Introduction
Ischemic stroke, which accounts for almost all of all strokes, is characterised by the blockage or narrowing of an artery that provides blood to the mind; subsequently, it’s related to excessive morbidity, excessive mortality, excessive recurrence fee, and a number of other issues.1 Also called Cerebral Artery Thrombosis, ischemic stroke happens due to plaque formation and build-up within the arteries (atherosclerosis [AS]), thereby triggering mind vitality metabolism problems, excitatory amino acid toxicity, oxidative/nitrification stress harm, irritation, cell apoptosis, autophagy, and a collection of pathological reactions.2 The mind is inclined to oxidative harm as a result of its excessive lipid content material and oxygen consumption.3 Most research have proven that oxidative stress and lipid peroxidation within the early stage of cerebral ischemia induce neuroinflammation, resulting in mind harm.4,5 Underneath regular physiological situations, oxygen-free radicals produced by mitochondria could be eradicated by antioxidant techniques, equivalent to superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) within the physique. Nevertheless, in cerebral ischemia, vitality depletion results in the buildup of lactic acid within the cells, thereby leading to acidosis and loss of life of neurons.6 Due to this fact, lowering neuronal accidents attributable to oxidative stress could be adopted as a promising technique for the therapy of stroke.
Huangqi Guizhi Wuwu Decoction (HGWD) is a classical Chinese language prescription described in Jingui yaolue, written by Zhang Zhongjing throughout the Jap Han Dynasty.7 It’s composed of Radix Astragali (Huangqi), Ramulus Cinnamomi (Guizhi), Paeoniae Radix Alba (Baishao), Zingiberis Rhizoma Recens (Shengjiang), and Jujubae Fructus (Dazhao). Medical research have proven that HGWD can promote the restoration of sufferers’ nerve perform, enhance cerebral blood circulation, and exert a number of therapeutic results.8 Nevertheless, the scientific foundation and potential pharmacological mechanisms of HGWD within the therapy of ischemic stroke stays unclear.
Trendy pharmacological research have demonstrated that the interplay between a drug and the goal protein (drug–receptor interplay) on the cell membrane is a vital step in figuring out the pharmacological exercise of a molecule.9 Cell membrane chromatography is a well-developed organic chromatographic approach during which cell membranes containing receptors are used because the stationary part and has been utilized for screening lively elements from complicated medicines, equivalent to conventional Chinese language medicines (TCM). This technique relies on the incubation of TCM extracts with reside cells adopted by solid-phase affinity chromatography.10 Our topic group has efficiently employed this technique to display six potential angiogenesis-promoting compounds from Buyang Huanwu Decoction in a earlier analysis.11,12
This research goals to determine the potential therapeutic elements of HGWD primarily based on the strategy of cell membrane solid-phase chromatography. First, an optimized solid-phase extraction (SPE) technique was used to pay attention the dissociation answer of HT22 cells handled with the HGWD extract, adopted by the identification of HT22-binding compounds by high-performance liquid chromatography-mass spectroscopy/mass spectroscopy (HPLC-MS/MS). Then, cell viability assay was utilized to judge the impact of the recognized compounds on defending HT22 cells from oxygen-glucose deprivation reperfusion (OGD/R) harm. Lastly, the potential mechanism of the lively components of HGWD for the therapy of stroke was elucidated by detecting the expression ranges of reactive oxygen species (ROS), SOD, and lactate dehydrogenase (LDH). The detailed workflow is depicted in Figure 1.
Determine 1 The workflow of the experimental procedures; *P<0.05 vs management group, #P < 0.05 vs OGD/R group. Abbreviations: HGWD, Huangqi Guizhi Wuwu Decoction; SPE, solid-phase extraction. |
Supplies and Strategies
Supplies
HGWD, composed of 5 Chinese language natural medicines (Table 1), was bought from Kangmei Pharmaceutical Co., Ltd (Guangzhou, China) and recognized by Professor Danyan Zhang from Guangzhou College of Chinese language Drugs (Guangzhou, China). Calycosin-7-O-glucoside, calycosin, formononetin, cinnamic alcohol, cinnamic acid, betaine, dl-2-phenylpropionic acid, 4-hydroxycinnamic acid, 6-methylcoumarin, wogonoside, and paeoniflorin have been bought from Chengdu Keloma Biotechnology Co., Ltd. (Chengdu, China). Nimodipine was bought from Shanghai Yuanye Bio-Know-how Co., Ltd. The purity of all of the reference requirements was above 98%. Bovine serum albumin (BSA), DMEM medium (Dulbecco’s Modified Eagle Medium), trypsin (1:250), and phosphate-buffered saline (PBS) buffer have been obtained from GIBCO (Invitrogen Company, Carlsbad, CA, USA). Stable-phase extraction (SPE) columns have been bought from Phenomenex Inc. (Torrance, CA, USA). Cell Counting Package-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). LDH, SOD, and ROS assay kits have been obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). HT22 cells have been obtained from Shanghai Kanglang Biotechnology Co., Ltd.
Desk 1 Parts of Huangqi Guizhi Wuwu Decoction (HGWD) |
Preparation of HGWD
The HGWD extract was ready in line with our earlier research.13 For this, Astragali Radix (9 g), Ramulus Cinnamomi (9 g), Paeoniae Radix Alba (9 g), Zingiberis Rhizoma Recens (18 g), and Jujubae Fructus (4 items) have been combined and immersed in distilled water (1:10 w/v) for 30 min and extracted by refluxing for 1 h. The obtained filtrate was mixed and concentrated to 50 mL, after which, the focus was adsorbed by D101 macroporous resin. Water-soluble elements have been eliminated with water. Then, 20%, 40%, and 70% ethanol eluates have been collected and concentrated beneath diminished stress to acquire a 1.08 g/mL pattern answer. The pattern was filtered by means of a 0.22 µm microporous membrane for additional evaluation.
High quality Management of HGWD
The standard management of HGWD was carried out with HPLC, and the content material of HGWD was decided as regards to paeoniflorin, calycosin-7-O-glucoside, and calycosin.
The next chromatographic situations have been used: Waters C18 column (4.6 mm × 250 mm, 5 µm); cell part, acetonitrile (A) and 0.025% formic acid in water (B); column temperature, 30 °C; circulation fee, 1.0 mL/min; and injection quantity, 10 µL. The elution situations are compiled in Table S1. The tactic was validated by way of specificity, precision, accuracy, extraction restoration, and stability.
Biospecific Stay-Cell-Primarily based Isolation of HGWD Parts
HT22 cells (Shanghai Kanglang Biotechnology Co., Ltd.) have been cultured in a ten cm tradition dish. When the expansion density reached 80%, HGWD extract was added to the HGWD group, and the identical quantity of medium was added to the management group. To facilitate the binding of lively elements to their corresponding receptors, each teams have been incubated at 37°C for 4 h. Afterward, the cells have been washed 5 occasions with PBS to take away unbound elements; the sure elements have been dissociated from HT22 by sodium citrate buffer (pH 4.0). The collected dissociation pattern was enriched and purified with an SPE column. Then, the eluents have been dried utilizing nitrogen, dissolved in 1 mL of fifty% methanol, and filtered by means of a 0.22 µm membrane for HPLC-MS/MS evaluation. The experiment was repeated 3 times.
HPLC-MS/MS
HPLC was carried out on an instrument geared up with an SIL-82A autosampler, LC-20A pump, and SPD-20A detector (Shimadzu, Japan). The next chromatographic situations have been used: Kinetex C18 column (50 mm × 2.1 mm, 2.6 µm); cell part A, acetonitrile, and cell part B, 0.025% formic acid in water; column temperature, 35 °C; circulation fee, 0.3 mL/min; and injection quantity, 3 µL. The elution situation was as follows: 10–30% A at 0–5.5 min, 30–60% A at 5.5–9 min, and 60–90% A at 9–15 min.
MS evaluation was carried out by AB SCIEX Triple TOF 5600+ system (Framingham, MA, USA). Following MS situations have been used: ESI ion supply, the scan vary of fifty–1200 Da, and scanning in optimistic and unfavourable ion mode. The optimum MS parameter settings have been as follows: ion spray voltage floating (ISVF), 4500 V; ion supply gasoline 1(GS1), 55 psi; ion supply gasoline 2(GS1), 55 psi; curtain gasoline (CUR), 35 psi; collision vitality (CE), 45 V; temperature, 600 °C. The outcomes have been analyzed utilizing Peak View software program to extract info, equivalent to fragment ions. The pattern info was additionally in contrast with customary supplies and literature for additional identification.
Institution of OGD/R Mannequin and Drug Remedy
HT22 cells have been cultured in a DMEM medium containing 10% FBS in an incubator (37 °C, 5% CO2). When the cell density reached 90%, the medium was eliminated, a glucose-free medium was added, and the plate was incubated in an incubator (95% N2 and 5% CO2) at 37 °C for 8 h to induce OGD/R damage. Then, the medium was changed with DMEM medium, and the cells have been additional incubated at 37 °C for six h. To find out the impact of the recognized elements on HT22 cells with OGD/R damage, experiment teams have been shaped with or with out recognized elements. Take a look at teams consisted of cells handled with calycosin-7-O-glucoside, calycosin, formononetin, cinnamic alcohol, cinnamic acid, betaine, dl-2-phenylpropionic acid, 4-hydroxycinnamic acid, 6-methylcoumarin, wogonoside, and paeoniflorin dissolved in DMSO (0.1%) and diluted with DMEM medium to acceptable concentrations. The management group was handled with DMEM medium (0.1% DMSO), whereas nimodipine was used because the optimistic management (5 µg/mL).
Cell Viability
To find out the viability of cells after treating with completely different medicine, CCK-8 equipment was employed. After therapy with completely different medicine, a whole medium containing 10% CCK-8 was added to HT22 cells (1 × 104cells per nicely) seeded into 96-well plates and incubated for two h. The OD worth at 450 nm of every group was measured utilizing a microplate reader (Thermo Scientific, Rockford, IL, United States). Cell viability was calculated primarily based on the next formulation: Cell viability = OD450 of the take a look at group/OD450 of the management group × 100%.
Launch Assay
The discharge of LDH was measured by an LDH assay equipment (Nanjing Jiancheng Institute of Organic Engineering, Nanjing, China). The cells have been first handled with OGD/R after which incubated with completely different medicine; the cell supernatant obtained after this step was incubated with substrate answer at 37 °C for 15 min. To this, 2,4-dinitrophenylhydrazine was added and incubated for one more 15 min. The absorbance sign was detected on a microplate reader at 450 nm. Every experiment was carried out in sextuplicate.
Detection of ROS
The degrees of ROS of every group have been evaluated by a equipment using the fluorescent probe 2ʹ,7ʹ-dichlorofluorescein-diacetate (DCFH-DA). The cells have been cultured in 96-well plates for twenty-four h and handled with OGD/R, apart from the conventional group. OGD/R-treated HT22 cells have been incubated with calycosin-7-O-glucoside (5,10, and 20 µg/mL), calycosin (0.5,1, and a couple of µg/mL), paeoniflorin (5,10, and 20 µg/mL), wogonoside (0.5,1, and a couple of µg/mL), and formononetin (5,10, and 20 µg/mL) for twenty-four h. Then, the DCFH-DA fluorescent probe working answer was added, and the plates have been incubated at 37 °C at nighttime for 30 min. The depth of ROS was measured at an OD of 525 nm. Every experiment was carried out in sextuplicate.
Dedication of SOD Content material
SODs are a bunch of detoxing enzymes as they’re highly effective antioxidants. This enzyme can scavenge free radicals, equivalent to superoxide anion, thereby defending cells from harm attributable to free radicals. The SOD exercise was measured using WST-1 that produces a water-soluble formazan dye upon discount with superoxide anion. The OGD/R-treated cells have been incubated with completely different medicine; the cell supernatant obtained after this step was incubated with enzyme working answer and substrate utility answer at 37°C for 20 min. The fluorescence depth was then detected utilizing a microplate reader at 450 nm. Every experiment was carried out in sextuplicate.
TUNEL Assay
The experiment was carried out in line with the producer’s directions utilizing the One Step TUNEL Apoptosis Assay Package (Beyotime, Shanghai, China). After drug therapy, HT22 cells have been mounted with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 for 20 min. Subsequent, the cells have been incubated with a fluorescein TUNEL-TdT response combination incubated at room temperature for 1 h at nighttime. After washing, the apoptotic cells have been detected by fluorescence microscope (Olympus Company) and analyzed through ImageJ evaluation software program. Every experiment was performed in triplicate.
Statistical Evaluation
All experiments have been repeated at the least 3 times. The generated information have been introduced as imply ±customary deviation (SD). Between-group comparisons of the outcomes have been made by Pupil’s t-test (P < 0.05 was thought of vital). Statistical evaluation was carried out utilizing SPSS 22.0 software program, and graphs have been plotted by GraphPad Prism 7.0 software program.
Outcomes and Dialogue
High quality Management of HGWD
The standard management checks, together with validation of specificity, linearity, and decrease restrict of quantitation (LLOQ), precision and accuracy, stability, and extraction restoration, have been executed. The standard chromatograms of calycosin-7-O-glucoside, calycosin, and paeoniflorin have been used as references (Figures 2 and 3). There was no interference within the corresponding absorption peaks, indicating that the strategy has good specificity. The calibration curves exhibited linearity over the focus vary of two.50–25.00 µg/mL for calycosin-7-O-glucoside, 0.25–2.51 µg/mL for calycosin, and 95.78–2394.50 µg/mL for paeoniflorin, with the correlation coefficients of calycosin-7-O-glucoside, calycosin, and paeoniflorin being y = 19113x-0905 (R2 = 0.9980), y = 26469x-2066.2 (R2 = 0.9998), and y = 813.34x-13,570 (R2 = 0.9990), respectively. The precision and accuracy outcomes of the three analytes demonstrated that the precision of the instrument was good (Table S2). The outcomes of stability are proven in Table S3. The pattern restoration take a look at outcomes confirmed that the typical recoveries have been 100.04%, 103.99%, and 106.47% for calycosin-7-O-glucoside, calycosin, and paeoniflorin, respectively, and RSD values have been 1.97%, 3.05%, and a couple of.58%, respectively (Table S4). Primarily based on the linear relationship, the typical contents of calycosin-7-O-glucoside, calycosin, and paeoniflorin in HGWD have been calculated to be 1884.33 µg, 250.41 µg, and 114,040.05 µg, respectively (Table S5).
Determine 2 The particular HPLC chromatograms of calycosin-7-O-glucoside and calycosin (1. HGWD with out Radix Astragali (unfavourable pattern); 2. HGWD; 3. Calycosin-7-O-glucoside; 4. Calycosin). |
Determine 3 The particular HPLC chromatograms of paeoniflorin (1. HGWD with out Paeoniae Radix Alba (unfavourable pattern); 2. HGWD; 3. paeoniflorin). |
Biospecific Stay-Cell-Primarily based Isolation and HPLC-MS/MS Identification of HGWD Parts
Biospecific live-cell-based isolation of lively compounds, which is the mix of life sciences and chromatographic separation, from medicinal extracts is an rising expertise with super prospects. Right here, dwelling cells are used because the stationary part, which might particularly bind to the lively components of TCM. The sure elements are eluted and decided with fashionable analytical methods, equivalent to HPLC or LC/MS.13 In contrast with Figure 4C, few peaks have been detected by HPLC-MS within the fifth wash answer and clean dissociation answer (Figure 4A and B). With the assistance of Peak View software program, a complete of 11 compounds have been recognized.14 Amongst them, eight compounds have been recognized beneath the optimistic ion mode, particularly calycosin-7-O-glucoside (C22H22O10), calycosin (C16H12O5), formononetin (C16H12O4), cinnamic alcohol (C9H10O), cinnamic acid (C9H8O2), betaine (C5H11NO2), dl-2-phenylpropionic acid (C9H10O2), and 6-methylcoumarin (C10H8O2); three elements have been recognized in unfavourable ion mode, together with 4-hydroxycinnamic acid (C9H8O3), wogonoside (C22H20O11), and paeoniflorin (C23H28O11). The secondary mass spectrum was proven in Figure 5 and Table 2. To additional decide the chemical buildings of the compounds, the information was in contrast with the usual product and literature. The MS information of calycosin-7-O-glucoside,14 calycosin,15 formononetin,16 cinnamic alcohol,17 cinnamic acid,18 betaine,19 wogonoside,20 and paeoniflorin21 have been according to earlier experiences. The buildings of dl-2-phenylpropionic acid, 6-methylcoumarin, and 4-hydroxycinnamic acid have been decided compared with the usual product. In brief, the eleven compounds that had a binding affinity to HT22 have been recognized as calycosin-7-O-glucoside, calycosin, formononetin, cinnamic alcohol, cinnamic acid, betaine, dl-2-phenylpropionic acid, 4-hydroxycinnamic acid, 6-methylcoumarin, wogonin, and paeoniflorin (Figure 6).
Desk 2 Fragment Ion Info of Affinity Part within the Dissociation Resolution |
Cell Viability Assay
The outcomes point out a major discount within the survival fee of the OGD/R group in contrast with the management group (P < 0.05), indicating that OGD/R precipitated harm to HT22 cells. After the intervention with completely different concentrations of calycosin-7-O-glucoside (1, 5, 10, and 20 µg/mL), the survival charges have been considerably completely different from that of the OGD/R group (P < 0.05), indicating that calycosin-7-O-glucoside might considerably alleviate the OGD/R-induced cell harm (Figure 7). As well as, calycosin (1 µg/mL), paeoniflorin (5–10 µg/mL), 4-hydroxycinnamic acid (0.1–20 µg/mL), formononetin (0.1–40 µg/mL), and wogonoside (0.1–20 µg/mL) might considerably improve the cell viability of OGD/R-treated cells (P < 0.05). Nevertheless, as in comparison with the OGD/R-treated group (P>0.05), no vital adjustments in survival charges have been noticed within the teams handled with cinnamic alcohol, cinnamic acid, betaine, dl-2-phenylpropionic acid, and 6-methylcoumarin. Research have proven that calycosin-7-O-glucoside can cut back the damage induced by OGD/R in HT22 cells by means of the SIRT1/FOXO1/PGC-1α pathway.22 Calycosin can enhance the ischemic stroke damage in rats by reworking microglia from a resting state to an lively “ameba-like state” by means of the BDNF/Trkb pathway.23 It may well additionally forestall autophagy, apoptosis, cell loss of life, and irritation attributable to oxidative stress, which is important for neuroprotection.24 Research have demonstrated that formononetin could improve neuronal differentiation and synaptic plasticity by means of PI3K/AKT/ERK pathway.25 Paeoniflorin is the lively part of the pink peony root, which might cut back cerebral ischemic harm by regulating the Ca2+/CaMKII/CREB signaling pathway.26 4-Hydroxycinnamic acid is a neuroprotective agent on account of its sturdy antioxidant and anti-apoptotic options.27 The outcomes of this research indicated that calycosin-7-O-glucoside, calycosin, paeoniflorin, 4-hydroxycinnamic acid, formononetin, and wogonoside, have a protecting impact on OGD/R-treated cells on the decided focus vary, whereas cinnamic alcohol, cinnamic acid, betaine, dl-2-phenylpropionic acid, and 6-methylcoumarin don’t have any such impact. Due to this fact, calycosin-7-O-glucoside, calycosin, paeoniflorin, 4-hydroxycinnamic acid, formononetin, and wogonoside will be the lively components of HGWD for selling nerve perform restoration after stroke.
The Protecting Results of Calycosin-7-O-Glucoside, Calycosin, Paeoniflorin, 4-Hydroxycinnamic Acid, Wogonoside, and Formononetin in HT22 Cells Towards OGD/R-Induced Harm
Underneath the pathological state of ischemia and hypoxia, extreme accumulation of lactic acid inhibits the exercise of LDH in cells, resulting in the loss of life of neurons.28 The outcomes of LDH content material dedication (Figure 8) point out that in contrast with the management group, the LDH exercise of the OGD/R-treated cells elevated considerably. In contrast with the OGD/R group (P<0.05), a major discount in LDH exercise was noticed after the therapy of OGD/R-treated cells with calycosin-7-O-glucoside, calycosin, paeoniflorin, 4-hydroxycinnamic acid, wogonoside, and formononetin.
Calycosin-7-O-Glucoside, Calycosin, Paeoniflorin, 4-Hydroxycinnamic Acid Wogonoside, and Formononetin Alleviates OGD/R-Induced Oxidative Stress
Oxidative stress is the imbalance of oxygen-containing free radicals and antioxidants within the physique, which implies that the elimination fee of ROS is way decrease than the manufacturing fee, resulting in lipid peroxidation and harm to tissues and organs. Oxidative stress attributable to reperfusion after cerebral ischemia is likely one of the pathological mechanisms of ischemic mind damage.3 The outcomes of the ROS content material dedication (Figure 9) point out that in contrast with the management group, the fluorescence depth of the OGD/R group was considerably increased (P < 0.05). Nevertheless, after drug intervention, the intracellular fluorescence depth of every group was considerably diminished in contrast with that of the OGD/R group (P < 0.05).
The outcomes of SOD content material dedication indicated that in contrast with the management group, the fluorescence depth of the OGD/R group was considerably increased (P<0.05), indicating that OGD/R precipitated oxidative stress harm. Additionally, the protecting impact of calycosin-7-O-glucoside and 4-hydroxycinnamic acid, wogonoside, and formononetin elevated with the rise of the drug focus, reflecting dose-dependency. Calycosin and paeoniflorin exerted the strongest protecting results on the focus of 1 µg/mL and 20 µg/mL, respectively (Figure 10). These outcomes confirmed that the above components might alleviate harm induced by oxidative stress, thereby exerting a neuroprotective impact towards OGD/R damage.
Calycosin-7-O-Glucoside, Calycosin, Paeoniflorin, 4-Hydroxycinnamic Acid Wogonoside, and Formononetin Induces Apoptosis
Tunel staining have been used to judge the impact of medicine on apoptosis in HT22 cells. Tunel staining confirmed that the apoptosis cell was considerably elevated in OGD/R group in contrast with the management group (P < 0.01), and after drug therapy, apoptosis cell was considerably diminished in contrast with the OGD/R group (P < 0.01) (Figure 11). These outcomes steered that calycosin-7-O-glucoside, calycosin, paeoniflorin, 4-hydroxycinnamic acid wogonoside, and formononetin have been capable of attenuate OGD/R-mediated apoptosis. Due to this fact, we hypothesized that the mechanism of motion of HGWD to advertise neurological restoration could also be associated to the regulation of apoptosis.
Conclusions
On this research, we established a cell membrane solid-phase chromatography technique to display eleven lively elements from HGWD. The survival fee of HT22 cells after OGD/R damage was investigated to discover the exercise of the recognized compounds. Amongst them, six elements had completely different levels of protecting impact on OGD/R-treated HT22 cells. The protecting impact was measured by the degrees of ROS, SOD and LDH and the diploma of apoptosis. The outcomes confirmed that calycosin-7-O-glucoside, calycosin, paeoniflorin, 4-hydroxycinnamic acid, wogonoside, and formononetin might alleviate the oxidative stress damage and apoptosis induced by OGD/R. Due to this fact, HGWD can promote neurological restoration after ischemic stroke by regulating oxidative stress and apoptosis. Present information on the therapy of ischemic stroke utilizing HGWD is majorly derived from medical analysis and never from experimental analysis. This experimental research, primarily based on cell membrane solid-phase chromatography, is the primary to discover the doable mechanisms of the therapeutic results of HGWD. Nevertheless, as a result of complexity of HGWD composition, some compounds with increased response values stay unidentified in line with the whole ion chromatogram of optimistic and unfavourable samples. Due to this fact, additional analysis is required for the identification of those compounds.
Abbreviations
HGWD, Huangqi Guizhi Wuwu Decoction; OGD/R, oxygen-glucose deprivation reperfusion; ROS, reactive oxygen species; SOD, superoxide dismutase, LDH, lactate dehydrogenase; TCM, conventional Chinese language medicines; BSA, bovine serum albumin; DMEM, Dulbecco’s Modified Eagle; PBS, phosphate-buffered saline; SPE, solid-phase extraction; CCK-8, Cell Counting Package-8.
Writer Contributions
All authors made a major contribution to the work reported, whether or not that’s within the conception, research design, execution, acquisition of information, evaluation, and interpretation, or in all these areas; took half in drafting, revising or critically reviewing the article; gave ultimate approval of the model to be revealed; have agreed on the journal to which the article has been submitted; and conform to be accountable for all features of the work.
Funding
This work was supported by the Nationwide Pure Science Basis of China (grant quantity 81373973), Pure Science Basis of China (grant quantity 81573872). All authors have declared no battle of curiosity.
Disclosure
The authors declare no conflicts of curiosity.
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