臺灣博碩士論文加值系統

苦蘵(Physalis angulate L.)是一種傳統中藥，常用於治療糖尿病、肝炎和氣喘。Physalin A為苦蘵的其中一種成分，chalcones為其主要的構成成分之一。利用脂多糖誘導老鼠巨噬細胞發炎與卡拉膠誘導老鼠後腳足腫脹，來探討Physalin A對抗發炎的影響。測試了Physalin A在脂多糖誘導老鼠巨噬細胞中細胞激素(IL-1β, IL-6, and TNF-α)、mitogen-activated protein kinase (MAPK) [JNK, ERK, and p38]、和NF-κB蛋白的表現。當脂多糖誘導巨噬細胞發炎時，Physalin A能以劑量依存性的方式顯著的抑制NO, PGE2, IL-1β, IL-6, and TNF-α的產生。在西方點墨法的實驗中，發現Physalin A抑制了脂多糖誘導聚吞噬細胞發炎中iNOS, COX-2和NF-κB蛋白的表現，並且也可以抑制IκB和JNK的磷酸化。在體內試驗方面，將卡拉膠注入老鼠腳足後，Physalin A可以抑制老鼠腳足腫的情形，也抑制了老鼠血清中NO和TNF-α的濃度，並且Physalin A能顯著抑制丙二醛(MDA)的濃度。Physalin A具有抗氧化活性並表現在提高抗氧化酵素超氧化物歧化酶, 觸酶, 麩胱甘呔過氧化酶的蛋白表現。Physalin A的抗發炎活性與減低JNK與IκB的磷酸化現象，和調控下游NF-κB, iNOS, 和COX-2的蛋白表象，這些蛋白的表現量與NO, PGE2, IL-1β, IL-6, 和TNF-α的表現量減低有關。本研究探討了Physalin A抗發炎的活性與透過下游調控nuclear factor κB和c-Jun N-terminal kinase pathway，與上游抗氧化酵素的調控，並作為一個具有潛力的抗發炎藥物。

Physalis angulata L. has been traditionally used in the supportive treatment of diabetes, hepatitis, and asthma. Physalin A is a major component of chalcones from Physalis angulata. The anti-inflammatory effects of Physalin A were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Physalin A was tested for its ability to reduce nitric oxide (NO) and prostaglandin E2 (PGE2) production, and the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expressions. Physalin A was tested in the inhibitor of mitogen-activated protein kinase (MAPK) [extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38], and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (Iκ-B) and IκB kinase (IKK) protein expressions in LPS-stimulated RAW 264.7 cells. In vitro tests, Physalin A significantly inhibited PGE2, NO, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) production in a dose-dependent manner in LPS-induced RAW 264.7 cells. Physalin A significantly blocked the protein expression of iNOS, COX-2, NF-κB and suppressed IκB and JNK phosphorylation in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. In vivo tests, Physalin A decreased the paw edema and suppressed the NO, malondialdehyde (MDA) and TNF-α level as did indomethacin (Indo) on serum after carrageenan injection in mice. That decreased activities of anti-oxidative enzymes such as superoxide dismutase, catalase, and glutathione peroxidase caused by LPS were reversed by Physalin A. These results indicate that the effect of anti-inflammation of Physalin A is through inhibition of MAPK, NF-κB and AP-1 activation and upregulation of anti-oxidative enzymes. This study also suggests that the potential utilization of Physalin A, as a lead for the development of anti-inflammatory drugs.

Table of Contents

Abstract in Chinese...................................................................................I
Abstract in English..................................................................................II
Acknowledgement..................................................................................IV
Table of Contents.....................................................................................V
List of Table............................................................................................IX
List of Figure..........................................................................................IX
List of Appendix.......................................................................................X
Introduction..............................................................................................1
Inflammation........................................................................................1
The role of NO and PGE2...................................................................1
The role of iNOS and COX-2 protein..................................................2
NF-κB transcriptional factor................................................................2
AP-1 transcriptional factor..................................................................3
MAPK pathway...................................................................................3
Toll-like receptor 4..............................................................................4
Oxidative stress....................................................................................5
Physalis angulata L. ...........................................................................6
Aim......................................................................................................6
Objective and Experimental Design.......................................................7
Materials and Methods............................................................................9
Reagents...............................................................................................9
Cell culture...........................................................................................9
Measurement of cell viability............................................................10
Measurement of nitric oxide..............................................................10
SDS–PAGE/immunoblot analysis.....................................................11
Real Time-PCR..................................................................................12
Enzyme-Linked Immunosorbent Assay (ELISA).............................12
Immunofluorescence staining............................................................14
Flow cytometry..................................................................................14
Animals..............................................................................................15
Carrageenan-induced Edema.............................................................15
MDA Assay.......................................................................................16
Measurement of catalase enzyme activity.........................................16
Measurement of superoxide dismutases enzyme activity..................17
Measurement of glutathione peroxidase activity...............................17
Statistical analysis..............................................................................18
Results......................................................................................................19
The effect of Physalin A on the cell viability, NO and PGE2 produciton of RAW 264.7 macrophage cells....................................19
Effect of Physalin A on protein expression of iNOS and COX-2 in LPS-induced RAW cells....................................................................20
Effects of Physalin A on LPS-induced expression of pro-inflammatory cytokines in RAW 264.7 cells..............................20
Effect of Physalin A on LPS-induced NF-κB activity in RAW 264.7 cells....................................................................................................21
Effect of Physalin A on LPS-induced AP-1 activation in RAW 264.7 cells....................................................................................................21
Effect of Physalin A on LPS-induced iNOS and COX-2 protein expression via inhibiting phosphorylation of MAPK in RAW 264.7 cells....................................................................................................22
Effect of Physalin A on LPS binding with TLR4 in RAW 264.7 Macrophages......................................................................................23
Effects of Physalin A in carrageenan-induced Mouse Paw Edema................................................................................................23
Effects of Physalin A on the NO Level in Carr-induced Mouse Paw Edema................................................................................................24
Effects of Physalin A on the TNF-α Level in Carr-induced Mouse Paw Edema........................................................................................24
Effects of Physalin A on the MDA Level in Carr-induced Mouse Paw Edema................................................................................................25
Effect of Physalin A on Activities of Antioxidant Enzyme in Carr-induced Mouse Paw Edema......................................................26
Discussion................................................................................................27
Reference.................................................................................................32
Tables.......................................................................................................38
Figures.....................................................................................................41
Appendix.................................................................................................56

List of Table

Table 1. Primer sequences of the tested genes.........................................38
Table 2. The table organized the antibodies used in Western blot...........................................................................................................38
Table 3. Effects of PA and indomethacin (Indo) on changes in CAT, SOD and GPx activities were studied on Carr-induced mice paw edema (5th hr).............................................................................................................40


List of Figure

Fig.1 The raw map of the study in vivo and in vitro.............................8
Fig.2 Chemical structure of Physalin A and the protocol of Physalin A from Physalis angulate of extraction and isolation..................................41
Fig.3 The effect of Physalin A on the cell viability, NO and PGE2 produciton of RAW 264.7 macrophage cells...........................................42
Fig.4 Effect of Physalin A on LPS-induced expression of iNOS and COX-2 in RAW 264.7 cells......................................................................43
Fig.5 Effect of Physalin A on LPS-induced expression of pro-inflammatory cytokines in RAW 264.7 cells.....................................45
Fig.6 Effect of Physalin A on NF-κB activation in LPS-stimulated RAW 264.7 cells.................................................................................................46
Fig.7 Effect of Physalin A on AP-1 activation in LPS-stimulated RAW 264.7 cells.................................................................................................48
Fig.8 Effect of Physalin A on MAPKs activation induced by LPS in RAW 264.7 cells......................................................................................49
Fig.9 Effect of Physalin A on LPS binding with TLR4 in of RAW 264.7 Macrophages.............................................................................................51
Fig.10 Effects of Physalin A and indomethacin on hind paw edema induced by Carrageenan mice...................................................................53
Fig.11 The proposed model of the molecular mechanisms by which Physalin A exerts its anti-inflammatory effects in LPS-stimulated RAW 264.7 cells.................................................................................................54

List of Appendix

Appendix 1. The principle of MTT assay.................................................55
Appendix 2. The principle of Griess reaciton...........................................55
Appendix 3. The flow chart of Western blot............................................56
Appendix 4. The flow chart of real-time PCR..........................................57
Appendix 5. The principle of ELISA assay..............................................58
Appendix 6. The principle of immunofluorescence staining...................58
Appendix 7. The principle of MDA assay................................................59
Appendix 8. The principle of catalase activity assay...............................59
Appendix 9. The principle of SOD activity assay....................................60
Appendix 10. The principle of glutathione peroxidase activity assay......60

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