Transferase-mediated dUTP Nick-end Labeling Staining Research Articles

Role of the PI3K/AKT signalling pathway in apoptotic cell death in the cerebral cortex of streptozotocin-induced diabetic rats.

Diabetes mellitus is associated with cognitive dysfunction. Numerous previous studies have shown that type 1 diabetes-induced hyperglycaemia causes structural brain damage, such as a decrease in whole-brain grey matter. The impact of diabetes mellitus on the cerebral cortex is poorly understood and requires further clarification. In the present study, diabetes was induced via an intraperitoneal injection of streptozotocin (50 mg/kg). Hematoxylin and eosin (H&E) staining was performed to detect the morphological changes in the cerebral cortex, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining was used to detect neuronal apoptosis and western blotting was performed to determine protein expression levels. Nine weeks after the induction of diabetes, the body weight was significantly lower and the blood glucose levels were significantly higher in the diabetic rats than in the control rats (P<0.05). H&E staining revealed nuclear chromatin condensation and cytoplasmic shrinkage in the cerebral cortex of the diabetic rats and TUNEL staining further indicated apoptotic changes in the cerebral cortex of the diabetic rats. The ratio of B-cell lymphoma 2 (Bcl-2) -associated X protein/Bcl-2 and the expression of cytochrome c and activated caspase-3 (cleaved caspase-3) were significantly increased, whereas the ratio of phosphorylated AKT/AKT was significantly decreased in the diabetic rats compared with that in the control rats (P<0.05). Taken together, these results suggested that diabetes mellitus may induce neuronal apoptosis in the cerebral cortex by downregulating AKT phosphorylation.

Study on the role of autophagy in heme oxygenase 1 preventing hepatic ischemia/reperfusion injury in rats

To identify the role of autopahgy in the protective mechanism of heme oxygenase 1 (HO-1) against hepatic ischemia/reperfusion (I/R) injury. Forty healthy male Sprague-Dawley (SD) rats were randomly (random number table) divided into five groups (n = 8 in each group), namely sham group, model group, cobalt protoporphyrin (CoPP) group, zinc protoporphyrin (ZnPP) group and 6-amino-3-methylpurine (3-MA) group. Partial hepatic I/R model was established by clamping the pedicles of left and median lobes for 1 hour and reopening for 6 hours in rats, and the rats in sham group were only received celiotomp without hepatic I/R. In the CoPP group, CoPP (a HO-1 inducer, 5 mg/kg) was administered i.p 24 hours before I/R. In the ZnPP or 3-MA group, besides pretreatment with CoPP, the rats were given ZnPP (a HO-1 inhibitor, 25 mg/kg) or 3-MA (an autophagy inhibitor, 30 mg/kg) i.p 1 hour before I/R. Serum alanine aminotransferase (ALT) was determined with automatic biochemistry analyzer. The hepatic pathological scores (PS) were determined under light microscope using hematoxylin-eosin (HE) staining. The hepatocyte apoptosis index (AI) was assessed with terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Autophagosomes in liver tissue were counted under electron microscope. The mRNA expressions of HO-1, caspase-3, Beclin-1 and Atg-5 in the liver were determined by reverse transcription-polymerase chain reaction (RT-PCR). The HO-1 activity was also measured by the generation of bilirubin with the method of double-wave spectrophotometry. Compared with the sham group, the level of serum ALT significantly increased in the I/R group (U/L: 560.3±73.6 vs. 49.1±13.8, P < 0.01), HE staining showed a severe hepatic injury (PS: 12.0±2.0 vs. 1.3±0.9, P < 0.01), TUNEL staining showed a higher hepatocytes apoptosis and the expression of caspase-3 significantly increased [AI: (19.38±3.07)% vs. (3.25±1.28)%, caspase-3 mRNA (2-ΔΔCt): 4.62±0.40 vs. 1.05±0.15, both P < 0.01]. However, there was no significant difference in the expression of HO-1 and the genes associated with autophagy between the two groups. In the CoPP group, the hepatic injury was blunted compared with that in the I/R group [ALT (U/L): 223.3±34.4 vs. 560.3±73.6, PS: 5.6±2.3 vs. 12.0±2.0, AI: (11.38±2.39)% vs. (19.38±3.07)%, caspase-3 mRNA (2-ΔΔCt): 2.42±0.33 vs. 4.62±0.40, all P < 0.01]. HO-1 was induced in the CoPP group and autophagy was also increased significantly after I/R when compared with those in the I/R group [HO-1 mRNA (2-ΔΔCt): 3.01±0.71 vs. 1.14±0.20, HO-1 activity (pmol×mg-1×h-1): 259±37 vs. 113±26, the number of autophagosomes: 8.75±0.87 vs. 1.25±0.71, Beclin-1 mRNA (2-ΔΔCt): 2.85±0.28 vs. 1.15±0.11, Atg-5 mRNA (2-ΔΔCt): 2.44±0.25 vs. 1.14±0.12, all P < 0.01]. In the ZnPP group, the activity of HO-1 was much lower than that in the CoPP group, and as a result autophagy was decreased and liver injury was increased. In the 3-MA group, although there was no difference in the activity of HO-1 compared with that in the CoPP group, autophagy was inhibited, and the protective effect of CoPP was eliminated. HO-1 could regulate the level of autophagy during liver I/R, and in turn autophagy might mediate the protective effects of HO-1 against liver I/R injury.

Resveratrol decreases FoXO protein expression through PI3K-Akt-dependent pathway inhibition in H₂O₂-treated synoviocytes.

The aim of this study was to investigate the effects of resveratrol (Res) on hydrogen peroxide (H₂O₂)-treated fibroblast-like synoviocytes (FLSs) in vitro. We studied the phosphoinositide 3-kinase (PI3K)-Akt pathway inhibition-mediated effects of Res on forkhead box O (FoXO) mRNA expression levels. FLS viability was determined by Cell Counting Kit-8 (CCK-8) assay, and FLS apoptosis was measured by terminal deoxyribo nucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and flow cytometry. FoXO1, FoXO3 and FoXO4 mRNA expression levels in FLSs were determined by RT-PCR, and p-Akt, Akt, p-FoXO1, FoXO1, p-FoXO3, FoXO3, Bcl-2 and Bax protein expression levels were determined by western blotting. Our results showed that low H₂O₂ concentrations (20 μM) can promote FLS growth and that Res significantly inhibited FLS activity. Moreover, Res significantly increased the number of apoptotic cells and the ratio of Bax/Bcl-2 protein expression in the group treated with Res compared with the group treated with H₂O₂ and LY294002, a PI3K inhibitor. Res also decreased FoXO1, FoXO3 and FoXO4 mRNA expression levels and p-Akt/Akt, p-FoXO1/FoXO1, p-FoXO3/FoXO3 protein expression levels. Taken together, these findings indicate that Res can induce apoptosis in H₂O₂-treated FLSs in part by inhibiting the PI3K-Akt signaling pathway.

Sestrin2 protects against acetaminophen-induced liver injury

Acetaminophen (APAP) overdose accounts for half of the cases of acute liver failure worldwide. We previously reported that Sestrin2 (Sesn2) protects against d-galactosamine/lipopolysaccharide-induced acute fulminant liver failure. In this study, we demonstrated that Sesn2 protects APAP-induced liver injury in mice, using a recombinant adenovirus encoding Sesn2 (Ad-Sesn2). First, we found that treatment of mice with toxic levels of APAP significantly reduced Sesn2 expression. Tail-vein injection with Ad-Sesn2 inhibited APAP-induced serum alanine aminotransferase and aspartate aminotransferase levels and markedly reduced hepatocyte degeneration and inflammatory cell infiltration. Additionally, APAP-induced glutathione depletion and reactive oxygen species generation were inhibited by Ad-Sesn2 treatment. Consistently, hepatic inflammatory gene expression and proinflammatory cytokine levels were also inhibited in Sesn2-infected mice, and we observed reduced APAP-mediated apoptotic signaling by terminal transferase-mediated dUTP nick-end labeling staining of the hepatic tissue. At a high dose of APAP, the mortality rate of Ad-Sesn2-infected mice was significantly lower than that of control mice. Furthermore, Sesn2 prevented APAP-induced damage through suppression of downstream mitogen-activated protein kinase pathway activation. Therefore, Sesn2 exerted a protective effect against APAP-induced acute liver damage by inhibiting oxidative stress and proinflammatory signaling.

A Model of Intrauterine Growth Restriction Was Induced by Bilateral Uterine Vessel Clamping in Pregnant Rats

Background: Intrauterine growth restriction (IUGR) is considered to be a major risk for cerebral palsy and disability after birth. However, IUGR lacks effective treatment strategies. We aimed to establish a rat model of bilateral uterine artery ischemia-reperfusion as a surgical model of IUGR.Methods: At the 17th day of pregnancy, six Sprague-Dawley (SD) rats were randomly divided into sham (NOR) and growth restriction (GR) groups. In the GR groups, four small artery clamps were used to occlude the uterine vessels near the lower and upper ends of the bilateral horns for 25 min; then, the clamps were removed, and the horns were placed back into the abdomen cavity. In the sham-operated animals, the uterine horns were exposed for 25 min without artery clamping. After labour and delivery, the death rates and weights during the first 8 days of newborn rats were recorded. A pathological examination was performed with hematoxylin-eosin staining (HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining (TUNEL) on 4-day-old and 8-day-old brain tissues of the newborn rats.Results: The mortality within the first 24 h of rats in the GR group were increased (66.7% vs 0.0%), and their daily weights within the first 8 postnatal days were decreased. In addition, the karyopyknosis percentages of 4-day-old and 8-day-old rats were increased (4-day-old: 45% vs 11%; 8-day-old: 34.7% vs 5.3%), and the numbers of TUNEL-positive cells were increased (4-day-old: 110 vs 12.7; 8-day-old: 146.7 vs 4).Conclusions: This model of foetal IUGR produced a decrease in weight, an increase in the death rate within 24 h and an increase in apoptosis in newborn rats. The establishment of the model was successful. Citation: Min Diao, Hao Li, Yi Qu, Hui Liu, Yu-Shan Ma, Xue-Mei Lin. A model of intrauterine growth restriction was induced by bilateral uterine vessel clamping in pregnant rats. J Anesth Perioper Med 2017;4: 101-7. doi:10.24015/JAPM.2017.0026This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Heat Acclimation Regulates the Autophagy-Lysosome Function to Protect Against Heat Stroke-Induced Brain Injury in Mice

Background/Aims: The mechanisms underlying the protective role of heat acclimation (HA) in heat stroke (HS)-induced brain injury are still unclear. The autophagy-lysosome pathway is known to pay an important role in protecting stressed or diseased cells from death. Nevertheless, whether autophagy and lysosomes are involved in HA-mediated neuroprotection following HS exposure remains unclear. Methods: The protective effects of HA were assessed by rectal temperature, hematoxylin-eosin staining, transmission electron microscopic analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, and Fluoro Jade B staining, after mice were subjected to HS. The effects of HA on autophagy and lysosomes were assessed in the presence of the autophagy inhibitor 3-methyladenine (3MA). Autophagy and lysosome-associated proteins were analysed by Western blotting. Results: We found that HA protected against HS-induced death and brain injury. HS can robustly induce autophagy and impair lysosome function. HA pre-conditioning significantly modulated the autophagy level, and improved lysosome function in HS mice. Furthermore, 3MA completely abolished the neuroprotective effect of HA on HS. Conclusion: HS may induce brain injury through lysosomal dysfunction and impaired autophagic flux. HA protected against HS-induced brain injury via a mechanism involving the autophagy-lysosome pathway.

Magnolol Reduces Renal Ischemia and Reperfusion Injury via Inhibition of Apoptosis.

Magnolol, a constituent of the bark of Magnolia officinalis, has been reported to decrease myocardial stunning and infarct size. In this study, we investigated whether magnolol can reduce renal ischemia and reperfusion (I/R) injury. Renal I/R, induced by a 60-min occlusion of bilateral renal arteries and a 24-h reperfusion, significantly increased blood urea nitrogen (BUN) and creatinine levels, and caused histological damage to the kidneys of rats. Apoptosis, as evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and caspase-3 activation, was significantly increased in the kidneys. Furthermore, serum levels of tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were significantly elevated, while the interleukin-10 (IL-10) level was suppressed. However, intravenous pretreatment with magnolol at doses of 0.003[Formula: see text]mg/kg and 0.006[Formula: see text]mg/kg 10[Formula: see text]min before renal I/R significantly limited the increases of BUN, creatinine, the histological damage, and apoptosis in the kidneys. The increases in TNF-[Formula: see text], IL-1β, and IL-6, and the decrease in IL-10 were also significantly inhibited. Additionally, magnolol increased Bcl-2 and decreased Bax in the kidneys. Phosphorylation of the prosurvival kinases, including Akt and extracellular signal-regulated kinases 1 and 2 (ERK1/2), was elevated, while phosphorylation of the pro-apoptotic mitogen-activated protein kinases, including p38 and c-Jun N-terminal kinase (JNK), was suppressed. In conclusion, magnolol reduces renal I/R injury. The underlying mechanisms for this effect might be related to the prevention of apoptosis, possibly via the inhibition of both extrinsic and intrinsic apoptotic pathways, including the reduction of TNF-[Formula: see text] production and the modulation of pro- and anti-apoptotic signaling elements.

Scutellarin suppresses growth and causes apoptosis of human colorectal cancer cells by regulating the p53 pathway

Scutellarin is a flavonoid isolated from a medicinal herb Scutellariabarbata D. Don and exerts therapeutic effects on cardiovascular diseases. However, it remains unclear whether Scutellarin exhibits anti‑tumor actions on human colon cancer. The current study aimed to investigate whether Scutellarin produces antiproliferative and pro‑apoptotic effects on HCT‑116 human colon cancer cells and to elucidate the mechanisms involved. Human colon cancer cells were exposed to different concentrations of Scutellarin, and cellular growth and apoptosis were evaluated by MTT assay, terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling (TUNEL) staining, western blot analysis and other assays. A cell viability assay demonstrated that Scutellarin treatment reduced the viability of HCT‑116 cells in a dose‑ and time‑dependent manner. TUNEL staining demonstrated that Scutellarin also induced apoptotic changes in HCT‑116 cells. The expression level of the anti‑apoptotic protein, Bcl‑2 apoptosis regulator (Bcl‑2), was reduced by Scutellarin in HCT‑116 cells, whereas the expression Bcl‑2 associated X apoptosis regulator (Bax) and the activation of caspase‑3 protein were increased by Scutellarin treatment. Further investigation revealed that Scutellarin significantly increased the phosphorylation of p53 protein in HCT‑116 cells. Additionally, suppression of p53 using a specific inhibitor, pifithrin‑α, abrogated the pro‑apoptotic effects of Scutellarin in HCT‑116 cells. Collectively, Scutellarin reduced the viability and induced apoptosis of human colon carcinoma cells, potentially by regulating p53 and Bcl‑2/Bax expression. These data suggested that Scutellarin may be useful as a promising anti‑tumor drug for treating colon cancer.

Argininosuccinate Synthetase 1 Loss in Invasive Bladder Cancer Regulates Survival through General Control Nonderepressible 2 Kinase–Mediated Eukaryotic Initiation Factor 2α Activity and Is Targetable by Pegylated Arginine Deiminase

Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2-dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.

Protective effects and mechanisms investigation of Kuntai capsule on the ovarian function of a novel model with accelerated aging ovaries

Ethnopharmacological relevanceKuntai capsule, a traditional Chinese medicine, has been widely used for the clinical treatment of menopausal syndrome. However, its mechanisms remain poorly understood. Considering that aging ovaries are the primary cause of menopause, this study was designed to investigate the effects and mechanisms of Kuntai capsule on ovarian function in a novel mice model with accelerated aging ovaries. Materials and methodsSeventy-five female C57BL/6 mice were chosen for this study. Fifteen of the mice were separated into the normal control group (NC). The remaining sixty were used to establish the novel accelerated aging ovary model by superovulation and oxidative stress and then by randomly dividing the mice into four equal groups. One group was considered the model group (Mod). The other three groups were treated with low (0.4g/kg), middle (0.8g/kg) and high (1.6g/kg) doses of Kuntai capsule intragastrically every day for 4 weeks. During the treatment, the body weight and fur condition of all mice were recorded. All the mice were forced to swim to record their exhaustive swimming time (EST), which measures their strength. Mice were then sacrificed for sampling. Ovarian reserve was evaluated using follicle counts and AMH expression. Ovarian function was evaluated using estrous cycle, sex hormone level and litter experiments. Ovarian follicles were categorized and counted to estimate ovarian reserve, and ovarian histologic sections were stained for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to detect apoptotic cells. The ultrastructure of ovarian cells was observed using transmission electron microscopy. Western blotting was used to measure expression of Bax, Bcl2, AMH and SOD2 protein. ResultsCompared with the NC GROUP, the Mod group clearly displayed worse fur condition and ovarian function. These situations showed some improvement after Kuntai capsule treatment. Specifically, the fur condition and the EST of the Kuntai capsule groups were superior to the fur condition and EST of the Mod group. In cases of damaged ovarian function, Kuntai capsule can regulate the estrous cycles, increase hormone secretion and fertility and significantly decrease atretic follicles. The transmission electron microscopy results revealed that Kuntai capsule rescued the ovarian ultrastructure of mice. TUNEL staining confirmed that the apoptotic cells were reduced after Kuntai capsule treatment. Western blotting revealed that Kuntai capsule can increase AMH, SOD2, and Bcl2 protein expression and decrease Bax expression. ConclusionsKuntai capsule may improve damaged ovarian function, which may be related to its antioxidant and anti-apoptosis effects.

Mild hypothermia protects rat neuronal injury after intracerebral hemorrhage via attenuating endoplasmic reticulum response induced neuron apoptosis

Background and purposeMild hypothermia has been proved to reduce global and focal cerebral ischemic injury in rodents by preventing cellular apoptosis through several pathways. However, whether hypothermia will be beneficial for intracerebral hemorrhage (ICH) and its underlying mechanisms haven’t reached a consensus. It has been implicated that endoplasmic reticulum (ER) stress plays a role in the secondary injury after ICH in rats. In this study, we aimed to investigate whether mild hypothermia would attenuate ICH induced neuronal injury via regulating ER stress. MethodsThe model of ICH was induced by injecting autologous blood (120μl) into the rat striatum. Rats were divided into sham, normothermic (NT) and hypothermic (HT) groups. HT group were subjected to mild hypothermia (33°C–35°C) for 2days starting from 6h after ICH. Neurological deficits were evaluated. The ER stress related proteins (GRP78, CHOP and p-eIF2α) and the apoptosis associated indicators (cleaved caspase3, Bcl-2 and Bim) around hematoma were assessed by western blot, qRT-PCR (quantificational real-time polymerase chain reaction), immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. ResultsNeurological deficits following ICH were reduced in HT group compared to NT group. Protein levels of GRP78, CHOP and p-eIF2α significantly increased after ICH in both NT and HT group compared to sham group, which was consistent with the trend of cleaved-caspase3 at protein level, and Bim, Bcl-2 at gene level. In comparison to NT group, GRP78, CHOP, p-eIF2α, cleaved caspase-3 and Bim all decreased, while Bcl-2 increased in HT group. Additionally, apoptotic cells detected by TUNEL staining significantly decreased in the HT group. ConclusionMild hypothermia could attenuate ICH caused neuron injury by decreasing ER response-induced neuron apoptosis.

Dexmedetomidine-Induced Neuroapoptosis Is Dependent on Its Cumulative Dose.

Dexmedetomidine (DEX) has inherent neuroprotective properties that have been attributed to the activation of prosurvival kinases. However, the impact of supraclinical doses of DEX on neuroapoptosis and neuronal viability has not been determined. Rat pups and primary neuronal cells were treated with DEX or ketamine (KET) alone or in combination. Neuroapoptosis was measured by cleaved-caspase-3 expression and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining in brain sections. Expression of prosurvival kinases was measured by Western blot. We measured the impact of DEX with and without α1-adrenergic receptor blockade on the viability of primary neuronal cell cultures. Increasing the cumulative dose of DEX resulted in elevated levels of neuroapoptosis in vivo. Low doses increased, whereas high dose decreased phosphorylation of the prosurvival kinases. KET alone and in combination with DEX produced a greater degree of apoptosis and reductions in expression of these protein kinases than DEX alone. Increasing concentrations of DEX decreased, while coadministration of an α1-adrenergic receptor blocker preserved neuronal viability in vitro. Although DEX is neuroprotective at clinical doses, high cumulative doses and concentrations induce neuroapoptosis, in vivo and in vitro, respectively. Because the current dosing schedules used in humans yield plasma levels that are substantially below concentrations that induce neurotoxicity, low-dose DEX should not be neurotoxic and has the potential to be a neuroprotective adjuvant.

Transfer of microRNA-486-5p from human endothelial colony forming cell–derived exosomes reduces ischemic kidney injury

Administration of human cord blood endothelial colony-forming cells (ECFCs) or their exosomes protects mice against kidney ischemia/reperfusion injury. Here we studied the microRNA (miRNA) content of ECFC exosomes and the role of miRNA transfer in kidney and endothelial cell protection. ECFC exosomes were enriched in miR-486-5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. In cultured endothelial cells exposed to hypoxia, incubation with ECFC exosomes increased miR-486-5p, decreased PTEN, and stimulated Aktphosphorylation. Exposure of hypoxic endothelial cellsto conditioned medium from ECFCs pretreated with anti-miR-486-5p blocked increases in miR-486-5p and phosphorylated Akt, restored expression of PTEN, and enhanced apoptosis. Coculture of endothelial cells with ECFCs enhanced endothelial miR-486-5p levels. Targeting of PTEN by miR-486-5p was observed in endothelial cells, and PTEN knockdown blocked apoptosis. In mice with ischemic kidney injury, infusion of ECFC exosomes induced potent functional and histologic protection, associated with increased kidney miR-486-5p levels, decreased PTEN, and activation of Akt. Infusion of exosomes from ECFCs transfected with anti-miR-486-5p had no protective effect. Thus, delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p targeting PTEN. Exosomes enriched in miR-486-5p could represent a therapeutic tool in acute kidney injury.

Influence of Light Emitting Diode-Derived Blue Light Overexposure on Mouse Ocular Surface.

PurposeTo investigate the influence of overexposure to light emitting diode (LED)-derived light with various wavelengths on mouse ocular surface.MethodsLEDs with various wavelengths were used to irradiate C57BL/6 mice at an energy dose of 50 J/cm2, twice a day, for 10 consecutive days. The red, green, and blue groups represented wavelengths of 630 nm, 525 nm, and 410 nm, respectively. The untouched group (UT) was not exposed to LED light and served as the untreated control. Tear volume, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured on days 1, 3, 5, 7, and 10. Levels of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in the cornea and conjunctiva using a multiplex immunobead assay at day 10. Levels of malondialdehyde (MDA) were measured with an enzyme-linked immunosorbent assay. Flow cytometry, 2’7’-dichlorofluorescein diacetate (DCF-DA) assay, histologic analysis, immunohistochemistry with 4-hydroxynonenal, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining were also performed.ResultsTBUT of the blue group showed significant decreases at days 7 and 10, compared with the UT and red groups. Corneal fluorescein staining scores significantly increased in the blue group when compared with UT, red, and green groups at days 5, 7, and 10. A significant increase in the corneal levels of IL-1β and IL-6 was observed in the blue group, compared with the other groups. The blue group showed significantly increased reactive oxygen species production in the DCF-DA assay and increased inflammatory T cells in the flow cytometry. A significantly increased TUNEL positive cells was identified in the blue group.ConclusionsOverexposure to blue light with short wavelengths can induce oxidative damage and apoptosis to the cornea, which may manifest as increased ocular surface inflammation and resultant dry eye.

Allicin improves cardiac function by protecting against apoptosis in rat model of myocardial infarction.

To study the effects of allicin on cardiac function and underlying mechanism in rat model of myocardial infarction (MI). Ninety-four Wistar rats were randomly assigned to 6 groups (n=14-16 per group): sham control group [underwent thoracotomy without left anterior descending (LAD) occlusion and only received an injection of the same amount of citrate buffer], MI control group (subjected to LAD occlusion and only received an injection of same amount of citrate buffer), positive control group (subjected to LAD occlusion and received an injection of diltiazem hydrochloride at the dose of 1.5 mg/kg), and MI + allicin groups (subjected to LAD occlusion and received an injection of allicin at the doses of 1.2, 1.8, and 3.6 mg/kg). All of the drugs were administered intraperitoneally daily for 21 days. The infarct area was measured by myocardial staining. Hematoxylin-eosin staining was used to observe the pathological changes. Cardiac function parameters were assessed by echocardiography. The myocardial apoptotic index was estimated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. The expression of Bax and Bcl-2 were detected by quantificational real-time polymerase chain reaction and Western blot. Treatment with allicin could attenuate the myocardial infarct area (P<0.05) and relieve the changes of the myocardium. The left ventricular anterior wall diastolic and systolic thicknesses were increased in the allicin-treated groups (P<0.05), while there was no signifificant difference in the left ventricular posterior wall diastolic and systolic thickness (P>0.05). The left ventricular internal diameter in systole, ejection fraction, fractional shortening, and stroke volume were dramatically elevated in allicin-treated rats (P<0.05). Allicin dose-dependently reduced creatine kinase and lactate dehydrogenase levels (P<0.05). The myocardial apoptotic index was also markedly lowered, and Bax expression was signifificantly decreased, whereas Bcl-2 expression exhibited an opposite trend in allicin-treated rats (P<0.05). Allicin appears to exert a cardioprotective effect that may be linked to blocking Bcl-2/Bax signaling pathway-denpendent apoptosis, further improving cardiac function.

Effect of baicalin on hippocampal damage in kainic acid-induced epileptic mice.

The aim of the present study was to determine the effect of baicalin on the expression of miR-497 and its target B-cell lymphoma-2 (Bcl-2) in the hippocampus of kainic acid (KA)-induced epileptic mice. To establish status epilepticus (SE), 0.1 µg/5 µl KA was injected into the lateral cerebral ventricle in mice, which then received an intraperitoneal injection of baicalin (100 mg/kg) after 1 and 8 h. Hematoxylin and eosin staining was used to observe the pathological changes in morphology and neuronal apoptosis was determined by terminal transferase-mediated dUTP nick end-labeling staining. Western blot analysis was used to detect the expression of Bcl-2 and cleaved caspase-3 proteins in the hippocampus, while reverse transcription-quantitative polymerase chain reaction was used to quantify hippocampal miR-497 expression. The results showed that baicalin significantly attenuated neuronal damage and apoptosis in the hippocampus 72 h after SE. In addition, baicalin decreased SE-induced expression of miR-497 and cleaved caspase-3 protein, while upregulating the expression of Bcl-2 protein. In conclusion, the present results suggest that baicalin possesses potent antiapoptotic properties and attenuates hippocampal injury in mice after SE, which may be associated with the downregulation of miR-497 and cleaved caspase-3 and the upregulation of Bcl-2.

House dust mite–induced asthma causes oxidative damage and DNA double-strand breaks in the lungs

Asthma is related to airway inflammation and oxidative stress. High levels of reactive oxygen and nitrogen species can induce cytotoxic DNA damage. Nevertheless, little is known about the possible role of allergen-induced DNA damage and DNA repair as modulators of asthma-associated pathology. We sought to study DNA damage and DNA damage responses induced by house dust mite (HDM) invivo and invitro. We measured DNA double-strand breaks (DSBs), DNA repair proteins, and apoptosis in an HDM-induced allergic asthma model and in lung samples from asthmatic patients. To study DNA repair, we treated mice with the DSB repair inhibitor NU7441. To study the direct DNA-damaging effect of HDM on human bronchial epithelial cells, we exposed BEAS-2B cells to HDM and measured DNA damage and reactive oxygen species levels. HDM challenge increased lung levels of oxidative damage to proteins (3-nitrotyrosine), lipids (8-isoprostane), and nucleic acid (8-oxoguanine). Immunohistochemical evidence for HDM-induced DNA DSBs was revealed by increased levels of the DSB marker γ Histone 2AX (H2AX)foci in bronchial epithelium. BEAS-2Bcells exposed to HDM showed enhanced DNA damage, as measured by using the comet assay and γH2AX staining. In lung tissue from human patients with asthma, we observed increased levels of DNA repair proteins and apoptosis, as shown by caspase-3 cleavage, caspase-activated DNase levels, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Notably, NU7441 augmented DNA damage and cytokine production in the bronchial epithelium and apoptosis in the allergic airway, implicating DSBs as an underlying driver of asthma pathophysiology. This work calls attention to reactive oxygen and nitrogen species and HDM-induced cytotoxicity and to a potential role for DNA repair as a modulator of asthma-associated pathophysiology.

In vivo synergistic antitumor effect and safety of siRNA and lonidamine dual-loaded hierarchical targeted nanoparticles

Based on development of nano-delivery system, co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) has exerted a promising advantage in cancer therapy. In this work, the superiority of synergistic therapy and safety of the hierarchical targeted co-delivery system loaded with siRNA and lonidamine (LND) were evaluated. The in vivo tumor accumulation ability and cancer growth inhibition effect of the polymer-blend nanocarriers were evaluated by a H22 subcutaneous sarcoma model. Moreover, hematoxylin and eosin (H&E) staining and transferase-mediated dUTP nick end-labeling (TUNEL) staining of tumor sections from each group were compared to assess the therapeutic efficacy. The dual-loaded nanocarriers had better tumor accumulation ability, remarkably inhibited growth of solid tumor in a synergistic manner, even significantly decreased hepatotoxicity of LND, and had good in vivo biocompatibility whereas LND alone showed serious hepatotoxicity. We believed that the dual-loaded hierarchical targeted delivery system with high effectiveness and biocompatibility would provide a promising approach for cancer combination therapy.

Neuroprotective effect of systemic and/or intravitreal rosuvastatin administration in rat glaucoma model.

To evaluate the neuroprotective effect of rosuvastatin, in a rat experimental glaucoma model. Ocular hypertension was induced in right eyes of Long-Evans rats (n=30) by cauterization of three episcleral veins. Left eyes were defined as controls. Rats were divided into five groups: oral rosuvastatin, intravitreal rosuvastatin, oral+intravitreal rosuvastatin, intravitreal sham and glaucoma without intervention. Rats were sacrificed at day 14. Retinal ganglion cell (RGC) number was assessed by histopathological analysis. Terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) staining and the expression of glial fibrillary acidic protein (GFAP) in RGC layer was also examined. A significant intraocular pressure (IOP) elevation was seen (P=0.002). Elevated IOP resulted in a significant decrease in number of RGCs in group 5 (70.33±8.2 cells/mm(2)) when compared with controls (92.50±13.72 cells/mm(2); P=0.03). The RGC number in group 1 (92.4±7.3 cells/mm(2)) was significantly higher than group 5 (P=0.03). The numbers of RGC in groups 2, 3 (57.3±8.2 cells/mm(2), 60.5±12.9 cells/mm(2)) were comparable with that of group 5 (P=0.18 and P=0.31). The apoptosis rates with TUNEL staining were also parallel to RGC number. Animals with experimentally induced glaucoma showed an increase in retinal GFAP immunoreactivity. Decrease in RGC loss and apoptosis suggest the neuroprotective potential of oral rosuvastatin treatment in a rat model of ocular hypertension. However intravitreal rosuvastatin showed a contrary effect and further studies are required.

Atorvastatin ameliorates early brain injury after subarachnoid hemorrhage via inhibition of AQP4 expression in rabbits.

The therapeutic effects of atorvastatin on early brain injury (EBI), cerebral edema and its association with aquaporin 4 (AQP4) were studied in rabbits after subarachnoid hemorrhage (SAH) using western blot analysis and the dry-wet method. Seventy-two healthy male New Zealand rabbits weighing between 2.5 and 3.2 kg were randomly divided into three groups: the SAH group (n=24), sham-operated group (n=24) and the SAH + atorvastatin group (n=24). A double SAH model was employed. The sham-operated group were injected with the same dose of saline solution, the SAH + atorvastatin group received atorvastatin 20 mg/kg/day after SAH. All rabbit brain samples were taken at 72 h after the SAH model was established successfully. Brain edema was detected using the dry-wet method after experimental SAH was induced; AQP4 and caspase-3 expression was measured by western blot analysis, and neuronal apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining at 72 h after SAH. The results indicated that brain edema and injury appeared soon after SAH, while brain edema and EBI were ameliorated and increased behavior scores were noted after prophylactic use of atorvastatin. Compared with the SAH group, the level of AQP4 and the cerebral content of water was significantly decreased (P<0.01) by atorvastatin, and TUNEL staining and studying the expression of caspase-3 showed that the apoptosis of neurons was reduced markedly both in the hippocampus and brain cortex by atorvastatin. The results suggest that atorvastatin ameliorated brain edema and EBI after SAH, which was related to its inhibition of AQP4 expression. Our findings provide evidence that atorvastatin is an effective and well-tolerated approach for treating SAH in various clinical settings.