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標題: 金線連糖苷對小鼠內毒素休克、風濕性關節炎和骨質疏鬆之改善作用
Ameliorative effects of kinsenoside on endotoxic shock, rheumatoid arthritis and osteoporosis in mice
作者: 蕭宏柏
Hsiao, Hung-Bo
關鍵字: kinsenoside;金線連糖苷抗發炎;inflammatory
出版社: 生命科學系所
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台灣金線連是台灣傳統且珍貴草藥而金線連糖苷為主要的活性成分。在我們先前的實驗當中發現,金線連糖苷可以抑制肝臟的巨噬細胞也就是Kupffer cells經過LPS刺激所產生的TNF-α。這些的結果我們猜測了金線連糖苷可能可以抑制了巨噬細胞的活化。因為巨噬細胞都會參予各種的發炎過程。因此我們在這裡去探討了金線連糖苷在一些疾病當中巨噬細胞參予的影響包含了內毒素休克、關節炎跟骨質疏鬆。我們獲得了以下的結果:
1. 在LPS刺激之下,金線連糖苷可以明顯的抑制腹腔巨噬細胞釋放發炎物質包含了NO、TNF-α、IL-1β、MCP-1跟MIF。抗發炎細胞激素IL-10卻是有增加的現象。在動物模式中,預防跟治療的處理方式證明了金線連糖苷可以增加ICR小鼠在LPS的致死劑量 (80毫克/公斤,腹腔注射)的存活率。LPS誘導急性發炎的模式當中,金線連糖苷組別在1小時內的時候可以降低血清中TNF-α、IL-1β和IL-10和MCP-1和MIF的濃度。在注射後 24小時內之後金線連糖苷可以促進血清中IL-10濃度增加。總之金線連糖苷可以抑制前趨發炎細胞激素的產生和增強抗發炎細胞激素的生成。
2. 在關節炎動物模式當中,服用金線連糖苷可以抑制脾臟細胞TNF-α,IFN-γ跟IL-17分泌,卻可以增強IL-4跟IL-10的表現。我們利用RT-PCR發現,金線連糖苷也減少TNF-α、IL-1β和MMP-9的表現,同時增加了IL-10的表現。我們發現金線連糖苷也可以增加CD4+ CD25+調節性T細胞的數目。在H&E染色、TRAP還有免疫組織化學染色顯示用金線連糖苷餵食後細胞浸潤明顯減少且減少骨質的破壞。
3. μCT分析之後,金線連糖苷可以減少去卵巢母鼠所造成的骨質流失。金線連糖苷也減少了血漿C-末端肽Ⅰ型膠原蛋白的濃度,但並沒有抑制血漿鹼性磷酸酶活性。RT-PCR分析還說明金線連糖苷降低了脛骨當中TRAP跟MMP-9的表現。金線連糖苷是透過抑制在NF-κB調控路徑並且抑制NFATc1的表現進而去減少蝕骨細胞的特定基因的表現,如TRAP、DC-STAMP、MMP-9和cathepsin K。
4. MMP-9在疾病方面扮演著一個重要的角色。更進一步發現了金線連糖苷可以抑制MMP-9的表現。RT-PCR和Western blot的分析說明了,經過RANKL的處理 Raw 264.7細胞在金線連糖苷可以明顯抑制MMP-9基因和蛋白的表現。在MMP-9的啟動子活性當中也發現,金線連糖苷可以明顯減少冷光酵素表現。這些的結果可以證明,在RANKL刺激的Raw 264.7細胞當中金線連糖苷能夠有效地抑制MMP-9的產生是透過抑制p38/AP-1信號和NF-κB和ROS。

Kinsenoside is a significant and active compound of the Anoectochilus formosanus (Orchidaceae), an important ethnomedicinal plant of Taiwan. In our previous works, we have reported kinsenoside inhibited the TNF-α secretion from Kupffer cells, hepatic macrophages, to LPS stimulation. These results suggest that kinsenoside may inhibit the activation of macrophages. Because macrophages participate in the various inflammatory process. Therefore in this study, we examined the effect of kinsenoside on the disease that including the participation of macrophages, sucsh as entotoxin shock, arthritis and osteoporosis. The following results were obtained.
1. In LPS-stimulated MPLMs, kinsenoside inhibited the inflammatory mediators, such as NO, TNF-α, IL-1β, MCP-1 and MIF production. In contrast, it stimulated anti-inflammatory cytokine IL-10 generation. In an animal model, both pre- and post-treatment of kinsenoside increased the survival rate of ICR mice challenged by LPS (80 mg/kg, i.p.). Pretreatment with kinsenoside decreased serum levels of TNF-α, IL-1β, IL-10, MCP-1 and MIF at 1 h after sublethal dose of LPS (40 mg/kg, i.p.) in mice. In contrast, kinsenoside enhanced serum IL-10 level at 24 h after LPS injection in mice. Kinsenoside inhibited the production of inflammatory mediators and enhanced anti-inflammatory cytokine generation.
2. In CIA mice, administration of kinsenoside significantly suppressed levels of TNF-α, IFN-γ and IL-17, but increased concentrations of IL-4 and IL-10 in the supernatants of each of the splenocytes. In RT-PCR analysis of mRNA expression in hind paws of CIA mice, kinsenoside decreased TNF-α, IL-1, and MMP-9 expression, and increased the expression of IL-10. H&E, TRAP and immunohistochemical staining indicated kinsenoside significantly reduced cellular infiltration and bone destruction. We also showed that kinsenoside enhanced the cell nimbers of CD4+ CD25+ Treg cells of splenocytes.
3. Microtomography scanning found that kinsenoside suppressed bone loss in OVX mice. Kinsenoside also decreased plasma CTx concentration but did not inhibit plasma ALP activities. RT-PCR analysis also showed that kinsenoside decreased the tibial mRNA expression of TRAP and MMP-9. In in vitro study, kinsenoside could inhibit RANKL-induced osteoclastogenesis in Raw 264.7 cells. Kinsenoside inhibited not only the RANKL-triggered nuclear translocations of NF-κB and NFATc1 but also the subsequent NFATc1 induction. NFATc1 promoted osteoclast-specific genes such as TRAP, DC-STAMP, and cathepsin K expression were inhibited by kinsenoside.
4. Since MMP-9 play an important role in several diseases. We further investigated the mechanism of kinsenoside to inhibit MMP-9 production. RT-PCR and Western blot analysis showed that kinsenoside significantly suppressed the mRNA and protein expression of inducible MMP-9 in RANKL-treated Raw 264.7 cells. Luciferase activity assay also showed that MMP-9 promoter activity was reduced by kinsenoside. Our data also showed that kinsenoside attenuated MMP-9 production via the inhibition of p38/AP-1 signaling, NF-κB and ROS activation in RANKL-treated Raw 264.7 cells.
In conclusion, our results demonstrated the beneficial effects of kinsenoside on endotoxic shock, CIA and osteoporosis in mice. Multiple mechanisms were involved in the ameliorative action of kinsenoside. Firstly, kinsenoside inhibited inflammatory mediators release via NF-κB pathway. Secondary, kinsenoside suppressed the MMP-9 secretion might be mediated through inhibition of MAPK pathway and ROS production.
其他識別: U0005-3101201122560300
Appears in Collections:生命科學系所

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