Isosamidin from Peucedanum japonicum Roots Prevents Methylglyoxal-Induced Glucotoxicity in Human Umbilical Vein Endothelial Cells via Suppression of ROS-Mediated Bax/Bcl-2

Methylglyoxal (MGO) is a highly reactive metabolite of glucose. Elevated levels of MGO induce the generation of reactive oxygen species (ROS) and cause cell death in endothelial cells. Vascular endothelial cell damage by ROS has been implicated in the progression of diabetic vascular complications, cardiovascular diseases, and atherosclerosis. In this study, the protective effect of isosamidin, isolated from Peucedanum japonicum roots, on MGO-induced apoptosis was investigated using human umbilical vein endothelial cells (HUVECs). Among the 20 compounds isolated from P. japonicum, isosamidin showed the highest effectiveness in inhibiting MGO-induced apoptosis of HUVECs. Pretreatment of HUVECs with isosamidin significantly prevented the generation of ROS and cell death induced by MGO. Isosamidin prevented MGO-induced apoptosis in HUVECs by downregulating the expression of Bax and upregulating the expression of Bcl-2. MGO treatment activated mitogen-activated protein kinases (MAPKs), such as p38, c-Jun N terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). In contrast, pretreatment with isosamidin strongly inhibited the activation of p38 and JNK. Furthermore, isosamidin caused the breakdown of the crosslinks of the MGO-derived advanced glycation end products (AGEs). These findings suggest that isosamidin from P. japonicum may be used as a preventive agent against MGO-mediated endothelial dysfunction in diabetes. However, further study of the therapeutic potential of isosamidin on endothelial dysfunction needs to explored in vivo models.


Extraction and Isolation
The extract of P. japonicum roots and its isolated twenty compounds were obtained and verified by Dr. Jinwoong Kim (Seoul National University). It was prepared as per the protocol of Kim et al. [24]. Firstly, the dried roots of P. japonicum (30.0 kg) were extracted with MeOH at room temperature (25 • C) in an ultrasonicator and filtered. Afterwards, the MeOH extract was partitioned with n-hexane, CHCl 3 , EtOAc, n-BuOH, and residual aqueous fractions. Kim et al. isolated twenty compounds from each subfraction according to the general analytical methods.

Cell Culture
HUVECs were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA; lot # 60319874) and maintained in EGM-2 medium, supplemented with 2% fetal bovine serum (FBS), at 37 • C in a humidified incubator containing 5% CO 2 . All cells used for the experiments were between passage number 5 and 8.

Cell Viability Analysis
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate cell viability. HUVECs were seeded at a density of 1.0 × 10 4 cells/well in 96-well plates; they were then treated with the test compounds for 1 h, followed by treatment with MGO for 24 h. Subsequently, the cells were incubated for 24 h at 37 • C. After incubation, the media containing the compounds was removed and the MTT solution was added; the cells were then incubated in a CO 2 incubator for 2 h at 37 • C. After removing the media, 100 µL of dimethyl sulfoxide was added to each well. Subsequently, the absorbance was measured at 570 nm using a VERSA max microplate reader (Molecular Devices, CA, USA).

LDH Production Assay
The LDH production was evaluated using the Pierce LDH cytotoxicity assay kit (Thermo Scientific, Waltham, MA, USA) according to protocol with as per manufacture's' instructions. HUVECs were seeded at a density of 1.0 × 10 4 cells/well in 96-well plates and incubated for 24 h, then treated with the isosamidin for 1 h, followed by treatment with MGO for 24 h at 37 • C. After incubation, the conditioned medium (50 µL) from the treated plates was transferred to a 96-well plate. Next, 50 µL of the substrate mixture was added to the 96-well plate. The plates were mixed by placing the plate on a shaker for 30 min in the dark. The absorbance was evaluated at 490 nm and 680 nm using a VERSA max microplate reader (Molecular Devices, San Jose, CA, USA).

Cell Apoptosis Assay
The effect of isosamidin on MGO-induced apoptosis in the HUVECs was analyzed by flow cytometry (FACSCalibur flow cytometer; Becton Dickinson, San Jose, CA, USA) using an Annexin V apoptosis detection kit (Santa Cruz Biotechnology, Dallas, CA, USA). Briefly, the cells were seeded at a density of 5.0 × 10 5 cells/well in a 6-well plate and incubated overnight at 37 • C. The cells were then treated with MGO (400 µM) and isosamidin (10 µM) for 24 h. Subsequently, the cells were harvested with trypsin-EDTA and washed with phosphate-buffered saline (PBS). The cells were then re-suspended in binding buffer with annexin V-FITC and propidium iodide (PI) and incubated at room temperature (25 • C) for 15 min in the dark. After incubation, 500 µL of PBS was added, and the percentage of apoptotic cells was analyzed by flow cytometry.

Measurement of Intracellular ROS
To determine the intracellular ROS scavenging activity of isosamidin, we used DCF-DA. HUVECs were seeded at a density of 1.0 × 10 5 cells/well in a 12-well plate and incubated for 24 h at 37 • C. The cells were pretreated with isosamidin (10 µM) for 1 h, followed by treatment with MGO for 2 h. The cells were then washed with PBS and incubated with DCF-DA (10 µM) for 30 min at 37 • C. Subsequently, the distribution of DCF fluorescence was photographed using a JuLI live-cell imaging system (NanoEnTek, Seoul, Korea).

Measurement of AGE Breakdown
We performed the TNBSA (2,4,6-trinitrobenzene sulfonic acid) assay to evaluate the ability of the isosamidin to cause the breakdown of MGO-AGEs according to the method described by Furlani et al. [30]. We mixed a 1 mg/mL sample of preformed MGO-AGEs solution with 100 µM and 400 µM of isosamidin. This mixture was then incubated at 37 • C for 24 h. After incubation, 4% NaHCO 3 (pH 8.5) and 0.1% TNBSA were added to each ep-tube for 2 h, at 37 • C; subsequently, the relative increase in the concentration of free amines was measured using a VERSA max microplate reader at 340 nm (Molecular Devices, CA, USA).

Western Blotting
To measure the protein the changes related to apoptosis and MAPKs, we performed Western blotting experiments in the HUVECs. After harvesting, the cells were lysed using PRO-PREP TM (iNtRON Biotechnology, Seongnam, Korea), containing phosphatase and protease inhibitors. The concentration of total protein extracts was measured using the Bradford assay. Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 1 h with 5% skim milk at room temperature and then incubated overnight with tubulin, p38, p-p38, JNK, p-JNK, ERK, p-ERK, Bcl-2, Bax, GLO-I, and Nrf2 primary antibodies at 4 • C. After 24 h, the membranes were conjugated for 1 h with secondary antibody at room temperature. additionally, washed and detected with TBST and ECL reagent (Millipore, Burlington, MA, USA). The bands were detected using a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA).

Statistical Analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA) in GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA), followed by a Bonferroni's post-hoc test. All data values were presented as the mean ± standard deviation (SD). A p-value < 0.05 was considered statistically significant.

Effect of Isosamidin on MGO-Induced Cell Death in HUVECs
To select the P. japonicum-derived compounds with the most protective effects against MGO-induced toxicity, we performed cell viability screening of 20 compounds (10 µM each) using the MTT assay. As it exhibited the highest cytoprotective activity, isosamidin was selected ( Table 1). The structure of the isosamidin is shown in Figure 1A. We investigated the effects of isosamidin on the viability of the HUVECs using the MTT assay. HUVECs were pretreated with 1, 5, and 10 µM of isosamidin for 1 h and then treated with 400 µM of MGO for 24 h. The MGO treatment markedly reduced the viability of the HUVECs, whereas pretreatment with isosamidin reversed this effect ( Figure 1B). In fact, pretreatment with isosamidin with a concentration of 1-10 µM increased the cell viability in a dose-independent manner. However, the other compounds tested had no effect on the cell viability of the HUVECs (Table 1). In addition, MGO (400 µM) treatment significantly increased the Antioxidants 2020, 9, 531 5 of 14 LDH production in the HUVECs. LDH production significantly decreased in several concentrations (5 and 10 µM) of isosamidin ( Figure 1C).  123 The percentage cell viability of each compound is presented as the mean ± SD of three independent experiments (*** p < 0.001 vs. MGO 400 µM).
Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 14 Table 1. Effect of the compounds isolated from the roots of P. japonicum on methylglyoxal (MGO)induced glucotoxicity in human umbilical vein endothelial cells (HUVECs).

Effect of Isosamidin on the Crosslinks in AGEs
The anti-glycation activity of isosamidin was measured using the TNBSA assay. Isosamidin (100 µM and 400 µM) significantly exhibited breaking activity of the MGO-BSA-AGE crosslink. As shown in Figure 1D, isosamidin showed 4.64% crosslink breaking abilities at a concentration of 400 µM.

Effect of Isosamidin on MGO-Induced Apoptosis in HUVECs
To examine whether isosamidin could reduce the MGO-induced cell apoptosis, we performed FACS analysis, using annexin V-FITC and PI double staining. As shown in Figure 2A,B, MGO treatment led to an increase in the number of early and late apoptotic cells. However, the MGO-induced increase in early and late apoptotic activity was decreased after pretreatment with isosamidin.

Effect of Isosamidin on the Crosslinks in AGEs
The anti-glycation activity of isosamidin was measured using the TNBSA assay. Isosamidin (100 µM and 400 µM) significantly exhibited breaking activity of the MGO-BSA-AGE crosslink. As shown in Figure 1D, isosamidin showed 4.64% crosslink breaking abilities at a concentration of 400 µM.

Effect of Isosamidin on MGO-Induced Apoptosis in HUVECs
To examine whether isosamidin could reduce the MGO-induced cell apoptosis, we performed FACS analysis, using annexin V-FITC and PI double staining. As shown in Figure 2A,B, MGO treatment led to an increase in the number of early and late apoptotic cells. However, the MGOinduced increase in early and late apoptotic activity was decreased after pretreatment with isosamidin.

Effect of Isosamidin on the Levels of Bax and Bcl-2
Western blotting was used to examine whether isosamidin could alter the MGO-induced expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins in HUVECs. As shown in Figure  2C-E, we observed that, compared to the control cells, MGO treatment resulted in a decrease in the

Effect of Isosamidin on the Levels of Bax and Bcl-2
Western blotting was used to examine whether isosamidin could alter the MGO-induced expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins in HUVECs. As shown in Figure 2C-E, we observed that, compared to the control cells, MGO treatment resulted in a decrease in the expression of the Bcl-2 protein and an increase in the expression of the Bax protein. In contrast, treatment with isosamidin led to a decrease in the levels of Bax and an increase in those of Bcl-2.

Effect of Isosamidin on MGO-Induced ROS Generation
The apoptosis of endothelial cells by intracellular ROS generation is a well-known phenomenon. Considering this, we further investigated whether the MGO-induced apoptosis in HUVECs is associated with increased ROS generation and whether isosamidin could decrease this ROS generation. As shown in Figure 3A, the intracellular level of ROS in the MGO-treated HUVECs increased significantly, whereas pretreatment with isosamidin markedly decreased the MGO-induced ROS generation.
Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 14 expression of the Bcl-2 protein and an increase in the expression of the Bax protein. In contrast, treatment with isosamidin led to a decrease in the levels of Bax and an increase in those of Bcl-2.

Effect of Isosamidin on MGO-Induced ROS Generation
The apoptosis of endothelial cells by intracellular ROS generation is a well-known phenomenon. Considering this, we further investigated whether the MGO-induced apoptosis in HUVECs is associated with increased ROS generation and whether isosamidin could decrease this ROS generation. As shown in Figure 3A, the intracellular level of ROS in the MGO-treated HUVECs increased significantly, whereas pretreatment with isosamidin markedly decreased the MGOinduced ROS generation.

Effect of Isosamidin on MAPK Activation
The MGO-induced apoptosis in endothelial cells is associated with phosphorylation-induced activation of MAPK. To assess the effect of isosamidin on MAPK signaling during MGO-induced apoptosis we examined the phosphorylation of p38, JNK, and ERK by Western blotting. As shown in Figure 3B-E, MGO treatment increased the phosphorylated forms of p38, JNK, and ERK, whereas the total protein levels of p38, JNK, and ERK remained unchanged. In contrast, pretreatment with isosamidin decreased the phosphorylation of p38 and JNK, whereas the phosphorylation protein levels of ERK remained unchanged.

Effect of Isosamidin on GLO-I and Nrf2 Expression
The effect of the isosamidin on GLO-I and Nrf2 protein expression was measured using Western blotting in MGO-induced HUVECs. It showed that the levels of GLO-I decreased and Nrf2 unchanged in HUVECs following MGO treatment ( Figure 4A). However, pretreatment with isosamidin increased the levels of GLO-I and Nrf2 in MGO-treated HUVECs ( Figure 4B). Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 14

Effect of Isosamidin on MAPK Activation
The MGO-induced apoptosis in endothelial cells is associated with phosphorylation-induced activation of MAPK. To assess the effect of isosamidin on MAPK signaling during MGO-induced apoptosis we examined the phosphorylation of p38, JNK, and ERK by Western blotting. As shown in Figure 3B-E, MGO treatment increased the phosphorylated forms of p38, JNK, and ERK, whereas the total protein levels of p38, JNK, and ERK remained unchanged. In contrast, pretreatment with isosamidin decreased the phosphorylation of p38 and JNK, whereas the phosphorylation protein levels of ERK remained unchanged.

Effect of Isosamidin on GLO-I and Nrf2 Expression
The effect of the isosamidin on GLO-I and Nrf2 protein expression was measured using Western blotting in MGO-induced HUVECs. It showed that the levels of GLO-I decreased and Nrf2 unchanged in HUVECs following MGO treatment ( Figure 4A). However, pretreatment with isosamidin increased the levels of GLO-I and Nrf2 in MGO-treated HUVECs ( Figure 4B).

Discussion
MGO is a toxic dicarbonyl compound associated with diabetic vascular complications; it is known to trigger cellular injury and apoptosis in endothelial cells. Apoptosis of endothelial cells plays a key role in the development of atherosclerosis and cardiovascular disease. In this study, we confirmed that isosamidin, one of the coumarin compounds isolated from the roots of P. japonicum, could protect against MGO-induced apoptosis and oxidative damage. To evaluate the anti-apoptotic effects of the coumarins obtained from P. japonicum, we pretreated the cells with 20 compounds

Discussion
MGO is a toxic dicarbonyl compound associated with diabetic vascular complications; it is known to trigger cellular injury and apoptosis in endothelial cells. Apoptosis of endothelial cells plays a key role in the development of atherosclerosis and cardiovascular disease. In this study, we confirmed that isosamidin, one of the coumarin compounds isolated from the roots of P. japonicum, could protect against MGO-induced apoptosis and oxidative damage. To evaluate the anti-apoptotic effects of the coumarins obtained from P. japonicum, we pretreated the cells with 20 compounds extracted from P. japonicum roots, at a concentration of 10 µM each for 1 h. In the present study, MGO treatment decreased the viability of HUVECs, thus indicating that MGO triggers direct cytotoxicity in HUVECs. We found that, out of all the compounds evaluated, only isosamidin had a potential protective effect on MGO-induced cell death (Table 1). However, pre-treatment of isosamidin concentration-independently (1, 10, and 100 µM) protected cell death in HUVECs ( Figure S1). We also observed that isosamidin inhibited the cellular toxicity and lactate dehydrogenase (LDH) production ( Figure 1C) observed in the MGO-induced apoptotic HUVECs. Resultantly, we selected 10 µM concentration of isosamidin for further mechanism study. In addition, annexin V-FITC/PI double staining demonstrated that MGO treatment increased apoptosis in HUVECs. Therefore, isosamidin protected the MGO-induced apoptotic cells (Figure 2A,B). We observed that isosamidin is effective in both the early and late stages of apoptosis. Hence, isosamidin appears to play a crucial role in the apoptosis induced by MGO in HUVECs.
Bcl-2 and Bax proteins, members of the Bcl-2 family, are involved in the modulation of cell apoptosis [31,32]. The induction of cell apoptosis can be determined by the alterations in the ratio of these two proteins. We found that exposure of HUVECs to MGO decreased the expression of Bcl-2 and increased the expression of Bax. However, pretreatment with isosamidin inhibited the MGO-induced apoptosis by decreasing the level of Bax and increasing the level of Bcl-2 ( Figure 2C-E). Therefore, isosamidin might influence MGO-induced cell apoptosis via regulation of Bcl-2 and Bax levels in HUVECs.
MGO is a highly reactive metabolite that forms advanced glycation end products (AGEs) via various metabolic pathways, including fragmentation of triose phosphates during glycolysis, ketone body metabolism, threonine catabolism, and lipid peroxidation [33][34][35][36][37][38]. MGO reacts directly with the free sulfhydryl (SH) group or amino residues (lysine, arginine, and cysteine) on proteins to form AGEs that affect protein function [39,40]. The MGO-induced glycated protein (carboxyethyl-lysine; CEL) leads to the subsequent activation of the receptor of AGEs (RAGE), which then initiates vascular complications [41,42]. Previous studies have reported that MGO can induce endothelial dysfunction and apoptosis, mainly via generation of ROS and high levels of AGEs [43,44]. Therefore, we investigated whether isosamidin could increase the breakdown of the crosslinks in MGO-AGEs, which would imply that it could improve AGE-related vascular endothelial dysfunction. Resultantly, isosamidin showed anti-glycation effects that resulted in the breakdown of the crosslinks in the MGO-induced AGEs ( Figure 1D) but it did not lead to a decrease in AGE formation ( Figure S2); however, further studies using HPLC and LC-MS/MS needs to be conducted to explore this effect.
Several studies have suggested that intracellular ROS generation can be increased by MGO; thus, this may play an important role in AGE-RAGE formation [45][46][47]. Furthermore, Deshpande et al. reported the ROS-mediated proteolytic cleavage of procaspase-3 in the mitochondria of HUVECs [48]. Therefore, using DCF-DA, we investigated whether isosamidin could alter the MGO-induced generation of ROS. Our results showed that isosamidin pretreatment significantly reduced the MGO-induced generation of ROS ( Figure 3A). Gupta et al. reported that the cell apoptosis induced by ROS generation was associated with the mitochondrial pathway and that Bcl-2 can regulate ROS signaling in the cells [49]. In another study, it was reported that the increase in Bax expression can upregulate intracellular ROS generation [50]. Therefore, this downregulation of the Bax/Bcl-2 ratio by isosamidin may be important for cell apoptosis, under the conditions of MGO-induced ROS generation in the HUVECs.
MAPK signaling is a key modulator of cell differentiation and cell apoptosis [51]. Many studies reported that phosphorylation of p38, JNK, and ERK leads to apoptosis [52][53][54]. Recently, several studies also reported that the activation of the MAPK family members, including JNK and p38, is associated with MGO-induced cytotoxicity [55,56]. In this study, we determined the effect of isosamidin on the MGO-induced inhibition of MAPK phosphorylation. We observed that pretreatment with isosamidin significantly inhibited the activation of p38 and JNK ( Figure 3B-E) in the MGO-activated MAPK signaling pathway. The results of the present study demonstrated that the inhibition of apoptosis by isosamidin was accompanied by the inhibition of MAPK activation. These data suggested that isosamidin could modulate the MAPK signaling pathways in MGO-treated HUVECs.
Detoxification enzymes, such as glyoxalase-I (GLO-I), are associated with the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway [57,58]. Nrf2 is an important component of the transcription factor regulating the expression of genes containing an ARE responsible for protection against oxidative stress and glutathione recycling [58]. Therefore, the development of GLO-I inducers via the activation and binding of Nrf2 to the GLO-I functional ARE is a promising strategy. This upregulation of GLO-I and Nrf2 expression by isosamidin may be vital for the detoxification of MGO-induced glucotoxicity in HUVECs.

Conclusions
Isosamidin from the roots of P. japonicum showed cytoprotective effects in HUVECs by reducing the MGO-induced apoptosis by regulating the generation of ROS and the apoptotic signaling cascades. It was demonstrated that isosamidin holds great potential as a cytoprotective agent against MGO-induced cell toxicity. In addition, it also possesses anti-glycation and anti-apoptosis effects ( Figure 5). These findings suggest that isosamidin from P. japonicum may play a protective role in MGO-and MGO-AGE-related endothelial dysfunction.
Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 14 data suggested that isosamidin could modulate the MAPK signaling pathways in MGO-treated HUVECs.
Detoxification enzymes, such as glyoxalase-I (GLO-I), are associated with the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway [57,58]. Nrf2 is an important component of the transcription factor regulating the expression of genes containing an ARE responsible for protection against oxidative stress and glutathione recycling [59]. Therefore, the development of GLO-I inducers via the activation and binding of Nrf2 to the GLO-I functional ARE is a promising strategy. This upregulation of GLO-I and Nrf2 expression by isosamidin may be vital for the detoxification of MGO-induced glucotoxicity in HUVECs.

Conclusions
Isosamidin from the roots of P. japonicum showed cytoprotective effects in HUVECs by reducing the MGO-induced apoptosis by regulating the generation of ROS and the apoptotic signaling cascades. It was demonstrated that isosamidin holds great potential as a cytoprotective agent against MGO-induced cell toxicity. In addition, it also possesses anti-glycation and anti-apoptosis effects ( Figure 5). These findings suggest that isosamidin from P. japonicum may play a protective role in MGO-and MGO-AGE-related endothelial dysfunction.

Conflicts of Interest:
The authors declare no conflict of interest.

Conflicts of Interest:
The authors declare no conflict of interest.