Curcumin could ameliorate diabetic vascular inflammation by decreasing ROS overproduction, leukocyte-endothelium interaction, and NOX2 expression Natchaya Wongeakin1, Parvapan Bhattarakosol2, Suthiluk Patumraj1 1Center of Excellence for Microcirculation, Department of Physiology, 2Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand . Email : natchayaww@gmail.com Abstract: Relative ICAM-1 expression (A) ICAM-1 expression ICAM-1 Β-actin DM DMCUR (B) ** The present study aimed to study the mechanisms of curcumin on preventing diabetes-induced leukocyte-endothelium interaction in association with its actions on NOX2 enzyme and Txnip expressions. Male Wistar rats were divided into three groups: control-treated with corn oil group (CON), diabetic-treated with corn oil group (DM; streptozotocin (STZ), i.v. 55 mg/kg BW), and diabetes-treated with curcumin (DMCUR; 300mg/kgBW started at 10 days after STZ-injection). The results showed that plasma glucose and HbA1c of DM and DMCUR were increased significantly as compared to CON (P < 0.001). Interestingly, the leukocyte adhesion, p47phox expression, and MDA level in DM were increased significantly as compared to CON (P < 0.05), but decreased significantly when compared to DMCUR (P < 0.05). It is concluded that curcumin could ameliorate diabetic vascular inflammation by decreasing ROS overproduction, reducing leukocyte-endothelium interaction, and also inhibiting NOX2 activation. ## Fig 2. The effect of curcumin on ICAM-1 expression in fundus extract of each group. A: The results are expressed as mean  SEM. B: The level of ICAM-1 was examined by Western Blot analysis. ** P < 0.001 compared with CON, ## P < 0.001 compared with DM Txnip expression (A) Relative Ttxnip expression (B) Introduction: CON DM * Recently, thioredoxin-interacting protein (Txnip) expression [1] and the activated NADPH oxidase enzyme (NOX2) are reported for developing ROS overproduction and causing vascular inflammation in diabetes [2]. From our previous studies [3-4], we found that curcumin could protect endothelial dysfunction closely related to the decreased ROS. Therefore, the present study was designed to further investigate the antioxidant effect of curcumin on prevention of diabetes-induced vascular inflammation in association with its actions on Txnip expressions and NOX2 enzyme activation. Txnip Β-actin Design and Methods: Fig 3. The effect of curcumin on Txnip expression in fundus extract of each group. A: The results are expressed as mean  SEM. B: The level of Txnip was examined by Western Blot analysis. * P < 0.05 compared with CON CON (Corn oil, n = 5) Male Wistar rats (Age 6 – 8 weeks) DM (Streptozotocin i.v.55mg/kgBW, n = 5) Relative p47phox expression * p47phox expression (A) (B) DMCUR (Curcumin 300mg/kgBW, n = 5) DM DMCUR # 12 weeks p47phox Iris blood perfusion (Laser doppler flowmeter) Iris leukocyte adhesion(Intravital fluorescent microscope [3]) Eye fundus MDA (TBARs assay) Txnip and p47phox expressions (Western Blot analysis) The assessment of Txnip and p47phox expression were determined by digital image software (Image J, National Institutes of Health (NIH), USA). Β-actin Fig 4. The effect of curcumin on p47phox expression in fundus extract of each group A: The results are expressed as mean  SEM. B: The level of p47phox was examined by Western Blot analysis. * P < 0.05 compared with CON, # P < 0.05 compared with DM Results: Table 1: This table shows the effects of curcumin on the physiological characteristics, biochemical parameters, lipid peroxidation, and hemodynamic changes Relative Txnip expression Relative p47phox expression Groups CON DM DMCUR Body weight (g) 424.0028.28 271.6713.22** 258.0017.64** MAP (mmHg) 124.779.60 113.566.98 112.804.99 Blood glucose (mg/dL) 172.8311.08 410.3316.77** 390.3311.59** HbA1c (%) 4.000.06 9.470.47** 9.300.21** Eye fundus MDA level (nmole/protein100µg) 5.171.05 11.690.22** 8.360.63# Mean Iris blood perfusion (AU) 870.2739.61 246.0026.99** 311.2831.13** Blood glucose (mg/dL) Leukocyte adhesion (cells/frame) Fig 5. The correlation of means blood glucose and means Txnip expression Fig 6. The correlation of means leukocyte-endothelium interaction and means p47phox expression Conclusion: ** P < 0.001 significant as compared to CON, # P < 0.05 significant as compared to DM In summary, our experiments demonstrated that curcumin supplementation (300mg/kgBW) can ameliorate diabetic vascular inflammation through decreasing ROS overproduction, reducing leukocyte-endothelium interaction associated with NOX2 enzyme activity suppression in diabetic rat model. Therefore our knowledge of this study should be a good evidence to suggest that NOX2 activitation is the important factor in diabetes-induced vascular inflammatory reactions (A) (B) Fig 1. The effect of curcumin on iris leukocyte-endothelium interaction in each group. A: The results are expressed as mean  SEM. B: Intravital microscopic demonstration of leukocyte adhesion in the postcapillary venule of iris in each group. ** P < 0.001 compared with CON, # P < 0.05 compared with DM Leukocyte adhesion (cells/frame) CON DM DMCUR 100 µm ** # leukocyte-endothelium References: 1. Shulze PC, et al. J Biol Chem. 2004; 279:30369-30374. 2. Omori K, et al. J Leukoc Biol. 2008;84:292-301. 3. Patumraj S, et al. Clin Hemorheol Microcirc. 2006;35(4):481-9. 4. Wongeakin N, et al. Journal of Vascular Research2006;43:32. Acknowledgements: The authors would like to acknowledge The Physiological Society for a travel grant to attend this meeting. Also this work was supported Ratchadapiseksompotch Research Fund, Faculty of Medicine, Chulalongkorn University.