in vivo 抗發炎活性評估

從細胞實驗中觀察到 FL1-FL7 可以抑制 heat-killed P. acnes induced THP-1 cell 生成的 IL-1β 和 IL-8，以 P. acnes 皮下注射鼠耳動物模式來檢視 樣品的抗發炎效果。為了確認樣品的安全性和 in vivo 實驗濃度，先以單純

注射FL1-FL7 至小鼠耳朵，根據小鼠外觀目測(外觀無紅腫等現象)選擇不造

成刺激的劑量，定20μg/10μL/ear 進行實驗。

將 P. acnes 活菌注射在 ICR 小鼠耳朵皮下組織中誘發小鼠耳朵紅腫發 炎。先進行預實驗，以注射 P. acnes 後不同時間點的外觀和促發炎細胞激素 變化來決定之後的刺激時間，實驗A 注射 P. acnes 6、12、24 小時後犧牲小 鼠，紀錄耳朵組織厚度變化，發現三個時間點都達到誘發小鼠耳朵腫脹的 效果(圖 4-14)，接著取下鼠耳組織均質，均質液以 ELISA 方法分析促發炎 細胞激素IL-6 和 IL-1β，發現 IL-1β 在刺激 12 小時後可生成最高的量(圖 4-15)，

而IL-6 則是刺激 6 小時後較高(圖 4-16)。我們選擇以刺激 12 小時作為後續

\實驗的模式。

接下來將 P. acnes 和樣品一起注射，實驗 B 注射 P. acnes 與 FL1-FL7 於12 小時後犧牲小鼠，從外觀上來看，FL1-FL7 皆比單純注射 P. acnes 組 紅腫程度較輕微(圖 4-17)；在厚度方面，FL1-FL7 皆能顯著降低 ICR 小鼠耳 朵的厚度，降低 P. acnes 刺激所造成之腫脹現象(圖 4-18、表 4-1)。之後將 鼠耳磨碎均質，組織均質液以ELISA 方法分析促發炎細胞激素 IL-6、IL-1β 和TNF-α，發現 FL1-FL7 對於 IL-1β、IL-6 和 TNF-α 皆有抑制的效果 (圖 4-19、圖 4-20 和圖 4-21)，FL1 -FL7 皆可顯著抑制 IL-6 和 TNF-α 的生成，

而FL3 -FL7 可顯著抑制 IL-1β 的生成。

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圖4-14 不同時間點 6、12、24 小時以 P. acnes 誘發 ICR 小鼠耳朵腫脹發炎 之影響

Figure 4-14 Time course of evaluation in vivo inflammatory activity of P.

acnes induced ear edema. Ears of ICR mice were injected intradermally with P.

acnes (6×10⁷ CFU per 10 μl in PBS) or PBS alone for 6, 12, 24 hours. The ear edema in mice was evaluated by measuring the ear thickness.Values are means

± SD.The data were evaluated for statistical significance with one-way ANOVA followed by LSD tests. Asterisk indicate significant difference from the control group(* p＜0.001). Control: PBS alone.

control 6h 12h 24h

Ear thickness (% of PBS injected-ear)

0 50 100 150 200 250

* *

*

with P. acnes injected

54

圖4-15 不同時間點 PBS 和 P. acnes 注射小鼠耳朵後組織均質液的 IL-1β cytokines 生成情形

Figure 4-15 Time course of IL-1β cytokines in homogenized ears after intradermally injection of PBS alone or P. acnes into the ears of ICR mice.

Ears of ICR mice were injected intradermally with P. acnes (6×10⁷ CFU per 10 μl in PBS) or PBS alone for 6, 12, 24 hours. Values are means ± SD.The data were evaluated for statistical significance with one-way ANOVA followed by LSD and Duncan’s multiple range tests. Pound sign indicates significant difference between the two bars(# p＜0.05). Control: PBS alone. Means with the same letter are not significantly different between P. acnes groups.

Differences were considered significant for p＜0.05.

IL-1 concentration (pg/mL)

0 200 400 600 800 1000

control P. acnes

6h 12h 24h

#

#

#

a

b c

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圖4-16 不同時間點 PBS 和 P. acnes 注射小鼠耳朵後組織均質液的 IL-6 cytokines 生成情形

Figure 4-16 Time course of IL-6 cytokines in homogenized ears after intradermally injection of PBS alone or P. acnes into the ears of ICR mice.

Ears of ICR mice were injected intradermally with P. acnes (6×10⁷ CFU per 10 μl in PBS) or PBS alone for 6, 12, 24 hours. Values are means ± SD.The data were evaluated for statistical significance with one-way ANOVA followed by LSD and Duncan’s multiple range tests. Pound sign indicates significant difference between the two bars(# p＜0.05). Control: PBS alone. Means with the same letter are not significantly different between P. acnes groups.

Differences were considered significant for p＜0.05.

IL-6 concentration (pg/mL)

0 100 200 300 400

6h 12h 24h

#

a b

c

#

control P. acnes

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圖4-17 P. acnes 及 FL1-FL7 注射後 12 小時小鼠耳朵的外觀變化情形 Figure 4-17 Effect of intradermally injected with P. acnes and FL1-FL7. P.

acnes (6×10⁷ CFU per 10 μL in PBS) and FL1-FL7(20μg/10μL) was

intradermally injected into the right ears of ICR mice for 12 hrs. Left ears did not inject anything.

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圖4-18、FL1-FL7 對 P. acnes 誘發 ICR 小鼠耳朵腫脹發炎之影響 Figure 4-18 Evaluation in vivo anti-inflammatory activity of FL1-FL7 on P.

acnes-induced ear edema. Ears of ICR mice were injected intradermally with P.

acnes (6×10⁷ CFU per 10 μl in PBS) and FL1-FL7 (20μg per 10 μL) or PBS alone for 12 hours. The ear edema in mice was evaluated by measuring the ear thickness.Values were means ± SD (n = 6). The data were evaluated for statistical significance with one-way ANOVA followed by LSD tests. Asterisk indicates significant difference from the DMSO group(*p＜0.001). DMSO: P.

acnes. Control: PBS alone.

Ear thickness (% of PBS injected-ear)

0 50 100 150 200 250

with P. acnes injected

FL1 FL2 FL3 FL4 FL5 FL6 FL7

DMSO (20g)

control

* *

*

*

*

* *

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抑制率(%)

P. acnes 0 ± 0.0 ^c

P. acnes + FL1 33.9 ± 6.4 ^{a, b}

P. acnes + FL2 27.8 ± 7.7 ^b

P. acnes + FL3 37.9 ± 5.8 ^{a, b}

P. acnes + FL4 36.3 ± 17.6

a, b

P. acnes + FL5 38.3 ± 16.9

a

P. acnes + FL6 30.7 ± 14.1 ^{a, b}

P. acnes + FL7 27.0 ± 8.6 ^b

表4-1 FL1-FL7 抑制 P. acnes 誘發 ICR 小鼠耳朵腫脹發炎之效果(% 抑制率) Table 4-1 % Inhibition of FL1-FL7 on P. acnes-induced ear edema in vivo anti-inflammatory activity. Ears of ICR mice were injected intradermally with P. acnes (6×10⁷ CFU per 10 μl in PBS) and FL1-FL7 (20μg per 10 μL). The ear edema in mice was evaluated by measuring the ear thickness. Values were means ± SD (n = 6). The data were evaluated for statistical significance with one-way ANOVA followed by Duncan’s multiple range tests. Means with the same letter are not significantly different between groups. Differences were considered significant for p＜0.05.

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圖4-19 FL1-FL7 對於 P. acnes 注射 12 小時後小鼠耳朵組織均質液的 IL-1β cytokines 生成情形

Figure 4-19 The effect of FL1-FL7 on IL-1β cytokines induced by

intradermally injection of P. acnes into the ear of ICR mice for 12 hrs. Ears of ICR mice were injected intradermally with P. acnes (6×10⁷ CFU per 10 μL in PBS) and FL1-FL7 (20μg per 10 μL) or PBS alone for 12 hours. Values are means ± SD (n = 6). The data were evaluated for statistical significance with one-way ANOVA followed by LSD and Duncan’s multiple range tests.

Asterisk indicates significant differences from the DMSO group(* p < 0.05, **

p < 0.01, ***p＜0.001). DMSO: positive control (with P.acnes), Control:

negative control (PBS alone). Means with the same letter are not significantly different between treatment groups. Differences were considered significant for p＜0.05.

control DMSO FL1 FL2 FL3 FL4 FL5 FL6 FL7 IL-1 concentration (pg/mL)

0

with P. acnes injected

(20g/10L)

60

圖4-20 FL1-FL7 對於 P. acnes 注射 12 小時後小鼠耳朵組織均質液的 IL-6 cytokines 生成情形

Figure 4-20 The effect of FL1-FL7 on IL-6 cytokines induced by P. acnes into the ear of ICR mice for 12 hrs. Ears of ICR mice were injected intradermally with P. acnes (6×10⁷ CFU per 10 μL in PBS) and FL1-FL7 (20μg per 10 μL) or PBS alone for 12 hours. Values are means ± SD (n = 6). The data were

evaluated for statistical significance with one-way ANOVA followed by LSD and Duncan’s multiple range tests. Asterisk indicates a significant difference from the DMSO group(*p＜0.001). DMSO: positive control (with P.acnes), Control: negative control (PBS alone). Means with the same letter are not significantly different between treatment groups. Differences were considered significant for p＜0.05.

control DMSO FL1 FL2 FL3 FL4 FL5 FL6 FL7

IL-6 concentration (pg/mL)

0

with P. acnes injected

(20g/10L)

61

圖4-21 FL1-FL7 對於 P. acnes 注射 12 小時後小鼠耳朵組織均質液的 TNF-α cytokines 生成情形

Figure 4-21 The effect of FL1-FL7 on TNF-α cytokines induced by P. acnes into the ear of ICR mice for 12 hrs. Ears of ICR mice were injected

intradermally with P. acnes (6×10⁷ CFU per 10 μL in PBS) and FL1-FL7 (20μg per 10 μL) or PBS alone for 12 hours. Values are means ± SD (n = 6).

The data were evaluated for statistical significance with one-way ANOVA followed by LSD and Duncan’s multiple range tests. Pound sign and asterisk indicate significant differences from the DMSO group(* p < 0.01, **p＜0.001).

DMSO: positive control (with P.acnes), Control: negative control (PBS alone).

Means with the same letter are not significantly different between treatment groups. Differences were considered significant for p＜0.05.

control DMSO FL1 FL2 FL3 FL4 FL5 FL6 FL7 TNF- concentration (pg/mL)

0

with P. acnes injected

(20g/10L)

**

a

**

a

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