第四章 結果
第二節 評估中鏈脂肪酸的抗發炎能力
壹、In vitro 抗發炎活性評估
一、 中鏈脂肪酸對於 P. acnes 誘導 THP-1 細胞
IL-8, TNF-, IL-1蛋 白質表現之影響
P. acnes 會刺激 THP-1 cell 產生促發炎細胞激素，造成發炎而惡化痤瘡，因 此試驗三種中鏈脂肪酸抑制促發炎細胞激素 IL-8、TNF-與 IL-1的能力。
將不同濃度的三種中鏈脂肪酸與 live P. acnes 200 g/mL 與 THP-1 cell 共同 培養 24 小時，收取上清液，以 ELISA 方法分析促發炎細胞激素 IL-8、TNF-與 IL-1的蛋白質表現量。實驗結果顯示，與 live P. acnes 共同培養會增加 IL-8、
TNF-與 IL-1的蛋白質表現量(圖 4-4、圖 4-5 和圖 4-6)。加入 capric acid, caprylic acid，於高濃度 100 µM 達顯著抑制效果；另外加入 caproic acid 則在 25, 50, 100 µM 的濃度下，皆可以顯著抑制由 live P. acnes 誘導的 IL-8 的蛋白質表現量 (圖 4-4)。
capric acid, caprylic acid, caproic acid 在高濃度 50, 100 µM 下具有降低 P.
acnes 誘導 THP-1 產生 TNF-的效果，其中以 caproic acid 具最佳抑制效果，抑 制程度達 92%；三種中鏈脂肪酸的皆可降低 IL-1的產生，其中 capric acid 抑制 程度達 51%，具最佳抑制 IL-1產生效果(圖 4-5 與圖 4-6)。
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圖 4-4 Capric acid, caprylic acid, caproic acid 抑制 live P. acnes 誘導 THP-1 細胞 之 IL-8 cytokines 生成
Figure 4. 4 Capric acid, caprylic acid and caproic acid inhibits live P. acnes-induced IL-8 protein production by THP-1 cells. THP-1 cells were incubated for 24 hr with live P. acnes suspension (200 μg/mL) in the presence of three concentrations of fatty acids from 25-100 μM. Control experiments were run without P. acnes suspension.
Values are means ± SD, n=3. The data were evaluated for statistical significance with one-way ANOVA followed by Duncan’s multiple range test. Means with the same letter are not significantly different. Differences were considered significant for p <
0.05.
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圖 4-5 Capric acid, caprylic acid, caproic acid 抑制 live P. acnes 誘導 THP-1 細胞 之 TNF- cytokines 生成
Figure 4. 5 Capric acid, caprylic acid and caproic acid inhibits live P.acnes-induced TNF-α protein production by THP-1 cells. THP-1 cells were incubated for 24 hr with live P. acnes suspension (200 μg/mL) in the presence of three concentrations of fatty acids from 25-100 μM. Control experiments were run without P. acnes suspension.
Values are means ± SD, n=3. The data were evaluated for statistical significance with one-way ANOVA followed by Duncan’s multiple range test. Means with the same letter are not significantly different. Differences were considered significant for p <
0.05.
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圖 4-6 Capric acid, caprylic acid, caproic acid 抑制 live P. acnes 誘導 THP-1 細胞 之 IL1- cytokines 生成
Figure 4. 6 Capric acid, caprylic acid and caproic acid inhibits live P.acnes-induced IL-1β protein production by THP-1 cells. THP-1 cells were incubated for 24 hr with live P. acnes suspension (200 μg/mL) in the presence of three concentrations of fatty acids from 25-100 μM. Control experiments were run without P. acnes suspension.
Values are means ± SD, n=3. The data were evaluated for statistical significance with one-way ANOVA followed by Duncan’s multiple range test. Means with the same letter are not significantly different. Differences were considered significant for p <
0.05.
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二、 中鏈脂肪酸對於 P. acnes 誘導 THP-1 細胞 IL-8, TNF-, IL-1mRNA 表現量之影響
將不同濃度的三種中鏈脂肪酸、live P. acnes 與 THP-1cells 共同培養 16 小時 後，抽取 RNA 以 quantitative-polymerase chain reaction 分析 IL-8, TNF-, IL-1
mRNA 的表現量。結果顯示，THP-1cells 與 live P. acnes 共同培養 16 小時後，其 IL-8, TNF-, IL-1mRNA 表現量顯著提高(圖 4-7、圖 4-8 和圖 4-9)。
capric acid, caprylic acid, caproic acid 於濃度 25, 50, 100 µM 的情況下，皆可 以顯著抑制 P. acnes 誘導 THP-1cells 產生的 IL-8 , TNF-, IL-1mRNA(圖 4-7、
圖 4-8 和圖 4-9)。
由三種中鏈脂肪酸抑制 P. acnes 誘導 IL-8 產生的實驗結果，capric acid,
caprylic acid, caproic acid 的 IC50分別為 22.9 M, 29.9 M, 43.74 M，發現 capric acid 的 IC50最小，表示 capric acid 在濃度 22.9 M 已擁有抑制半數 IL-8 產生的效 果。所以後續將選擇 capric acid 為樣品，繼續探討其影響促發炎細胞激素產生的 作用機制(圖 4-10)。
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圖 4-7 Capric acid, caprylic acid, caproic acid 對 P.acnes 刺激 THP-1 產生 IL-8 mRNA 的影響
Figure 4. 7 Capric acid, caprylic acid and caproic acid inhibits live P.acnes-induced IL-8 mRNA production by THP-1 cells. THP-1 cells were incubated for 16 hr with live P. acnes suspension (200 μg/mL) in the presence of three concentrations of fatty acids from 25-100 μM. Control experiments were run without P. acnes suspension.
Values are means ± SD, n=3. The data were evaluated for statistical significance with one-way ANOVA followed by Duncan’s multiple range test. Means with the same letter are not significantly different. Differences were considered significant for p <
0.05.
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圖 4-8 Capric acid, caprylic acid, caproic acid 對 P.acnes 刺激 THP-1 產生 TNF-
mRNA 的影響
Figure 4. 8 Capric acid, caprylic acid and caproic acid inhibits live P.acnes-induced TNF- mRNA production by THP-1 cells. THP-1 cells were incubated for 16 hr with live P. acnes suspension (200 μg/mL) in the presence of three concentrations of fatty acids from 25-100 μM. Control experiments were run without P. acnes suspension.
Values are means ± SD, n=3. The data were evaluated for statistical significance with one-way ANOVA followed by Duncan’s multiple range test. Means with the same letter are not significantly different. Differences were considered significant for p <
0.05.
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圖 4-9 Capric acid, caprylic acid, caproic acid 對 P.acnes 刺激 THP-1 產生 IL-1mRNA 的影響
Figure 4. 9 Capric acid, caprylic acid and caproic acid inhibits live P.acnes-induced IL-1 mRNA production by THP-1 cells. THP-1 cells were incubated for 16 hr with live P. acnes suspension (200 μg/mL) in the presence of three concentrations of fatty acids from 25-100 μM. Control experiments were run without P. acnes suspension.
Values are means ± SD, n=3. The data were evaluated for statistical significance with one-way ANOVA followed by Duncan’s multiple range test. Means with the same letter are not significantly different. Differences were considered significant for p <
0.05.
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圖 4-10 Capric acid, caprylic acid, caproic acid 對 live P. acnes 誘導 THP-1 細胞 之 IL-8 生成效果
Figure 4-10 Capric acid, caprylic acid and caproic acid inhibits live P. acnes-induced IL-8 protein production by THP-1 cell. The paraments IC50 coefficient along with the SD are shown on the respective graphs.
(A) capric acid
(B) caprylic acid
(C) caproic acid
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貳、In vivo anti-inflammatory effect
本實驗先前實驗結果顯示，capric acid 能夠減少 P. acnes 引起的發炎反應，
也能減少小鼠耳朵腫脹的程度。因此我們試驗並比較同為中鏈脂肪酸的 capric acid, caprylic acid 和 caproic acid 在 in vivo 的抗發炎效果。
一、 In vivo 抗發炎實驗結果
參考 Nakatsuji 等人(2009)的研究，先單純注射三種中鏈脂肪酸至小鼠耳朵，
根據小鼠耳朵外觀目測與組織切片選擇不造成刺激的劑量(外觀無紅腫等現 象)，選定 8 μg/10 μL 進行實驗。
從外觀與組織切片結果顯示注射 live P. acnes 會引起小鼠耳朵明顯的紅腫，
而測量耳朵厚度的結果顯示與 PBS control 組相比，小鼠耳朵腫脹程度約為 2 倍，
而 capric acid 能夠降低腫脹厚度約 7-23%、caprylic acid 能夠降低 20.4-41.3%和 caproic acid 能夠降低 37.5-49.6%。以 4mm biopsy punch 取等面積耳朵組織秤重，
結果顯示注射 P. acnes 會造成小鼠耳朵重量約增加 2 倍，而 capric acid 能夠減少 重量約 9.1-30.5%、caprylic acid 能夠降低 28.4-44.7%和 caproic acid 能夠降低 34.3-54.8% (圖 4-11)。
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(A)
PBS- control P. acnes P. acnes + capric acid - 8 μg
P. acnes + caprylic acid - 8 μg P. acnes + caproic acid - 8 μg
圖 4-11 Capric acid, caprylic acid, caproic acid 對 P. acnes 引起小鼠耳朵腫脹與重 量變化之影響
Figure 4.11 Evaluation in vivo anti-inflammatory activity of capric acid, caprylic acid and caproic acid on P. acnes-induced inflammation. Ears of ICR mice were injected intradermally with P. acnes (6×107 CFU per 10 μl in PBS) with PBS alone or with capric acid, caprylic acid and caproic acid (8 μg per 10 μL in 5% DMSO in PBS).
After 24 hours, observed by hematoxylin and eosin (H&E) staining (A). The increase in ear thickness was measured using a micro caliper after bacterial injection (B). The ear was punched with a 4 mm biopsy punch after injection and weighed the increase of swelling weight (C). Data are representative of six separate experiments with similar results.Scale bar = 100 μm.
(B) (C)
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參、中鏈脂肪酸對於 P. acnes-induced MAPK pathway and NF-B 磷酸化之影響
在過去 in vitro 的實驗中 lauric acid(C12:0)具有抗菌較果且也具有抑制 P.
acnes 生長的能力，並且在 in vivo 實驗中對於 P. acnes 誘發的發炎反應也具有抑 制的效果(Nakatsuji, et al., 2009)。本實驗此次實驗結果顯示，capric acid, caprylic acid, caproic acid 能夠減少 P. acnes 引起的發炎反應，也能減少小鼠耳朵腫脹的程 度；capric acid IC50 最小，所以後續選擇 capric acid 為樣品，lauric acid 為對照 組，探討 capric acid 和 lauric acid 對 P. acnes-induced MAPK pathway and NF-B 磷酸化之影響。
此部分路徑實驗由本實驗室黃文程進行分析，實驗方法參考(連, 2010)。
一、mitogen-activated protein kinases (MAPK) pathway活化分析
MAPK pathway 為真核細胞中參與調控免疫反應的訊息傳遞路徑，可將細胞 外的訊息傳遞至細胞核內，調控 DNA 的表現或是蛋白質的轉譯作用，使細胞產 生各種不同的反應，在發炎反應時更是扮演了相當重要的角色。為探討 capric acid 和 lauric acid 抑制發炎反應是否經由抑制 MAPK pathway 活化的途徑，利用 western blot 分析 pathway 蛋白質磷酸化的表現。
將人類巨噬細胞 THP-1 與中鏈脂肪酸、P. acnes 共培養 2 小時後，p38, ERK, 化的表現並無顯著影響。capric acid(C10:0)則是在 25、50、100 M 三個濃度下，
皆可以顯著降低 p38, ERK, JNK 蛋白質磷酸化的表現。
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圖 4-12 Capric acid, lauric acid 對 P. acnes 誘導 p38 蛋白質磷酸化表現量之影響
Figure 4.12 Inhibitory effects of capric acid and lauric acid on P. acnes-induced phospho-p38 protein levels in THP-1 cells. Capric acid treatment with P. acnes and THP-1 cells for 2 h. Amounts of phospho-p38 protein were quantified using total-p38 as a loading control and are expressed relative to those control. Data are expressed as the means ± SD of indepent three tests asterisks indicate a significant difference compared with the P. acnes-treated control group, *p<0.05. DMSO: Positive control (P. acnes (+)), Control: Native control (P. acnes (-) ).
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Figure 4.13 Inhibitory effects of capric acid and lauric acid on P. acnes-induced phospho-ERK protein levels in THP-1 cells. Capric acid treatment with P. acnes and THP-1 cells for 2 h. Amounts of phospho-ERK protein were quantified using
total-ERK as a loading control and are expressed relative to those control. Data are expressed as the means ± SD of indepent three tests asterisks indicate a significant difference compared with the P. acnes-treated control group, *p<0.05. DMSO:
Positive control (P. acnes (+)), Control: Native control (P. acnes (-) ).
phospho -ERK
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圖 4-14 Capric acid, lauric acid 對 P. acnes 誘導 JNK 蛋白質磷酸化表現量之影響
Figure 4.14 Inhibitory effects of capric acid and lauric acid on P. acnes-induced phospho-JNK protein levels in THP-1 cells. Capric acid treatment with P. acnes and THP-1 cells for 2 h. Amounts of phospho-JNK protein were quantified using
total-JNK as a loading control and are expressed relative to those control. Data are expressed as the means ± SD of indepent three tests asterisks indicate a significant difference compared with the P. acnes-treated control group, *p<0.05. DMSO:
Positive control (P. acnes (+)), Control: Native control (P. acnes (-) ).
phospho-JNK
control DMSO 25 50 100 25 50 100
capric acid (M)
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二、轉錄因子 nuclear factor-kappa B (NF-κB)活化分析
NF-κB 為轉錄因子，調控多種發炎和免疫相關基因的表現。細胞未受刺激 時，NF-κB dimers 因與抑制蛋白 IκB 結合所以呈現去活化狀態，但若細胞受到外 界壓力刺激，抑制蛋白 IκB 受磷酸化而與 NF-κB 分開，使得 NF-κB 活化進入細 胞核內，調控下游促發炎細胞激素的基因表現。
本實驗室先前的研究發現，將 P. acnes (200 μg/mL)與 THP-1 細胞共同培養 16 小時，會顯著增加 THP-1 細胞核內的 NF-κB/p65 含量(連, 2010)，確定了 NF-κB 參與 P. acnes 啟動宿主細胞的免疫反應。為探討 capric acid, lauric acid 抑制發炎 反應是否經由抑制 NF-κB 活化的途徑，利用 NF-κB/p65 ActivELISA kit 測定細胞 核內 NF-κB (p65)蛋白質含量，以反應細胞核內 NF-κB 活化情形。
將 P. acnes (200 μg/mL)與不同濃度的樣品和 THP-1 細胞共同培養 16 小時，
之後收集細胞萃取核蛋白，再利用 NF-κB/p65 ActivELISA kit 測定核蛋白
NF-κB/p65 含量。結果顯示 capric acid 在三個濃度皆能夠降低 P. acnes 誘導 THP-1 細胞中 NF-κB 的活化 (圖 4-15)。
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圖 4-15 capric acid 和 lauric acid NFκB 分析結果
Figure 4.15 Effects of capric acid and lauric acid on P. acnes-induced nuclear NFκB protein level in THP-1 cells. THP-1 cells stimulated with live P. acnes (200 μg/mL) for 16 hr in the presence of different concentrations of capric acid and lauric acid from 25-100 μM. Control experiments were run without P. acnes suspension. NF-κB
protein concentration was analyzed using NF-κB/p65 ActivELISA kit to detect the active form of the p65 subunit. Data are presented as the means ± SD. Significantly difference is indicated by *p＜.05.
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第三節 capric acid 對 pretreated AA 刺激 P. acnes- induced IL-8 之影響
AA 可藉由磷脂質酶 A2 （ phospholipase A2）作用，而由細胞膜磷脂產生。
在 AA 代謝路徑中，經由 5-lipoxygenase 而生成 LTB4除了會增加白血球、巨噬細
胞趨化聚積外，目前更有研究發現 LTB4在痤瘡發炎過程著實扮演重要的角色
(Zouboulis, 2009a)。另有研究發現，在病人痤瘡病灶 5-LOX, COX-2 活性上升，
此外，也發現皮脂腺的 IL-6 和 IL-8 的表現量也提高(Alestas, et al., 2006)。
一、 建立 pretreated AA 刺激 P. acnes 誘導 IL-8 產生的模式
為探討 AA 在發炎痤瘡所扮演的角色，首先建立並觀察 pretreated AA 對刺激 P. acnes 誘導 IL-8 的影響。將 AA（100 M）先與 THP-1 cells 共培養 16 小時後，
分別加入 AA（100 M）、P. acnes（200 g/mL）後再培養 24 小時，收取上清液，
以 ELISA 方法分析促發炎細胞激素 IL-8 的蛋白質表現量，並與未 pretreated AA 組別比較。結果顯示，單獨的 AA 不會增加 THP-1 cell IL-8 的產生，單獨的 P.acnes 會誘使 IL-8 增加生成；pretreated AA 後的組別比未 pretreated AA 的組別顯著增 加 IL-8 生成。而會對 P.acnes 所誘使 IL-8 的產生具有加成作用，在 pretreated AA 後 IL-8 比未 pretreated AA 明顯增加(圖 4-16)。
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圖 4-16 pretreated AA 對 P. acnes 刺激 THP-1 產生 IL-8 的影響
Figure 4.16 Effects of pretreatment with AA on live P.acnes-induced IL-8 protein production by THP-1 cell. THP-1 cell pretreatment with AA for 16 hr and stimulated with live P. acnes (200 μg/mL), AA , AA/ P. acnes for 24 hr. Control experiments were run without P. acnes suspension. Data are presented as the means ± SD.
Significant difference compared with AA-treated group*p＜.05.
*
*
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二、capric acid 對於 pretreated AA 刺激 P. acnes 誘導 IL-8 產生之 影響
痤瘡的致病機制相當複雜，可能與毛囊過度角質化、皮脂分泌和 P.acnes(痤 瘡桿菌)聚積刺激產生促發炎細胞激素引起發炎反應相關。於前面的結果發現，
pretreated AA 會使得 IL-8 分泌增加，顯示 AA 可能會經由刺激 THP-1cells 提高 促發炎細胞激素 IL-8 的生成，這樣可能加重痤瘡的發炎情況。所以另外也觀察 AA 對於人類皮脂腺細胞 SZ95 在 IL-8 分泌之影響和 capric acid 對於 pretreated AA
pretreated AA 會使得 IL-8 分泌增加，顯示 AA 可能會經由刺激 THP-1cells 提高 促發炎細胞激素 IL-8 的生成，這樣可能加重痤瘡的發炎情況。所以另外也觀察 AA 對於人類皮脂腺細胞 SZ95 在 IL-8 分泌之影響和 capric acid 對於 pretreated AA