| 摘要: | 類風濕性關節炎 (Rheumatoid arthritis; RA) 是一種自體免疫失調疾病會導致關節疼痛、關節腫漲和血管翳 (Pannus) 的形成。先前的研究中指出介白素1-β (Interleukin 1 beta; IL-1β) 和腫瘤壞死因子α (Tumor necrosis factor-alpha; TNF-α) 是造成類風濕性關節炎最主要的促發炎因子。此外褪黑激素 (Melatonin) 是調控生物重要機轉的賀爾蒙，具有抗氧化與抑制發炎反應的作用，然而目前褪黑激素在RA如何抑制發炎反應的機轉尚未被釐清。
滑液纖維母細胞是滑液囊組織中含量最多的細胞，MH7A細胞株是類似纖維母細胞的細胞株，因此將利用此細胞株做後續實驗。我們發現褪黑激素會藉由MT1接受器進入細胞進而減少IL-1β和TNF-α的表現且對細胞沒有毒性。此外，我們篩選了許多的訊息傳遞路徑，發現褪黑激素會抑制PI3K、AKT和ERK磷酸化，進而降低TNF-α和IL-1β的表現。
NF-κB是同時結合在TNF-α 和IL-1β上的轉錄因子，研究結果顯示在給予褪黑激素後，會降低pp65、pIkkα/β、pIκBα表現，減少NF-κB入核binding到TNF-α和IL-1β的promotor上。另一方面，目前已經知道miRNA參與在許多的疾病當中，因此，在分析過許多miRNA資料庫後，發現其中miRNA-3150a-3p同時帶有TNF-α 和IL-1β mRNA的3' UTR，給予褪黑激素會促進miRNA-3150a-3p的表現。在給予 PI3K、AKT和ERK活化劑後，發現會瓦解miRNA-3150a-3p中的TNF-α 和IL-1β的3' UTR，因此推論miRNA-3150a-3p參與在PI3K、AKT和ERK的訊息路徑中。
在動物實驗當中我們利用關節炎動物模型(collagen induced arthritis; CIA) model，發現在不同濃度的褪黑激素治療下CIA老鼠在骨表面較完整以及腳掌的腫脹程度有減輕。同時，在不同濃度褪黑激素治療下CIA老鼠，分析老鼠的血清，發現體內的TNF- α和IL-1β表現量皆有降低。綜合以上結論，褪黑激素會透過MT1接受器抑制PI3K、AKT和ERK的磷酸化造成TNF-α 和IL-1β的表現量減少，進而減少NF-κB表現和增加miRNA-3150a-3p表現來調控此訊息傳遞路徑，最後在動物實驗當中能夠有效改善CIA老鼠的關節嚴重程度。因此，褪黑激素是一種有潛力又安全治療類風濕性關節炎的藥物。
Rheumatoid arthritis (RA) is one of the autoimmune diseases which results in pain, swelling as well as stiffness of the joints leading to the pannus formation. Two important pro-inflammatory cytokines, interleukin 1 beta (IL-1b) and tumor necrosis factor-alpha (TNF-a), play important role in RA. Moreover, melatonin showed significant anti-inflammatory and anti-oxidant activity. However, the effect of melatonin in arthritis has not been completely elucidated. Synovial membrane is rich in fibroblast-like synoviocyte and MH7a is one of the fibroblast-like synoviocyte cell line, so MH7a is applied in the following experiments. Our findings revealed that treatment of melatonin with MH7a cell inhibited the IL-1b and TNF-a mRNA and protein expression but did not influence viability of cells via MT1 receptor. Our lab selected some pathways that are inhibited by melatonin, and these are PI3K、AKT and ERK phosphorylation and inhibit the expression of TNF-a and IL-1b in cells. NF-κB is a protein complex that controls transcription of DNA and TNF-a as well as IL-1b are the cytokines that NF-κB producing. Data shows that when the concentration of melatonin is higher, the expression of pp65、pIkkα/β、pIκBα is lower and inhibit NF-κB into the nuclear to bind on the promotor of IL-1b and TNF-a. On the other hand, we knew miRNA involve in many disease. Therefore, after analyzing the database we found miRNA3150a-3p included the 3' UTR of IL-1b and TNF-a, and the expression of miRNA3150a-3p increased while treating melatonin. After treating the activators of PI3K、AKT and ERK, the 3' UTR of IL-1b and TNF-a break down, so we assume miRNA-3150a-3p involve in the PI3K、AKT and ERK signaling pathway. In animal experiments, We also found that the group of melatonin-treating RA mouse have the better recovery of bone surface and the less swelling in the paw. Finally, we analysis the serum of RA patients, and we found that the concentration of TNF-a and IL-1b in RA patients is higher than the healthy people. Sum up all the data, melatonin regulate the signaling pathway by inhibiting PI3K、AKT and ERK phosphorylation via MT1 receptor and resulting in decreasing the expression of IL-1b and TNF-a and then the expression of NF-κB decreased and the expression of miRNA-3150a-3p increased. Last, melatonin improves the recovery level in RA mouse. Overall, melatonin can be a potential therapy to the Rheumatoid arthritis. |
