mm&sup2;) radiolúcidas periapicais. Aos 120 dias após a obturação doa canais radiculares os animais foram eutanasiados e os espécimes foram processados histotecnicamente, corados com HE e avaliados com microscopia de luz convencional e de fluorescência (infiltrado inflamatório, espessamento do ligamento periodontal e reabsorção de tecidos mineralizados). Os resultados obtidos foram analisados estatisticamente utilizando os testes quiquadrado, Fisher, Anova e pós teste de Tukey, com nível de significância de 5% em todos os testes. De acordo com os resultados radiográficos, o percentual médio de redução da área radiolúcida (± erro padrão da média) foi semelhante (p>0,05) nos grupos EGCG em veículo aquoso (64,57%; ±7,514); pasta à base de EGCG (59,95%; ±8,023) e pasta à base de hidróxido de cálcio (58,65%; ±6,192). Todos foram superiores ao grupo tratado em sessão única (19,49%; ±2,881) (p<0,01). A análise histopatológica mostrou que os grupos que receberam aplicação de curativo de demora com EGCG ou hidróxido de cálcio, apresentaram reaparação da lesão periapical com semelhança em todos os parâmetros avaliados. Por outro lado, o tratamento em sessão única resultou em manutenção da lesão periapical, com maior espessura do ligamento periodontal (p<0,001), persistência de infiltrado inflamatório moderado ou severo (p<0,01) e presença de reabsorção óssea e cementária (p<0,0001). Foi possível concluir que a pasta à base de EGCG proporcionou o reparo de lesões periapicais, constituindo possível medicação intracanal alternativa / Green tea has been used in the prevention and treatment of various infectious and immunoinflammatory diseases, since it has beneficial effects due to the presence of Epigallocatechin-3-gallate (EGCG). This catequin present anti-inflammatory, antimicrobial, antioxidant and mineralizing role that could be usefull in the treatment of periapical disease. The aim of the present study was to evaluate the effect of the intracanal dressing with an EGCG paste-based im periapical lesions experimentaly induced in dogs. A total of 80 dog premolar roots with complete rhizogenesis and experimentally induced periapical lesions were randomly divided into 4 groups submitted to different endodontic treatment protocols:1) intracanal dressing with EGCG based paste; 2) intracanal dressing with EGCG in aqueous vehicle; 3) intracanal dressing with based paste on calcium hydroxide (Calen paste); 4) single session endodontic treatment. To evaluate the tissue response, the teeth were evaluated radiographically and histopathologically. Radiographic images are selected before 120 days after treatment, were photographed and scanned for the program Image J 1.28 for the measurement of periapical radiolucent areas (mm2). To 120 days after the root canal filling animals were euthanasied and the specimens processed, stained in the rotin HE and evaluated under conventional and fluorescence microscopy (inflammatory infiltrate, periodontal ligament space and mineralized tissue resorption). The results were compared statistically using chisquare, Fisher, Anova and Tukey post-test, with a significance level of 5% in all tests. According to the radiographic results, the mean percentage reduction of the radiolucent area (standard pattern of the mean) was similar (p> 0.05) in the EGCG groups in aqueous vehicle (64.57%; ± 7.514); EGCG based paste (59.95%, ± 8.023) and calcium hydroxide based paste (58.65%, ± 6.192). All groups were higher for the group treated in single session (19.49%, ± 2.881) (p <0.01). The histopathological analysis showed that the groups that received the prescription of intracanal dressings with EGCG or calcium hydroxide, resulted in periapical lesion repair and were similar in all the evaluated parameters. However, treatment in single session did not repair the periapical lesion resulting in greatest measure of the periodontal ligament (p <0.001), persistence of moderate or severe inflammatory infiltrate (p <0.01) and presence of bone and cementum resorption (p <0.0001). It is possible to conclude that EGCG paste-based allows the periapical lesions repair, constituting possible alternative intracanal medication

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed Jan. 29, 2010). Includes bibliographical references (p. 55-60).

Endothelial inflammation is a pivotal early event in the development of atherosclerosis. Long term exposure to cardiovascular risk factors will ultimately exhaust those protective anti-inflammatory factors such as the heme oxygenase (HO) system. The HO system plays a critical role in cellular and tissue self-defense against oxidative stress and inflammation. Caveolae are membrane domains and are particularly abundant in endothelial cells, where they are believed to play a major role in the regulation of endothelial vesicular trafficking as well as the uptake of lipids and related lipophilic compounds, possibly including bioactive food components such as flavonoids. Research in this dissertation addresses the role of HO-1 and caveolae on dietary flavonoid epigallocatechin gallate (EGCG) mediated protection against pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and linoleic acid-induced activation of endothelial cells. The data support the hypothesis that EGCG protects against TNF-α-induced monocyte recruitment and adhesion partially through the induction of HO-1 and bilirubin. The observed anti-inflammatory effects of EGCG are mimicked by the HO-1 inducer cobalt protoporphyrin (CoPP) and abolished by HO-1 gene silencing. Nrf2 is the major transcription factor of phase II antioxidant enzymes including HO-1. Results clearly show that EGCG-induced HO-1 expression and subsequent bilirubin productions are dependent on functional Nrf2. EGCG also can down-regulate the base-line level of caveolin-1. Furthermore, silencing of the caveolin-1 gene can markedly down-regulate linoleic acid-induced COX-2 and MCP-1, indicating that caveolae may be a critical platform regulating inflammatory signaling pathways. Similar to EGCG treatment, silencing of caveolin-1 can also result in the activation of Nrf2, up-regulation of HO-1 and bilirubin. This may be one of the mechanisms to explain the protection effect of caveolin-1 gene silencing against endothelial inflammation. Moreover, EGCG rapidly accumulates in caveolae, which is associated with caveolin-1 displacement from the plasma membrane towards the cytosol. Caveolin-1 gene silencing can significantly reduce the uptake of EGCG in endothelial cells within 30 min. These data suggest that caveolae may play a role in the uptake and transport of EGCG in endothelial cells. These studies provide a novel target through which EGCG functions to protect against inflammatory diseases such as atherosclerosis.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Additional advisors: Ada Elgavish, Vithal Ghanta, Hui-Chen Hsu, Thane Wibbels. Description based on contents viewed June 11, 2008; title from title screen. Includes bibliographical references.

Universiẗat, Diss., 2004--München.

No description available.

Background: Fetal alcohol spectrum disorder (FASD) is a challenging public health problem. Previous studies have found an association between FASD and oxidative stress. In the present study, we assessed the role of oxidative stress in ethanol-induced embryonic damage and the effect of (-)-epigallocatechin-3-gallate (EGCG), a powerful antioxidant extracted from green tea, on the development of FASD in a murine model.Methods: Pregnant female mice were given intraperitoneal ethanol (25%, 0.005 to 0.02 ml/g) on gestational day 8 (G8) to establish the FASD model. On G10.25, mice were sacrificed and embryos were collected and photographed to determine head length (HL), head width (HW), and crown rump length (CRL). For mice given EGCG, administration was through a feeding tube on G7 and G8 (dose: 200, 300, or 400 mg/kg/d, the total amount for a day was divided into 2 equal portions). G10.25 embryos were evaluated morphologically. Brain tissues of G9.25 embryos were used for RT-PCR and western blotting of neural marker genes and proteins and detection of oxidative stress indicators.Results: Administration of ethanol to pregnant mice on G8 led to the retardation of embryonic growth and down-regulation of neural marker genes. In addition, administration of ethanol (0.02 ml/g) led to the elevation of oxidative stress indicators [hydrogen peroxide (H2O2) and malondialdehyde (MDA)]. Administration of EGCG on G7 and G8 along with ethanol on G8 ameliorated the ethanol-induced growth retardation. Mice given EGCG (400 mg/kg/d) along with ethanol had embryo sizes and neural marker genes expression similar to the normal controls. Furthermore, EGCG (400 mg/kg on G7 and G8) inhibited the increase in H2O2 and MDA.Conclusions: In a murine model, oxidative stress appears to play an important role in ethanol-induced embryonic growth retardation. EGCG can prevent some of the embryonic injuries caused by ethanol.

Duchenne muscular dystrophy is a debilitating genetic disorder characterized by severe muscle wasting and early death in affected boys. The primary cause of this disease is mutations in the dystrophin gene resulting in the loss of the dystrophin protein from the plasma membrane of muscle fibers. In the absence of dystrophin, muscles undergo massive muscle degeneration and inflammation. Inflammation is believed to contribute substantially to dystrophic muscle pathology. The transcription factor NF-κB regulates inflammatory gene expression and provides a logical target for therapeutic treatments. Green tea extract and its primary polyphenol, epigallocatechin gallate, have been shown to have anti-inflammatory properties and to improve dystrophic muscle pathology. The purpose of these studies was to determine if dietary treatment with green tea extract or epigallocatechin gallate administered prior to disease onset could reduce dystrophic muscle pathology during the early disease time course and identify potential mechanisms through which NF-κB may be involved. Green tea extract has been shown to decrease muscle pathology and increase muscle function in mdx mice, a dystrophic mouse model. These changes have been attributed to the antioxidant potential of epigallocatechin gallate; however, other mechanisms such as suppression of the inflammatory response have not been evaluated. In the studies reported herein, both green tea extract and epigallocatechin gallate significantly decreased muscle pathology in mdx mice when provided in their diets prior to disease onset. In green tea extract (0.25% and 0.5%) treated mdx mice, serum creatine kinase, a systemic marker of muscle damage, was decreased by 85% at age 42 days. Normal fiber morphology in the tibialis anterior muscle was increased by 32% at this age (P≤0.05). The primary histopathological change was a 21% decrease in regenerating fibers (P≤0.05). NF-κB staining in central nuclei of regenerating fibers was decreased by 34% (P≤0.05). In epigallocatechin gallate (0.1%) treated mdx mice, serum creatine kinase was unchanged; however, normal fiber morphology in the tibialis anterior was increased by 20% at ages 28 and 42 days (P≤0.05). At age 42 days, the primary histopathological change was a 21% decrease in regenerating fibers (P≤0.05). NF-κB staining in central nuclei of regenerating muscle fibers was decreased by 21% at this age (P≤0.05). Epigallocatechin gallate appears to be the primary polyphenol of green tea extract responsible for many of the beneficial changes in dystrophic muscle. These data suggest that both green tea extract and epigallocatechin gallate decrease NF-κB activity in regenerating fibers resulting in reduced muscle pathology. Complimentary and alternative medicine approaches, including the use of green tea, provide important therapeutic options for ameliorating Duchenne muscular dystrophy. Green tea extract and epigallocatechin gallate are effective at decreasing muscle pathology potentially by reducing NF-κB activity in regenerating fibers in mdx mice. Use of these botanicals appears to elicit a beneficial response in dystrophic muscle that may ultimately lead to effective therapies for patients with this incurable disease. / Ph. D.