利用幹細胞分泌體調節粒線體動態失衡所造成的巨噬細胞極化藉以改善痛風

Abstract

痛風性關節炎是由單鈉尿酸鹽結晶(monosodium urate crystals, MSU)的堆積所造成的，而巨噬細胞在急性痛風性關節炎中扮演很重要的角色。活化的巨噬細胞通常被分成兩大類：典型活化巨噬細胞(classically activated macrophages, M1)與另類活化巨噬細胞(alternatively activated macrophages, M2)，而他們分別具有促發炎與抗發炎的功效。粒線體動態被視為維持正常細胞功能的一個機制，在先前的研究中，幹細胞能旁分泌(paracrine)出生長因子等進而影響周圍的細胞。將這些幹細胞旁分泌出來的分泌體(secretome)收集成條件培養基(conditioned medium)做後續實驗，而為了能使幹細胞條件培養基(stem cell conditioned medium, SCM)能更加有效，會將幹細胞預先培養在含有發炎因子的環境中數小時，使其提早適應病理環境藉以強化他，並收集強化幹細胞條件培養基(strengthened stem cell conditioned medium, SSCM)。高尿酸血症所引起的痛風性關節炎由於發作時只能利用一些化學藥物來消炎止痛，而且這些藥物會導致胃腸道與腎臟的毒性，所以本研究想利用幹細胞的分泌體來進行治療。在細胞實驗中，我們利用不同濃度的MSU結晶刺激巨噬細胞，結果顯示巨噬細胞的ROS含量、發炎現象隨著結晶濃度的增加而隨之增加，粒線體動態則是隨著結晶濃度的增加而愈趨向融合(fusion)。在經過發炎環境的預處理後，幹細胞CXCR4的蛋白質表現量上升，證實能透過這條路徑強化幹細胞，且強化幹細胞conditioned medium能使發炎現象下降並改善粒線體動態的不平衡，也能夠使M1巨噬細胞的數量下降。在動物實驗中，強化幹細胞conditioned medium能夠使關節中細胞浸潤的現象降低，並能夠使軟骨組織的排列較為緊密且量多，而粒線體動態也有改善的現象。因此強化幹細胞conditioned medium對於痛風有改善的潛力。

Macrophage plays an important role in acute gouty arthritis caused by deposition of monosodium urate crystals (MSU). Activated macrophages are commonly classified into two types of polarization: classically activated macrophages (M1) and alternatively activated macrophages (M2), and they have proinflammatory and anti-inflammatory ability, respectively. Mitochondrial dynamics is thought to be one of key mechanisms to maintain a normal function of cells. In previous studies, stem cells can paracrine growth factors and other factors to affect the surrounding cells. We collected the secretome from these stem cells into the stem cell conditioned medium (SCM) for follow-up experiments. In order to make the effects of SCM more effective, pretreating stem cells into inflammatory environment for few hours and let them adapt to the pathology environment in advance, and collecting the strengthened stem cell conditioned medium (SSCM). Gouty arthritis caused by hyperuricemia can only use some chemical drugs to reduce inflammation and pain, but these drugs will cause gastrointestinal and kidney toxicity. This study aimed to use the secretome secreted by stem cells to treat gouty arthritis. In the cell experiment, we used different concentrations of MSU crystals to stimulate macrophages, and the results showed that the activation of ROS and inflammation for macrophages were proportional to MSU crystals concentration. With the higher the concentration of MSU crystals, the mitochondrial dynamics tend to fusion in activated macrophages. After the pretreatment of the inflammatory environment, the protein expression of stem cells CXCR4 increased, which proved that stem cells can be strengthened through this pathway. After strengthening, the stem cell conditioned medium not only can reduce inflammation and improve the imbalance of mitochondrial dynamics but also reduce the number of M1 macrophage. In animal experiments, the strengthened stem cell conditioned medium can reduce the cell infiltration in the joints, and not only can make the cartilage tissues more tight and dense but also improve the imbalance of mitochondrial dynamics. Therefore, the strengthened stem cell conditioned medium has the potential to improve gouty arthritis in clinical.