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Salvia miltiorrhiza prevents methylglyoxal-induced glucotoxicity via the regulation of 3 apoptosis-related pathways and the glyoxalase system in human umbilical vein 4 endothelial cells

Methylglyoxal (MGO), which is produced as a byproduct of glucose metabolism, is the 22 leading to diabetic cardiovascular complications. Salvia miltiorrhiza Bunge (Lamiaceae) has 23 been reported as a potential plant to control diabetes and cardiovascular disease. However, no 24 report exists on the effect of Salvia miltiorrhiza Bunge extract (SME) on MGO-induced 25 glucotoxicity in human umbilical vein endothelial cells (HUVECs). We demonstrated the protective effects of SME (1, 5, and 10 µ g/mL) and its components 27 against MGO-induced endothelial dysfunction in HUVECs. Cytotoxicity was evaluated using 28 the several in vitro experiments. Additionally, the protein expression of receptor of advanced 29 glycation end-products (RAGE), mitogen-activated protein kinase (MAPK) pathway and 30 glyoxalase system were measured. Then, the inhibitory effects of SME and its main 31 components on MGO-induced oxidative stress, radical scavenging, formation of MGO- 32 derived advanced glycation end products (AGEs), and MGO-AGEs crosslinking were 33 evaluated. that S. miltiorrhiza is a potential natural resource for the treatment of MGO- induced vascular endothelial dysfunction.


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With the increasing prevalence of diabetes, diabetic complications are emerging as a huge 50 social issue, especially diabetic cardiovascular complications. Diabetic cardiovascular 51 complications are the main origin of morbidity and mortality among diabetic patients; they 52 affect almost one-third of patients with diabetes, regardless of the type of diabetes 1) . 53 Although the pathogenesis of diabetic cardiovascular complications is not yet fully 54 understood, it is now established that endothelial dysfunction is a crucial factor for the 55 advance of diabetic cardiovascular complications 2) . Endothelial dysfunction refers to the 56 malfunction of vascular endothelial cells. Vascular endothelial cells regulate hemodynamics 57 through constriction or vasodilation, control vessel wall permeability, and seize circulating 58 leukocytes, playing a vital role in inflammation 2, 3) . When these vascular endothelial cells are 59 exposed to certain conditions, such as hyperglycemia, insulin resistance, or mechanical 60 stretch, cell damage is induced by apoptosis, mitogen-activated protein kinase (MAPK) 61 signaling activation, and reaction oxygen species (ROS) overproduction, which eventually 62 leads to endothelial dysfunction [2][3][4] . Recently, hyperglycemia-induced endothelial 63 dysfunction has been shown to originate from dicarbonyl stress 5) . In hyperglycemia and 64 hyperfructosemia, sugar reacts with proteins and forms a Schiff base. Following this, the generated as a by-product of glycolysis by the fragmentation of glyceraldehyde-3-phosphate 74 and dihydroxyacetone phosphate 4) . Endothelial cells generate 85% of their ATP through 75 glycolysis; thus, they are prone to have higher intracellular MGO concentrations when 76 exposed to hyperglycemia 8) . This high concentration of MGO exacerbates inflammation and 77 apoptosis in vascular complications by affecting endothelial cells, such as inducing the 78 activation of the MAPK signaling pathway, which induces cell death 11) , and overproduction 79 of ROS generation, which damages endothelial cells 4,12) . Hanssen et al. reported the 80 relationship between plasma MGO levels and cardiovascular disease in patients with type 1 81 and type 2 diabetes mellitus 10) . Under normal conditions, MGO is formed but detoxified via 82 a nuclear factor erythroid-2-related factor 2 (Nrf2) and glyoxalase 1 (GLO-1) interaction. 83 However, in chronic hyperglycemia, MGO suppresses Nrf2 and leads to the downregulation 84 of the glyoxalase defense system 8) . Therefore, new solutions, such as Glo-1 inducers and 85 anti-glycative agents, can be proposed to treat endothelial dysfunction and reduce the 86 incidence of cardiovascular diseases. 87 Salvia miltiorrhiza Bunge (Lamiaceae) is widely distributed in Asia. The dried root of S. 88 miltiorrhiza has been traditionally used to treat various cardiovascular diseases, such as 89 cerebrovascular diseases and coronary heart disease, including Parkinson's and Alzheimer's 90 diseases 13) . More than 100 compounds from S. miltiorrhiza have been identified and non-91 polar diterpenoid and hydrophilic phenolic compounds have been studied for their anti-92 diabetic and anti-cardiovascular effects 14) . Among them, tanshinone I, tanshinone IIA, 93 salvianolic acid A, and salvianolic acid B has been reported to be effective in various in vitro 94 and in vivo diabetic models 15,16) . Particularly, salvianolic acid A have prevented early-stage 95 diabetic nephropathy via the amelioration of glomerular endothelial hyperpermeability in 96 type 2 diabetes mellitus rat model via AGE-RAGE associated signaling pathways 17) . 97 Additionally, tanshinone 2A has protected brain microvascular endothelial cells from   Cell toxicity and morphological investigation. Cell toxicity was evaluated using an MTT 133 assay 9) . Cells were seeded into 96-well plates (1.0 × 10 4 cells/well) for 24 h. After 134 incubation, the cells were treated with several concentrations (1, 5, and 10 μg/mL) of SME 135 for 1 h, followed via 500 μM MGO for 24 h. After incubation, the MTT solution was mixed 136 to each well for 2 h. Then, the medium was suctioned and DMSO added to each well. The  Intracellular ROS exposure. HUVECs were seeded into 6-well plates (3.0 × 10 5 cells/well) 142 and incubated at 37 °C in 5% CO 2 for 24h. After incubation, cells were treated with SME for 143 1 h, followed by 500 μM MGO treatment for 2 h. Cells were treated with 20 μM DCF-DA for 144 15 min in the supplement-free medium in the dark. The cells were observed using a JuLi live-145 cell imaging machine (NanoEnTek, Seoul, Korea).

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Intracellular SOD1, SOD2, CAT, and GSH-Px levels. HUVECs were seeded into 6-well 147 plates (3.0 × 10 5 cells/well) and incubated at 37 °C in 5% CO 2 for 24h. After incubation, 148 cells were treated with SME for 1 h, followed by 500 μM MGO treatment for 1 h. Cell 149 lysates were prepared by using Seoul,Korea;cat no. 150 17081), and then protein concentration were quantified using the Bradford assay protocol 5) .

SME inhibits MGO-induced necrosis and apoptosis in HUVECs. To investigate whether
242 MGO-induced cell toxicity is related to necrosis and apoptosis, we used a flow cytometry 243 technique based on FITC/PI staining. The MGO treatment group indicated a significant 244 increase in the density of early and late apoptotic cells ( Fig. 1E and 1F). However, SME pre-245 treatment decreased the density of apoptotic cells in HUVECs ( Fig. 1E and 1F). In addition, 246 the pre-treatment with 5 ( * P < 0.05) and 10 μg/mL ( *** P < 0.001) of SME decreased the ratio 247 of Bax/Bcl-2 and cleaved poly (ADP-ribose) polymerase (PARP)/PARP ( Fig. 1G and 1H).

SME inhibits MGO-induced RAGE expression in HUVECs. It was well reported that 249
AGEs and RAGE interaction contributed to accelerate the inflammation, carcinogenesis and 250 atherosclerosis 25) . Thus, we investigated the effect of SME on the levels of RAGE expression 251 in MGO-treated HUVECs ( Fig. 2A and 2B). The MGO treatment group showed significantly 252 increased the expressed levels of RAGE protein ( ### P < 0.001) in comparison with control 253 group. Meanwhile, SME ( *** P < 0.001) strongly suppressed the levels of RAGE protein 254 expression at 10 µg/mL concentration, as similar that of positive control group ( *** P < 0.001). 255 SME upregulates MGO-induced glyoxalase system in HUVECs. To evaluate whether the 256 effects of MGO on the glyoxalase system are associated with Glo-1 and Nrf2, we performed 257 western blotting. The MGO treatment group ( # P < 0.05) showed significantly decreased Glo-258 1 and Nrf2 protein expression in comparison with control group, but SME increased Glo-1 259 and Nrf2 expression in a concentration-dependent manner ( Fig. 2C and 2D).

SME decreases MGO-induced ROS production and MAPK signaling in HUVECs.
We 261 evaluated whether the SME pre-treatment can decrease production of ROS in HUVECs or 262 not. ROS production was quantified using DCF-DA staining and a JuLi live-cell imaging 263 machine. MGO treatment significantly increased ROS generation, whereas SME pre-  HUVECs. We found that each antioxidant enzymes significantly decreased by treating MGO 275 in comparison with control, but they were restored with SME pre-treatment (Fig. 4). This 276 result suggest that SME may prevent MGO-induced cell apoptosis mediated by ROS stress.

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We conducted an MGO-AGEs formation assay by quantifying fluorescence. SME (1, 5, and 279 10 mg/mL) co-treatment significantly inhibited the formation of MGO-BSA or -fibrinogen in 280 a concentration-dependent manner, comparing with control ( Fig. 5A and 5B). Moreover, to 281 investigate whether SME could break preformed MGO-BSA-AGEs or MGO-fibrinogen-

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AGEs crosslinks, a TNBSA assay was performed to evaluate the number of remaining free 283 amines. SME significantly broke the MGO-BSA-AGEs or -fibrinogen-AGEs crosslink in a 284 cocnentration-independent manner in comparison that of control ( Fig. 5C and 5D).

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These two proteins are involved in intrinsic cell death pathways, which known as the 328 mitochondrial pathway 4) . Mitochondria may play key roles in activating apoptosis in 329 mammalian cells. PARP is an enzyme that normally repairs DNA but, when apoptosis begins, 330 it becomes a substrate for caspases. Cleaved PARP is a family of proteins involved in cellular 331 process such as DNA repair and programmed cell death, forms as a product of the caspase 332 reaction. Therefore, it is as a key indicator of cell apoptosis in HUVECs 4, 11) .

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In the present study, MGO increased the ratio of Bax/Bcl-2 and cleaved PARP/PARP 334 compared with the control, and SME was concentration-dependently decreased the ratio of 335 Bax/Bcl-2 and cleaved PARP/PARP ( Fig. 1G and 1H). Namely, SME significantly may

Biological and Pharmaceutical Bulletin Advance Publication
Annexin V-FITC/PI double staining apoptosis assay, our results suggested that SME 338 ameliorates the mitochondrial pathway of apoptosis. We detected early apoptotic HUVEC 339 cells by MGO pre-treatment and, under the same conditions, SME treatment significantly 340 decreased the number of apoptotic cells compared with the control group ( Fig. 1E and 1F).

Biological and Pharmaceutical Bulletin Advance Publication
concentration-dependent manner ( Fig. 3A and 3B). Additionally, SME pre-treatment reduced 362 the phosphorylation of p38, JNK, and ERK ( Fig. 3C and 3D). These experimental results 363 suggest that SME may have a protective effect against MGO-induced apoptosis via the 364 suppression of MAPKs signaling activation in HUVECs.

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Our in vitro study showed that MGO induced significant damage to HUVECs through 366 decreased cell viability, increased ROS production, and cell apoptosis, In addition, we found 367 in this work that MAPK pathways were activated by MGO, whereas treatment with SME  Indeed, our present data displayed that oxidative stress significantly increased in HUVECs 375 by treating MGO (Fig. 3A and 3B). It is several ways that MGO could increase the level of 376 intracellular ROS. First, MGO-derived AGEs stimulate the generation of superoxide anion 377 and hydrogen peroxide, and also the glutathione was used in MGO detoxification. Second, 378 the MGO may inactivate the antioxidant enzymes that scavenge the ROS such as SOD1, 379 SOD2, and GSH-Px 31) . These results showed that SME significantly inhibited ROS 380 generation, and increased the activities of antioxidant enzymes (e.g., SOD1, SOD2, CAT, and 381 GSH-Px) in HUVECs (Fig. 4). Also, we examined the radical scavenging effects of SME and 382 its bioactive compounds. As a result, SME and its bioactive compounds showed radical 383 scavenging ability (i.e., ABTS and DPPH radicals) ( Table 2). SME can contribute to the 384 amelioration of oxidative stress by reducing the production of ROS or quenching the formed 385 Biological and Pharmaceutical Bulletin Advance Publication radicals. This antioxidant activity might be owing to its abundant compounds, such as 386 rosmarinic acid and salvianolic acid B in SME (Fig.3, Fig.4, and Table 2).

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Meanwhile, RAGE, a receptor for AGEs, is expressed in cell surface of several types 388 including endothelial cells and immune cells 32) . The interactions of AGEs and RAGE 389 initiates the inflammation and cell death responses 25,32) . Thus, one of the major roles RAGE 390 is involved in glycotoxins-induced inflammation and apoptosis signaling. In this work, we 391 found that the expressed level of RAGE was increased by treating 500 µM MGO, whereas 392 SME pre-treatment significantly reversed it in a concentration-dependent manner (See Fig.2A 393 and 2B). As a result, our data support that the protective effect of SME was due to down-394 regulate the RAGE pathway, and also it blocked the MGO induced apoptosis and 395 inflammation in HUVECs ( Fig.1 and 3). indicate that SME prevents MGO-induced HUVECs apoptosis via decreasing oxidative stress, 405 inhibiting MAPK pathways and RAGE expression. According to our results and previous 406 studies, the ability of SME to prevent MGO-induced oxidative stress and apoptosis might be 407 protective against development of diabetic complications. We can also expect the potential 408 Biological and Pharmaceutical Bulletin Advance Publication activity of SME against age-related chronic diseases such as obesity, neurodegenerative 409 disease, and cancer 8) .

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To identify the bioactive compounds in SME, we tested the anti-AGE activities of . To quantify the content of each component in SME, we performed HPLC analysis and 414 observed that, among these compounds, salvianolic acid B was the most abundant in SME 415 (Table. 1 and Fig. 4S). An AGE formation assay was conducted for each compound. with control, but the other components did not show it (Fig. 6B). In the MGO-BSA-AGEs 420 breaker assay, rosmarinic acid, dihydrotanshinone, and tanshinone IIA showed breaking 421 ability in the listed order (Fig. 6C) and all five components in SME significantly broke the 422 MGO-fibrinogen-AGE crosslinking, comparing with control (Fig. 6D). Interestingly, 423 salvianolic acid B inhibited MGO-derived AGEs formation and dihydrotanshinone interfered 424 with MGO-AGEs formation and breaking. However, tanshinone I had no significant effects 425 on the in vitro anti-glycation assay. Although these three tanshinone type compounds are 426 similar in structure, but they showed the quite different in anti-glucotoxicity activities.

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Therefore, further research on the structure-activity relationship (SAR) on anti-glucotoxicity 428 of tanshinone derivatives are needed.

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Taken together, our results strongly suggest that S. miltiorrhiza extract (SME) may protect In summary, our results demonstrated that the extract of S. miltiorrhiza (SME) prevented 439 HUVECs against MGO-treated apoptosis via the modulation of MAPKs, Bax/Bcl-2 ratio, and 440 PARP-related apoptosis pathways. We also showed that SME not only modulated the cell 441 apoptosis pathway but also enhanced the glyoxalase system (Glo-1 and Nrf2). SME also 442 decreased the production of ROS in a concentration-dependent manner and showed 443 antioxidant activity. Furthermore, SME and its phytochemicals, dihydrotanshinone,      whereas SME (blue arrow) prevented the MGO-endothelial dysfunction via several pathways.