Curcumin supplementation could improve diabetes-induced endothelial dysfunction associated with decreased vascular superoxide production and PKC inhibition

Abstract

Background: Curcumin, an Asian spice and food-coloring agent, is known for its anti-oxidant properties. We propose that curcumin can improve diabetes-induced endothelial dysfunction through superoxide reduction.

Methods: Diabetes (DM) was induced in rats by streptozotocin (STZ). Daily curcumin oral feeding was started six weeks after the STZ injection. Twelve weeks after STZ injection, mesenteric arteriolar responses were recorded in real time using intravital fluorescence videomicroscopy. Superoxide and vascular protein kinase C (PKC-βII) were examined by hydroethidine and immunofluorescence, respectively.

Results: The dilatory response to acetylcholine (ACh) significantly decreased in DM arterioles as compared to control arterioles. There was no difference among groups when sodium nitroprusside (SNP) was used. ACh responses were significantly improved by both low and high doses (30 and 300 mg/kg, respectively) of curcumin supplementation. An oxygen radical-sensitive fluorescent probe, hydroethidine, was used to detect intracellular superoxide anion (O2●-) production. O2●- production was markedly increased in DM arterioles, but it was significantly reduced by supplementation of either low or high doses of curcumin. In addition, with a high dose of curcumin, diabetes-induced vascular PKC-βII expression was diminished.

Conclusion: Therefore, it is suggested that curcumin supplementation could improve diabetes-induced endothelial dysfunction significantly in relation to its potential to decrease superoxide production and PKC inhibition.

Figure 1

Ethidium bromide-positive nuclei. Number of ethidium bromide (EB)-positive nuclei from the selected arteriolar wall. The white lines depict the 100-micron vascular length where EB-positive nuclei were counted. (Bar represents 50 μm).

Figure 2

Acetylcholine-induced arteriolar vasodilation. Acetylcholine-induced changes in mesenteric arteriolar diameters from control (con), diabetes (DM) and curcumin-treated groups (DM+cur30, DM+cur300 and con+cur300). Data are means ± SEM (n = 5 for each group). NS, not significant different compared to control arterioles; ** P < 0.01, significant difference compared to control arterioles; †† P < 0.01, significant difference compared to diabetic arterioles.

Figure 3

Sodium nitroprusside-induced arteriolar vasodilation. Sodium nitroprusside-induced changes in mesenteric arteriolar diameter from control (con), diabetes (DM) and curcumin-treated groups (con+cur300, DM+cur30, DM+cur300). Data are means ± SEM (n = 5 for each group). NS, no significant difference compared to control arterioles.

Figure 4

Number of ethidium bromide-positive nuclei. Histogram showing the ethidium bromide-positive nuclei along the mesenteric arterioles of rats that were untreated diabetics (DM), diabetics treated with low curcumin (DM+cur30), diabetics treated with high curcumin (DM+cur300), controls (con) or controls treated with curcumin (con+cur300) rats. Data are expressed as mean ± SEM (n = 5 for each group). NS, no significant difference compared to control arterioles; **P < 0.01 and *P < 0.05, significant difference compared to control arterioles; †† P < 0.01, significant difference compared to diabetic arterioles.

Figure 5

Immunofluorescent staining for PKC-βII. PKC-βII and TRITC signals from immunofluorescent staining of mesenteric arteries. Microvessels with diameter of approximately 100 μm were fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. They were later deparaffinized in xylene and rehydrated in a mixture of ethanol and distilled water. Antigens were unmasked using sodium citrate (10 mmol/L, pH 6.0), followed by exposure to a microwave heat source. Samples were then incubated at room temperature for 60 minutes with anti-PKC-βII at 1:100. Sections were washed in PBS and incubated with swine anti-rabbit IgG-TRITC (1:50 in PBS) for 30 minutes at room temperature. Immunofluorescent staining of small mesenteric arteries displayed a strong signal for PKC-βII in DM rats (Figure 5C). In contrast, the TRITC signals of anti-PKC-βII antibodies were weak in the controls and DM+cur rat vessels (Figure 5 B and D, respectively). The negative control displayed a minimal detectable fluorescence when the secondary antibodies were used alone (Figure 5A).

Figure 6

Relationship between ethidium bromide-positive nuclei and % changes in ACh-induced arteriolar vasodilation. Relationship between EB-positive nuclei per 100 μm vessel length and percentage of ACh-induced change in arteriolar diameter for diabetes (DM), diabetes treated with 30 and 300 mg/kg curcumin (DM+cur30 and DM+cur300, respectively), control (con) and control treated with 300 mg/kg curcumin (con+cur300).