The results of the current study indicate that dietary supplementation with purple corn anthocyanins has the potential to improve meat quality and prevent lipid oxidation by promoting antioxidant status and regulating related antioxidant gene expression, as well as improving polyunsaturated fatty acid concentrations in the longissimus thoracis et lumborum muscle of growing goats.Currently, unsaturated fatty acids (UFAs) in goat meat have gained popularity among consumers based on the benefits to human health. However, UFAs are prone to lipid oxidation. Purple corn is rich in anthocyanins, which have a wide range of biological activities, such as antioxidation, scavenging free radicals, and preventing lipid peroxidation. This study hypothesized that purple corn anthocyanin can improve meat quality and prevent lipid oxidation by promoting antioxidant status and UFA levels in p > 0.05) the dry matter intake, average daily gain, or feed conversion ratio compared to the control group. The addition of PCP reduced (p < 0.05) shear force in the longissimus thoracis et lumborum muscle (LTL) during the growth phase of the goats. Goats receiving PCP showed higher (p < 0.05) levels of reduced glutathione, 2,2-diphenyl-1-picrylhydrazyl scavenging activity and peroxidase in LTL compared to the control. Moreover, compared to the control, the PCP group displayed lower (p < 0.05) concentrations of 12:0, C16:0, and total saturated fatty acids, but increased (p < 0.05) concentrations of various unsaturated fatty acids, including C18:1n9, C20:3n6, C20:4n6, C18:2n6 cis, C20:3n6, C22:5n3, C22:6n3, and total polyunsaturated fatty acids (PUFAs). The abundance of nuclear factor, erythroid 2 like 2, superoxide dismutase 1, glutathione peroxidase 1, and catalase was upregulated (p < 0.05) in the LTL of goats receiving 0.5 g/d PCP in comparison to the other groups. Collectively, result of the current study indicated that PCP anthocyanin could be used as a source of natural functional additive because anthocyanin-rich PCP has the potential to improve meat quality and enhance muscle antioxidant status as well as improve the proportions of PUFAs in goat muscle.This study was conducted to examine the effect of purple corn anthocyanin on performance, meat quality, muscle antioxidant activity, antioxidant gene expression, and fatty acid profiles in goats. The feeding trial period lasted 74 d. The adaptation period was 14 d, and the formal experimental period was 60 d. Eighteen Qianbei-pockmarked goats  were randomly allotted into three equal groups, including a control with no purple corn pigment (PCP) and groups receiving either 0.5 g/d PCP or 1.0 g/d PCP. The inclusion of PCP did not affect ( Consumers are increasingly interested in obtaining high-quality meat products with good nutritional profiles and health value . RuminanDietary supplementation of antioxidants can be transferred to the muscle, where they work with natural defence systems and thus counteract pro-oxidants . AlthougAnthocyanins are large polyphenol compounds that have strong natural antioxidation properties and widely exist in plants and food . In addilongissimus thoracis et lumborum muscle (LTL) in goats.Additionally, we previously confirmed that purple corn pigment (PCP) could maintain PUFA levels in milk during the storage period ,15. HencThe PCP was purchased from Nanjing Herd Source Biotechnology Co., Ltd. . The anthocyanin composition of PCP extracted using 1.5 M HCl dissolved in a 95% ethanol solution (ethanol:HCl = 85:15) was determined by high performance liquid chromatography tandem mass spectrometry, according to Tian et al. . The antad libitum intake and 10% refusals on an as-fed basis.The feeding trial was conducted at Fuxing Husbandry Co., Ltd., Guizhou, China . The feeding trial period lasted 74 d. The adaptation period was 14 d. Thus, the formal experimental period was 60 d. Eighteen Qianbei-pockmarked wether goats , 21.38 ± 1.61 kg; mean ± standard deviation) were randomly divided into three groups, including a control with no PCP (CON) and treatments receiving either 0.5 g/d PCP (LA) or 1.0 g/d PCP (HA), using a completely randomized design. The Qianbei-pockmarked goat is one of main goat breeds in the Guizhou province in China and has strong tolerance to the inclement environment, roughage resistance, good meat quality, and has deep academic research and practical application value. The level of PCP used in this study was based on Tian et al. . BrieflyTM analyser . Organic matter and hemicellulose were obtained by 100 minus the ash and NDF minus ADF, respectively  × 100. For drip loss, approximately 10 g and 3-cm length cubes of LTL were manually trimmed, weighed, suspended in a fishhook in an inflated plastic bag, sealed, and stored for 24 h at 4 °C. Each sample was weighed after removing the fishhook and dried on filter paper. Drip loss was calculated as follows: Drip loss (%) = [/initial weight] × 100. For cooking loss, approximately 100 g of LTL was weighed without fascia, epimysium, or fat. The sample was placed in a steamer and cooked for 30 min, and then the steam meat sample was weighed after cooling at 4 °C for 2 h. Cooking loss was calculated as follows: Cooking loss (%) = (fresh weight − cooked weight)/fresh weight × 100. Meat colour (Opto), defined as the L-value (L*) in the Cielab system, was detected at 45 min after slaughter using Opto-Star equipment . The shear force of the sample cores was measured perpendicularly to the direction of the muscle fibre using a digital display tenderness meter  with a load cell of 15 kg and a 200-mm/min crosshead speed. Each index was run in three replicates.All animals were slaughtered at the end of the experiment, and carcasses were processed as described by Danforth . The lef® ultrasonic cleaner . The homogenate was centrifuged at 4000× g for 10 min at 4 °C, and the supernatant was transferred to a 1.5-mL tube and stored at −80 °C.The muscle sample was washed with precooled phosphate buffered solution  to remove residual blood, weighed, and then cut up. The chopped tissues and PBS were added to a glass homogenizer at a ratio of 1:9 and thoroughly ground on ice. The homogenate was ultrasonicated using a Bransonic2−), and hydroxyl FR (OH) were determined using commercially available kits from Nanjing Jiangcheng Bioengineering Institute . The above parameters were detected by spectrophotometry, and the absorbance was analysed using a microplate reader . All of the measurement procedures strictly followed the manufacturers’ recommendations. An FR parameter of 2,2-diphenyl-1-picrylhydrazyl  scavenging activity was measured by spectrophotometry [g for 10 min at 4 °C, and then 200 μL of the supernatant was immediately transferred into a 96-well plate  and incubated in the dark at 37 °C for 30 min. The absorbance was analysed at a wavelength of 517 nm using a microplate reader . DPPH scavenging activity was calculated using the following equation: DPPH scavenging activity (%) = (Ac − As) × 100/Ac, where Ac is the absorbance of the control and As is the absorbance of the sample.Total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GPX), reduced glutathione (GSH), catalase (CAT), peroxidase (POD), malondialdehyde (MDA), nitric oxide (NO), superoxide anion , and the supernatant was transferred to a 2-mL centrifuge tube. The sample was esterified by 0.8 mL of 2% sodium hydroxide-methanol solution, and a reflux condenser was connected. After cooling to room temperature, 1 mL n-heptane was added, and the tube was thoroughly mixed, left to stand for 5 min, and centrifuged at 10,000× g at 4 °C for 5 min. Then, 100 mg of anhydrous sodium sulfate powder was added after the supernatant was transferred to a 2-mL tube with sufficient mixing and left to stand for 5 min, and the supernatant was transferred to a 1.5-mL tube and stored at −20 °C until measurement. Individual FAs were detected using gas chromatography . The GC conditions were as follows: separation of FA by a strong polar stationary phase of a polydicyanopropyl siloxane capillary column (100 m × 0.25 mm × 0.20 µm), the injection volume was 1 µL, the injection port temperature was 270 °C, and the detector temperature was 280 °C. The temperature program included: an initial temperature of 100 °C for 13 min with a 10 °C/min rise to 180 °C, and this temperature was held for 6 min, a 1 °C/min rise to 200 °C, and this temperature was held for 20 min, then, a 4 °C/min rise to 230 °C, and this temperature was held for 10.5 min. The split ratio was 100:1, and nitrogen gas was used as the carrier. Individual FAs were detected from the chromatogram peak areas, and the data were expressed in grams per 100 g of FAs.The FAs were extracted from the LTL according to the Chinese standard GB 5009.168-2016 , with a The total RNA was extracted as follows: First, 100 mg of each LTL sample was weighed and moved into a 1.5-mL sterilized tube, 1 mL of RNA extraction solution  was added, and the tube was shaken vigorously before running in a homogenizer. Second, the total RNA was dissolved with HyPureTM Molecular Biology Grade Water. Third, the RNA concentration and purity were measured using a NanoDrop 2000 machine . Complementary deoxyribonucleic acid (cDNA) synthesis was determined by a commercial cDNA synthesis kit .Nrf2), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), and catalase (CAT). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene in the current trial. All cDNA samples were assayed using quantitative real-time polymerase chain reaction (qPCR) amplifications by a StepOnePlus™ real-time PCR system . The volume of the 15 μL reaction volume was as follows: 7.5 μL of 2 × SYBR Green qPCR master mix, 1.5 μL of 2.5 μM forward and reverse primers, 2.0 μL of cDNA, and 4.0 μL of ddH2O. The cycling conditions were as follows: 10 min at 95 °C for pre-denaturation, 40 cycles of 15 s at 95 °C for denaturation, and 30 s at 60 °C for extension.As shown in ij = µ + τi + εij, where Yij is the observation j (j = 1 to 6) in treatment i, µ is the overall mean, τi is the effect of the treatment (denoted an unknown parameter), and εij is the random error with a mean of 0 and variance σ2 [p < 0.05.The sample size was calculated by Statistical Analysis System 9.1.3 software . Each animal was treated as an experimental unit. All of the data analyses were performed using SAS 9.1.3 by the one-way ANOVA model: Yiance σ2 . The sigp > 0.05) in DMI, BWC, ADG, or FCR were observed among the treatment groups, whereas the g/kg BW0.75 (%) of the two treatments was higher (p < 0.05) than that of the control (No differences ( control .p > 0.05) pH45min, pH24h, percentage of water loss, drip loss, cooking loss, or meat colour. However, the feeding of PCP could result in a significant (p < 0.05) decrease in muscle shear force value compared to the control group (The addition of PCP did not affect (ol group .p < 0.05) than those in the control group (p < 0.05) POD activity than the other two groups. The feeding of 1.0 g/d of PCP exhibited higher (p < 0.05) O2− and OH levels compared to the other groups.The GSH, CAT, and DPPH scavenging activities in the HA group were greater (ol group . The LA p < 0.05) levels of C12:0, C16:0, and total SFA than LA group. In terms of individual UFAs, C14:1, C16:1, C18:1n9 cis, C20:1, C18:2n6 trans, C18:3n3, C18:3n6, and C20:2 did not differ (p > 0.05) among all groups. However, the inclusion of PCP in goats resulted in an increase (p < 0.05) in individual UFAs for C18:1n9 trans and C20:3n6. Supplementation with 1.0 g/d of PCP led to an increase (p < 0.05) in the C20:4n6 level compared to the other two groups. The C18:2n6 cis, C20:3n6, C22:5n3, C22:6n3, and total PUFA levels were higher (p < 0.05) in goats that received 0.5 g/d PCP in comparison to the control group.p < 0.05) in the LTL of goats receiving 0.5 g/d PCP relative to the other groups. In addition, goats fed the control diet showed decreased CAT mRNA abundance (p < 0.05) compared to those fed the 0.5 g/d PCP diet.As shown in 0.75 [The DMI level is an important prerequisite for the growth performance of ruminants . Anthocy0.75 .Natural dietary antioxidant extracts may contain tenderising compounds, which can increase meat tenderness . It has 2− and OH concentrations in LTL, perhaps due to PUFAs being unable to tightly bind owing to the bending of the carbon chain in the presence of double bonds, leading to PUFAs with increased susceptibility to lipid oxidation. Moreover, goats receiving 1.0 g/d PCP may act as a “pro-oxidant” in the body, leading to high FR concentrations in muscle [The OS is the main cause of various metabolic diseases because it directly affects animal performance and causes a decline in the health of ruminants . Indeed,n muscle . Our resn muscle , who pren muscle  suggesten muscle  showed ttrans C18:1 in dairy cows. Hence, supplementation of PCP in the basal diet for growing goats led to a decline in SFA content, perhaps because anthocyanins could shift the relative abundance of rumen microflora in goats [Ruminants have a high SFA content because of the extensive microbial biohydrogenation in the rumen. PUFAs are considered as being important compounds for consumers’ nutrition and health . Vasta ain goats . Consistin goats  found thcis-9, trans-11, C20:5n-3, DHA, and total PUFA levels in milk. Collectively, dietary supplementation with PCP anthocyanins could enhance the antioxidant activity and increase PUFA concentrations in muscle, subsequently improving meat quality of goats.Lipid oxidation of meat includes three steps: FR initiation, reproduction, and termination. PUFAs oxidize lipid radicals, followed by the oxidation of peroxyl radicals with oxygen that then change FA hydroperoxide with hydrogen, thereby negatively affecting the body’s antioxidant enzymes . AntioxiNrf2 is a sensor protein of xenobiotic toxic substances and OS because it plays an important role in participating in cellular antioxidant stress and the major defence mechanism induced by xenobiotic toxic substances [Nrf2-activating agents that can directly modify sensor cysteines present in kelch-like ECH protein (Keap1), thereby activating Nrf2 and regulating the expression of cytoprotective proteins with anti-inflammatory and antioxidant functions [Nrf2 dissociation and phase II detoxification or antioxidant enzymes, such as SOD, GPX, and CAT [Nrf2 signalling pathway. As a result, the feeding of 0.5 g/d PCP can increase Nrf2 expression, thereby increasing the expression of SOD1, GPX1, and CAT in the LTL of goats. Consistent with our results, Tian et al. [Nrf2 and GPX1 genes in the mammary gland of Saanen dairy goats. Of interest, a high dose of anthocyanins might have side effects on the body by acting as a “pro-oxidant” that generates toxic oxygen [bstances . Variousbstances ,55. Anthunctions . Indeed, and CAT . Liu et  and CAT  demonstrn et al.  showed tc oxygen . Hence, The results of the current study indicate that PCP can be used as a source of natural functional additive for growing goats, because (1) PCP anthocyanins can improve meat quality, (2) PCP anthocyanins has the potential to enhance muscle antioxidant status and regulate muscle related antioxidant gene expression, and (3) PCP anthocyanins can improve muscle PUFA concentrations. However, the underlying mechanisms require further investigation. Studies on the bioavailability of anthocyanin in the body that elucidate the mechanism by which anthocyanin improves the antioxidant status and prevents lipid oxidation of ruminants are warranted.


Zymography in situ indicated that MMPs had a variable, hydrogel concentration-dependent influence on ASCs-secreted MMPs. In 5% GelMA, ASCs showed a high and sustained expression of MMPs, while, in 10% and 15% GelMA, such expression was almost null. ASCs morphology based on F-actin staining showed that increasing GelMA concentrations inhibit their spreading. Scanning electron microscopy (SEM) showed that hydrogel ultrastructure in terms of pore density, pore size, and percentage porosity were not consistently influenced by cells. Interestingly, changes in ultrastructural parameters were detected also in cell-free materials, albeit without a clear trend. We conclude that hydrogel concentration and its underlying mechanics influenced MMP expression by ASCs. The exact MMPs that respond to these mechanical cues should be defined in follow-up experiments.Adipose tissue-derived stromal cells (ASCs) are of interest in tissue engineering and regenerative medicine (TERM) due to their easy acquisition, multipotency, and secretion of a host of factors that promote regeneration. Retention of ASCs in or around lesions is poor following direct administration. Therefore, for TERM applications, ASCs can be ‘immobilized’ via their incorporation into hydrogels such as gelatine methacryloyl (GelMA). Tweaking GelMA concentration is a common approach to approximate the mechanical properties found in organs or tissues that need repair. Distinct hydrogel mechanics influence the ability of a cell to spread, migrate, proliferate, and secrete trophic factors. Mesenchymal cells such as ASCs are potent remodellers of the extracellular matrix (ECM). Not only do ASCs deposit components, they also secrete matrix metalloproteases (MMPs) which degrade ECM. In this work, we investigated if GelMA polymer concentration influenced the expression of active MMPs by ASCs. In addition, MMPs’ presence was interrogated with regard to ASCs morphology and changes in hydrogel ultrastructure. For this, immortalised ASCs were embedded in 5%, 10%, and 15% ( Adipose tissue-derived stromal cells (ASCs) are plastic adherent cells that express surface markers common to mesenchymal stromal/stem cells (MSCs) ,2,3. ASCASCs can be combined with carrier biomaterials such as hydrogels to tackle the before-mentioned disadvantages ,14,15,16Tweaking hydrogel concentrations is a common approach to approximate the mechanical properties of native tissues for TERM purposes ,36,37. WThus, in this work, we investigated if the expression of ASCs-derived MMPs would be altered by hydrogel concentration and its related mechanics, namely elastic modulus  and stress relaxation. We also investigated if MMPs expression could be reflected in hydrogel swelling ratio and ultrastructure changes after 14 days. We also evaluated ASCs’ morphology in histological stains. If GelMA-ASCs constructs are aimed for clinical use, their characterisation in vitro is relevant to know the end product before patient applications.® > 600 MW, Leverkusen, Germany). Gelatine (100 g) was dissolved in 1× Dulbecco’s phosphate-buffered saline  and 0.6 mL of Methacrylic anhydride  was added per gram of gelatine at 40 °C with gentle stirring for three hours. The solution was diluted in an equal volume of 1× DPBS, centrifuged at 2000× g for 5 min., and the supernatant decanted into a 14 kDa dialysis tube . The tube was dialysed for a week at 30 °C with demi-water that was replaced twice daily. Once the dialysis was completed, the solution was lyophilised in a Free Zone® 2.5 Plus freeze dryer  at −80 °C until dry. The degree of functionalisation (DoF) was determined through 1H-Nuclear Magnetic Resonance (1H-NMR), as described previously [w/v) concentrations. All working solutions were mixed with the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate  at a concentration of 0.5% (w/v) at 50 °C. Once dissolved, the working solutions were filter-sterilised with 0.2 µm polyethersulfone membrane filters  and stored in the dark at −20 °C until further use.GelMA was synthesised by a one-pot method using medical grade gelatine type A, 99.8 kDa MW, 262 g Bloom  in growth media composed of high glucose Dulbecco’s modified Eagle medium  containing 10% Fetal Bovine Serum , 1% Penicillin-Streptomycin  and 2 mM L-glutamine .Immortalised ASCs (iADSC13)  were cul5 ASCs were resuspended in 350 µL of either 5%, 10%, or 15% (w/v) GelMA working solutions and thawed at 37 °C. The GelMA-ASC suspensions were cast in 48-well plates (Corning®) and photopolymerised at 7 mW/cm2 for 5 min. with a UV/blue light lamp (405 nm). All gels, including cell-free (acellular) hydrogels, were cultured in growth media for 1 d, 7 d, and 14 d at 37 °C, 5% CO2 (Mycoplasma spp. from passages 19 to 22 were employed in these experiments.All cells were harvested with 0.5% trypsin-EDTA (Sigma–Aldrich) after reaching 80% confluence and counted with an automated cell counter . Aliquots of 7 × 10, 5% CO2 a. Only cex BP 340–380 nm/λem BP 450–490 nm) and TXR (λex BP 540–580 nm/λem BP 593–668 nm) at 20× magnification.Cell-loaded hydrogels of 1 d, 7 d, and 14 d were fixed with 2% formalin for 24 h, followed by a series of ethanol dehydration and paraffin wax embedded b. Sectioex BP 460–500 nm/λem BP 512–542 nm) and DAPI (λex BP 340–380 nm/λem BP 450–490 nm) at 20× magnification.Cell-loaded and cell-free hydrogels were snap-frozen in liquid nitrogen and cryosectioned (4 µm thick) for experiments c. EvaluaWs) on a scale . All samples were freeze-dried for 24 h and weighed again to determine the dry weight (Wd). The swelling ratio was calculated as shown in Equation (1):Cell-loaded and cell-free GelMA hydrogels of 1 d, 7 d, and 14 d were carefully removed from the 48-well plates and weighed (ε) deformation rate of 20%·s−1. The elastic modulus was determined during the first second of compression. For the stress relaxation percentage, the strain rate (ε) of 0.2 s−1 was kept constant for 50 s and calculated as the difference between t = 0 and t = 50 s. Data derived from three independent experiments were analysed with MatLab 2018 .The 5%, 10%, and 15% GelMA hydrogels were swollen in 1 X DPBS for 24 h before mechanical testing on the Low-Load Compression Tester (LLCT) ,45. The ® water, snap-frozen in liquid nitrogen, and freeze-dried for 24 h. The lyophilised hydrogels were mounted on top of 0.5″ pin stubs , placed inside a Leica EM SCD050 sputter coater device , and rinsed and coated with Au-Pd one day before scanning electron microscopy (SEM) imaging. Hydrogels were visualised with Zeiss Supra 55 STEM  at 2500× magnification, 3.0 kV, and Z = 40.0 mm . All data were searched for outliers using the robust regression and outlier removal (ROUT) test and analysed for normality using Shapiro–Wilk and D’Agostino and Pearson tests ,49,50. TCells were visible in all hydrogel sections with areas of clustered cells randomly distributed (Morphology of ASCs differed across GelMA concentrations and time points . In 5% GThe staining time (1 h) was kept consistent for all GelMA concentrations and the staining intensity of PSR increased alongside hydrogel concentration comparable to the H&E staining . PSR allAll ASCs secreted active MMPs irrespective of GelMA concentration after 1 d a. At 14 The 5% GelMA had the highest swelling ratio of all concentrations, followed by 10% and 15%. ASCs did not change the GelMA swelling properties when compared across timepoints and cell-free material .Increasing hydrogel concentration led to a significant, near-linear increase in elastic modulus . The elastic modulus of 5%, 10%, and 15% GelMA hydrogels were 5.2 ± 1.7 kPa, 55.6 ± 8.4 kPa, and 161.1 ± 11.5 kPa, respectively. Regarding hydrogel viscoelasticity, the 5% GelMA (8.2 ± 3.2%) had a greater stress relaxation % than 10% GelMA (5.5 ± 0.8%) but not more than 15% GelMA (6.3 ± 1.0). No differences between 10% and 15% were found.The surface ultrastructure of cell-loaded and cell-free GelMA hydrogels was visualised with SEM . The anaIn 10% GelMA, cell-loaded material had a greater pore density after 14 d compared to 1 d and 7 d and its respective control. In contrast, the average pore size decreased after 14 d in culture. Interestingly, differences in pore density and average pore size were detected in cell-free 10% GelMA.In 15% GelMA, pore density increased in cell-loaded hydrogels after 14 d. Compared to cell-loaded 1 d, the average pore size increased at 7 d but decreased at 14 d.Modulating hydrogel concentration is a common approach to tailor GelMA-ASCs’ hydrogels for TERM applications. In this work, we found that increasing hydrogel concentration led to an inhibition of active MMPs after 14 d in cell culture. The availability of active MMPs seems to be a spatiotemporal regulated process influenced by hydrogel concentration and mechanics. Such spatiotemporal regulation of MMPs is described in in vivo models with pathological conditions but is less studied in 3D in vitro models ,52,53.MMPs facilitate cell spreading by degrading the immediate microenvironment and reducing the mechanical constraint from the pericellular region . Hence, ASCs’ spreading follows a clear trend regarding hydrogel concentration, where cell spreading is facilitated at lower polymer concentrations—where the elastic modulus is lower and exhibits higher stress relaxation. Thus, MMPs expression does not follow the same trend as spreading in terms of hydrogel concentration. Although GelMA has MMP degradation motifs, a lack of sustained expression suggests that MMPs’ release and activity are influenced by the pericellular mechanics. In our model, increasing hydrogel concentration led to a near-linear increase in the hydrogel elastic modulus. Reportedly, mechanics downregulated MMPs through TIMPs, albeit in 2D hydrogel models . PreviouThe relation between hydrogel concentration and the inhibition of MMPs is of clinical relevance. GelMA hydrogels are well suited as carrier biomaterial to deliver ASCs on site of injection to decrease osteoarthritis or to heal osteochondral defects ,62. On oPolymer degradation also impacts hydrogel mechanics, although most studies characterise such a phenomenon in cell-free materials ,64,65. SInterestingly, cell-free GelMA also changed in terms of ultrastructure parameters across timepoints, and it is unclear if such changes are due to artefacts during specimen preparation for SEM, hydrogel contraction during culture, or additional changes in hydrogel conformation. Overall, data on the hydrogel structure in its native wet state is scarce and even less present when describing cell-loaded materials. We also recognise the additional limitations of our current work. For example, matrix deposition, such as ASCs-derived collagen, was not discernible in PSR stain and might require further histological assessments. Evidence indicates that hydrogel concentration influences MMPs’ expression, such as MMP-2 and MMP-9 ; howeverActive MMP expression by ASCs is a regulated spatiotemporal process influenced by hydrogel concentration and its underlying mechanics. Hence, we observe that an increased GelMA hydrogel stiffness inhibits MMPs activity in a 3D microenvironment. A more gradual trend was seen in terms of the function of ASCs, i.e., less spreading and collagen deposition observed in the highest concentration hydrogels.


RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified  An elevated level of circulating low-density lipoprotein (LDL) cholesterol is a major risk factor for atherosclerotic cardiovascular diseases, including myocardial infarction and stroke , 5, 6, 7RAB10 decreased cellular endocytosis of LDL but increased accumulation of another endocytic cargo, transferrin. The receptors for LDL (LDLR) and transferrin receptor (TFR) are both endocytosed from the cell surface via clathrin-coated vesicles and transported through intracellular recycling pathways  as previously described . Virus pCells were seeded in 6-well plates to achieve ∼70%–80% confluence on the day of analysis. For uptake assays, cells were washed with serum-free DMEM and then incubated in DMEM containing either 4 μg/ml DyLight550-conjugated LDL  or 5 μg/ml Alexa Fluor 555-conjugated transferrin (ThermoFisher Scientific) at 37°C for 1 h or 30 min, respectively. Cells were harvested with TrypLE express (ThermoFisher Scientific), washed with ice cold PBS, resuspended in 150ul of ice-cold PBS, and analyzed with a Bio-Rad Ze5 flow cytometer. Data analysis was performed with FlowJo (FlowJo).Cells were cultured at 37°C in 10 cm dish until 70%–80% confluent. Cells collected with trypLE express were washed in PBS and then lysed in RIPA lysis and extraction buffer (Thermo Scientific) containing complete protease inhibitor cocktail (Roche). After brief sonication, lysed cell suspensions were rotated at 4°C for 1 h for protein extraction followed by centrifugation at 15000g. Protein concentration was determined with the Bio-Rad DC assay kit , and SDS-PAGE was performed using NuPAGE™ 4%–12%, Bis-Tris, mini protein gels (ThermoFisher Scientific # NP0321BOX) according to manufacturer’s instruction. Western blot transfer was done into nitrocellulose membrane (Thermo Scientific #IB23002) using the iBlot 2 Dry Blotting System (Thermo Scientific).6 cells in 1 ml blocking buffer and incubated for 30 min with end-over-end rotation at 4°C. After centrifugation at 400g for 5 min, cells were resuspended in fluorescently labeled LDLR antibody or TFR antibody diluted in 100 μl blocking buffer and incubated for 1 h in the dark at 4°C. Cells were then washed three times with ice-cold PBS, resuspended in 150 μl cold PBS for final analysis by flow cytometry (Bio-Rad ZE5). For quantification of total cellular LDLR or TFR, harvested cells were fixed with 2% PFA for 10 min followed by PBS wash and permeabilization with 500 μl of 0.5% saponin in PBS before proceeding with staining for LDLR and TFR.HuH7 cells cultured in 6-well plates were prepared for analysis at 70%–80% confluence. For surface staining, collected cells were washed three times with ice-cold blocking buffer , resuspended at approximately 10Cells cultured on poly-D-lysine–coated glass coverslips  were fixed in 2% paraformaldehyde for 15 min in the dark at room temperature. After washing three times with PBS, cells were then permeabilized with 0.1% saponin in PBS for 5 min, incubated for 1 h in blocking buffer (PBS with 4% FBS and 40 mM glycine), stained with primary antibody at indicated dilutions in PBS with 4% FBS for 1 h, washed with PBS three times, stained with secondary antibody at indicated dilutions in PBS with 4% FBS for 1 h, and washed with PBS three times. Coverslips were mounted on glass slides with ProLong Diamond antifade mounting reagent (Invitrogen). Images were acquired with a NIKON A1 standard sensitivity confocal microscope with 60X (NA51.4) oil objective. Colocalization quantification was done using the open-source Fiji (Image J) software. Mander's coefficient and Pearson's coefficient were calculated using JACop in Image J. A total of 10–30 cells from 2 to 3 biological replicates were analyzed. For all quantitative analysis, the observer was blinded to cell genotype.An assay for transferrin receptor endocytosis was adapted from previous reports . BrieflyAn assay for LDLR endocytosis was adapted from previous reports , 23. BriAn assay for transferrin recycling was adapted from a previous report . BrieflyRAB10 gene and confirmed efficient depletion of RAB10 protein by immunoblotting  had no effect.To test the influence of RAB10 on LDL uptake, we generated RAB10-deficient HuH7 cells by CRISPR-mediated disruption of the blotting A. Consisblotting , RAB10-ting gRNA B, C. To activity B, C. We activity A , or GDP-locked (T23N) RAB10 expressed in a clonal cell line deleted for endogenous RAB10 D. ConsisRAB10-targeted cells detected by immunoblotting and flow cytometry (RAB10-targeted cells revealed increased staining for both LDLR and TFR that remained distributed in punctae (By confocal imaging, we observed that a subset of RAB10 colocalized with both LDLR and TFR in intracellular punctae A, B. We ytometry A–C, F–H, punctae . Colocal punctae C, H, con punctae A, H, the punctae B, H, the punctae E, H, the punctae F, H, or  punctae G, H. Sig punctae D, H.Fig.RAB10-targeted cells revealed a significant increase in colocalization with RAB4 (A similar analysis of TFR redistribution in ith RAB4 B, G and ith RAB4 A, G. In ith RAB4 C, D, G. ith RAB4 E, G or tith RAB4 F, G. TheRAB10-targeted cells could be due to an influence on endocytosis or recycling. To distinguish between these possibilities, we first examined endocytosis of TFR in complex with fluorescently labeled transferrin in RAB10-targeted and control cells using a previously reported approach (RAB10-targeted cells by flow cytometry (The redistribution of LDLR and TFR from the plasma membrane to endosomes in approach , 35. Briapproach A, consisytometry J. For thytometry B.Fig. 6RIn contrast to the TFR–transferrin complex, LDLR-LDL dissociates in the acidic environment of early endosomes with subsequent LDL degradation , 29, whiRAB10-targeting resulted in a significant delay in TFR recycling, with 50% of the intracellular transferrin–TFR complexes recycled within 8 min for control cells compared to 15–20 min for RAB10-targeted cells (In our assay of TFR endocytosis B, we noted cells E.Recycling of endocytosed membrane proteins to the cell surface plays an important role in maintaining the composition of the plasma membrane and the physiologic functions of the recycled proteins , 40, 41.Rab GTPases have previously been reported to play broad roles in the regulation of vesicular trafficking. We recently identified the small GTPase RAB10 as a putative modifier of cellular LDL and transferrin uptake . In the Several lines of evidence support a model in which RAB10 promotes the recycling of both LDLR and TFR. First, RAB10 depletion caused a decrease in the amount of both receptors on the cell surface without a corresponding decrease in gene expression . Second,Previous studies have revealed heterogeneity in the recycling of different receptors, with some, including LDLR, transported along a RAB11-mediated slow recycling pathway involving the endocytic recycling compartment, while others, including TFR, utilize both RAB11- and RAB4-mediated rapid recycling pathways , 42, 43.EXOC1, EXOC2, EXOC3, EXOC4, EXOC7, and EXOC8 that phenocopied RAB10, with depletion of either protein resulting in decreased LDL uptake and increased transferrin accumulation. Association of RAB10 with the exocyst complex has also been reported in renal epithelial cells (Small GTPases function as molecular switches, cycling between a GDP-bound inactive state and GTP-bound active state that mediates recruitment of effector proteins to membranes. Intriguingly, GTP-bound RAB10 has previously been demonstrated to mediate the insulin-stimulated transport of GLUT4-containing vesicles to the plasma membrane via its recruitment of the exocyst membrane tethering complex . Our prial cells . Taken tal cells  also ideal cells . This scal cells . A recenal cells . Taken tC. elegans (RAB10 has also been implicated in diverse areas of membrane trafficking in different cell types, including formation of noncanonical macropinosome tubules in macrophages , vesicle elegans , and Gol elegans . Consist elegans , 51, 52. elegans . This laAll data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The phase composition, morphology, fluorescence, and biosafety of the resulting samples were characterized. Moreover, the in vivo fluorescent bioimaging application of Eu/Gd:FAP nanocrystals was evaluated in mice with subcutaneously transplanted tumors. The results showed that the Eu/Gd:FAP nanocrystals were short rod-like particles with a size of 59.27 ± 13.34 nm × 18.69 ± 3.32 nm. With an increasing F substitution content, the Eu/Gd:FAP nanocrystals displayed a decreased size and enhanced fluorescence emission. Eu/Gd:FAP nanocrystals did not show hemolysis and cytotoxicity, indicating good biocompatibility. In vivo fluorescent bioimaging study demonstrated that Eu/Gd:FAP nanocrystals could be used as a bioimaging agent and displayed stable fluorescence emitting in tumors, indicating an accumulation in tumor tissue due to the passive targeting ability. In addition, any adverse effects of Eu/Gd:FAP nanocrystals on major organs were not observed. This study shows that biocompatible rare earth co-doped FAP nanocrystals have the potential to be used as a bioimaging agent in vivo.In this study, Eu The calcium phosphate apatite is the main inorganic component of the bones and teeth of mammals, which mainly exists as hydroxyapatite (HAP) in vivo ,2. With e (ClAP) ,4,5. Cale (ClAP) ,7,8. Thee (ClAP) ,13,14,15e (ClAP) ,18,19,20− ions with lower vibration energy can significantly avoid the quenching effect of the -OH group to promote the fluorescence conversion of rare earth elements [In addition, the apatite lattice is very suitable for the doping of rare earth elements and can provide a suitable crystal field environment for promoting the fluorescence emission of rare earth ions located at the divalent cation sites of apatite ,23,24,25elements ,29,30,312+. In addition, utilizing the energy transfer between the co-doped rare earth elements is an alternative way to enhance the fluorescence [3+ (excited state) to Eu3+ (ground state). Eu3+ ions are activated to the energy level that is matched with the 6GJ energy level of Gd3+. The Eu3+ ions could generate gradual energy level transitions to the 5HJ, 5D0, and 7FJ of Eu3+ by radiation transition with low energy loss. Therefore, rare earth element co-doping is considered to be an effective method of improving the luminescence-emission intensity based on energy transfer and electron transfer processes [The high fluorescence of rare-earth-doped FAP is the first prerequisite for its use as a bioimaging agent. This can be improved by promoting the thermal diffusion of rare earth ions into the crystallographic site of Carescence ,29. For rocesses . Of courrocesses . The hydrocesses ,34.3+/Gd3+ co-doped fluoroapatite (Eu/Gd:FAP) nanocrystals were synthesized by the hydrothermal method based on the effect of energy transfer from Gd3+ to Eu3+. The phase composition, morphology, fluorescence, hemolysis, and cytotoxicity of the resulting samples were characterized by the varied fluoride ion (F−) proportions of apatite. The in vivo fluorescent bioimaging application of Eu/Gd:FAP nanocrystals was further carried out in mice with subcutaneously transplanted tumors, with the aim of revealing their potential in the bioimaging field.Herein, the Eu2·2H2O, >99.9%), disodium hydrogen phosphate dodecahydrate , europium nitrate hexahydrate (Eu(NO3)3·6H2O, >99.9%), gadolinium chloride hexahydrate (Gd(NO3)3·6H2O, >99.9%), and sodium fluoride  were purchased from Sinopharm Chemical Reagent Co., Ltd. Ultrapure water  was used in the experiments. Human hepatocellular carcinomas (HepG2) and human normal hepatocyte cells (L02) were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd. The BALB/c-nu mice and the rabbit were purchased from the Animal Experiment Center of Wuhan University.Calcium chloride dihydrate (CaCl3+/Gd3+ co-doped FAP (Eu/Gd:FAP) nanocrystals were synthesized by the hydrothermal method. The initial compounds were separately dispersed in deionized water 3·6H2O, Gd(NO3)3·6H2O, Na2HPO4·12H2O, NaF). First, Na2HPO4·12H2O and NaF were dissolved together in water to achieve the mixing solution A of PO43− (0.24 mol/L) and F− (0.08 mol/L). CaCl2·2H2O, Eu(NO3)3·6H2O, and Gd(NO3)3·6H2O were dissolved together in water to achieve the mixing solution B of Ca2+ (0.386 mol/L), Eu3+ (0.008 mol/L), and Gd3+ (0.006 mol/L). Then, solution A (15 mL) was poured into solution B (15 mL) and strongly stirred, and the NH3·H2O solution was added to the above suspension with a resulting pH of about 9–9.5. Thereupon, the suspension was transferred into a stainless-steel autoclave and treated at different temperatures  for 60 min. After cooling to room temperature, the resulting product was separated via centrifugation under 2000 rpm and washed with ultra-pure water three times. Finally, the product was dried using a freeze-drying method. In addition, the molar ratios of (Ca + Eu + Gd)/P, Eu/(Ca + Eu + Gd), and Gd/(Ca + Eu + Gd) were 1.67, 0.02, and 0.015, respectively.The EuR) of luminescence intensity:I1 is the Eu/Gd:HAP emission peak intensity, and I2 is the Eu/Gd:FAP emission peak intensity.The morphology and elemental composition were investigated by using a scanning electron microscope  with energy dispersive spectroscopy (EDS) and a high-resolution transmission electron microscope . Crystalline phases and the phase composition were analyzed using a powder X-ray diffractometer . Fourier Transform Infrared  Spectroscopy was used to identify the chemical structure and phase composition of the samples. The luminescence property was recorded with luminescence spectra using a 970CTR luminescence spectrophotometer . The equation calculates the relative enhancement ratio (OD). Then, the RBCs were incubated with deionized water and 0.9 wt% NaCl for the control groups (positive and negative). The above results were used to calculate hemolysis % using the following equation:The hemolysis test was conducted using heparinized rabbit red blood cells (RBCs), and a 5% RBC suspension was prepared in a 0.9 wt% NaCl solution. The Eu/Gd:FAP suspension (1 mL) 0.9 wt% NaCl solution pre-incubated the RBC suspension. After that, the mixture was incubated at 37 °C for 1 h, and the supernatant was separated with centrifugation to check the light absorbance value  and calculated using the following equation:In vivo bioimaging was carried out using BALB/c-nu mice (about 20 g) with a subcutaneous transplantation of a HepG2 tumor. Eu/Gd/FAP powder was dispersed into ultrapure water as 1 mg/1 mL and injected into nude mice through a tail vein injection. An in vivo imaging test was conducted with a visible light imaging system  after 0.5 h, 1 h, 2 h, and 4 h. The mice in the control group were injected with the same volume of normal saline. Then, the tissues  were taken out and washed three times with PBS solution. The procedure was continued for the tissue samples for imaging.−1 and 631 cm−1 (-OH stretching vibration and bending vibration), 1100 cm−1 and 1030 cm−1 (ν3 PO43− asymmetric stretching), 603 cm−1, 564 cm−1 (ν4 PO43− antisymmetric stretching), and 962 cm−1 (ν1 PO43− asymmetric stretching). The ionic radius of the F− ions are smaller than that of the -OH group, but its electronegativity is higher than the latter. The crystallinity increase  and which means the sample was partially carbonized.The ratios of F substituted -OH are (i) 0%, (ii) 20%, (iii) 50%, and (iv) 100%, and for Eu/Gd substituted Ca is 2%/1.5%. The XRD results a show thincrease a, and th3+/Gd3+ (2%/1.5%) co-doped HAP  and (211) lattice planes with an interplanar spacing of 3.53 Å and 2.86 Å are assigned in high-resolution TEM images inset in b,c.6 electronic configuration and a strong red luminescence in the visible spectral region. The intensive excitation wavelength is centered at 394 nm in 5L6←7F0 energy level transition [5D0→7F1, 5D0→7F2, 5D0→7F3, and 5D0→7F4 energy level transitions, respectively. Luminescence emission intensity would slightly decline (in The fluorescence properties of rare earth elements would be significantly promoted by doping into an apatite lattice, which could provide a hydrophobic environment from fluorescence quenching and a lowered crystal symmetry environment for higher fluorescence emission. The trivalent europium ion has been a common choice in past research for a lanthanide fluorescence probe with a special 4fansition . The strcline in e due to 5D0→7F1 transition, magnetic dipole transition at 591 nm, is an invariable integrated intensity less influenced by Eu3+ surroundings. While, the 5D0→7F2 transition is extremely dependent on the vicinity of Eu3+ ions, the so-called hypersensitive transition. In general, the symmetry/asymmetry for Eu3+ can be instructed by the ratio of I617/I591 [591 and I617 refer to the intensity of 5D0→7F2 and 5D0→7F1 transition, respectively. The I617/I591 value is listed in 3+ to the Ca2+ sites in the crystal lattice [The 617/I591 , where IAs shown in In vivo fluorescent imaging was conducted, as shown in 3+ asymmetrical surroundings. The biosafety was verified by conducting hemolysis and cytotoxicity tests, and the tissue sections also showed no damage or abnormality. In comparison to healthy organs, Eu/Gd:FAP has a much higher accumulation in tumors, with a stronger and longer radiance signal. These findings contribute in several ways to our understanding of rare earth doped FAP and provide the basis for further research. More research is required to examine the long-term efficacy and safety of the material.In summary, we successfully prepared Eu/Gd co-doped fluoroapatite nanocrystals as a luminescent-enhancing agent. Due to F substitution, the resulting Eu/Ga:FAP has a smaller aspect ratio, higher crystallinity, and luminescence emission, suggesting the transformation of the main crystalline phase from HAP to FAP. With increasing hydrothermal temperatures, the crystallinity of Eu/Ga:FAP is further improved. Moreover, the fluorescence properties are significantly enhanced as a result of Eu


The magnitude of changes in GLU (peak after EC1: 26 ± 10% vs. EC2: 7 ± 6%), insulin (46 ± 27% vs. 15 ± 8%), HOMA (86 ± 48% vs. 24 ± 15%), TC (−20 ± 5% vs. −6 ± 4%), TG (−32 ± 11% vs. −6 ± 3%), LDHC (−47 ± 15% vs. −12 ± 9%), HDLC (35 ± 26% vs. 7 ± 4%), and TC/HDLC ratio (−139 ± 13% vs. −11 ± 7%) were significantly greater after EC1 than EC2. Peak plasma CK activity was significantly (p < 0.05) correlated with the peak changes in blood insulin sensitivity and lipid markers for the combined data of EC1 and EC2. These results suggest that the greater the magnitude of muscle damage, the greater the magnitude of changes in the insulin sensitivity to a negative direction and lipid markers to a positive direction.An acute bout of eccentric exercise affects insulin sensitivity and lipid profile, but how the magnitude of muscle damage affects them is not clear. We compared changes in blood insulin sensitivity and lipid markers after the first (EC1) and second (EC2) eccentric exercise bouts. Fifteen sedentary young men performed arm, leg and trunk muscle eccentric exercises, and repeated them 2 weeks later. Fasting blood samples were taken before, 2 h and 1–5 days after each exercise bout to analyze plasma creatine kinase (CK) activity, serum glucose (GLU), insulin, homeostasis model assessment (HOMA), triacylglycerols (TG), total (TC) and low- (LDLC) and high-density lipoprotein cholesterol (HDLC) concentrations as well as TC/HDLC ratio. Changes in these measures were compared between bouts and relationships to peak plasma CK activity were analyzed. Plasma CK activity increased ( Resistance exercise training induces not only muscle adaptations such as increases in muscle strength and mass, but also positive effects on insulin sensitivity and blood lipid profile . Generalo/s) eccentric contractions of the trunk extensors significantly increased resting serum glucose concentration by more than 10% and decreased TG by more than 11% at 2–3 days post-exercise, but no such effect was observed after concentric contractions that did not induce muscle damage. Thus, it is necessary to understand the effects of muscle damage on insulin sensitivity and blood lipid profile. In fact, in vivo performed every 7 days between bouts. It is possible that membrane integrity is strengthened even after the initial eccentric exercise bout, reducing the negative effects of eccentric exercise-induced muscle damage on glucose transporters and intracellular glucose disposal. It is well known that the magnitude of muscle damage is substantially attenuated when the same eccentric exercise is repeated within several weeks, which is known as the repeated bout effect  strength, delayed onset of muscle soreness (DOMS), plasma CK activity and myoglobin concentration . It is iTherefore, the purpose of the present study was to investigate the hypothesis that changes in insulin sensitivity and lipid markers after the first bout of whole-body eccentric exercise would be greater than those after the second bout of the same exercise performed 2 weeks later. The present study measured serum glucose, insulin, homeostasis model assessment (HOMA), TG, total (TC) and low- (LDLC) and high-density lipoprotein cholesterols (HDLC) concentrations and TC/HDLC ratio to investigate changes in these measures following the first and second eccentric exercise bouts and their relationships to peak plasma CK activity.The details of the study except for the blood measures specific to the present study can be found in the previous study . BrieflyThe participants performed two bouts of nine eccentric exercises (EC1 and EC2) consisting of arm curl (target muscles: elbow flexors), arm extension (elbow extensors), chest press , leg extension (knee extensors), leg curl (knee flexors), standing calf raise (plantar flexors), lat pulldown (latissimus), abdominal crunch (abdominis) and back extension (erector spinae) on nine different resistance training machines , with 2 weeks between bouts . For eacThe blood analyses consisted of plasma CK activity, serum glucose, insulin, HOMA, TG, TC, LDLC, HDLC concentrations as well as TC/HDLC ratio. The blood samples were collected after 10-hour of fasting between 7:00–9:00 a.m. except for the time point of 2-hour post-exercise. Blood sample was drawn by a standard venipuncture technique from the cubital fossa region of the arm to two 5-ml tubes; one containing ethylenediaminetetraacetic acid  and another serum separation tube. The blood was clotted at room temperature for the serum sample, and the tubes were centrifuged at 3,000 rpm for 10-minute to obtain plasma and serum, respectively, and the samples were separated to several tubes and stored at −80°C until analyses.Plasma CK activity was analyzed spectrophotometrically by an automated clinical chemistry analyzer  using a commercially available test kit e.g., . Serum gBlood lipid profile parameters consisted of serum TG, TC, HDLC, LDLC concentrations, and TC/LDHC ratio e.g., . These mThe test–retest reliability of each measure was determined by coefficient of variation (CV) can be found in the previous studies . The CV 2) were calculated as measures of effect size, and they were considered ∼0.02 as small effect, ∼0.13 as medium effect and >0.26 as large effect (n = 15) and EC2 (n = 15) separately, and EC1 and EC2 by pooling them together (N = 30). A significance level was set at p ≤ 0.05 for all analyses. The data were presented as mean ± standard deviation (SD).Baseline values of the blood measures before EC1 and EC2 were compared by paired t-tests. Changes in plasma CK activity, and serum glucose, insulin, HOMA, TG, TC, HDLC and LDLC concentrations as well as TC/LDHC ratio over time were compared between EC1 and EC2 by a two-way repeated-measures analysis of variance (ANOVA). When a significant interaction effect was found, a Tukey’s post-hoc test was performed. Eta-squared values (ηe effect . Additiop > 0.05) in the baseline values of serum TG, TC, HDLC and LDHC concentrations as well as TC/HDLC ratio were evident between bouts (p < 0.05) smaller than those before EC1  (No significant differences (en bouts . However ± 0.46) .2 = 0.787, EC2: η2 = 0.686), insulin , HOMA , TG , TC , HDLC  and LDHC  concentrations, and TC/HDLC ratio  changed significantly (p < 0.001) following both bouts (2 = 0.663), insulin (η2 = 0.639), HOMA (η2 = 0.754), TG (η2 = 0.762), TC (η2 = 0.669), HDLC (η2 = 0.663), LDHC (η2 = 0.780), and TC/HDLC ratio (η2 = 0.708) were significantly (p < 0.001) smaller after EC2 than EC1.th bouts . Howevern = 15), a significant (p < 0.01) correlation was found between the peak plasma CK activity and peak glucose (r = 0.793), insulin (r = 0.965), HOMA (r = 0.944), TC (r = −0.755), TG (r = −0.723), HDLC (r = 0.830) and LDLC (r = −0.778) concentrations as well as TC/HDLC ratio (r = −0.882). As for EC2 only (n = 15), a significant (p < 0.05) correlation was also observed between the peak plasma CK activity and peak glucose (r = 0.530), insulin (r = 0.540), HOMA (r = 0.569), TG (r = −0.538), HDLC concentrations (r = 0.555), but no significant correction was found between the peak plasma CK activity and peak TC  and LDLC concentrations  as well as TC/HDLC ratio . When pooling the data from EC1 and EC2 (N = 30), significant (p < 0.001) correlations were still evident between the peak plasma CK activity and peak glucose (r = 0.880), insulin (r = 0.938), HOMA (r = 0.943), TG (r = −0.895), TC (r = −0.856), HDLC (r = 0.888) and LDHC (r = −0.881) concentrations, as well as TC/HDLC ratio (r = −0.919) after EC1 and EC2.The main findings of the present study were follows. 1) Changes in all insulin sensitivity  and lipid markers  were significantly greater following EC1 than EC2, although the changes after EC2 were still significant . 2) The The magnitude of peak increases in glucose (26%), insulin (46%) and HOMA (86%) after EC1 was significantly greater than that after EC2  as shown in As shown in Changes in TC (−20%), TG (−32%), HDLC (35%), LDHC (−47%), TC/HDLC ratio (−39%) following EC1 were significantly greater than those after EC2  as shown in As shown in It is interesting that the effects of muscle damage on the lipid markers were the direction of favorable responses, in contrast to the glucose markers that showed unfavorable responses. It should be noted that the favorable or unfavorable changes after eccentric exercise found in the present study were acute responses, and the variables of both insulin sensitivity and lipid profile markers returned to the pre-exercise levels in 2 weeks before performing EC2 . Thus, eSeveral studies have shown that eccentric exercise training without muscle damage improved insulin sensitivity and blood lipid profile . For exaThere were several limitations in the present study. Firstly, the present study used relatively smaller number of young health sedentary men, thus the findings of the present study could not be generalized to other populations. Secondly, body composition, daily food intake and physical activity levels were not monitored in the present study. It is possible that these factors also affected the outcome measures. Thirdly, the present study only analyzed blood insulin sensitivity and lipid profile markers in relation to plasma CK activity as a muscle damage marker. It is interesting to investigate the relationships between other markers of muscle damage such as muscle function and muscle soreness and blood insulin sensitivity and lipid profile markers. However, it appears that plasma CK activity represent muscle plasma membrane damage well. Muscle biopsy technique to examine histological changes in muscle fibers and measuring energy markers such as adiponectin are necessary to understand the mechanisms underpinning the effects of muscle damage on glucose and lipid metabolism. Fourthly, there was no control or comparison group  in the present study. Thus, it is not known whether the changes in the blood insulin sensitivity and lipid profile markers were peculiar to eccentric exercise, and how whole-body concentric exercises affect the markers. Lastly, it is not known whether the whole-body eccentric exercises are more effective than other eccentric exercises, when they are performed regularly for a longer period of time , to improve insulin sensitivity and lipid profile.The present study showed the greater the magnitude of muscle damage induced by eccentric exercise, the more negative effects on the insulin sensitivity markers but the more favorable effects on the blood lipid profile markers. Further studies are warranted to investigate how muscle damage affected them, the mechanisms underpinning the repeated bout effect on the insulin sensitivity and lipid markers, and the effects of whole-body eccentric exercise training on obesity, metabolic syndrome, type 2 diabetes mellitus and other metabolic diseases.


Tg (Tg = 110 °C) of poly (PCTT) renders it useful as a substituent for PET in higher temperature applications. This work links thermal ageing and mechanical performance of a commercial PCTT plastic after exposure to 40–80 °C for up to 2950 h. No chemical or conformational changes were found while pronounced physical ageing, measured as enthalpic relaxation, caused yield hardening (28% increase in yield strength) and embrittlement (80% decrease in toughness). Enthalpic relaxation increased with temperature and time to 3.8 J g−1 and correlated to the determined toughness and yield strength. Finally, a 9% increase in Young's modulus was observed independent of temperature and with no correlation to enthalpic relaxation. Enthalpic relaxation followed Vogel–Fulcher–Tammann behaviour, while yield strength and charpy v-notch toughness followed Arrhenius behaviour enabling prediction of the different properties with time and temperature.Successfully evaluating plastic lifetime requires understanding of the relationships between polymer dynamics and mechanical performance as a function of thermal ageing. The relatively high  co-2,2,4,4-tetramethyl-1,3-cyclobutanediol terephthalate) shows physical ageing, without chemical degradation, resulting in 80% impact toughness decrease.Poly(1,4-cyclohexylenedimethylene- Polyesters are challenged in high-temperature environments due to their vulnerability to thermally induced mechanical changes which limits service life. These mechanical changes can be subdivided into chemical and physical ageing. Chemical aging e.g. hydrolysis and oxidation can cause polymer cleavage and/or cross-linking.5 Excessive cross-linking often results in embrittlement where a reduction in molecular mass reduces mechanical, thermal and rheological properties rendering the plastic defective.6 Physical ageing arises from super-cooling amorphous materials and is the subsequent time and temperature-dependent drift towards equilibrium.7 It is a general effect, influencing the amorphous phases in all polymer materials decreasing the ductile properties.8–10 This polymeric compaction by segmental polymer movement results in reduced free volume, and thus increases density of the amorphous phases.11,12 Physical ageing is only observed below the glass transition temperature  where the amorphous phases are solidified out of equilibrium. Thus, heating the material above Tg erases physical ageing, presented as enthalpic relaxation, determinable by differential scanning calorimetry (DSC).13 However, below Tg the driving force of physical ageing is the temperature difference between the exposed temperature (Te) and Tg14 implying that higher service temperature (below Tg) causes faster physical aging.Increased awareness of environmental impact from plastics has intensified the research into bio-based and recyclable polymeric materials.Tg–Te is a common strategy for physical aging inhibition, other inhibiting methods include increasing crystallinity, increasing molecular weight and addition of hydrogen-bonding additives.14,15 Increasing crystallinity reduces the size of the amorphous phases and restricts polymeric movement as a larger fraction of the amorphous phases are restricted by the crystal–amorphous interface. This mobility restriction of the polymer decreases the rate of enthalpic relaxation.16 Physical aging rate reduction has a nonlinear relationship with crystallinity in e.g. poly (PET) an increase in crystallinity from 19–21% causes 29% decrease in enthalpic relaxation rate while an increase from 38–48% causes 23% decrease in enthalpic relaxation rate.16 It could be argued that moisture acting as plasticiser increases physical aging rate, however, it is demonstrated in PET that moisture splits enthalpic relaxation into two peaks, but not consequently accelerating it.17,18While maximizing 19 The activation energy is the strength of temperature dependence, calculated from time–temperature superpositions . This is particularly useful when predicting properties of commodity materials, as they are typically used at various temperature ranges. However, when approaching Tg the relaxation times of the amorphous phase, such as enthalpic relaxation, is known to follow a Vogel–Fulcher–Tammann (VFT) behaviour as given in 20R0 is the rate factor, Te is the exposed temperature, T∞ is a material parameter or Vogel temperature and B is a shape parameter. The shape parameter B determines the degree of temperature dependence like the activation energy in Arrhenius factorisation. This accelerated behaviour further stress why Tg is an important property in evaluating the durability of commodity polymers in varying temperature environments, even when below Tg.Temperature acceleration is commonly evaluated by Arrhenius-factorisation, expressed by an activation energy.Tg = 75 °C) products are challenged by physical aging in elevated temperature applications.16 Mechanical implications of physical ageing for PET is observed as a decrease in essential work of fracture and increase in yield strength.21 These changes in mechanical properties causes physical ageing to be considered the most significant ageing kinetic in PET that limits its service life.22,23 PETG (Tg = 78 °C) is synthesised as PET but substituting a fraction of the ethylene glycol with 1,4-cyclohexanedimethanol (CHDM) causes the end-product to be more ductile but Tg only increases by ∼4 °C.24,25 A potential material could be poly  synthesised by replacing the ethylene in PETG with the rigid 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD) causing Tg to increase significantly, ∼35 °C.26 Thus, PCTT could be suitable for high temperature applications in comparison to PET and PETG. At present there is no literature or studies examining the chemical and mechanical properties of PCTT during thermal aging at temperatures below Tg.PET  for various periods (until 2950 h) and subsequent changes to chemical, thermal and mechanical properties were determined. Chemical changes were investigated by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and nuclear magnetic resonance spectroscopy (NMR) and morphological changes were evaluated by DSC where enthalpic relaxation was used to determine parameters for a VFT-model. Mechanical properties were evaluated by tensile and impact testing and used to determine parameters for an Arrhenius model.2.2.1.PCTT  was dried at 85 °C for six hours (to ∼0.03 wt% water) before injection moulding  at 260 °C into an 80 °C mould. 248 dumbbell-shaped tensile specimens  located in an environmentally controlled room  were kept at 40, 60 and 80 °C, respectively. One set was tested immediately as reference (0 h). 10 sets were placed in each oven and extracted after 8, 18, 43, 67, 95, 262, 418, 1127, 1605 and 2950 h. Three times six v-notch charpy specimens were heat-treated at 70 °C, 60% RH in a humidity chamber  and were extracted after 18, 43 and 67 h. After extraction all sets were stored for at least 48 h prior measurements for temperature and surface humidity equilibration.2.3.27 Young's modulus determination was between 0.05 to 0.25% elongation at 1 mm min−1 while yield strength, ultimate tensile strength, stress and strain at break were evaluated at 100 mm min−1 above 0.25% elongation.Test specimens were mounted with the inlet pointing outward in the bottom grip in a universal testing machine  fitted with a 25 mm gauge length clip on extensometer  all controlled by testXpert II . Tensile tests and data extractions were performed according to ISO 527-1.2.4.Charpy specimens were cut  according to ISO 179-1  type A-n2.5.∅4 mm cylindrical sample was punched from a tensile specimen and placed in a pan , covered with a lid  crimped , weighed  and inserted into the DSC . Temperature was swept twice from 20 °C to 300 °C and back to 20 °C at 20 °C min−1. DSC was applied to quantify enthalpic relaxation by integration of the endothermic peak at Tg.29 Enthalpic relaxation (ΔH(Tg)) was determined in Universal Analysis 2000  by numerical integration with a linear extrapolation from the constant heat flow plateau after Tg subtracted the unaged ΔH(Tg) (t = 0 h)  . Onset T2.6.aT) and reciprocal exposed temperature (1/Te). Yield strength and enthalpic relaxation shift factors were determined from superposing the logarithmic function, y = a + b log(x), to 40 °C. Charpy v-notch shift factors were determined from when toughness was halved and time-superposing to when 60 °C was halved. Yield strength and charpy v-notch toughness was fit with Arrhenius and enthalpic relaxation to the VFT equation.Arrhenius and VFT plots were created from shift factors  below the 10×  lens where it was imaged  and saved by software .2.8.−1 with avg. of 8 scans, wavelength dependent penetration depth and baseline were corrected in OMNIC . Bands 2934 cm−1, 1719 cm−1, 1452 cm−1, 1018 cm−1, 874 cm−1, and 729 cm−1 were integrated and normalized by the integrated area of the band at 874 cm−1 as a function of time at 40, 60, and 80 °C.Spectra were collected on the tensile specimen grip section on FT-IR  equipped with ZnSe ATR  from 500–4000 cm2.9.3  at 40 °C assisted by ultrasound  for 2 h. NMR spectrometer  recorded HCQS, NOESY, 16 scan 1H and 1024 scan 13C spectra with subsequent data-treatment in Mnova .PCTT samples were dissolved in 0.8 mL solution of 25 vol% trifluoroacetic acid  in CDCl3.3.1.trans, and cis atoms, respectively.The NMR assignment and calculations will follow the nomenclature given in i.e. terephthalic acid (TPA), 1,4-cyclohexanedimethanol (CHDM)24 and 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD).3013C NMR spectra of virgin PCTT is presented in PCTT was characterised by comparing the NMR spectra to analysis of the PCTT monomers, 13C NMR in NTT, NTC, NCT, and NCC) and clear splitting of the cis and trans configuration of the CHDM (i.e. 5′/5′′ and 6′/6′′) and TMCD (i.e. 12′/12′′) units was seen. 1H–1H COSY NMR  at 4.05 ppm has 1H–1H cross-peaks at 1.73, 1.66 and 4.85 ppm which are assigned to the equatorial, axial methine proton (5*) and hydroxyl proton (13*). TMCD contains no proton-containing adjacent groups, and thus HSQC is required to support the previous 13C NMR to 1H NMR analysis.The signals from proton-containing adjacent groups  allows f1H NMR and 13C NMR signals shown in cis and trans conformation in CHDM and correlate with the previous 13C NMR assignment of TMCD.30 Also, the methyl and methine end-groups present cross-signals in 1H–13C NMR at 4.05 ppm–53.42 ppm and 1.73/1.66 ppm–22.00 ppm, respectively. Lastly, no cross-signal is presented at the hydroxyl group at 4.85 ppm, confirming the CHDM end-group assignment. Full assignment from 1H and 13C NMR is presented in Table S1 ESI.†The correlation between i.e. that CHDM is the only end-group of linear polymers, then the average degree of polymerization of the CHDM (C) unit, nC, can be estimated from 1H NMR by dividing the methyl cis  and trans  peaks, by the end-group . TMCD (T) average degree of polymerization, nT, can similarly be calculated using the methine cis  and trans  peaks by the end-group  as given in I4 and I10 are the integrals of their respective peaks in 1H NMR.Assuming linearity M̄n, can be found as given in MC and MT are the molecular weights of the CHDM and TMCD repeating units (both 274 g mol−1), respectively.From the degree of polymerization, the average number molecular weight, fT/C) was calculated from the carbonyl 13C-NMR CHDM  and TMCD  peaks as given in TMCD/CHDM ratio  were calculated from the methine cyclic carbon peak in CHDM  and TMCD  as given in 13C NMR peak at 133.7 ppm and calculated from the concentration of TMCD and CHDM as given in Nij and Xi is the dyad sequence mole fraction and monomer mole fraction, respectively.Mole fraction of the Dyad sequences was determined from the benzene terephthalic acid 1H, 13C, COSY, HSQC and M̄n), and monomer fractions  and dyads were found. None of these values showed significant change at the 80 °C heat-treatment temperature over time  PCTT sample is given in ATR-FTIR spectra of reference  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretching is assigned to the band at 1719 cm−1 and C–O at 1267 cm−1. Hexane C–H is assigned to bending at 1452 cm−1 and stretching at 1018 cm−1.31 Aromatic C–H bending is assigned to the bands at 873 cm−1 while 1,4-para-substituted aromatic C–H out of plane stretch is assigned to the 729 cm−1 band.32,33 Which in combination verified the chemical composition. Ester C−1 CH2/CH3 stretching vibrations. Hence, ATR-FTIR analysis show no chemical change over the examined time and heat-treatment temperature range.3.3.Young's modulus is shown in From 3.4.Charpy v-notch specimen testing presented impact toughness decrease as a function of time and treatment temperature as shown in i.e. from yielding fracture to brittle fracture. This transition occurred earlier with higher treatment temperature, demonstrating a ductile-to-brittle transition of heat-treated PCTT with time and accelerated by temperature. However, samples heat-treated at 40 °C did not drop below half the initial toughness, and thus did not transition from ductile-to-brittle within the investigated time range.From Picturing the fracture surfaces of the heat-treated charpy v-notch bar from the ductile and brittle fracture are shown in A ductile  and a br3.5.H(Tg)) determined by DSC is shown in Enthalpic relaxation  of tensile testing while brittle results were clearly obtained during the higher strain rates of impact testing (3000 s−1)  .3639 Also, the enthalpic relaxation at 70 °C fits the VFT model in the investigated temperature-range  is temperature-independent.40 Although care needs to be taken when extrapolating outside  the investigated temperature range42 parameters for prediction of mechanical and thermal behaviour are determined and can be applied in prediction of PCTT performance resulting from different temperature and time conditions.Charpy v-notch toughness and especially yield strength did not express significant VFT behaviour, but Arrhenius behaviour in the investigated temperature range . Although below There are no conflicts to declare.RA-009-C9RA00925F-s001


ZnO NPs strongly inhibited cell proliferation and MTT reduction at 10 and 20 μg/cm2 after 72 h of treatment. ZnO NPs at 20 μg/cm2 elevated DCF fluorescence, indicating alterations in the cellular redox state associated with changes in ΔΨm and cell death. ZnO NPs also reduced the intracellular expression of troponin I and atrial natriuretic peptide. ZnO NPs are toxic for cardiac cells; therefore, consumption of products containing them could cause heart damage and the development of cardiovascular diseases.Zinc oxide nanoparticles (ZnO NPs) are widely used in the cosmetic industry. They are nano-optical and nano-electrical devices, and their antimicrobial properties are applied in food packaging and medicine. ZnO NPs penetrate the body through inhalation, oral, and dermal exposure and spread through circulation to various systems and organs. Since the cardiovascular system is one of the most vulnerable systems, in this work, we studied ZnO NPs toxicity in H9c2 rat cardiomyoblasts. Cardiac cells were exposed to different concentrations of ZnO NPs, and then the morphology, proliferation, viability, mitochondrial membrane potential (ΔΨm), redox state, and protein expression were measured. Transmission electron microscopy (TEM) and hematoxylin–eosin (HE) staining showed strong morphological damage. ZnO NPs were not observed inside cells, suggesting that Zn Zinc oxide nanoparticles (ZnO NPs) constitute a white thermostable material and are the most widely manufactured nanoparticles for personal care products including pigments, coatings, sensors, antibacterial creams, electronic gadgets, catalysts, food additives, and biomedical applications, among others ,2. Due tSince the main route of nanoparticle distribution is through systemic circulation, accounting for the accumulation of ZnO NPs in the heart, exposure to this material has been widely correlated with cardiovascular diseases . Oral adThe results obtained from the X-ray Powder Diffraction (XRD) pattern showed that the crystalline phases correspond to zinc oxide, according to the Joint Committee on Powder Diffraction Standards (JCPDS) card 01-080-7099 . On the 2 (2. At higher concentrations (≥ 10 μg/cm2), numerous large vesicles containing dense material were observed. Organelle disintegration was detected in the mitochondria and endoplasmic reticulum as well as the disruption of the cytoplasmic membrane and collapsed nuclei with a low density of nuclear material. These results indicated that ZnO NPs induced strong cellular damage. H9c2 cells were exposed to different concentrations of ZnO NPs for 24 h, and cellular uptake was evaluated by TEM. Cellular structures, such as the nucleus, nucleoli, mitochondria, and endoplasmic reticulum, showed no alterations in nonexposed cells. Nanoparticles were not observed inside the cells; however, important morphological changes were perceived at 5 μg/cm2 . Multipl2 of ZnO NPs. The most significant morphological changes were obtained at 20 μg/cm2, including compact nuclei, cytoplasm with limited stellar extensions, a plasma membrane rupture, and cytoplasm leakage, indicating strong cellular damage.ZnO NPs-derived changes in cellular structure were identified by hematoxylin–eosin (HE) staining. Nonexposed cells displayed the fusiform morphology of epithelial appearance with elongated and abundant cytoplasm, central and round nuclei, and several mitoses . An impo2 of ZnO NPs after 48 h of treatment, with the maximum effect (>50% of inhibition) at 72 h , the cell damage was so strong that the cellular redox state could not be determined.Since ZnO NPs decreased cell proliferation and MTT reduction, the changes in the cellular redox state were examined . After 12  significantly reduced intracellular troponin I and atrial natriuretic peptide expression by 27% and 20%, respectively  internalization as whole NPs and 2) internalization as free Zntoxicity ,22. One toxicity . Likewisn2+ ions . These r2+ ion concentration, generating intracellular ROS, plasma membrane leakage, mitochondrial dysfunction, and cell death [Morphological alterations induced by ZnO NPs were linked to decreased cell proliferation and MTT reduction , oxidatill death . In mousll death .Oxidative stress has been described as a key component of ZnO NPs toxicity . ZnO NPsWe did not detect mitophagy after ZnO NPs exposure (24 h); however, severe damage overcame cellular defenses, resulting in nuclear and mitochondrial alterations and causing apoptosis and necrosis. Interestingly, our results in fluorescence microscopy revealed apoptosis and necrosis in exposed cells, whereas flow cytometry detected only apoptosis . The dif2, thus we suppose that intracellular levels decreased while secreted proteins increased. This result supports the strong injury caused by ZnO NPs in cardiac cells. Markers of cardiac injury include the natriuretic peptides released from myocytes in cardiac atria and ventricles in response to increased wall tension and stretch to reduce blood pressure, cardiac hypertrophy, and ventricular fibrosis ,36. On t2 (33 and 66 µg/mL) were the most toxic to H9c2 cells. A pharmacokinetic study of rats that were orally exposed to single-dose ZnO NPs showed that 50 mg/Kg ZnO NPs displayed the maximum plasma concentration which was near 100 µg/mL after 6 h [Remarkably, the ZnO NPs concentrations of 10 and 20 μg/cmfter 6 h . Despitefter 6 h .2 NPs) and silver (AgNPs). Previous works using human endothelial cells, an excellent model for studying toxicity in the cardiovascular system, have compared the cytotoxicity of several metallic NPs, showing that ZnO NPs were more cytotoxic than Fe3O4, Al2O3, AgNPs, and TiO2 NPs [2 NPs inhibit proliferation and induce apoptotic and necrotic death in human umbilical vein endothelial cells (HUVEC). Anyway, they provoked endothelial dysfunction and activation, mediating the release of inflammatory cytokines, the adhesion of monocytes, and the increased expression of adhesion molecules [2 NPs and AgNPs has also been determined in cardiomyocytes. Rat and human cardiomyocytes exposed to 100 μg/mL and 10 μg/mL of TiO2 NPs reduced the contraction amplitude and the beating rate, respectively [2 NPs has also been associated with arrhythmic events [Similar toxic effects induced by ZnO NPs in H9c2 cells have been described in vitro in metal-based NPs such as titanium dioxide  high glucose, Fetal Bovine Serum (FBS), antibiotic–antimycotic solution (Anti-Anti 100X), and 0.25% trypsin-EDTA solution were purchased from Thermo Fisher Scientific . Western blot reagents were acquired from Bio-Rad . Antibodies against β-actin, troponin I, and atrial natriuretic peptide were purchased from Santa Cruz Biotechnology . The autophagy assay kit was supplied by ABCAM . Annexin-V-FLUOS staining kit was provided by Roche . Annexin-V-FITC was provided by Biolegend , and Annexin-V buffer and Propidium Iodine were supplied by BD . Sterile plastic material for tissue culture and 8-well tissue culture chambers were obtained from Sarstedt . MitoTracker Green FM and LysoTracker were from Invitrogen . ZnO nanopowder < 50 nm particle size (BET), >97% (cat. No. 677450), and all other reagents were purchased from Sigma-Aldrich . ® CRL-1446). Cells were cultured in DMEM high glucose supplemented with 10% FBS and Anti–Anti solution (1×). Cells were maintained at 37 °C under a humidified atmosphere with 5% CO2.Cells derived from embryonic rat ventricular tissue (H9c2) were obtained from the American Type Culture Collection . Both suspensions were vortexed at 60 Hz for 10 min for subsequent reading on the Zetasizer Nano-ZS90 equipment. To identify the phases of ZnO NPs crystals, an XRD pattern was performed. For this, a Siemens D5000 diffractometer was used with Cu Kα radiation (λ = 1.541874 Angstrom) in the range of 20–90° in 2-Theta at a scanning step of 0.020°/1.2 s at room temperature. ZnO NPs were also analyzed by TEM to determine the nanoparticle size. NPs were examined in a JEOL transmission electron microscope model ARM200F, using copper Agar brand grids with formvar cover and carbon type A as well as with an acceleration voltage of 200 kV. 2) for 24 h. After treatment, H9c2 cells were fixed with 2.5% glutaraldehyde-paraformaldehyde in phosphate buffer solution (PBS) (pH 7.2) for 45 min. Afterwards, fixation cells were placed in 1% osmium tetroxide for 1 h. Then, cells were dehydrated through graded series of alcohols and embedded in Epon 812 epoxy resin. Then, 60-nm-thin sections were obtained with a diamond knife on Ultracut-R ultramicrotome; mounted on copper grids; and impregnated with heavy metals, lead nitrate, and uranyl acetate. Grids were examined in a JEOL 10/10 transmission electron microscope at 60 kV and an AMT Camera System. ZnO NPs internalization was detected by TEM. Cells were cultured with ZnO NPs  were seeded on 8-well tissue culture chambers; exposed to 2.5, 5, 10, and 20 μg/cm2 over 48 h; stained with hematoxylin–eosin (HE); analyzed with a microscope Olympus BX51 with 20X objective; and photographed with the camera Q Imaging MicroPublisher 5.0 RTV.For morphological analysis, H9c2 cells (20 × 103/well) were cultured in 96-well plates and exposed to different concentrations of ZnO NPs  over 24, 48, and 72 h. No exposed cells were used as a negative control. After exposure, cells were fixed with 1.1% glutaraldehyde for 10 min, washed twice with water, and stained with 0.1% crystal violet solution for 20 min. Incorporated crystal violet was solubilized in 100 μL/well of acetic acid (10%), and optical density at 590 nm was measured in a microplate spectrophotometer . To assay viability, 20 μL/well of MTT (5 μg/mL) were added, and cells were incubated for 4 h at 37 °C. Then the medium was removed, formazan crystals were dissolved with acid isopropanol (0.04 N HCl), and optical density was read at 570 nm.Proliferation and viability were evaluated by crystal violet staining and MTT reduction respectively, as previously described by our work group . H9c2 ce2DCFDA) into 2,7-dichlorodihydrofluorescein (DCF) which is a redox indicator probe [3) were cultured with ZnO NPs  for 1, 3, and 6 h in 56-mm glass Petri dishes. After treatment, cells were trypsinized and incubated with H2DCFDA (10 μM) for 30 min, and they were washed twice with PBS. Fluorescence was quantified in a FACSAria flow cytometer . The cellular redox state was measured by oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (Hor probe . H9c2 ce6) were treated with ZnO NPs  for 1 h. Then, cells were washed with PBS and incubated with Rh123 (5 μg/mL) for 15 min. After incubation, cells were washed with PBS and analyzed in a FACSAria flow cytometer . Data was processed using FlowJo 8.7 software (Stanford University).Oxidative stress was also tested through ΔΨm changes based on the fluorescent dye rhodamine 123 (Rh123). For this, cell suspensions (1 × 104) were cultured in 35-mm glass-bottomed Petri dishes  with ZnO NPs  for 6 h. To detect nuclei, mitochondria, or lysosomes, cells were pre-incubated with 0.4 µM Bis-Benzimide H 33342 trihydrochloride (Hoechst), 0.5 µM MitoTracker Green (MTG), and 0.5 µM LysoTracker Red (LTR), respectively, for 30 min at 37 °C in DMEM without phenol red. Epifluorescence images were taken with the EVOS FL  cell-imaging microscope at 60X magnification.To assay mitophagy , H9c2 rat cardiomyoblasts (50 × 103/well) were cultured in 6-well plates and treated with 2.5, 5, 10, and 20 μg/cm2 of ZnO NPs for 24 and 48 h. Afterwards, exposure cells were analyzed by fluorescence microscopy in a Floid Cell Imaging Station . For this, the culture medium was discarded, and cells were washed with PBS and subsequently incubated with 100 μL of annexin-V-Fluos labeling solution (20 μL propidium iodide plus 20 μL annexin-V-Fluos in 1 mL of incubation buffer) for 15 min then analyzed immediately. The annexin-V-Fluos staining kit was used to determine cell death by microscopy. H9c2 cells . For flow cytometry, cells were washed with PBS, trysinized, incubated with 100 μL of annexin-V-Fluos labeling solution, and immediately analyzed. Data was processed using FlowJo 8.7 software (Stanford University).Apoptosis and necrosis were also examined through flow cytometry, using dual staining with annexin V-FITC and propidium iodide, according to the manufacturer’s instructions. Briefly, 1 × 102) for 48 h, and total proteins were isolated with a lysis extraction buffer. Proteins were quantified using Bio-Rad protein assay dye reagent concentrate. Proteins (30 μg) were separated through electrophoresis, using 8% SDS-polyacrylamide gels; transferred to PVDF membranes; blocked; and incubated with primary antibodies against troponin I, atrial natriuretic peptide, and β-actin as a load control  overnight. Afterwards, membranes were washed three times with Tris Buffered Saline with Tween (TBS-T), incubated with the secondary antibody (diluted 1:2500) for 1 h, and washed again three times with TBS-T. Proteins were detected with the SuperSignal® system and the ChemiDocTM MP Imaging System (Bio-Rad). Densitometric analysis for protein quantification was carried out with the Image Lab™ V 4.0 Software (Bio-Rad).Western blot analysis was performed as described previously by our work group . Cells wt-tests were performed, and p < 0.05 was considered significant. For western blot analysis, multiple comparisons were based on one-way analysis of variance (ANOVA), followed by Turkey’s pairwise comparison as a post hoc test in Prism 5.01.To quantify and compare the patterns of actin structures, a nonparametric Kruskal–Wallis test was performed using the Prism 7.0a software . In order to determine statistical differences in all assays, Student’s ZnO NPs induce severe damage to cardiomyocytes, characterized by lower cell proliferation, higher oxidative stress, cell death, and strong morphological changes with cellular disruption; therefore, the consumption of products containing ZnO NPs could cause the development of organ failure and cardiovascular diseases.