具降NF-kappaB轉錄活性之食材對高油飲食小鼠血糖及發炎介質的影響

Abstract

本研究欲探討在高油飲食誘發小鼠肥胖的模式中，經由飲食介入具降低NF-kappaB活性的食材，對肥胖小鼠胰島素抗性及發炎狀況的影響。本研究先以帶有3倍NF-kappaB結合位的質體轉染至RAW264.7細胞株，篩選具有降低NF-kappaB轉錄活性之待測樣品，與C57BL/6J小鼠初代腹腔或脾臟細胞共同培養48小時，挑選出具有降低初代腹腔細胞TNF-alpha及初代脾臟細胞IFN-gamma分泌的樣品，包括藥物sodiun salicylate及rosiglitazone，以及食材苦瓜、GMI和f-GMI。 第一批動物實驗探討篩出的食材對血糖的影響。第一階段餵食C57BL/6J小鼠AIN-76，同時給予待測樣品苦瓜(BGP組)及不同劑量之GMI (G1x、G4x、G20x)。餵食五週後中劑量GMI (G4x組)的血糖顯著下降，六週後低劑量G1x組的血糖也有下降的趨勢。接著將飼料換成30%奶油飲食，為第二階段。繼續餵食待測樣品，但發現沒有明顯的降血糖效果。BGP組雖然無法改善禁食血糖，但顯著降低OGTT的AUC及HOMA-IR index。高劑量G40x組顯著使HOMA-IR index上升。經過四週的wash out期後，將餵食樣品改為f-GMI，繼續第三階段。餵食四週f-GMI無明顯降血糖效果。 BGP組在第三階段餵食兩週後即有顯著降低血糖的效果，並降低OGTT的AUC和HOMA-IR index。 第二批動物實驗自C57BL/6J小鼠六週齡時開始餵食AIN-76 (Basal組)或30%高油飲食(HF組)，餵食九週後HF組的體重、血糖及血脂均顯著高於Basal組。再將HF組分為七組，分別為HF組，Salicylate組(佔飼料的0.7%)，Rosi (rosiglitazone)組(佔飼料的0.005%)，BGP組(飼料中含10%苦瓜粉)，及G6x、G12x、G18x組。 實驗結果發現，抗發炎藥物salicylate及BGP組可降低高油飲食小鼠的體重，並且兩組皆可降低餵食五週後之禁食血糖。BGP及Rosi組也可降低血脂、OGTT的AUC、血清胰島素含量、以及HOMA-IR index。G12x組可降低餵食兩週及五週的禁食血糖，但餵食五週後，G18x組的禁食血糖有增高的趨勢。脂肪組織中，Rosi、salicylate、G6x及G12x組可降低MCP-1及F4/80 mRNA的表現量，減少巨噬細胞聚集至脂肪組織的能力。Rosi、BGP、salicylate、G6x及G18x組可增加M2巨噬細胞指標IL-10或arginase的表現量，具有抗發炎能力。Rosi、BGP、及G18x組增加TNF-alpha的表現，並且Basal及BGP組IL-1beta表現量上升，顯示這幾組可能對脂肪組織有促發炎的作用，或是這兩個細胞激素可能具有其他功能。Salicylate、G6x、G12x、及G18x組顯著降低肝臟CRP的含量。BGP組降低肝臟IL-6含量，但在肝臟及血清皆提升MCP-1的濃度，並且增加血清中CRP的含量，顯示苦瓜可能具有促發炎的作用。 由本研究細胞實驗所篩選出具抗發炎潛力之待測樣品餵食高油飲食小鼠，發現可降低禁食血糖或HOMA-IR index的樣品皆可有效減少肥胖小鼠脂肪組織內F4/80及MCP-1的表現，減少巨噬細胞的聚集，並增加M2巨噬細胞指標IL-10及arginase的表現量，而具有抗發炎的作用。並且改善胰島素抗性效果最好的苦瓜也可減少肝臟IL-6、血清NEFA和leptin的含量，達成降低發炎反應的效果。顯示本研究以細胞模式篩選具抗發炎潛力之樣品，可藉由降低高油飲食小鼠體內發炎的指標，而促使胰島素敏感性。但若能配合其他細胞模式或指標輔助但若能配合其他細胞模式或指標輔助篩選，可能有較確切地初篩效果。

The goal of this study was to investigate whether dietary factors prescreened by NF-kappaB transactivation assay could alleviate the development of insulin resistance and inflammatory state. We employed a NF-kappaB promoted luciferase reporter gene plasmid to transfect RAW264.7 macrophage cell lines for pre-screening of anti-inflammatory activity. Proinflammatory cytokine productions from LPS-stimulated primary peritoneal macrophages and ConA-stimulated primary splenocytes isolated from C57BL/6 mice were also measured. The results showed that drugs, including sodium salicylate and rosiglitazone, and dietary factors, including bitter gourd, GMI, and f-GMI, inhibited NF-kappaB-dependent luciferase activity, TNF-alpha secretion from peritoneal macrophages, and IFN-gamma secretion from splenocytes. To further investigate the hypoglycemic effect in vivo, in StageⅠ, five groups of 12-week-old C57BL/6 mice were fed with AIN-76 diets supplemented with tested samples, including BGP (bitter gourd powder) and different doses of GMI (G1x, G4x, and G20x). After 5 weeks of feeding, G4x group had significantly lower serum glucose level compared to the control, and after 6 weeks of feeding, G1x group also had lower fasting glucose. In StageⅡ, a 30% high fat diet was applied to increase the body weight of mice. After 8 weeks of high-fat diet feeding, no tested sample had hypoglycemic effects on mice, but BGP group had significantly lower AUC of OGTT and HOMA-IR index, whereas G40x group elevated HOMA-IR index. After 4-week wash out period, we switched the tested samples to f-GMI, and continued with StageⅢ of the experiment. After 4 weeks of f-GMI feeding, no hypoglycemic effect was observed. However, the serum glucose levels of BGP group significantly lowered after 2 weeks into StageⅢ, and the AUC of OGTT and HOMA-IR index were also lowered after 3 weeks of feeding. To further investigate the hypoglycemic effect of f-GMI, 6-week-old C57BL/6J mice were divided randomly into two groups: the Basal group was fed AIN-76 diet, whereas the HF (high fat) group was fed with a 30% butter diet. After 9 weeks of induction, the HF group was subdivided into the following 7 groups: containing HF, salicylate (0.7% sodium salicylate in diet), Rosi (0.005% rosiglitazone in diet), BGP (5% BGP in diet), G6x (0.4 mg/mouse/day), G12x (0.8 mg/mouse/day), and G18x (1.2 mg/mouse/day). After 5 weeks of feeding, BGP, salicylate, and G12x group were shown to be effective in ameliorating the HF diet-induced hyperglycemia, whereas G18x group led to an increase in serum glucose. BGP and Rosi group also decreased hyperleptinemia, AUC of OGTT, and HOMA-IR index. In the adipose tissue of high fat-fed mice, Rosi, salicylate, G6x, and G12x group decreased MCP-1 and F4/80 mRNA expression, resulting in reduced macrophage infiltration in adipose tissue. Furthermore, Rosi, BGP, salicylate, G6x, and G18x group increased the mRNA levels of M2 macrophage markers, such as IL-10 and arginase, and tended to induce M2 macrophage polarization. In addition, Rosi, BGP, and G18x group had higher TNF-alpha mRNA levels, while Basal and BGP group also upregulated IL-1beta mRNA expression, which suggest that these samples might have proinflammatory effect in the adipose tissue of diet-induced obese mice. Moreover, salicylate, G6x, G12x, and G18x significantly reduced CRP levels in liver. BGP group decreased IL-6 content in liver, but raised MCP-1 levels in liver and serum. BGP also increased CRP concentration in serum. The results showed that bitter gourd might have proinflammatory effect in high-fat fed C57BL/6J mice. Administering the tested samples prescreened by NF-kappaB transactivation assay to high-fat fed C57BL/6J mice showed lower fasting glucose and anti-inflammatory effect through not only reduced F4/80 and MCP-1 mRNA levels, but also increased the levels of M2 macrophage markers. Moreover, BGP also had anti-inflammatory activity by reducing IL-6 levels in liver, leptin and NEFA levels in serum. In conclusion, dietary factors prescreened by NF-kappaB transactivation assay may alleviate the development of insulin resistance through reducing inflammatory state.