The Aim Of The Present Study Was To Investigate The Effects Of Blue Honeysuckle Extract

The aim of the present study was to investigate the effects of blue honeysuckle extract (BHE), which contains high level of phenolic compounds, on endotoxin-induced uveitis (EIU). Male Lewis rats were randomly divided into 5 groups with 14 rats in each (eight rats for collection of aqueous humor, six rats for histologic examination). EIU was induced by a footpad injection of lipopolysaccharide (LPS). 1, 10, or 100 mg of BHE was injected intravenously immediately after LPS injection. The aqueous humor was collected at 24 h after LPS injection, the number of infiltrating cells, protein concentration, nitric oxide (NO), tumor necrosis factor (TNF)-a, and prostaglandin (PG)-E2 levels in the aqueous humor were determined. Some eyes were enucleated for histologic examination and immunohistochemical analysis. Immunohistochemical staining with a monoclonal antibody against activated nuclear factor (NF)-kB was performed to evaluate the effect of BHE on NF-kB activation. To further clarify the anti-inflammatory effect, RAW264.7 cells (a mouse macrophage cell line) were stimulated with LPS in the presence or absence of BHE and its major phenolics, cyanidin 3glucoside (C3G), cyanidin 3-rutinoside (C3R), chlorogenic acid (CA). Expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were analyzed by Western blot method. BHE treatment significantly reduced the inflammatory cell infiltration, the protein concentration, the levels of NO, TNF-a and PGE2 in the aqueous humor and improved histologic status of the ocular tissue. * The number of activated NF-kB-positive cells was lower in the iris-ciliary body treated with BHE at 3 h after LPS injection. BHE significantly suppressed the production of NO, PGE2 and TNF-a in the culture medium as well as the expression of iNOS and COX-2 by LPSstimulated RAW264.7 cells in a dose-dependent fashion. C3G, C3R and CA showed no or weak inhibitory effects on the level of inflammatory mediators and the expression of iNOS and COX-2.* These results suggest that BHE attenuates the degree of inflammation in eyes with EIU by inhibiting the NF-kB dependent signaling pathway and the subsequent production of proinflammatory mediators.

摘要：

本研究的目的是調查包含多量酚類化合物藍色忍冬屬植物萃取物(BHE)，在內 毒素影響葡萄膜炎的作用 (EIU)。馬律劉易斯鼠任意地被劃分了成每5個小組， 每組14隻鼠 (八隻鼠為水性體液彙集，六隻鼠為組織測試)。利用足蹼注射脂肪 多醣類(LPS)導致了EIU。注射LPS後，再將1、10、100 mg的BHE進行靜脈注射 水性體液彙集組注射LPS 24H之後，觀察滲入細胞的數量、蛋白質含量、硝酸氧 化物(NO)、腫瘤壞死因素(TNF)- α、和前列腺素(PG)-E2。有些觀點是用來解釋組 織測試和免疫組織化學的分析。免疫組織化學的染色劑與單細胞繁殖的抗體抵抗 活化的核因素(NF)-kb，評估BHE 的作用影響NF-kb活性。進一步評估抗發炎效 果，而刺激RAW264.7細胞 (老鼠巨噬細胞細胞線)的LPS主要是受到BHE 和它 的主要酚醛樹脂、cyanidin 3 glucoside (C3G)、cyanidin 3 rutinoside (C3R)、綠原酸酸(CA)的存在或缺乏影響。表示可誘導氧化氮合成酶 (iNOS)和 cyclooxygenase-2 (COX-2) ，並且能利用西方點墨法分析。BHE在水性體液 彙集組的治療作用極大減少了發炎細胞滲透活動、蛋白質含量、NO濃度、TNF-a and PGE2，且明顯地改善了組織狀態。BHE 在培養基有效地壓制NO、PGE2和 TNF-α的產生，並且與RAW264.7細胞反應，大大降低了 iNOS 和COX-2的濃 度。然而C3G 、C3R 和CA在抑制 iNOS和COX-2的效果，顯示出沒有或微弱的 抑制作用。結果顯示，藉著抑制 NF kb依賴信號路和發炎因子的產生，BHE會 隨著EIU在眼睛的發炎程度而降低。

Endotoxin-induced uveitis (EIU) is an animal model of acute ocular inflammation, produced by injection of endotoxin, the lipopolysaccharide (LPS) component of gram-negative bacterial cell wall (Rosenbaum et al., 1980). EIU is characterized by protein leakage in the anterior chamber and by infiltration of macrophages and neutrophils into the eye (Li et al., 1995), (Chan et al., 2000). In Lewis rats with EIU, acute inflammation develops mainly in the anterior chamber (iridocyclitis) and inflammatory cells may also infiltrate the vitreous and retina (Tuaillon et al., 2002). Numerous data indicate that cytokines such as tumor necrosis factor (TNF)-a play an essential role in the development of EIU (de Vos et al., 1994),(Planck et al., 1994). The inducible nitric oxide synthase-nitric oxide (iNOS-NO) pathway and inducible cyclooxygenase-prostaglandin E2 (COX-2-PGE2) pathway are also involved in the pathogenesis of EIU in rats. The overproduction of iNOS, COX-2, TNF-a and other cytokines is mediated through nuclear factor (NF)-kB (Baeuerle and Henkel, 1994; Pahl, 1999; Celec, 2004). NF-kB is one of the most ubiquitous transcription factors that regulate gene expressions involved in cellular proliferation, inflammatory responses, and cell adhesion. Upon stimulation with a wide variety of stimuli including LPS, ＊NF-kB translocates from the cytoplasm to the nucleus where the NF-kB positively regulates the expression of genes involved in the immune and inflammatory responses, including iNOS, COX-2, and TNF-(a (Pahl, 1999; Baldwin, 1996). Ohta et al. (Ohta et al., 2002) demonstrated for the first time a

significant upregulation of activated NF-kB in the iris-ciliary body (ICB) during EIU.＊ Blue honeysuckle or sweet berry honeysuckle (Lonicera caerulea L.) fruits are widely harvested in Russia, China, and Japan. Fruit shapes are oval to long and dark navy blue to purple in color. Their flavor is similar to that of bilberries, black currants, and blueberries. Blue honeysuckle extract *(BHE) contains high level of phenolic compounds, which have been reported to have multiple biological properties, including antioxidant (Kahkonen et al., 2001; Wang and Lin, 2000), antimicrobial (Puupponen-Pimia et al., 2001; Zhu et al., 2004) and anti-inflammatory properties (Jiang and Dusting, 2003; Park et al., 2004).* Polyphenolic compounds have been regarded as a potential novel, safe, and nontoxic strategy for the modulation of inflammation dependent on the NF-kB pathway. It has been widely shown that many kinds of polyphenolic compounds present significant anti-inflammatory effects in vitro and in vivo* The purpose of the present study was to investigate the effects of BHE on EIU in rats. We investigated the effects of BHE on cellular infiltration, protein extravasation and levels of TNF-a, PGE2 and NO. To clarify the anti-inflammatory effect, we investigated the activation of NF-kB in ICB of the rats treated with BHE. In addition, we also investigated the levels of NO, PGE2 and TNF-a and the expression of iNOS and COX-2 in RAW 264.7 cells treated with BHE.

前言： (EIU)，一種動物的眼睛經內毒素而導致嚴重的發炎症狀。(LPS) 是由革蘭氏陰 性的細菌細胞壁所組成，釋放出的內毒素會對人體造成危害 。EIU的特徵是蛋白 質會滲漏到前庭，以及巨噬細胞與嗜中性白血球滲透到眼睛。在感染EIU的老鼠 中，發炎症狀主要顯現在前庭(虹膜睫狀體炎)，且發炎細胞也會滲入玻璃體和 視網膜。眾多的資料表明，腫瘤壞死因素(TNF)-a的細胞分裂是EIU發展的根本。 iNOS-NO 與 COX-2-PGE2 也被發現與EIU發病原理有關，而iNOS 、COX-2 、 TNF-α 的產生是由(NF) kb來調控。 * NF κb 是調控基因表達被介入在多孔的擴散、激動反應, 和細胞黏附力的最普 遍存在的副本因素的當中一個。在刺激與各種各樣的刺激包括LPS, NF kb 改變 的位置從細胞質對NF kb 正面地調控基因表示被介入在免疫的中堅力量並且激 動反應, 包括iNOS, COX-2, 和TNF-α 第一次展示了被激活的NF kb 的一重大 upregulation 在虹膜ciliary 身體(ICB) 在EIU 期間。* 藍色忍冬屬植物或甜莓果忍冬屬植物(Lonicera caerulea L ) 果子廣泛被收穫 在俄國、中國, 和日本。果子形狀是長卵形，而顏色是藍紫色。他們的味道類似越 桔、黑醋栗，和藍莓。藍色忍冬屬植物萃取物(BHE) 包含高水平酚類化合物，報 告中有多生物特性，包括抗氧化、抗菌、抗發炎等。 * 多數的酚類化合物被認為是一種新穎、安全、和無副作用的藥方，尤其在發炎 症狀中來調控NF kb。在很多報告中顯示，許多種酚類化合物在試管內和在體內 有效地抗發炎作用。* 本研究的目的在利用EIU的老鼠研究BHE的作用。我們發現BHE影響了細胞的滲 透作用、蛋白質外滲、TNF-a、PGE2與NO濃度。為了證實其抗發炎反應，我們在 BHE處理過的老鼠虹膜睫體調查NF kb的活化作用。另外，也在BHE處理的 RAW264.7細胞觀察了NO、PGE2、TNF-a、COX-2和iNOS的濃度。

在本研究中，BHE治療極大減少了LPS導致的EIU多孔濾滲和蛋白質彙集。結果 顯示，BHE劑量越大，越是能壓制EIU的發炎反應。 由結果可得知，EIU的產生與嗜中性白血球所誘導的發炎因子有關係，例如： TNF-a, PGE2 and NO。 在許多發炎疾病中TNF-a常被發現，這種物質是由人體巨噬細胞所分泌的一種 細胞激素（cytokine），TNF-a會藉由NF kB活化來促進發炎反應。本研究的結果 顯示，BHE成功地壓制LPS引起的TNF-a 與 NF kb 的活化作用。除TNF-a以外， NF kb也能調控iNOS 和COX-2，兩者可以各自地誘導NO和PGE2。在此研究中， TNF-a、NO和PGE2 皆被BHE 抑制並且有效地降低濃度，這資料說明了，抗發炎 反應的原因是BHE抑制了這些發炎因子的生產。此外，巨噬細胞跟EIU發病也有 關係，經過LPS注射後，巨噬細胞和單核白血球是最初進入視覺組織的發炎細 胞(接下來是多形核白血球和T細胞)，兩者皆產生不同的細胞激素與其他的發炎 因子。因此，我們在試管內利用RAW264.7細胞(老鼠巨噬細胞細胞線)研究了 BHE的抗發炎反應。酵素iNOS與細胞激素、LPS作用後會在巨噬細胞、嗜中性白血 球和內皮細胞內產生NO。在經過LPS刺激的RAW264.7細胞裡，發現了BHE能抑 制iNOS 和COX-2 的表現，以及其產生的NO和PGE2。因此，顯示了BHE藉由抑 制iNOS 和COX-2 的表現以抵制NO和PGE2的生產。結果建議，BHE抑制iNOS和 COX-2 的表現是一個重要的抗發炎機制。然而，TNF-a 、iNOS 和COX-2 的表現 量全部由NF kb調控。因此，抑制NF kb的活化可用來解釋BHE 在EIU中的治療作 用。＜BHE 包含植物化學物質的一個不同的範圍。＞大多數植物萃取物的生物活 動歸因於它的酚類化合物，我們拿BHE在試管內三種抗發炎作用的主要酚類化 合物來做比較，沒有任何一種的抗發炎作用比BHE的效果好。＜phytochemicals 複雜混合物在BHE 可能提供抗發炎作用主要通過疊加性並且/或者協同作用的作 用的組合。＞ 總之,，這項研究顯示， BHE有效地抑制EIU在老鼠眼睛組織中的 發炎作用。＜可能的機制為這個BHE 的作用也許取決於他們的能力禁止NF kb 的活化作用和proinflammatory 斡旋人的隨後生產譬如TNF-a 、PGE2 和沒有。 ＞

結論： In the present study, treatment with BHE significantly reduced LPS induced cellular infiltration and protein concentration in the aqueous humor as well as histopathologic manifestation of EIU. EIU is believed to result from the release of a variety of mediators such as TNF-a, PGE2 and NO by activated inflammatory cells (Smith et al., 1998). TNF-a is known to play a key role in many inflammatory diseases, as it promotes inflammation through the induction of other proinflammatory cytokines and sustains inflammation by facilitating the infiltration of leukocytes. It has been reported that uveitis attacks in Behc¸et’s disease were remitted by administration of anti-TNF-a antibody, and in general, symptoms ameliorated remarkably (Nakamura and Ohno, 2005). TNF-a is activated through NF-kB, also activates NF-kB, thus promoting its own secretion and generating a positive loop that amplifies the cytokine cascade and the inflammatory response (Baeuerle and Henkel, 1994; Baldwin, 1996; Barnes and Karin, 1997). The results of the present study indicate that BHE treatment suppresses the LPS-induced elevation of TNF-a in the aqueous humor as well as the activation of NF-kB in ICB, therefore, it is reasonable to conclude that the positive loop between TNF-a and NF-kB is interfered by BHE. Besides TNF-a, NF-kB also regulates the expression of iNOS and COX-2, which is responsible to the production of NO and PGE2, respectively. ＊NO and PGE2 are important inflammatory mediators involved in the pathogenesis of EIU and both of them are considered to contribute to the breakdown of the blood-aqueous barrier during EIUreported that NO and PGE2 have additive effects in EIU in rabbits, and that the inhibition of both pathways would improve the therapeutical management of uveitis.＊ In the present study, like TNF-a, level of NO and PGE2 in aqueous humor was also reduced by BHE administration. These data indicated that the suppression of inflammatory manifestation by BHE treatment may mainly be attributed to the inhibition of these inflammatory mediators production. Macrophages play a key role in the pathogenesis of EIU.

After LPS injection, macrophages and monocytes are the earliest inflammatory cells to invade the ocular tissues followed by polymorphonuclear leukocytes and T cells (McMenamin and Crewe, 1995). Macrophages/monocytes could play a prominent role in EIU by synthesizing different cytokines and other inflammatory mediators (Pouvreau et al., 1998). So, we investigated the in vitro anti-inflammtory effects of BHE by using a mouse macrophage cell line, RAW264.7 cell. The enzyme iNOS, which has the capacity to sustain production of NO, presents in macrophages, neutrophils and endothelial cells following transcriptional induction by LPS or cytokines. ＊ It has been implicated that both inflammatory and resident ocular cells are involved in iNOS expression during EIU . COX-2 is responsible for the production of large amounts of proinflammatory PGs at the inflammatory sites in animals as well as patients with inflammatory diseases (Seibert et al., 1994; Masferrer et al., 1994). ＊ We have found BHE can inhibit the expression of iNOS and COX-2 as well as their products, NO and PGE2, in LPS-stimulated RAW264.7 cells. Thus, it appears that BHE suppresses the production of NO and PGE2 by blocking the expression of iNOS and COX-2 protein, respectively. The results suggested that the blocking of iNOS and COX-2 proteins expression is one of the anti-inflammatory mechanisms of BHE. The expressions of TNF-a, iNOS and COX-2 are all regulated by NF-kB. ＊Most genes encoding adhesion molecules, cytokines such as TNF-a, and other proinflammatory proteins such as iNOS and COX-2 have functional NF-kB binding elements in their promoter regions＊ (Baeuerle and Henkel, 1994; Baldwin, 1996). Thus, the inhibition of NF-kB activation can help to explain the beneficial effect of BHE in EIU treatment. BHE contains a diverse range of phytochemicals. Most of the biological activity of plant extracts has been attributed to its phenolic compounds. We compared the in vitro antiinflammatory effects of three kinds of its major phenolic compounds with that of BHE. None of them showed antiinflammatory effects as strong as BHE. ＊These results suggest

that complex mixtures of phytochemicals in BHE maybe provide anti-inflammatory effects mainly through a combination of additive and/or synergistic effects.＊ In summary, this study indicates that BHE shows a significant anti-ocular inflammatory effect on EIU in rat. The possible mechanisms for this effect of BHE may depend on their ability to inhibit the activation of NF-kB and the subsequent production of proinflammatory mediators such as TNF-a, PGE2 and NO.

2. Materials and methods 2.1. Preparation of the ethanol extract of blue honeysuckle Blue honeysuckle fruit (541.3 g) harvested in Hokkaido, Japan was homogenized with 50% (w/w) ethanol. The homogenized sample was filtered under reduced pressure and extracted again with an equal volume of 50% (w/w) ethanol. The filtrate of the extract was concentrated and dried with a vacuo and a freeze-dryer, respectively. 55.3 g of dried extract was obtained. The BHE preparation was LPS free. The composition of the extract measured by Folin–Denis assay and high-performance liquid chromatography was as follows: 1) sugars 48.9%; sorbitol 21.8%, glucose 15.2%, fructose 11.9%, 2) organic acid 24.1%; citric acid 3.7%, malic acid 18.0%, unknown 2.4%, 3) polyphenolics 12.2%; antocyanin 9.2%, quercetin 1.8%, unknown 1.2% and 4) soluble fiber 14.8%.

藍色忍冬屬植物對氨基苯甲酸二萃取物的準備 藍色忍冬屬植物果子(541.3 g) 收穫了在北海道, 日本均勻了與50% (w/w) 對 氨基苯甲酸二。均勻的樣品在減壓下被過濾了得和再被提取了以50% (w/w) 對 氨基苯甲酸二的相等的容量。萃取物的濾出液被集中了和被烘乾了與vacuo 和 冷凍烘乾機, 各自地。55.3 g 乾萃取物被獲得了。BHE 準備任意是LPS 。 萃取物的構成測量了由Folin Folin-Denis 分析用試樣並且高性能液相色譜是如 下: 1) 糖48.9%; 山梨糖醇21.8%, 葡萄糖15.2%, 果糖11.9%, 2) 有機酸 24.1%; 檸檬酸3.7%, 蘋果酸18.0%, 未知數2.4%, 3) polyphenolics 12.2%; antocyanin 9.2%, 五¥黃酮1.8%, 未知數1.2% 和4) 可溶解纖維

14.8% 。

2.2. Animal groups and EIU Eight-week-old male Lewis rats (180–220 g) were randomly divided into 5 groups with 14 rats in each (eight rats for collection of aqueous humor, six rats for histologic examination). EIU was induced by a footpad injection of 200 mg LPS from Salmonella typhimurium (Sigma, St. Louis, MO, USA) that had been diluted in 0.1 mL phosphate-buffered saline (PBS, pH 7.4). Rats were injected intravenously with 1, 10, or 100 mg BHE diluted in 0.1 mL PBS containing 0.1% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) immediately after LPS injection. Rats of control group were injected with 0.1 mL PBS containing 0.1% DMSO and without LPS injection. All experimental animalswere treated in accordance with theARVOStatement for use of animals in Ophthalmic and vision research.

2.2. 動物小組和EIU 八星期的公劉易斯鼠(180-220 g) 任意地被劃分了成5 個小組與14 隻鼠在每 個(八隻鼠為含水幽默的彙集, 六隻鼠為histologic 考試) 。EIU 通過200 毫克 LPS 的攔路賊射入導致了從被稀釋了在0.1機器語言磷酸緩衝鹽(PBS, 酸鹼度 7.4) 。 鼠靜脈內被注射了與1, 10, 或100 毫克BHE 被稀釋在0.1 機器語言PBS 包含 0.1% 二甲基sulfoxide (DMSO, 斯格碼, 聖路易斯, MO, 美國) 在LPS 射入之 後。控制群鼠被注射了以0.1 機器語言PBS 包含0.1% DMSO 和沒有LPS 射入。

所有實驗性animalswere 對待了與theARVOStatement 符合至於對動物的 使用在眼科和視覺研究。

2.3. Number of infiltrating cells and protein concentration in aqueous humor At 24 h after LPS injection, the rats were euthanized and the aqueous humor was collected immediately from both eyes by an anterior chamber puncture (15–20 mL/rat) using a 30-gauge needle under the surgical microscope. The aqueous humor was then accurately diluted 10-fold with PBS (pH 7.4). For cell counting, the aqueous humor sample was suspended in an equal amount of Tu¨rk stain solution, and the cells were counted, using a hemocytometer under a light microscope. The number of cells per field (an equivalent of 0.1 mL) was manually counted, and the number of cells per micro-liter was obtained by averaging the results of four fields from each sample. The total protein concentration in the aqueous humor samples was measured with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). The aqueous humor samples were stored in ice water until testing, cell counts and total protein concentrations were measured on the day of sample collection.

2.3. 滲入細胞和蛋白質含量的數字在含水幽默 在LPS 射入24H以後，將老鼠安樂死並且立刻從體液測試組的兩隻眼睛，在外

科顯微鏡下使用30口徑的測量針在前庭穿孔(15-20 mL/rat)。含水幽默用PBS (酸鹼度7.4) 10-fold 準確地然後被稀釋了。為細胞計數, 含水幽默樣品暫停了 在相等的相當數量Tu.rk 汙點解答, 並且細胞計數了, 使用一個血細胞計在一個 光學顯微鏡下。細胞的數量每領域(0.1 機器語言等值) 手工計數了, 和細胞的數 量每微升由平均為獲得了四個領域的結果從各個樣品。總蛋白含量含量在含水幽 默樣品被測量了與bicinchoninic 酸蛋白質分析用試樣成套工具(皮爾斯, Rockford, IL, 美國) 。含水幽默樣品被存放了在冰水裡直到測試, 細胞計數並且 總蛋白含量含量被測量了在樣品彙集的那天。 Turk stain solution 白血球計數亦用前述之血球計算器，計算盤

利用血球計數盤在顯微鏡下計算細胞活性。

2.4. Cell culture and LPS stimulation RAW 264.7, a mouse macrophage cell line, was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 2 mM glutamine, antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) and 10% heat-inactivated fetal bovine serum (Gibco-BRL, Grand Island,NY, USA) andmaintained at 37 8C in a humidified incubator containing 5% CO2. RAW cells were seeded onto a 24-well plate (3.2!105 cells/well). These cells were stimulated with 1 mg/ml LPS for 24 h in the presence or absence of 1, 10, 100 mg/ml of BHE or 100 mg/ml of C3G, C3R, CA, unless otherwise stated. LPS fromS. typhimurium were purchased from Sigma (St. Louis, MO, USA). C3G, C3R and CA obtained from Funacoshi Co. Ltd (Osaka, Japan) were LPS free.

2.4. 細胞培養和LPS 刺激 未加工264.7, 老鼠巨噬細胞細胞線, 被獲得了從美國型文化彙集(Manassas, VA, 美國) 。細胞被開化了在RPMI-1640 媒介(斯格碼, 聖路易斯, MO, 美國) 用 2 毫米被補充glutamine, 抗生素(100 U/mL 青黴素和100 U/mL 肺病特效藥) 並且10% 熱被撤消的胎兒遲鈍的清液(Gibco-BRL, 盛大海島, NY, 美國)

andmaintained 在37 8C 在一個被濕潤的孵養器包含5% 二氧化碳。未加工的 細胞播種了24-well 板材(3.2!105 cells/well) 。這些細胞被刺激了與1 mg/ml LPS 為24 h 在出現或缺乏BHE 1, 10,100 mg/ml 或C3G, C3R, 加州100 mg/ml, 除非另外說明。LPS fromS. typhimurium 被購買了從斯格碼(聖路易 斯, MO, 美國) 。C3G 、C3R 和加州被獲得從Funacoshi 有限公司(大阪, 日本) Co. 任意是LPS 。

2.5. Determination of NOlevels in aqueous humor and inmedium The total level of nitrate plus nitrite in the aqueous humor and medium was measured by using a total nitrite colorimetric assay kit (Oxis International, Portland, OR, USA), according to the manufacturer’s instruction. The absorbance values were measured at 540 nm in the microtiter plate reader.

2.5. 決心of NO levels 在含水幽默和 inmedium 硝酸鹽的總水平加上亞硝酸鹽在含水幽默和 媒介由使用測量了總亞硝酸鹽比色法分析用試樣成套工具(Oxis 國際, 波特蘭, 俄勒岡, 美國), 根據製造商的指示。absorbance 價值被測量了在540 毫微米在microtiter 板材讀者。

2.6. Levels of TNF-a and PGE2 in aqueous humor and in medium The levels of TNF-a and PGE2 in the aqueous humor and in

the medium were assessed with commercially available ELISA kits (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. The ELISA assay was performed in duplicate.

2.6. TNF-a 和PGE2 的水平在含水幽默和在媒介 TNF-a 和PGE2 的水平在含水幽默和在媒介被估計了與商業可利用的ELISA 成 套工具(R&D 系統, 米尼亞波尼斯, MN, 美國), 根據製造商的指示。ELISA 分析用試樣一式兩份執行了得。

2.7. Histopathologic evaluation Rats were euthanized 24 h after LPS injection. The eyes were enucleated immediately and stored in 10% buffered formalin for 24–48 h, and then the eyes were embedded in paraffin. 5 mm sagittal sections were cut near the optic nerve head and stained with hematoxylin and eosin (H–E). Anterior chamber, ICB, vitreous and retina were observed by a masked ocular pathologist under light microscopy. The histopathologic evaluation of inflammation was scored on grades of 0–3. A 0 grade represents no infiltrating cells. A grade 1 represents mild cell infiltration. A grade 2 represents moderate cell infiltration. A grade 3 represents severe cell infiltration with severe anterior chamber exudate.

2.7. Histopathologic 組織分析 評估鼠是euthanized 24 h 在LPS 射入以後。眼睛是立刻enucleated 並且存 放在10% 緩衝了甲醛水為24-48 h, 並且眼睛然後被埋置了在石蠟裡。5 毫米瀘 頂骨矢狀合縫的部分被削減了在視覺神經頭附近和被弄髒了與hematoxylin 和 四溴熒光素(他) 。先前分庭, ICB, 玻璃和視網膜由一位被掩沒的視覺病理學家觀 察了在光學顯微學之下。炎症的histopathologic 評估被計分了在等級0-3 。一 個0 個等級不代表滲入細胞。等級1 代表溫和的細胞濾滲。等級2 代表適度細胞 濾滲。等級3 代表嚴厲細胞濾滲嚴厲先前分庭滲出液。

ICB = 虹膜睫狀體

2.8. Immunohistochemical studies for NF-kB At 3 h after LPS injection, rats were anesthetized and the eyes were fixed by an intracardiac perfusion of 4% paraformaldehyde in 0.1 M PBS. The eyes were enucleated and immersed in the same fixative for 12 h. Then, embedment and section were performed as described above. The sections were rinsed in PBS twice and incubated with normal goat serum and then with the p65 antibody (Santa Cruz, c-20, CA, USA). Binding of the primary antisera was localized using Cy-3 conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA, USA). Nuclei were then stained with PBS containing YO-PRO-1 (Molecular Probes, Eugene, OR, USA) for 5 min. The sections were examined by laser scanning confocal microscopy (MRC-1024: Bio-Rad, Richmond, CA, USA; and LSM 510: Carl Zeiss, Oberkochen, Germany). Within each ICB sample, two areas were randomly photographed and the number of activated NF-kB positive cells was counted by a masked researcher. The results of two areas were averaged for each sample and in each group. This analysis was performed in the six eyes of three rats in each group.

2.8. Immunohistochemical 研究為NF kb 在3 h 在LPS 射入, 鼠以後被麻醉了並且眼睛由4% 多聚甲醛一心臟內的灌注固 定了在0.1 M PBS 。眼睛是enucleated 和浸沒了在同樣固定劑為12 h 。然後, embedment 和部分如上所述執行了得。部分兩次被漂洗了在PBS 和被孵化了 與正常山羊清液和然後與p65 抗體(聖克魯斯, c-20 、加州, 美國) 。束縛主要抗 血清地方化了使用Cy-3 被共軛的山羊反兔子IgG (傑克遜免役研究實驗室, 公司, 西部樹叢, PA, 美國) 。中堅力量然後被弄髒了與PBS 包含YO-PRO-1 (分子探針, Eugene, 或, 美國) 5 分鐘。部分由laser 審查了掃描confocal 顯微學(MRC-1024: 生物隨機存取 磁碟, 里士滿, 加州, 美國; 並且LSM 510: 卡爾・Zeiss, Oberkochen, 德國) 。 在各個ICB 樣品之內, 二個區域任意地被拍攝了並且被激活的NF kb 正面細胞 的數量由一位被掩沒的研究員計數了。二個區域的結果平均為各個樣品和在各個 小組。這分析執行了在三隻鼠的六隻眼睛在各個小組。

2.9. Western blot analysis RAW cells were washed with ice-cold PBS and then lysed in cold NP-40 lysis buffer (50 mM Tris-Cl [pH 7.6], 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml each of leupeptin, aprotinin, and pepstatin) for 15 min at 4 8C. Plates were then scraped, and crude lysates were cleared by centrifugation at 14,000 g for 10 min at 4 8C. Aliquots of the lysates were diluted with 2!SDS sample buffer and boiled for 2 min. Lysates were separated on 10% SDS-polyacrylamide gels, and transferred to immobilon polyvinylidene difluoride membranes. The membranes were then incubated in blocking solution (3% wt/vol dried low-fat milk and 0.1% vol/vol Tween 20 in PBS). Subsequently, the membranes were incubated with anti-COX-2 antibody (Alexis Biochemicals, Carlsbad, CA, USA), antiiNOS antibody (Upstate, Lake Placid, NY, USA), anti-atubulin antibody (Lab Vision, Westinghouse Drive Fremont, CA, USA). The membranes were then probed with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ, USA) and visualized by chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA).

2.9. 西部汙點分析

未加工的細胞被洗滌了與冰冷PBS 和然後被溶解了在冷的NP-40 病勢漸退緩衝 (50 毫米Tris Cl [ 酸鹼度7.6 ], 150 毫米NaCl, 10% 丙三醇, 1% NP-40, 1 毫米phenylmethylsulfonyl 氟化物, 和1 mg/ml 每個leupeptin 、aprotinin, 和pepstatin) 15 分鐘在4 8C 。板材然後被刮了, 並且粗暴lysates 被離心法 清除了在14,000 g 10 分鐘在4 8C 。lysates 的整除數用2!SDS 樣品緩衝被 稀釋了和煮沸了2 分鐘。Lysates 被分離了在10% SDS polyacrylamide 膠凝 體, 和轉移了到immobilon 聚偏乙烯difluoride 膜。膜然後被孵化了在阻攔解 答(3% wt/vol 低脂肪牛奶粉和0.1% vol/vol 非離子活性劑20 在PBS) 。隨後, 膜被孵化了與anti-COX-2 抗體 (Alexis Biochemicals, Carlsbad, 加州, 美國), antiiNOS 抗體(Upstate, 湖安詳, NY, 美國), anti-atubulin 抗體(實驗室視覺, 西屋電器驅動Fremont, 加州, 美國) 。膜被探查了與辣根peroxidase 被共軛 的次要抗體(Amersham Biosciences, Piscataway, NJ, 美國) 並且由化合光 (Amersham Biosciences, Piscataway 然後形象化, NJ, 美國) 。 2.10. Cell Viability Cell viability was determined by MTT assay after 24 h exposure to various concentrations of BHE. 5 mg/ml of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Chemicon, Temecula, CA, USA) was added to 0.1 mL of cell suspension for 4 h, and the formazan formed was then dissolved in isopropanol. Optical density was measured using a plate reader at 570 nm. 2.10. 細胞存活

細胞在經過BHE注射24H後，由MTT化驗分析。 3-(4,5- dimethylthiazol 2 yl) 2,5 二苯基tetrazolium 溴化物5 mg/ml 增 加了來0.1 機器語言細胞懸浮為4 h, 並且formazan 被形成然後被溶化了在異 丙醇裡。光學密度被測量了使用一個板材讀者在570 毫微米。 Fig. 1. Effect of BHE on cellular infiltration (A) and protein concentration (B) in the aqueous humor 24 h after LPS injection. Data are the meanGS.D. (nZ8). **Significantly different from LPS group (p!0.01). 圖1. BHE 的作用在多孔的濾滲(a) 和蛋白質含量(b) 在含水幽默24 h 在LPS 射入以後。資料是meanGS.D. (nZ8) 。** 顯著與LPS 小組不同(p!0.01) 。

Fig. 2. Histologic evaluation of EIU treated with BHE. A: histologic changes in the anterior segment 24 h after LPS injection. (a) control group: rats were not injected with LPS. (b) LPS group: rats were injected with LPS and 0.1 mL PBS. (c) BHE-treated group: rats were injected with LPS and 100 mg BHE diluted in 0.1 mL PBS. AC, anterior chamber; CB, ciliary body; HE staining; original magnification, !200. Arrows, inflammatory cells. B: effect of various dosage of BHE on histologic score of EIU. The score is the meanGS.D. (nZ6). *p!0.05, ** p!0.01 Significantly different from LPS group. 圖2. EIU 的Histologic 評估對待了與BHE 。A: histologic 變化在先前段24 h 上在LPS 射入以後。(a) 控制群: 鼠未被注射與LPS 。(b) LPS 小組: 鼠被注射了 以LPS 和0.1 機器語言PBS 。(c) BHE 被對待的小組: 鼠被注射了與LPS 和100 毫克BHE 被稀釋在0.1 機器語言PBS 。AC, 先前分庭; CB, 虹膜睫狀體; 他弄髒; 原始的放大,!200. 箭頭, 激動細胞。B: BHE 各種各樣的劑量的作用在EIU histologic 比分。比分是meanGS.D. (nZ6) 。* p!0.05, ** p!0.01 顯著與LPS 小組不同。 Fig. 3. Effect of BHE on LPS-induced NO (A), TNF-a (B) and PGE2 (C) concentrations in the aqueous humor 24 h after LPS injection. Data are the meanGS.D. (nZ8). **Significantly different from LPS group (p!0.01). ND not detected. 圖3. BHE 的作用不LPS 導致(a) 、TNF-a (b) 和PGE2 (c) 集中在含水幽默24 h 在LPS 射入以後。資料是meanGS.D. (nZ8) 。** 顯著與LPS 小組不同 (p!0.01) 。ND 沒被查出。 Fig. 4. Effect of BHE on LPS-inducedNO (A), TNF-a (B) and PGE2 (C) concentrations in LPSstimulatedRAWcells. RAWcells treated with various concentrations of BHE or 100 mg/ml of C3G, C3R, CA were incubated with LPS (1 mg/ml) for 24 h. Experimental data are expressed as meanGS.D. (nZ8). * p!0.05, ** p!0.01, compared with LPS group. ND not detected.

圖4. BHE 的作用在Lp inducedNO (a), TNF-a (b) 和PGE2 (c) 集中在Lp

stimulatedRAWcells.RAWcells 被對待以BHE 或C3G, C3R, 加州100 mg/ml 的各種各樣的集中被孵化了與LPS (1 mg/ml) 為實驗性資料被表達作 為meanGS.D. 的24 h. (nZ8) 。* p!0.05, ** p!0.01, 比較LPS 小組。ND 沒 被查出。 Fig. 5. Effect of BHE on NF-kB p-65 activation in the ICB 3 h after LPS injection. A: immunohistochemistry of NF-kB p-65 (red) in the ICB. Dualimmunofluorescence labeling showed the colocalization (yellow) in nuclei (green). (a) control group: Rats were not injected with LPS. (b) LPS group: rats were injected with LPS and 0.1 mL PBS. (c) BHE-treated group: rats were injected with LPS and 100 mg of BHE diluted in 0.1 mL PBS. Magnification:!400. Arrows, activated NF-kB positive cells. B: Quantitative analysis of NF-kB-positive cells in the ICB. Data are the meanGS.D. (nZ3). **Significantly different from LPS group (p!0.01) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

圖5. BHE 的作用在NF kb p-65 活化作用在ICB 3 h 在LPS射入以後。 A:immunohistochemistry NF kb p-65 (紅色) 在ICB 。 Dualimmunofluorescence 標記顯示了colocalization (黃色) 在細胞 (綠色) 。(a) 控制群: 鼠未被注射與LPS 。(b) LPS 小組: 鼠被注射了以LPS 和0.1 機器 語言PBS 。(c) BHE 被對待的小組: 鼠被注射了與BHE LPS 和100 毫克被稀釋 在0.1 機器語言PBS 。Magnification:!400 。箭頭, 被激活的NF kb 正面細胞。 B: 對NF kB 正面細胞的定量分析在ICB 。資料是meanGS.D. (nZ3) 。**顯著與 LPS 小組不同(p!0.01) (為參考的解釋在顏色的在這個圖傳奇, 讀者提到這篇文 章的網版本) 。

Fig. 6. Inhibition of LPS-induced iNOS protein and COX-2 protein by BHE in raw macrophages. Raw cells treated with various concentrations of BHE or 100 mg/ml of C3G, C3R, CA were incubated with LPS (1 mg/ml) for 24 h. Expression of iNOS protein and COX-2 protein were detected by western blot. Equal loading of proteins was verified by western blot analysis of a-tublin. This experiment was repeated three times with similar results. Lane 1, control; Lane 2, LPS; Lane 3, 1 mg/ml BHE; Lane 4, 10 mg/ml BHE; Lane 5, 100 mg/ml BHE; Lane 6, 100 mg/ml C3G; Lane 7, 100 mg/ml C3R; Lane 8, 100 mg/ml CA.

圖6. LPS 導致的iNOS 蛋白質和COX-2 蛋白質的禁止由BHE 在未加工的巨噬 細胞。未加工的細胞被對待以BHE 或C3G, C3R, 加州100 mg/ml 的各種各樣 的集中被孵化了與LPS (1 mg/ml) 為iNOS 蛋白質24 h. 表示並且COX-2 蛋白

質由西部汙點查出了。蛋白質相等的裝貨由對a-tublin 的西部汙點分析核實了。 這個實驗被重覆了三次以近似結果。車道1, 控制; 車道2, LPS; 車道3, 1 mg/ml BHE; 車道4, 10 mg/ml BHE; 車道5, 100 mg/ml BHE; 車道6, 100 mg/ml C3G; 車道7, 100 mg/ml C3R; 車道 8, 100 mg/ml 加州。 3.1. Effect of BHE on cellular infiltration and protein concentration in aqueous humor In the LPS group, the number of inflammatory cells in aqueous humor 24 h after LPS administration was 22.06G 7.26!105cells/ml (nZ8). Treatment with 100 mg of BHE significantly reduced the number of inflammatory cells (p!0.01), whereas 1 or 10 mg of BHE didn’t result in a reduction in the cell count. On the other hand, no infiltrating cell was detected in the aqueous humor of control group (Fig. 1(A)). The protein concentration in the groups treated with BHE was significantly lower than that in the LPS group, and the decrease showed dose-dependent fashion (Fig. 1(B)).

3.1. BHE 的作用在多孔的濾滲和蛋白質集中在含水幽默在LPS 小組, 激動細胞 的數量在含水幽默24 h 在LPS 管理以後是22.06G 7.26!105cells/ml (nZ8) 。治療與100 BHE 毫克極大減少了激動細胞的數量(p!0.01), 但是1 或10 BHE 毫克沒有導致對細胞計數的減少。另一方面, 滲入細胞未被查出在含水幽默控制 群(圖1(A)) 。蛋白質含量在小組比那對待了與BHE 顯著低在LPS 小組, 並且減 退顯示了dose-dependent 時尚(圖1(B)) 。 3.2. Histopathological findings Significant reductions of inflammation were observed in eyes treated with 100 mg BHE compared to LPS group (p!0.01, nZ6; Fig. 2(A)). The histopathological findings in 10 mg of BHE-treated were milder than that in LPS group, whereas treatment with 1 mg of BHE didn’t decrease the grading compared to LPS group (Fig. 2(B)).

3.2. Histopathological 炎症的研究結果重大減少被觀察了對眼睛被對待與 100 毫克BHE 與LPS 小組(p!0.01 比較, nZ6; 圖2(A)) 。histopathological 研究結果在10 裡毫克BHE 對待比那溫和的在LPS 小組, 但是治療與BHE 1 毫 克沒有減少分級與LPS 比較小組(圖2(B)) 。

3.3. Effect of BHE on NO, TNF-a and PGE2 in aqueous humor Considerable production of NO, TNF-a and PGE2 in the aqueous humor was seen in LPS group. BHE treatment significantly reduced their concentrations in aqueous humor with a dose-dependent fashion (p!0.01, nZ8; Fig. 3(A)–(C)).

BHE作用在沒有、TNF-a和PGE2 在含水幽默可觀的生產沒有, TNF-a 和PGE2 在含水幽默看了在LPS 小組。BHE 治療極大減少了他們的集中在含水幽默以 dose-dependent 時尚(p!0.01, nZ8; 圖3(A) (C))。 3.4. Effect of BHE on NO, TNF-a and PGE2 in medium of RAW264.7 cell Treatment with 1, 10 or 100 mg/ml of BHE reduced significantly the NO, TNF-a and PGE2 production compared to the LPS group. C3G or C3R (100 mg/ml each) also reduced NO production to the comparable level of 1 mg/ml BHE-treated group, whereas100 mg/ml of CA showed no inhibitory effects on the NO production (Fig. 4(A)). Treatment with 100 mg/ml of C3G also reduced PGE2 concentration. The same dose of C3R or CA exerted no influence on the PGE2 production by LPS-stimulated RAW cells (Fig. 4(C)). C3R or CA (100 mg/ml) showed no inhibition effects on the TNF-a level, whereas 100 mg/ml of C3G augmented TNF-a production by LPS-stimulated RAW cells (Fig. 4(B)). BHE did not decrease the cell viability of RAW264.7 cells when these cells were incubated with 100 mg/ml BHE for 24 h (data not shown).

3.4. BHE 的作用在沒有、TNF-a 和PGE2 在RAW264.7 細胞治療媒介與BHE 1, 10 或100 mg/ml 極大減少了沒有、TNF-a 和PGE2 生產與LPS 小組比較。C3G 或C3R (100 mg/ml 每個) 並且沒有使生產降低到 1 個mg/ml BHE 被對待的小組, whereas100 的可比較的水平mg/ml 加州顯 示了對沒有生產的禁止作用(圖4(A)) 。治療與C3G 100 mg/ml 並且減少了 PGE2 集中。C3R 或加州同樣藥量沒有施加對PGE2 生產的影響由LPS 被刺激 的未加工的細胞(圖4(C)) 。C3R 或加州(100mg/ml) 沒有顯示禁止作用在TNFa 水平上, 但是C3G 100 mg/ml 增添了TNF-a 生產由LPS 被刺激的未加工的

細胞(圖4(B)) 。BHE 沒有減少RAW264.7 細胞的細胞生活能力當這些細胞被孵 化了與100 mg/ml BHE 為24 h (資料沒被顯示)。 3.5. Immunohistochemistry of NF-kB p-65 in the ICB after LPS injection Three hours after injection of LPS, activated NF-kB p65 immunoreactivity was strongly expressed in the ICB. In contrast, the numbers of activated NF-kB-positive cells were lower in the ICB of rats treated with 100 mg of BHE. Normal controls showed only background levels (Fig. 5(A)). To obtain a quantitative measure of NF-kB activity in the ICB, the active NF-kB-positive cells were counted. In the control group, no active NF-kB-positive cells were detected in the ICB. LPS injection resulted in a marked increase in the percentages of active NF-kB-positive cells in ICB at 3 h (25.70G4.02%). In the BHE group, the percentages of active NF-kB-positive cells decreased significantly (12.60G1.52%, p!0.01; Fig. 5(B)).

3.5. NF kb Immunohistochemistry p-65 在ICB 在LPS 射入以後三個小時 在LPS 的射入以後, 被激活的NF kb p65 immunoreactivity 用ICB 強烈被表 達了。相反, 被激活的NF kB 正面細胞的數量是低在鼠ICB 被對待與100 BHE 毫克。正常控制顯示了唯一背景水平(圖5(A))。獲得NF kb 活動一個定量措施在 ICB, 活躍NF kB 正面細胞計數。在控制群, 活躍NF kB 正面細胞未被查出在ICB 。LPS 射入導致在活躍NF kB 正面細胞的百分比的明顯增量在ICB 在3 h (25.70G4.02%) 。在BHE 小組, 活躍NF kB 正面細胞的百分比極大減少了 (12.60G1.52%, p!0.01; 圖5(B)) 。 3.6. Expression of iNOS protein and COX-2 protein No iNOS or COX-2 signals were detected in unstimulated RAW cells (Fig. 6, Lane 1). LPS stimulation induced considerable expression of iNOS and COX-2 proteins (Fig. 6, Lane 2). BHE inhibited the expression of both iNOS and COX-2 proteins in a dose-dependent fashion (Fig. 6, Lane 3–5). However, 100 mg/ml of C3G, C3R or CA showed no effects on the expression of either iNOS or COX-2 proteins (Fig. 6, Lane 6–8).

3.6. iNOS 蛋白質和COX-2 蛋白質表示沒有iNOS 或COX-2 信號被查出了在 unstimulated 未加工的細胞(圖6, 車道1) 裡。LPS 刺激導致可觀的表示iNOS

和COX-2 蛋白質(圖6, 車道2) 。BHE 禁止了iNOS 和COX-2 表示蛋白質dosedependent 時尚(圖6, 車道3-5) 。但是, C3G 、C3R 或加州100 mg/ml 沒有 顯示對表示的作用或iNOS 或COX-2 蛋白質(圖6, 車道6-8) 。