TW201600089A - Zhankuic acid A and analogs thereof and their use as an anti-inflammatory agent - Google Patents

Zhankuic acid A and analogs thereof and their use as an anti-inflammatory agent Download PDF

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TW201600089A
TW201600089A TW103122167A TW103122167A TW201600089A TW 201600089 A TW201600089 A TW 201600089A TW 103122167 A TW103122167 A TW 103122167A TW 103122167 A TW103122167 A TW 103122167A TW 201600089 A TW201600089 A TW 201600089A
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zaa
lps
mice
binding
salmonella
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吳天賞
吳昭良
蕭璦莉
陳宇楓
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國立成功大學
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

Zhankuic acid A (ZAA) is the major pharmacologically active compound of Taiwanofungus camphoratus. We analyzed the structure of human TLR4/MD-2 complex with ZAA by X-score and HotLig modeling approaches. Two antibodies against MD-2 were used to verify the MD-2/ZAA interaction. The inflammation and survival of the mice pretreated with ZAA and injected with LPS were monitored. The modeling structure shows that ZAA binds the MD-2 hydrophobic pocket exclusively via specific molecular recognition; the contact interface is dominated by hydrophobic interactions. Binding of ZAA to MD-2 reduced antibody recognition to native MD-2, similar to the effect of LPS binding. Furthermore, ZAA significantly ameliorated LPS-induced endotoxemia and Salmonella-induced diarrhea in mice. Our results indicate that ZAA, which can compete with LPS for binding to MD-2 as a TLR4/MD-2 antagonist, is a potential therapeutic agent for Gram-negative bacterial infections.

Description

樟芝酸A及其類似物及作為抗炎劑的用途 Anthraquinone A and its analogues and use as anti-inflammatory agents

本發明係關於一種樟芝酸A及其類似物的新穎用途,其係使用作為抗炎劑。 The present invention relates to a novel use of anthraquinone A and its analogs as an anti-inflammatory agent.

脂多醣(LPS)係一種由兩親組分脂質A、親水性多醣核心及O-抗原最外區段構成的醣脂質內毒素。LPS通常存在於多種革蘭陰性細菌的外膜中(1)。哺乳動物的免疫系統將LPS識別為外源分子,此係對宿主警示出有侵襲性革蘭陰性細菌感染的可能性之第一步驟。在哺乳動物中,CD14及TLR4/MD-2複體參與LPS的細胞識別(2,3)。TLR4/MD-2複體與LPS結合觸發TLR4/MAPK發信途徑,其包括p38、ERK及JNK(4)。LPS亦在靜止細胞中引起炎性反應,其係透過IκBα之磷酸化及隨後降解而促進NF-κB之核轉移及經NF-κB刺激的炎性基因之表現性,諸如誘導型一氧化氮合成酶(iNOS)、TNF-α及IL-6(5,6)。因此,在循環中高LPS位準可導致嚴重的敗血病,一種危及生命的炎性併發症。但是,對LPS媒介 性炎性症狀尚未獲得有效處理。 Lipopolysaccharide (LPS) is a glycolipid endotoxin composed of an amphiphilic component lipid A, a hydrophilic polysaccharide core, and an outermost segment of the O-antigen. LPS is usually present in the outer membrane of a variety of Gram-negative bacteria (1). The mammalian immune system recognizes LPS as a foreign molecule, which is the first step in alerting the host to the possibility of an invasive Gram-negative bacterial infection. In mammals, CD14 and TLR4/MD-2 complexes are involved in cellular recognition of LPS (2, 3). Binding of the TLR4/MD-2 complex to LPS triggers the TLR4/MAPK signaling pathway, which includes p38, ERK and JNK (4). LPS also causes an inflammatory response in quiescent cells, which promotes nuclear transfer of NF-κB and NF-κB-stimulated inflammatory genes through phosphorylation and subsequent degradation of IκBα, such as inducible nitric oxide synthesis. Enzymes (iNOS), TNF-α and IL-6 (5,6). Therefore, high LPS levels in the circulation can lead to severe septicemia, a life-threatening inflammatory complication. However, for LPS media Sexual inflammatory symptoms have not been effectively treated.

牛樟薄孔菌(Taiwanofungus camphoratus)(牛樟(stout camphor)真菌)係一種僅生長在牛樟樹(Cinnamomum kanehirai)的內部心材壁上之寄生性真菌。此真菌已經廣泛使用在中藥中來治療藥物中毒、腹瀉、腹痛、高血壓及癌(7)。牛樟薄孔菌(T.camphoratus)的甲醇萃取物在小神經膠質細胞中透過抑制iNOS及環氧酶-2(COX2)表現性具有抗炎性活性(8)。已經顯示出在牛樟薄孔菌的子實體中之主要藥理活性化合物ZAA防止因人類嗜中性白血球的炎性反應且沒有施加明顯的細胞毒害性(9)。再者,ZAA亦藉由抑制LPS引起的NO製造而顯示出強效的抗炎性活性(10)。但是,藉由ZAA調節及改善發炎之機制尚未充分地闡明。 Taiwanofungus camphoratus (stout camphor fungus) is a parasitic fungus that grows only on the inner heartwood wall of the burdock tree (Cinnamomum kanehirai). This fungus has been widely used in traditional Chinese medicine to treat drug poisoning, diarrhea, abdominal pain, high blood pressure and cancer (7). The methanol extract of T. camphoratus has anti-inflammatory activity in microglia by inhibiting the expression of iNOS and cyclooxygenase-2 (COX2) (8). It has been shown that the main pharmacologically active compound ZAA in the fruiting bodies of Burdock burdock prevents inflammatory reactions due to human neutrophils and does not exert significant cytotoxicity (9). Furthermore, ZAA also exhibits potent anti-inflammatory activity by inhibiting the production of NO by LPS (10). However, the mechanism by which ZAA regulates and improves inflammation has not been fully elucidated.

在本發明中,我們使用沙門氏豬霍亂桿菌(S.choleraesuis)引起腹瀉的老鼠模型來研究ZAA透過阻斷LPS作用減低產生自革蘭陰性細菌感染的發炎之能力。我們使用X-score及HotLig模型方法(11,12)顯示出ZAA可作用為MD-2的配體,因此抑制LPS/MD-2交互作用。再者,ZAA試管內及活體內抑制NF-κB發信途徑及減低TNF-α及IL-6位準。腹膜內給藥ZAA保護老鼠對抗由LPS引起的肺及腎臟損傷及由沙門氏豬霍亂桿菌引起的腹瀉。亦研究一系列的ZAA類似物及發現在由LPS引起的TNF-α表現性上具有抗炎性活性。 In the present invention, we used a mouse model of diarrhea caused by S. choleraesuis to study the ability of ZAA to reduce inflammation caused by Gram-negative bacterial infection by blocking LPS. We used X-score and HotLig model methods (11, 12) to show that ZAA can act as a ligand for MD-2, thus inhibiting LPS/MD-2 interaction. Furthermore, ZAA inhibits the NF-κB signaling pathway and reduces the levels of TNF-α and IL-6 in vitro and in vivo. Intraperitoneal administration of ZAA protects mice against lung and kidney damage caused by LPS and diarrhea caused by Salmonella typhimurium. A series of ZAA analogs have also been studied and found to have anti-inflammatory activity in the expression of TNF-[alpha] caused by LPS.

本發明建議ZAA及一系列的ZAA類似物可有潛 力地作用為治療藥物來防範由革蘭陰性細菌感染所造成的炎性疾病。 The present invention suggests that ZAA and a series of ZAA analogs can have potential Acting as a therapeutic drug to prevent inflammatory diseases caused by Gram-negative bacterial infections.

根據本發明的一個態樣,本發明提供一種治療一對象的炎性疾病之方法,其包括將一具有下列化學式(I)之化合物或其醫藥上可接受的鹽給藥至需要該治療的對象: 其中R1係=O;R2係=O、OCHO或OH;R3係H或OH;R4係-C(=CH2)-C(CH3)H-(C=O)ORa,其中Ra係H或C1-4烷基,或R4係-(C=O)Rb,其中Rb係C1-4烷基;R5係=O、OH或H;及R6係H或OH。 According to an aspect of the present invention, the present invention provides a method for treating an inflammatory disease in a subject, which comprises administering a compound having the following formula (I) or a pharmaceutically acceptable salt thereof to a subject in need of such treatment : Wherein R 1 is =O; R 2 is =O, OCHO or OH; R 3 is H or OH; R 4 is -C(=CH 2 )-C(CH 3 )H-(C=O)OR a , Wherein R a is H or C 1-4 alkyl, or R 4 is -(C=O)R b , wherein R b is C 1-4 alkyl; R 5 is =O, OH or H; and R 6 H or OH.

根據本發明的另一個態樣,本發明提供一種具有式(I)之化合物或其醫藥上可接受的鹽之用途,其係使用在藥物製造中作為活性成份用以治療一對象的炎性疾病。 According to another aspect of the present invention, there is provided a use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof for use as an active ingredient in the manufacture of a medicament for the treatment of a subject's inflammatory disease .

較佳的是,該炎性疾病係LPS媒介性炎性症狀。 Preferably, the inflammatory disease is a mediating inflammatory condition of LPS.

較佳的是,該炎性疾病係由細菌感染及更佳為革蘭陰性細菌感染造成。 Preferably, the inflammatory disease is caused by a bacterial infection and more preferably a Gram-negative bacterial infection.

較佳的是,該炎性疾病包含由LPS引起的肺損傷或由LPS引起的腎臟損傷。 Preferably, the inflammatory disease comprises lung damage caused by LPS or kidney damage caused by LPS.

較佳的是,該炎性疾病包含腹瀉。 Preferably, the inflammatory disease comprises diarrhea.

較佳的是,該炎性疾病包含腸炎。 Preferably, the inflammatory disease comprises enteritis.

較佳的是,該具有式(I)的化合物係選自於由下列所組成之群: Preferably, the compound of formula (I) is selected from the group consisting of:

圖1。ZAA在經LPS及IFN-γ刺激的鼠巨噬細胞中抑制製造出發炎相關分子。(A)鼠腹腔巨噬細胞係使用或不使用ZAA預處理1小時,接著以LPS(0.5微克/毫升)培養24小時。讓總細胞溶成物接受免疫印漬用以偵測COX2及iNOS。COX2及iNOS蛋白質的相對表現性位準係藉由密度分析與ImageJ軟體定量及根據β-肌動蛋白基準帶常態化。(B)鼠腹腔巨噬細胞係以ZAA預處理1小時,接著以LPS(0.5微克/毫升)或IFN-γ(50奈克/毫升)培養24小時。在培養媒質中的亞硝酸鹽之存在係藉由Griess試驗分析及係使用作為NO位準之指示(n=4;*p<0.05及***p<0.001對經LPS或IFN-γ誘發的細胞)。(C)Raw264.7細胞係以pNFκB-Luc及pβ-肌動蛋白-LacZ質體共轉染。在48小時後,該等細胞係使用或不使用ZAA處理1小時,然後以LPS(0.5微克/毫升)處理24小時。採集總細胞溶成物,及決定其發光酶活性及以β-半乳糖苷酶活性為基礎常態化。該等值係平均±SD(n=4;*p<0.05及**p<0.01對經LPS刺激的細胞)。(D及E)ZAA在經LPS刺激的Raw264.7細胞中抑制NF-κB、MAPK及Akt發信途徑。該等細胞係以ZAA處 理1小時,接著以LPS(0.5微克/毫升)刺激30分鐘。藉由免疫印漬檢驗總細胞溶成物之所指示出的蛋白質。在D中於墨點下的數字及在E中的墨點下之表中所顯示出的那些代表藉由密度分析與ImageJ軟體定量及根據β-肌動蛋白基準帶常態化之相對表現性位準。在三個各自獨立的實驗中獲得類似結果。N.D.,無法偵測。 figure 1. ZAA inhibits the production of inflammatory related molecules in LPS and IFN-γ stimulated murine macrophages. (A) Murine peritoneal macrophage cell line was pretreated with or without ZAA for 1 hour, followed by incubation with LPS (0.5 μg/ml) for 24 hours. The total cell lysate is subjected to immunoblotting for detection of COX2 and iNOS. The relative expression levels of COX2 and iNOS proteins were quantified by density analysis and ImageJ software and normalized according to the β-actin reference band. (B) The murine peritoneal macrophage cell line was pretreated with ZAA for 1 hour, followed by incubation with LPS (0.5 μg/ml) or IFN-γ (50 Ng/ml) for 24 hours. The presence of nitrite in the culture medium was analyzed by Griess test and used as an indicator of NO level (n=4; *p<0.05 and ***p<0.001 for LPS or IFN-γ induced). cell). (C) The Raw264.7 cell line was co-transfected with pNFκB-Luc and pβ-actin-LacZ plastids. After 48 hours, the cell lines were treated with or without ZAA for 1 hour and then treated with LPS (0.5 μg/ml) for 24 hours. Total cell solubilized material was collected, and its luciferase activity was determined and normalized based on β-galactosidase activity. The values are mean ± SD (n=4; *p<0.05 and **p<0.01 vs. LPS-stimulated cells). (D and E) ZAA inhibits NF-κB, MAPK and Akt signaling pathways in LPS-stimulated Raw264.7 cells. These cell lines are at ZAA After 1 hour, it was stimulated with LPS (0.5 μg/ml) for 30 minutes. The protein indicated by the total cell lysate was examined by immunoblotting. The numbers shown in D under the ink dots and those shown in the table under the ink dots in E represent the relative performance of the density by the density analysis and ImageJ software and the normalization according to the β-actin reference band. quasi. Similar results were obtained in three separate experiments. N.D., unable to detect.

圖2。ZAA與MD-2的疏水性結合袋交互作用。(A)ZAA假設有一相配的組態以安置進否則會由LPS佔據的結合袋中。一起以MD-2的修剪表面模型(clipped surface model)(PDB登錄:3FXI)與緞帶模型表示來描出埋在該MD-2蛋白質內之LPS結合袋。以品紅顯示出的胺基酸Gly110-Asn158係存在於MD-2胺基酸110-160的免疫性胜肽片段內,其在MD-2的緞帶模型中係繞著ZAA結合位置。Gly110及Asn158係由箭號指示出。(B)顯示出在該MD-2結合袋內包含ZAA結合至該疏水性胺基酸殘基的交互作用。(C)顯示出在該3FXI MD-2模型的複體結構內,該對接(docked)的ZAA構形體(以黑色描出)係疊加在該LPS的終端碳鏈上。(D)以LPS(1或10微克)或ZAA培養重組的人類MD-2蛋白質(0.15微克)3小時,讓其接受非變性PAGE(native PAGE),及以對抗MD-2的不同抗原決定位(胺基酸110-160及2-160)之抗體進行免疫印漬。(E)以LPS(10微克)或ZAA培養重組的人類TLR4/MD-2複體(1微克)3小時,讓其接受非變性PAGE,及以對抗MD-2(胺基酸110-160)的抗體進行免疫印漬。在三個各自獨立的實驗中獲得類似結果。 figure 2. ZAA interacts with the hydrophobic binding pocket of MD-2. (A) ZAA assumes a matching configuration to fit into the binding pocket that would otherwise be occupied by the LPS. The LPS binding pockets embedded in the MD-2 protein were modeled together with a MD-2 clipped surface model (PDB registration: 3FXI) and a ribbon model representation. The amino acid Gly110-Asn158, which is shown in magenta, is present in the immunogenic peptide fragment of MD-2 amino acid 110-160, which is in the ribbon-mode of MD-2 around the ZAA binding site. Gly110 and Asn158 are indicated by arrows. (B) shows the interaction of the binding of ZAA to the hydrophobic amino acid residue in the MD-2 binding pocket. (C) shows that within the complex structure of the 3FXI MD-2 model, the docked ZAA configuration (depicted in black) is superimposed on the terminal carbon chain of the LPS. (D) Recombinant human MD-2 protein (0.15 μg) was cultured with LPS (1 or 10 μg) or ZAA for 3 hours, subjected to non-denaturing PAGE (native PAGE), and to anti-MD-2 different epitopes The antibodies (amino acids 110-160 and 2-160) were immunostained. (E) Recombinant human TLR4/MD-2 complex (1 μg) was cultured with LPS (10 μg) or ZAA for 3 hours, subjected to non-denaturing PAGE, and against MD-2 (amino acid 110-160) The antibody is immunologically printed. Similar results were obtained in three separate experiments.

圖3。ZAA在經LPS或沙門氏豬霍亂桿菌處理的Raw264.7細胞及老鼠中抑制TNF-α及IL-6製造。(A及C)在96井板中,以ZAA培養Raw264.7細胞(2×104細胞/井)1小時,接著以LPS(0.25或0.5微克/毫升)(A)或沙門氏豬霍亂桿菌(2×103CFU/井)(C)處理。藉由ELISA評估其各別在4及6小時後所收集之上層液的TNF-α及IL-6位準。(B)C3H/HeJ及C3H/HeN老鼠係以2毫克/公斤ZAA腹膜內預處理30分鐘,接著腹膜內注射LPS(4毫克/公斤)。(D)C57BL/6老鼠係以10毫克/公斤ZAA預處理30分鐘,接著口服給藥沙門氏豬霍亂桿菌(2×109CFU/老鼠)。在6小時後,藉由ELISA測量在血漿中之TNF-α及IL-6的位準。該等值係平均±SD(n=6-8;*p<0.05,**p<0.01,及***p<0.001)。在至少三個各自獨立的實驗中獲得類似結果。S.C.,沙門氏豬霍亂桿菌。 image 3. ZAA inhibits the production of TNF-α and IL-6 in Raw264.7 cells treated with LPS or Salmonella cholerae and in mice. (A and C) Rab264.7 cells (2 x 10 4 cells/well) were cultured in Z-well plates for 1 hour in ZAS, followed by LPS (0.25 or 0.5 μg/ml) (A) or Salmonella cholerae (2 × 10 3 CFU / well) (C) treatment. The TNF-α and IL-6 levels of the supernatant collected after 4 and 6 hours were evaluated by ELISA. (B) C3H/HeJ and C3H/HeN mice were pre-treated with 2 mg/kg ZAA intraperitoneally for 30 minutes followed by intraperitoneal injection of LPS (4 mg/kg). (D) C57BL/6 mice were pretreated with 10 mg/kg ZAA for 30 minutes followed by oral administration of Salmonella cholerae (2 x 109 CFU/mouse). After 6 hours, the levels of TNF-α and IL-6 in plasma were measured by ELISA. The values are mean ± SD (n = 6-8; *p < 0.05, **p < 0.01, and *** p < 0.001). Similar results were obtained in at least three separate experiments. SC, Salmonella cholera.

圖4。ZAA減低在老鼠中由LPS引起的病理改變。(A-D)C3H/HeN及C3H/HeJ老鼠係以ZAA(20毫克/公斤)或媒劑腹膜內預處理30分鐘,然後腹膜內注射LPS(4毫克/公斤)。在10小時後,犧牲老鼠及移出其肺及腎臟組織。(A)顯示出肺及腎臟組織的蘇木紫伊紅染色切片之典型微觀影像。(B)藉由光學顯微鏡(原始倍率×200)觀察在每個肺泡中浸潤的PMNs數目。對每隻老鼠計數每載片在四個隨意選擇的視野中之PMNs數目及對肺泡數目常態化。(C及D)ZAA減低BUN(C)及血清肌酸酐(D)的位準。在B-D中所顯示的值係平均±SD(n=10;**p<0.01及***p<0.001)。(E)對已經以ZAA(2或10毫克/公斤)或媒劑腹膜內預處理30分鐘的C3H/HeN及 C3H/HeJ老鼠腹膜內注射致死劑量的LPS(20毫克/公斤)。監視存活時間及顯示出在四組中的Kaplan-Meier存活曲線(n=10;***p<0.001對經LPS處理的C3H/HeN老鼠)。在至少三個各自獨立的實驗中獲得類似結果。 Figure 4. ZAA reduces pathological changes caused by LPS in mice. (A-D) C3H/HeN and C3H/HeJ mice were pre-treated with ZAA (20 mg/kg) or vehicle for 30 minutes intraperitoneally, followed by intraperitoneal injection of LPS (4 mg/kg). After 10 hours, the mice were sacrificed and their lung and kidney tissues were removed. (A) A typical microscopic image showing a stained section of hematoxylin and eosin in lung and kidney tissues. (B) The number of PMNs infiltrated in each alveolar was observed by an optical microscope (original magnification × 200). The number of PMNs in each of the four randomly selected fields of view and the number of alveolar cells were normalized for each mouse. (C and D) ZAA reduces the level of BUN (C) and serum creatinine (D). The values shown in B-D are mean ± SD (n = 10; **p < 0.01 and *** p < 0.001). (E) C3H/HeN that has been pre-treated with ZAA (2 or 10 mg/kg) or vehicle for 30 minutes. C3H/HeJ mice were injected intraperitoneally with a lethal dose of LPS (20 mg/kg). Survival time was monitored and Kaplan-Meier survival curves in four groups were shown (n=10; ***p<0.001 vs. LPS-treated C3H/HeN mice). Similar results were obtained in at least three separate experiments.

圖5。ZAA改善由沙門氏豬霍亂桿菌引起的腹瀉、體重減輕及胃腸道感染。(A及B)已經以ZAA(2或10毫克/公斤)或媒劑腹膜內預處理30分鐘的C57BL/6老鼠口服給藥抗卡那黴素性沙門氏豬霍亂桿菌(2×109CFU/老鼠)。在2天後,以0-3標度評分腹瀉(0=正常丸粒,1=稍微鬆散的糞便,2=鬆散糞便,及3=水狀腹瀉)(A)。每2天記錄體重2週(n=9-12;**p<0.01及***p<0.001)(B)。(C)在沙門氏豬霍亂桿菌感染後,在24-小時區間處收集糞便樣品直到96小時,及評估可存活的細菌CFU計數(n=10;**p<0.01及***p<0.001)。(D及E)已經以ZAA(2毫克/公斤)或媒劑口服處理30分鐘的C57BL/6老鼠口服給藥經pCMV-Luc轉化的沙門氏豬霍亂桿菌(2×108CFU/老鼠)。在48小時後,在注射D-螢光素後,進行老鼠的生物發光成像。整體影像係顯示在D中。光子通量標度係顯示在右邊上。(E)生物螢光成像的量化資料。輻射量值係以平均±SD(***p<0.001)表示。S.C.,沙門氏豬霍亂桿菌。 Figure 5. ZAA improves diarrhea, weight loss and gastrointestinal infections caused by Salmonella typhimurium. (A and B) Oral anti-kanamycin-resistant Salmonella cholerae (2×10 9 CFU) was administered orally in C57BL/6 mice pretreated with ZAA (2 or 10 mg/kg) or vehicle for 30 minutes. /mouse). After 2 days, diarrhea was scored on a scale of 0-3 (0 = normal pellets, 1 = slightly loose feces, 2 = loose feces, and 3 = watery diarrhea) (A). Body weight was recorded every 2 days for 2 weeks (n=9-12; **p<0.01 and ***p<0.001) (B). (C) After the Salmonella typhimurium infection, the stool samples were collected at 24-hour intervals until 96 hours, and the viable bacterial CFU counts were evaluated (n=10; **p<0.01 and ***p<0.001) ). (D and E) C57BL/6 mice that had been orally treated with ZAA (2 mg/kg) or vehicle for 30 minutes were orally administered with pCMV-Luc-transformed Salmonella cholerae (2 x 10 8 CFU/mouse). After 48 hours, bioluminescence imaging of the mice was performed after injection of D-luciferin. The overall image is displayed in D. The photon flux scale is shown on the right. (E) Quantitative data for bioluminescence imaging. The amount of radiation is expressed as mean ± SD (*** p < 0.001). SC, Salmonella cholera.

圖6。ZAA對鼠腹腔巨噬細胞不為細胞毒素。(A)以多種ZAA濃度培養細胞72小時。細胞生存能力係藉由四唑鎓比色法(MTS)及磺醯羅丹明(sulphorhodamine)B(SRB)試驗決定。用於MTS及SRB試驗的吸收度係各別在490及590奈米下測量。(B)以多種ZAA濃度培養細胞1小時,接著以LPS(0.5微 克/毫升)刺激72小時。細胞生存能力係藉由SRB試驗決定。該等值係至少三個各自獨立的實驗之平均±SD(*p<0.05對未處理的細胞)。 Figure 6. ZAA is not a cytotoxin to rat peritoneal macrophages. (A) Cells were cultured for 72 hours at various ZAA concentrations. Cell viability was determined by tetrazolium colorimetric assay (MTS) and sulphorhodamine B (SRB) assay. The absorbances used in the MTS and SRB tests were measured at 490 and 590 nm, respectively. (B) Cells were cultured for 1 hour at various ZAA concentrations followed by LPS (0.5 micro G/ml) stimulated for 72 hours. Cell viability is determined by the SRB test. The values are the mean ± SD of at least three independent experiments (*p < 0.05 versus untreated cells).

圖7。ZAA無法抑制沙門氏豬霍亂桿菌複製。沙門氏豬霍亂桿菌係於多種ZAA濃度或提供作為正對照的安比西林存在下,在LB肉湯中培養24或48小時。細菌生長係藉由測量在600奈米處的吸收度評估及以至少三個各自獨立的實驗之平均±SD表示。(***p<0.001對未處理的細胞)。 Figure 7. ZAA is unable to inhibit the replication of Salmonella choleraesuis. Salmonella typhimurium is cultured in LB broth for 24 or 48 hours in the presence of various ZAA concentrations or in the presence of ampicillin as a positive control. Bacterial growth was assessed by measuring the absorbance at 600 nm and by the mean ± SD of at least three independent experiments. (***p<0.001 vs untreated cells).

發明之詳細說明 Detailed description of the invention

在下列實施例中所使用的縮寫係如下:MD-2,髓樣分化因子(myeloid differentiation factor)-2;ZAA,樟芝酸A;CD14,分化簇14;iNOS,誘導型一氧化氮合成酶;fMLP,N-甲醯基-甲硫胺醯基-白胺醯基-苯丙胺酸;PMA,佛波醇-12-肉豆蔻酸酯-13-醋酸酯;及ROS,活性含氧物。 The abbreviations used in the following examples are as follows: MD-2, myeloid differentiation factor-2; ZAA, anthuric acid A; CD14, differentiation cluster 14; iNOS, inducible nitric oxide synthase ; fMLP, N-methyl decyl-methionine decyl-alkamine-phenylalanine; PMA, phorbol-12-myristate-13-acetate; and ROS, active oxygenates.

實施例: Example:

材料及方法 Materials and methods

細胞、細菌及老鼠 Cells, bacteria and mice

Raw264.7鼠巨噬細胞的細胞株及減弱型沙門氏 腸道亞種腸道血清型豬霍亂桿菌(Salmonella enterica subsp.enterica serovar Choleraesuis)(沙門氏豬霍亂桿菌)(13)係從生物資源保存及研究中心(Bioresource Collection and Research Center)(新竹(Hsinchu),台灣(Taiwan))獲得。公的C3H/HeJ、C3H/HeN及C57BL/6老鼠(8至10週大)係從台灣國家實驗動物中心(National Laboratory Animal Center,Taiwan)(台北(Taipei),台灣)獲得。 Raw264.7 murine macrophage cell line and attenuated Salmonella Intestinal subspecies intestinal serotype Salmonella enterica subsp.enterica serovar Choleraesuis (S. cholerae) (13) from the Bioresource Collection and Research Center (Hsinchu) , Taiwan (Taiwan) obtained. Male C3H/HeJ, C3H/HeN, and C57BL/6 mice (8 to 10 weeks old) were obtained from the National Laboratory Animal Center (Taiwan) (Taipei, Taiwan).

質體及試劑 Platinum and reagents

NFκB報導質體p-NFκB-Luc係從Promega(Madison,WI)購買。pβ-肌動蛋白-LacZ質體係衍生自pFRL2質體(14),藉由以由β-肌動蛋白啟動子驅動的β-半乳糖苷酶表現組合體(expression cassette)置換由CMV啟動子驅動的螢火蟲發光酶表現組合體。該pCMV-Luc報導質體係從Addgene(Cambridge,MA)獲得。包括抗卡那黴素性基因的pEGFP-N1(△EGFP)質體係衍生自pEGFP-N1,藉由刪除EGFP編碼區。抗COX2、iNOS及TLR4的抗體係從Santa Cruz(Santa Cruz,CA)購買。抗IkBα、ERK、JNK、Akt及p38、和磷醯基(p)-IkB激酶(IKK)α/β(pIKKα/β)、pNF-κBp65、pERK、pJNK、pAkt及pp38的抗體係從Cell Signaling(Danvers,MA)購得。 NFκB reported that the plastid p-NFκB-Luc was purchased from Promega (Madison, WI). The pβ-actin-LacZ system is derived from the pFRL2 plastid (14) and is driven by the CMV promoter by a β-galactosidase expression assembly driven by the β-actin promoter. The firefly luminescent enzyme expression assembly. The pCMV-Luc reporter system was obtained from Addgene (Cambridge, MA). The pEGFP-N1 (ΔEGFP) quality system including the kanamycin-resistant gene was derived from pEGFP-N1 by deleting the EGFP coding region. Anti-COX2, iNOS and TLR4 anti-systems were purchased from Santa Cruz (Santa Cruz, CA). Anti-IkBα, ERK, JNK, Akt and p38, and phosphonium (p)-IkB kinase (IKK) α/β (pIKKα/β), pNF-κBp65, pERK, pJNK, pAkt and pp38 anti-systems from Cell Signaling (Danvers, MA) purchased.

真菌化合物之萃取及分離 Extraction and separation of fungal compounds

ZAA係如先前描述般分離自牛樟薄孔菌(10,15)。將該化合物以濃度2毫克/毫升溶解在40%環糊精 (Sigma-Aldrich,St.Louis,MO)中使用作為原料溶液,貯存在-20℃下,及在每次實驗前以細胞培養媒質稀釋。在全部實驗中所使用的最後環糊精濃度係低於0.2%。 ZAA was isolated from Boletus edulis (10, 15) as previously described. Dissolve the compound in 40% cyclodextrin at a concentration of 2 mg/ml (Sigma-Aldrich, St. Louis, MO) was used as a raw material solution, stored at -20 ° C, and diluted with cell culture medium before each experiment. The final cyclodextrin concentration used in all experiments was less than 0.2%.

抗炎性分子之試驗 Anti-inflammatory molecule test

對C57BL/6老鼠腹膜內注射3%氫硫乙酸鹽,及晚後72小時收集其腹膜巨噬細胞。巨噬細胞係在37℃下於5% CO2的濕潤環境中以DMEM培養,以10% FBS及50微克/毫升正大黴素補充。該等細胞係使用或不使用ZAA預處理1小時,然後以LPS(Sigma-Aldrich;0.5微克/毫升)或IFN-γ(PeproTech,Rocky Hill,NJ;50奈克/毫升)培養24小時。讓該等細胞溶成物接受SDS-PAGE用以偵測COX2及iNOS表現性。如先前描述般,藉由Griess試驗(Sigma-Aldrich)分析存在於該培養媒質中之亞硝酸鹽(NO的代謝物)(16)。 C57BL/6 mice were injected intraperitoneally with 3% hydrogen thioacetate and their peritoneal macrophages were collected 72 hours later. Macrophage cell lines were cultured in DMEM at 37 ° C in a humidified environment of 5% CO 2 supplemented with 10% FBS and 50 μg/ml gentamicin. These cell lines were pretreated with or without ZAA for 1 hour and then incubated with LPS (Sigma-Aldrich; 0.5 μg/ml) or IFN-γ (PeproTech, Rocky Hill, NJ; 50 Ng/ml) for 24 hours. These cell lysates were subjected to SDS-PAGE to detect COX2 and iNOS expression. The nitrite (metabolite of NO) present in the culture medium was analyzed by the Griess test (Sigma-Aldrich) as previously described (16).

免疫印漬分析 Immunoprinting analysis

Raw264.7細胞係使用或不使用多種ZAA濃度處理1小時,接著以LPS(1微克/毫升)刺激30分鐘及在RIPA溶菌緩衝液(lysis buffer)(50mM Tris-HCl,150mM NaCl,2mM EDTA,pH 8.0,1mM Na3VO4,20微克/毫升亮肽素(leupeptin),20微克/毫升抗蛋白酶肽,1mM PMSF,及50mM NaF)中均化。該細胞溶成物係藉由以抗pIKKα/β、IkBα、pNF-κBp65、pERK、ERK、pJNK、JNK、pAkt、Akt、pp38、p38及β-肌動蛋白的一級抗體,接著適當的二級抗體進行免疫 印漬來分析。使用增強化學發光(ECL)試劑盒(Pierce Biotechnology,Rockford,IL)偵測免疫反應性蛋白質帶。該蛋白質帶的相對強度係對著β-肌動蛋白常態化及使用Image J軟體(可在http://rsb.info.nih.gov/ij/處獲得)定量。 The Raw264.7 cell line was treated with or without various ZAA concentrations for 1 hour, followed by stimulation with LPS (1 μg/ml) for 30 minutes and in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1 mM Na 3 VO 4 , 20 μg/ml leupeptin, 20 μg/ml anti-protease peptide, 1 mM PMSF, and 50 mM NaF) were homogenized. The cell lysate is obtained by a primary antibody against pIKKα/β, IkBα, pNF-κBp65, pERK, ERK, pJNK, JNK, pAkt, Akt, pp38, p38 and β-actin, followed by an appropriate secondary Antibodies were immunoblotted for analysis. Immunoreactive protein bands were detected using an Enhanced Chemiluminescence (ECL) kit (Pierce Biotechnology, Rockford, IL). The relative intensity of this protein band was quantified against β-actin normalization and using Image J software (available at http://rsb.info.nih.gov/ij/).

報導子試驗 Reporter test

在24井板中培養之次融合的Raw264.7細胞係使用Neon Transfection System(Invitrogen,Carlsbad,CA),根據製造商的協定,以p-NFκB-Luc及pβ-肌動蛋白-LacZ質體共轉染。轉染後48小時,將細胞培養在含或不含ZAA(0.5或10M)的無血清DMEM中1小時,然後以LPS(1微克/毫升)處理24小時。採集細胞溶成物及藉由雙光路發光酶及β-半乳糖苷酶報導子基因試驗系統(Tropix,Bedford,MA)決定其發光酶活性。相對發光酶活性係以發光酶活性除以β-半乳糖苷酶活性來測量以常態化轉染效率。 The secondary fused Raw264.7 cell line cultured in a 24-well plate was treated with the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol, with p-NFκB-Luc and pβ-actin-LacZ plastids. Transfection. Forty-eight hours after transfection, cells were cultured in serum-free DMEM with or without ZAA (0.5 or 10 M) for 1 hour and then treated with LPS (1 μg/ml) for 24 hours. Cell lysates were collected and their luminescent enzyme activities were determined by the dual-lumen luminescent enzyme and β-galactosidase reporter gene assay system (Tropix, Bedford, MA). The relative luciferase activity was measured by dividing the luciferase activity by the β-galactosidase activity to normalize the transfection efficiency.

分子對接(molecular docking) Molecular docking

使用Dock 5.1軟體進行可撓分子對接(17)。對用於力場計算的蛋白質模型施加Kollam部分電荷。藉由Marvin 5.2.2(可在http://www.chemaxon.com處獲得)及Ballon 0.6軟體產生能量最佳化的小分子三維座標(18)。額外地,藉由施用OpenBabel 2.2.3軟體計算Gasteiger部分電荷(19)。設定用於Dock程式的參數,以在該MD-2結合袋中迭代產生1000個定向及200個構形體。對接的構形體藉由HotLig再評分及歸類 以預測蛋白質-配體交互作用。HotLig係一種分子表面取向性評分函數,其施用蛋白質的Connolly表面來評估分子交互作用。首先,藉由在HotLig包裹中的工具PscanMS計算蛋白質的Connolly表面;然後,輸入該經對接的配體構形體以分析分子交互作用及計算束縛能分數。使用Chimera軟體進行提供分子模型用的圖形(20)。 Flexible Docking (17) using Dock 5.1 software. A Kollam partial charge is applied to the protein model used for force field calculations. Energy-optimized small molecule three-dimensional coordinates (18) were generated by Marvin 5.2.2 (available at http://www.chemaxon.com) and Ballon 0.6 software. Additionally, the Gasteiger partial charge (19) was calculated by applying OpenBabel 2.2.3 software. The parameters for the Dock program are set to iteratively generate 1000 orientations and 200 configurations in the MD-2 binding pocket. Docked configurations are re-rated and categorized by HotLig To predict protein-ligand interactions. HotLig is a molecular surface orientation scoring function that applies the Connolly surface of a protein to assess molecular interactions. First, the Connolly surface of the protein was calculated by the tool PscanMS in the HotLig package; then, the docked ligand configuration was input to analyze the molecular interaction and calculate the binding energy score. A graphic for providing a molecular model (20) was performed using Chimera software.

非變性PAGE Nondenaturing PAGE

對試管內結合分析來說,對預定量的LPS或ZAA進行聲波處理3分鐘,及在37℃下以重組的人類MD-2(R & D,Minneapolis,MN;0.15微克)或重組的人類TLR4/MD-2複體(R & D;1微克)培養3小時。讓樣品接受非變性PAGE,及藉由免疫印漬與二種抗-MD-2抗體偵測經TLR4連結的或自由態MD-2的位準,其中該二種抗體係抗MD-2胺基酸110-60的兔多株抗體(abcam,Cambridge,MA)及抗MD-2胺基酸2-160的老鼠單株抗體(abcam)。訊號係經由ECL偵測。 For in-tube binding assays, a predetermined amount of LPS or ZAA was sonicated for 3 minutes, and at 37 °C with recombinant human MD-2 (R & D, Minneapolis, MN; 0.15 μg) or recombinant human TLR4 /MD-2 complex (R &D; 1 μg) was incubated for 3 hours. The sample is subjected to non-denaturing PAGE, and the TLR4-linked or free-state MD-2 level is detected by immunoblotting with two anti-MD-2 antibodies, wherein the two anti-MD-2 amine groups are resistant. Rabbit 110-60 antibody (abcam, Cambridge, MA) and mouse monoclonal antibody (abcam) against MD-2 amino acid 2-160. The signal is detected by ECL.

用於細胞素表現性的ELISA ELISA for cytokine expression

Raw264.7細胞係使用或不使用多種ZAA濃度培養1小時,接著以LPS(0.25或0.5微克/毫升)或沙門氏豬霍亂桿菌[2×103個菌落形成單位(CFU)/井]處理4及6小時,藉由ELISA試劑盒(R & D)偵測各別在上層液中的TNF-α及IL-6位準。 Raw264.7 cell line was incubated with or without various ZAA concentrations for 1 hour, followed by LPS (0.25 or 0.5 μg/ml) or Salmonella cholerae [2×10 3 colony forming units (CFU)/well] And 6 hours, the TNF-α and IL-6 levels in the supernatant were detected by ELISA kit (R & D).

由LPS或沙門氏豬霍亂桿菌引起的炎性反應及腹瀉之老鼠模型 Mouse model of inflammatory response and diarrhea caused by LPS or Salmonella cholerae

老鼠係以ZAA(20毫克/公斤)或媒劑(0.2%環糊精,在生理食鹽水中)腹膜內預處理30分鐘,接著腹膜內注射LPS(4毫克/公斤)。在6小時後,藉由ELISA測量在血漿中的細胞素之表現性位準。在LPS處理後十小時,犧牲老鼠,及切除器官並將其固定在福馬林中。將肺及腎臟埋入石蠟中,切片及以蘇木紫及伊紅染色。收集血清樣品用以決定血脲氮(BUN)及血清肌酸酐位準。將經pEGFP-N1(△EGFP)轉化的沙門氏豬霍亂桿菌(2×109CFU/老鼠)口服給藥至含或不含ZAA(2或10毫克/公斤)口服預處理的老鼠。6小時後,收集血清樣品用於藉由ELISA決定TNF-α及IL-6位準。在沙門氏豬霍亂桿菌感染後,在24-小時區間處收集糞便樣品直到96小時。在37℃下培養過夜後,藉由在含卡那黴素的瓊脂板上平板接種一系列稀釋的糞便樣品及計數菌落來定量在糞便中的沙門氏豬霍亂桿菌。每日監視老鼠的腹瀉症狀及體重減輕。腹瀉係根據0-3的腹瀉定義來計分(0=正常丸粒,1=稍微鬆散的糞便,2=鬆散糞便及3=水狀腹瀉),如先前描述(21)。該實驗方法堅持台灣動物保護條例細則(the rules of Animal Protection Act of Taiwan)及由國立成功大學(National Cheng Kung University)的實驗動物照護及使用委員會(Laboratory Animal Care and Use Committee)批准。 Mice were pretreated intraperitoneally with ZAA (20 mg/kg) or vehicle (0.2% cyclodextrin in physiological saline) for 30 minutes followed by intraperitoneal injection of LPS (4 mg/kg). After 6 hours, the expression level of cytokines in plasma was measured by ELISA. Ten hours after LPS treatment, the mice were sacrificed and the organs were excised and fixed in the formalin. The lungs and kidneys were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Serum samples were collected to determine blood urea nitrogen (BUN) and serum creatinine levels. Salmonella cholerae (2 x 109 CFU/mouse) transformed with pEGFP-N1 (ΔEGFP) was orally administered to mice orally pretreated with or without ZAA (2 or 10 mg/kg). After 6 hours, serum samples were collected for determination of TNF-[alpha] and IL-6 levels by ELISA. After infection with Salmonella cholerae, the stool samples were collected at 24-hour intervals until 96 hours. After overnight incubation at 37 ° C, Salmonella cholerae in the feces was quantified by plating a series of diluted stool samples on a kanamycin-containing agar plate and counting colonies. Daily monitoring of diarrhea symptoms and weight loss in mice. Diarrhea was scored according to the definition of diarrhea 0-3 (0 = normal pellets, 1 = slightly loose feces, 2 = loose feces and 3 = watery diarrhea) as previously described (21). The experimental method adheres to the rules of Animal Protection Act of Taiwan and is approved by the Laboratory Animal Care and Use Committee of National Cheng Kung University.

由LPS引起的敗血病模型 Septicemia model caused by LPS

C3H/HeN及C3H/HeJ老鼠係以ZAA(2或10毫克/公斤)或媒劑腹膜內預處理。在30分鐘後,對其腹膜內給藥LPS(20毫克/公斤)。每2-4小時監視老鼠一次直到在未經ZAA處理及經LPS處理的群組中之全部C3H/HeN老鼠斷氣。 C3H/HeN and C3H/HeJ mice were pretreated intraperitoneally with ZAA (2 or 10 mg/kg) or vehicle. After 30 minutes, LPS (20 mg/kg) was administered intraperitoneally. Mice were monitored every 2-4 hours until all C3H/HeN mice in the group not treated with ZAA and treated with LPS were deflated.

發光酶基底之非侵入性生物發光成像 Non-invasive bioluminescence imaging of luminescent enzyme substrates

C57BL/6老鼠係口服提供ZAA(2毫克/公斤)或媒劑,30分鐘後,接著口服給藥經pCMV-Luc轉化的沙門氏豬霍亂桿菌(2×108CFU/老鼠)。在48小時後,老鼠係腹膜內注射D-螢光素鉀鹽(Promega,2.5毫克在100微升中)。然後,它們係以2%異氟烷麻醉。使用IVIS-200系統及其整合的採集及分析軟體(Living Image V.2.50)(Perkin Elmer,Fosty City,CA)進行活體內生物發光成像及訊號量化。 C57BL/6 mice were orally administered with ZAA (2 mg/kg) or vehicle, and 30 minutes later, pCMV-Luc-transformed Salmonella cholerae (2 x 10 8 CFU/mouse) was orally administered. After 48 hours, the mice were injected intraperitoneally with D-luciferin potassium salt (Promega, 2.5 mg in 100 microliters). They were then anesthetized with 2% isoflurane. In vivo bioluminescence imaging and signal quantification were performed using the IVIS-200 system and its integrated acquisition and analysis software (Living Image V. 2.50) (Perkin Elmer, Fosty City, CA).

統計分析 Statistical Analysis

結果係以平均±標準偏差(SD)呈現。使用Student未成對T檢定及SigmaPlotTM軟體(Systat)分析統計差異。p值小於0.05視為統計顯著。 Results are presented as mean ± standard deviation (SD). Student's unpaired T test and SigmaPlot TM software (Systat) analyze the statistical differences. A p value of less than 0.05 is considered statistically significant.

結果 result

ZAA劑量相依性抑制iNOS、COX2及NO之製造 ZAA dose dependence inhibits the production of iNOS, COX2 and NO

首先,我們研究從牛樟薄孔菌純化出的ZAA之抗炎性性質。在發炎期間,由iNOS及COX2產生大量前炎性介體、NO及前列腺素E2(PGE2)。ZAA以劑量相依的方式下調由LPS在 鼠腹腔巨噬細胞中所引起的COX2及iNOS位準(圖1A),和抑制由LPS及IFN-γ所引起的NO製造(圖1B)。但是,最高30M的ZAA處理72小時在固有或經LPS活化的巨噬細胞上不施加任何細胞毒素效應,如藉由四唑鎓比色法(MTS)及磺醯羅丹明B(SRB)試驗決定(22),如顯示在圖6中。 First, we studied the anti-inflammatory properties of ZAA purified from the genus Burdock. During inflammation, a large amount of pro-inflammatory mediators, NO and prostaglandin E2 (PGE2) are produced by iNOS and COX2. ZAA is down-regulated by LPS in a dose-dependent manner COX2 and iNOS levels induced in murine peritoneal macrophages (Fig. 1A), and inhibition of NO production by LPS and IFN-γ (Fig. 1B). However, up to 30M of ZAA treatment for 72 hours did not exert any cytotoxic effects on intrinsic or LPS-activated macrophages, as determined by tetrazolium colorimetric (MTS) and sulfopyramine B (SRB) assays. (22), as shown in Figure 6.

ZAA阻斷由LPS引起的NF-κB、MAPK及Akt發信途徑 ZAA blocks NF-κB, MAPK and Akt signaling pathways induced by LPS

為了研究ZAA在經LPS刺激的NF-κB發信中之抑制角色,首先我們偵測其在NF-κB的轉錄活性上之效應。圖1C顯示出ZAA在Raw264.7細胞中抑制NF-κB媒介性轉錄活性,如藉由發光酶報導子試驗決定。再者,LPS處理刺激NF-κBp65磷酸化,其明顯由ZAA防止(圖1D)。類似地,ERK、JNK、p38及Akt之LPS引起的磷酸化亦由ZAA抑制(圖1E)。這些結果共同強烈地建議ZAA可抑制經LPS刺激的NF-κB、MAPK及Akt發信途徑。 To investigate the inhibitory role of ZAA in LPS-stimulated NF-κB signaling, we first examined its effect on the transcriptional activity of NF-κB. Figure 1C shows that ZAA inhibits NF-κB vector transcriptional activity in Raw264.7 cells, as determined by the luminescent enzyme reporter assay. Furthermore, LPS treatment stimulated NF-κBp65 phosphorylation, which was clearly prevented by ZAA (Fig. 1D). Similarly, phosphorylation by LPS of ERK, JNK, p38 and Akt was also inhibited by ZAA (Fig. 1E). These results together strongly suggest that ZAA inhibits LPS-stimulated NF-κB, MAPK and Akt signaling pathways.

ZAA與MD-2的疏水性袋交互作用以阻斷LPS作用 ZAA interacts with the hydrophobic pocket of MD-2 to block LPS

已經闡明MD-2與TLR4的細胞外區段結合可觸發LPS媒介性反應(23,24)。為了研究ZAA是否藉由與LPS對MD-2的結合進行競爭而中斷TLR4發信,我們施用在矽片中(in silico)的分子對接分析來模擬在ZAA與MD-2間之交互作用。先前研究已顯示出知識基礎的評分函數係對預測蛋白質-配體交互作用的較好方法,然而經驗基礎的評分函數係對預測配體-結合親和力更有效(25,26)。因此,我們使用新的知 識基礎的評分程式HotLig來預測在ZAA與MD-2間之分子交互作用。再者,我們施用經驗基礎的評分程式X-score來預測其結合親和力。對配體結合位置的預測來說,HotLig顯示出有約85%~90%的成功比例(12)。另一方面,當與許多其它熟知的評分程式比較時,經報導X-score係評等蛋白質-配體親和力的最好評分函數(25,26)。圖2A一起呈現出MD-2的修剪表面模型(PDB登錄:3FXI)與緞帶模型,以描出埋在MD-2蛋白質內之LPS結合袋。值得注意的是,ZAA假設有一相配的組態以安置進先前已鑑定的LPS結合袋中。圖2B顯示出在ZAA與MD-2構成LPS結合袋的疏水性胺基酸殘基(例如,Ile、Val、Phe、Leu及Tyr)間之交互作用。ZAA明確地藉由疏水性交互作用(在圖2B中的半圓形輻射線符號)接觸該口袋,沒有氫鍵。額外地,如顯示在圖2C中,受MD-2束縛的ZAA(以黑色描出)及LPS(在3FXI MD-2結構內的共結晶配體)之分子疊加意謂著ZAA可佔據否則會由LPS的終端碳鏈佔據之空間。 It has been demonstrated that binding of MD-2 to the extracellular domain of TLR4 triggers LPS vector responses (23, 24). To investigate whether ZAA interrupted TLR4 signaling by competing with LPS binding to MD-2, we applied a molecular docking analysis in in silico to simulate the interaction between ZAA and MD-2. Previous studies have shown that a knowledge-based scoring function is a good method for predicting protein-ligand interactions, whereas empirically based scoring functions are more effective at predicting ligand-binding affinities (25, 26). So we use new knowledge The basic scoring program HotLig is used to predict the molecular interaction between ZAA and MD-2. Furthermore, we applied the empirically based scoring program X-score to predict its binding affinity. For the prediction of ligand binding position, HotLig showed a success rate of about 85% to 90% (12). On the other hand, X-score is reported as the best scoring function for protein-ligand affinity (25, 26) when compared to many other well-known scoring programs. Figure 2A together presents a trimmed surface model of MD-2 (PDB Login: 3FXI) and a ribbon model to depict LPS binding pockets embedded in MD-2 protein. It is worth noting that ZAA assumes a matching configuration for placement in previously identified LPS binding pockets. Figure 2B shows the interaction between ZAA and MD-2 forming hydrophobic amino acid residues (e.g., Ile, Val, Phe, Leu, and Tyr) of the LPS binding pocket. ZAA explicitly contacts the pocket by hydrophobic interaction (the semicircular radiant symbol in Figure 2B), with no hydrogen bonds. Additionally, as shown in Figure 2C, the molecular overlap of the ZAA-bound ZAA (depicted in black) and LPS (the co-crystallized ligand in the 3FXI MD-2 structure) means that the ZAA can be occupied otherwise. LPS's terminal carbon chain occupies space.

為了評估ZAA及LPS對MD-2之潛在結合親和力,在經由HotLig產生結合位置預測後,使用X-score評分函數進行共通評分分析(表1)。ZAA所預測的pKd(平均HPScore、HMScore及HSScore)係7.83,同時LPS係5.83(表2)。因此,我們假設ZAA之相配的分子組態與明顯的疏水性交互作用效應耦合可提供足夠的結合力,以安定MD-2/ZAA複體。因此,ZAA可藉由與LPS競爭對MD-2之結合而干擾LPS/TLR4發信。 To assess the potential binding affinities of ZAA and LPS for MD-2, a common score analysis was performed using the X-score scoring function after binding position prediction was generated via HotLig (Table 1). The pKd (average HPScore, HMScore, and HSScore) predicted by ZAA was 7.83, while the LPS was 5.83 (Table 2). Therefore, we hypothesized that the matching molecular configuration of ZAA with the apparent hydrophobic interaction effect provides sufficient binding to stabilize the MD-2/ZAA complex. Therefore, ZAA can interfere with LPS/TLR4 signaling by competing with LPS for the combination of MD-2.

Mw,分子量;LogP,分配係數。HPScore=C0,1+CVDW,1*(VDW)+CHB,1*(H-鍵結)+CHP*(疏水性對)+CRT,1*(轉子(rotor)) HMScore=C0,2+CVDW,2*(VDW)+CHB,2*(H-鍵結)+CHM*(疏水性相配)+CRT,2*(轉子) HSScore=C0,3+CVDW,3*(VDW)+CHB,3*(H-鍵結)+CHS*(疏水性表面)+CRT,3*(轉子) X-Score=(HPScore+HMScore+HSScore)/3 Mw, molecular weight; LogP, partition coefficient. HPScore=C0,1+CVDW,1*(VDW)+CHB,1*(H-bonding)+CHP*(hydrophobic pair)+CRT,1*(rotor) HMScore=C0,2+CVDW , 2*(VDW)+CHB, 2*(H-bond)+CHM*(hydrophobic matching)+CRT, 2*(rotor) HSScore=C0,3+CVDW,3*(VDW)+CHB,3 *(H-bond)+CHS*(hydrophobic surface)+CRT, 3*(rotor) X-Score=(HPScore+HMScore+HSScore)/3

先前研究已闡明LPS對MD-2及TLR4/MD-2複體具有類似的親和力,此建議MD-2係主要LPS結合組分(27)。再者,可溶的重組MD-2之單體形式結合LPS產生穩定 的MD-2/LPS複體;此複體足以引發TLR4相依性活化(28,29)。其次,我們以二種具有不同抗原決定位的抗MD-2抗體進行免疫電泳,其中一種係抗MD-2胺基酸110-160的多株抗體,及其它係抗MD-2胺基酸2-160的單株抗體。ZAA結合至人類MD-2減低二者抗體對非變性MD-2之識別,類似於LPS結合的效應(圖2D)。然後,使用重組的人類TLR4/MD-2蛋白質複體來測量ZAA的結合效率。圖2E顯示出10微克ZAA係大約相等於10微克LPS。因此,ZAA可透過與抗體的競爭性結合或造成MD-2蛋白質的構形改變來干擾抗MD-2抗體對MD-2的識別。 Previous studies have demonstrated that LPS has similar affinities for MD-2 and TLR4/MD-2 complexes, suggesting that MD-2 is the major LPS binding component (27). Furthermore, the monomeric form of soluble recombinant MD-2 is stable in combination with LPS. The MD-2/LPS complex; this complex is sufficient to initiate TLR4-dependent activation (28, 29). Secondly, we performed immunoelectrophoresis with two anti-MD-2 antibodies with different epitopes, one of which was an antibody against MD-2 amino acid 110-160, and the other was anti-MD-2 amino acid 2 -160 monoclonal antibodies. ZAA binding to human MD-2 reduces the recognition of non-denatured MD-2 by both antibodies, similar to the effect of LPS binding (Fig. 2D). The recombinant human TLR4/MD-2 protein complex was then used to measure the binding efficiency of ZAA. Figure 2E shows that 10 micrograms of ZAA is approximately equal to 10 micrograms of LPS. Thus, ZAA can interfere with the recognition of MD-2 by anti-MD-2 antibodies by competitive binding to antibodies or by conformational changes in MD-2 proteins.

ZAA減低由LPS及沙門氏豬霍亂桿菌引起的前炎性細胞素製造 ZAA reduces the production of pro-inflammatory cytokines caused by LPS and Salmonella cholerae

因為LPS引發前炎性細胞素製造,其次,我們比較ZAA在試管內及活體內由LPS及沙門氏豬霍亂桿菌引起的TNF-α及IL-6製造上的影響。ZAA在二種測試濃度下抑制在Raw264.7巨噬細胞中由LPS引起的TNF-α及IL-6製造(圖3A)。為了闡明活體內是否亦觀察到此影響,在注射LPS前30分鐘,將ZAA給藥至老鼠。雖然ZAA明顯減低在C3H/HeN老鼠中的TNF-α及IL-6製造,在TLR4發信缺陷型C3H/HeJ老鼠中觀察到更弱的反應(圖3B),此建議ZAA之作用對LPS/TLR4/MD-2途徑係選擇性。 Because LPS triggered the production of pro-inflammatory cytokines, secondly, we compared the effects of ZAA on the production of TNF-α and IL-6 caused by LPS and Salmonella typhimurium in vitro and in vivo. ZAA inhibited the production of TNF-[alpha] and IL-6 by LPS in Raw264.7 macrophages at two concentrations tested (Fig. 3A). To clarify whether this effect was also observed in vivo, ZAA was administered to mice 30 minutes before LPS injection. Although ZAA significantly reduced TNF-α and IL-6 production in C3H/HeN mice, a weaker response was observed in TLR4 signaling-deficient C3H/HeJ mice (Fig. 3B), suggesting that ZAA acts on LPS/ The TLR4/MD-2 pathway is selective.

其次,我們探索ZAA試管內及活體內二者在沙門氏豬霍亂桿菌媒介性前炎性細胞素製造上之效應。我們的 結果顯示出ZAA預處理1小時有效地在Raw264.7細胞(圖3C)及C57BL/6老鼠(圖3D)中抑制由沙門氏豬霍亂桿菌引起的TNF-α及IL-6製造。 Secondly, we explored the effects of both ZAA in vitro and in vivo on the production of vector-induced pro-inflammatory cytokines in Salmonella typhimurium. our The results showed that ZAA pretreatment for 1 hour effectively inhibited the production of TNF-α and IL-6 by Salmonella cholerae in Raw264.7 cells (Fig. 3C) and C57BL/6 mice (Fig. 3D).

ZAA減弱由LPS引起的肺及腎臟損傷及致死率 ZAA attenuates lung and kidney damage and mortality caused by LPS

因為ZAA抑制LPS引起的前炎性細胞素製造及發信途徑,然後我們探索ZAA活體內減低由LPS導致的器官病理及致死率。我們決定在肺及腎臟組織中由於其建構性TLR4表現性的發炎反應(30,31)。在將LPS給藥至C3H/HeN老鼠後,在肺中之多形核白血球(PMNs)的浸潤提高;但是,ZAA處理明顯防止由LPS引起的PMNs肺累積(圖4A及4B)。類似地,由LPS引起的腎絲球腎炎程度在經ZAA預處理的C3H/HeN老鼠中明顯減低(圖4A)。與腎功能衰竭一致,BUN及血清肌酸酐位準在LPS給藥後增加。ZAA顯著減低腎臟損傷標誌二者之製造(圖4C及4D)。值得注意的是,LPS處理不在TLR4發信缺陷型C3H/HeJ老鼠中引發任何病理改變(圖4A-4D)。 Because ZAA inhibits LPS-induced proinflammatory cytokine production and signaling pathways, we then explored that ZAA reduces organ pathology and mortality caused by LPS in vivo. We decided to express inflammatory responses in lung and kidney tissues due to their constructive TLR4 (30, 31). Infiltration of polymorphonuclear leukocytes (PMNs) in the lung was increased following administration of LPS to C3H/HeN mice; however, ZAA treatment significantly prevented lung accumulation of PMNs caused by LPS (Figs. 4A and 4B). Similarly, the extent of renal glomerulonephritis caused by LPS was significantly reduced in ZAA pretreated C3H/HeN mice (Fig. 4A). Consistent with renal failure, BUN and serum creatinine levels increased after LPS administration. ZAA significantly reduced the manufacture of both kidney damage markers (Figures 4C and 4D). Notably, LPS treatment did not elicit any pathological changes in TLR4 signaling-deficient C3H/HeJ mice (Figures 4A-4D).

為了評估ZAA對抗由LPS引起的致死率之保護效力,C3H/HeN及C3H/HeJ老鼠係以ZAA或媒劑處理及以LPS激發免疫反應。ZAA明顯保護C3H/HeN老鼠對抗致死率及改善在內毒素血症期間的存活(圖4E),此建議ZAA一般可具有對抗由LPS引起的敗血病及革蘭陰性細菌感染之治療潛力。 To assess the protective efficacy of ZAA against lethality caused by LPS, C3H/HeN and C3H/HeJ mice were treated with ZAA or vehicle and challenged with LPS. ZAA clearly protects C3H/HeN mice against lethality and improves survival during endotoxemia (Fig. 4E), suggesting that ZAA generally has therapeutic potential against septicemia and Gram-negative bacterial infections caused by LPS.

ZAA改善由沙門氏豬霍亂桿菌感染的老鼠之臨床症狀 ZAA improves clinical symptoms in mice infected with Salmonella cholerae

TLR4在對抗沙門氏菌感染的宿主防禦反應中扮演明顯角色(32,33)。先前研究已顯示出缺乏TLRs特別是TLR4的老鼠更抗沙門氏菌感染(32,34),此建議在細菌外膜上封鎖LPS,因此TLR4/MD-2交互作用係有希望的抗菌對策。為了進一步證實ZAA活體內的抗炎性性質,以ZAA處理受沙門氏豬霍亂桿菌感染的C57BL/6老鼠,及各別監視腹瀉及體重2天及2週。圖5A顯示出ZAA預處理減低在受感染的老鼠中之腹瀉計分。ZAA處理(10毫克/公斤)戲劇性減弱體重減輕(圖5B),造成在2週內返回正常體重。如顯示在圖5C中,在未經處理的老鼠之糞便中的細菌負載隨著時間逐漸遞減。值得注意的是,糞便的細菌負載以劑量及時間相依的方式在經ZAA處理的老鼠中戲劇性減低。在細菌感染後96小時處,在以10毫克/公斤的ZAA處理之老鼠中的糞便細菌位準係探測不到。再者,受攜帶pCMV-Luc質體的沙門氏豬霍亂桿菌感染之老鼠在以ZAA口服處理後具有比媒劑更弱的生物發光(圖5D及5E)。一起採用這些結果,其顯示出ZAA可有效抑制沙門氏豬霍亂桿菌感染及減弱老鼠的臨床症狀。 TLR4 plays a prominent role in host defense responses against Salmonella infection (32, 33). Previous studies have shown that mice lacking TLRs, particularly TLR4, are more resistant to Salmonella infection (32, 34), suggesting that LPS is blocked on the bacterial outer membrane, so TLR4/MD-2 interaction is a promising antibacterial response. To further confirm the anti-inflammatory properties of ZAA in vivo, C57BL/6 mice infected with Salmonella cholerae were treated with ZAA, and diarrhea and body weight were monitored separately for 2 and 2 weeks. Figure 5A shows that ZAA pretreatment reduces diarrhea scores in infected mice. ZAA treatment (10 mg/kg) dramatically reduced weight loss (Figure 5B), resulting in a return to normal body weight within 2 weeks. As shown in Figure 5C, the bacterial load in the feces of untreated mice gradually decreased over time. It is worth noting that the bacterial load of feces was dramatically reduced in dose- and time-dependent manner in ZAA-treated mice. At 96 hours after bacterial infection, fecal bacterial levels in mice treated with 10 mg/kg of ZAA were not detected. Furthermore, mice infected with Salmonella cholerae carrying the pCMV-Luc plastid had a weaker bioluminescence than the vehicle after oral treatment with ZAA (Figs. 5D and 5E). Together with these results, it was shown that ZAA is effective in inhibiting Salmonella cholerae infection and attenuating clinical symptoms in mice.

討論 discuss

在本發明中,我們使用二種不同模型方法及抗體識別以第一時間顯示出ZAA與MD-2的疏水性袋交互作用而阻斷LPS作用。我們發現ZAA可作用為MD-2的配體,因此抑制LPS與MD-2的交互作用。我們亦顯示出ZAA的全身性 給藥保護老鼠遠離由LPS引起的肺及腎臟損傷及由沙門氏菌引起的腸炎及體重減輕。我們的結果指示出ZAA擁有抗炎性活性及可係一種用於敗血病性休克的潛在治療藥物。 In the present invention, we used two different model methods and antibody recognition to show the interaction of ZAA with the hydrophobic pocket of MD-2 at the first time to block the LPS effect. We found that ZAA acts as a ligand for MD-2, thus inhibiting the interaction of LPS with MD-2. We also show the generality of ZAA The administration protects the mice from lung and kidney damage caused by LPS and enteritis and weight loss caused by Salmonella. Our results indicate that ZAA has anti-inflammatory activity and can be a potential therapeutic for septic shock.

牛樟薄孔菌的子實體之甲醇萃取物抑制在經LPS/IFN-γ活化的小神經膠質中之COX2、iNOS及TNF-α製造(8),此建議其抗炎性性質可歸因於ERK、JNK及NF-κB磷酸化之抑制。假定ZAA係在子實體中的主要藥理活性化合物,本發明研究ZAA的抗炎性性質。先前研究已顯露出ZAA抑制在經fMLP或PMA活化的人類外周嗜中性白血球(peripheral human neutrophils)中的ROS製造(9)。類似地,我們闡明ZAA調節在經活化的鼠巨噬細胞中之NO製造及減弱前炎性介體(例如iNOS、COX2、TNF-α及IL-6)的表現性。 The methanol extract of the fruiting body of Burdock burdock inhibits the production of COX2, iNOS and TNF-α in LPS/IFN-γ activated microglia (8), suggesting that its anti-inflammatory properties can be attributed to Inhibition of phosphorylation of ERK, JNK and NF-κB. The present invention investigates the anti-inflammatory properties of ZAA assuming that ZAA is the major pharmacologically active compound in the fruiting body. Previous studies have revealed that ZAA inhibits ROS production in human peripheral neutrophils activated by fMLP or PMA (9). Similarly, we demonstrate that ZAA regulates the production of NO in activated murine macrophages and attenuates the expression of pro-inflammatory mediators such as iNOS, COX2, TNF-[alpha], and IL-6.

對抗TLR4/MD-2複體的抗體已顯示出用於處理由LPS喚起的急性炎性症狀之效力(35,36)。最近亦已探索能阻斷TLR4/MD-2雜二聚體形成及起始發炎的分子。例如,依立托倫(eritoran)係一種合成的四醯基化的脂質A,其與LPS競爭在MD-2中的相同結合位置及減弱該經LPS活化的受體複體之形成,其相繼地抑制穿過漿膜的信號傳輸(37)。不幸的是,依立托倫的階段III研究顯示出在治療與安慰劑組間無明顯差異(38)。最近,亦已報導出與細菌脂質A的結構不相關之天然及合成的化學物質係MD-2取向性LPS拮抗劑(39)。結合MD-2口袋的機制可分成三種一般型式:(1)對進入MD-2口袋的競爭[例如,雙-ANS(1-苯胺基萘8-磺酸鹽)及太平洋紫杉醇(paclitaxel)];(2)與在MD-2口袋內的Cys133殘基之共價 交互作用(例如,N-芘馬來醯亞胺、金諾芬(auranofin)及JTT-705);及(3)在該口袋的底部內部部分之嘴處的線性配向(例如,JSH、薑黃色素、黃腐醇(xanthohumol)及異黃腐醇(isoxanthohumol))。ZAA與LPS競爭進入MD-2口袋中,因此ZAA的治療效力可大概藉由增加其在血液中的溶解度,或藉由提高其經由LPS結合蛋白質識別把MD-2口袋作為標的的能力而改善。 Antibodies against the TLR4/MD-2 complex have been shown to be effective in treating acute inflammatory conditions evoked by LPS (35, 36). Molecules that block the formation of TLR4/MD-2 heterodimers and initiate inflammation have also recently been explored. For example, eritoran is a synthetic tetra-negative lipid A that competes with LPS for the same binding position in MD-2 and attenuates the formation of the LPS-activated receptor complex, which is successive Signal transmission through the serosa is suppressed (37). Unfortunately, eritoran's Phase III study showed no significant differences between the treatment and placebo groups (38). Recently, natural and synthetic chemical substances MD-2 oriented LPS antagonists (39) which are not related to the structure of bacterial lipid A have also been reported. The mechanism of binding to the MD-2 pocket can be divided into three general types: (1) competition for entry into the MD-2 pocket [eg, bis-ANS (1-anilinophthalene 8-sulfonate) and paclitaxel]; (2) covalent with the Cys133 residue in the MD-2 pocket Interactions (eg, N-芘马醯亚胺, auranofin, and JTT-705); and (3) linear alignment at the mouth of the inner portion of the bottom of the pocket (eg, JSH, curcumin) , xanthohumol (xanhohumol) and isoxanthohumol (isoxanthohumol). ZAA competes with LPS for entry into the MD-2 pocket, so the therapeutic efficacy of ZAA can be improved, probably by increasing its solubility in the blood, or by increasing its ability to target the MD-2 pocket via LPS binding protein recognition.

沙門氏菌係與人類的菌血症、傷寒熱及腸炎相關。在沙門氏菌發病原理上的大部分研究已使用在老鼠中的沙門氏鼠傷寒腸道型亞種腸道血清型桿菌(S.enterica subsp.enterica serovar Typhimurium)(鼠傷寒沙門氏桿菌(S.typhimurium))感染模型。雖然鼠傷寒沙門氏桿菌感染在老鼠中產生傷寒熱狀疾病,此微生物在人類中專門造成腸炎(40)。因為受鼠傷寒沙門氏桿菌感染的老鼠不發展出腹瀉,老鼠傷寒模型非為研究由沙門氏菌感染造成的腸炎之好的模型。另一方面,沙門氏豬霍亂桿菌在天然宿主的豬中產生腸炎及菌血症二者,但是其亦在人類中造成疾病(41)。再者,感染沙門氏豬霍亂桿菌可在老鼠中導致菌血症及死亡(42,43)。因此,感染沙門氏豬霍亂桿菌的老鼠可提供作為由沙門氏菌引起的菌血症及腸炎之合適的模型。因為我們已使用由LPS引起的敗血病模型來顯示出ZAA預處理在致死性LPS激發免疫前可改善老鼠的存活(圖4E),想要使用沙門氏豬霍亂桿菌感染模型來進一步研究ZAA在改善老鼠腸炎上的效應。在本發明中,我們使用減弱型沙門氏豬霍亂桿菌株,其係用於豬的疫 苗候選物。老鼠以此病株感染導致腹瀉及體重減輕,使得其合適於研究ZAA在沙門氏菌感染上的影響。我們的結果顯露出就腹瀉嚴重性、體重改變及糞便的細菌負載而論,ZAA預處理改善受沙門氏豬霍亂桿菌感染的老鼠之臨床症狀。 Salmonella is associated with human bacteremia, typhoid fever and enteritis. Most studies on the pathogenesis of Salmonella have used S. enterica subsp. enterica serovar Typhimurium (S. typhimurium) in mice. ) infection model. Although Salmonella typhimurium infection produces typhoid fever in mice, this microorganism specifically causes enteritis in humans (40). Because mice infected with Salmonella typhimurium did not develop diarrhea, the model of typhoid fever was not a good model for studying enteritis caused by Salmonella infection. On the other hand, Salmonella cholerae produces both enteritis and bacteremia in natural host pigs, but it also causes disease in humans (41). Furthermore, infection with Salmonella cholerae can cause bacteremia and death in mice (42, 43). Therefore, mice infected with Salmonella cholerae can provide a suitable model for bacteremia and enteritis caused by Salmonella. Because we have used the septicemia model caused by LPS to show that ZAA pretreatment can improve survival in mice before lethal LPS stimulation (Fig. 4E), we want to use the Salmonella porcine infection model to further study ZAA. Improve the effect of intestinal inflammation in mice. In the present invention, we use attenuated Salmonella cholera strains for use in swine plague Seedling candidates. Infection with this disease caused diarrhea and weight loss, making it suitable for studying the effects of ZAA on Salmonella infection. Our results revealed that ZAA pretreatment improved the clinical symptoms of mice infected with Salmonella choleraesuis in terms of diarrhea severity, weight changes, and bacterial load in feces.

總而言之,我們顯示出ZAA有效改善產生自由LPS或沙門氏豬霍亂桿菌引起的實驗性內毒素血症之炎性反應,大概由於其與在巨噬細胞中的MD-2口袋之特定的交互作用。再者,發炎之緩和可歸因於在糞便中的沙門氏菌位準減低。但是,ZAA在沙門氏菌之生長上無直接抑制效應(圖7)。再者,我們的觀察強烈支持ZAA可調整由沙門氏豬霍亂桿菌引起的腹瀉而沒有激起許多抗生素常見的副作用(即,有害免疫反應可歸因於殺死有益的細菌)之假設。總之,考慮到上述資料及在表3中所列出的支持資料,本發明對ZAA及其類似物作為用於LPS媒介性感染的抗發炎藥之機制提供新的見解。 In conclusion, we show that ZAA effectively improves the inflammatory response to experimental endotoxemia caused by free LPS or Salmonella cholerae, presumably due to its specific interaction with the MD-2 pocket in macrophages. Furthermore, the mitigation of inflammation can be attributed to a reduction in the level of Salmonella in the feces. However, ZAA has no direct inhibitory effect on the growth of Salmonella (Figure 7). Furthermore, our observations strongly support the assumption that ZAA can modulate diarrhea caused by Salmonella cholerae without the common side effects of many antibiotics (ie, the harmful immune response can be attributed to the killing of beneficial bacteria). In summary, in view of the above information and the supporting materials listed in Table 3, the present invention provides new insights into the mechanism by which ZAA and its analogs act as anti-inflammatory drugs for LPS vector infection.

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Claims (9)

一種治療在一對象中的炎性疾病之方法,其包括將一具有下列化學式(I)的化合物或其醫藥上可接受的鹽給藥至需要該治療之對象: 其中R1係=O;R2係=O、OCHO或OH;R3係H或OH;R4係-C(=CH2)-C(CH3)H-(C=O)ORa,其中Ra係H或C1-4烷基,或R4係-(C=O)Rb,其中Rb係C1-4烷基;R5係=O、OH或H;及R6係H或OH。 A method of treating an inflammatory disease in a subject comprising administering a compound having the following formula (I) or a pharmaceutically acceptable salt thereof to a subject in need of such treatment: Wherein R 1 is =O; R 2 is =O, OCHO or OH; R 3 is H or OH; R 4 is -C(=CH 2 )-C(CH 3 )H-(C=O)OR a , Wherein R a is H or C 1-4 alkyl, or R 4 is -(C=O)R b , wherein R b is C 1-4 alkyl; R 5 is =O, OH or H; and R 6 H or OH. 如請求項1之方法,其中該炎性疾病係LPS媒介性炎性症狀。 The method of claim 1, wherein the inflammatory disease is a mediating inflammatory symptom of LPS. 如請求項1之方法,其中該炎性疾病係由細菌感染造成。 The method of claim 1, wherein the inflammatory disease is caused by a bacterial infection. 如請求項3之方法,其中該細菌感染係由革蘭陰性細菌造成。 The method of claim 3, wherein the bacterial infection is caused by Gram-negative bacteria. 如請求項1之方法,其中該炎性疾病包含由LPS引起的肺 損傷或由LPS引起的腎臟損傷。 The method of claim 1, wherein the inflammatory disease comprises a lung caused by LPS Injury or kidney damage caused by LPS. 如請求項1之方法,其中該炎性疾病包含腹瀉。 The method of claim 1, wherein the inflammatory disease comprises diarrhea. 如請求項1之方法,其中該炎性疾病包含腸炎。 The method of claim 1, wherein the inflammatory disease comprises enteritis. 如請求項1之方法,其中該具有式(I)的化合物係樟芝酸A。 The method of claim 1, wherein the compound of formula (I) is anocetin A. 如請求項1之方法,其中該具有式(I)的化合物係選自於由下列所組成之群: The method of claim 1, wherein the compound of formula (I) is selected from the group consisting of:
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105919063A (en) * 2016-04-29 2016-09-07 上海理工大学 Antrodia camphorata spore powder and use thereof
WO2021218974A1 (en) * 2020-04-28 2021-11-04 Alps Biotech Co., Ltd. Uses of antrodia cinnamomea extract in manufacturing products for reducing expression and treating associated diseases of angiotensin converting enzyme 2

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105919063A (en) * 2016-04-29 2016-09-07 上海理工大学 Antrodia camphorata spore powder and use thereof
WO2021218974A1 (en) * 2020-04-28 2021-11-04 Alps Biotech Co., Ltd. Uses of antrodia cinnamomea extract in manufacturing products for reducing expression and treating associated diseases of angiotensin converting enzyme 2
CN115697364A (en) * 2020-04-28 2023-02-03 浩峰生物科技股份有限公司 Application of Antrodia camphorata extract in preparation of products for reducing expression of angiotensin converting enzyme 2 and treating related diseases thereof

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