molecules Article
Protective Mechanism of the Antioxidant Baicalein toward Hydroxyl Radical-Treated Bone Marrow-Derived Mesenchymal Stem Cells Yage Tian 1,2,† , Xican Li 3,4, *,†,‡ and Dongfeng Chen 1,5, * ID 1 2 3
4 5
* † ‡
ID
, Hong Xie 3,4 , Xiaozhen Wang 3 , Yulu Xie 3,4 , Chuanbing Chen 3
School of Basic Medical Science, Guangzhou University of Chinese Medicine, Guangzhou 510006, China;
[email protected] International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China;
[email protected] (H.X.);
[email protected] (X.W.);
[email protected] (Y.X.);
[email protected] (C.C.) Innovative Research & Development Laboratory of TCM, Guangzhou University of Chinese Medicine, Guangzhou 510006, China The Research Center of Basic Integrative Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China Correspondence:
[email protected] or
[email protected] (X.L.);
[email protected] (D.C.); Tel.: +86-20-3935-8076 (X.L.) These authors contributed equally to this work. Address: School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Waihuan East Road No. 232, Guangzhou Higher Education Mega Center, Guangzhou510006, China. Homepage: www.researchgate.net/profile/Xican_Li.
Received: 5 December 2017; Accepted: 17 January 2018; Published: 20 January 2018
Abstract: Our study explores the antioxidant and cytoprotective effects of baicalein and further discusses the possible mechanisms. A methyl thiazolyl tetrazolium (MTT) assay revealed that baicalein could considerably enhance the viability of hydroxyl radical-treated bone marrow-mesenchymal stem cells (bmMSCs) at 37–370 µM. The highest viability rate was 120.4%. In subsequent studies, baicalein was observed to effectively scavenge hydroxyl radical and PTIO• radicals, reducing Fe3+ and Cu2+ ions. In the Fe2+ -chelating UV-vis spectra, mixing of baicalein with Fe2+ yielded two evident redshifts (275 → 279 nm and 324 → 352 nm) and a broad absorption peak (λmax ≈ 650 nm, ε = 1.6 × 103 L mol−1 ·cm−1 ). Finally, we compared the Fe2+ -chelating UV-vis spectra of baicalein and its analogues, including 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, catechol, pyrogallol, and chrysin. This analysis revealed that the 4-keto group of the C-ring played a role. The 5,6,7-trihydroxy-group (pyrogallol group) in the A-ring served as an auxochrome, enhancing the absorbance of the UV-vis spectra and deepening the color of the Fe2+ -complex. We concluded that baicalein, as an effective hydroxyl radical-scavenger, can protect bmMSCs from hydroxyl radical-mediated oxidative stress. Its hydroxyl radical-scavenging effects are likely exerted via two pathways: direct scavenging of hydroxyl radicals, possibly through electron transfer, and indirect inhibition of hydroxyl radical generation via Fe2+ chelation through the 4-keto-5,6,7-trihydroxy groups. Keywords: baicalein; antioxidant mechanism
5,6,7-trihydroxyflavone;
Molecules 2018, 23, 223; doi:10.3390/molecules23010223
e-transfer;
bmMSCs;
Fe2+ -chelating;
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Introduction 1. 1. Introduction Hydrogen peroxide Ocan ) can cross cell membranes freely and often foundininbiological biological 2O22) 2 cross cell membranes freely and areare often found Hydrogen peroxide (H(H 2+ ), it will undergo the Fenton reaction (Fe2+ + H O → systems [1]. When mixed with ferrous iron (Fe 22 → 2 systems [1]. When mixed with ferrous iron (Fe2+), it will undergo the Fenton reaction (Fe2+ + H2O 3+ + •OH + HO− ), generating a hydroxyl radical (•OH), well-known as the most harmful reactive Fe 3+ − Fe + •OH + HO ), generating a hydroxyl radical (•OH), well-known as the most harmful reactive oxygen species (ROS). Hydroxylradicals radicalscan canattack attackallalltypes types cellular biomolecules(especially (especiallyDNA) DNA) oxygen species (ROS). Hydroxyl ofof cellular biomolecules to cause oxidative stress [2,3]. to cause oxidative stress [2,3]. Oxidativestress stressnot notonly onlylowers lowersviability viabilityofofbone bonemarrow-derived marrow-derivedmesenchymal mesenchymalstem stemcells cells Oxidative (bmMSCs), but also induces their differentiation [3,4]. It is reported that mild oxidative stress can induce (bmMSCs), but also induces their differentiation [3,4]. It is reported that mild oxidative stress can differentiation of bmMSCs to adipose cells, but not but to osteose nerve or cells [5,6].cells Thus, ironThus, overload, induce differentiation of bmMSCs to adipose cells, not to or osteose nerve [5,6]. iron a cause of a•OH radical generation, can usually lead to bone loss These detrimental overload, cause of •OH radical generation, can usually lead to [7]. bone loss aforementioned [7]. These aforementioned effects of oxidative stress currently limit the clinical application of bmMSC transplantation detrimental effects of oxidative stress currently limit the clinical application of bmMSCfor neurodegenerative diseases (e.g., Parkinson’s [8] and bone diseases (e.g.,bone osteoporosis) [9,10]. transplantation for neurodegenerative diseases disease) (e.g., Parkinson’s disease) [8] and diseases (e.g., As such, scientists are searching for an effective antioxidant from natural products [11] or synthetic osteoporosis) [9,10]. As such, scientists are searching for an effective antioxidant from natural compounds [12,13] to relieve •OH radical-mediated oxidative stress, to improve bmMSCs viability products [11] or synthetic compounds [12,13] to relieve •OH radical-mediated oxidative stress, to enough for the clinical application [14]. improve bmMSCs viability enough for the clinical application [14]. Baicalein(Figure (Figure1),1),a anatural naturalflavonoid flavonoidoccurring occurringinintraditional traditionalChinese Chineseherbal herbalmedicine, medicine, Baicalein Scutellaria baicalensis Georgi, is predicted to be useful as an antioxidant. Recently, baicalein has been Scutellaria baicalensis Georgi, is predicted to be useful as an antioxidant. Recently, baicalein has been demonstrated suppress the early stages adipogenesis [15,16] and regulate bone formation [17]. demonstrated toto suppress the early stages ofof adipogenesis [15,16] and regulate bone formation [17]. In addition, baicalein has been reported to protect HS-SY5Y cells from hydrogen peroxide-induced In addition, baicalein has been reported to protect HS-SY5Y cells from hydrogen peroxide-induced oxidative stress [18] and attenuateneurological neurologicalinjury injuryininrats rats[19]. [19]. oxidative stress [18] and toto attenuate
Figure 1. Structure (A) and preferential conformation-based ball-and-stick model (B) of baicalein Figure 1. Structure (A) and preferential conformation-based ball-and-stick model (B) of baicalein (5,6,7-trihydroxyflavone). (5,6,7-trihydroxyflavone).
Nevertheless, direct evidence evidencefor forthe the beneficial effects of baicalein toward •OHNevertheless,there thereisis no no direct beneficial effects of baicalein toward •OH-treated treated bmMSCs. This study therefore used a methyl thiazolyl tetrazolium (MTT) assay to assess its bmMSCs. This study therefore used a methyl thiazolyl tetrazolium (MTT) assay to assess its protective protective effects •towards •OH-treated thus providing information use of baicalein effects towards OH-treated bmMSCs,bmMSCs, thus providing information for the usefor ofthe baicalein in bmMSC intransplantation bmMSC transplantation technology. technology. More importantly, therethere are some regardingregarding the antioxidation mechanisms mechanisms (especially More importantly, are disputes some disputes the antioxidation 2+ Fe(especially -chelation) baicalein. Yoshino and colleagues have suggested thatsuggested baicalein could inhibit •OH 2+ -chelation) Feof of baicalein. Yoshino and colleagues have that baicalein could radical generation [20], while Shieh and colleagues have reported that baicalein could could not inhibit •OH radical generation [20], while Shieh and colleagues have reported that baicalein 2+ scavenge •OH •[21]. Regarding Fe -chelation chemistry, RenRen andand colleagues stated that thethe 4-keto not scavenge OH [21]. Regarding Fe2+ -chelation chemistry, colleagues stated that 4-keto group of flavonoids plays a critical role in this process [22], while Perez and colleagues argued that group of flavonoids plays a critical role in this process [22], while Perez and colleagues argued that 2+-chelation mainly occurred at the 6,7-dihydroxyl groups in flavonoids [23]. Thus, our study used FeFe 2+ -chelation mainly occurred at the 6,7-dihydroxyl groups in flavonoids [23]. Thus, our study various chemical approaches to explore the possible antioxidation mechanisms, especially the Fe2+the used various chemical approaches to explore the possible antioxidation mechanisms, especially chelation pathway. We believe that this study will help settle the above disputes. 2+ Fe -chelation pathway. We believe that this study will help settle the above disputes. 2. 2. Results and Discussion Results and Discussion AsAsseen in in Figure 2, in model group, bmMSCs damaged by •OH showedshowed only seen Figure 2, the in the model group, bmMSCs damaged by radicals •OH radicals 52.9 ± 12.0% However, when when damaged bmMSCs were were further treated with with baicalein at only 52.9 ± viability. 12.0% viability. However, damaged bmMSCs further treated baicalein 10–100 µg/mL (37–370 µM), the viability was completely restored, and further increased to at 10–100 µg/mL (37–370 µM), the viability was completely restored, and further increased 120.7 ± 4.3% (baicalein group). ThisThis result suggests thatthat baicalein protects •OH to 120.7 ± 4.3% (baicalein group). result suggests baicalein protects •OHradical-treated radical-treated bmMSCs, asas supported byby previous bmMSCs, supported previousstudies studies[24,25]. [24,25].
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Figure 2. bmMSCs. Cell viability was assessed using Figure2. 2. Protective Protective effect effect of of baicalein baicalein toward toward •OH-treated bmMSCs.Cell Cellviability viabilitywas wasassessed assessedusing using Figure Protective effect of baicalein toward ••OH-treated OH-treated bmMSCs. the MTT method. •OH radicals were generated by addition of FeCl 2 (100 µM) followed by the MTT method. •OH radicals were generated by addition of FeCl 2 (100 µM) followed by the MTT method. •OH radicals were generated by addition of FeCl2 (100 µM) followed by H2 O2 H 2 (50 µM). The control group was cultured in medium only, while the model group was treated H22O O 2 (50The µM). The control group was cultured in medium only, model treated (50 µM). control group was cultured in medium only, while the while modelthe group wasgroup treatedwas with •OH with •OH radicals. The baicalein group was treated by •OH followed by baicalein. Each value is with •OH radicals. The baicalein group was treated by •OH followed by baicalein. Each value radicals. The baicalein group was treated by •OH followed by baicalein. Each value is expressed as theis expressed as the mean ± SD, n = 3; * Significant difference vs. model group, p < 0.05. bmMSCs, bone expressed ± SD, n difference = 3; * Significant difference group, p bone < 0.05. bmMSCs, bone mean ± SD,asnthe = 3;mean * Significant vs. model group, vs. p < model 0.05. bmMSCs, marrow-derived marrow-derived mesenchymal stem MTT, methyl marrow-derived mesenchymal stem cells; cells; MTT,tetrazolium. methyl thiazolyl thiazolyl tetrazolium. tetrazolium. mesenchymal stem cells; MTT, methyl thiazolyl
The largely due to attack, as radicals are more toxic damage to tothe thecells cellsmay maybe largely due to •OH attack, as •OH radicals are much The damage to the cells may bebe largely due to •OH •OH attack, as •OH •OH radicals are much much moremore toxic than H 2O2H alone. Correspondingly, the above cytoprotective effects of baicalein are attributable toxic than O alone. Correspondingly, the above cytoprotective effects of baicalein are attributable than H2O2 alone. to 2 2 Correspondingly, the above cytoprotective effects of baicalein are attributable to its •OH-scavenging ability deoxyribose to •OH-scavenging ability[26,27]. [26,27].In fact,in the•OH-scavenging •OH-scavenging assay assay based based on itsits •OH-scavenging ability [26,27]. InInfact, fact, ininthe the •OH-scavenging assay on deoxyribose deoxyribose degradation, effective dose-dependent degradation, baicalein was observed to have have an response (Figure (Figure 3A). 3A). degradation, baicalein baicalein was was observed observed to have an an effective effective dose-dependent dose-dependent response The IC 50, Trolox/IC 50, Baicalein value (1.46) in Table 1 suggests that baicalein is a stronger •OH-scavenger The IC /IC value (1.46) in Table 1 suggests that baicalein is a stronger • OH-scavenger The IC50, 50, Trolox Trolox/IC50, value (1.46) in Table 1 suggests that baicalein is a stronger •OH-scavenger 50,Baicalein Baicalein than data appear to the opinions of [20], and than the standard antioxidant,Trolox. Trolox.Our Our data appear to support opinions of Yoshino than the standard antioxidant, antioxidant, Trolox. Our data appear to support support thethe opinions of Yoshino Yoshino [20],[20], and disagree with those of [21]. and disagree those of Shieh disagree withwith those of Shieh Shieh [21].[21].
Figure Dose-response curves of in the assay (A) and in Figure3.3. 3.Dose-response Dose-response curves of baicalein baicalein the •OH-scavenging •OH-scavenging and in PTIO•PTIO•Figure curves of baicalein in thein •OH-scavenging assay (A)assay and in(A) PTIO •-scavenging scavenging assay (B). PTIO•-scavenging assay was conducted at pH 5.0, 6.0, 7.4, 8.0, and 9.0. Trolox scavenging assay (B). PTIO•-scavenging assay was conducted at pH 5.0, 6.0, 7.4, 8.0, and 9.0. Trolox assay (B). PTIO•-scavenging assay was conducted at pH 5.0, 6.0, 7.4, 8.0, and 9.0. Trolox served as served as positive control. Each value is expressed as the mean ± SD, n = 3. served as positive control. Each value is expressed as the mean ± SD, n = 3. positive control. Each value is expressed as the mean ± SD, n = 3. 50 values of baicalein and Trolox in various assays. Table 1. IC 50 values values of of baicalein baicalein and and Trolox Troloxin invarious variousassays. assays. Table1. 1.IC IC50 Table
Assays Assays
Assays
•OH-scavenging •OH-scavenging • OH-scavenging PTIO• PTIO• scavenging scavenging ** PTIO• scavenging * 2+ reducing Cu 2+ reducing 2+ reducing CuCu FRAP FRAP FRAP
Baicalein Baicalein
Baicalein μM μM µM
a 93.7 93.7 ±± 1.6 1.6 a
Trolox Trolox Trolox μM μM µM b 137.6 137.6 ±± 3.6 3.6 b 137.6 ± 3.6bb b 384.2 ± 23.3 384.2 ± 23.3 b 384.2 ± 23.3 b 44.6 ±± 1.5 b b 44.6 1.51.5 44.6 ± a 12.0 ±± 0.0 a a 12.0 0.00.0 12.0 ±
Ratio Ratio value value Ratio value 50, Trolox /IC Baicalein IC 50, Baicalein IC50, Trolox/IC50, IC50, Trolox /IC50, Baicalein 1.46 1.46 1.46 2.03 2.03 2.03 2.34 2.34 2.34 0.96 0.96 0.96
a 93.7 ±±1.6 a 188.7 188.7 ± 13.1 13.1a a 188.7 ± 13.1 a 19.0 0.1 19.0±±±0.1 0.1a a 19.0 a 11.1 0.0 11.1±±±0.0 0.0a a 11.1 IC value was defined as concentration of (relative reducing power), IC505050value valuewas was defined as the the final final concentration of 50% 50% radical-scavenging radical-scavenging (relative reducing power), IC defined as the final concentration of 50% radical-scavenging (relative reducing power), was calculated was calculated by linear regression analysis, and is expressed as the mean ± SD (n = 3). by linear regressionby analysis, is expressed as the mean ± SD (n = 3). Linear was± analyzed Origin was calculated linearand regression analysis, and is expressed asregression the mean SD (n =using 3). Linear Linear 6.0 professional software. Theusing mean Origin values with different superscripts (a orThe b) inmean the same row, are significantly regression was analyzed 6.0 professional software. values with different regression was analyzed using Origin 6.0 professional software. The mean values with different different (p < 0.05). * The assay was conducted at pH 7.4. The ratio value is defined as IC50, Trolox /IC50, Baicalein . superscripts (a b) row, are significantly different superscripts (a or orantioxidant b) in in the the same same significantly different (p (p
