translocation of NF-κB p65 and p50 in IL-1 β-stimulated HUVECs
3.3.5 Carnosic acid inhibits IL-1 β-induced ROS in HUVECs
translocation of p65 and p50 (Fig. 3-6, 3-7). In addition, pretreatment of HUVECs with 20 μmol/l carnosic acid was more effective on decreasing the nuclear translocation of p65 and p50 than that of 10 μmol/l carnosic acid.
3.3.5 Carnosic acid inhibits IL-1 β-induced ROS in HUVECs
To study the effect of carnosic acid on IL-1 β-induced ROS production in HUVECs, cells were pretreated with 10 and 20 μmol/l carnosic acid for 18 h and then stimulated by IL-1 β (10 ng/ml) for 6 h. HUVECs were labeled with H2O2-sensitive fluorescent probe and detected by fluorescence microplate reader. The production of ROS induced by IL-1 β was increased and significantly decreased after pretreatment with 10 and 20 μmol/l carnosic acid (Fig. 3-8 A & B). In addition, pretreatment of HUVECs with 20 μmol/l carnosic acid was more effective on decreasing the production of ROS than 10 μmol/l carnosic acid.
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3.4 Discussion
Expression of cell adhesion molecules (CAM) by the endothelium and the attachment of monocytes to endothelium may play a major role in the early atherogenic process. Carnosic acid is an antioxidant polyphenolderived from Sage (Salvia oddicinalis) and Rosemary (Rosmarinus officinalis). Previous studies showed that Carnosic acid is a lipophilicantioxidant that scavenges singlet oxygen, hydroxyl radicals,and lipid peroxyl radicals, therefore, it suggested that carnosic acid could prevent the peroxidation of lipid and disruption of biological membranes (Aruoma et al., 1992; Haraguchiet al., 1995). In the present study, we found that carnosic acid had antioxidative effect in vitro. In addition, we also found that carnosic acid significantly suppressed IL-1 β-induced intracellular ROS production, the activation of redox-sensitive transcription factors NF-κB p50, p65, the expression of ICAM-1, VCAM-1 and E-selectin; and the adhesiveness to a human monocytic cell line (U937). These results demonstrated that carnosic acid had inhibitory effect on proanthersclerotic mechanism in vitro.
Previous studies showed that sage and rosemary extracts contained 0.29 % and 0.1-0.5 % of carnosic acid, respectively (Huang et al., 1994; Kiyofumi et al., 2004). In the present study, we found that 20 μmol/l carnosic acid did not have any significant effect on the viability of HUVECs from MTT test. Previous study also showed that 20 μmol/l carnosic acid did not have any significant effect on the viability of HepG2 cells (Costa et al., 2006). Therefore, according to the MTT test, we chose 10 and 20 μmol/l carnosic acid to do all the experiments.
Adhesion and transendothelial migration of monocytes into the surrounding tissues are crucial steps in inflammation, immunity, and atherogenesis (Springer, 1994; Li et al.,
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1993; Jang et al., 1994). Vascular endothelial cells play an active role in this process by expressing cell adhesion molecules which enhance the adhesion of monocytes to the endothelium (Alexiou et al., 2001). In the present study, we found that control group showed minimal binding to U937 cells, but adhesion increased when the HUVECs were treated with IL-1 β. Pretreatment with 10 and 20 μmol/l carnosic acid reduced the number of U937 cells adhering to IL-1 β-stimulated HUVECs. A similar result was seen when HUVECs were pretreated with other polyphenolic compound, such as vitamin E (40 μmol/l), probucol (50 μmol/l) or tea flavonoid (60 μmol/l), these polyphenolic compound reduced monocytes adhesion to endothelial cells (Islam et al., 1998; Zapolska-Downar et al., 2001; Ludwig et al., 2004).
The process by which monocytes become adherent to the endothelium is the result of complex choreography requiring the sequential, yet overlapping, functions of many classes of adhesion molecules (Munro, 1993; Price & Loscalzo, 1999). Tethering and rolling, the first steps of monocyte adhesion to the endothelial surface, appear to depend on the interaction of P- and E- selectin with carbohydrate ligands on leukocytes. Firm adhesion follows if the monocytes encounter activating signals while rolling along the endothelium; this is facilitated by interaction of very late antigen-4 (VLA-4) with VCAM-1 or lymphocyte function antigen-1 (LFA-1) with ICAM-1 (Marlin & Springer, 1987; Elices et al., 1990). Previous study showed that adhesion molecules are strong predictors of atherosclerotic lesion development and future cardiovascular events (Blankenberg et al., 2003). It has been reported that expression of adhesion molecules on HUVECs is increased or induced by stimulation with inflammatory cytokines, including TNF-α, IL-1 β, IL-4, and IL-13 (Schleimer & Rutledge, 1986). In the present study, we found that the expression of ICAM-1, VCAM-1 and E-selectin induced by IL-1β were
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increased and the pretreatment with carnosic acid could decreased the inducetion of expression by IL-1 β in HUVECs. These results indicated that carnosic acid could inhibit the rolling, tethering and firm adhesion of the monocytes on the vascular wall.
The translocation of the transcription factor, NF-κB, is involved in the signal transduction pathways for IL-1β-induced adhesion molecule expression (Lenardo &
Baltimore, 1989). The activated form of NF-κB is a heterodimer, which usually consists of two proteins, a p65 (also called relA) subunit and a p50 subunit (Baldwin, 1996).
Udalova et al., 2000 described that the p50–p65 heterodimers are involved in enhancing the transcription of adhesion molecules, cytokines and chemokines. In the present study, we examined whether the inhibitory effect of carnosic acid on the cytokine-induced expression of adhesion molecules is medicated via NF-κB, therefore, we measured the nuclear translocation of p65 and p50 protein of the NF-κB family. We found that the nuclear translocation of p65 and p50 induced by IL-1 β were increased and the pretreatment with carnosic acid could decreased the inducetion of the nuclear translocation by IL-1 β in HUVECs. Therefore, we confirmed that carnosic acid has an anti-inflammatory effect through the partial interference of NF-κB activation.
Harrison et al., 2003 described that ROS play a central roles in the pathogenesisof endothelial dysfunction and atherosclerosis. Previous study indicated that the activation mechanisms of NF-κB nuclear translocation has been suggested to involve ROS (Schreck et al., 1991&1992; Suzuki et al., 1993). In the present study, we found that the production
of ROS induced by IL-1 β were increased and the pretreatment with carnosic acid could decreased the inducetion of expression by IL-1 β in HUVECs. Previous studies showed that the activation of NF-κB could be inhibited by different antioxidants, it is strongly suggested that endogenous ROS may play an important role in these redox-sensitive
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transcription pathways in atherogenesis (Manna et al., 1992; Muller et al., 1994). Based on the present results, we proposed that the inhibitory effect of carnosic acid on adhesion molecules expression and NF-κB activation may be due to its antioxidant and anti-inflammatory properties and that it may act by directly scavenging free radicals. In the present study, we also found that carnosic acid was approximately 5-6-folds more potent than Trolox in antioxidative ability and it also could scavenge DPPH, alkoxyl radical (RO‧) and peroxyl radical (ROO‧) (Appendix 1). Since atherosclerosis is a chronic inflammatory disease associated with increased oxidative stress in the vascular endothelium, it is possible that the antiatherogenic effects of carnosic acid may due to its antioxidative properties.
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3.5 Conclusion
In the present study, we found that carnosic acid inhibited monocyte adhesion to endothelial cell, the expression of adhesion molecules, the translocation of NF-κB and the production of ROS in HUVECs. These findings may provide a rationale for the in vitro antiatherosclerosis effect of carnosic acid and support its potential use in the prevention of atherosclerosis.
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Fig. 3-1
Figure 3-1. Cytotoxic effect of carnosic acid on HUVECs with MTT test.
HUVECs were treated with various concentrations (0~60 μmol/l) of carnosic acid for 24 h i n 1 0 % F B S - M 1 9 9 . R e s u l t s w e r e f r o m f o u r e x p e r i m e n t s a n d expressed as mean ± S.D. *p < 0.05 compared with control.
*
*
0 20 40 60 80 100 120
0 5 10 20 40 60
Carnosic acid (μmol/l)
% of control
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Fig. 3-2
(A)
C IL-1
IL-1+ CA 10 IL-1+ CA 20
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Fig. 3-2
(B)
Figure 3-2. Effect of carnosic acid on IL-1 β-induced adhesion of U-937 cells to HUVECs. (A) Representative images of the reduction of IL-1 β-induced adhesion of U-937 cells to HUVECs monolayers after pretreatment of 10 and 20 μmol/l carnosic acid for 18 hours. (B) HUVECs were pretreated with 10 and 20 μmol/l carnosic acid for 18 hours and induced by IL-1 β (10 ng/ml) for 6 hours. Fluorescence-labeled U-937 cell were added to the HUVECs monolayer and allowed to adhere for 30 min. Values are mean ± S.D., n=3. a-d Means with different letters are significantly different at p < 0.05.
Abbreviation: C (control), IL-1 (IL-1 β), CA10 (carnosic acid 10 μmol/l), CA 20 (carnosic acid 20 μmol/l).
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Fig. 3-3
(A)
(B)
Figure 3-3. Effect of carnosic acid on the protein levels of ICAM-1 in cultured HUVECs. HUVECs were pretreated with 10 and 20 μmol/l carnosic acid for 18 hours and induced by IL-1 β (10 ng/ml) for 6 hours. (A) Representative images of the reduction of IL-1 β-induced the expression of ICAM-1 by carnosic acid in HUVECs. (B) Densitometric analysis was conducted with image analysis system software to quantify Western blot data. The summarized data (mean ± S.D.) from 3 separate experiments is shown in the bar graph. a-c Means with different letters are significantly different at p <
0.05.Abbreviation: C (control), IL-1 (IL-1 β), CA10 (carnosic acid 10 μmol/l), CA 20 (carnosic acid 20 μmol/l).
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Fig 3-4
(A)
(B)
Figure 3-4. Effect of carnosic acid on the protein levels of VCAM-1 in cultured HUVECs. HUVECs were pretreated with 10 and 20 μmol/l carnosic acid for 18 hours and induced by IL-1 β (10 ng/ml) for 6 hours. (A) Representative images of the reduction of IL-1 β-induced the expression of VCAM-1 by carnosic acid in HUVECs. (B) Densitometric analysis was conducted with image analysis system software. to quantify Western blot data. The summarized data (mean ± S.D.) from 3 separate experiments is shown in the bar graph. a-d Means with different letters are significantly different at p <
0.05.Abbreviation: C (control), IL-1 (IL-1 β), CA10 (carnosic acid 10 μmol/l), CA 20 (carnosic acid 20 μmol/l).
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Fig 3-5
(A)(B)
Figure 3-5. Effect of carnosic acid on the protein levels of E-selectin in cultured HUVECs. HUVECs were pretreated with 10 and 20 μmol/l carnosic acid for 18 hours and induced by IL-1 β (10 ng/ml) for 6 hours. (A) Representative images of the reduction of IL-1 β-induced the expression of E-selectin by carnosic acid in HUVECs. (B) Densitometric analysis was conducted with image analysis system software. to quantify Western blot data. The summarized data (mean ± S.D.) from 3 separate experiments is shown in the bar graph. a-c Means with different letters are significantly different at p <
0.05.Abbreviation: C (control), IL-1 (IL-1 β), CA10 (carnosic acid 10 μmol/l), CA 20 (carnosic acid 20 μmol/l).
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Fig 3-6
Figure 3-6. Effect of carnosic acid on IL-1 β-induce activation of NF-κB p65.
HUVECs were pretreated with 10 μmol/l and 20 μmol/l carnosic acid for 18 hours and then induced by IL-1 β (10ng/ml) for 6 hours. Nuclear extractes were prepared and analyzed for activation of NF-κB. Ten micrograms of nuclear protein was used in each experiment. Values are mean ± S.D., n=3. a-d Means with different letters are significantly different at p < 0.05. Abbreviation: C (control), IL-1 (IL-1 β), CA10 (carnosic acid 10 μmol/l), CA 20 (carnosic acid 20 μmol/l).
d
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Fig 3-7
Figure 3-7. Effect of carnosic acid on IL-1 β-induce activation of NF-κB p50.
HUVECs were pretreated with 10 μmol/l and 20 μmol/l carnosic acid for 18 hours and then induced by IL-1 β (10ng/ml) for 6 hours. Nuclear extractes were prepared and analyzed for activation of NF-κB. Ten micrograms of nuclear protein was used in each experiment. Values are mean ± S.D., n=3. a-d Means with different letters are significantly different at p < 0.05. Abbreviation: C (control), IL-1 (IL-1 β), CA10 (carnosic acid 10 μmol/l), CA 20 (carnosic acid 20 μmol/l).
d
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Fig 3-8
(A)
C IL-1
IL-1 + CA 10 IL-1 + CA 20
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Fig 3-8
(B)Figure 3-8. Effect of carnosic acid on IL-1 β-induce ROS production in HUVECs.
(A) Fluorescent images showed the reduction of IL-1 β-induced ROS production in HUVECs after pretreatment of 10 and 20 μmol/l carnosic acid for 18 hours. (B) HUVECs were pretreated with 10 μmol/l and 20 μmol/l carnosic acid for 18 hours and then induced by IL-1 β (10ng/ml) for 6 hours. HUVECs were labeled with H2O2-sensitive fluorescent probe and detected by fluorescence microplate reader. Values are mean ± S.D., n=3. a-c Means with different letters are significantly different at p < 0.05. Abbreviation: C (control), IL-1 (IL-1 β), CA10 (carnosic acid 10 μmol/l), CA 20 (carnosic acid 20 μmol/l).
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Chapter 4
Summary
In summary, curcumin and carnosic acid could inhibit IL-1β-induced ICAM-1, VCAM-1 and E-selectin expression in HUVEC through a mechanism that involves NF-κB. It reduced the binding of human monocytic cell line U937 to IL-1β-induced HUVECs, which might be due to its antioxidant and anti-inflammatory properties. Our results suggested that curcumin and carnosic acid inhibited the expression of molecules involved in the inflammatory process, therefore, they could play an important role in the prevention of atherosclerosis.
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Figure 4-1. The role of curcumin and carnosic acid in the prevention of atherosclerosis.