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Journal of Huazhong University of Science and Technology Med Sci DOI 10.1007/s 11596-007-0207-x Journal of Huazhong University of Science and Technology

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(2): 138-141, 2007 ［27 Med Sci］ 27 (2): 2007

Effects of Andrographitis Paniculata Extracts on the Expression of CD40 in Endothelial Cells

张洁

李树生

万磊

ZHANG Jie ( ), LI Shusheng ( )#, WAN Lei ( ) Department of Emergency, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Summary: In order to investigate the expression of CD40 in endothelial cells (ECs) in a variety of injured conditions and the interventional role of Andrographitis Paniculata isolate (API0134), the thoracic aorta ECs of guinea pigs were cultured in vitro until the third passage, incubated in the presence of media containing xanthine oxidase (XO) and xanthine (Xan) which produced oxygen free radical (OFR group); oxidized-LDL (ox-LDL group); XO, Xan and API0134 (OFR+API0134 group); or ox-LDL and API0134 (ox-LDL+API0134 group). The expression of CD40 in ECs was detected by immunofluorescence assay and reverse transcription-PCR (RT-PCR). The results showed as compared with the control group, the expression of CD40 in ECs in OFR group and ox-LDL group was increased (P<0.01), but attenuated significantly in OFR+ API0134 group and ox-LDL+API0134 group (P<0.05). It was suggested that API0134 could protect atherosclerosis by inhibiting the expression of CD40 molecule in injured ECs. Key words API0134; endothelial cells; CD40; atherosclerosis

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Endothelial dysfunction elicited by oxidized (ox) LDL and oxygen free radical (OFR) plays a critical role in the pathogenesis of atherosclerosis. Ox-LDL and OFR change the secretory activities of the endothelium and cause it to become dysfunctional. Ox-LDL and OFR inhibit the expression of constitutive endothelial nitric oxide synthase, induce the expression of adhesion molecules on the endothelium, and facilitates inflammatory cells to adhere to the intima. Ox-LDL increases free radical generation, release of the cytokines TNF- and interleukin-6, monocyte chemoattractant protein [MCP]-1, interleukin-8, angiotensin , and the expression of adhesion molecules; all of these contribute to the inflammatory process and initiate and/or accelerate atherosclerosis. Recently, it has been reported that during the inflammatory response of main cells in the atherosclerotic plaque, CD40 was expressed in these cells such as endothelial cells (ECs), smooth muscle cells etc. There is emerging evidence that CD40/CD40L activation is a critical inflammatory signal in atherosclerosis. Ox-LDL may trigger CD40/CD40L signaling and initiate and augment progression of atherosclerosis[1]. Many studies have revealed the ECs dysfunction could make advancement in atherosclerosis by promoting the interaction between CD40 and CD40L which further induced adhesion molecule. Our research team has discovered that Andrographitis Paniculata isolate (API0134) could protect ECs, resist atherosclerosis and prevent restenosis after angioplasty. In the present study, the expression of CD40 molecule in normal arterial ECs and injured ECs

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ZHANG Jie, female born in 1979, Postgraduate Student # Corresponding author Email: [email protected]

and the effect of API0134 on the expression of CD40 in atherosclerotic plaque were observed in order to provide the experimental information for the treatment of atherosclerosis in clinical practice. 1 MATERIALS AND METHODS 1.1 Materials Male adult guinea pigs, aged 15 weeks, weighing 250–350 g, were provided by the Animal Center of Tongji Medical College, HUST, Wuhan, China. Both M199 and 0.125% trypsin were both from Gibco Co., USA. Fetal calf serum was from Huaxi Biology Research Institute (China). Both factor kit and reverse transcription-PCR (RT-PCR) kit were both from Sigma Co., USA. CD40 antibody labeled by PE was from Institute of Biological Products, USA. API0134 was from Department of Pharmacy, Tongji Hospital, China. Ox-LDL was obtained as previously reported[2, 3]. 1.2 Cell Culture and Grouping According to the previously described method with a little modification[4, 5], guinea pigs were killed by bloodletting and the thoracic aorta was removed under asepsis. Guinea pigs aorta ECs were isolated, digested with 0.125% trypsin and maintained in M199 supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. ECs were incubated in a humidified incubator at 37°C (95% room air, 5% CO2) until the third passage. When ECs were confluent to a single layer (7 to 8 days), they were trypsinized and seeded in 6-well plates. factor in ECs was detected by ABC assay[6]. After a 48-h incubation at 37°C, cells reached confluence and incubated for 24 h in the presence of media containing 200 µmol/L xanthine oxidase

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(XO) and 10 µmol/L xanthine (Xan); 0.1 mg/mL ox-LDL; 200 µmol/L XO, 10 µmol/L Xan and 50 mg/L API0134; 0.1 mg/mL ox-LDL and 50 mg/L API0134, as previously described[7]. The cultured ECs without any stimulant served as controls. Before the experiment, all cells in 5 groups were maintained in M199 without new-born serum for 24 h. 1.3 Measurement of Immunofluorescence According to the instructions of the kit, the ECs were added with 50 µL CD40-PE labeled antibody and incubated at 4°C for 30 min. Subsequently, the ECs were washed with PBS twice. The fluorescent density was analyzed according to the concentration of antibody added.. 1.4 Detection of CD40 Expression by Reverse Transcription-PCR (RT-PCR) Total RNA was isolated from cell cultures with Trizol reagent. The expression of CD40 mRNA was detected by using RT-PCR technique. Expression of mRNA for β-actin was used as an internal standard. One microgram of total RNA was reversely transcribed using the RT-PCR kit according to the manufacturer’s protocol. PCR was done in a 25-µL reaction mixture containing 5 µL of cDNA template, 1-PCR buffer, 1.5 mmol/L MgCl2, 0.8 mmol/L deoxynucleotide triphosphates, 1 U Taq DNA polymerase, and 100 nmol/L of each primer for CD40 (sense primer, 5'-TGCCAGCCAGGACAGAAA CT-3'; antisense primer, 5'-GGGACCACAGACAACA TCAG-3') or for β-actin (sense primers, 5'-CCTTCCTG GGCATGGAGTCCTG-3'; antisense primer, 5'-GGAGC AATGATGATCTTGATCTTC-3'), as previously reported[8]. Expected amplicon sizes were 420 bp for CD40

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and 205 bp for β-actin. The conditions for PCR were as follows: 30 cycles at 94°C for 30 s, 56°C for 50 s, and 72°C for 30 s and final extension for 10 min at 72°C. PCR products were resolved in 1.5% agarose gels stained with ethidium bromide. The negative film of mRNA was quantitated by image analysis system, and the measuring unit was integral absorbance (A). 1.4 Statistical Analysis Results were expressed as ±s. Data were analyzed by SPSS12.0. All data were determined out of n=5 to 7 independent experiments at different days. A P<0.05 was considered significant. 2 RESULTS 2.1 Determination of Modified Degree of ox-LDL The malonaldehyde level of LDL oxidized by CuSO4 was 9.132±0.285 nmol/mg protein, which was significantly higher than that of unoxidized natural LDL (nLDL) (2.154±0.101 nmol/mg protein, P<0.05). After agarose electrophoresis for 30 min at 100 V, in relative to nLDL, the electrophoretic mobility of ox-LDL was 86, and nLDL was 1. 2.2 CD40 Fluorescence Examination Under the fluorescence microscopy, it was found clearly that the CD40 was expressed on the surface of human ECs with red fluorescence (fig. 1A-E). As compared with control group, the immunofluorescent intensity in OFR group and ox-LDL group was enhanced, but that in OFR+API0134 group and ox-LDL+API0134 group attenuated significantly.

Fig. 1 Expression of CD40 in ECs in each group (Fluorescent staining×200) A: Normal ECs; B: OFR group; C: ox-LDL group; D: OFR+API0134 group; E: ox-LDL+API0134 group

2.3 Detection of the CD40 Expression in ECs by RT-PCR As compared with the control group, the relative

intensity of CD40 mRNA in OFR group and ox-LDL group was increased, but that in OFR+API0134 group and ox-LDL+API0134 group was reduced significantly (table

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Journal of Huazhong University of Science and Technology

1, fig. 2). Table 1 The expression of CD40 mRNA in ECs in each s) group (

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Groups Control OFR

CD40 mRNA/β-actin mRNA 0.21±0.14

0.66±0.21**▲

ox-LDL

0.72±0.19**●

OFR+API0134

0.38±0.11*

ox-LDL+API0134 0.53±0.16* * P<0.01, P<0.05, as compared with control group; ▲P<0.05, OFR group vs OFR+API0134 group; ●P<0.05 Ox-LDL group vs ox-LDL+API0134 group, **

Fig. 2 The expression of CD40 in ECs in each group M: Marker; 1: ox-LDL group; 2: OFR group; 3: API0134+ox-LDL group; 4: API0134+OFR group; 5: control group

3 DISCUSSION CD40 is a 50-kD (1 kD=0.9992 ku) integral member of the tumor necrosis factor receptor family. An important role for CD40L-CD40 signaling in atherosclerosis has been reported. In atherosclerotic plaques of mice and humans, CD40L and CD40 are present on vascular smooth muscle cells (VSMCs), ECs, macrophages, and T lymphocytes. Under normal conditions, there are only a few CD40 molecules expressed in arterial ECs. However, in the atherosclerotic plaque, there are more CD40 molecules expressed, mainly in the shoulder of atherosclerotic plaque and the juncture between the normal tissue and the plaque[9]. It has been shown that CD40-soluble CD40 ligand (sCD40L) interactions might constitute an important mediator for vascular inflammation. Activation of vascular cells via CD40-CD40L signaling pathway has been shown to induce inflammatory responses with expression of adhesion molecules, secretion of pro-inflammatory cytokines (IL-1, IL-3, TNF-α, GM-CSF, and particularly IFN-γ), matrix metalloproteinases, tissue factor and chemokines, molecules considered as crucial players in atherogenesis. Increasing evidence supports the central role of the CD40-CD40L signaling pathway in atherosclerosis. CD40-CD40L blockade has been shown to prevent atherosclerotic plaque progression, promote plaque stability and prevent transplant associated vasculopathy, an accelerated form of atherosclerosis[10]. Early pathological study suggested that CD40 might take part in the progress of atherosclerosis, and may create a proinflammatory and prothrombotic milieu for aggravating atherosclerosis and instability of atherosclerotic plaques. Indeed, CD40-CD40L interactions have been implicated in a wide range of chronic inflammatory conditions, including arthritis, atherosclerotic disease, and allograft rejection[11]. In summary, it is now clear that atherosclerosis depends on

［Med Sci］ 27 (2): 2007

the immune system and chronic inflammation. The initial cause or causes for the nidus of inflammation and endothelial cell dysfunction are unknown but may be immune or infectious or may result from traumas or disordered physiology. API0134 is an ingredient of Chinese medicine Andrographitis extracted by stratography. Previously, our study indicated that API0134 resisted lipid oxidization, and could eliminate cytotoxic effect in ECs induced by ox-LDL. And it was discovered API0134 had antioxidnt and protective effects on ECs in animal model[12, 13]. In this study, ECs were injured by oxygen free radicals and low density lipoprotein to establish the model of ECs injury. Immunofluorescence and RT-PCR techniques revealed that as compared with control group the expression of CD40 was increased obviously in the injured ECs (ox-LDL group and OFR group, P<0.01). The expression of CD40 was reduced in groups (API0134+ox-LDL group and API0134+OFR group, P<0.05). As compared with the corresponding groups, the expression of CD40 in the API0134-treated groups was decreased significantly (P<0.05). In conclusion, the present study suggested that ox-LDL and OFR could induce the increased expression of CD40 in ECs, and API0134 could antagonize the activating effect of ox-LDL and OFR and inhibit the expression of CD40 in ECs. API0134 could protect ECs by inhibiting the interaction between CD40 and CD40L. The exact mechanisms need further research. But we could presume that the membrane-binding CD40 and sCD40L serum concentration might be useful clinical markers of arteriosclerosis, and that therapeutic modalities by down-regulating CD40-CD40L interaction may represent a new therapeutic approach for the treatment of arteriosclerosis. REFERENCES 1 Li D Y, Li L, Chen H J et al. LOX-1, an oxidized LDL endothelial receptor, induces CD40/CD40L signaling in human coronary artery endothelial cells. Art Thromb Vasc Biol (Chinese), 2003,23(5):816-821 2 Zhang L H, Liu B W. Separation of human serum lipoproteins by one-step ultracentrifugation. Acta Biochim Biophys (Chinese), 1989,21:257-260 3 Yan X H, Ouyang J P, Tu S Z et al. Effects of Angelica Sinensis on oxLDL - induced decrease of NO release from human endothelial cells and increase of ICAM-1 expression on the surface of human endothelial cells. Chin J Pathophysiol (Chinese), 2000,16(1):51-59 4 Yan P H, Li F Z. Culture and identification of rat aortic endothelial cells. Bull Acad Mili Med Sci (Chinese), 1993,17(2):124-126 5 Lai Y, Shen W J, Li Y P et al. Culture of endotheliocytes derived from guinea-pig common carotid artery and establishment of guinea-pig donor model. J WCUMS (Chinese), 2000,30(3):341-343 6 Zhao X L, Liu S J, Sun B C et al. Application of immunohistochemistry technique in detecting cultured endothelial cells. J Clin Exp Pathol (Chinese), 1997,13(1): 82 7 Chen T P, Song X M, Li Z P et al. Protective Effect of Chitin on human umbilical vein endothelial cells (HUVECs) injured by Oxygen Free Radicals in vitro. Chin J Mari Drugs (Chinese), 1998,65(1):28-30 8 Yan J C, Wu Z G, Zhong Q R et al. CD40 and CD40 ligand coexpression on human endothelial cells and in human atherosclerosis plaque lesions. Chin J Pathophysiol

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(Chinese), 2002,19(8):1021-1024 Cao D L, Yang X J. CD40 ligand and atherosclerosis. Clinical Focus (Chinese), 2004,19(1):45-47 10 Lin R, Liu J T, Gan W J et al. C-reactive protein-induced expression of CD40-CD40L and the effect of Lovastatin and Fenofibrate on it in human vascular endothelial cells. Biol Pharm Bull, 2004,27(10):1537-1543 11 Mach F, Schonbeck U, Sukhova G K et al. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implication for CD40-CD40 ligand in atherosclerosis. Proc Natl Acad 9

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Sci USA, 1997,94:1931-1936 12 Wang H W, Zhao H Y, Ma B X. Protective effects of API0134 on endothelial cells damaged by oxidatively modified low density lipoprotein. Pharmacol Clin Chin Mater Med (Chinese), 1996,(1):27 13 Wang D W, Zhao H Y. Prevention of atherosclerosis arterial stenosis and restenosis after angioplarty with andrographin paniculate nees and fish oil. Chin Med J (Chinese), 1994,107(6):454-459 (Received Nov. 17, 2006)