Baicalein is an available anti-atherosclerotic compound through modulation of nitric oxide-related mechanism under oxLDL exposure

Abstract

OxLDL facilitate reactive oxygen species (ROS) formation and up-regulation of the executioner caspase-3 via the mitochondrial apoptotic pathway involves several critical steps in human endothelial cells. Previous studies reported that oxLDL-facilitated endothelial oxidative stress is associated with impairment of eNOS and up-regulation of inducible nitric oxide synthase (iNOS). Baicalein is the most abundant component that has anti-HIV, anti-tumor, anti-oxidant and free radical scavenging functions. In this present study, we shown that baicalein hinibits oxLDL-caused endothelial dysfunction through suppression of endothelial inflammation and oxidative stress that causes to cellular apoptosis. Specifically, baicalein reduces the elevation of ROS concentration, which subsequently inhibits the oxLDL-decreased expression of anti-oxidant enzymes, enriches the bioavailability of NO, stabilizes the mitochondrial membrane, thereby inhibiting the discharge of cytochrome c from mitochondria, a molecule required for the activation of the pro-apoptotic protein caspase 3. However, inhibition of eNOS impairs the anti-apoptotic and anti-inflammatory effects of baicalein. These results provide new insight into the possible molecular mechanisms by which baicalein protects against atherogenesis by NO-related pathways.

Keywords: Gerotarget; baicalein; endothelial cells; nitric oxide; oxidized low-density lipoprotein; reactive oxygen species.

Figure 1. Effects of baicalein on oxLDL-induced endothelial cell death

HUVECs were incubated with oxLDL (150 μg/ml) in the absence or presence of indicated concentrations of baicalein. Photomicrographs from phase-contrast microscopy A. Viability was determined via MTT assay B. and LDH release C. HUVECs were incubated with oxLDL in the absence (middle) or presence (right) of baicalein for 24 h. Late apoptotic death of oxLDL-exposed HUVECs was evaluated using the TUNEL assay D., E. The values represent means±SEM from three separate experiments. #P < 0.05 vs. control; *P < 0.05 vs. oxLDL treatment.

Figure 2. The protective effects of baicalein on oxLDL-mediated ROS generation in HUVECs

After preincubation for 2 hrs with the indicated concentrations of baicalein (2.5-20 μM), HUVECs were incubated with the DHE for 1 hr, followed by treatment with oxLDL. A. Fluorescence images exhibited the ROS level in control cells (left) and HUVECs stimulated with oxLDL (middle) in the presence of baicalein (right). B. Fluorescence intensity of HUVECs was measured with a fluorescence microplate reader. Fluorescence distribution of DHE oxidation was expressed as a percentage of increased intensity. The activity of C. SOD and D. catalase in HUVECs stimulated with oxLDL in the absence or presence of indicated concentrations of baicalein were determined. E. HUVECs were incubated with oxLDL (150 μg/ml) in the absence or presence of baicalein. In some groups, SOD inhibitor (DDTC) was treated before expose to oxLDL and baicalein. Viability was determined via MTT assay. Data are expressed as the mean±S.E. of three independent analyses. #P < .05 vs. untreated control; *P < 0.05 vs. oxLDL treatment. &P < .05 vs. oxLDL+ baicalein treatment.

Figure 3. Effects of baicalein on oxLDL up-regulated iNOS, down-regulated eNOS, nitrotyrosine protein expression and oxLDL-enhanced nitrite accumulation

The protein expression of iNOS, eNOS A. and nitrotyrosine D. were analyzed by Western blot pretreated with baicalein (20 μM) for 2 hours followed by oxLDL(150 μg/ml) for 24 hours in HUVECs. Anti-β-actin antibody was used for normalization of cytosolic proteins. E. Content of NO was assayed using Griess reagent. Data of bar figure represent mean±SEM of 3 independent analyses. # P < 0.05 compared with control and *P < 0.05 compared with oxLDL-stimulated HUVECs.

Figure 4. Effects of baicalein on oxLDL-induced NF-κB activation

HUVECs were pretreated with each inhibitor 1 hr before incubated with oxLDL. Nucleic proteins were extracted for nuclear translocation assay of NF-κBp65 A. Effects of baicalein on COX-2 levels were assessed by Western blotting. B., C. The values represent means±S.E. from three separate experiments. #P < .05 vs. untreated control; *P < 0.05 vs. oxLDL treatment. &P < .05 vs. oxLDL+ baicalein treatment.

Figure 5. Effect of baicalein on mitochondrial transmembrane permeability transition and apoptosis

Cells were incubated with 20 μM baicalein for 2 hours and then incubated with 150 μg/ml oxLDL for an additional 24 hours. A. The change in mitochondrial membrane potential was assessed based on the signal intensity from monomeric and J-aggregate JC-1 fluorescence as described in materials and methods. (left) No treatment; (middle) oxLDL; and (right) oxLDL + baicalein. B. JC-1 fluorescence was confirmed by flow cytometry. Green fluorescence intensity indicates the cells with low ΔΨm, while the red fluorescence intensity indicated the cells with stable ΔΨm. Effects of baicalein on dephosphorylation of p53, analysis of Bcl-2 family proteins, and mitochondrial cytochrome c release in response to oxLDL and baicalein. HUVECs were incubated with 150 mg/ml oxLDL in the absence or presence of indicated concentrations of baicalein for 24 hrs. Representative, Western blots C. and summary data D.,E.,F. show that oxLDL upregulated pro-apoptotic (Bax) proteins and downregulated anti-apoptotic (Bcl-2) proteins and increased the abundance of cytochrome c in the cytosolic fraction, whereas baicalein pretreatment suppressed these apoptosis-provoking alterations. Results were subjected to densitometric analysis. Data of bar figure represent means±SEM of 3 independent analyses. # P < 0.05 compared with control and *P < 0.05 compared with oxLDL-stimulated HUVECs.

Figure 6. Effects of baicalein on oxLDL-induced caspase 3 activation

A. HUVECs were incubated for 1 hr with indicated concentrations of baicalein, followed by exposure to oxLDL for another 24 hrs. Fluorescent images show the activated caspase 3 level in control cells (left), HUVECs stimulated with oxLDL (middle), and in the presence of baicalein (right). B. Fluorescence intensity of cells was measured by flow cytometry. C.The activity of caspase 3 was measured by EnzCaspase-3 assay kit. Data are expressed as the mean±S.E. of three independent analyses. #P < .05 vs. untreated control; *P < .05 vs. oxLDL treatment. &P < .05 vs. oxLDL+ baicalein treatment.