Dissertations / Theses on the topic 'Antifibrotics'

Systemic sclerosis (SSc) – is one of the most complicated and fatal systemic diseases, and the lack of effective therapy is very evident. The tyrosine kinase inhibitor imatinib mesylate (IM) was shown to inhibit TGF-β and PDGF signaling pathways and prevent the development of dermal fibrosis upon challenge with bleomycin in murine model of SSc. The aim of therapy is not only to stop disease progression, but even induce regression of preexisting fibrosis. On other hand, blocking TGF-β and PDGF signaling in angiogenesis might worsen the vascular manifestations of SSc. We found important to evaluate effectiveness of IM for the treatment of pre-established tissue fibrosis and to exclude that the anti-fibrotic effects of IM are complicated by inhibitory effects on endothelial cell functions. Aim of the study: assess the effect of IM on the process of fibrosis and endothelium in experimental models of systemic sclerosis and cell cultures. Objectives of the study: assess the effectiveness of IM on murine models of established fibrosis; evaluate if IM has an effect on basal functions of endothelial cells; assess effect of IM on the process of angiogenesis. We have shown that IM exerts potent antifibrotic effects in two different models of SSc. Imatinib was effective for prevention of fibrosis and for treatment of established dermal fibrosis. We’ve demonstrated that IM does not inhibit major functions of endothelial cells. Thus, IM might not augment further the preexisting vascular... [to full text]
Sisteminė sklerozė (SSc) – viena sunkiausių ir fatališkiausių autoimuninių sisteminių reumatinių ligų, o bazinių vaistų stygius, šiai ligai gydyti, itin didelis. Į onkologinę klinikinę praktiką įdiegtas tirozinkinazių inhibitorius – imatinibo mezilatas(IM). IM blokuoja TGF-β ir PDGF intraląstelinio signalo perdavimą ir taip sąlygoja fibrozės prevenciją SSc pelių modelyje. Mums buvo svarbu išsiaiškinti, ar imatinibas gali turėti įtakos ne tik prevencijai, bet ir susiformavusiai fibrozei. Be to TGF-β ir PDGF blokavimas angiogenezėje, galėtų riboti daug žadančio fibrozės inhibitoriaus IM naudojimą gydant SSc. Darbo tikslas: įvertinti imatinibo mezilato poveikį fibrozės procesui ir endoteliui sisteminės sklerozės eksperimentiniuose modeliuose ir ląstelių kultūrose. Darbo uždaviniai: įvertinti imatinibo efektyvumą neuždegiminiame SSc modelyje ir patikrinti imatinibo mezilato efektyvumą uždegiminiame suformuotos fibrozės modelyje; ištirti, ar terapinės imatinibo mezilato koncentracijos daro neigiamą poveikį gyvybinėms endotelio funkcijoms; įvertinti imatinibo mezilato poveikį angiogenezės etapams. Mūsų gauti duomenys rodo, kad: IM ne tik sustabdė bet ir paskatino jau egzistuojančios (bleomicino sukeltos) odos firbrozės regresiją; IM ryškiai sumažino poodžio ir odos storį, bei normalizavo miofibroblastų skaičių Tsk-1 pelėse; IM neturėjo poveikio endotelio ląstelių bazinėms funkcijoms; IM neturėjo neigiamo poveikio angiogenezės etapams.

Fibrosis is a complex chronic disease characterized by a persistent repair response. Its pathogenesis is poorly understood but it is known to require the activity of TGFbeta and extracellular matrix (ECM) tension, both of which drive fibroblasts to transition into a myofibroblast phenotype (FMT). As the effector cells of repair, myofibroblasts migrate to the site of injury to deposit excessive amounts of type I collagen as well as other matrix proteins and stimulate high levels of contraction. Such actions are vital for wound closure and re-establishing the normal tissue structure however, limit normal tissue repair and remodeling when left uncontrolled. The cellular and molecular mechanisms regulating fibrosis are not well defined but hallmarks of the myofibroblast phenotype include major cytoskeletal rearrangements, formation of alpha-smooth muscle actin (alpha-sma)-rich stress fibers, and mature focal adhesions (FAs). Prominent signalling pathways that support myofibroblastic differentiation include TGFβ-dependent signalling, Wnt/beta-catenin, Ras/Raf/MAP kinase and Rho-Associated Kinase (ROCK)-dependent pathways. Resolution of the myofibroblast phenotype is vital to prevent continual wound repair, otherwise further deposition of ECM proteins substantiate tissue fibrosis. It is well known that PGE2 inhibits human FMT through mechanisms that are vaguely known. Our laboratory has created a strategy to better understand the signalling pathways in which the antifibrotic mediator PGE2 prevents myofibroblast development. Our work describes a draft of the complete PGE2-dependent phosphoproteome/GalphaS signalosome in fibroblast-like cell phenotypes (by PhosphoScanTM, Cell Signalling), which include proteins associated with cellular differentiation and cytoskeletal/adhesion structure. Furthermore, we have identified the specific amino acid sequence of novel phosphorylation sites, vital information towards the control process of a protein's activity. Examples of PGE2-induced phosphoproteins associated with myofibroblastic activities include beta-catenin (differentiation), RIN1 (Ras-inhibitor) (migration), ROCK2 (contraction), as well as various proteins upstream and downstream of ROCK. Giving weight to our strategy, mutational analysis of the ROCK2 serine 1379 phosphosite prevented PGE2 inhibition of the enzyme, potentially inhibiting myofibroblast contraction. Furthermore, mutation of the RIN1 serine 291 and 292 phosphosites prevented the inhibition of PGE2 on TGFβ induced migration, another vital characteristic of myofibroblasts. Other observations included a marked reversal of cytoskeletal protein phosphorylation, loss of stress fiber/FA formation, suppression of alpha smooth muscle actin (alpha-sma) expression, and blockade of the FMT through activation of the "phospholipase/cyclooxygenase/mPGE synthase/prostaglandin E2 biosynthetic axis" in human fibroblasts. Ultimately, the results will encourage PGE2 mimetics as potential therapeutics for the treatment of fibrosis.
La fibrose est une maladie chronique complexe caractérisée par une réaction de réparation persistante. Sa pathogénie est mal comprise mais on la connaît pour exiger l'activité de TGFbeta et une tension extracellulaire de la matrice (ECM), qui dirige des fibroblastes dans une transition vers un phénotype de myofibroblast (FMT). Comme cellules effectrices de la réparation, les myofibroblasts migrent au site des dommages pour déposer des quantités excessives de collagène de type I aussi bien que d'autres protéines de matrice et pour stimuler de hauts niveaux de contraction. De telles actions sont essentielles pour la fermeture de blessure et le rétablissement de la structure normale du tissu cependant, laissé incontrôlé elle limite la réparation normale et le remodelage du tissu. Les mécanismes cellulaires et moléculaires réglant la fibrose ne sont pas bien définis mais les marqueurs du phénotype de myofibroblastes incluent des réarrangements cytosquelettiques importants, la formation des fibres de stress riches en alpha-actine du muscle lisses (alpha-sma), et des adhérences focales (FAs). Les voies importantes de signalisation qui soutiennent la différenciation myofibroblastique incluent une signalisation TGFbeta-dépendante, la Wnt/beta-catenin, la kinase de Ras/Raf/MAP et les voies dépendantes de la kinase Rho-Associée (ROCK). La résolution du phénotype de myofibroblast est essentielle pour empêcher la réparation continuelle de la blessure, autrement davantage de dépôt des protéines extracellulaire de la matrice explique la fibrose du tissu. Il est bien connu que la PGE2 empêche FMT humain par des mécanismes qui sont vaguement connus. Notre laboratoire a créé une stratégie pour mieux comprendre les voies de signalisation dans lesquelles la PGE2, un médiateur antifibrotique , empêche le développement de myofibroblast. Notre travail décrit un croquis complet du phosphoprotéome/GalphaS signalosome dépendante de la PGE2 dans les cellules avec le phénotype fibroblaste (par PhosphoScanTM, signalisation cellulaires), qui incluent des protéines liées à la différenciation cellulaire et à la structure cytosquelettique/adhérence. En outre, nous avons identifié la séquence des acides aminés spécifique de sites inédits de phosphorylation, une information indispensable vers le procédé de contrôle de l'activité d'une protéine. Des exemples de phosphoprotéines induites par la PGE2 liées aux activités myofibroblastic incluent le beta-cateninix(différenciation), le RIN1 (Ras-inhibiteur) (migration), le ROCK2 (contraction), aussi bien que les diverses protéines en amont et en aval de la ROCK. Donnant de la considération à notre stratégie, l'analyse mutationnelle du phosphosite ROCK2 a empêché l'inhibition de l'enzyme par la PGE2, inhibant potentiellement la contraction myofibroblastique. En outre, la mutation du phosphosite RIN1 a empêché l'inhibition de la PGE2 sur la migration induite par le TGFbeta, une autre caractéristique essentielle des myofibroblasts. D'autres observations inclus une inversion marquée de la phosphorylation des protéines du cytosquelette, la perte de formation de fibre de stress et des adhérences focales, la suppression de l'expression de l'alpha-actine du muscle lisses (alpha-sma), et le blocus du FMT par l'activation « de l'axe de biosynthèse des phospholipase/cyclooxygenase/mPGE synthase/prostaglandine E2» dans les fibroblastes humains. Finalement, les résultats encourageront la PGE2 en tant que cible thérapeutique potentielle pour le traitement de la fibrose.

The calcineurin inhibitors, cyclosporine and tacrolimus, are pivotal immunosuppressants in renal transplantation, but produce a cascade of acute (functional) and chronic (fibrotic) injurious events that contribute to chronic allograft nephropathy (CAN). Using the rat salt depletion model of calcineurin inhibitor toxicity, this study aimed to examine the effects of clinically relevant combinations of cyclosporine, tacrolimus and sirolimus on renal functional, structural and molecular markers of injury. Further, the effect of pirfenidone when added to these drug combinations was examined. There were differences in the effects of cyclosporine and tacrolimus on functional and molecular variables, with tacrolimus displaying more favourable results. As sole therapy, sirolimus had no effect on renal function or messenger RNA expression. Deterioration in renal function and a deleterious effect on molecular markers of fibrosis were seen when cyclosporine and sirolimus were combined at high doses; at lower doses, favourable outcomes for these end-points were elicited. When sirolimus and tacrolimus were combined, renal function worsened and the beneficial molecular effects of tacrolimus were reversed. Pirfenidone's actions were non-dose dependent and beneficial effects for renal function and molecular markers were demonstrated. The effect of pirfenidone on renal function has not previously been described. There were no differences in urinary protein or interstitial fibrosis measurements across the groups. Without fibrosis, it is impossible to say whether pirfenidone acted in a truly antifibrotic manner. However, the molecular changes possibly represent interim markers of fibrosis, suggesting such an effect may have been developing.

Orientador: James Venturini
Resumo: Paracoccidioidomicose (PCM) é uma micose sistêmica causada por fungos do gênero Paracoccidioides; suas principais formas clínicas são aguda/subaguda, crônica e residual. A PCM é uma doença restrita a países da América Latina com maior incidência no Brasil, especialmente entre os trabalhadores rurais. A maioria dos pacientes com a forma crônica da doença, mesmo após tratamento eficaz, apresentam sequelas, incluindo fibrose pulmonar e adrenal. Os problemas sociais, econômicos e psicológicos desencadeados pela fibrose pulmonar são subestimados; além disso, a fibrose na PCM permanece negligenciada, uma vez que não há tratamento. Dessa forma, o estudo teve por objetivo investigar a influência de drogas com potencial antifibrótico (pentoxifilina - PTX, azitromicina - AZT e talidomida - Thal) associadas aos tratamentos antifúngicos com itraconazol - ITC e cotrimoxazol - CMX em modelo murino de PCM pulmonar. Para tanto, camundongos BALB/c machos foram inoculados com leveduras do isolado 326 de P. brasiliensis e após 8 semanas de infecção foi dado início aos esquemas terapêuticos: PTX/ITC, PTX/CMX, AZT/ITC, AZT/CMX, Thal/ITC e Thal/CMX. Após 8 semanas de tratamento, os animais foram eutanasiados a fim de se avaliar a deposição de fibras colágenas, produção de hidroxiprolina, recuperação de fungos viáveis e a porcentagem das áreas com lesão nos pulmões e peso corporal. Visando identificar os mecanismos envolvidos foi avaliada a produção de TGF-β1, CCL3, IFN-γ, TNF-α, IL-10, VEGF, IL-6,... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of Paracoccidioides genus; the main clinical forms are acute/subacute, chronic and residual. PCM is a disease restricted to Latin American countries with a higher incidence in Brazil, especially among rural workers. Most patients with the chronic form, even after effective treatment, present sequelae, including pulmonary and adrenal fibrosis. The social, economic and psychological problems triggered by pulmonary sequels are underestimated. In addition, fibrosis in PCM remains neglected, since there is no treatment. The aim of this study was to investigate the influence of antifibrotic drugs (pentoxifylline - PTX, azithromycin - AZT and thalidomide - Thal) associated with antifungal treatments with itraconazole - ITC and cotrimoxazole - CMX in a murine model of pulmonary PCM. Male BALB/c mice were inoculated with P. brasiliensis “isolated 326” and after 8 weeks of infection the treatment were started: PTX/ITC, PTX/CMX, AZT/ITC, AZT/CMX, Thal/ITC and Thal/CMX. After 8 weeks of treatment, the mice were euthanized in order to evaluate the deposition of collagen fibers, hydroxyproline production, recovery of viable fungi and the percentage of areas with injury in lung and body weight. In order to identify the mechanisms involved, the production of TGF-β1, CCL3, IFN-γ, TNF-α, IL-10, VEGF, IL-6, IL-1β, IL-17 and IL-2 in the lung homogenate was evaluated. Our findings revealed that infected mice treated with PTX/ITC s... (Complete abstract click electronic access below)
Mestre

Atrial fibrillation (AF) is the commonest sustained arrhythmia in humans and is responsible for a significant socioeconomic burden. Affected individuals can suffer significant symptoms and are at risk of potentially life-threatening complications. Obesity is increasingly recognised as risk factor for the development of this arrhythmia. Weight fluctuation is common during attempted weight loss and has detrimental cardiovascular effects in human cohort studies, including patients with AF. However, the pathophysiological mechanisms by which this occurs are unclear. The first aim of this thesis is to characterise the electrophysiological effects of weight fluctuation using an obese ovine model. Previous studies have demonstrated that obesity promotes the development of atrial fibrosis as well as the upregulation of profibrotic factors in atrial tissue. The second major aim of this thesis is to investigate the effect of blockade of these profibrotic receptors on obesity-related atrial remodelling. Chapter 2 describes the use a fluctuating weight model in order to study the electrophysiological changes over time. Weight fluctuation was associated with progressive changes in atrial electrophysiology. This group demonstrated reduction in conduction velocity when compared to a lean control group, particularly following a second cycle of weight gain followed by weight loss. These changes were less severe when compared to an obese group. Additionally, the changes in conduction were more heterogeneous than in animals with persistent obesity. This resulted in an increased propensity to AF when compared with lean controls. Chapter 3 investigates the role of endothelin receptor blockade in the prevention of atrial substrate in obesity. Obesity was again induced in ovine subjects and two groups were compared. One was treated with the endothelin receptor antagonist (ERA) bosentan whilst the other acted as a control group. Animals treated with bosentan had attenuation of obesity-related conduction slowing. This was seen on both endocardial and epicardial surfaces. Importantly, there was no effect on either haemodynamics or refractory periods. AF inducibility was also reduced by ERA treatment. Examination of atrial demonstrated reduced fibrosis and downregulation of pro-fibrotic factors with ERA treatment. Importantly, this effect was independent of the TGF-β pathway. Chapter 4 examines the effect of the TGF-β receptor antagonist tranilast on the obese ovine atrium. A similar model of induced obesity was used to compare tranilast treatment with a control group. Animals receiving tranilast demonstrated attenuation of conduction slowing. Endo and epicardial mapping showed this slowing was heterogeneous across atrial sites, perhaps suggesting a predominantly local mechanism in the development of these electrophysiological changes.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2017

碩士
國立陽明大學
臨床醫學研究所
94
Abstract Hepatic fibrosis is a would-healing response during liver injury. Activation of hepatic stellate cells (HSCs) is a critical event in the pathogenesis of liver fibrosis. Activated HSCs are the predominant source of the increased extracellular matrix proteins. Tumor necrosis factor-alpha (TNF-��) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, ��-N-phthalidoglutarimide, (C13H10N2)4, has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-�� effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on rat hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-��1 (TGF-��1) or TNF-��. The inhibitory effects of thalidomide on the NF�羠 signaling cascade and fibrosis markers including ��-smooth muscle actin (��-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats. The rats were randomly assigned to 1 of 5 groups: control rats, DMN rats receiving vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100 - 800 nM) concentration-dependently inhibited NF�羠 transcriptional activity induced by TNF-��, including IKK�� expression and I�羠�� phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-��1-induced ��-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 + 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 + 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that ��-SMA and NF�羠-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-��1, ��-SMA, collagen 1��2, TNF-�� and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TGF-��1 and TNF-��, and ameliorated liver fibrosis in DMN-intoxicated rats.

Systemic scleroderma (SSc) is a systemic connective tissue disease affecting skin and internal organs. The pathogenesis of SSc is characterized by inflammation, vasculopathy and fibrosis. No agent has been proven effective in the treatment of SSc. There is a lack of suitable biomarkers for monitoring the disease activity or the response to the treatment of SSc. Therefore our aim was to analyse the extracellular levels of S100A4, Hsp90 (Heat shock protein 90) and IL-35 (interleukin-35) in SSc. S100A4 and Hsp90 have been initially studied in tumours; in some of them considered as suitable prognostic markers and candidates for future therapies. We have recently described the profibrotic role of S100A4 and Hsp90 in the pathogenesis of SSc. Our results showed that inactivation of S100A4 and Hsp90 effectively prevented the development of experimental skin fibrosis. This was consequently confirmed by the analysis of S100A4 and Hsp90 in the peripheral blood of patients with SSc, where significant associations with disease activity and organ involvement were detected. IL-35 may become another potential biomarker of SSc. We detected increased expression of IL-35 in the affected skin, dermal fibroblasts and in serum of patients with SSc. Moreover, the main profibrotic mediator transforming growth factor...

碩士
國立高雄海洋科技大學
水產食品科學研究所
104
Tetrahydrocurcuin (THC), a major metabolite of curcumin, with high bioavailability and strong antioxidant property. According to a previous study, THC exhibited in vivo hepatoprotective and antifibrotic effects against liver injury induced by dimethylnitrosamine (DMN). In the present study, the protective effect and molecular mechanism of THC was investigated on carbontetrachloride (CCl4)-induced liver fibrosis in Sprague-Dawley (SD) rats. Our data showed that the administration of THC at doses of 10 and 50 mg/kg significantly reduced the elevated serum levels of alanine aminotransferase (ALT), aspartate amino-transferase (AST) in CCl4-treated rats. The histopathological examination showed that the administration of THC at doses of 10 and 50 mg/kg decreasing in regions of hepatic degeneration, inflammatory cell infiltration, hepatocyte ballooning, bridging firbrosis and apoptosis in CCl4-treated rats. THC also decreasd the protein and mRNA expression of -smooth muscle actin (α-SMA) and collagen I in CCl4-treated rats via blocked TGF-β/Smad pathway and TAK1/JNK/p38 pathway. In addition, the administration of THC at doses of 10 and 50 mg/kg significantly reduced apoptosis of hepatic cells, decreasd the protein expression of activated casepase-3 and cleaved of PARP in CCl4-treated rats. Furthermore, THC reduced the marker (LC3) of autophagy and α-SMA protein expression in CCl4-treated rats by double staining immune-histochemistry. TGF-β1-activated HSC-T6 cells were used to investigate the in vitro effects of THC. THC blocked phosphorylation of Smad2 and α-SMA protein expression in TGF-β1 activated HSC-T6. These results suggested that THC attenuated CCl4-induced liver fibrosis which may reversal of activated HSC-T6 cells to a inactivated-like phenotype. The results of the study reveal that THC shows more pronounced protective effect than curcumin against CCl4-induced liver fibrosis, and it also has developed into a health food in future.

碩士
國立陽明大學
傳統醫藥學研究所
92
Studies on the Antifibrotic Activity of Salvia miltiorrhiza in Rat Primary Hepatic Stellate Cells Abstract Activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis, in which oxidative stress has been implicated. Transforming growth factor-β1（TGF-β1）is known to be the most potent factor to induce HSCs activation and accumulation of exrtacellular matrix（ECM）. Salvia miltiorrhiza (SM), a Chinese traditional herb, was reported to have effect on blood circulation and anti-lipid peroxidative activity. The aim of this study is to investigate whether SM could inhibit HSCs activation by carbon tetrachloride and TGF-β1,with primary stellate cells from Sprgue-Dawely rats as a cellular model. Firstly, MDA（malondialdehyde）-TBA assay was used to evaluate the effects of SM on CCl4-induced lipid peroxidation. Then, HSCs were stimulated with TGF-β1, and the production of α-smooth muscle actin (α-SMA) and collagen measured by ELISA. Results showed that SM, partial purified（PSA）and its active principles, salvianolic acid A (Sal A) and salvianolic acid B（Sal B） significantly inhibited MDA formation. Their EC50 values were 74.2 ± 9.5 μg/ml, 5.61 ± 1.0 μg/ml, 5.7 ± 0.8 μM and 3.9 ± 0.5 μM, respectively. Both α-SMA and collagen expression could also be suppressed by Sm, Sal A and Sal B at 500 μg/ml, 20 μM and 280 μM, respectively. Furthermore, we have measured reactive oxygen species（ROS） by CM-H2DCFDA probe under CCl4 and TGF-β1challenge. According to our data, Sm, Sal A and Sal B at 50 μg/ml, 5 μM and 17.5 μM, Sal A and Sal B at 5 μM and 35 μM respectively could scavenge ROS formation stimulated separately by CCl4 and TGF-β1. In the last part, stellate cells separately isolated from bile duct ligation（BDL）and sham rats were stimulated by CCl4 to observe the effects of SM. The results showed that CCl4 would significantly induce ROS formation in HSCs from sham and BDL rats at 1.6 times and 1.2 times, respatively and SM could still reduce ROS formation in these cells. Our results showed that SM inhibited MDA, α-SMA, collagen and ROS formation in stimulated HSCs. Moreover we demonstrated that SM scavenged ROS formation in HSCs from normal and BDL rats. It suggested that SM might attenuate hepatic fibrosis in vivo via ROS scavenging .

碩士
慈濟大學
醫學生物技術碩士班
107
Schistosomiasis is an important tropical disease caused by the parasite, Schistosoma. Eggs of the Schistosoma are the main cause of symptoms in human. The eggs first trapped in the liver, which the immune cells cannot clear the eggs. It then cause inflammation, granuloma and at last, hepatic fibrosis. Casticin, an active compound found in Artemisia annua, has been used as a Chinese medicine for headache, cold and eye pain for many years. Studies also confirmed its effect in anti-prolactinemia, anti-tumor, anti-inflammation, neuron protection and immune regulation. More recently, Casticin has been found to reduce the severity of hepatic fibrosis caused by bile duct ligation (BDL) in mice. Furthermore, it can reduce inflammation by downregulation of inflammasomes. In our study, we used Schistosoma masoni to induce hepatic fibrosis in mice and treat it with Casticin for 2 weeks.Blood results first showed that Casticin does not affect liver function in mice. Then, SEM studies showed that the surface of the adult worm experienced damage after Casticin treatment. Number of adult worm also reduced after treatment, although number of eggs did not have significant change. qRT-PCR results showed an increase mRNA expression of TGF-β1, TNF-α, PDGFA, α-SMA, COL1A1, MMP-2, MMP-9, IL-1β and CCL2 in mice infected with S. mansoni for 10 weeks. However, treatment of Casticin for 2 continuous weeks starting at week 8 showed a downregulation of the above genes. Next, we used Western blotting to analyze fibrosis-related protein, results showed an increase expression of α-SMA and collagen type I at week 10 post-infection, and while after treatment of Casticin, the expression decreased. Immunohistochemistry staining showed that expression of TGF-β1, α-SMA, collagen type I, fibronectin and IL-1β increase at week 10 post-infection and decreased after treatment of Casticin for 2 weeks. Generally speaking, our study confirmed that Casticin can significantly inhibit hepatic fibrosis in mice induced by S. mansoni.

Conclusion. The present results indicate that, baicalein with optimal dosage of 30 muM suppressed collagen deposition in AngII stimulated SHR CF cultures. In animal model of hypertension, high dose of baicalein feeding for 12 week showed optimal antifibrotic effect in hypertensive hearts. (Abstract shortened by UMI.)
For in-vivo study, comparing to control group, HW/BW (x1000) of SHR was significantly reduced in 12 weeks-high dose baicalein and (-0.78+/-0.23, p=0.014) 12 weeks-Valsartan group (-0.71+/-0.22, p=0.021), however, no significant change was observed in the LW/BW ratio.
In Blood pressure control, no effects on attenuation of SBP were observed after 4 weeks and 12 weeks daily administration of baicalein, only 12 weeks feeding of Valsartan significantly down-regulated the systolic blood pressure by -19.25+/-10.09 mmHg, p=0.049.
In the in-vivo study, SHR was used as a model of genetic hypertension. The objectives were: firstly, to determine the efficacy of baicalein in the prevention of myocardial fibrosis (interstitial fibrosis) in SHR, & compared with WKY rats as normal controls. Secondly, to determine if over-expression of pro-collagen I (and III, if any) gene in the ventricles could be normalized by baicalein. Thirdly, to determine if left ventricular hypertrophy in SHR is improved by baicalein. Furthermore, to determine if blood pressure and blood biochemistry parameters (plasma level of brain natriuretic peptides (BNP), and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) level could be alternated by baicalein. Besides, to determine the body weight (BW), heart weight to body ratio (HW/BW), liver weight to body weight ratio (LW/BW), serum AST and ALT level could be alternated by baicalein. Finally to evaluate by echocardiography if there are changes of ivss and ivsd in SHR after administration of baicalein.
Keywords. baicalein, wogonin, collagen, cardiac fibrosis, hypertension
Objectives. In the in-vitro study, cardiac fibroblast culture was prepared from neonatal SHR and WKY rats. The objectives were multi-fold: firstly, to determine over-expression of pro-collagen I mRNA (and III, if any) in cardiac fibroblasts cultures could be normalized by baicalein and wogonin after AngII activation. Secondly, to evaluate the efficacy of baicalein and wogonin on the suppression of total collagen protein production in cardiac fibroblasts cultures after AngII activation. Thirdly, to evaluate the mechanism (in protein level) of baicalein and wogonin on regulating collagen deposition in cardiac fibroblasts after AngII activation. Furthermore, to determine if there were any effects on cytotoxicity and membrane integrity of baicalein and wogonin towards cardiac fibroblasts cultures. Finally, to determine the optimal concentration of baicalein and wogonin for the above actions in-vitro.
Results. For in-vitro study, incubation of AngII resulted in significant up-regulation of COL-I and COL-III mRNA and total collagen protein production. Addition of either baicalein or wogonin significantly suppressed the mRNA synthesis and total collagen protein in CF with an optimal dosage of 30 muM. No effects on viability and membrane integrity were observed on baicalein and wogonin towards cardiac fibroblasts cultures.
Kong, Kam Chuen Ebenezer.
Advisers: Cheuk-Man Yu; Gabriel W. K. Yip.
Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0242.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 176-204).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.

碩士
國立中興大學
獸醫學系
92
The traditional Chinese herbs have been use to treate human hepatic fibrosis for a long time. In this study, we wanted to investigate anti-fibrotic effect and machenism of the traditional Chinese herb－Modified Chai-Hu-Shu-Gan-Tong on CCl4-induced hepatic fibrosis in Sprague-Dawley rats, have been given orally CCl4 (l.0ml/kg) twice a week for 8 weeks. There were divided into four groups, including normal control group (not treated with CCl4) , negative group to be only treated with CCl4, and two experimental groups to be treated with CCl4 and low (1.26g/kg), high (6.30 g/kg) dose of the herb, respectively. Evaluated their therapeutic effects include:1) measurement of the liver-specific cytosolic enzyme activities (AST and ALT), 2) gross lesion finding and liver, spleen and kidney weight/body weight ration, 3) histopathological score of hepatocytic degeneration and liver fibrosis, 4) determination of liver collagen content, 5) immunostaining against α-smooth muscle actin to count the number of activated stellate cells, 6) the content of α-smooth muscle actin were assayed by western blot., and 7) gelatinases (MMP-2, MMP-9) were assayed by zympgraphy. The results show that high dose group of herb treatment can significantly decreased the content of AST, ALT (p<0.05), and liver, kidney weight/body weight ration (p<0.05), liver collagen accumulation was markly reduced (p<0.05), the degree of hepatocytic degeneration and liver fibrosis were more mild (p<0.001, p<0.05). By the way, the numbers of α-smooth muscle actin positive stellate cells were significantly decreased (p<0.01), the α-smooth muscle actin protein expression was markly reduced (p<0.001) to be also high dose of herb treatment, and the expression of MMP-2 was markly reduced (p<0.05) to be also high dose of herb treatment. These results show that this traditional Chinese herb has hepato-protective and anti-fibrotic effects in CCl4-induced liver fibrosis rat model. Anti-fibrotic mechanism may be inhibit hepatic stellate cells proliferation and activation.

碩士
國立陽明大學
傳統醫藥學研究所
90
Studies on the antifibrotic activity of hepatoprotective Chinese medicinal herbs in rat hepatocytes and stellate cells. National Yang-Ming University, Institute of Traditional Medicine Miao-Hua Luo Abstract High morbidity and mortality have threatened many patients with chronic hepatic disease in Taiwan. Hepatic fibrosis is a wound healing and scarring process, which arises in response to liver damage. Liver fibrosis is reversible, whereas cirrhosis, the end-stage consequence of fibrosis is generally irreversible. Previously, studies have suggested that oxidative damage of cholestatic liver injury and hepatic fibrogenesis in the pathogenesis. Therefore, exploitation of antioxidants for prevention and treatment of liver fibrosis has opened a new route for drug development. The future prospects for effective antifibrotic treatment will be a better promise than ever for millions of patients with chronic liver disease. Salvia miltiorrhiza (SM), a Chinese medicinal herb with anti-lipid-peroxidative activity, has been reported to protect rat liver against oxidative stress damage. This study is aimed to investigate the hepatoprotective effects of SM and some hepatopretective Chinese medicinal herbs against CCl4 or D-galactosamine in the primary cultured rat hepatocytes. The potential of SM to inhibit TGF-1 induced activation in the primary culture of rat hepatic stellate cells will also be investigated. Primary cultured hepatocytes were isolated from 8 weeks old Sprague-Dawley rats with 0.02% collagenase. The viabilities of hepatocytes were above 85% and yields were about 8-10  107 cells per liver. Silymarin, SM and hepatoprotective Chinese medicinal herbs inhibited MDA formation induced by CCl4. Inhibitory effect of silymarin was 102.1  1.6%. About 64.6  1.1% of SM crude extract showed better inhibitory effects than other hepatoprotective Chinese medicinal herbs such as 60.9  3.4% of Ganoderma lucidum crude extract and 62.6  1.4% of Scutellaria baicalensis crude extract. 10 g/ml of SM partial purified extracts could show the same antioxidative effect with salvianolic acid B (Sal B). The EC50 values were evaluated by AST values which showed the hepatoprotective activities : SM partial purified extracts (44 g/ml)  Sal B (70 M; 50 g/ml)＞SM crude extract (390 g/ml)＞silymarin (640 g/ml). 25 g/ml SM partial purified extracts (viability: 70.0  6.2%), Sal A (viability: 51.9  1.2%), Sal B (84.1  2.6%), Sal C (89.1  2.1%) also could increase the viability of hepatocytes reduced by D-galactosamine (viability: 41.9  0.6%). High concentration (25-75 mg/ml) of Sal B, the major phenolic compound of SM, did not show cytotoxicity in rat hepatocytes. Primary cultured rat hepatic stellate cells (HSC) were isolated and cultured from 12 weeks old Sprague-Dawley rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz. The viability of HSC were above 90% and yields were about 4-8  106 cells per liver. HSC was identified by using morphological observation and desmin-immunostaining techniques. SM partial purified and Sal B (44 g/ml) could inhibit the expression of -SMA (smooth muscle actin) induced by 1 ng/ml TGF-1 in HSC. And 44 g/ml Sal B did not reduce viability of HSC. SM partial purified extracts (44 g/ml) could not reduce the expression of procollagen I, which was induced by 1 ng/ml TGF-1 in HSC. This study concluded that SM and some hepatoprotective Chinese medicinal herbs could protect rat hepatocytes and resist from CCl4 induced oxidative damage. SM also could inhibit hepatotoxicity of D-galactosamine. SM partial purified extracts and Sal B inhibited the expression of -SMA on HSC.

碩士
國立宜蘭大學
生物技術與動物科學系生物技術碩士班
101
Chronic and cirrhosis of liver diseases are two of the top ten causes of death in recent years. For all of these diseases there is a common pathologic mechanism that leads to fibrosis. Many studies are being made to find a treatment or amelioration of liver fibrosis drugs. Propolis is a complex mixture of which the main bioactive compound is polyphenolic. Several studies show that propolis has antibacterial, antiviral, antioxidant, anti-inflammatory and anti-tumor activity. Prenylflavanones is main bioactive of Taiwanese green propolis, from which propolins are isolated. In this study, we use propolin G which is isolated from Taiwanese green propolis and Taiwanese green propolis to vestigate the anti liver fibrosis. In liver, active hepatic stellate cells are the key fibrogenic cells, and as liver injury gets worse, α-smooth muscle actin will increase and result to extracellular matrix excessive accumulation leading to fibrosis. In this study, we use rat hepatic stellate HSC-T6 cell lines to investigate how Taiwanese green Taiwanese green propolis and propolin G influences cells. By using TGF-β1, we stimulate cell activation and treat Taiwanese green propolis or propolin G to investigate how possible mechanism of protein presents. The result showed that Taiwanese green propolis or propolin G changed the morphology and reduced α-SMA and collagen presents, and caused growth inhibition of HSC-T6 cell line. Furthermore, Taiwanese green propolis or propolin G induced apoptosis associated protein expression (cleaved caspase-3 and cleaved caspase-7) and pJNK activity, but reduced pSmad2/3 expression. Therefore, Taiwanese green propolis and propolin G can induce cell death in activated HSCs through apoptosis to reduce liver fibrosis.

碩士
國立臺灣大學
食品科技研究所
92
Hepatic fibrosis is a wound-healing response to chronic liver injury, which may lead to cirrhosis and liver failure if persisted. Previous studies have indicated that transforming growth factor-β1 (TGF-β1) plays a crucial role in the pathogenesis of liver fibrosis. It induces the phenotypic transition of hepatic stellate cells to proliferating myofibroblast-like cells, enhances the production of extracellular matrix components, and induces apoptotic cell death in hepatocytes. Previously, studies have suggested reactive oxygen species may contribute to both the onset and the progression of fibrosis. Therefore, exploitation of antioxidants for prevention and treatment of liver fibrosis has opened a new way. Recent scientific studies have suggested that the polysaccharides (glucans) and triterpenes in Ganoderma lucidum and isoflavones in leguminous plants, such as black bean and Astragalus membranaceus, have antioxidative activities. Using black bean and A. membranaceus as part of the fermentation medium, isoflavones might be transformed into the more bioavailable form (aglycone) by microbial enzymatic hydrolysis of the glycosidic linkage. The polysaccharides (glucans) might also undergo enzymatic degradation into smaller molecules with higher hydrophilicity and bioavailability. The objectives of this research are to investigate the antifibrotic activities of the fermentation products by using black bean and A. membranaceus as part of liquid fermentation medium of G. lucidum under various fermentation conditions and to evaluate the relationship between the oxidative stress and antifibrotic activity. Human hepatoma cell line (Hep 3B) and rat hepatic stellate cell line (HSC-T6) treated with TGF-β1 were used as the models. Results showed that the recovery effects of TGF-β1 induced oxidative damage exhibited by most filtrates of broth were better than the filtrates of broth removal of polysaccharides and the hot water extracts of mycelia were all better than the ethanol and methanol extracts in Hep 3B cell model. Among all the fermentation filtrates of broth of G. lucidum, GL-2b, which was the fermentation broth of 5% black bean and 2% A. membranaceus fermented at 24 ℃ for 11 days with agitation speed 50 rpm and aeration rate 0.375 vvm, gave the best recovery effect. GL-5mHW, the hot water extract of mycelium by fermentation with 5% black bean and 2% A. membranaceus in the medium of G. lucidum at 30 ℃with agitation speed 50 rpm and aeration rate 0.75 vvm for 11days, showed the best recovery effect among all the mycelia samples. However, the cell cycle analysis of GL-5mHW detected by flow cytometer did not show apoptotic inhibition. In the HSC-T6 cell model system, only the hot water extracts of the raw materials of fermentation (black bean and A. membranaceus), and the fruiting body of G. lucidum showed significant inhibition in α-smooth muscle actin (α-SMA) expression (p<0.05). None of the fermentation products had any significant inhibitory effect toward α-SMA expression.