Pharmacological Reports Copyright © 2012 2012, 64, 11891199 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
Oroxylin A, a classical natural product, shows a novel inhibitory effect on angiogenesis induced by lipopolysaccharide
Xiuming Song1, Yan Chen1, Yajing Sun1, Biqi Lin2, Yansu Qin1, Hui Hui1, Zhiyu Li1, Qidong You1, Na Lu1, Qinglong Guo1
1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, P.R. China
2 School of Pharmacy, China Pharmaceutical University, Nanjing 210009, P.R. China
Correspondence: Na Lu, e-mail: [email protected]; Qinglong Guo, e-mail: [email protected]
Abstract: Background: There is an obvious relationship among angiogenesis and inflammation. From previous study, we learn that oroxylin A possesses anti-angiogenic activity in vitro and in ovo. It also has an inhibitory effect on inflammation. But whether oroxylin A sup- presses the inflammation-induced angiogenesis is still unknown. Our present study focuses on the role of oroxylin A in targeting LPS-induced angiogenesis, inflammatory and related pathways. Methods: The effects of oroxylin A on angiogenesis were investigated by transwell assay, tube formation assay, rat aortic ring assay and chorioallantoic membrane (CAM) model. Western blotting analysis was used to detect the expression of certain proteins. Results: We found that oroxylin A inhibited LPS-induced migration and tube formation of human umbilical vein endothelial cells (HUVECs), as well as microvessel sprouting from rat aotric ring in vitro and the angiogenesis of chicken chorioallantoic membrane (CAM) model in ovo. The results also indicated that oroxylin A could inhibit the expression of LPS acceptor toll-like receptor 4 (TLR4) and the activities of its downstream mitogen-activated protein kinases (MAPKs), including reducing expressions of the phosphorylation of JNK, p38, and ERK. Moreover, oroxylin A prevented NF-kB dimers from translocating to the nucleus. Conclusions: Taken together, oroxylin A can suppress the angiogenesis induced by LPS and it may affect the LPS/TLR4 signaling pathway.
Key words: Scutellaria baicalensis Georgi (Lamiaceae), oroxylin A, lipopolysaccharide, angiogenesis, toll like receptor 4 (TLR4)
Abbreviations: CAM – chorioallantoic membrane, DMSO – Introduction dimethyl sulfoxide, ERK – extracellular signal-regulated ki- nases, FBS – fetal bovine serum, HUVECs – human umbilical vein endothelial cells, IKK – IkB kinase, JNKs – c-Jun N- Angiogenesis occurs in many physiological and terminal kinases, LPS – lipopolysaccharide, MAPKs – mito- gen-activated protein kinases, p38 – p38 mitogen-activated pathological processes such as reproductive functions protein kinases, TLRs – toll-like receptors in adults, wound healing, tumor development, some
Pharmacological Reports, 2012, 64, 11891199 1189 chronic inflammatory diseases and so on [23]. A link In the present study, we isolated oroxylin A from Scutel- between angiogenesis and inflammation has been laria baicalensis Georgi and identified its structure. The ef- strongly supported. It seems that most inflammations, fect of oroxylin A on LPS-induced angiogenesis in vitro especially chronic inflammations, are linked to angio- and in ovo was also examined, which depended on TLR4, genesis [2, 10]. In inflammations, vascular remodel- phosphorylation of MAPKs and activation of NF-kB. ing occurs and contributes to inflammatory diseases, proceeding to other diseases for the next step. Chronic inflammation also promotes angiogenesis by certain inflammatory factors. Angiogenesis, a significant risk Materials and Methods factor for the development of inflammations, plays an important role in inflammatory diseases. Thus, target- Cell lines, chemicals and biochemicals ing angiogenesis may be effective in the development of some diseases caused by inflammation. Human umbilical vein endothelial cells (HUVECs) Lipopolysaccharide (LPS), an important structural were isolated from human umbilical cord veins by component of the outer membrane of Gram-negative collagenase treatment as described previously [9]. bacteria, acts as a mediator in inflammatory responses The harvested cells were grown in medium 199 and it also induces angiogenesis [19] as an exogenous (Gibco, Grand Island, NY) containing endothelial cell factor in several chronic inflammatory diseases. LPS growth supplement (ECGS, 30 µg/ml; Sigma, St. promotes angiogenesis in many stages in which the Louis, MO), epidermal growth factor (EGF, 10 ng/ml; toll-like receptors (TLRs) play a central role. TLRs Sigma, St. Louis, MO), 20% fetal bovine serum (FBS, are receptors involved in the immune response and Gibco, Grand Island, NY), 100 U/ml penicillin, and can recognize LPS as their ligand [16]. LPS/TLR in- 100 µg/ml streptomycin, pH 7.4. The cells were incu- tracellular signaling pathway depends on the activity bated in a humidified atmosphere of 95% air + 5% of tumor necrosis factor (TNF) receptor associated CO2 at 37°C. After 3–5 passages, HUVECs were col- factor 6 (TRAF6) [19]. Different TLRs use different lected for use in all experiments. combinations of adaptor proteins to determine down- The drug was dissolved in dimethyl sulfoxide stream signaling and TLR4 is critical for signal trans- (DMSO) as a stock solution, stored at –20°C, and di- duction utilizing all the adaptor proteins in the path- way [14]. In the process of angiogenesis stimulated luted with medium before experiments. The final by LPS, the activation of TLR4 can induce the up- DMSO concentration did not exceed 0.1% throughout regulation of chemokines and adhesion molecules in the study. The control groups were treated with 0.1% Escheri- MAPKs pathway and stimulate NF-kB leading to the DMSO in the corresponding experiments. chia coli synthesis and secretion of various cytokines, metabo- (055:B5) LPS was supplied by Sigma lites, and enzymes that in turn mediate their different Chemical Co. (St. Louis, MO). Primary antibodies for biological effects [22]. TLR4 (H-80), p38, p-p38, p-ERK1/2, JNK, p-JNK, The Chinese herbs, Huang-Chi, Scutellaria bai- NF-kB, IkB, lamin A and b-actin were obtained from calensis Georgi, has been widely used to treat several Santa Cruz Biotechnology (Santa Cruz, CA). Primary diseases such as inflammation, diarrhea, and pyro- antibody for ERK1/2 and p-IKK were from Bioworld genic infections and so on. Oroxylin A is one of the (St. Louis Park, MN). Primary antibody for IKK and p-IkB were from Cellsignaling (Danvers, MA). major flavonoids which are the most common active TM ingredients in the root of Scutellaria baicalensis IRDye 800 conjugated secondary antibodies were Georgi [11]. It has been widely studied and proved to obtained from Rockland Inc. (Philadelphia, PA). possess a wide spectrum of pharmacological proper- ties, such as pro-apoptotic [12] and anti-invasive [15, Plant material 25] activities. Several previous reports suggested that oroxylin A was a potential antiinflammatory agent [11]. The roots of Scutellaria baicalensis Georgi (Radix But whether oroxylin A can inhibit LPS-mediated in- Scutellariae) were purchased from Bozhou, Anhui, duction of angiogenesis in some chronic inflamma- China (latitude: 33° 52’ 38’’ N, longitude: 115° 46’ tory diseases is still unknown. What’s more, the mo- 13’’ E). A voucher specimen was deposited at Depart- lecular mechanisms remain poorly understood and ment of Natural Medicinal Chemistry and identified warrant further investigation. by Dr. F. Feng, China Pharmaceutical University.
1190 Pharmacological Reports, 2012, 64, 11891199 Oroxylin A inhibits LPS induced angiogenesis Xiuming Song et al.
Extraction and isolation for 60 min. These cells were harvested after trypsin treatment and suspended in medium containing 1% Oroxylin A was isolated and purified from Radix Scu- FBS before seeding and planting onto matrigel and tellariae according to the established methods [24] with then LPS (1 µg/ml) was added. Following 8 h of incu- slight modifications. In brief, dried Scutellaria bai- bation, the plate was examined for capillary tube for- calensis Georgi roots were chopped into small pieces, mation under an inverted microscope and photo- immersed, and extracted with 10-fold v/w acetone graphed and tubular structures were quantified by twice at room temperature for 2 weeks. After filtration, manual counting the tube numbers, and five randomly the residues were reflux-extracted with 4-fold v/w of chosen fields were analyzed for each well. 50% aqueous ethanol twice for 6 h. The acetone ex- tracts were subjected to column chromatography on Rat aortic ring assay silica gel eluted with CHCl3 and CHCl3-MeOH (9:1, v/v), and rechromatographed on silica gel eluted with Rat aortic ring assay was performed as described pre- hexane-acetone (1:1, v/v) to yield oroxylin A. viously [1]. The thoracic aorta was dissected from male Sprague-Dawley rats (6 weeks old) and cut into Identification and content determination 1-mm long rings and set in 24-well plates. The disks were treated as described previously [1]. Clotting me- The structure of oroxylin A was identified using IR, UV, + 1 dia contained M199 (M199 with 100 U/ml penicillin TOF/MS, and H-NMR. The data were consistent with and 100 µg/ml streptomycin) plus 0.3% fibrinogen that had been reported before [7]. The purity level of and 0.5% e-amino-n-caproic acid (ACA; Sigma, St. oroxylin A exceeded 99% as determined by HPLC with Louis, MO). Growth media consisted of M199+ with Evaporative Light Scattering Detector (HPLC ELSD). 20% FBS and 0.5% ACA. Then, the growth media were added to the wells with or without 1 µg/ml LPS Endothelial cell migration assay and various concentrations of oroxylin A (8, 40 and 200 µM). Plates were then stored in incubator at 37°C Chemotactic motility of HUVECs was assayed using and 5% CO2. After 7 days, the sprouting microvessels a Transwell chamber (Millicell, Billerico, MA) as de- in five randomly chosen fields were counted and pho- scribed previously [18]. Briefly, cells were incubated tographed under a microscope for each group. in M199 containing 1% FBS in the absence or pres- ence of oroxylin A (1, 10 and 100 µM) for 60 min. Chicken chorioallantoic membrane (CAM) assay Then, cells were trypsinized and suspended at a final 5 concentration of 5 × 10 cells/ml in M199 containing Anti-angiogenic effect of oroxylin A on CAM was as- 1% FBS. The fresh M199 medium containing 1% sayed according to the method described with modifi- FBS and 1 µg/ml LPS was placed in the lower wells. cation [13]. Briefly, fertilized chicken eggs were incu- Cell suspension was loaded into each of the upper bated at 37°C for 9 days. After this incubation, a small wells. After 4 h, cells were fixed with methanol, stain- hole was punched on the broad side of the egg, and ing with crystal violet, releasing the bound dye with a window was carefully created through the egg shell. 33% glacial acetic acid, and measuring the optical Sterilized filter paper disks (5 × 5 mm) saturated with density (OD) of the solution at 570 nm by using an or without LPS (1 µg/ml) and oroxylin A (1.5, 15 and enzyme immunosorbent assay reader. 150 ng/egg) were placed on the CAMs. They were then incubated at 37°C for another 2 days. After 48 h Tube formation assay of incubation, shells of fertilized chicken eggs were opened. Then, an appropriate volume of 10% fat An in vitro capillary tube formation assay was per- emulsion (Intralipose, 10%) was injected into the em- formed as described earlier [18]. Briefly, a 96-well bryo chorioallantois for observing the density and plate was coated with 80 µl matrigel (Becton Dickin- length of vessels toward the CAM face. Neovascular son, Bedford, MA), which was allowed to solidify zones under the filter paper disks were observed and and polymerize at 37°C for 30 min. HUVECs were photographed with a digital camera at 5× magnifica- incubated in medium containing 1% FBS in the ab- tion. The number of newly growth vessels were sence or presence of oroxylin A (1, 10 and 100 µM) counted on digitalized pictures.
Pharmacological Reports, 2012, 64, 11891199 1191 Western blot analysis
Human umbilical vein endothelial cells were pre- treated for 2 h with various concentrations of oroxylin A (1, 10 and 100 µM) and stimulated with LPS (1 µg/ml) for 60 min. After stimulation, cells were collected and lysed in lysis buffer [50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% (m/v) NP-40, 0.2 mM PMSF, 0.1 mM NaF and 1 mM DTT], and lysates were clarified by centrifugation at 4°C for 15 min at 13,000 × g. The concentration of protein in the supernatants was detected using bicinchoninic acid (BCA) assay with a Varioskan multimode micro- plate spectrophotometer (Thermo, Marietta, OH). Fig. 1. Effect of oroxylin A on LPS-stimulated migration of HUVECs. Then, equal amount of total proteins was separated by Inhibition of LPS-stimulated migration of HUVECs by oroxylin A. SDS-PAGE and transferred onto the PVDF mem- Optical density (OD) of the solution was measured at 570 nm. For each oroxylin A-treated group, asterisk indicates values significantly branes (Millipore, Billerica, MA). The blots were in- different from the LPS-treated control group, * p < 0.05, ** p < 0.01 cubated with specific antibodies against indicated pri- mary antibodies overnight at 4°C followed by IRDyeTM800 conjugated secondary antibody for 1 h at 37°C. Detection was performed by the Odyssey Infra- red Imaging System (LI-COR Inc., Lincoln, NE). Oroxylin A suppresses LPS-stimulated tube formation of HUVECs
Statistical analysis Tube formation assay was performed to evaluate the ef- All data in different experimental groups were expressed fects of oroxylin A on LPS-induced tube formation of as the mean ± SEM. These data shown in the study were HUVECs on a matrigel substratum. As shown in Figure obtained in at least three independent experiments. Sta- 2, LPS promoted formation of capillary-like tubes, and tistical analyses were performed using an unpaired, the number of tubes was much larger than control group. two-tailed Student’s t-test. All comparisons were made Tube formation of cells treated with oroxylin A before relative to LPS-treated groups; p value less than 0.05 seeding and plating onto matrigel was inhibited in was considered as statistically significant. a concentration-dependent manner. Quantitative analysis revealed that treatment with oroxylin A resulted in 27%, 65% and 80% inhibition of LPS-induced tube formation at 1, 10, and 100 µM, respectively. Results Oroxylin A inhibits LPS-stimulated angiogenesis in vitro Oroxylin A reduces LPS-induced migration of HUVECs Several stages in angiogenesis, including endothelial Transwell assay was used to evaluate the effect of orox- cell proliferation, migration, and tube formation, can ylin A on the migration of HUVECs. As shown in be mimicked in the rat aortic ring model. We utilized Figure 1, LPS stimulated HUVECs migration compared the model to evaluate the effect of oroxylin A on to the control. When treated with 1, 10 and 100 µM LPS-induced angiogenesis. As shown in Figure 3, oroxylin A, fewer cells migrated to the lower side of the oroxylin A at 8, 40 and 200 µM resulted in the inhibi- filter in the Transwell Chamber. Oroxylin A signifi- tion of microvessel growth by 17%, 48% and 79%, re- cantly inhibited the migration of HUVECs in a con- spectively. At the tested concentrations, oroxylin A centration-dependent manner. The inhibition of migra- suppressed the formation of microvessel outgrowth tion is reduced by 11%, 44% and 59% at 1, 10 and 100 from explants of rat aorta in a concentration-depend- µM, respectively, compared with the LPS group. ent manner.
1192 Pharmacological Reports, 2012, 64, 11891199 Oroxylin A inhibits LPS induced angiogenesis Xiuming Song et al.
Fig. 2. Effect of oroxylin A on LPS-stimulated tube formation of HUVECs. (A) Control (0.1% DMSO). (B) LPS. (C) 1 µM oroxylin A plus LPS. (D) 10 µM oroxylin A plus LPS. (E) 100 µM oroxylin A plus LPS. Magnification: 100´.(F) Inhibition of LPS-stimulated tube formation of HUVECs by oroxylin A. For each oroxylin A-treated group, asterisk indicates values significantly different from the LPS-treated control group, p < 0.05
Oroxylin A inhibits LPS-stimulated angiogenesis Influence of oroxylin A on TLR4 and its signaling in ovo pathways
To study the anti-angiogenesis capacity of oroxylin A In this part, western blotting analysis was utilized to in ovo, CAM model was applied. From this model, we evaluate the molecular mechanism of oroxylin A an- investigated the process of new blood vessel forma- tagonizing LPS-induced angiogenesis in HUVECs. tion and vessel responses to anti-angiogenic agents. LPS promotes angiogenesis through activation of its As shown in Figure 4, more and thicker new blood cell surface receptor TLR4. So we investigated the ef- vessels were formed in the LPS group compared with fects of oroxylin A on the interaction between LPS the control group. When oroxylin A was added, the and TLR4 as well as the activation of its downstream quantity of vessels was reduced by 10%, 35% and signaling molecules including ERK1/2, JNK and p38 50% at 1, 10, and 100 µM, respectively. It displayed MAPKs. As shown in Figure 5, level of TLR4 was that oroxylin A inhibited the in ovo angiogenesis in apparently induced by addition of exogenous LPS to a concentration-dependent manner. HUVECs. When the HUVECs were pre-treated with
Pharmacological Reports, 2012, 64, 11891199 1193 oroxylin A, the up-regulation of TLR4 induced by Oroxylin A inhibits the IKK/NF-kB pathway LPS was reduced significantly. We also observed the total steady state protein levels remained unchanged, To study the role of oroxylin A in the IKK/NF-kB while the phosphorylated protein levels of ERK1/2, pathway, western blotting analysis was utilized to ex- JNK and p38 MAPKs were decreased in a concentra- amine the effects of oroxylin A on LPS-induced nu- tion-dependent manner after the treatment with orox- clear translocation of NF-kB. As shown in Figure 6, ylin A. To sum up, the results suggested that TLR4 oroxylin A inhibited NF-kB nuclear translocation in- might be blocked by oroxylin A, leading to interrup- duced by LPS in a concentration-dependent manner. tion of LPS-triggered signaling pathway. When the HUVECs were pre-treated with oroxylin A,
Fig. 3. Effect of oroxylin A on LPS-stimulated rat aortic ring microvessel sprouting. (A) Control (0.1% DMSO). (B) LPS. (C) 8 µM oroxylin A plus LPS. (D) 40 µM oroxylin A plus LPS. (E) 200 µM oroxylin A plus LPS. Magnification: 200´.(F) Inhibition of neovessel formation from the rat aortic rings by oroxylin A. For each oroxylin A-treated group, asterisk indicates values significantly different from the LPS-treated control group, p < 0.05
1194 Pharmacological Reports, 2012, 64, 11891199 Oroxylin A inhibits LPS induced angiogenesis Xiuming Song et al.
the cytosolic IkB degradation and IkB kinase (IKK) proved to have inhibitory effect on inflammation [11]. phosphorylation induced by LPS were inhibited in It also inhibited LPS-induced iNOS and COX-2 gene a concentration-dependent manner. To sum up, the re- expression by blocking NF-kB activation [3]. Orox- sults suggested that oroxylin A could influence ylin A has been shown to be a promising candidate for IKK/NF-kB pathway, which may be involved in inflammation. Since a close relationship among angio- LPS-triggered angiogenesis. genesis and inflammation has been substantiated [4, 6, 17], in the present study we investigated if oroxylin A could inhibit vascular remodeling caused by certain in- flammation factor and have a potential ability to slow down inflammation progression sequentially. Discussion Angiogenesis is fundamental to many physiologic and pathologic processes such as wound healing, re- Oroxylin A is a flavonoid compound originated from productive functions in adults, cancer, ischemic dis- Scutellaria baicalensis Georgi. Oroxylin A has been eases, and chronic inflammation [20]. It is a multistep
Fig. 4. Effect of oroxylin A on LPS-stimulated new blood vessel formation in CAMs. (A) Control (0.1% DMSO). (B) LPS. (C) 1.5 ng oroxylin A plus LPS. (D) 15 ng oroxylin A plus LPS. (E) 150 ng oroxylin A plus LPS. (F) Quantification of neovascularization of the CAMs. For each oroxylin A-treated group, asterisk indicates values significantly different from the LPS-treated control group, p < 0.05
Pharmacological Reports, 2012, 64, 11891199 1195 Fig. 5. Effect of oroxylin A on LPS-stimulated activation of TLR4 and its signaling pathway. (A) Western blotting assays were used to examine the expression of TLR4 and phosphorylation of ERK1/2, JNK and p38 MAPKs. (B) Quantification analysis gave the relative ratios of TLR4/b- actin, phosphorylated JNK/total JNK, phosphorylated ERK1/2/total ERK1/2, and phosphorylated p38 MAPK/total p38 MAPK. For each oroxylin A-treated group, asterisk indicates values significantly different from the LPS-treated control group, p < 0.05
1196 Pharmacological Reports, 2012, 64, 11891199 Oroxylin A inhibits LPS induced angiogenesis Xiuming Song et al.
Fig. 6. Effect of oroxylin A on LPS-stimulated NF-kB nuclear translocation, IKK phosphorylation, IkB phosphorylation and IkB degradation. (A) Western blotting assays were used to examine the level of NF-kB in cytoplasm and nuclear, respectively. (B) Western blotting assays were used to examine the level of IKK, p-IKK, IkB and p-IkB. Quantification analysis gave the relative ratios of NF-kB/lamin A, NF-kB/b-actin, phos- phorylated IKK/total IKK, IkB/b-actin, and phosphorylated IkB/b-actin. For each oroxylin A-treated group, asterisk indicates values significantly different from the LPS-treated control group, * p < 0.05, ** p < 0.01
Pharmacological Reports, 2012, 64, 11891199 1197 process of sprouting of new capillaries from preexist- role in LPS-induced angiogenesis due to its ability to ing blood vessels involving the proliferation and mi- induce transcription of pro-inflammatory genes [19]. gration of endothelial cells, remodeling of the extra- In our study, we found oroxylin A could prevent cellular matrix, maturation of the newly blood vessels NF-kB dimers from translocating to the nucleus due and all kinds of cytokine production [8]. Angiogene- to the inhibition of IKK phosphorylation and the sub- sis and inflammation complement each other. Devel- sequent IkB degradation. This may prevent certain opment of angiogenesis is a multistep process which target genes downstream NF-kB from being activated can be activated by inflammatory agents. Inflamma- in angiogenesis. Our findings suggested that inhibi- tion promotes angiogenesis by many ways and it fur- tory effect of oroxylin A on LPS-induced angiogene- ther potentiates endothelial proliferation and angio- sis in inflammation was also due to inhibition of genesis, while angiogenesis also contributes to the pa- NF-kB pathway. Taken the results together, we con- thology of inflammations. Vascular remodeling occurs jecture that oroxylin A can influence LPS-induced in many chronic inflammatory disorders, such as in- angiogenesis by inhibiting the expression of TLR4, flammatory bowel disease [21]. phosphorylation of MAPKs and activation of NF-kB. In our study, transwell assay and tube formation as- In summary, we demonstrated that oroxylin A in- say were employed to demonstrate the effects of orox- hibited angiogenesis induced by LPS in vitro and in ylin A on LPS-induced migration and tube formation ovo through down-regulating the activation of of HUVECs in vitro. The results showed that oroxylin LPS/TLR4 signaling pathway. Oroxylin A, as a novel A remarkably suppressed the LPS-stimulated migra- angiogenesis inhibitor, probably is beneficial in the tion and tube formation of HUVECs. Rat aortic ring treatment of inflammatory disorders. Our findings assay was used to investigate if oroxylin A could in- provide new insights into the molecular mechanisms hibit the microvessels sprouting induced by LPS from of oroxylin A-induced suppression of angiogenesis rat aorta. Then, we used CAM model to illustrate that and suggest possible applications for oroxylin A in in- oroxylin A inhibited new vessel formation and vascu- flammation diseases. lar remodeling in ovo. Our results demonstrated that oroxylin A could inhibit the microvessel outgrowth from rat aorta in vitro and it also had anti-angiogenic Conflict of interest: activity in ovo. These results implied that oroxylin None declared. A could act as a probable anti-angiogenic compound Acknowledgments: in inflammation. This work was supported by the National Natural Science Foundation of China (No. 30973556 and 81001452), Program LPS, a potent inflammatory mediator, plays an im- for Changjiang Scholars and Innovative Research Team in portant role in inflammation and indeed has angio- University (IRT1193), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University genic potential [5]. Its activities are transmitted (No. JKGZ201101), the National Science & Technology Major through TLRs. In fact, LPS directly causes endothe- Project (No. 2012ZX09304-001) and Natural Science Foundation of Jiangsu province (No. BK2009297 and No. BK2010432). lial sprouting and angiogenesis utilizing the stimula- tion of TLRs and TRAF6 [19]. Involvement of vari- ous intracellular signaling pathways, such as MAPKs, have been reported downstream TLR4. Activation of MAPKs induces certain effects in LPS-induced References: angiogenesis. Therefore, further experiments were performed to determine whether oroxylin A could 1. Berger AC, Wang XQ, Zalatoris A, Cenna J, Watson JC: affect activation of MAPKs. When HUVECs were A murine model of ex vivo angiogenesis using aortic pretreated with oroxylin A, the upregulated expres- disks grown in fibrin clot. Microvasc Res, 2004, 68, 179–187. sions of TLR4 and phosphorylated JNK, ERK and 2. Bonnet CS, Walsh DA: Osteoarthritis, angiogenesis and p38 MAPKs induced by LPS were blocked, without inflammation. Rheumatology (Oxford), 2005, 44, 7–16. affecting their total proteins. This indicated that the 3. Chen Y, Yang L, Lee TJ: Oroxylin A inhibition of inhibitive effects of oroxylin A on the responsibility lipopolysaccharide-induced iNOS and COX-2 gene ex- pression via suppression of nuclear factor-kappaB activa- of HUVECs to LPS might derive partly from inhibit- tion. Biochem Pharmacol, 2000, 59, 1445–1457. ing TLR4 signal transduction. As one of the most im- 4. Clavel G, Valvason C, Yamaoka K, Lemeiter D, Laroche portant transcription factors, NF-kB plays a pivotal L, Boissier MC, Bessis N: Relationship between angio-
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