We aim to investigate the effects of curcumin on preventing diabetes-induced vascular inflammation in association with its actions on Txnip, ICAM-1, and NOX2 enzyme expressions. Male Wistar rats were divided into four groups: control (CON), diabetic (DM; streptozotocin (STZ), i.v. 55 mg/kg BW), control-treated with curcumin (CONCUR; 300 mg/kg BW), and diabetes treated with curcumin (DMCUR; 300 mg/kg BW). 12th week after STZ injection, iris blood perfusion, leukocyte adhesion, Txnip, p47phox, and malondialdehyde (MDA) levels were determined by using laser Doppler, intravital fluorescent confocal microscopy, Western Blot analysis, and TBAR assay, respectively. The iris blood perfusion of DM and DMCUR was decreased significantly compared to CON and CONCUR
Diabetes-induced hyperglycemia can disturb the vascular homeostasis that is characterized by decreased vascular blood perfusion, increased vascular permeability, and enhanced vascular inflammation, leading to diabetic vascular complications. Several studies [
Hyperglycemia could induce high-level thioredoxin-interacting protein (Txnip) expression [
Through the other pathway, hyperglycemia-induced ROS can stimulate NF-
Recently, the use of natural plant products has gained more attention among scientists in order to prevent diabetic vascular complications. Curcumin is one of the polyphenol compounds which exhibit antioxidant, anti-inflammatory, antitumorigenic, and antimicrobial properties. With its antioxidant activity, curcumin has direct effect to scavenge free radicals by redox activity [
Based on these reviews, we hypothesized that the curcumin supplementation which is able to reduce leukocytes adhesion may be achieved by its antioxidative effects on Txnip and ICAM-1 expression and also the inhibition of phagocytic/leukocyte action of NOX2-generated
Male Wistar rats (220–250 g) were obtained from National Laboratory Animal Center, Salaya Campus, Mahidol University, Thailand
On the 12th week after STZ injection, the rat was anesthetized with pentobarbital sodium (60 mg/kg BW i.p.), the rats were kept warm at 37°C using warming pad, and a tracheotomy was performed. A jugular vein and carotid artery were cannulated with polyethylene tube for injection of fluorescence tracers and for recording of systolic and diastolic blood pressure, respectively. The blood pressure was measured by using Statham pressure transducer connected to the Polygraph system (Nihon Koden, Japan). Mean arterial blood pressure was calculated from diastolic pressure + 1/3(systolic-diastolic).
The right iris blood perfusion was measured using the Laser Doppler Blood Perfusion Monitoring (Perimed AB, Sweden) with the optic needle probe (0.1 millimeter). The needle probe was fixed perpendicularly about 1 mm above iris. Eight different measurement points of iris around pupil were performed at each time and the mean of iris blood perfusion was determined for each rat.
The iris blood vessel was used to observe instead of retina because the microcirculation in the iris is quite properly to get the good image of leukocyte-endothelial cell interaction and to assess blood perfusion. Besides, the diabetic iridopathy is commonly used clinically to indicate the progression of the diabetic microvascular complications, particularly, in association with advanced proliferative diabetic retinopathy by using fluorescein angiography of the iris [
During the experiment, the real time image of iris blood vessel was recorded by an epi-illumination fluorescence video microscopy system (Optiphot 2, Nikon, Japan) equipped with a 100 W mercury lamp, CCD camera (Hamamatsu C2400, Japan), a video recorder (VC-S5, Sharp, Japan) with a video timer (VTG-33, For-A, Japan), and 20x objective lens (CF Plan Fluor, Nikon, Japan).
Leukocyte adhesion in postcapillary venules of iris was determined by rhodamine-6G (R6G) which can label mitochondria especially in leukocyte (Sigma, USA) at 0.15 mg/kg BW following the method described by Jariyapongskul et al. [
The parameters for metabolic changes were blood glucose and HbA1c. All these were determined at the end of the experiments by collected blood sample from abdominal aorta under anesthesia. Blood glucose and HbA1c were measured using enzymatic method and turbidimetric immunoinhibition method, respectively (Bangkok RIA Laboratory Co., Bangkok, Thailand).
In this study fundus malondialdehyde level (MDA), product of lipid peroxidation, was used as indicator of oxygen free radicals in fundus. Thiobarbituric acid (TBA) assay is a commonly used method to determine malondialdehyde. This method is based on the reaction of TBA and MDA. The lipid material is simply heated with TBA under acidic condition, and the formation of a pink color is measured at or close to 532 nm. The pink color is formed by reaction of one molecule of MDA with two molecules of TBA [
After collecting blood, the eyeball was collected immediately by enucleating from the orbital cavity. Lens and eye fluid were excised from the eye ball; the eyeball without lens and fluids is called fundus. The fundus was washed in phosphate buffer (0.1 M, pH 7.4). Then, the fundus was chopped with fine scissors and was directly in lysis buffer containing 1X RIPA buffer (250
The protein expressions of Txnip, ICAM-1, and p47phox were performed with 80
All data were presented as means ± SEM (standard error of mean). For comparison among groups of rats, two-way analysis of variance (two-way ANOVA) was used and followed by unpaired
As shown in Table
Values are means ± SEM.
Group | BW (g) | MAP (mmHg) |
---|---|---|
12-week CON ( |
424.00 ± 28.28 | 124.77 ± 9.60 |
12-week CONCUR ( |
480.67 ± 17.26 | 132.58 ± 5.50 |
12-week DM ( |
|
|
12-week DMCUR ( |
|
|
Blood glucose levels (BG) of DM group and DMCUR groups were significantly elevated as compared to CON group and CONCUR group (
Plasma glycosylated hemoglobin levels of DM group and DMCUR group were significantly elevated as compared to CON group and CONCUR group (
Values are means ± SEM.
Group | BG (mg/dL) | Hemoglobin A1c (%) |
---|---|---|
12-week CON ( |
|
|
12-week CONCUR ( |
|
|
12-week DM ( |
|
9.47±0.47** |
12-week DMCUR ( |
|
|
By using laser Doppler flowmetry, the regional iris blood perfusion of each rat was evaluated from eight different points measured around the pupil as described previously. Mean regional iris blood perfusion in each group was summarized in Figure
The effects of curcumin on mean regional iris blood perfusion in each group. Control = CON, control treated with curcumin = CONCUR; curcumin 300 mg/kg BW, started at 10 days after STZ injection, diabetic = DM; streptozotocin (STZ), i.v. 55 mg/kg BW, diabetes treated with curcumin = DMCUR; curcumin 300 mg/kg BW. Data are means ± SEM (
The number of leukocytes was counted as adherent one that remained stationary for equal to or longer than 30 seconds. The leukocyte adhesion was counted per field of view totally of postcapillary venule (diameter 20–30
In the present video microscopic visualization showed clear image of leukocyte adhering to the endothelium of postcapillary venule in the different monitored views of iris of each rat (Figure
Intravital microscopic demonstration of leukocyte adhesion in the postcapillary venule of iris in CON group (a), CONCUR group (b), DM group (c), and DMCUR group (d). White dots represent leukocyte stained by rhodamine-6G i.v. injection. (Scale bar represents 100
The effects of curcumin on leukocyte-endothelium interaction. Data are means ± SEM (
The number of leukocyte adhesions was significantly increased in DM group as compared to CON group and CONCUR group (
In this study fundus malondialdehyde level (MDA), product of lipid peroxidation, was used as indicator of oxygen free radicals in fundus. MDA level was significantly elevated in DM group as compared to CON group and CONCUR group (
The effects of curcumin on fundus MDA level. Control = CON, control treated with curcumin = CONCUR; curcumin 300 mg/kg BW, started at 10 days after STZ injection, diabetic = DM; streptozotocin (STZ), i.v. 55 mg/kg BW, diabetes treated with curcumin = DMCUR; curcumin 300 mg/kg BW. Data are means ± SEM (
The level of Txnip was significantly elevated in DM group and DMCUR group as compared to CON group (
The effect of curcumin on Txnip expression. Control = CON, control treated with curcumin = CONCUR; curcumin 300 mg/kg BW, started at 10 days after STZ injection, diabetic = DM; streptozotocin (STZ), i.v. 55 mg/kg BW, diabetes treated with curcumin = DMCUR; curcumin 300 mg/kg BW. Data are means ± SEM (CON and DMCUR groups;
The level of ICAM-1 was significantly elevated in DM group as compared to CON group (
The effect of curcumin on ICAM-1 expression. Control = CON, control treated with curcumin = CONCUR; curcumin 300 mg/kg BW, started at 10 days after STZ injection, diabetic = DM; streptozotocin (STZ), i.v. 55 mg/kg BW, diabetes treated with curcumin = DMCUR; curcumin 300 mg/kg BW. Data are means ± SEM (
The level of p47phox was significantly elevated in DM group as compared to CON group and CONCUR group (
The effect of curcumin on p47phox expression. Control = CON, control treated with curcumin = CONCUR; curcumin 300 mg/kg BW, started at 10 days after STZ injection, diabetic = DM; streptozotocin (STZ), i.v. 55 mg/kg BW, diabetes treated with curcumin = DMCUR; curcumin 300 mg/kg BW. Data are means ± SEM (CON, CONCUR, and DM groups;
From Figure
The association between the biochemical and functional data parameters was analyzed by using two-tailed Pearson’s correlation analysis with
This experiment demonstrated that curcumin supplementation can attenuate leukocyte-endothelium interaction in association with the decreased ROS overproduction. In addition, curcumin can also suppress NOX2 enzyme expression in vascular inflammatory process in diabetic rats. However, the curcumin supplementation (300 mg/kg BW) could not show the significant inhibition of the hyperglycemia-enhanced Txnip expression when compared to the DM group.
From the previous studies, hyperglycemia was reported as an important factor to induce Txnip expression to increase. Moreover, the hyperglycemia-enhanced Txnip expression has been noted to play a critical role in progression of diabetic vascular complications as well [
It is noted that Txnip expression in DMCUR group has tended to slightly reduce down to 14.46% compared to DM group (Figure
In addition to hyperglycemia-regulated Txnip overexpression, hyperglycemia also can activate many sources to generate ROS such as mitochondria electron transport chain, NADPH oxidase, xanthine oxidase, endothelial NO synthase, and cytochrome p450 [
The experiments confirmed that hyperglycemia promotes ROS overproduction resulting oxidative stress to damage macromolecules such as lipid which is in apparently the increasing of fundus MDA level in DM group. However, the fundus MDA level in DMCUR group was significantly decreased down to 28% as compared to DM group (
Both Txnip expression and ROS overproduction could impair endothelial function that causes vascular homeostasis alteration including the abnormal vascular blood flow resistance. In this experiment, blood perfusion in iris was chosen to represent one of the alterations of vascular homeostasis. The results showed that iris blood perfusion (IBP) in DM and DMCUR groups was significantly decreased as compared to CON group, though IBP in DMCUR had tended to increase up to 4.78% as compared to DM group. However, these results did not show the significant difference similar to the previous studies [
Furthermore, our results have shown that the low dose of curcumin supplementation could significantly decrease ICAM-1 expression down to 52.17% and the number of leukocyte-endothelium interactions was down to 55.68% as compared to the CON group (Figures
From Figure
Our findings indicated that the p47phox expression in DMCUR group was significantly decreased down to 41.38% as compared to DM group. At this point, it may imply that when curcumin could decrease ICAM-1 and NOX2 enzyme expressions, it will inhibit the leukocyte adhesion and prevent vascular inflammation in consequence. Moreover the data from our study also demonstrated the correlation between the plasma MDA level and the endothelial functions significantly (Figure
In summary, our experiments demonstrated that low dose curcumin supplementation (300 mg/kg BW) can ameliorate diabetic vascular inflammation through the decrease in ROS overproduction, reducing leukocyte-endothelium interaction associated with decreased ICAM-1 and NOX2 enzyme expressions in diabetic rat model. Therefore the findings of this study should be a good evidence to suggest that the low dose of curcumin might offer a novel therapeutic strategy for treatment of the diabetic retinopathy associated with vascular inflammation in the future.
Diabetes mellitus
Iris blood perfusion
Intercellular adhesion molecule 1
Malondialdehyde levels
Mean arterial blood pressure
NADPH oxidase enzyme
Rhodamine-6G
Region of interest
Reactive oxygen species
Streptozotocin
Superoxide
Thioredoxin-interacting protein.
The authors have no conflict of interests to report.
This study was supported by Ratchadapiseksompotch Fund of the Faculty of Medicine and the 90th Anniversary Research Fund, the Graduate School, Chulalongkorn University, Thailand.