Sesamol及Cinnamophilin抑制基質金屬蛋白酶
                              保護軟骨細胞的作用機轉探討

在骨關節炎(osteoarthritis)及風濕性關炎(rheumatoid arthritis) 病人之骨關節滑(synovial fluid)發現升高的細胞激素(interleukin-1)及腫瘤壞死因子(tumor necrosis factor-α)可誘發不同之基質金屬蛋白酵素Matrix Metalloproteinases (MMPs)，進而分解軟骨基質及抑制軟骨細胞基質蛋白之表現。基質金屬蛋白酵素對於正常生理具有胚胎發育性之再塑作用，同時在許多發炎疾病如缺血性腦傷害症、急性呼吸窘迫症、類風濕性關節炎及多發性硬化症都扮演著重要的角色。軟骨細胞並非持續性(constitutive)產生基質金屬蛋白酵素，而是受特定外來發炎介質(如細胞激素)所刺激活化，再經細胞內訊息傳遞，經分裂素活化蛋白酵素(MAPKs：JNK、ERK)或NFκB等基因轉錄活化過程，以產生與釋放不同含量之基質金屬蛋白酵素。芝麻酚是ㄧ種強效的酚類抗氧化物，俱有抗癌、保護肝臟、和抗老化的作用。Cinnamophilin (MA-1)是由天然植物菲律賓樟樹(Cinnamomum philippinense)中所粹取出來的成分是一強而有效之自然抗氧化劑與自由基截取劑，且可抑制缺血後之血小板聚集，是以有潛力降低缺血後的能量流失。本研究的目的是要探討天然物芝麻酚sesamol與cinnamophilin 皆具有明顯抑制 MMPs 活化之作用並探討其作用機轉。我們以人類軟骨細胞 (SW1353) 為實驗細胞，分別以不同濃度的TNF-α或IL-1β處理24小時後，以電泳酵素分析法(gelatin zymography) 評估可發現到人類軟骨細胞能誘發大量 MMPs 的活性，其中更以 MMP-9為甚。尤其當TNF-α或IL-1β刺激濃度分別30 ng/ml與30ng/ml，細胞數為1×106 時，此分解gelatin的效果最為明顯適當。隨後的實驗便以此為試驗條件，的確在電泳酵素分析法下觀察到sesamol有意義地依濃度效應抑制TNF-α 或IL-1β誘發人類軟骨細胞之 MMP-9活性。另外以細胞存活率測定 (MTT assay) 發現 sesamol 與 cinnamophilin 的抑制作用並非源自細胞之損壞或死亡。以西方點墨法 (Western blot) 的實驗發現在PMA 的刺激下，MMP-1 和MMP-13的酵素蛋白表現量隨sesamol的濃度增加而降低，故此證明天然物成分是作用在 MMP-1 and MMP-13 蛋白質表現的層面，但MMP-1和 MMP-13表現不是由TNF-α或IL-1β誘發。TNF-α?z?n?過-β or PMA 的刺激下，MMP-1 and MMP-13 的酵素蛋白表現量隨sesamol 與 cinnamophilin 的濃度增加而降低，故此證明天然物成分是作用在 MMP-1 and MMP-13 蛋白質表現的層面。由此我們更深入瞭解細胞轉錄 (transcription) 之影響程度。接下來我們探討sesamol與 cinnamophilin在訊息傳遞中作用機轉的途徑。由實驗結果發現，sesamol會明顯抑制由TNF-α或PMA所刺激導致的 Inhibitor- κB-α (IκB-α)的降解作用，使nuclear factor-κB (NF- κB)無法進入細胞核中與特定MMP 相關DNA 序列接合。同時sesamol抑制PMA引起的ERK1/2或p38燐酸化也抑制IL-1β引起的p38燐酸化。在monosodium iodoacetate(MIA)誘發老鼠退化性關節炎時sesamol有效抑制MMP-1與MMP-9。Sesamol不僅在細胞實驗中經由抑制IκB-α的降解作用或ERK1/2或p38燐酸化抑制基質金屬蛋白酵素，且在誘發老鼠退化性關節炎時sesamol有效抑制基質金屬蛋白酵素，展現其對防止退化性關節炎的潛力。Cinnamophilin亦有意義地依濃度效應抑制IL-1β誘發人類軟骨細胞之 MMP-1和MMP13活性，在西方點墨法 (Western blot) 的實驗發現在IL-1β 的刺激下，MMP-1 和MMP-13的酵素蛋白表現量cinnamophilin的濃度增加而降低。在mitogen-actived protein kinase (MAPKs) 方面， Cinnamophilin 能抑制由IL-1β所刺激導致的 c-Jun-NH2-terminal kindase (JNK) 表現，且Cinnamophilin抑制IL-1β刺激導致的inhibitor- κB-α (IκB-α) 的降解作用，使 nuclear factor-κB (NF- κB) 無法進入細胞核中與特定 MMP 相關 DNA 序列接合。Cinnamophilin明顯抑制軟骨細胞 IL-1β刺激c-jun燐酸化Cinnamophilin不僅經由NF-κB或ERK/p38 MAPKs且由p-c-jun路徑抑制軟骨細胞經由IL-1β刺激MMP-1 和MMP-13表現。以上表現cinnamophilin 具有抑制基質金屬蛋白酵素而保護軟骨。

關鍵字

細胞激素；腫瘤壞死因子；芝麻酚；基質金屬蛋白；酵素；分裂素活化蛋白酵素

並列摘要

Cartilage loss is a hallmark of arthritis. Over-expression of matrix metalloproteinases (MMPs) is a major pathological factor to cause cartilage destruction in osteoarthritis (OA). We hypothesized that two natural products of East Asian countries, namely sesamol and cinnamophilin would limit cartilage degradation by respectively suppressing MMPs production. Sesamol, derived from sesame seed oil, resistant to oxidative deterioration than other vegetable oils and the other product of cinnamophilin (Cinn), derived from Cinnamomum philippinense, possessed antioxidant and free radical-scavenging effects. In this study, Sesamol significantly attenuated TNF-α- and IL-1β-induced gelatinolysis and expression of MMP-9 in a concentration-dependent manner in SW1353 cells. Additionally, both MMP-1 and -13 stimulated by PMA were inhibited by sesamol. Both sesamol and Cinn. significantly attenuated IL-1β-induced expression of MMPs-1 and 13 in a concentration-dependent manner in SW1353 cells. On the other hand, the NF-κB signaling activation through IκB kinase (IKK)-α/β, and p-65 phosphorylation was restored by these two products upon stimulation. Furthermore, these bioactive compounds exerted the reduction on induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 MAPKs, and c-Jun N-terminal kinase (JNK). Activation of phosphorylated (p)-c-Jun in chondrocytes was significantly inhibited by cinnamophilin. In this study, it was interesting to evaluate whether sesamol down-regulate MMP expression in the joint cartilage of monosodium iodoacetate (MIA) induced OA in rats. The results revealed that sesamol prevented the expression of MMP-1 and -9 in the cartilages of MIA-induced OA in rats. These results indicate that both sesamol and cinnamophilin inhibited MMPs expressions in stimulated chondrocytes through either NF-κB or ERK/p38 MAPK downregulation and/or suppressing p-c-Jun pathways. In conclusion, this study provides strong evidence that both sesamol and cinnamophilin inhibits MMP-1, and -13 expressions in stimulated chondrocytes, at least in part, via inhibiting NF-κB, ERK/JNK, and p38 MAPK activation. Thus, these results suggest that inhibiting MMP-1 and 13 activities of chondrocytes may be beneficial for protecting cartilage degradation. In this respect, both sesamol and cinnamophilin may be a good source of potential agents for the development of therapeutics for destructive joint diseases.

並列關鍵字

matrix metalloproteinases ； Sesamol ； Cinnamomum

參考文獻

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國際替代計量

Sesamol及Cinnamophilin抑制基質金屬蛋白酶保護軟骨細胞的作用機轉探討

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