Herbal formula Xinshuitong capsule exerts its cardioprotective effects via mitochondria in the hypoxia-reoxygenated human cardiomyocytes

Herbal formula Xinshuitong capsule exerts its cardioprotective effects via mitochondria in the... Background: The collapse of mitochondrial membrane potential (ΔΨm) resulted in the cell apoptosis and heart failure. Xinshuitong Capsule (XST) could ameliorate left ventricular ejection fraction (LVEF), New York Heart Association (NYHA) classes and the quality of life in patients with chronic heart failure in our clinical study, however, its cardioprotective mechanisms remain unclear. Methods: Primary human cardiomyocytes were subjected to hypoxia-reoxygenation and treated with XST200, 400 and 600 μg/ml. The model group was free of XST and the control group was cultured in normal conditions. Cell viability, ΔΨm, the activity of mitochondrial respiratory chain complexes, ATPase activity, reactive oxygen species (ROS) and apoptosis cells were determined in all the groups. Results: The cell viability in the XST-treated groups was significantly higher than that in the model group (P < 0.05). Coupled with the restoration of the ΔΨm, the number of polarized cells increased dose dependently in the XST- treated groups. XST also restored the lost activities of mitochondrial respiratory chain complexes I-IV induced by the oxidative stress. The total of mitochondrial ATPase activity was significantly elevated at XST400 and 600 μg/ ml compared to the model group (P < 0.05). The levels of mitochondrial ROS and the number of apoptosis cells declined in the XST-treated groups compared to those in the model group (P < 0.05). Conclusions: XST, via restoration of ΔΨm and the mitochondrial respiratory chain complexes I-IV activities, and suppression of mitochondrial ROS generation and the apoptosis cells, maintained the integrity of the mitochondrial membrane to exert its cardioprotective effects in the hypoxia-reoxygenated human cardiomyocytes. Keywords: Xinshuitong capsule, Mitochondrial potential, Hypoxia-reoxygenated human cardiomyocytes Background and uncoupling. Thus, MPTP opening and mitochondrial Mitochondria, as the power house of the heart, are highly ΔΨm collapse have been regarded as a primary mediator of packed in the cardiomyocytes. Cardiac cells under pro- apoptosis in the ischemia-reperfusion heart injury [2, 3]. longed hypoxia condition have been shown with the open- The mitochondrial electron transport chain (ETC) is ing of mitochondrial permeability transition pore (MPTP) found in the inner membrane, where it serves as the site [1]. Due to the opening of MPTP causes a transient hyper- of oxidative phosphorylation through the use of ATP polarization, followed by depolarization, and subsequently synthase. During this chemical process, ROS can be the collapse of the mitochondrial membrane potential formed as a byproduct of normal cellular aerobic metab- (ΔΨm), which is characterized by mitochondrial swelling olism in the heart [4, 5]. Thus, the major process from which the heart derives sufficient energy can also result in the production of ROS [5]. On the other hand, ROS * Correspondence: tchunj@126.com; chen.wl@163.com can depress the activity of mitochondrial ETC and alter Chunjiang Tan and Wenlie Chen contributed equally to this work. Fujian Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 2 of 10 ion pump function in heart [6]. Mounting evidence has ml) and incubated for 4 h at 37 °C. Followed by removing strongly implicated ROS signaling in the genesis of car- MTT and oscillating for 10 min, cell viability was estimated diac hypertrophy [7–9]. Therefore, maintaining the in- at absorbance 570 nm by a Tecan Infinite M200 Pro micro- tegrity of the mitochondrial membrane, enhancing plate reader (Tecan, Mannedorf, Swizerland). antioxidant defense may be a therapeutic method for the protection of cardiomyocytes against the injury of ische- Mitochondrial ΔΨm detected by JC-1 staining mia or hypoxia. As JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimida- Xinshuitong Capsule (XST, awarded the Invention Patent zolyl-carbocyanine iodide, Beyotime, China) is a lipophilic of the People’s Republic of China, No.ZL201210197892.X), a fluorescentcationthatisincorporatedintothe mitochon- Chinese herbal medicine formula for chronic heart failure drial membrane, where it can form aggregates due to the (CHF), which consists of Astragali radix, Pseudostellariae state of the mitochondrial ΔΨm. This aggregation changes radix and Salviae miltiorrhizae radix et al., has the effects of the fluorescence properties of JC-1 from green to orange benefiting Qi and Yang, activating blood and eliminating sta- fluorescence as the ΔΨm increased. After treatment, cells sis, and inducing diuresis to alleviate edema. Our previous were harvested, re-suspended and incubated with 10 μg/ml clinical study showed that the CHF patients, who received JC-1 at 37 °C for 30 min as before [13, 14]. The cells were XST treatment (3 capsules, tid.), were significantly amelio- then washed and centrifuged, the intact living cells stained rated in left ventricular ejection fraction (LVEF), New York the mitochondria with JC-1 would exhibit a pronounced Heart Association (NYHA) classes, the symptoms and the orange fluorescence, however, the cells with a breakdown quality of life compared to the control group [10]. In vitro, of ΔΨm showed a decrease of the orange fluorescence (or a XST-treated hypoxia-reoxygenated human cardiomyocytes increase of the green fluorescence). Thus, the intact and in- showed more tolerant to hypoxia stress. The cells exhibited jured cells could be distinguished, and the cell populations more regular shape and size than the control [11]. However, will be counted according to the different fluorescence by the drug’s cardioprotective mechanisms remain elusive, es- the flow cytometry (BD Biosciences, CA) (JC-1 green: Ex/ pecially, its actions on MPTP and mitochondrial apoptosis Em = 485/525 nm; JC-1 red: Ex/Em =535/590 nm). pathway. Thus, in current experiments, mitochondrial ΔΨm Similarly, the fluorescence intensity of JC-1 as the index and mitochondrial mass, the activities of the mitochondrial of ΔΨm alterations could be detected by a confocal micro- ETC and the mitochondrial ATPase, and their associations scope (Carl Zeiss AG, Oberkochen, Germany), and the ra- with ROS levels and apoptosis cells will be studied in the tio of red/green fluorescence intensity is indicated as the hypoxia-reoxygenated cardiomyocytes. alterations of mitochondrial ΔΨm. For quantification of mitochondrial mass, we used Mito- Methods tracker Green probe (Molecular Probes), which preferen- Hypoxia-reoxygenated cell model tially accumulates in mitochondria regardless of the Primary human cardiomyocyte (HCM) was purchased mitochondrial membrane potential and provides an accur- from American Science Cell Research Laboratories (San ate assessment of mitochondrial mass. Firstly, the cells were Diego. USA). When the cells reached 80–90% conflu- washed with PBS and incubated at 37 °C for 30 min with ence, they ere placed on a 96-well plate or a petri dish at 100 nM MitoTracker Green FM (Molecular Probes) and a density of 0.75 × 105cells/ml in a hypoxia chamber then harvested using trypsin/EDTA and re-suspended in (80%N ,10%H ,10%CO and 0.2%O ) for 12 h,11 follow- PBS. Fluorescence intensity was detected with excitation 2 2 2 2 ing by 2 h reoxygenation. During the process of hypoxia and emission wavelengths of 490 and 516 nm, respectively, and reoxygenation, the study group were exposed to the and values were corrected for total protein (mg/ml). water-extract of XST (200, 400 and 600 μg/ml, respect- ively), while the model group was cultured in the identi- Determination of the activities of mitochondrial cal conditions free of XST treatment, and the control respiratory chain complexes group was cultured in normal conditions. The drug’s According to manufacturer’s instructions, mitochondrial low and high concentrations used in the current experi- isolation was performed at 4 °C using a Kit for cultured ment were comparable to the human serum levels in the mammal cell (Thermo Scientific Rockford. USA). previous clinical study. The activities of mitochondrial respiratory chain com- plexes were analyzed by spectrophotometer (Secomam, Cell viability assay Domont,France) as described before [13]. Briefly, com- As described before [12], cell viability was estimated by the plex I (NADH dehydrogenase, EC 1.6.5.3) enzyme activ- assay of 3-[4,5-dimethylthiazol-2-yl]-diphenyl-tetrazolium ity was measured as a decline in absorbance from bromide (MTT, Sigma-Aldrich). Briefly, after treatment, the NADH oxidation by decylubiquinone before and after cells were washed twice with phosphate-buffered saline adding rotenone (St. Louis, MO, USA). Complex II (PBS, pH 7.4), and then added 100ul MTT in PBS (0.5 mg/ (succinate dehydrogenase, EC 1.3.5.1) activity was Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 3 of 10 determined as a function of the decrease in absorb- addedat40units/mltoconvertallsuperoxideinto H O . 2 2 ance from 2, 6-dichloroindophenol reduction. Com- Resorufin formation (Amplex Red oxidation by H O )was 2 2 plex III (ubiquinone cytochrome c oxidoreductase, EC detected at an excitation/emission wavelength of 545/ 1.10.2.2) activity was calculated as a function of in- 590 nm using a spectrophotometer (Secomam, Domont, crease in absorbance from cytochrome c reduction. France). Readings of resorufin formation were recorded And complex IV (cytochrome c oxidoreductase, EC every 5 min for 30 min, and a slope (i.e., rate of formation) 1.9.3.1) activity was measured as a function of the de- was produced. The slope obtained was converted into the crease in absorbance from cytochrome c oxidation. rate of H O production with a standard curve. The assay 2 2 Mitochondrial complexes activities were normalized was done at 37 °C in 96-well plates using succinate. The to whole mitochondrial protein content and expressed data was converted to nmol/mg protein/minute. as arbitrary units. Quantitative assessment of apoptosis cells by flow Determination of mitochondrial total ATPase activity cytometry Cell mitochondria and submitochondrial particles As described before [17], Annexin V-APC/7-AAD Apop- were prepared as described before [14]. Briefly, the tosis Detection Kits (Becton-Dickinson Biosciences) mitochondrial particles were incubated at 37 °C for were used to detect apoptosis cells. The cells stained 60 minina0.5mlmediumcontaining2mmol/l with annexinV+/7-AAD- were considered apoptosis ATP, 100 mmol/l NaCl, 20 mmol/l KCL, 5 mmol/l cells, and the percentage of apoptosis cells was deter- MgCl , 1 mmol/l EDTA in 50 mmol/l Tris–HCl (pH mined by flow cytometry. = 7.0). The tubes were chilled immediately and cen- trifuged at 200×g for 10 min. Inorganic phosphate Statistical analysis liberated in the supernatant was calculated as an in- Software of SPSS Version 19.0 was used for statistical dication of ATPase activity according to Fiske and analysis. Numerical data are expressed as means ± SD. Subbarow [15]. Protein determination was carried The significance of differences was examined using the out in accordance with Lowry [16] with crystalline ANOVA method. Results with P<0.05 were considered bovine serum albumin as a standard. to be significant. Determination of mitochondrial ROS Results As described before [13], mitochondrial ROS production XST increased the viability of hypoxia-reoxygenated HCM was determined using Amplex Red (Molecular Probes, Eu- As shown in Fig. 1, the cell viability in the XST-treated 200, gene,OR, USA).Briefly,superoxidedismutase (SOD) was 400 and 600 μg/ml group were 77, 81 and 84%, Fig. 1 Bar graphs showed the cell viability in the three XST-treated groups were significantly increased compared with the model group. No difference was found between the XST-treated groups or the XST-treated groups compared to the control group. All samples were checked in three independent experiments with three replicates each. Data are represented as the mean ± SD ( P<0.05 vs. each of the three XST-treated groups or the control group) Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 4 of 10 respectively, which showed a significant difference XST dose-dependently increased the number of polarized than the model group (42.20%, P<0.05).However,the cells cell viability exhibited no difference between the three JC-1 is capable of entering selectively into mitochondria, dosages of XST (P >0.05). Under the light micro- and the color of the dye changes reversibly from green scope, cells in the three XST-treated groups and the to orange as the mitochondrial membrane becomes control group grew similarly well, and the cells were more polarized. Based upon the specific fluorescent like in size and shape. By contrast, most of the cells characteristics, the cells could be classified into two in the model group showed swelling and was out of groups of cells by the flow cytometer, and the two kinds of the regular shape and size (figures not shown). The fluorescence were indicated as the two populations of cells. data indicated that XST could protect the cells against Quantitative assessment was reflected by the dot plots as hypoxia-induced injury. indicated in Fig. 2 The ratio of red/green fluorescence, as Fig. 2 The cell suspensions were analyzed and sorted with flow cytometry according to the different intensity of red and green fluorescence in the five groups. X and Y axes indicated the logarithm of the relative intensity of green and red fluorescence, respectively. Bar graphs. a showed the quantitative assessment of the red/green fluorescence intensity ratio. All samples were checked in three independent experiments with three * ** replicates each. Data are represented as the mean ± SD ( P<0.05 vs. XST 400 and 600 μg/ml, P<0.001 vs. the control group) Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 5 of 10 the index of cell populations, showed a dose-dependent in- crease in the three XST-treated groups. A significant differ- ence was noted at XST400 and XST600ug/ml compared to the model group (P < 0.05), suggesting that XST could in- crease the polarized cell populations (Fig. 2a). XST dose dependently restored the loss of ΔΨm and mitochondrial mass induced by hypoxia The intensity of red fluorescence of JC-1 aggregates de- tected by confocal laser scanning microscopy decreased in the model group, however, XST could dose dependently increase the fluorescence, indicating that the drug could restore the loss of Δψm induced by hypoxia as showed in Fig. 3. Further, the red/green fluorescence ratio in XST-treated 200, 400 and 600μg/ml groups was 55, 81 and 85%, respectively, a significant difference was found in the XST-treated groups compared to the model group (18%). However, no difference was found between XST400, XST600 ug/ml and the control (P > 0.05) as indi- cated by the bar graph Fig. 3a. Further, an accurate assessment of mitochondrial mass was conducted, showing the green fluorescence increased in the XST-treated groups (Fig. 4). A significant elevation was noted in XST-400 and 600μg/ml groups compared to that in the model group, suggesting the drug prevented the loss of mitochondria in the hypoxia cells. The data was in line with the status of mitochondrial Δψm detected in above ex- periments. Additionally, morphological observations showed the cells in the XST-treated groups were more uniform in shape and size compared to those in the model group. XST restored the mitochondrial electron transport chain complexes activities ** The activities of mitochondria complexes I, II, III, and IV were assessed by spectrophotometric methods. As showed in Fig. 5, the activities of complexes I- IV were reduced in varying degrees in the model group. XST could dose de- Control Model XST200µg/ml XST400µg/ml XST600µg/ml pendently restore the activities of complexes I, II and III, Fig. 3 The mitochondrial Δψ detected by confocal laser scanning but the activity of complex IV showed no difference among microscopy showed that the intensity of red fluorescence of JC-1 XST 200, 400 and 600 μg/ml groups. Complexes I-IV activ- aggregates dropped by more than 80% in the model group compared to the control group. XST dose dependent increase in the ities were significantly elevated in XST 200 and 400 μg/ml red fluorescence was noted, indicating that the drug could restore groups than those in the model group (P < 0.05); in the the loss of mitochondrial Δψ induced by hypoxia. Morphologically, XST 600 μg/ml group, the activities of complexes I-IV re- the cells in the XST-treated groups were more regular in size and stored nearly to the normal levels. The data indicated that shape than those in the model group (× 400). Bar graphs showed the drug could dose dependently restore the activities of that the red/green fluorescence intensity ratio increased in a XST dose dependent manner. All samples were checked in three the mitochondrial electron transport chain complexes I-IV. independent experiments with three replicates each. Data are represented as the mean ± SD ( P<0.05 vs. XST 200, 400 or 600μg/ ** XST increased mitochondrial total ATPase activity in the ml, P<0.001 vs. the control group) hypoxia-reoxygenated HCM Mitochondrial total ATPase activity was determined by es- timating the amount of ATP hydrolyzed in terms of inor- decreased about 70% compared to the control (P < 0.05), ganic phosphorus (Pi) liberated in the cell supernatant. As however, the activity increased about 40, 52 and 60% in the showninFig. 6, the ATPase activity in the model group XST-treated 200, 400 and 600 μg/ml groups compared to Red/Green fluorescence intensity ratio Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 6 of 10 Fig. 4 Mitochondrial content (using MitoTracker Green) was detected in the five groups, showing the green fluorescence increased in a XST dose dependent manner. Quantitative assessment showed that XST-treated groups were significantly greater than that in the control (bar graphs (a). All samples were checked in three independent experiments with three replicates each. Data are represented as the mean ± SD ( P<0.05 vs. XST ** 400 and 600 μg/ml, P<0.001 vs. the control group) the model group (P < 0.05), which indicated that XST dose dependently increased the mitochondrial ATPase activity induced by hypoxia. The XST-induced elevation of ATPase activity was correlated with the increase in mitochondrial Δψm and the activities of mitochondrial complexes I-IV in the XST-treated groups (Figs. 3 and 5). XST decreasedmitochondrial ROS production in the hypoxia-reoxygenated HCM As showninFig. 7, the mitochondrial ROS in the model group was about three times higher than that in the control group (P<0.05), however, all the three XST-treated groups exhibited a significant decrease in ROS levels compared to the model group (P<0.05), no difference was noted between the three dosages of XST-treated groups (P > 0.05). Previ- ous studies reported that the increase in mitochondrial Δψm and ATPase activity led to the decrease in mitochon- drial ROS production [18]. Here, we confirmed that the XST-induced increase in Δψm and ATPase activity was coupled with a decrease in ROS. The data suggested that the three dosages of XST had the similar inhibitory effects on the production of mitochondrial ROS in hypoxia-reoxygenated HCM. Fig. 5 The activities of complexes I-IV were decreased variable in the XST decreased the apoptosis cells in the hypoxia- model group, XST could dose dependently restore the activities of reoxygenated HCM complexes I, II and III, but complex IV showed no difference among As detected by flow cytometry, apoptosis cells, which the three XST-treated groups. All samples were checked in three independent experiments with three replicates each. Data are stained with annexinV+/7-AAD-, were significantly in- represented as the mean ± SD ( P < 0.05 vs. XST 200 μg/ml or creased in the model group than those in the control ** ※ XST400μg/ml, P < 0.001 vs. the control or XST 600 μg/ml, P < 0.05 group (P < 0.05) (Fig. 8). XST treatment could signifi- vs. XST 400 μg/ml or XST600μg/ml) cantly decrease the apoptosis cells; however, no Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 7 of 10 Fig. 6 Mitochondrial ATPase activity in cardiomyocytes was determined by estimating the amount of ATP hydrolysis in terms of inorganic phosphorus (Pi) liberated in the cell supernatant. A significant decrease was found in the model group compared to that in the control. Three XST-treated groups exhibited a dose-dependent increase in the activity as showed in bar graphs. All samples were checked in three independent * ※ experiments with three replicates each. Data are represented as the mean ± SD ( P < 0.05 vs.XST 400 and 600 μg/ml, P < 0.05 vs. 600 μg/ml) difference was noted between the three XST-treated function. The mitochondrion is very susceptible to dam- groups (P > 0.05). The quantitative assessment of the age mediated by ischemia or ischemia/reperfusion. The apoptosis cells was indicated by bar graphs Fig. 8a. damaged mitochondria cause a depletion in ATP and a release of cytochrome c, which leads to activation of cas- Discussion pases and onset of apoptosis [19–21]. As such, maintain- Mitochondria, as the dominant source of heart ATP, ing mitochondrial homeostasis is critical to cell survival. represent approximately one-third of the mass of the Current experiments showed that XST could maintain heart and play a critical role in maintaining cellular the activities of mitochondrial electron transport chain Fig. 7 Mitochondrial ROS in the model group showed significantly higher than that in the control group, however, ROS levels exhibited a significant decrease in the XST-treated groups compared to those in the model group. No difference was noted between the three XST-treated groups. All samples were tested in three independent experiments with three replicates each. Data are represented as the mean ± SD. ( P<0.05 vs. each XST-treated group or the control group) Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 8 of 10 + − Fig. 8 Five groups were subjected to an assessment of apoptosis cells in the typical diagrams by flow cytometry (annexinV /7-AAD ). The + − percentage of annexinV /7-AAD , as the index of the apoptosis cells, increased in the model group. XST-treated groups declined the apoptosis cells as indicated by the quantitative bar graph (a). All samples were tested in three independent experiments with three replicates each. Data are represented as the mean ± SD ( P < 0.05 vs. the control or the XST-treated groups) complexes I-IV, and restore the mitochondrial Δψm and cell apoptosis. The data may partly shed a light on mitochondria mass induced by oxidative stress in the the drug’s therapeutic effects on the CHF patients. cardiomyocytes. Further, the drug could activate mito- Previous studies showed that an elevated ΔΨmwas as- chondrial total ATPase to supply cells energy to raise sociated with the enhanced mitochondrial ROS forma- the cell viability in the anoxic conditions. On the other tion [18, 23], and a slight decrease in ΔΨm could hand, XST could suppress the mitochondrial ROS gener- prevent ROS formation without seriously compromising ation and attenuate the ROS-induced damage to the cellular energetics [18, 24, 25]. However, in the isolated cells, which may partly account for this drug’s inhibition energized cardiac mitochondria, which were induced by of apoptosis and the increase in cell viability. The hypoxia, caused the inhibition of the activity of the elec- current data suggested that XST exerted its cardiopro- tron transport chain, and this mild decrease in ΔΨm led tective effects via maintaining the integrity of mitochon- to ROS formation during reoxygenation [26]. The col- dria in the hypoxia-reoxygenated HCM. lapse of mitochondrial ΔΨm, regarded as an index of Our previous clinical study showed that the CHF mitochondrial inner integrity, would go hand in hand patients treated with XST (0.3 g each capsule, three with the dysfunctions of the mitochondrial respiratory capsules each time, tid.) showed significant improve- chain and a decline of ATPase activity, which led to the ment in LVEF, NYHA classes, as well as the symp- elevation of mitochondrial ROS formation and cell apop- toms (dyspnea, edema and etc.) and the patients’ tosis. The phenomena were supported in the current ex- qualityoflife[22]. Further, in vitro experiments veri- periments as showed in Figs. 3, 5 and 7. XST treatment fied that dosages 200-600 μg/ml had the optimal cell could restore the loss of ΔΨm and repaired the dysfunc- protective effects in the oxidative stress conditions tions of the mitochondrial respiratory chain. Addition- [11]. Based on the previous data, we used these dos- ally, XST could activate ATPase and suppress ROS ages in the current study, which confirmed that XST formation, resulted in the decline in apoptosis cells in could restore the ΔΨm and mitochondria mass to the hypoxia-reoxygenated HCM. These data suggested elevate the polarized cell populations. Treated at that XST possibly exerted its cardioprotective effects XST600μg/ml, the activities of respiratory chain com- partly through mitochondria. Mitochondrial respiratory plexes I-IV regained their activities nearly to the nor- chain, especially, complex I, III and III, were seen as the mal levels, and the total of mitochondrial ATPase prime source of ROS [27–30]. ROS generations are de- activity was raised by XST. On the other hand, XST creased when the available electrons are limited and po- suppressed the generation of mitochondrial ROS and tential energy for the transfer is low [30]. Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 9 of 10 Recent study found that when the electron transport Availability of data and materials The datasets used and/or analysed during the current study available from chain functions of complexes I, III and III are at the sub the corresponding author on reasonable request. optimal level, the rate of mitochondrial free radical pro- duction is inversely increasing proportional to the rate Authors’ contributions of electron transport [31], suggesting that the elevated CT Data collection and writing the paper, JZ and YW Drug quality inspection, JZH Cell experiments Cell viability assay, ΔΨm detection, mitochondrial ROS levels found under these conditions may originate respiratory chain complexes, total ATPase activity, mitochondrial ROS, extra-mitochondrially or are attributed to the defective apoptosis cells, WC Study design and experimental director, All authors antioxidant defense. Our experiments showed that have read and approved the final manuscript to submitted to this journal. XST-induced elevation of mitochondrial respiratory Ethics approval and consent to participate chain complexes activities was inversely correlated with Not applicable. the levels of mitochondria-derived ROS in the XST-treated groups (Figs. 5 and 7). Possibly, the drug Competing interests suppressed ROS generation extra-mitochondrially, or The authors declare that they have no competing interests. through the antioxidant defense or other mechanisms. For example, other powerful sources of mitochondrial Publisher’sNote ROS production are represented by the mitochondrial Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. NO synthase [32, 33], and the byproducts of several cel- lular enzymes including NADPH oxidases, xanthine oxi- Received: 14 March 2018 Accepted: 21 May 2018 dase [22, 23, 34]. 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Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch Biochem Biophys. 1996;328:85–92. 33. Dröge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002;82:47–95. 34. Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol. 2003;552:335–44. 35. Mathur A, Hong Y, Kemp BK. Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes. Cardiovasc Res. 2000;46:126–38. 36. Suh DH, Kim MK, Kim HS. Mitochondrial permeability transition pore as a selective target for anti-cancer therapy. Front Oncol. 2013;3:41. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Complementary and Alternative Medicine Springer Journals

Herbal formula Xinshuitong capsule exerts its cardioprotective effects via mitochondria in the hypoxia-reoxygenated human cardiomyocytes

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Medicine & Public Health; Complementary & Alternative Medicine; Internal Medicine; Chiropractic Medicine
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Abstract

Background: The collapse of mitochondrial membrane potential (ΔΨm) resulted in the cell apoptosis and heart failure. Xinshuitong Capsule (XST) could ameliorate left ventricular ejection fraction (LVEF), New York Heart Association (NYHA) classes and the quality of life in patients with chronic heart failure in our clinical study, however, its cardioprotective mechanisms remain unclear. Methods: Primary human cardiomyocytes were subjected to hypoxia-reoxygenation and treated with XST200, 400 and 600 μg/ml. The model group was free of XST and the control group was cultured in normal conditions. Cell viability, ΔΨm, the activity of mitochondrial respiratory chain complexes, ATPase activity, reactive oxygen species (ROS) and apoptosis cells were determined in all the groups. Results: The cell viability in the XST-treated groups was significantly higher than that in the model group (P < 0.05). Coupled with the restoration of the ΔΨm, the number of polarized cells increased dose dependently in the XST- treated groups. XST also restored the lost activities of mitochondrial respiratory chain complexes I-IV induced by the oxidative stress. The total of mitochondrial ATPase activity was significantly elevated at XST400 and 600 μg/ ml compared to the model group (P < 0.05). The levels of mitochondrial ROS and the number of apoptosis cells declined in the XST-treated groups compared to those in the model group (P < 0.05). Conclusions: XST, via restoration of ΔΨm and the mitochondrial respiratory chain complexes I-IV activities, and suppression of mitochondrial ROS generation and the apoptosis cells, maintained the integrity of the mitochondrial membrane to exert its cardioprotective effects in the hypoxia-reoxygenated human cardiomyocytes. Keywords: Xinshuitong capsule, Mitochondrial potential, Hypoxia-reoxygenated human cardiomyocytes Background and uncoupling. Thus, MPTP opening and mitochondrial Mitochondria, as the power house of the heart, are highly ΔΨm collapse have been regarded as a primary mediator of packed in the cardiomyocytes. Cardiac cells under pro- apoptosis in the ischemia-reperfusion heart injury [2, 3]. longed hypoxia condition have been shown with the open- The mitochondrial electron transport chain (ETC) is ing of mitochondrial permeability transition pore (MPTP) found in the inner membrane, where it serves as the site [1]. Due to the opening of MPTP causes a transient hyper- of oxidative phosphorylation through the use of ATP polarization, followed by depolarization, and subsequently synthase. During this chemical process, ROS can be the collapse of the mitochondrial membrane potential formed as a byproduct of normal cellular aerobic metab- (ΔΨm), which is characterized by mitochondrial swelling olism in the heart [4, 5]. Thus, the major process from which the heart derives sufficient energy can also result in the production of ROS [5]. On the other hand, ROS * Correspondence: tchunj@126.com; chen.wl@163.com can depress the activity of mitochondrial ETC and alter Chunjiang Tan and Wenlie Chen contributed equally to this work. Fujian Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 2 of 10 ion pump function in heart [6]. Mounting evidence has ml) and incubated for 4 h at 37 °C. Followed by removing strongly implicated ROS signaling in the genesis of car- MTT and oscillating for 10 min, cell viability was estimated diac hypertrophy [7–9]. Therefore, maintaining the in- at absorbance 570 nm by a Tecan Infinite M200 Pro micro- tegrity of the mitochondrial membrane, enhancing plate reader (Tecan, Mannedorf, Swizerland). antioxidant defense may be a therapeutic method for the protection of cardiomyocytes against the injury of ische- Mitochondrial ΔΨm detected by JC-1 staining mia or hypoxia. As JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimida- Xinshuitong Capsule (XST, awarded the Invention Patent zolyl-carbocyanine iodide, Beyotime, China) is a lipophilic of the People’s Republic of China, No.ZL201210197892.X), a fluorescentcationthatisincorporatedintothe mitochon- Chinese herbal medicine formula for chronic heart failure drial membrane, where it can form aggregates due to the (CHF), which consists of Astragali radix, Pseudostellariae state of the mitochondrial ΔΨm. This aggregation changes radix and Salviae miltiorrhizae radix et al., has the effects of the fluorescence properties of JC-1 from green to orange benefiting Qi and Yang, activating blood and eliminating sta- fluorescence as the ΔΨm increased. After treatment, cells sis, and inducing diuresis to alleviate edema. Our previous were harvested, re-suspended and incubated with 10 μg/ml clinical study showed that the CHF patients, who received JC-1 at 37 °C for 30 min as before [13, 14]. The cells were XST treatment (3 capsules, tid.), were significantly amelio- then washed and centrifuged, the intact living cells stained rated in left ventricular ejection fraction (LVEF), New York the mitochondria with JC-1 would exhibit a pronounced Heart Association (NYHA) classes, the symptoms and the orange fluorescence, however, the cells with a breakdown quality of life compared to the control group [10]. In vitro, of ΔΨm showed a decrease of the orange fluorescence (or a XST-treated hypoxia-reoxygenated human cardiomyocytes increase of the green fluorescence). Thus, the intact and in- showed more tolerant to hypoxia stress. The cells exhibited jured cells could be distinguished, and the cell populations more regular shape and size than the control [11]. However, will be counted according to the different fluorescence by the drug’s cardioprotective mechanisms remain elusive, es- the flow cytometry (BD Biosciences, CA) (JC-1 green: Ex/ pecially, its actions on MPTP and mitochondrial apoptosis Em = 485/525 nm; JC-1 red: Ex/Em =535/590 nm). pathway. Thus, in current experiments, mitochondrial ΔΨm Similarly, the fluorescence intensity of JC-1 as the index and mitochondrial mass, the activities of the mitochondrial of ΔΨm alterations could be detected by a confocal micro- ETC and the mitochondrial ATPase, and their associations scope (Carl Zeiss AG, Oberkochen, Germany), and the ra- with ROS levels and apoptosis cells will be studied in the tio of red/green fluorescence intensity is indicated as the hypoxia-reoxygenated cardiomyocytes. alterations of mitochondrial ΔΨm. For quantification of mitochondrial mass, we used Mito- Methods tracker Green probe (Molecular Probes), which preferen- Hypoxia-reoxygenated cell model tially accumulates in mitochondria regardless of the Primary human cardiomyocyte (HCM) was purchased mitochondrial membrane potential and provides an accur- from American Science Cell Research Laboratories (San ate assessment of mitochondrial mass. Firstly, the cells were Diego. USA). When the cells reached 80–90% conflu- washed with PBS and incubated at 37 °C for 30 min with ence, they ere placed on a 96-well plate or a petri dish at 100 nM MitoTracker Green FM (Molecular Probes) and a density of 0.75 × 105cells/ml in a hypoxia chamber then harvested using trypsin/EDTA and re-suspended in (80%N ,10%H ,10%CO and 0.2%O ) for 12 h,11 follow- PBS. Fluorescence intensity was detected with excitation 2 2 2 2 ing by 2 h reoxygenation. During the process of hypoxia and emission wavelengths of 490 and 516 nm, respectively, and reoxygenation, the study group were exposed to the and values were corrected for total protein (mg/ml). water-extract of XST (200, 400 and 600 μg/ml, respect- ively), while the model group was cultured in the identi- Determination of the activities of mitochondrial cal conditions free of XST treatment, and the control respiratory chain complexes group was cultured in normal conditions. The drug’s According to manufacturer’s instructions, mitochondrial low and high concentrations used in the current experi- isolation was performed at 4 °C using a Kit for cultured ment were comparable to the human serum levels in the mammal cell (Thermo Scientific Rockford. USA). previous clinical study. The activities of mitochondrial respiratory chain com- plexes were analyzed by spectrophotometer (Secomam, Cell viability assay Domont,France) as described before [13]. Briefly, com- As described before [12], cell viability was estimated by the plex I (NADH dehydrogenase, EC 1.6.5.3) enzyme activ- assay of 3-[4,5-dimethylthiazol-2-yl]-diphenyl-tetrazolium ity was measured as a decline in absorbance from bromide (MTT, Sigma-Aldrich). Briefly, after treatment, the NADH oxidation by decylubiquinone before and after cells were washed twice with phosphate-buffered saline adding rotenone (St. Louis, MO, USA). Complex II (PBS, pH 7.4), and then added 100ul MTT in PBS (0.5 mg/ (succinate dehydrogenase, EC 1.3.5.1) activity was Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 3 of 10 determined as a function of the decrease in absorb- addedat40units/mltoconvertallsuperoxideinto H O . 2 2 ance from 2, 6-dichloroindophenol reduction. Com- Resorufin formation (Amplex Red oxidation by H O )was 2 2 plex III (ubiquinone cytochrome c oxidoreductase, EC detected at an excitation/emission wavelength of 545/ 1.10.2.2) activity was calculated as a function of in- 590 nm using a spectrophotometer (Secomam, Domont, crease in absorbance from cytochrome c reduction. France). Readings of resorufin formation were recorded And complex IV (cytochrome c oxidoreductase, EC every 5 min for 30 min, and a slope (i.e., rate of formation) 1.9.3.1) activity was measured as a function of the de- was produced. The slope obtained was converted into the crease in absorbance from cytochrome c oxidation. rate of H O production with a standard curve. The assay 2 2 Mitochondrial complexes activities were normalized was done at 37 °C in 96-well plates using succinate. The to whole mitochondrial protein content and expressed data was converted to nmol/mg protein/minute. as arbitrary units. Quantitative assessment of apoptosis cells by flow Determination of mitochondrial total ATPase activity cytometry Cell mitochondria and submitochondrial particles As described before [17], Annexin V-APC/7-AAD Apop- were prepared as described before [14]. Briefly, the tosis Detection Kits (Becton-Dickinson Biosciences) mitochondrial particles were incubated at 37 °C for were used to detect apoptosis cells. The cells stained 60 minina0.5mlmediumcontaining2mmol/l with annexinV+/7-AAD- were considered apoptosis ATP, 100 mmol/l NaCl, 20 mmol/l KCL, 5 mmol/l cells, and the percentage of apoptosis cells was deter- MgCl , 1 mmol/l EDTA in 50 mmol/l Tris–HCl (pH mined by flow cytometry. = 7.0). The tubes were chilled immediately and cen- trifuged at 200×g for 10 min. Inorganic phosphate Statistical analysis liberated in the supernatant was calculated as an in- Software of SPSS Version 19.0 was used for statistical dication of ATPase activity according to Fiske and analysis. Numerical data are expressed as means ± SD. Subbarow [15]. Protein determination was carried The significance of differences was examined using the out in accordance with Lowry [16] with crystalline ANOVA method. Results with P<0.05 were considered bovine serum albumin as a standard. to be significant. Determination of mitochondrial ROS Results As described before [13], mitochondrial ROS production XST increased the viability of hypoxia-reoxygenated HCM was determined using Amplex Red (Molecular Probes, Eu- As shown in Fig. 1, the cell viability in the XST-treated 200, gene,OR, USA).Briefly,superoxidedismutase (SOD) was 400 and 600 μg/ml group were 77, 81 and 84%, Fig. 1 Bar graphs showed the cell viability in the three XST-treated groups were significantly increased compared with the model group. No difference was found between the XST-treated groups or the XST-treated groups compared to the control group. All samples were checked in three independent experiments with three replicates each. Data are represented as the mean ± SD ( P<0.05 vs. each of the three XST-treated groups or the control group) Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 4 of 10 respectively, which showed a significant difference XST dose-dependently increased the number of polarized than the model group (42.20%, P<0.05).However,the cells cell viability exhibited no difference between the three JC-1 is capable of entering selectively into mitochondria, dosages of XST (P >0.05). Under the light micro- and the color of the dye changes reversibly from green scope, cells in the three XST-treated groups and the to orange as the mitochondrial membrane becomes control group grew similarly well, and the cells were more polarized. Based upon the specific fluorescent like in size and shape. By contrast, most of the cells characteristics, the cells could be classified into two in the model group showed swelling and was out of groups of cells by the flow cytometer, and the two kinds of the regular shape and size (figures not shown). The fluorescence were indicated as the two populations of cells. data indicated that XST could protect the cells against Quantitative assessment was reflected by the dot plots as hypoxia-induced injury. indicated in Fig. 2 The ratio of red/green fluorescence, as Fig. 2 The cell suspensions were analyzed and sorted with flow cytometry according to the different intensity of red and green fluorescence in the five groups. X and Y axes indicated the logarithm of the relative intensity of green and red fluorescence, respectively. Bar graphs. a showed the quantitative assessment of the red/green fluorescence intensity ratio. All samples were checked in three independent experiments with three * ** replicates each. Data are represented as the mean ± SD ( P<0.05 vs. XST 400 and 600 μg/ml, P<0.001 vs. the control group) Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 5 of 10 the index of cell populations, showed a dose-dependent in- crease in the three XST-treated groups. A significant differ- ence was noted at XST400 and XST600ug/ml compared to the model group (P < 0.05), suggesting that XST could in- crease the polarized cell populations (Fig. 2a). XST dose dependently restored the loss of ΔΨm and mitochondrial mass induced by hypoxia The intensity of red fluorescence of JC-1 aggregates de- tected by confocal laser scanning microscopy decreased in the model group, however, XST could dose dependently increase the fluorescence, indicating that the drug could restore the loss of Δψm induced by hypoxia as showed in Fig. 3. Further, the red/green fluorescence ratio in XST-treated 200, 400 and 600μg/ml groups was 55, 81 and 85%, respectively, a significant difference was found in the XST-treated groups compared to the model group (18%). However, no difference was found between XST400, XST600 ug/ml and the control (P > 0.05) as indi- cated by the bar graph Fig. 3a. Further, an accurate assessment of mitochondrial mass was conducted, showing the green fluorescence increased in the XST-treated groups (Fig. 4). A significant elevation was noted in XST-400 and 600μg/ml groups compared to that in the model group, suggesting the drug prevented the loss of mitochondria in the hypoxia cells. The data was in line with the status of mitochondrial Δψm detected in above ex- periments. Additionally, morphological observations showed the cells in the XST-treated groups were more uniform in shape and size compared to those in the model group. XST restored the mitochondrial electron transport chain complexes activities ** The activities of mitochondria complexes I, II, III, and IV were assessed by spectrophotometric methods. As showed in Fig. 5, the activities of complexes I- IV were reduced in varying degrees in the model group. XST could dose de- Control Model XST200µg/ml XST400µg/ml XST600µg/ml pendently restore the activities of complexes I, II and III, Fig. 3 The mitochondrial Δψ detected by confocal laser scanning but the activity of complex IV showed no difference among microscopy showed that the intensity of red fluorescence of JC-1 XST 200, 400 and 600 μg/ml groups. Complexes I-IV activ- aggregates dropped by more than 80% in the model group compared to the control group. XST dose dependent increase in the ities were significantly elevated in XST 200 and 400 μg/ml red fluorescence was noted, indicating that the drug could restore groups than those in the model group (P < 0.05); in the the loss of mitochondrial Δψ induced by hypoxia. Morphologically, XST 600 μg/ml group, the activities of complexes I-IV re- the cells in the XST-treated groups were more regular in size and stored nearly to the normal levels. The data indicated that shape than those in the model group (× 400). Bar graphs showed the drug could dose dependently restore the activities of that the red/green fluorescence intensity ratio increased in a XST dose dependent manner. All samples were checked in three the mitochondrial electron transport chain complexes I-IV. independent experiments with three replicates each. Data are represented as the mean ± SD ( P<0.05 vs. XST 200, 400 or 600μg/ ** XST increased mitochondrial total ATPase activity in the ml, P<0.001 vs. the control group) hypoxia-reoxygenated HCM Mitochondrial total ATPase activity was determined by es- timating the amount of ATP hydrolyzed in terms of inor- decreased about 70% compared to the control (P < 0.05), ganic phosphorus (Pi) liberated in the cell supernatant. As however, the activity increased about 40, 52 and 60% in the showninFig. 6, the ATPase activity in the model group XST-treated 200, 400 and 600 μg/ml groups compared to Red/Green fluorescence intensity ratio Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 6 of 10 Fig. 4 Mitochondrial content (using MitoTracker Green) was detected in the five groups, showing the green fluorescence increased in a XST dose dependent manner. Quantitative assessment showed that XST-treated groups were significantly greater than that in the control (bar graphs (a). All samples were checked in three independent experiments with three replicates each. Data are represented as the mean ± SD ( P<0.05 vs. XST ** 400 and 600 μg/ml, P<0.001 vs. the control group) the model group (P < 0.05), which indicated that XST dose dependently increased the mitochondrial ATPase activity induced by hypoxia. The XST-induced elevation of ATPase activity was correlated with the increase in mitochondrial Δψm and the activities of mitochondrial complexes I-IV in the XST-treated groups (Figs. 3 and 5). XST decreasedmitochondrial ROS production in the hypoxia-reoxygenated HCM As showninFig. 7, the mitochondrial ROS in the model group was about three times higher than that in the control group (P<0.05), however, all the three XST-treated groups exhibited a significant decrease in ROS levels compared to the model group (P<0.05), no difference was noted between the three dosages of XST-treated groups (P > 0.05). Previ- ous studies reported that the increase in mitochondrial Δψm and ATPase activity led to the decrease in mitochon- drial ROS production [18]. Here, we confirmed that the XST-induced increase in Δψm and ATPase activity was coupled with a decrease in ROS. The data suggested that the three dosages of XST had the similar inhibitory effects on the production of mitochondrial ROS in hypoxia-reoxygenated HCM. Fig. 5 The activities of complexes I-IV were decreased variable in the XST decreased the apoptosis cells in the hypoxia- model group, XST could dose dependently restore the activities of reoxygenated HCM complexes I, II and III, but complex IV showed no difference among As detected by flow cytometry, apoptosis cells, which the three XST-treated groups. All samples were checked in three independent experiments with three replicates each. Data are stained with annexinV+/7-AAD-, were significantly in- represented as the mean ± SD ( P < 0.05 vs. XST 200 μg/ml or creased in the model group than those in the control ** ※ XST400μg/ml, P < 0.001 vs. the control or XST 600 μg/ml, P < 0.05 group (P < 0.05) (Fig. 8). XST treatment could signifi- vs. XST 400 μg/ml or XST600μg/ml) cantly decrease the apoptosis cells; however, no Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 7 of 10 Fig. 6 Mitochondrial ATPase activity in cardiomyocytes was determined by estimating the amount of ATP hydrolysis in terms of inorganic phosphorus (Pi) liberated in the cell supernatant. A significant decrease was found in the model group compared to that in the control. Three XST-treated groups exhibited a dose-dependent increase in the activity as showed in bar graphs. All samples were checked in three independent * ※ experiments with three replicates each. Data are represented as the mean ± SD ( P < 0.05 vs.XST 400 and 600 μg/ml, P < 0.05 vs. 600 μg/ml) difference was noted between the three XST-treated function. The mitochondrion is very susceptible to dam- groups (P > 0.05). The quantitative assessment of the age mediated by ischemia or ischemia/reperfusion. The apoptosis cells was indicated by bar graphs Fig. 8a. damaged mitochondria cause a depletion in ATP and a release of cytochrome c, which leads to activation of cas- Discussion pases and onset of apoptosis [19–21]. As such, maintain- Mitochondria, as the dominant source of heart ATP, ing mitochondrial homeostasis is critical to cell survival. represent approximately one-third of the mass of the Current experiments showed that XST could maintain heart and play a critical role in maintaining cellular the activities of mitochondrial electron transport chain Fig. 7 Mitochondrial ROS in the model group showed significantly higher than that in the control group, however, ROS levels exhibited a significant decrease in the XST-treated groups compared to those in the model group. No difference was noted between the three XST-treated groups. All samples were tested in three independent experiments with three replicates each. Data are represented as the mean ± SD. ( P<0.05 vs. each XST-treated group or the control group) Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 8 of 10 + − Fig. 8 Five groups were subjected to an assessment of apoptosis cells in the typical diagrams by flow cytometry (annexinV /7-AAD ). The + − percentage of annexinV /7-AAD , as the index of the apoptosis cells, increased in the model group. XST-treated groups declined the apoptosis cells as indicated by the quantitative bar graph (a). All samples were tested in three independent experiments with three replicates each. Data are represented as the mean ± SD ( P < 0.05 vs. the control or the XST-treated groups) complexes I-IV, and restore the mitochondrial Δψm and cell apoptosis. The data may partly shed a light on mitochondria mass induced by oxidative stress in the the drug’s therapeutic effects on the CHF patients. cardiomyocytes. Further, the drug could activate mito- Previous studies showed that an elevated ΔΨmwas as- chondrial total ATPase to supply cells energy to raise sociated with the enhanced mitochondrial ROS forma- the cell viability in the anoxic conditions. On the other tion [18, 23], and a slight decrease in ΔΨm could hand, XST could suppress the mitochondrial ROS gener- prevent ROS formation without seriously compromising ation and attenuate the ROS-induced damage to the cellular energetics [18, 24, 25]. However, in the isolated cells, which may partly account for this drug’s inhibition energized cardiac mitochondria, which were induced by of apoptosis and the increase in cell viability. The hypoxia, caused the inhibition of the activity of the elec- current data suggested that XST exerted its cardiopro- tron transport chain, and this mild decrease in ΔΨm led tective effects via maintaining the integrity of mitochon- to ROS formation during reoxygenation [26]. The col- dria in the hypoxia-reoxygenated HCM. lapse of mitochondrial ΔΨm, regarded as an index of Our previous clinical study showed that the CHF mitochondrial inner integrity, would go hand in hand patients treated with XST (0.3 g each capsule, three with the dysfunctions of the mitochondrial respiratory capsules each time, tid.) showed significant improve- chain and a decline of ATPase activity, which led to the ment in LVEF, NYHA classes, as well as the symp- elevation of mitochondrial ROS formation and cell apop- toms (dyspnea, edema and etc.) and the patients’ tosis. The phenomena were supported in the current ex- qualityoflife[22]. Further, in vitro experiments veri- periments as showed in Figs. 3, 5 and 7. XST treatment fied that dosages 200-600 μg/ml had the optimal cell could restore the loss of ΔΨm and repaired the dysfunc- protective effects in the oxidative stress conditions tions of the mitochondrial respiratory chain. Addition- [11]. Based on the previous data, we used these dos- ally, XST could activate ATPase and suppress ROS ages in the current study, which confirmed that XST formation, resulted in the decline in apoptosis cells in could restore the ΔΨm and mitochondria mass to the hypoxia-reoxygenated HCM. These data suggested elevate the polarized cell populations. Treated at that XST possibly exerted its cardioprotective effects XST600μg/ml, the activities of respiratory chain com- partly through mitochondria. Mitochondrial respiratory plexes I-IV regained their activities nearly to the nor- chain, especially, complex I, III and III, were seen as the mal levels, and the total of mitochondrial ATPase prime source of ROS [27–30]. ROS generations are de- activity was raised by XST. On the other hand, XST creased when the available electrons are limited and po- suppressed the generation of mitochondrial ROS and tential energy for the transfer is low [30]. Tan et al. BMC Complementary and Alternative Medicine (2018) 18:170 Page 9 of 10 Recent study found that when the electron transport Availability of data and materials The datasets used and/or analysed during the current study available from chain functions of complexes I, III and III are at the sub the corresponding author on reasonable request. optimal level, the rate of mitochondrial free radical pro- duction is inversely increasing proportional to the rate Authors’ contributions of electron transport [31], suggesting that the elevated CT Data collection and writing the paper, JZ and YW Drug quality inspection, JZH Cell experiments Cell viability assay, ΔΨm detection, mitochondrial ROS levels found under these conditions may originate respiratory chain complexes, total ATPase activity, mitochondrial ROS, extra-mitochondrially or are attributed to the defective apoptosis cells, WC Study design and experimental director, All authors antioxidant defense. Our experiments showed that have read and approved the final manuscript to submitted to this journal. XST-induced elevation of mitochondrial respiratory Ethics approval and consent to participate chain complexes activities was inversely correlated with Not applicable. the levels of mitochondria-derived ROS in the XST-treated groups (Figs. 5 and 7). Possibly, the drug Competing interests suppressed ROS generation extra-mitochondrially, or The authors declare that they have no competing interests. through the antioxidant defense or other mechanisms. For example, other powerful sources of mitochondrial Publisher’sNote ROS production are represented by the mitochondrial Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. NO synthase [32, 33], and the byproducts of several cel- lular enzymes including NADPH oxidases, xanthine oxi- Received: 14 March 2018 Accepted: 21 May 2018 dase [22, 23, 34]. 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BMC Complementary and Alternative MedicineSpringer Journals

Published: May 31, 2018

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