Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

by Koo Po Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 120-130). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (Chinese version) --- p.iv / Content --- p.vii / List of Abbreviations --- p.xi / List of Figures and Tables --- p.xiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Saponins --- p.1 / Chapter 1.2 --- Structure of Saponin --- p.2 / Chapter 1.2.1 --- Triterpene Class --- p.2 / Chapter 1.2.2 --- Steroid Class --- p.3 / Chapter 1.2.2.1 --- Spirostanol Glycoside --- p.4 / Chapter 1.2.2.2 --- Furostanol Glycoside --- p.4 / Chapter 1.2.3 --- Steroid Alkaloid Class --- p.5 / Chapter 1.3 --- Steroidal Saponin as Anti-Tumor Drug --- p.5 / Chapter 1.4 --- Possible Anti-Tumor Action Mechanisms of Steroid Saponin --- p.6 / Chapter 1.4.1 --- Direct Cytotoxic and Growth Inhibitory Effects --- p.7 / Chapter 1.4.2 --- Immune-Modulatory Effects --- p.8 / Chapter 1.5 --- Possible Anti-Carcinogenicity Action Mechanism of Saponin --- p.9 / Chapter 1.5.1 --- Saponin Binding to Bile Acids --- p.9 / Chapter 1.6 --- Saponin as Cardioactive Drug --- p.9 / Chapter 1.7 --- Liver Cancer --- p.10 / Chapter 1.7.1 --- Prevalence of Hepatocellular Carcinoma (HCC) --- p.11 / Chapter 1.8 --- Coronary Heart Disease (CHD) --- p.12 / Chapter 1.8.1 --- Prevalence and Risk Factors of CHD --- p.12 / Chapter 1.9 --- Diosgenin --- p.14 / Chapter 1.10 --- Hong Kong (HK) Products --- p.15 / Chapter 1.10.1 --- HK-18 (Polyphyllin D) --- p.15 / Chapter 1.11 --- DI AO XIN XUE KANG (DI AO) --- p.17 / Chapter 1.12 --- Aims of My Project --- p.20 / Chapter 1.12.1 --- In Vitro Study of the Effect of HK-18 on Human Hepatocellular Carcinoma Cell Line (HepG2) --- p.21 / Chapter 1.12.2 --- In Vivo Study of the Effect of HK-18 by Human Liver Tumor HepG2 Cells-Bearing Nude Mice Model --- p.21 / Chapter 1.12.3 --- In Vitro Study of the Effect of HK-18 on Multidrug- Resistant Human Hepatocellular Carcinoma Cell Line (R-HepG2) --- p.22 / Chapter 1.12.4 --- Myocardial Ischemia-Reperfusion (IR) Injury in Isolated- Perfused Rat Heart Model --- p.23 / Chapter 1.12.5 --- Effect of DI AO Pretreatment on Global IR Injury --- p.26 / Chapter 1.12.6 --- Effect of DI AO Pretreatment on Isoproterenol-Induced Myocardial Injury in Rats --- p.26 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Cell Lines and Culture Medium / Chapter 2.1.1.1 --- Cell Lines --- p.28 / Chapter 2.1.1.2 --- Culture Medium --- p.29 / Chapter 2.1.2 --- Chemicals --- p.30 / Chapter 2.1.3 --- Buffers and Reagents --- p.31 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- In Vitro Studies --- p.33 / Chapter 2.2.1.1 --- In Vitro Cytotoxicity --- p.33 / Chapter 2.2.1.2 --- Cell Cycle Analysis by Flow Cytometry --- p.34 / Chapter 2.2.1.3 --- Maintenance of P-glycoprotein in R-HepG2 cells by Doxorubicin and HK-18 --- p.35 / Chapter 2.2.1.4 --- Assessment of DNA Fragmentation --- p.36 / Chapter 2.2.2 --- In Vivo Assessment of the Anti-Tumor Activity of HK-18 --- p.37 / Chapter 2.2.2.1 --- Animals and Tumor Inoculation --- p.37 / Chapter 2.2.2.2 --- Drug Administration --- p.37 / Chapter 2.2.2.3 --- Assessment of the Tumor Size and Tumor Weight --- p.38 / Chapter 2.2.2.4 --- Plasma Preparation --- p.38 / Chapter 2.2.2.5 --- Measurement of the Plasma Enzyme Activity --- p.39 / Chapter 2.2.3 --- Isoproterenol (ISO)-Induced Myocardial Injury (Rat Model) --- p.40 / Chapter 2.2.3.1 --- Animals --- p.40 / Chapter 2.2.3.2 --- Drug Preparations --- p.40 / Chapter 2.2.3.3 --- Animal Treatment --- p.41 / Chapter 2.2.3.4 --- Preparation of Myocardial Tissue Homogenate --- p.41 / Chapter 2.2.3.5 --- Preparation of Cytosolic Fraction of Heart Homogenates --- p.42 / Chapter 2.2.3.6 --- Myocardial Antioxidant Enzyme Activity --- p.42 / Chapter 2.2.3.6.1 --- Glutathione Reductase (GRD) --- p.42 / Chapter 2.2.3.6.2 --- Glutathione S-Transferases (GST) --- p.43 / Chapter 2.2.3.7 --- Myocardial Antioxidant Capacity --- p.43 / Chapter 2.2.3.7.1 --- Myocardial Malondialdehyde (MDA) Content --- p.43 / Chapter 2.2.3.7.2 --- Myocardial Thiol Content --- p.44 / Chapter 2.2.3.7.3 --- Tert-Butylhydroperoxide (tBHP)-Induced Thiol Depletion --- p.45 / Chapter 2.2.3.7.4 --- TBHP-Induced Thiobarbituric Acid-Reactive Substances (TBARS) Formation --- p.45 / Chapter 2.2.4 --- Myocardial Ischemia-Reperfusion (IR) Injury --- p.46 / Chapter 2.2.4.1 --- Langendorff Isolated Perfused Rat Heart --- p.46 / Chapter 2.2.4.1.1 --- Preparation of Perfusion Buffer --- p.46 / Chapter 2.2.4.1.2 --- Preparation of Isolated Rat Heart --- p.47 / Chapter 2.2.4.1.3 --- Myocardial Global Ischemia-Reperfusion Injury --- p.49 / Chapter 2.2.4.1.4 --- Contractile Force Recovery --- p.49 / Chapter 2.2.5 --- Statistical Analysis --- p.50 / Chapter Chapter 3 --- Study of HK-18 on Anti-Tumor Effect / Chapter 3.1 --- In Vitro Study of HK-18 on Human Hepatoma Carcinoma Cell Line (HepG2) --- p.51 / Chapter 3.1.1 --- The Effect of HK-18 on Cell Proliferation of HepG2 Cells by MTT Assay --- p.52 / Chapter 3.1.2 --- DNA Fragmentation Assay --- p.54 / Chapter 3.1.3 --- The Effect of HK-18 on Cell Cycle Phase Distribution --- p.57 / Chapter 3.2 --- In Vivo Study of HK-18 on HepG2-Inoculated Nude Mice --- p.61 / Chapter 3.2.1 --- Assessment of the Anti-Tumor Activity of HK-18 --- p.61 / Chapter 3.2.2 --- The Effect of HK-18 Towards Heart Tissue --- p.65 / Chapter 3.2.3 --- In Vitro Study of HK-18 on Multidrug Resistant Cell Line (R-HepG2) --- p.68 / Chapter 3.2.4 --- The Comparison of the Cytotoxicity of DOX on the Parental Cells and Resistant Cells of HepG2 --- p.69 / Chapter 3.2.5 --- The Effect of HK-18 on Cell Proliferation of R-HepG2 Cells by MTT Assay --- p.72 / Chapter 3.2.6 --- DNA Fragmentation Assay --- p.74 / Chapter 3.2.7 --- The Effect of HK-18 on Cell Cycle Phase Distribution --- p.77 / Chapter 3.2.8 --- The Relationship Between HK-18 and P-glycoprotein --- p.80 / Chapter Chapter 4 --- Study of the Cardioprotective Effect of DI AO / Chapter 4.1 --- Myocardial Ischemia-Reperfusion (IR) Injury in Isolated- Perfused Rat Heart --- p.82 / Chapter 4.1.1 --- Time Course of Global Ischemia-Reperfusion-Induced LDH Leakage --- p.82 / Chapter 4.1.2 --- Effect of DI AO Pretreatment on Global IR Injury --- p.85 / Chapter 4.1.2.1 --- LDH Leakage --- p.85 / Chapter 4.1.2.2 --- Contractile Force --- p.87 / Chapter 4.2 --- Isoproterenol-Induced Myocardial Injury in Rats --- p.89 / Chapter 4.2.1 --- Effect of DI AO Pretreatment --- p.89 / Chapter 4.2.2 --- Alternations in the Activity of Myocardial Antioxidant Enzymes --- p.91 / Chapter 4.2.3 --- Alternations in Myocardial Antioxidant Capacity --- p.94 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- The Significance of the Study of Saponin in the Treatment of Liver Cancer and Heart Injury --- p.96 / Chapter 5.2 --- Effect of HK-18 on Human Hepatocellular Carcinoma Cell --- p.101 / Chapter 5.3 --- Mechanism Study of Anti-Tumor Effect of HK-18 --- p.102 / Chapter 5.4 --- Cytotoxicity of HK-18 Toward Normal Tissue --- p.105 / Chapter 5.5 --- Effect of HK-18 on Multidrug Resistant Human Hepatocellular Carcinoma / Chapter 5.6 --- Protective Effect of DI AO Against Isoproterenol (ISO)- Induced Myocardial Injury --- p.110 / Chapter 5.7 --- Cardioprotective Effect of DI AO Against Ischemia- Reperfusion (IR) Injury --- p.111 / Chapter 5.8 --- Effect of DI AO Pretreatment on Myocardial Antioxidant Enzymes Activities and Antioxidant Capacity --- p.113 / Chapter 5.9 --- Conclusion and Future Prospect --- p.117 / Chapter Chapter 6 --- References --- p.121

INTRODUÇÃO: Líquen plano (LP) é uma doença mucocutânea de natureza inflamatória crônica de etiologia ainda desconhecida. Alterações na resposta imune inata, como aos padrões moleculares associados à patógenos (PAMPs) e padrões moleculares associados ao dano (DAMPs) podem levar à inflamação crônica e contribuir com a patogênese do LP. OBJETIVO: Avaliar o efeito da ativação via o DAMP S100A8 e o receptor Toll-like 4 (TLR-4) em células Natural killer (NK) e TCD8 citotóxicas e suas subpopulações de memória/efetoras em pacientes com LP. MÉTODOS: Foram selecionados 25 pacientes com LP (22 mulheres, 3 homens) com idade média de 43,46 anos ± 8,46 e um grupo controle com 25 indivíduos (22 mulheres, 3 homens) com idade média de 42 anos ± 5,5. A determinação transcricional e da expressão por imunohistoquimica dos DAMPs S100A8, HMGB-1 e de TLR-4 e RAGE foi realizada em biópsias de lesões cutâneas de indivíduos com LP, e os níveis séricos de S100A8, HMGB-1, MICA e MICB foram determinados por ELISA. As células mononucleares (CMNs) de sangue periférico foram avaliadas por citometria de fluxo quanto a frequência de TNF, IL-1beta e o marcador de desgranulação CD107a em células TCD8+ e células NK CD56+ e suas subpopulações. A avaliação da via de sinalização de TLR em células TCD8+ purificadas e ativadas com S100A8 foi analisada por PCR array e a determinação da expressão de mRNA dos componentes do inflamassoma em células TCD8+ ativadas com S100A8 por PCR em tempo real. RESULTADOS: Foi evidenciado nos indivíduos com LP elevada expressão da proteína S100A8 nas lesões cutâneas e de HMGB-1, TLR-4 e RAGE na derme, em paralelo ao aumento da expressão de mRNAs para S100A8 e S100A9 e diminuição de RAGE. Além disto, uma elevação dos níveis séricos do dímero S100A8/A9 foi detectada nos pacientes comparados aos controles, ao contrário do DAMP HMGB-1 que mostrou níveis similares em ambos os grupos. A influência do S100A8 em células TCD8+ e células NK, foi analisada em CMNs pela ativação com o lipopolissacáride e a proteína recombinante S100A8, ambos ligantes de TLR-4. Nos indivíduos com LP foi detectado aumento da resposta citotóxica de linfócitos TCD8+ e células NK CD56bright pela expressão do marcador de desgranulação CD107a por citometria de fluxo. A proteína S100A8 foi capaz de induzir a expressão de genes pró-inflamatórios como IL-1beta, TNF e IL-6 em células TCD8+ de pacientes com LP em contraste com os indivíduos saudáveis que mostraram expressão IL-10 e IFN tipo I. As células TCD8+ de indivíduos com LP ativadas ou não com S100A8 expressam transcritos de NLRP1, NLRP3 e AIM-2 e produzem IL-1beta em níveis similares a controles saudáveis. Além disso, células TCD8+ ativadas com S100A8 mostraram aumento de expressão TLR3, TLR5, TLR7 e TLR8 na doença comparada às biopsias de controles. O aumento da resposta TCD8+ citotóxica foi principalmente mediado pelo subtipo de memória efetora (TEM, CCR7- CD45RA-). Elevação basal da expressão do receptor ativador NKG2D e inibidor NKG2A foi observado em células NK CD56dim nos indivíduos com LP e um nível similar do ligante solúvel MICB em ambos os grupos. CONCLUSÃO: Estes resultados evidenciam que componentes da imunidade inata, como a proteína S100A8 pode contribuir na manutenção do perfil inflamatório do LP / BACKGROUND: Lichen planus (LP) is a mucocutaneous inflammatory chronic disease of unknown etiology. Alterations in the innate immune response such as the pathogen-associated molecular pattern (PAMPs) and damage-associated molecular pattern (DAMPs) can lead to chronic inflammation and contribute to the pathogenesis of LP. OBJECTIVE: Evaluate the effect of the activation trough the DAMP S100A8 and the Toll-like receptor 4 (TLR-4) on the Natural killer cells (NK) and cytotoxic TCD8 cells and their memory / effector subsets in LP disease. METHODS: We selected 25 patients with LP (22 women, 3 men) with a mean age of 43.46 years ± 8.46 and a control group of 25 subjects (22 women, 3 men) with a mean age of 42 ± 5, 5. The transcriptional determination and protein expression by immunohistochemistry of DAMPs, S100A8 and HMGB-1 as well as TLR-4 and RAGE was performed on biopsies of skin lesions from patients with LP, and serum levels of S100A8, HMGB-1, MICA and MICB were determined by ELISA. Peripheral blood mononuclear cells (PBMCs) were assessed by flow cytometry to evaluate the frequency of TNF, IL-1beta and the degranulation marker CD107a in CD8+ T cells and CD56 + NK cells and their subsets. The evaluation of the TLR signaling pathway in purified CD8 + T cells activated with S100A8 were analyzed by PCR array and the determination of mRNA expression of inflammasome components on CD8 + T cells activated by S100A8 was measured by real time PCR. RESULTS: It was shown in the LP individuals an increased expression of the S100A8 protein in the cutaneous lesions and HMGB-1, TLR-4 and RAGE in the dermis, in parallel to increased level of mRNAs for S100A8 and S100A9 and decreased expression of RAGE. Moerover, increased serum levels of the dimer S100A8 / A9 was detected in patients compared to controls, in contrast to DAMP HMGB1 that revealed similar levels in both groups. The influence of S100A8 in CD8 + T cells and NK cells, was analyzed in PBMC activating with lipopolysaccharide and recombinant protein S100A8, both ligands of TLR-4. It was detected in LP individuals, an increased cytotoxic response of CD8+ T lymphocytes and CD56bright NK cells trough CD107a degranulation marker expression. The S100A8 protein was able to induce the pro-inflammatory genes expressions such as IL-1beta, TNF and IL-6 in CD8 + T cells of LP patients in contrast to healthy subjects who promoted IL-10 expression and type I IFN. CD8 + T cells of LP individuals activated or not with S100A8 are able to express NLRP1, NLRP3 and AIM-2 and IL-1beta production at similar levels to healthy controls. Moreover, CD8 + T cells activated with S100A8 showed increased expression of TLR3, TLR5, TLR7 and TLR8 in LP compared to biopsies from healthy controls. The increased CD8 + T cells cytotoxic response was mediated by the subtype of effector memory (TEM CD45RA- CCR7). The increased baseline expression of activating receptor NKG2D and the inhibitory NKG2A in the NK CD56dim cells in LP individulas, and the similar level of MICB soluble in both groups. CONCLUSION: These results shows that innate immunity components, such as S100A8 protein may contribute to the maintenance of LP inflammatory profile

INTRODUÇÃO: Líquen plano (LP) é uma doença mucocutânea de natureza inflamatória crônica de etiologia ainda desconhecida. Alterações na resposta imune inata, como aos padrões moleculares associados à patógenos (PAMPs) e padrões moleculares associados ao dano (DAMPs) podem levar à inflamação crônica e contribuir com a patogênese do LP. OBJETIVO: Avaliar o efeito da ativação via o DAMP S100A8 e o receptor Toll-like 4 (TLR-4) em células Natural killer (NK) e TCD8 citotóxicas e suas subpopulações de memória/efetoras em pacientes com LP. MÉTODOS: Foram selecionados 25 pacientes com LP (22 mulheres, 3 homens) com idade média de 43,46 anos ± 8,46 e um grupo controle com 25 indivíduos (22 mulheres, 3 homens) com idade média de 42 anos ± 5,5. A determinação transcricional e da expressão por imunohistoquimica dos DAMPs S100A8, HMGB-1 e de TLR-4 e RAGE foi realizada em biópsias de lesões cutâneas de indivíduos com LP, e os níveis séricos de S100A8, HMGB-1, MICA e MICB foram determinados por ELISA. As células mononucleares (CMNs) de sangue periférico foram avaliadas por citometria de fluxo quanto a frequência de TNF, IL-1beta e o marcador de desgranulação CD107a em células TCD8+ e células NK CD56+ e suas subpopulações. A avaliação da via de sinalização de TLR em células TCD8+ purificadas e ativadas com S100A8 foi analisada por PCR array e a determinação da expressão de mRNA dos componentes do inflamassoma em células TCD8+ ativadas com S100A8 por PCR em tempo real. RESULTADOS: Foi evidenciado nos indivíduos com LP elevada expressão da proteína S100A8 nas lesões cutâneas e de HMGB-1, TLR-4 e RAGE na derme, em paralelo ao aumento da expressão de mRNAs para S100A8 e S100A9 e diminuição de RAGE. Além disto, uma elevação dos níveis séricos do dímero S100A8/A9 foi detectada nos pacientes comparados aos controles, ao contrário do DAMP HMGB-1 que mostrou níveis similares em ambos os grupos. A influência do S100A8 em células TCD8+ e células NK, foi analisada em CMNs pela ativação com o lipopolissacáride e a proteína recombinante S100A8, ambos ligantes de TLR-4. Nos indivíduos com LP foi detectado aumento da resposta citotóxica de linfócitos TCD8+ e células NK CD56bright pela expressão do marcador de desgranulação CD107a por citometria de fluxo. A proteína S100A8 foi capaz de induzir a expressão de genes pró-inflamatórios como IL-1beta, TNF e IL-6 em células TCD8+ de pacientes com LP em contraste com os indivíduos saudáveis que mostraram expressão IL-10 e IFN tipo I. As células TCD8+ de indivíduos com LP ativadas ou não com S100A8 expressam transcritos de NLRP1, NLRP3 e AIM-2 e produzem IL-1beta em níveis similares a controles saudáveis. Além disso, células TCD8+ ativadas com S100A8 mostraram aumento de expressão TLR3, TLR5, TLR7 e TLR8 na doença comparada às biopsias de controles. O aumento da resposta TCD8+ citotóxica foi principalmente mediado pelo subtipo de memória efetora (TEM, CCR7- CD45RA-). Elevação basal da expressão do receptor ativador NKG2D e inibidor NKG2A foi observado em células NK CD56dim nos indivíduos com LP e um nível similar do ligante solúvel MICB em ambos os grupos. CONCLUSÃO: Estes resultados evidenciam que componentes da imunidade inata, como a proteína S100A8 pode contribuir na manutenção do perfil inflamatório do LP / BACKGROUND: Lichen planus (LP) is a mucocutaneous inflammatory chronic disease of unknown etiology. Alterations in the innate immune response such as the pathogen-associated molecular pattern (PAMPs) and damage-associated molecular pattern (DAMPs) can lead to chronic inflammation and contribute to the pathogenesis of LP. OBJECTIVE: Evaluate the effect of the activation trough the DAMP S100A8 and the Toll-like receptor 4 (TLR-4) on the Natural killer cells (NK) and cytotoxic TCD8 cells and their memory / effector subsets in LP disease. METHODS: We selected 25 patients with LP (22 women, 3 men) with a mean age of 43.46 years ± 8.46 and a control group of 25 subjects (22 women, 3 men) with a mean age of 42 ± 5, 5. The transcriptional determination and protein expression by immunohistochemistry of DAMPs, S100A8 and HMGB-1 as well as TLR-4 and RAGE was performed on biopsies of skin lesions from patients with LP, and serum levels of S100A8, HMGB-1, MICA and MICB were determined by ELISA. Peripheral blood mononuclear cells (PBMCs) were assessed by flow cytometry to evaluate the frequency of TNF, IL-1beta and the degranulation marker CD107a in CD8+ T cells and CD56 + NK cells and their subsets. The evaluation of the TLR signaling pathway in purified CD8 + T cells activated with S100A8 were analyzed by PCR array and the determination of mRNA expression of inflammasome components on CD8 + T cells activated by S100A8 was measured by real time PCR. RESULTS: It was shown in the LP individuals an increased expression of the S100A8 protein in the cutaneous lesions and HMGB-1, TLR-4 and RAGE in the dermis, in parallel to increased level of mRNAs for S100A8 and S100A9 and decreased expression of RAGE. Moerover, increased serum levels of the dimer S100A8 / A9 was detected in patients compared to controls, in contrast to DAMP HMGB1 that revealed similar levels in both groups. The influence of S100A8 in CD8 + T cells and NK cells, was analyzed in PBMC activating with lipopolysaccharide and recombinant protein S100A8, both ligands of TLR-4. It was detected in LP individuals, an increased cytotoxic response of CD8+ T lymphocytes and CD56bright NK cells trough CD107a degranulation marker expression. The S100A8 protein was able to induce the pro-inflammatory genes expressions such as IL-1beta, TNF and IL-6 in CD8 + T cells of LP patients in contrast to healthy subjects who promoted IL-10 expression and type I IFN. CD8 + T cells of LP individuals activated or not with S100A8 are able to express NLRP1, NLRP3 and AIM-2 and IL-1beta production at similar levels to healthy controls. Moreover, CD8 + T cells activated with S100A8 showed increased expression of TLR3, TLR5, TLR7 and TLR8 in LP compared to biopsies from healthy controls. The increased CD8 + T cells cytotoxic response was mediated by the subtype of effector memory (TEM CD45RA- CCR7). The increased baseline expression of activating receptor NKG2D and the inhibitory NKG2A in the NK CD56dim cells in LP individulas, and the similar level of MICB soluble in both groups. CONCLUSION: These results shows that innate immunity components, such as S100A8 protein may contribute to the maintenance of LP inflammatory profile