Raw264.7巨噬細胞株預處理山苦瓜萃物影響3T3-L1脂肪細胞株的發炎介質分泌和脂質代謝

Abstract

肥胖已成為近年來重要的健康議題之一，肥胖者的脂肪組織被發現有脂肪細胞過度肥大以及巨噬細胞浸潤的情形，造成組織的慢性發炎，更進一步引起糖尿病、高血壓、高血脂等代謝異常的疾病。苦瓜為亞熱帶地區經常使用的草藥與食材，被發現具有降血糖、抗發炎以及降低體脂的功效。本實驗利用山苦瓜乙酸乙酯及水萃物，以Raw264.7巨噬細胞與3T3-L1脂肪細胞作為平台，探討山苦瓜萃物對於發炎反應以及脂質代謝的影響，評估山苦瓜是否具有改善肥胖以及肥胖引起的發炎反應。首先以山苦瓜萃物處理成熟脂肪細胞，觀察脂肪細胞脂解作用以及脂質代謝相關基因的表現，並測定上清液中細胞激素的分泌。接著探討山苦瓜萃物對巨噬細胞促發炎激素分泌的影響，最後將Raw264.7細胞以山苦瓜萃物預處理後再與3T3-L1共培養，觀察山苦瓜萃物是否透過影響巨噬細胞，進而改變脂肪細胞的發炎反應以及脂質代謝。 給予分化成熟3T3-L1脂肪細胞苦瓜乙酸乙酯萃物，可降低發炎激素MCP-1以及IL-6的分泌，減少細胞內脂質的累積，並增加脂肪酸氧化相關基因的表現。而以苦瓜水萃物處理的脂肪細胞，發炎激素分泌量上升，脂解作用增加，脂質生成及脂肪酸氧化的相關基因表現顯著下降。另一方面，山苦瓜乙酸乙酯萃物可降低巨噬細胞在LPS刺激下促發炎激素TNF-α及IL-6的分泌，相反的，山苦瓜水萃物會促進巨噬細胞促發炎激素的產生。綜合上述，山苦瓜乙酸乙酯萃物具有抗發炎的功效，相反的，水萃物則有促發炎的效果，而兩種萃物皆增加脂解作用的進行，可能具有減重的潛力。 進而將Raw264.7細胞以山苦瓜萃物預處理24小時，清洗細胞去除萃物後再與分化成熟3T3-L1脂肪細胞共培養，發現先給予Raw264.7細胞山苦瓜乙酸乙酯或水萃物，可減少共培養刺激之下巨噬細胞TNF-α的分泌，而山苦瓜萃物透過改變巨噬細胞也降低了脂肪細胞MCP-1的釋放以及兩者IL-6的分泌，並抑制脂肪細胞脂解作用的進行，減少引起發炎的游離脂肪酸，脂肪細胞發炎基因 COX-2的表現也被抑制，顯示在此細胞模式之下，山苦瓜萃物可透過影響巨噬細胞進而改變脂肪細胞的發炎反應與脂質代謝，具有潛力改善因肥胖而引起的慢性發炎。

Obesity is often accompanied by several disorders, such as diabetes, hyperlipidemia, and hypertension. There is considerable evidence that obesity is a state of chronic low grade inflammation. Obese adipose tissue exhibits the inflammatory changes characterized by adipocyte hypertrophy and increased infiltration of macrophages. Bitter gourd (Momordica Charantia) is a tropical vegetable that has also been used in a traditional medicine for treating diabetes. In this study, the ethyl acetate (EA) and water extracts (WE) of bitter gourd powder (BGP) were used to determine the anti-obesity and anti-inflammatory effects on 3T3-L1 adipocytes, Raw264.7 macrophages and their interaction. The pro-inflammatory cytokine secretions were examined after 24 hours treatment of BGP extracts. We showed that BGP-EA decreased the production of MCP-1 and IL-6 in 3T3-L1 adipocytes. TNF-α and IL-6 secretions in LPS-stimulated Raw264.7 was also inhibited. Inversely, BGP-WE elevated the production of pro-inflammatory cytokines both in 3T3-L1 and LPS-stimulated Raw264.7. On the other hand, Both BGP extracts stimulate basal lipolysis of 3T3-L1 after 24 hours treatment. The BGP-EA promoted the mRNA expression of β-oxidation factors. Unlike BGP-EA, BGP-WE suppressed the expression of lipogenic and β-oxidation factors in 3T3-L1. This study provides evidence for the anti-inflammatory effect of BGP-EA but pro-inflammatory effect of BGP-WE on macrophages and adipocytes. Increased lipolysis also suggested the anti-obesity effect of BGP extracts. To further investigate the interaction between adipocytes and macrophages, Raw 264.7 cells were pretreated with different concentrations of BGP-EA and BGP-WE then cocultured with 3T3-L1. Coculture of 3T3-L1 adipocytes and RAW 264.7 macrophages markedly enhanced lipolysis of adipocytes and the production of TNF-α, MCP-1 and IL-6 compared with control cultures. However, the secretion of these cytokines was decreased and lipolysis was inhibited when Raw264.7 cells were pretreated with BGP extracts. The mRNA expression of pro-inflammatory factors COX-2 was also diminished in 3T3-L1 adipocytes which were cocultured with BGP extracts-pretreated Raw264.7 cells. The inflammatory changes and the disorder of lipid metabolism induced by the interaction between adipocytes and macrophages were improved by the pretreatment of Raw264.7 with BGP extracts. The BGP may have potential to ameliorate the inflammation in obese adipose tissue.