Transferase-mediated dUTP Nick-end Labeling Assay Research Articles

Korean red ginseng decreases 1-methyl-4-phenylpyridinium-induced mitophagy in SH-SY5Y cells

ObjectiveMitophagy is known to contribute towards progression of Parkinson’s disease. Korean red ginseng (KRG) is a widely used medicinal herb in East Asia, and recent studies have reported that KRG prevents 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell death. This study was undertaken to investigate whether KRG suppresses MPP+-induced apoptosis and mitophagy. MethodsSH-SY5Y cells were incubated with KRG for 24 h, and subsequently exposed to MPP+. The MPP+-induced cell death was confirmed with the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Changes in the structure and function of mitochondria were confirmed using mitotracker, MitoSOX red mitochondrial superoxide indicator, parkin, and phosphatase and tensin homolog deleted on chromosome ten-induced putative kinase 1 (PINK1) immunofluorescent staining. Western blotting was performed to evaluate the expression of apoptosis-related factors in whole cells, including Bax, Bcl-2 and cleaved caspase-3, and mitophagy-related factors in the mitochondrial fraction, including cytochrome c, parkin, PINK1, translocase of the outer membrane 20 (TOM20), p62 and Beclin 1. ResultsMPP+ induced cell death by cytochrome c release and caspase-3 activation; however, this effect was suppressed by KRG’s regulation of the expressions of Bcl-2 and Bax. Moreover, MPP+ exposure increased the mitochondrial expressions of parkin, PINK1, Beclin 1 and p62, and decreased TOM20, cytochrome c and Bcl-2 expressions. These MPP+-induced changes in the mitochondrial fraction were attenuated by treatment with KRG. ConclusionKRG effectively prevents MPP+-induced SH-SY5Y cell death by regulating cytochrome c release from mitochondria and PINK1/parkin-mediated mitophagy, through regulation of the Bcl-2 family.

Combination of Melatonin and Zoledronic Acid Suppressed the Giant Cell Tumor of Bone in vitro and in vivo.

Melatonin (Mlt) confers potential antitumor effects in various types of cancer. However, to the best of our knowledge, the role of Mlt in the giant cell tumor of bone (GCTB) remains unknown. Moreover, further research is required to assess whether Mlt can enhance the therapeutic effect of zoledronic acid (Zol), a commonly used anti-GCTB drug. In this research, we investigated the effects of Mlt, Zol, and the combination of these two drugs on GCTB cells’ characteristics, including cell proliferation, apoptosis, osteogenic differentiation, migration, and invasion. The cell counting kit-8 (CCK-8) assay, colony formation assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL), alkaline phosphatase (ALP) staining, alizarin red staining (ARS), scratch wound healing assay, and transwell experiment were performed, respectively. Our results showed that Mlt could effectively inhibit the proliferation, migration, and invasion of GCTB cells, as well as promote the apoptosis and osteogenic differentiation of tumor cells. Of note, a stronger antitumor effect was observed when Mlt was combined with Zol treatment. This therapeutic effect might be achieved by inhibiting the activation of both the Hippo and NF-κB pathways. In conclusion, our study suggests that Mlt can be a new treatment for GCTB, which could further enhance the antitumor effect of Zol.

The cyto-protective effects of LH on ovarian reserve and female fertility during exposure to gonadotoxic alkylating agents in an adult mouse model.

STUDY QUESTIONDoes LH protect mouse oocytes and female fertility from alkylating chemotherapy?SUMMARY ANSWERLH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan.WHAT IS KNOWN ALREADYChemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment.STUDY DESIGN, SIZE, DURATIONThis experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment.PARTICIPANTS/MATERIALS, SETTING, METHODSIn the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis.MAIN RESULTS AND THE ROLE OF CHANCELH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation.WIDER IMPLICATIONS OF THE FINDINGSThe current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest.

Cardiotonic Activity of Daidzein Against Doxorubicin-Induced Congestive Cardiac Failure in Rats

Despite significant medical advances, congestive heart failure remains associated with high mortality rate. Therefore, there is a continuing need for better therapeutics. In this study, we have examined the effect of daidzein against congestive heart failure induced by administration of doxorubicin. Blood pressure, electrocardiogram, B-type natriuretic peptide, and markers of oxidative stress were assessed as measures of cardioprotective effect of daidzein. To further assess the mechanism, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, Western blot assay, and histopathology were performed. Daidzein reduced blood pressure, reversed the altered pattern of electrocardiogram, levels of B-type natriuretic peptide, and oxidative stress markers in doxorubicin-induced cardiotoxicity in rats. Furthermore, treatment with daidzein ameliorated apoptosis and expression of proinflammatory proteins and oxidative pathway.

Circular RNA circ-FAM158A promotes retinoblastoma progression by regulating miR-138–5p/SLC7A5 axis

BackgroundMounting evidence has shown that circular RNAs (circRNAs) have vital roles in human cancers, including retinoblastoma (RB). The purpose of this study was to investigate the exact roles and underlying mechanism of circRNA ER membrane protein complex subunit 9 (circ-FAM158A) in RB. MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was carried out to determine the expression levels of circ-FAM158A, miR-138–5p and solute carrier family 7 member 5 (SLC7A5). Cell proliferation was evaluated by Cell counting Kit-8 (CCK-8) assay and colony formation assay. Flow cytometry analysis was applied to determine cell cycle distribution and apoptosis rate. Transwell assay was conducted to assess cell migration and invasion. The interaction between miR-138–5p and circ-FAM158A or SLC7A5 was predicted by starBase v2.0 and confirmed by dual-luciferase reporter assay. Western blot assay was performed to examine the protein expression of SLC7A5. The mice xenograft model was established, immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assays were conducted to confirm the role of circ-FAM158A in vivo. ResultsCirc-FAM158A and SLC7A5 were overexpressed and miR-138–5p was downregulated in RB tissues and cells. Circ-FAM158A knockdown inhibited RB cell proliferation, metastasis, and promoted apoptosis in vitro and in vivo. MiR-138–5p was a direct target of circ-FAM158A, and miR-138–5p inhibition reversed the inhibitory effect of circ-FAM158A silence on the progression of RB cells. Additionally, SLC7A5 was identified as a target of miR-138–5p, and SLC7A5 overexpression abated the anti-tumor roles of miR-138–5p in RB cells. Besides, circ-FAM158A positively regulated SLC7A5 expression by sponging miR-138–5p. ConclusionCirc-FAM158A knockdown inhibited the progression of RB by regulating miR-138–5p/SLC7A5 axis, which provided new insights into the pathogenesis of RB.

LncRNA MIR155HG Accelerates the Progression of Sepsis via Upregulating MEF2A by Sponging miR-194-5p.

Long noncoding RNA MIR155HG exerts important effects in the progression of multiple diseases. This study investigated the functions of MIR155HG in sepsis development. Blood samples were collected from 28 patients with sepsis and 28 without sepsis. The murine cardiac muscle cell line (HL-1) and macrophage cell line (RAW 264.7) treated with lipopolysaccharide (LPS) were used as the in vitro sepsis models. The levels of MIR155HG, miR-194-5p, and MEF2A were determined using real-time-quantitative polymerase chain reaction. Cell counting kit-8 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were used to assess cell viability and apoptosis, respectively. The association between miR-194-5p and MIR155HG or MEF2A was confirmed using a dual-luciferase reporter assay. The levels of inflammatory cytokines were detected using enzyme-linked immunosorbent assay (ELISA). In this study, we demonstrated that MIR155HG expression was significantly increased in sepsis blood samples, RAW 264.7, and HL-1 cells treated with LPS. Silencing of MIR155HG promoted cell viability and obstructed cell apoptosis and inflammation of RAW 264.7 and HL-1 cells treated with LPS. MiR-194-5p depletion abrogated cell viability promotion and suppressive effect on cell apoptosis and inflammation caused by MIR155HG knockdown. In addition, MIR155HG upregulated MEF2A through interaction with miR-194-5p. Finally, rescue assays indicated that MEF2A overexpression abolished the inhibitory effect on sepsis progression induced by MIR155HG deletion. In conclusion, MIR155HG promotes sepsis progression in an in vitro sepsis model by modulating the miR-194-5p/MEF2A axis. This discovery provides a promising biomarker for sepsis therapy.

Long noncoding RNA SOX2OT silencing alleviates cerebral ischemia-reperfusion injury via miR-135a-5p-mediated NR3C2 inhibition

ObjectiveThis study is aimed to investigate the role of the long noncoding RNA SOX2 overlapping transcript (SOX2OT) in cerebral ischemia-reperfusion injury (CIRI) and the underlying regulatory mechanisms. MethodsThe oxygen-glucose deprivation/reoxygenation (OGD/R)-treated PC12 cells and middle cerebral artery occlusion/reperfusion (MCAO/R)-treated rats were established to simulate CIRI condition in vitro and in vivo. Quantitative real-time polymerase chain reaction was performed to detect the expression of SOX2OT, microRNA-135a-5p (miR-135a-5p), and nuclear receptor subfamily 3 group C member 2 (NR3C2). The cell viability and apoptosis were analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) or interleukin (IL)-1β and IL-6 were used to evaluate the oxidative stress or inflammation. Dual-luciferase reporter assay was conducted to validate the interactions among SOX2OT, miR-135a-5p, and NR3C2. Additionally, neurological deficit scores (NDS), infarct volume, and brain edema were used to assess brain impairments in vivo. ResultsThe expression of SOX2OT and NR3C2 was increased, while miR-135a-5p was decreased in OGD/R-treated PC12 cells. SOX2OT silencing repressed the levels of LDH, MDA, ROS, IL-1β, IL-6, reduced the numbers of TUNEL positive cells, and elevated viability and SOD level in OGD/R-treated PC12 cells. Besides, SOX2OT targeted miR-135a-5p, and miR-135a-5p targeted NR3C2. Both miR-135a-5p downregulation and NR3C2 upregulation reversed the suppressive effects of SOX2OT knockdown on oxidative stress, apoptosis, and inflammation of OGD/R-treated PC12 cells. Furthermore, injection of sh-SOX2OT reduced the NDS, cerebral infarct, and cerebral edema in MCAO/R-treated rats. ConclusionsSilencing of SOX2OT attenuated CIRI via regulation of the miR-135a-5p/NR3C2 axis, which may provide a novel therapeutic target for CIRI.

Mild therapeutic hypothermia improves neurological outcomes in a rat model of cardiac arrest

Cardiac arrest (CA) is the leading cause of death in humans. Research has shown that mild therapeutic hypothermia (MTH) can reduce neurological sequelae and mortality after CA. Nevertheless, the mechanism remains unclear. This study aimed to determine whether MTH promotes neurogenesis, attenuates neuronal damage, and inhibits apoptosis of neurons in rats after CA. Sprague–Dawley rats were divided into the normothermia and mild hypothermia groups. The rats in the normothermia and hypothermia groups were exposed to 2 h of normothermia (36–37℃) and hypothermia (32–33℃), respectively, immediately after resuscitation from 5 min of asphyxial CA. Corresponding control groups not subjected to CA were included. On days 1–6, 5-bromodeoxyuridine (BrdU) 100 mg/kg/day was administered intraperitoneally. The animals were euthanized 1 week after CA. Compared with the normothermia group, the hypothermia group showed a significant increase in the number of doublecortin (DCX) immune-positive cells in the subgranular zone of the hippocampus 1 week after CA. Neurogenesis was assessed using double immunofluorescent labeling of BrdU with neuronal-specific nuclear protein (NeuN)/DCX. There was no marked change in the number of newborn mature (BrdU+-NeuN+) neurons, though there was a significant increase in the number of newborn immature (BrdU+-DCX+) neurons in the hypothermia than in the normothermia group 1 week after CA. Neuronal injury and apoptosis in the CA1 region of the hippocampus, assessed using NeuN immunofluorescence and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays, were significantly reduced in the hypothermia group 1 week after CA. Moreover, mild hypothermia increased the expression of cold-shock protein RNA-binding motif protein 3 (RBM3) in the early stage (24 h/48 h) after CA. These results suggested that mild hypothermia promotes generation of neuronal cells, reduces neuronal injury, and inhibits apoptosis of neurons, which may be related to RBM3 expression.

Effect of resveratrol on mouse ovarian vitrification and transplantation

BackgroundAfter ovarian tissue transplantation, ischemia-reperfusion injury and free radicals cause follicle depletion and apoptosis. Therefore, the use of antioxidants to reduce the production of free radicals is an important method to address the consequences of ischemia-reperfusion injury. Resveratrol is a natural active polyphenol compound with anti-inflammatory, antitumor, strong antioxidant and anti-free radical properties. The aim of this study was to investigate whether resveratrol could improve the effect of autologous ovarian transplantation after cryopreserve-thawn mouse ovarian tissue.MethodsWhole-ovary vitrification and autotransplantation models were used to investigate the effects of resveratrol. Six-week-old female mice from the Institute of Cancer Research (ICR) were subjected to vitrification. All ovaries were preserved in liquid nitrogen for 1 week before being thawed. After thawing, ovarian tissues were autotransplanted in the bilateral kidney capsules. Mice (n = 72) were randomly divided into four groups to determine the optimal concentration of resveratrol (experiment I).Treatments were given as follows: saline, 5 mg/kg resveratrol, 15 mg/kg resveratrol and 45 mg/kg resveratrol, which were administered orally for one week. Grafted ovaries were collected for analysis on days 3, 7, and 21 after transplantation. Ovarian follicle morphology was assessed by hematoxylin and eosin staining. Serum FSH and E2 levels were measured to estimate the transplanted ovarian reserve and endocrine function. Other mice were randomly divided into two groups—saline and 45 mg/kg resveratrol to further evaluate the effect of resveratrol and explore the mechanisms underlying this effect (experiment II). Ovarian follicle apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. Immunohistochemistry, qRT-PCR and western blotting (MDA, SOD, NF-κB, IL-6 and SIRT1) were used to explore the mechanisms of resveratrol. Moreover, oocytes derived from autotransplanted ovaries at 21 days were cultured and fertilized in vitro.ResultsThe proportions of morphologically normal (G1) follicles at 3, 7 and 21 days were significantly higher in the 45 mg/kg resveratrol group than in the saline group. The TUNEL-stained follicles (%) at 7 days were significantly decreased in the 45 mg/kg resveratrol group compared with the saline group. Western blot analysis revealed that SOD2 and SIRT1 levels were significantly higher in the 45 mg/kg resveratrol group than in the saline group at day 7 and that MDA and NF-κB levels were lower in the saline group on day 3. Likewise, IL-6 was lower in the saline group on day 7. These results are basically consistent with the qRT-PCR results. In addition, the mean number of retrieved oocytes and fertilization and cleavage were significantly increased in the 45 mg/kg resveratrol group compared with the saline group.ConclusionsAdministration of resveratrol could improve the quality of cryopreserved mouse ovarian tissue after transplantation and the embryo outcome, through anti-inflammatory and antioxidative mechanisms.

Embryo Buoyancy and Cell Death Gene Expression During Embryogenesis of Yellow-Tail Kingfish Seriola lalandi.

In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.

Effects of carnosine and hypothermia combination therapy on hypoxic-ischemic brain injury in neonatal rats.

BackgroundCarnosine has antioxidative and neuroprotective properties against hypoxic-ischemic (HI) brain injury. Hypothermia is used as a therapeutic tool for HI encephalopathy in newborn infants with perinatal asphyxia. However, the combined effects of these therapies are unknown.PurposeHere we investigated the effects of combined carnosine and hypothermia therapy on HI brain injury in neonatal rats.MethodsPostnatal day 7 (P7) rats were subjected to HI brain injury and randomly assigned to 4 groups: vehicle; carnosine alone; vehicle and hypothermia; and carnosine and hypothermia. Carnosine (250 mg/kg) was intraperitoneally administered at 3 points: immediately following HI injury, 24 hours later, and 48 hours later. Hypothermia was performed by placing the rats in a chamber maintained at 27°C for 3 hours to induce whole-body cooling. Sham-treated rats were also included as a normal control. The rats were euthanized for experiments at P10, P14, and P35. Histological and morphological analyses, in situ zymography, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, and immunofluorescence studies were conducted to investigate the neuroprotective effects of the various interventional treatments.ResultsVehicle-treated P10 rats with HI injury showed an increased infarct volume compared to sham-treated rats during the triphenyltetrazolium chloride staining study. Hematoxylin and eosin staining revealed that vehicle-treated P35 rats with HI injury had decreased brain volume in the affected hemisphere. Compared to the vehicle group, carnosine and hypothermia alone did not result in any protective effects against HI brain injury. However, a combination of carnosine and hypothermia effectively reduced the extent of brain damage. The results of in situ zymography, TUNEL assays, and immunofluorescence studies showed that neuroprotective effects were achieved with combination therapy only.ConclusionCarnosine and hypothermia may have synergistic neuroprotective effects against brain damage following HI injury.

DNA fragmentation of sperm in automobile painters.

Reproductive health is a major concern for workers constantly exposed to chemicals at work, especially in developing countries. The problems mainly stemmed from chronic exposure to the chemicals contained in paint thinner, which is mainly toluene, used during automobile painting. Unfortunately, in Indonesia, there is no study on the reproductive health of male workers in automobile workshops, especially in relation to spermatogenesis. This study aimed to explore the possibility of reproductive problems, namely spermatozoa DNA fragmentation, among male workers in an automobile workshop in Malang. We recruited workers who have worked as automobile painters for years at an automobile workshop in Malang and have been chronically exposed to thinner. Semen samples were collected from 15 automobile workshop workers who were married and had children. Twelve office workers were used as the control group. The two groups of semen samples were tested with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay combined with propidium iodide and analyzed by flow cytometry. The results showed a significant difference between DNA fragmentation of the control group and workshop workers at 14.9% and 47.66%, respectively. Risk factor analysis showed a significant increase in risk factors (p = 0.001). Taken together, chronic exposure to paint thinner in those engaged in painting at the automobile workshop increased the risk of DNA fragmentation in spermatozoa. Our study highlighted the importance of health education for the risked workers on minimization of exposure, which could be recommended to the health policy makers.

Combined Administration of Metformin and Atorvastatin Attenuates Diabetic Cardiomyopathy by Inhibiting Inflammation, Apoptosis, and Oxidative Stress in Type 2 Diabetic Mice.

Diabetic cardiomyopathy (DCM), a common complication of diabetes mellitus, may eventually leads to irreversible heart failure. Metformin is the cornerstone of diabetes therapy, especially for type 2 diabetes. Statins are widely used to reduce the risk of cardiovascular diseases. In this study, we aimed to investigate whether the combined administration of metformin and atorvastatin could achieve superior protective effects on DCM and to elucidate its molecular mechanism. Here, db/db mice (9–10 weeks old) were randomly divided into four groups, including sterile water group (DM), metformin group (MET, 200 mg/kg/day), atorvastatin group (AVS, 10 mg/kg/day), and combination therapy group (MET + AVS). Mice were treated with different drugs via gavage once per day for 3 months. After 3 months of treatment, the pathological changes (inflammation, fibrosis, hypertrophy, and oxidative stress makers) were detected by histopathological techniques, as well as Western blotting. The H9C2 cardiomyocytes were treated with palmitate (PAL) to mimic diabetic condition. The cells were divided into control group, PAL treatment group, MET + PAL treatment group, AVS + PAL treatment group, and MET + AVS + PAL treatment group. The effects of MET and AVS on the cell viability and inflammation of H9C2 cells subjected to PAL condition were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, immunofluorescence staining, and Western blotting. Both MET and AVS prevented diabetes-induced fibrosis, hypertrophy, and inflammation. The combination therapy showed superior effects in protecting myocardial tissue against diabetes-induced injury. Mechanistically, the combination therapy significantly inhibited oxidative stress and the expression levels of inflammation-related proteins, e.g., NLRP3, caspase-1, interleukin-1β (IL-1β), Toll-like receptor 4 (TLR4), and P-p65/p65, in both cardiac tissues and H9C2 cells. TUNEL assay showed that the combination therapy significantly attenuated the apoptosis of cardiomyocytes; decreased the expression level of pro-apoptotic-related proteins, such as cleaved caspase-3 and BAX; and enhanced the expression level of anti-apoptotic protein (Bcl-2). Furthermore, the combination therapy remarkably upregulated the expression levels of 5′-AMP-activated protein kinase (AMPK) and SIRT1. Our findings indicated that the anti-inflammation and anti-apoptosis effects of the combination therapy may be related to activation of AMPK/SIRT1 signaling pathway.

In vitro maturation on ovarian granulosa cells encapsulated in agarose matrix improves developmental competence of porcine oocytes

In vivo, mammalian oocytes are surrounded by granulosa cells (GCs) that exist in a three-dimensional (3D) microenvironment with soft stiffness. The GCs play an important role for the in vivo growth and development of oocytes, through bidirectional communication between oocytes and GCs. To mimic the cellular microenvironment of a 3D organized follicle, this study designed a co-culture system using porcine ovarian GCs (pGCs) encapsulated in agarose matrix for in vitro maturation (IVM) of pig oocytes. We report the effects of our newly designed co-culture system on IVM and development of pig oocytes. Immature cumulus-oocyte-complexes (COCs) were matured on a 1% (w/v) agarose matrix encapsulated without or with pGCs. The number of pGCs within the agarose matrix was optimized by analyzing the in vitro development of parthenogenetic embryos. Moreover, the role of the ovarian stromal pGCs as feeder cells was assessed by analyzing the PA embryonic development. Subsequently, the effect of pGCs encapsulated in a 3D agarose matrix was evaluated for the developmental competence of pig oocytes by analyzing blastocyst formation after parthenogenetic activation (PA), intra-oocyte GSH and ROS contents, expression levels of BMP15 and BAX, TUNEL (terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling) assay, protein expression levels of BMP15, and intra-oocyte ATP levels. The optimized number of pGCs (5 × 104 cells/well) in a 3D agarose matrix led to a significantly higher blastocyst formation, increased BMP15 gene and protein expression, and intra-oocyte ATP levels; moreover, it induced significantly lower intra-oocyte ROS contents, pro-apoptotic BAX gene expression, and apoptotic index, compared to control. Our results demonstrate that application of pGCs as feeder cells encapsulated in the agarose matrix for IVM effectively increases the developmental competence of porcine oocytes.

Programmed cell death during the formation of rhytidome and interxylary cork in roots of Astragalus membranaceus (Leguminosae).

Programmed cell death (PCD) plays a critical role throughout the lives of plants, it is regarded as a highly regulated and active process of plant cell death during the times of biotic or abiotic stress. This study aims to provide developmental anatomical characteristics of the interxylary cork formation in the roots of Astragalus. membranaceus var. mongholicus, and to subsequently show cytomorphological evidence that PCD is involved in the development of rhytidome and interxylary cork. The developmental anatomy of rhytidome and interxylary cork of the perennial fresh main root of A. membranaceus var. mongholicus was studied using light microscopy, whereas the PCD in the development of rhytidome and interxylary cork was studied using fluorescence microscopy and transmission electron microscopy. Histologically, it was observed that the parenchyma cells of secondary phloem and xylem in roots recovered their meristematic ability, and later developed into rhytidome and interxylary cork. Cytologically, ultrastructural characteristics such as nucleus malformation, vacuole disappearance, mitochondrial degeneration, and vesicle filling were observed. In roots, the nucleus of the phloem parenchyma cells were terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive from the pre-rhytidome stage to the formation of rhytidome stage and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)-negative during the mature rhytidome stage. The TUNEL assay of the xylem parenchyma cells showed positive characteristics from the early stage of interxylary cork formation to the interxylary cork formation stage, whereas DAPI-negative characteristics were observed in the mature interxylary cork. Gel electrophoresis showed that DNA cleavage was random. Our results indicated that the formation of the rhytidome and interxylary cork involved the PCD process.

Chrysoeriol promotes functional neurological recovery in a rat model of cerebral ischemia

Background: Stroke is among the leading causes of death worldwide, but current treatment options for cerebral ischemia are limited. Chrysoeriol is a natural isolated agent with bioactive pharmacological properties. Materials and Methods: Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) surgery and chrysoeriol was administrated for the subsequent 3 days. Neurological deficit scores, apoptosis, oxidant stress, and inflammatory cytokines were then measured and the effects on the Wnt/β-catenin pathway were evaluated. Results: Behavioral tests revealed that chrysoeriol promoted recovery from neurological deficits in rats. Hematoxylin and eosin staining demonstrated that chrysoeriol alleviated neurological damage. Triphenyl tetrazolium chloride staining demonstrated that chrysoeriol reduced the area of ischemia. An enzyme-linked immune sorbent assay (ELISA) demonstrated that chrysoeriol inhibited excessive pro-inflammatory cytokine production (tumor necrosis factor-α, interleukin [IL]-1β, and IL-6) and regulated oxidative stress (malondialdehyde, superoxide dismutase, and glutathione). Caspase-3 immunofluorescence and a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay showed that chrysoeriol reduced neuronal apoptosis. The ELISA assay and Nissl staining showed that chrysoeriol promoted neuronal survival and growth. Western blotting and an immunofluorescence assay demonstrated that the Wnt/β-catenin signaling pathway plays a crucial role in the neuronal effects of chrysoeriol. The Wnt inhibitor, Dickkopf-related protein-1, validated the role of the Wnt/β-catenin signaling pathway in the neuroprotective effects of chrysoeriol. Conclusion: Chrysoeriol exerts neuroprotective effects in a rat model of MCAO. These effects are mediated by the Wnt/β-catenin signaling pathway.

The aqueous extract of brucea javanica reduces tumorigenicity of human lung cancer tumorspheres.

Aim: Therapy to overcome drug resistance by modulating epidermal growth factor receptor (EGFR) is a viable approach to suppress the proliferation of human non-small cell lung cancer (NSCLC) cells. A previous study demonstrated that the seeds of an aqueous Brucea javanica (BJ) (L.) Merr (Simaroubaceae) extract containing quassinoid mixtures effectively inhibited the growth and alleviated tumorigenesis in H1975 cells of NSCLC by targeting T790M/L858R EGFR. This study aimed to further determine whether the aqueous BJ extract affects the enriched H1975 spheroids in suspension culture and mouse xenograft tumor models. Methods: The spheroids of NSCLC adenocarcinoma H1975 cells were enriched in a serum-free media. The growth rate of sphere propagation by aqueous BJ extract was determined in suspended culture and in colony-formation assay. BJ extract was fed orally to nude mice bearing xenograft tumors. The resected tumors were analyzed by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and proliferating cell nuclear antigen assessment. Various markers were used to determine the pluripotency of tumors from mice treated with different concentrations of BJ extract. Results: BJ extract was demonstrated to be effective against the propagation of the enriched spheroids. In animal models, oral administration of the aqueous BJ extract reduced spheroid tumorigenicity. The alleviated growth of the established xenograft tumors can be attributed to the reduced drug resistance and induced apoptosis without distinct adverse effects. More evidence supports activated apoptotic death attenuated spheroid stemness of tumors. Conclusion: As an effective treatment regime to assuage lung cancer, the indigenous BJ extract promises to obliterate drug resistance and the growth of cancer stem cell tumors from NSCLC cells harboring T790M/L858R EGFR.

Salidroside induces apoptosis and autophagy in gastric cancer cells via regulation of mitogen-activated protein kinases signaling pathway

Background: Salidroside, an active ingredient of Rhodiola rosea, exhibits antiproliferative effect in gastric cancer (GC) cells. However, the involvement of salidroside in apoptosis and autophagy of GC cells has not been elucidated. Materials and Methods: Cell viability of BGC-823 cells was assessed by CCK-8 assay, and apoptosis was analyzed with terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay and flow cytometry. The apoptosis- and autophagy-associated proteins and mitogen-activated protein kinases (MAPKs) were determined by Western blot analysis. Autophagy was evaluated by green fluorescent protein-fused LC3 punctate formation. Results: Salidroside inhibited proliferation and promoted apoptosis and autophagy of BGC-823 cells in a dose-dependent manner. In addition, salidroside enhanced the phosphorylation of p38 MAPK, ERK1/2, and JNK. Furthermore, inhibition of MAPKs significantly abolished the effects of salidroside in BGC-823 cells. Conclusion: Salidroside induced apoptosis and autophagy of BGC-823 cells via activation of MAPK signaling pathway.

Dexmedetomidine Inhibits Inflammation to Alleviate Early Neuronal Injury via TLR4/NF-κB Pathway in Rats with Traumatic Brain Injury.

Purpose - This study aims to explore the potential mechanism of dexmedetomidine in terms of inhibiting inflammation to alleviate early neuronal injury via TLR4/NF-κB pathway in rats with traumatic brain injury. Methods - The model of brain injury was established in rats. After the model was established, the rats were randomly divided into five groups: Sham, Sham + DEX, TBI, TBI + vehicle, and TBI + DEX. Each group included 10 rats. The water content in the brain tissue was measured. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were performed on histopathological tissue sections to evaluate neuronal apoptosis. Enzyme-linked immunosorbent assay and PCR were applied to detect the levels of the inflammatory factors, TNF-α, IL-1β, IL-6, and NF-κB. Results - TBI-challenged rats exhibited significant neuronal apoptosis, which was characterized via the wet-to-dry weight ratio, neurobehavioral functions, TUNEL assay results, and the levels of cleaved caspase-3, Bax upregulation, and Bcl-2, which were attenuated by DEX. Western blot, immunohistochemistry, and PCR results revealed that DEX promoted TLR4 expression and upregulated expression of the TLR4 downstream factors, HO-1 and NQO-1. Furthermore, DEX treatment markedly prevented the downregulation of inflammatory response factors, TNF-α, IL-1β and NF-κB, and IL-6. Conclusion - Dexmedetomidine is able to inhibit inflammation and attenuate early neuronal injury in rats with acute brain injury, which may act on TLR4/NF-κB pathway.

MiR-204 reduces apoptosis in rats with myocardial infarction by targeting SIRT1/p53 signaling pathway.

The aim of this study was to investigate the influence of micro ribonucleic acid (miR)-204 on rats with myocardial infarction by targeting the silent information regulator 1 (SIRT1)/p53 signaling pathway. A total of 36 Sprague-Dawley rats were randomly divided into three groups, including: sham-operation group (n=12), model group (n=12) and miR-204 mimics group (n=12). The rats in the sham-operation group only underwent thoracotomy, without myocardial infarction injury. Meanwhile, the rats in model group and miR-204 mimics group were utilized to establish the models of myocardial infarction, and then, intervened with normal saline and miR-204 mimics, respectively. The morphology of myocardial tissues was observed via hematoxylin-eosin (HE) staining. Immunofluorescence was performed to detect the expression of Caspase-3. Target genes of miR-204 were analyzed using bioanalysis software. Western blotting (WB) assay was applied to measure the relative protein expression of SIRT1. MiR-204 expression and the messenger RNA (mRNA) expressions of SIRT1 and p53 were measured via quantitative Polymerase Chain Reaction (qPCR). Furthermore, cell apoptosis was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. HE staining showed that the morphology of myocardial tissues was normal in sham-operation group. Severe myocardial tissue injury was visible in model group, and the injury was relieved in miR-204 mimics group when compared with model group. The results manifested that the positive expression of Caspase-3 in cardiac tissues increased remarkably in the model group and miR-204 mimics group in comparison with sham-operation group (p<0.05). Meanwhile, it was evidently lower in miR-204 mimics group than model group (p<0.05). Based on the analysis via bioanalysis software, SIRT1 was the target gene of miR-204. WB results revealed that the relative protein expression level of SIRT1 was elevated notably in the other two groups compared with the 2sham-operation group (p<0.05). However, it was markedly lowered in miR-204 mimics group in contrast with model group (p<0.05). QRT-PCR results demonstrated that the model group and miR-204 mimics group exhibited distinctly lower expression of miR-204 but higher mRNA expressions of SIRT1 and p53 than sham-operation group (p<0.05). However, miR-204 mimics group exhibited prominently higher expression of miR-204 but lower mRNA expressions of SIRT1 and p53 than model group (p<0.05). Finally, the results of TUNEL assay demonstrated that the apoptosis rate increased remarkably in the model group and miR-204 mimics group when compared with sham-operation group (p<0.05). However, it decreased notably in miR-204 mimics group in comparison with model group (p<0.05). MiR-204 reduces the apoptosis level in rats with myocardial infarction via targeted inhibition of the SIRT1/p53 signaling pathway.