Number Of TUNEL-positive Cells Research Articles

Guhong injection promotes post-stroke functional recovery via attenuating cortical inflammation and apoptosis in subacute stage of ischemic stroke

BackgroundAs a leading cause of death and disability, alternative therapies for stroke are still limited by its complicated pathophysiological manifestations. Guhong injection (GHI), consisting of safflower aqueous extract and aceglutamide, has been widely applied for the clinical treatment of cerebrovascular diseases, especially ischemic stroke and post-stroke recovery, in China. Recently, a series of studies have reported the positive effect of GHI against cerebral ischemia/reperfusion injury via targeting various molecular mechanisms. However, questions remain on whether treatment with GHI contributes to better functional recovery after stroke and if so, the potential mechanisms and active substances. PurposeThe aim of this work was to explore the potential therapeutic possibilities of GHI for the neurological and behavioral recovery after stroke and to investigate the underlying molecular mechanisms as well as active substances. MethodsThe neural and motor deficits as well as cortical lesions after GHI treatment were investigated in a mouse model of transient ischemic stroke. Based on the substance identification of GHI, network pharmacology combined with an experimental verification method was used to systematically decipher the biological processes and signaling pathways closely related to GHI intervention in response to post-stroke functional outcomes. Subsequently, ingenuity pathway analysis (IPA) analysis was performed to determine the anti-stroke active substances targeting to the hub targets involved in the significant molecular pathways regulated by GHI treatment. ResultsTherapeutically, administration of GHI observably ameliorated the post-stroke recovery of neural and locomotor function as well as reduced infarct volume and histopathological damage to the cerebral cortex in subacute stroke mice. According to 26 identified or tentatively characterized substances in GHI, the compound-target-pathway network was built. Bioinformatics analysis suggested that inflammatory and apoptotic pathways were tightly associated with the anti-stroke effect of GHI. Based on protein-protein interaction network analysis, the hub targets (such as NF-κB p65, TNF-α, IL-6, IL-1β, Bax, Bcl-2, and Caspase-3) involved in inflammation and apoptosis were selected. On the one hand, immunofluorescence and ELISA results showed that GHI (10 ml/kg) treatment obviously reduced NF-κB p65 nuclear translocation as well as decreased the abnormally elevated concentrations of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in damaged cortex tissues. On the other hand, GHI (10 ml/kg) treatment significantly downregulated the number of TUNEL-positive apoptotic cells in ischemic cortex and effectively restored the abnormal expression of Bax, Bcl-2, and Caspase-3. Based on the results of IPA, hydroxysafflor yellow A, baicalin, scutellarin, gallic acid, syringin, chlorogenic acid, kaempferol, kaempferol-3-O-β-rutinoside, and rutin acted synergistically on core targets, which could be considered as the active substances of GHI. ConclusionOverall, the current findings showed that the beneficial action of GHI on improving post-stroke functional recovery of subacute stroke mice partly via the modulation of cortical inflammation and apoptosis. These findings not only provide a reliable reference for the clinical application of GHI, but also shed light on a promising alternative therapeutic strategy for ischemic stroke patients.

High-frequency repetitive transcranial magnetic stimulation mitigates depression-like behaviors in CUMS-induced rats via FGF2/FGFR1/p-ERK signaling pathway

High-frequency repetitive transcranial magnetic stimulation (rTMS) is a widely used and effective biological treatment for depression. Although previous studies have shown that astrocyte function may be modified by rTMS, the specific neurobiological mechanisms underlying its antidepressant action are not clear. Substantial evidence has accumulated indicating that neurotrophin dysfunction and neuronal apoptosis play a role in the development of depression. To evaluate this hypothesis, we applied a chronical unpredictable mild stress (CUMS) protocol to induce depression-like behaviors in rats, followed by the delivery of 10-Hz rTMS for 3 weeks. Behavioral outcome measures consisted of a sucrose preference test, forced swimming test, and open field test. Histological analysis focused on apoptosis, expression of GFAP and FGF2, and FGF2 pathway-related proteins. The results showed that after rTMS treatment, the rats’ sucrose preference increased, open field performance improved while the immobility time of forced swimming decreased. The behavioral changes seen in rTMS treated rats were accompanied by marked reductions in the number of TUNEL-positive neural cells and the level of expression of BAX and by an increase in Bcl2. Furthermore, the expression of GFAP and FGF2 was increased, along with activation of FGF2 downstream pathway. These results suggest that rTMS treatment can improve depression-like behavior, attenuate neural apoptosis, and reverse reduction of astrocytes in a rat model of depression. We hypothesize that the therapeutic action of rTMS in CUMS-induced rats is linked to the activation of the FGF2/FGFR1/p-ERK signaling pathway.

Treadmill exercise ameliorates ethanol with lipopolysaccharide and carbon tetrachloride-mediated liver injury in mice.

We reported that application of ethanol with lipopolysaccharide (LPS) and carbon tetrachloride (CCl4) enhanced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level. In the current experiment, the protective effect of treadmill running on liver injury caused by ethanol with LPS and CCl4 in mice was studied. Liver injury severity was determined by measuring ALT and AST level in the blood. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, immunohistochemistry for caspase-3, and Western blotting for Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) were performed to indicate hepatocyte apoptosis. In addition, to understand the mechanism, 5′-adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was studied by Western blotting. Treadmill exercise ameliorated ethanol with LPS and CCl4-mediated elevation of ALT and AST level. Treadmill exercise suppressed ethanol with LPS and CCl4-mediated elevation of the TUNEL-positive cell number and cleaved caspase-3 expression. Treadmill exercise suppressed ethanol with LPS and CCl4-mediated elevation of Bax expression and increased Bcl-2 expression suppressed by application of ethanol with LPS and CCl4. Treadmill exercise enhanced AMPK phosphorylation which was suppressed by application of ethanol with LPS and CCl4. Treadmill exercise has the effect of reducing liver damage caused by alcohol and or drug addiction.

A sheep model of chronic cervical compressive myelopathy via an implantable wireless compression device

PurposeThis study aimed to establish an animal model in which we can precisely displace the spinal cord and therefore mimic the chronic spinal compression of cervical spondylotic myelopathy.MethodsIn vivo intervertebral compression devices (IVCDs) connected with subcutaneous control modules (SCCMs) were implanted into the C2-3 intervertebral disk spaces of sheep and connected by Bluetooth to an in vitro control system. Sixteen sheep were divided into four groups: (Group A) control; (Group B) 10-week progressive compression, then held; (Group C) 20-week progressive compression, then held; and (Group D) 20-week progressive compression, then decompression. Electrophysiological analysis (latency and amplitude of the N1-P1-N2 wave in somatosensory evoked potentials, SEP), behavioral changes (Tarlov score), imaging test (encroachment ratio (ER) of intraspinal invasion determined by X-ray and CT scan), and histological examinations (hematoxylin and eosin, Nissl, and TUNEL staining) were performed to assess the efficacy of our model.ResultsTarlov scores gradually decreased as compression increased with time and partially recovered after decompression. The Pearson correlation coefficient between ER and time was r = 0.993 (p < 0.001) in Group B at 10 weeks and Groups C and D at 20 weeks. And ER was negatively correlated with the Tarlov score (r = -0.878, p < 0.001). As compression progressed, the SEP latency was significantly extended (p < 0.001), and the amplitude significantly decreased (p < 0.001), while they were both partially restored after decompression. The number of abnormal motor neurons and TUNEL-positive cells increased significantly (p < 0.001) with compression.ConclusionOur implantable and wireless intervertebral compression model demonstrated outstanding controllability and reproducibility in simulating chronic cervical spinal cord compression in animals.

Diet-induced maternal obesity and overnutrition cause a decrease in the sperm quality of the offspring

The present work aimed to study the changes caused by maternal obesity and overnutrition in both the quality and function of spermatozoa of the offspring. To this end, female rats received either a standard or cafeteria diet from 22 days of age until the weaning of their offspring, and the male offspring from rats fed the standard and cafeteria diet (OSD and OCD respectively) were used. Different endpoints in the offspring, as body weight, weight gain, and glycemia were recorded and the testes were removed at 60 days of age. Different spermatozoa parameters, such as mitochondrial function, functional integrity of the sperm plasma membrane, capacitation, and acrosome status, were evaluated. The OCD group was heavier than the OSD group and exhibited lower testis and epididymal indices. The OCD group also showed a decrease in the ability of the sperm tail to react in the presence of a hypoosmotic solution, deficiency in sperm mitochondrial function, a lower percentage of spermatozoa without acrosome when exposed to a capacitation medium, and a higher number of abnormal metaphases. In addition, compared with OSD, OCD rats had a higher number of TUNEL-positive cells in the histological sections of the testis, and greater presence of reactive oxygen species in the spermatozoa, evaluated by a fluorescent probe. However, the OCD group displayed lower protein levels of cytochrome c and caspase-3 in testis tissue than the control group. These results suggest that maternal obesity and overnutrition program the offspring to develop poor sperm quality and function, which may imply a condition of subfertility.

Apoptosis and blood-testis barrier disruption during male reproductive dysfunction induced by PAHs of different molecular weights

The association between polycyclic aromatic hydrocarbons (PAHs) and male reproductive dysfunction has attracted increasing attention. The purpose of this study was to compare the male reproductive toxicity of multiple PAHs and to investigate the underlying molecular mechanisms. TM4 cells (mouse testicular Sertoli cells, SCs) were treated with benzo(a)pyrene (BaP), pyrene (Py), fluoranthene (Fl) and phenanthrene (Phe) (0, 0.1, 1, 10, 50, or 100 μM) for varying time points (4, 12, 24, or 48 h), and male C57BL/6 mice were administered BaP and Py (0, 10, 50, or 100 mg/kg body weight) for 14 days based on the cell experimental results. Histopathological examination, western blotting, ELISA, biochemical assays, RT–PCR, flow cytometry, JC-1 staining and trans-epithelium electrical resistance (TEER) measurements were used to assess apoptosis, blood-testis barrier (BTB) integrity, intracellular calcium ([Ca2+]i) concentrations and oxidative stress (OS). The results revealed that the mRNA levels and enzymatic activities of CYP450 and GST family members; levels of ROS, MDA, cleaved caspase 3 (c-caspase 3), caspase 9, Bax, and cytochrome C (CytC); and numbers of TUNEL-positive cells were significantly increased by BaP and Py, while levels of AhR, GSH, SOD, CAT, Bcl-2 and ΔΨm were decreased. Additionally, BaP and Py notably interfered with tight junctions (TJs) and adherens junctions (AJs) in the BTB. Intriguingly, BaP, but not Py, induced [Ca2+]i overload and gap junction (GJ) destruction. There was no dramatic effect of Fl and/or Phe on any of the above parameters except that slight cytotoxicity was observed with higher doses of Fl. Collectively, these findings showed that BaP and Py elicited SC apoptosis and BTB disruption involving mitochondrial dysfunction and OS, but [Ca2+]i fluctuation and GJ injury were only observed with BaP-induced reproductive toxicity. The male reproductive toxicity of the selected PAHs was ranked in the order of BaP > Py > Fl > Phe.

Sex-related differential susceptibility to ponatinib cardiotoxicity and differential modulation of the Notch1 signalling pathway in a murine model.

Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. We assessed mechanisms of sex‐related PON‐induced cardiotoxicity and identified rescue strategies in a murine model. PON+scrambled siRNA‐treated male mice had a higher number of TUNEL‐positive cells (%TdT+6.12 ± 0.17), higher percentage of SA‐β‐gal‐positive senescent cardiac area (%SA‐β‐gal 1.41 ± 0.59) and a lower reactivity degree (RD) for the survival marker Bmi1 [Abs (OD) 5000 ± 703] compared to female (%TdT+3.75 ± 0.35; %SA‐β‐gal 0.77 ± 0.02; Bmi1 [Abs (OD) 8567 ± 2173]. Proteomics analysis of cardiac tissue showed downstream activation of cell death in PON+siRNA scrambled compared to vehicle or PON+siRNA‐Notch1‐treated male mice. Upstream analysis showed beta‐oestradiol activation, and downstream analysis showed activation of cell survival and inhibition of cell death in PON+scrambled siRNA compared to vehicle or PON+siRNA‐Notch1‐treated female mice. PON+scrambled siRNA‐treated mice also had a downregulation of cardiac actin—more marked in males—and vessel density—more marked in females. Female hearts showed greater cardiac fibrosis than their male counterparts at baseline, with no significant change after PON treatment. PON+siRNA‐scrambled mice had less fibrosis than vehicle or PON+siRNA‐Notch1‐treated mice. The left ventricular systolic dysfunction showed by PON+scrambled siRNA‐treated mice (male %EF 28 ± 9; female %EF 36 ± 7) was reversed in both PON+siRNA‐Notch1‐treated male (%EF 53 ± 9) and female mice (%EF 52 ± 8). We report sex‐related differential susceptibility and Notch1 modulation in PON‐induced cardiotoxicity. This can help to identify biomarkers and potential mechanisms underlying sex‐related differences in PON‐induced cardiotoxicity.

Minocycline improves learning and memory functions in ischemic stroke rats via reduction of cerebral ischemia-induced neuroinflammation and apoptosis

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To study the effect minocycline on learning and memory functions in ischemic stroke rats, and the underlying mechanism.&#x0D; Methods: 60 adult male SD rats were randomly divided into control group, ischemic brain damage (6 and 24 h MACO) groups; and 6 and 24 h minocycline groups, with 12 rats in each group. The volume of cerebral infarction, neuronal cell apoptosis, NF-κB protein expression, learning and memory ability, and the number of Iba-1+CD206-positive cells, and CD206/CD68 mRNA expressions in sham group, 6 h MACO group and 6 h minocycline group were determined and compared.&#x0D; Results: The number of iba-1 +CD206-positive cells, expression level of CD206 mRNA, frequency of platform crossing, and percentage of third quadrant route in 6 h minocycline group were significantly higher than the corresponding values in 6 h MACO group. However, the cerebral infarction volume, number of Nini-positive cells, and the NF- B protein expression levels were markedly reduced, relative to corresponding values in 6 h MACO rats. The number of iba-1+CD206-positive cells was significantly lower in 6 h MACO rats than in sham rats, while the expression level of CD68 mRNA was significantly higher (p &lt; 0.05). The number of TUNEL-positive cells in 6 and 24 h minocycline groups were markedly lower than that in 6 h MACO group (p &lt; 0.05).&#x0D; Conclusion: Minocycline improves learning and memory of ischemic stroke rats by relieving the neuroinflammation induced by cerebral ischemia and cell apoptosis. Thus, the compound can be further developed for management learning and memory deficits in stroke patients.&#x0D; &#x0D; &#x0D; &#x0D;

Cognitive impairment persists at least 1 year after juvenile rats are treated with methotrexate

Methotrexate (MTX) is widely employed for children with cancer, but is also associated with persistent cognitive deficits among survivors. The present study investigated the mechanisms behind long-term cognitive dysfunction after juvenile animals are treated with MTX. Male and female Long-Evans rats were treated with a combination of 6 systemic doses (0.5 mg/kg/dose intraperitoneally) and 4 intrathecal doses (1 mg/kg) beginning at post-natal age 3 weeks, a schedule designed to mimic repeated exposure given to children with leukemia. Behavioral testing was conducted at 60–61 weeks of age, followed by analysis of brain histolopathology. This MTX regimen had no acute toxicity and no effect on growth. The spatial memory and visual memory deficits observed at 13 and 17 weeks of age persisted 1 year after MTX exposure in both females and males. Significantly decreased cell proliferation and increased hippocampal microglial activation were observed in MTX-treated females when compared to the controls, with a similar trend in the male groups. In addition, MTX treatment significantly increased the number of TUNEL positive cells in the periventricular area. Our study demonstrates that a clinically relevant regimen of systemic and intrathecal MTX induces persistent deficits in cognition, lasting approximately 1 year after the last injection. The mechanisms behind MTX-induced deficits are likely multifactorial, including suppression of neurogenesis, microglial activation, and increased brain cell apoptosis. Our study suggests female and male animals differ in susceptibility to MTX-induced neurotoxicity and provides insights for developing therapeutic approaches to prevent treatment related cognitive impairment among children with ALL.

SUMOylation of Nuclear γ-Actin by SUMO2 supports DNA Damage Repair against Myocardial Ischemia-Reperfusion Injury

Myocardial infarction triggers oxidative DNA damage, apoptosis and adverse cardiac remodeling in the heart. Small ubiquitin-like modifier (SUMO) proteins mediate post-translational SUMOylation of the cardiac proteins in response to oxidative stress signals. Upregulation of isoform SUMO2 could attenuate myocardial injury via increasing protein SUMOylation. The present study aimed to discover the identity and cardioprotective activities of SUMOylated proteins. A plasmid vector for expressing N-Strep-SUMO2 protein was generated and introduced into H9c2 rat cardiomyocytes. The SUMOylated proteins were isolated with Strep-Tactin® agarose beads and identified by MALDI-TOF-MS technology. As a result, γ-actin was identified from a predominant protein band of ~42 kDa and verified by Western blotting. The roles of SUMO2 and γ-actin SUMOylation were subsequently determined in a mouse model of myocardial infarction induced by ligating left anterior descending coronary artery and H9c2 cells challenged by hypoxia-reoxygenation. In vitro lentiviral-mediated SUMO2 expression in H9c2 cells were used to explore the role of SUMOylation of γ-actin. SUMOylation of γ-actin by SUMO2 was proven to be a new cardioprotective mechanism from the following aspects: 1) SUMO2 overexpression reduced the number of TUNEL positive cells, the levels of 8-OHdG and p-γ-H2ax while promoted the nuclear deposition of γ-actin in mouse model and H9c2 cell model of myocardial infarction; 2) SUMO-2 silencing decreased the levels of nuclear γ-actin and SUMOylation while exacerbated DNA damage; 3) Mutated γ-actin (K68R/K284R) void of SUMOylation sites failed to protect cardiomyocytes against hypoxia-reoxygenation challenge. The present study suggested that SUMO2 upregulation promoted DNA damage repair and attenuated myocardial injury via increasing SUMOylation of γ-actin in the cell nucleus.

Neuroprotective Effects of Cordyceps cicadae (Ascomycetes) Mycelium Extract in the Rat Model of Optic Nerve Crush.

Cordyceps cicadae mycelium is an herbal medicine used to provide anti-inflammatory and antiapoptotic actions. However, little is known about the role of C. cicadae mycelium in neuroprotection. This study aimed to investigate the neuroprotective effects of C. cicadae mycelium extract (CCME) in the optic nerve crush (ONC) model. The optic nerves of adult male Wistar rats (aged 7-8 weeks) were crushed by a standardized method. Rats were divided equally into three groups: 1) a sham-operated group (sham), 2) a phosphate buffered saline-treated control group (crush), and 3) a CCME-treated group (CCME) that received CCME once daily for 7 consecutive days at doses of 100 mg/kg before ONC. Two weeks after ONC in rats, retinal ganglion cell (RGC) density and visual function were determined by using retrograde labeling with FluoroGold and flash visual evoked potentials. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry of ED1 (a marker of macrophage/microglia) were used to evaluate the antiapoptotic and anti-inflammatory effects of CCME in the optic nerve section. The P1-N2 amplitude and RGC density in the CCME-treated group were higher than those in the ONC control (crush) group by 5.15- and 3.13-fold, respectively. The numbers of TUNEL-positive cells and ED1-positive cells in the CCME-treated group were reduced by 4.38- and 6.63-fold, respectively, compared to those in the crush group. Oral administration of CCME provided neuroprotective effects in the ONC model via antiapoptotic and anti-inflammatory actions, which provides a potential treatment for patient with traumatic optic neuropathy.

Quercetin-loaded on whey protein improves male fertility parameters and atherogenic indices of rats on a western-style diet

The western-style diet, characterized by high fat-high fructose (HF/HFr) intake, induces alteration in spermatogenesis and testicular functions by atherogenic indices derangement and oxidative stress. The antioxidant properties of Quercetin (Quer) are proved; however, it has some adverse effects on male fertility parameters. So, this study aimed to investigate the protective effect of a biocompatible carrier (whey protein coated with black seed oil) as a new drug delivery system to encapsulate the Quer on atherogenic indices, insulin sensitivity, sperm parameters, and testicular function alterations induced by a western-style diet in male rats. Abnormal testicular epithelium with detached and swelled germ cells, alterations in the ratio of the spermatogenic cells, increased percentage of apoptotic cells (TUNEL-positive), and abnormalities in sperm parameters were observed in the male rats on a western-style diet. Treated groups with the free Pro-gel (whey protein gel) and Pro-gel-Quer (Quer loaded on whey protein gel) showed significant recovery of the germ cells and reduction in the TUNEL-positive cell number. There was a significant increase in the atherogenic index TG/HDL-C, serum FBS, insulin, and HOMA-IR, but the decrease in testosterone levels in the model groups (p < 0.05). However, these parameters reached near to the control in the Pro-gel-Quer-treated one compared with the free Quer and Pro-gel groups. Nutritional transition to the HF-HFr diets has deleterious effects on the testicular function and sperm parameters. Quer, as an antioxidant, loaded on whey protein is a new biocompatible antioxidant-therapy in male fertility and lipid profile complications induced by the western-style diet.

Chloroquine Combined With Rapamycin Arrests Tumor Growth in a Patient-derived Orthotopic Xenograft (PDOX) Mouse Model of Dedifferentiated Liposarcoma.

Dedifferentiated liposarcoma (DDLS) is a type of soft-tissue sarcoma with a poor prognosis due to distant metastasis and resistance to chemotherapy. The antimalarial drug chloroquine (CQ) can induce apoptosis in cancer cells. CQ in combination with rapamycin (RAPA), an mTOR inhibitor, has shown efficacy on osteosarcoma and other types of cancer. In the present study the efficacy of RAPA combined with CQ on the treatment of a DDLS patient-derived orthotopic xenograft (PDOX) model was investigated. A patient-derived DDLS was transplanted into the left retroperitoneum of nude mice to establish a DDLS PDOX nude-mouse model. The mice were randomly divided as follows: untreated control group; CQ group; RAPA group; combined CQ and RAPA group (n=7 for all groups). During the treatment period, tumor volume was measured every 3-4 days with calipers. After 2 weeks treatment, the mice were sacrificed, and H&E staining was performed for histological evaluation. The TUNEL assay was performed to detect apoptosis. The combination of CQ and RAPA arrested tumor growth in the DDLS PDOX compared to the untreated control (p=0.009) and was significantly more effective than RAPA alone (p=0.009). RAPA alone slowed tumor growth, but the difference was not statistically significant (p>0.05). CQ was not active alone (p>0.05). The number of apoptotic TUNEL-positive cells was significantly higher in the CQ plus RAPA group than in the other groups (p=0.02). Combination therapy with CQ and RAPA arrested tumor growth in a DDLS PDOX model by inducing apoptosis.

The following was originally published in ACUPUNCTURE &amp; ELECTRO-THERAPEUTICS RES., INT. J., Vol. 46, pp. 357-369, 2021

Background: Electroacupuncture (EA) pre-treatment can play a significant neuroprotective role in rats with cerebral ischaemia and reperfusion (CIR) injury, but the specific mechanism needs to be further elucidated.&lt;br/&gt; Objective: To investigate the effects of EA pre-treatment on apoptosis-related proteins Bax, Bcl-2, Cytochrome c (cyt c), cleaved caspase-9 and -3 in rats with CIR injury.&lt;br/&gt; Methods: CIR injury was induced using middle cerebral artery occlusion (MCAO) ischaemia-reperfusion. Thirty-six male Specific Pathogen Free (SPF) Sprague-Dawley rats were randomly divided into three groups: Sham (n=12), MCAO (n=12), and the EA+MCAO group (n=12). EA+MCAO group rats received electroacupuncture at DU20 for 2 consecutive weeks before MCAO model preparation. After 24 h of reperfusion, neurological deficits were evaluated by modified neurological severity scores. Brain infarct volumes were examined by 2,3,5-triphenyltetrazolium chloride staining. Expression of Bax, Bcl-2, cyt c, cleaved caspase-9 and -3 in the ischaemic penumbra were detected by Western blotting, and apoptosis evaluated by TUNEL staining.&lt;br/&gt; Results: After perfusion for 24 h, compared with the MCAO group, the neurological deficit scores and brain infarct volumes in the EA+MCAO group were significantly decreased (P &lt;0.05), as was the level of Bax (P &lt;0.05) or the Bax/Bcl-2 ratio (P &lt;0.05), levels of cyt c, cleaved caspase-9 and -3 also decreased (P &lt;0.05), like the number of TUNEL-positive cells (P &lt;0.05), in contrast, the level of Bcl-2.&lt;br/&gt; Conclusion: EA pre-treatment exerts an anti-apoptotic effect through Bax-to-Bcl-2 ratio downregulation, blockage of mitochondrial cyt c release to the cytosol, and reduction of caspase-9 and -3 expression in rats with CIR injury.

12 Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of notch signalling in a muine model

Abstract Aims Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. Although sex is a risk factor for PON-induced cardiotoxicity in humans, little is known about its mechanisms, in general, and sex-related mechanisms in particular. To determine the mechanisms of sex-related PON-induced cardiotoxicity and identify potential rescue strategies in a murine model. Methods Twenty-four-month-old male and female C57B5 mice were treated with 3 mg/kg/day of PON or vehicle via oral gavage for 28 days, with/without siRNA-Notch1 or siRNA-scrambled via tail vein every 3 days. Results PON + scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells, a higher percentage of senescence-associated β-galactosidase positive senescent cardiac areas, as well as a lower reactivity degree for the survival marker Bmi1 than female counterparts. Proteomics analysis of cardiac tissue showed upstream activation of nitric oxide synthase (NOS) type 2, downstream activation of cell death and production of reactive oxygen species in PON + scrambled siRNA- compared to vehicle or PON + Notch1 siRNA-treated male mice. Upstream analysis showed beta-estradiol activation, while downstream analysis showed activation of cell survival and inhibition of cell death in PON + scrambled siRNA compared to vehicle-treated female mice. PON + scrambled siRNA-treated mice also showed a down-regulation of cardiac actin, which was more marked in male; as well as vessel density, which was more marked in female mice. Female hearts showed a greater extent of cardiac fibrosis than male counterparts at baseline, with no significant changes after PON treatment. In contrast, PON + scrambled siRNA-treated mice had less fibrosis than vehicle or PON + Notch1-siRNA-treated mice. Left ventricular systolic dysfunction shown in PON + scrambled siRNA-treated male mice and—to a lesser extent—by female mice was similarly reversed in both PON + Notch1-siRNA-treated male and female mice (Table 1). Conclusions We found a sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can improve our understanding of sex-related differences and help identify biomarkers in PON cardiotoxicity.

Treatment With Glycogen Synthase Kinase 3β Inhibitor Decreases Apoptotic and Autophagic Reactions in Rat Rotator Cuff Tears.

Background:Apoptosis and autophagy are known to be correlated with the extent of damage in torn rotator cuffs, and there is no biological evidence for self-recovery or healing of the rotator cuff tear.Purpose:To establish in a rat model of partial- and full-thickness rotator cuff tears how a glycogen synthase kinase 3β (GSK-3β) inhibitor affects the expression of apoptotic and autophagic markers.Study Design:Controlled laboratory study.Methods:Twelve-week-old Sprague Dawley rats were divided into 3 groups (n = 16 per group). Group 1 acted as the control, with no treatment; group 2 received partial-thickness (right side) and full-thickness (left side) rotator cuff tears only; and group 3 received the same rotator cuff injuries, with GSK-3β inhibitor injected afterward. The tendons from each group were harvested 42 days after surgery. Evaluation of gene expression, immunohistochemistry, and TUNEL staining (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) were performed for the following markers: caspases 3, 8, and 9 as well as Bcl-2 (B-cell lymphoma 2); BAX (Bcl-2-associated X protein); beclin 1; p53; and GSK-3β; which represented apoptotic and autophagic reactions. Statistical analysis was performed using 1-way analysis of variance.Results:In the group 2 rats with partial- and full-thickness tears, there were significant increases in the mRNA levels (fold changes) of all 8 markers as compared with group 1 (control). All these increased markers showed significant downregulation by the GSK-3β inhibitor in partial-thickness tears. However, the response to the GSK-3β inhibitor in full-thickness tears was not as prominent as in partial-thickness tears. The number of TUNEL-positive cells in group 2 (partial, 35.08% ± 1.625% [mean ± SE]; full, 46.92% ± 1.319%) was significantly higher than in group 1 (18.02% ± 1.036%; P < .01) and group 3 (partial, 28.04% ± 2.607% [P < .01]; full, 38.97% ± 2.772% [P < .01]), and immunohistochemistry revealed increased expression of all the markers in group 2 as compared with control.Conclusion:The apoptotic and autophagic activity induced in a rat model of an acute rotator cuff tear was downregulated after treatment with a GSK-3β inhibitor, particularly with partial-thickness rotator cuff tears.Clinical Relevance:A GSK-3β inhibitor may be able to modulate deterioration in a torn rotator cuff.

Single neonatal irradiation induces long-term gene expression changes in the thyroid gland, which may be involved in the tumorigenesis

Exposure to ionizing radiation in childhood has been recognized as a risk factor for thyroid cancer. We previously demonstrated that neonatal X-irradiation induced specific deformation of the thyroid follicles. Here, we further analyzed this model to understand the possible relationship with thyroid carcinogenesis. Wistar rats were subjected to cervical X-irradiation at different ages of 1–8 weeks old and at different doses of 1.5–12 Gy. For tumor promotion, rats were fed with an iodine-deficient diet (IDD). In cervically X-irradiated neonatal rats, the size of thyroid follicles decreased, accompanied by an increase in the number of TUNEL-positive cells. Fas and Lgals3 mRNA levels increased, while Mct8 and Lat4 expressions decreased. The co-administration of IDD induced the proliferation and the upregulation in Lgals3 expression, resulting in thyroid adenoma development at 28 weeks post-exposure. Our data demonstrated that single neonatal X-irradiation induced continuous apoptotic activity in the thyroid with the long-term alternation in Fas, Mct8, Lat4, and Lgals3 mRNA expressions. Some of these changes were similar to those induced by IDD, suggesting that neonatal X-irradiation may partially act as a thyroid tumor promoter. These radiation-induced thyroidal changes may be enhanced by the combined treatment with IDD, resulting in the early development of thyroid adenoma.

Warming moxibustion attenuates inflammation and cartilage degradation in experimental rabbit knee osteoarthriti.

To explore the molecular mechanism of warming moxibustion (WM) in knee osteoarthritis (KOA). The knee joints of 40 New Zealand rabbits were placed in a plaster cast in an extended position to establish a KOA model. The animals were randomly divided into four groups: the control group, model group, WM group, and diclofenac (DF) group. Hematoxylin-eosin staining and the modified Mankin score were applied to evaluate the histopathological changes. Chondrocyte apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Western blotting and real-time quantitative polymerase chain reaction were performed to measure the expression of interleukin-1α (IL-1β), prostaglandin E receptor 3 (PTGER3), a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5 (ADAMTS-5), matrix metalloproteinase-13 (MMP-13), and C-terminal telopeptides of collagen type II (CTX-II) in cartilage tissues of the different groups. The concentrations of IL-1β, PTGER3, and CTX-II in serum were detected by the enzyme-linked immunosorbent assay. Rabbits with KOA in the WM and DF groups showed significantly reduced cartilage erosion and Mankin scores, compared with the untreated rabbits. The number of TUNEL-positive cells observed in the WM group was much fewer than that in the model group. The expression of PTGER3, MMP-13, CTX-II IL-1β, and ADAMTS-5 in cartilage tissues was remarkably downregulated following therapy with WM and DF. Moreover, a marked reduction was observed in the serum levels of IL-1β, PTGER3, and CTX-II in the WM and DF groups. WM exerts favorable therapeutic effects on articular injuries of KOA by regulating the expression of inflammatory and cartilage degradation-related cytokines.

Lead Exposure in Developmental Ages Promotes Aβ Accumulation by Disturbing Aβ Transportation in Blood-Cerebrospinal Fluid Barrier/Blood-Brain Barriers and Impairing Aβ Clearance in the Liver.

Environmental lead exposure is closely related to the progression of Alzheimer's disease (AD). Our previous study has shown that exposure to lead could result in the cholesterol unbalance and increase amyloid-beta (Aβ) generation in the brain. However, the potential effect of lead exposure on Aβ transportation is poorly reported. In this study, we sought to explore whether lead exposure in developmental ages impaired the integrity of BCSFB and BBB, two highly vascularized structures in the brain in a rat model. The Aβ clearance in the liver was also assessed. Our results showed that lead treatment in developmental ages increased the number of TUNEL-positive apoptotic cells in rat choroid plexus and microvessels. Moreover, lead exposure markedly increased pro-inflammatory factors expression including TNF-α and IL-1β in rat choroid plexus and microvessels. Interestingly, lead treatment increased the expression of AQP-1 and reduced the expression of TTR, two key proteins associated with the functions of choroid plexus and microvessels. Additionally, the expressions of ABCB1, LRP-1, and RAGE, three major receptors responsible for Aβ transportation, were disturbed by developmental lead exposure. All these pathologies resulted in Aβ1-40 deposition within BCSFB and BBB and malfunctions of these two vascularized structures. Finally, we found that lead treatment remarkably inhibited the gene expression of LRP-1, which is responsible for Aβ endocytosis, in the liver tissue of the rat model. Collectively, our results provide the first evidence that developmental lead exposure induces Aβ deposition in BCSFB and BBB and impairs Aβ clearance in the liver, which would ultimately disturb Aβ transportation via choroid plexus/brain microvessels and facilitate Aβ deposition in the brain.

High Glucose-Induced Apoptosis Is Linked to Mitochondrial Connexin 43 Level in RRECs: Implications for Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the most common causes of vision loss and blindness among the working-age population. High glucose (HG)-induced decrease in mitochondrial connexin 43 (mtCx43) level is known to promote mitochondrial fragmentation, cytochrome c release, and apoptosis in retinal endothelial cells associated with DR. In this study, we investigated whether counteracting HG-induced decrease in mtCx43 level would preserve mitochondrial integrity and prevent apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM glucose) or HG (30 mM glucose) medium for 7 days. In parallel, cells grown in HG were transfected with Cx43 plasmid, or empty vector (EV), as control. Western blot (WB) analysis showed a significant decrease in mtCx43 level concomitant with increased cleaved caspase-3, Bax, cleaved PARP, and mitochondrial fragmentation in cells grown in HG condition compared to those grown in N medium. When cells grown in HG were transfected with Cx43 plasmid, mtCx43 level was significantly increased and resulted in reduced cleaved caspase-3, Bax, cleaved PARP and preservation of mitochondrial morphology with a significant decrease in the number of TUNEL-positive cells compared to those grown in HG alone. Findings from the study indicate a novel role for mtCx43 in regulating apoptosis and that maintenance of mtCx43 level could be useful in preventing HG-induced apoptosis by reducing mitochondrial fragmentation associated with retinal vascular cell loss in DR.