Curcumin inhibits IL-1 β-induced ROS production in HUVECs

To study the effect of curcumin on IL-1 β-induced ROS production in HUVECs, cells were pretreated with 10 and 20 μmol/l curcumin 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

42

curcumin (Fig. 2-8 A & B). In addition, pretreatment of HUVECs with 20 μmol/l curcumin was more effective on decreasing the production of ROS than 10 μmol/l curcumin.

43

2.4 Discussion

The expression of cell adhesion molecules by the endothelium and the attachment of monocytes to endothelium may play a major role in the early atherogenic process.

Curcumin is the principal curcuminoid of the Indian curry spice turmeric. The curcuminoids are polyphenols and are responsible for the yellow color of turmeric.

Previous studies indicated that curcumin exhibits a variety of pharmacological effects including antitumor, anti-inflammtory and anti-infectious activities (Mazumder et al., 1995; Ruby et al., 1995; Surh, 2002). In the present study, we found that curcumin had antioxidative effect in vitro. In addition, we also found that curcumin could suppressed IL-1 β-induced intracellular ROS production, the activation of redox-sensitive transcription factors NF-κB p50, p65, the expression of VCAM-1, ICAM-1 and E-selectin; and the adhesiveness to a human monocytic cell line (U937) in HUVECs.

These results demonstrated that curcumin had inhibitory effect on proanthersclerotic mechanism in vitro.

Curcumin is the most active component of turmeric which contains 2 to 5 % of curcumin. Commercial curcumin is usually isolated from the rhizome of turmeric which contains three major curcuminoids (approximately 77 % curcumin, 17 % demethoxy -curcumin, and 3 % bisdemethoxycurcumin) (Bharat et al., 2005). There are several good sources of curcumin with purity ranging from 60 to 98 % (Bharat et al., 2005). Previous study indicated that the serum concentration was 1.77±1.87 μmol/l after the intake of 8 g curcumin in human (Cheng et al., 2001), therefore, curcumin was absorbable in digestive tract in human. In the present study, we found that 20 μmol/l curcumin did not have any significant effect on the viability of HUVECs from MTT test, therefore, we chose 10 and

44

20 μmol/l curcumin to do all the experiments.

One of the earliest events in atherogenesis is the adhesion of monocytes to the endothelium, followed by their infiltration and differentiation into macrophages. In the present study, we found that the 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 curcumin reduced the number of U937 cells adhering to IL-1 β-stimulated HUVECs. A similar resulet aws 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 leukocytes encounter activating signals while rolling along the endothelium, and is facilitated by the 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 was increased by IL-1 β

45

and the pretreatment with curcumin could decreased the inducetion of expression by IL-1 β in HUVECs. These results indicated that curcumin could inhibit the rolling, tethering and firm adhesion of the monocytes on the vascular wall.

NF-κB is a redox-sensitive transcription factor which mediates cell migration, endothelial cell activation and the balance between cell proliferation and apoptosis (Tak

& Firestein, 2001). The activation of NF-κB in endothelial cells is associated with the activation of genes responsible for an increased transcription of adhesion molecules, cytokines and chemokines (True et al., 2000; Valen et al., 2001; Thornburg et al., 2003;

Hatada et al., 2003). 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. In the present study, we examined whether the inhibitory effect of curcumin 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 was increased and the pretreatment with curcumin could decreased the inducetion of translocation by IL-1 β in HUVECs. Previous studies also showed that curcumin could inhibit the activation of NF-κB in human lung epithelial cells and human myelomonoblastic leukemia cells (Sanjaya & Bharat, 1995; Shishodia et al., 2003). Therefore, we confirmed that curcumin has an anti-inflammatory effect through the partial interference of NF-κB activation.

Several studies have indicated that ROS are implicated in the activation of NF-κB (Muller et al., 1997). In the present study, we found that the production of ROS induced by IL-1 β was increased and the pretreatment with curcumin could decreased the

46

inducetion of expression by IL-1 β in HUVECs. Previous studies showed that antioxidants such as PDTC and NAC could inhibit the activation of NF-κB, it is strongly suggested that endogenous ROS may play an important role in these redox-sensitive transcription pathways in atherogenesis (Schrect et al., 1992; Weber et al., 1994). Based on the present results, we proposed that the inhibitory effect of curcumin 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 found that curcumin was approximately 2-3-folds more potent than Trolox in antioxidative ability and it also could scavenge DPPH, alkoxyl radical (RO‧) and peroxyl radical (ROO‧) (Table 2-1). Since atherosclerosis is a chronic inflammatory disease associated with increased oxidative stress in the vascular endothelium, it is possible that the antiatherogenic effects of curcumin may due to its antioxidative properties.

47

2.5 Conclusion

In the present study, we found that curcumin 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 curcumin and support its potential use in the prevention of atherosclerosis.

48

Table 2-1. Antioxidative capacities of curcumin in vitro.

Inhibition of LDL DPPH radicals TEAC Oxidation scavenging ability

(IC50, μmol/l) (IC50, μmol/l)

curcumin 2.56±0.12 45.8±0.1 2.88±0.04

All values are mean ± S.D. IC50 values were obtained from the concentration response curves; n =3.

49

Fig. 2-1

Figure 2-1. Cytotoxic effect of curcumin on HUVECs with MTT test.

HUVECs were treated with verious concentration (0~50 μmol/l) of curcumin for 24 h i n 1 0 % F B S - M 1 9 9 . V a l u e s a r e m e a n ± S . D . , n = 4 . * p < 0 . 0 5 compared with control .

* * *

0 20 40 60 80 100 120

0 10 20 30 40 50

curcumin (μmol/l)

% of c ont ro l

50

Fig. 2-2

(A)

C IL-1

IL-1+CU 10 IL-1+CU 20

51

Fig. 2-2

(B)

Figure 2-2. Effect of curcumin 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 curcumin for 18 hours. (B) HUVECs were pretreated with 10 and 20 μmol/l curcumin 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), CU10 (curcumin 10 μmol/l), CU20 (curcumin 20 μmol/l).

d

52

Fig. 2-3

(A)

(B)

Figure 2-3. Effect of curcumin on the protein levels of ICAM-1 in cultured HUVECs.

HUVECs were pretreated with 10 and 20 μmol/l curcumin 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 curcumin 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-dMeans with different letters are significantly different at p < 0.05.Abbreviation:

C (control), IL-1 (IL-1 β), CU10 (curcumin 10 μmol/l), CU20 (curcumin 20 μmol/l).

ICAM-1

53

Fig. 2-4

(A)

(B)

Figure 2-4. Effect of curcumin on the protein levels of VCAM-1 in cultured HUVECs. HUVECs were pretreated with 10 and 20 μmol/l curcumin 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 curcumin 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-cMeans with different letters are significantly different at p < 0.05.Abbreviation:

C (control), IL-1 (IL-1 β), CU10 (curcumin 10 μmol/l), CU20 (curcumin 20 μmol/l).

VCAM-1

54

Fig. 2-5

(A)

(B)

Figure 2-5. Effect of curcumin on the protein levels of E-selectin in cultured HUVECs. HUVECs were pretreated with 10 and 20 μmol/l curcumin 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 curcumin 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-cMeans with different letters are significantly different at p < 0.05.Abbreviation:

C (control), IL-1 (IL-1 β), CU10 (curcumin 10 μmol/l), CU20 (curcumin 20 μmol/l).

E-selectin

55

Fig. 2-6

Figure 2-6. Effect of curcumin on IL-1 β-induced activation of NF-κB p65.

HUVECs were pretreated with 10 μmol/l and 20 μmol/l curcumin for 18 hours and then induced by IL-1 β (10 ng/ml) for 6 hours. Nuclear extractes were prepared and analyzed for the 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), CU10 (curcumin 10 μmol/l), CU20 (curcumin 20 μmol/l).

d

56

Fig. 2-7

Figure 2-7. Effect of curcumin on IL-1 β-induced activation of NF-κB p50.

HUVECs were pretreated with 10 μmol/l and 20 μmol/l curcumin 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), CU10 (curcumin 10 μmol/l), CU20 (curcumin 20 μmol/l).

d

57

Fig. 2-8

C IL-1

IL-1 + CU 10 IL-1 + CU 20

58

Fig. 2-8

(B)

Figure 2-8. Effect of curcumin on IL-1 β-induced 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 curcumin for 18 hours. (B) HUVECs were pretreated with 10 μmol/l and 20 μmol/l curcumin 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), CU10 (curcumin 10 μmol/l), CU20 (curcumin 20 μmol/l).

c

59

Chapter 3

Experimentation II

Carnosic acid attenuates the expression of adhesion molecules by