Methylglyoxal (MGO) is a highly reactive metabolite of glucose which is known to cause damage and induce apoptosis in endothelial cells. Endothelial cell damage is implicated in the progression of diabetes-associated complications and atherosclerosis. Hypericin, a naphthodianthrone isolated from
Hypericin, a powerful naturally occurring photosensitizer, is produced by
Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is an intermediate product formed during glycation of proteins by glucose and its formation involves many pathways consisting of enzymatic and non-enzymatic reactions (Yim
MGO and 2′,7′-dichlorofluorescein diacetate (DCF-DA) were obtained from Sigma (St. Louis, MO, USA). Hypericin was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Endothelial growth medium (EGM-2) was purchased from Lonza (Walkersville, MD, USA). p38, phospho-p38, ERK1/2, phospho-ERK1/2, JNK, and phospho-JNK were purchased from Cell Signaling Technology (Danvers, MA, USA). Bcl-2, Bax and p53 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
The human umbilical vein endothelial cell line (HUVECs, Lot # 60319874) was purchased from the American Type Culture Collection (ATCC, VA, USA). HUVECs were cultured under standard cell culture conditions (37°C in a humidified incubator containing 5% CO2) in EGM-2 supplemented with 4% FBS. The passage number of all the cells used was between 5 and 8.
Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HUVECs were seeded in 96-well plates at 1.0×104 cells/well and incubated for 24 h at 37°C. The cells were then pretreated with hypericin for 1 h, followed by treatment with MGO for 24 h. Morphological changes in the HUVECs were observed with an IncuCyte ZOOM imaging system (Essen Bioscience, MI, USA) 24 h after incubation with MGO. MTT solution was added at a final concentration of 0.1 mg/ml. This was followed by a 2 h incubation in the CO2 incubator at 37°C. The medium was gently removed and the formazan crystals were dissolved in 100 μl/well dimethyl sulfoxide. The absorbance at 570 nm was measured using a microplate reader (Molecular Devices, CA, USA).
DCF-DA probe was used to measure the intracellular ROS scavenging activity of hypericin. Briefly, 2.0×105 cells were seeded in a 12-well plate and incubated overnight at 37°C. After 24 h, cells were pre-incubated with hypericin for 30 min, followed by incubation with MGO for 1 h. The medium was removed and the cells were washed with PBS. Then, medium containing 10 μM DCF-DA was added for 20 min at 37°C. After washing with PBS, cells were photographed using a JuLI live-cell imaging system (NanoEnTek, Seoul, Korea). The fluorescence intensity was assessed using the computer software program Image J software (NIH, Bethesda, MD, USA) not by human eyes, to avoid potential subjective errors.
Changes in the levels of proteins related to MAPKs and apoptosis in HUVECs were evaluated by Western blotting. HUVECs were lysed in PRO-PREPTM (iNtRON Biotechnology, Seongnam, Korea) containing phosphatase inhibitor. Total protein concentration was determined using the Bradford assay. Samples containing equal amounts of proteins were separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. Blots were blocked in 5% skim milk for 1 h at room temperature and exposed to the primary antibodies overnight at 4°C. They were then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Densities of the resulting bands were detected with ECL reagents using a ChemiDoc XRS+imaging system (Bio-Rad, CA, USA).
The AGEs formation assay was used to investigate inhibition of protein glycation; the protocol was slightly modified from that published by Kiho
Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Values are given as mean ± S.D. Statistical analysis of results was performed using one-way ANOVA followed by Bonferroni’s test. A
MGO-induced cell morphological changes in HUVECs after treatment with hypericin were observed. As shown in Fig. 1A, the percentage of apototic cells was increased (43%) in HUVEC incubated for 24 h in the EGM-2 media containing 400 μM MGO. In addition to decrease in cell number, typical morphological features such as shrinkage, fragmentation and rounding of the cell were observed at this time. But, treatment with hypericin rescued the morphological changes such as a loss of confluence and decreased the number of floating cell fragments induced by MGO. To determine the effect of hypericin on MGO-induced cytotoxicity in HUVECs, we performed an MTT assay. HUVEC cell viability was significantly reduced after MGO treatment, but hypericin treatment restored cell viability (Fig. 1B). Moreover, pretreatment with 0.5 μM hypericin increased cell viability.
It is known that MGO can increase intracellular ROS levels and may induce cell death. Therefore, we measured ROS formation in HUVECs following treatment with MGO. We also estimated the inhibitory effect of hypericin on ROS formation in HUVECs following treatment with MGO. We measured the antioxidative effect of hypericin using DCF-DA. As shown in Fig. 1C, we found that pretreatment with hypericin significantly reduced ROS generation in MGO treated HUVECs.
Next, we investigated whether MGO can affect expression of apoptosis related proteins such as. Bcl-2, Bax and p53 in HUVECs. As shown in Fig. 1D, treatment with MGO decreased Bcl-2 protein expression, but enhanced protein expressions of Bax and p53 in HUVECs. Also, treatment with hypericin has downregulated the expression of Bax and p53 and upregulated the expression of Bcl-2. These data suggest that hypericin may prevent MGO-induced apoptosis in HUVECs.
Activation of MAPK plays an important role in MGO-induced apoptosis in various types of cells. In this study, we investigated three proteins in MAPK subfamilies, p38, ERK1/2, and JNK, in MGO-treated HUVECs, and evaluated the effect of hypericin on activation of MAPK cascades induced by MGO. As shown in Fig. 2, there was a significant increase in p38, ERK1/2, and JNK activation in MGO-treated cells, whereas pretreatment with hypericin prior to MGO stimulation reduced the activation of all three MAPKs.
The AGEs formation assay was used to measure fluorescence at an excitation wavelength of 355 nm and an emission wavelength of 460 nm; aminoguanidine was used as a positive control. As shown in Table 1, we found that formation of AGEs was significantly increased in cells treated with BSA-MGO. Hypericin (10 μM) significantly attenuated the formation of AGEs. However, 1 μM hypericin did not produce a significant difference in AGEs formation.
MGO is one of the most significant reactive carbonyl species formed by the triose phosphate glycolytic intermediates of glucose metabolism (Lo
It is well known that intracellular ROS is generated in vascular endothelial cells in response to MGO treatment (Figarola
Alteration of the ratio of the expression of one or more members of the Bcl-2 protein family is significant in determining whether apoptosis occurs, because Bcl-2 family member proteins play critical roles in regulating the process of apoptosis (Murphy
In endothelial cells, MGO may induce cytotoxicity via activation of several key molecules including ERK, JNK, and p38, which are involved in the MAPK signaling pathway (Akhand
To investigate the protective effect of hypericin against MGO-induced apoptosis, we performed annexin V-FITC/PI assay using flow cytometry. However, PI baseline was shifted to the necrosis zone, since hypericin is a red-colored naphthodianthrone derivative. So we could not get the reliable annexin V-FITC/PI assay data. However, the proteins expressions of Bcl-2, Bax, p53 and MAPKs by treatment of MGO were changed. Furthermore, many previously studies reported that MGO-mediated diabetic vascular complications might be due to apoptosis in endothelial cells (Phalitakul
MGO, a dicarbonyl compound, is more reactive than are reducing sugars such as glucose. This highly reactive compound is a major precursor in the formation of AGEs (Desai and Wu, 2007). One of the major AGEs adducts, Nε-(carboxymethyl) lysine, is formed from MGO (Thornalley, 1996). In endothelial cells, increased levels of AGEs cause oxidative stress, mitochondrial dysfunction, cellular dysfunction, and, ultimately, cell death (Li
In conclusion, our results support the notion that hypericin protects against MGO-induced apoptosis in cultured HUVECs via scavenging of ROS and reducing the activation of cell apoptosis via the MAPK pathway. Therefore, our results suggest that hypericin may be a useful tool to prevent or reverse MGO-induced vascular damage.
This research was supported by iPET (Korean Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry of Agriculture, Food and Rural Affairs (NO. 115045-3).
This research was supported by High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (114006041HD020).