單磷酸腺苷激酶活化劑Energi-704抑制脂多糖誘發大鼠巨噬細胞RAW264.7發炎反應之分子機制研究

在體外、體內的研究指出，活化型的腺苷酸活化蛋白激酶(AMPK)可抑制由脂多醣(LPS)所引起的發炎反應，而Energi-704已知是AMPK的活性劑，初步被證實可減輕皮膚受傷所引起的發炎反應。然而，Energi-704 是否在活化型的巨噬細胞中，也具有抗發炎的活性，以及在發炎反應中扮演重要角色仍然不清楚。因此,本篇研究主要探討Energi-704在LPS刺激小鼠巨噬細胞 RAW264.7上的影響，以及相關的反應機制。以RAW264.7細胞做為本篇實驗的model,前處理Eanergi-704濃度範圍(0、0.1、0.5、1 μM)，隨後加入LPS(1 μg/mL)，再分別收集細胞與培養液。利用RT-PCR與qPCR分析mRNA的表現量，以西方墨點法、使用專一性抗體測定酵素的活性，NO與ROS分析辦法，各別使用Griess reagent 與DCFH-DA。實驗結果顯示，Energi-704顯著抑制RAW264.7由LPS引起促發炎激素mRNA的表現(TNF-α、 IL-1β、IL-6、 MCP-1、iNOS 與 COX-2 )，此外，Energi-704也減少被刺激細胞所產生的NO與ROS，再進一步做了研究，實驗顯示在Energi-704活化AMPK的同時，也會降低PI3K/AKT 與mTOR 的活性。總結實驗數據，Energi-704能有效抑制發炎物質的表現，也能減少NO、ROS的產量，證實Anergi-704具有抗發炎的活性，能抵制PI3K/AKT，而mTOR信號也可能參與在其中。

關鍵字

發炎；單磷酸腺苷激酶活化劑；巨噬細胞；脂多糖

並列摘要

Activation of Adenosine monophosphate kinase (AMPK) has been reported to inhibit inflammatory responses induced by lipopolysaccharide (LPS) in vitro and in vivo. An AMPK activator Energi-704 has been demonstrated to ameliorate inflammatory responses of injured skin. However, whether Energi-704 possesses anti-inflammatory activity on activated macrophage, a main player in inflammatory responses, still remain sketchy. Therefore, the present study was aimed to investigate effects of Energi-704 on LPS-stimulated murine macrophage RAW264.7 and the underlying mechanisms.RAW 264.7 cells were used as cell model. After treated with Energi-704 at serial concentrations (0, 0.1, 0.5 and 1 μM) and followed LPS (1 μg/mL), the cells and the cultured medium were separately collected. Expression level of mRNA was analyzed by RT-PCR and real-time quantitative PCR (qPCR). Kinase activation was demonstrated by immunoblot using specific antibodies. Nitric oxide (NO) and reactive oxygen species (ROS) were determined by using Griess reagent and 2’,7’-dichlorofluorescein diacetate (DCFH-DA), respectively. Energi-704 significantly suppressed mRNA levels of pro-inflammatory TNF-α, IL-1β, IL-6, MCP-1, iNOS and COX-2 in RAW264.7 cells stimulated with LPS. In addition, Energi-704 also reduced production of NO and ROS by the stimulated cells. Further investigation showed that Energi-704 reduced activation of PI3K/AKT and mTOR on the activation of AMPK.Our findings show that the Energi-704 robustly inhibited expression of pro-inflammatory components and reduced production of NO and ROS, suggesting Energi-704 possesses potent anti-inflammatory activity against LPS-induced inflammatory responses. Moreover, Energi-704 suppresses LPS-induced inflammatory response by inhibition of phosphatidylinositol 3-kinase/Akt activation and mTOR signaling is possible involved in the anti-inflammatory activity of Energi-704.

並列關鍵字

inflammation ； n AMPK activator ； RAW264.7 ； LPS

參考文獻

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單磷酸腺苷激酶活化劑Energi-704抑制脂多糖誘發大鼠巨噬細胞RAW264.7發炎反應之分子機制研究

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