Deoxynucleotidyl Transferase-mediated dUTP-biotin Nick End Research Articles

Repair Effects of KGF on Ischemia-Reperfusion–Induced Flap Injury via Activating Nrf2 Signaling

BackgroundIschemia-reperfusion (IR) injury is a main cause to and the mechanism of necrosis after flap transplantation. Researches were hardly conducted on the role and possible mechanism of keratinocyte growth factor (KGF) in association with IR flap injury. Materials and methodsA CoCl2-stimulated hypoxia cell model was established to investigate the effects of KGF on cell viability, apoptosis, cell cycle, and reactive oxygen species level. The experiments were performed by cell counting kit-8 and flow cytometry as required. Meanwhile, the expressions of cell cycle-related and nuclear factor E2–related factor 2 (Nrf2) signaling–related genes were determined using quantitative real-time PCR and Western blot. The right dorsolateral areas of Institute of Cancer Research mice were marked as flaps, the pedicle of which formed an IR process through clamping and loosening. Tissue morphologies were observed using hematoxylin and eosin staining 24 h after the surgery. The effects of KGF on cell apoptosis and associated genes expressions were studied by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, immunohistochemistry, and Western blot. ResultsHaCAT cells treated with 40 μM CoCl2 could not only reduce cell viability, promote cell apoptosis, arrest G1 phase of cell cycle and increase the activity of reactive oxygen species but also downregulate the expressions of c-myc, c-fos, transforming growth factor-α, Nrf2, heme oxygenase-1, and gamma-glutamyl cysteine synthetase. Additional recombinant human KGF, on one hand, could protect the cells from hypoxia injury. On the other hand, recombinant human KGF could significantly inhibit cell apoptosis, increase KGF activity, and increase the Nrf2, heme oxygenase-1, and gamma-glutamyl cysteine synthetase proteins levels in IR flap tissues. ConclusionsKGF played an important role in protecting mice flaps from IR injury, and the possible mechanism was involved in activating the Nrf2 signaling.

Radix Kansui Stir-Fried with Vinegar Reduces Radix Kansui-Related Hepatotoxicity in Mice via Mitochondrial Pathway.

To investigate the mechanism of Radix Kansui (RK) stir-fried with vinegar (VRK) decreased hepatotoxicity in mice. According to a random number table, 40 mice were randomly divided into negative control group (0.5% carboxymethylcellulose sodium, 20 mL/kg), positive control group (0.1% mixture of carbon tetrachloride in soybean oil, 20 mL/kg), RK group (the ethyl acetate extracts of RK, 250 g crude drug/kg) and VRK group (the ethyl acetate extracts of VRK, 250 g crude drug/kg) with 10 mice per group. All mice were administered orally by gavage daily for 7 continuous days. The morphology of liver tissues was examined to assess the liver injury by a transmission electron microscope. Hepatocyte apoptosis in vivo was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling (TUNEL) assay. Immunohistochemical technique was adopted to detect the expression of particular antiapoptotic and proapoptotic proteins in the mitochondrial pathways, including B-cell lymphoma (Bcl-2) and caspase-3, as well as the expression of inflammatory mediators, including nuclear factor kappa B (NF- κ B) and intercellular adhesion molecule-1 (ICAM-1). Liver injury and hepatocyte apoptosis were observed in RK mice, and the liver injury were significantly reduced in VRK-treated mice. In immunohistochemistry study, compared with the negative control group, RK inhibited dramatically the Bcl-2 protein expression and significantly increased the expression of caspase-3, NF- κ B and ICAM-1 (all P<0.01). Compared with the RK group, VRK group induced significant increase on Bcl-2 protein expression, and decreased the caspase-3, NF- κ B and ICAM-1 protein expression (P<0.05 or P<0.01). The mechanism of reduced hepatotoxicity of VRK may be associated with the reduced inflammation, regulation of antiapoptotic and proapoptotic mediators in the mitochondrial pathway.

D-Limonene protects PC12 cells against corticosterone-induced neurotoxicity by activating the AMPK pathway

The stress-induced hormone corticosterone initiates oxidative stress and inflammatory responses, culminating in cell apoptosis and neurological changes. We assessed the effects of d-Limonene on a PC12 cellular model of corticosterone-induced neurotoxicity, and whether these effects involved the AMP-activated protein kinase (AMPKα) pathway. PC12 cells were treated with corticosterone with or without d-limonene for 24 h. Western blots were performed to measure activation of AMPK pathway members [Silent mating type information regulation 2 homolog-1 (SIRT1), AMPKα, and nuclear factor (NFκB)], reactive oxygen species, inflammatory cytokines, and markers of apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was used to measure cell death after treatment. d-Limonene reversed the effects of corticosterone on PC12 cells: it decreased the levels of malondialdehyde (MDA) and nitric oxide (NO), activities of NADPH oxidase (p67-phox and p47-phox), expression of pro-inflammatory markers [inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α)], and expression of pro-apoptotic proteins [Bcl2 associated with X protein (Bax) and cleaved caspase-3)]. d-Limonene also increased levels of the antioxidant enzymes superoxide dismutase 1 (SOD1) and heme oxygenase 1 (HO-1) and the anti-apoptotic protein Bcl-2 while decreasing the number of TUNEL-positive cells. d-limonene significantly activated AMPKα and suppressed NF-κB nuclear translocation through up-regulation of SIRT1. Addition of compound C, an AMPK inhibitor, severely weakened these neuroprotective effects of d-limonene. d-Limonene has a neuroprotective effect on corticosterone-induced PC12 cell injury induced by activating the AMPKα signaling pathway, and thereby inhibiting reactive oxygen species and inflammatory factors. These data suggest that d-limonene might protect against neuronal death to improve depressive symptoms.

HO-1 protects the nerves of rats with cerebral hemorrhage by regulating the PI3K/AKT signaling pathway.

Objective: This study aimed to investigate the neuroprotective effect of heme oxygenase-1 (HO-1) on the PI3K/AKT signaling pathway in rats with cerebral hemorrhage.Materials and methods: Adult male Sprague-Dawley rats were randomly divided into: a sham group, a model group and an HO-1 inhibitor group (ZnPP group). Functional defects after surgery were scored according to the Longa5 standard. Hemotoxylin and eosin staining was used to detect whether the model was constructed successfully. Superoxide dismutase (SOD) vitality and malondialdehyde (MDA) content were calculated by the xanthine oxidase method and thiobarbituric acid method, respectively. Blood-brain barrier permeability was measured by Evans Blue. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The expression of Bcl-2 and BAX was evaluated by immunohistochemistry and the expression of PI3K, p-PI3K, AKT and p-AKT was tested by Western blotting.Results: The rat intracerebral hemorrhage model was successfully constructed. Compared with the model group, the bleeding in the ZnPP group was more serious, the cell edema and deformation were aggravated, and the neurological deficit score in the rat was significantly increased. In addition, the content of Evans blue, MDA, the number of apoptotic cells, the water content of brain tissue and the expression of BAX were significantly increased, while the SOD activity and the expressions of Bcl-2, p-PI3K and p-AKT protein were decreased.Conclusion: HO-1 could protect the nerves of rats with cerebral hemorrhage by regulating the PI3K/AKT signaling pathway.

CD8 T cell-derived perforin aggravates secondary spinal cord injury through destroying the blood-spinal cord barrier

Perforin plays an important role in autoimmune and infectious diseases, but its function in immune inflammatory responses after spinal cord injury (SCI) has received insufficient attention. The goal of this study is to determine the influence of perforin after spinal cord injury (SCI) on secondary inflammation. Compared recovery from SCI in perforin knockout (Prf1−/−) and wild-type(WT)mice, WT mice had significantly lower the Basso mouse score (BMS), CatWalk XT, and motor-evoked potentials (MEPs) than Prf1−/− mice. Spinal cord lesions were also more obvious through glial fibrillary acidic protein (GFAP), Nissl, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Furthermore, the blood-spinal cord barrier (BSCB) disruption was more severe and inflammatory cytokine levels were higher. Flow cytometry indicated that perforin mainly originated from CD8 T cells. With flow cytometry and enzyme-linked immunosorbent assay (ELISA), human cerebrospinal fluid (CSF) yielded similar results. Together, this study firstly demonstrated that CD8 T cell-derived perforin is detrimental to SCI recovery in the mouse model. Mechanistically, this effect occurs because perforin increases BSCB permeability, causing inflammatory cells and related cytokines to infiltrate and disrupt the nervous system.

Retracted: Protection of miR-19b in hypoxia/reoxygenation-induced injury by targeting PTEN.

To study the role and mechanism of microRNA 19b (miR-19b) in hypoxia/reoxygenation (H/R)-induced injury by targeting PTEN. PC12 and BV2 cells induced by H/R were treated with miR-19b mimics/inhibitors or small interfering PTEN (si-PTEN), respectively. Lactate dehydrogenase (LDH) level, malondialdehyde (MDA), and superoxide dismutase (SOD) content was detected. Besides, cell viability and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Hoechst33342 staining, and flow cytometry, whereas mitochondrial membrane potential (MMP) tested by JC-1 assay, and reactive oxygen species (ROS) evaluated by the dichloro-dihydro-fluorescein diacetate assay. The ischemia/reperfusion (I/R) rats model was used to investigate the effects of miR-19b in vivo test. The infarct area and apoptosis rates in brain tissues were detected by 2,3,5-triphenyltetrazolium chloride and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, respectively. miR-19b and PTEN/PI3K/Akt pathway-related proteins were detected by quantitative reverse-transcription polymerase chain reaction and western blot analysis. miR-19b mimics could reduce LDH, MDA, and ROS levels and decline cell apoptosis, but enhance the viability, MMP, and SOD activity with decreased PTEN and cleaved caspase, as well as increased p-Akt/Akt and Bcl-2/Bax ratios in H/R-induced PC12 and BV2 cells. However, miR-19b inhibitors led to completely opposite results to aggravate H/R-induced cell injury. Meanwhile, si-PTEN could reverse the effect of miR-19b inhibitors on H/R-induced injury. Moreover, treatment with miR-19b agomir after I/R in vivo sufficiently decreased infarct area and reduced apoptosis rates by targeting PTEN through the regulation of the PI3K/Akt pathway. miR-19b could inhibit oxidative stress, enhance cell MMP, promote cell survival, and inhibit cell apoptosis by targeting PTEN via the regulation of the PI3K/Akt pathway, thus playing the neuronal protective effects.

LINC-PINT Activates the Mitogen-Activated Protein Kinase Pathway to Promote Acute Myocardial Infarction by Regulating miR-208a-3p.

This study is performed to explore the differential expression of long intergenic non-coding-p53 induced non-coding transcript, miR-208a-3p and JUN in acute myocardial infarction (AMI) and their potential mechanisms. Methods and Results: Gene Expression Omnibus, R software, Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analysis were used for analyzing the differentially expressed genes (DEGs) and pathways. The differential expressions of LINC-PINT and miR-208a-3p were examined by qRT-PCR. The expressions of JUN and the mitogen-activated protein kinase (MAPK) pathway-related proteins were analyzed by Western blot. The triphenyltetrazolium chloride (TTC) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining methods were used to measure the myocardial infarction size and tissue apoptosis respectively. The targeted relationships between miR-208a-3p and LINC-PINT or JUN were confirmed using a dual luciferase reporter assay. DEGs were significantly enriched in the MAPK signaling pathway. LINC-PINT could sponge miR-208a-3p, which targeted and regulated JUN. LINC-PINT and JUN were confirmed to be overexpressed in AMI tissues. Silencing LINC-PINT and JUN could exert a protective influence against AMI. The expression of miR-208a-3p was significantly decreased in AMI tissues, and miR-208a-3p reduced myocardial ischemia-reperfusion injury and apoptosis. Downregulation of LINC-PINT facilitated miR-208a-3p expression and suppressed the protein level of JUN, contributing to the inactivation of the MAPK pathway in the AMI tissues and thus generating protective effects. Knockdown of LINC-PINT inactivated the MAPK pathway by releasing miR-208a-3p and suppressing the JUN, protecting the injury during the process of AMI.

Effects of propofol pretreatment on myocardial cell apoptosis and SERCA2 expression in rats with hepatic ischemia/reperfusion

IntroductionHepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. ObjectiveTo investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. MethodsMale Sprague-Dawley rats (n=18) were randomly allocated into three groups: Sham Group (Group S, n=6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n=6) and Propofol Group (Group P, n=6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30min and reperfusion for 4h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10min before ischemia with 120mg.kg−1, following by continuous infusion at 20mg.kg−1.h−1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. ResultsApoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p<0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p<0.01). ConclusionsPropofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.

Flurbiprofen axetil attenuates cerebral ischemia/reperfusion injury by reducing inflammation in a rat model of transient global cerebral ischemia/reperfusion.

Ischemic stroke has been ranked as the second cause of death in patients worldwide. Inflammation which is activated during cerebral ischemia/reperfusion (I/R) is an important mechanism leading to brain injury. The present study aimed to investigate the effect of flurbiprofen axetil on cerebral I/R injury and the role of inflammation in this process. Rats were subjected to sham operation or global cerebral I/R with or without flurbiprofen axetil (5 or 10 mg/kg). Global cerebral ischemia was achieved by occlusion of bilateral common carotid arteries combined with hypotension for 20 min followed by reperfusion for 72 h. Then the neurological deficit score, hippocampal cell apoptosis, levels of aquaporin (AQP) 4, AQP9, intercellular cell adhesion molecule-1 (ICAM-1), nuclear factor-κB (NF-κB), tumor necrosis factor (TNF-α), interleukin-1 β (IL-1β), thromboxane B2 (TXB2), and 6-keto-PGI1α were assessed. After reperfusion, neurological deficit score was significantly increased accompanied by severe neuronal damage (exacerbated morphological deficit, increased terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL)-positive cells and cleaved caspase-3 protein expression in hippocampal CA1 region). Cerebral I/R injury also enhanced expressions of TNF-α, IL-1β, NF-κB, AQP4 and AQP9 as well as TXB2 and TXB2/6-keto-PGI1α. All these changes were reversed by pretreatment with flurbiprofen axetil. Flurbiprofen axetil protects the brain from cerebral I/R injury through reducing inflammation and brain edema.

Effect of endogenous sulfur dioxide on the apoptosis induced by cobalt chloride in the human pulmonary arterial endothelial cells

Objective To explore the effect of endogenous sulfur dioxide (SO2) on the apoptosis induced by cobalt chloride (CoCl2) in the human pulmonary arterial endothelial cells (HPAECs). Methods CoCl2 was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1(AAT1), and the key enzyme generating endogenous SO2 were over-expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups: vehicle group, vehicle+ CoCl2 group, AAT1 group and AAT1+ CoCl2 group.The expressions of AAT1, B-cell lymphoma-2 (bcl-2), bcl-associated X protein (bax), Caspase-3 and activated Caspase-3 (cleaved Caspase-3) in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Results There were significant differences in the endogenous SO2 content, the expre-ssions of AAT1 and bcl-2, and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were (F=147.364, 23.738, 6.521, 64.884, all P 0.05). Compared with vehicle group, AAT activity [(0.96±0.24) Carmen′s unit/μg vs.(2.21±0.60) Carmen′s unit/μg], endogenous SO2 content (40.71±7.72 vs.105.60±16.20) and bcl-2 expression (0.59±0.19 vs.1.02±0.20) in the HPAECs of vehicle+ CoCl2 group were significantly decreased, while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (1.56±0.25 vs.0.95±0.13) were significantly increased (all P 0.05). The SO2 content (351.50±42.43 vs.105.60±16.20) and AAT1 expression (1.22±0.33 vs.0.53±0.11) in the HPAECs of AAT1 group were higher than those of vehicle group (all P 0.05). Moreover, no difference was observed in the HPAECs apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (0.51±0.17 vs.0.50±0.11) between the two AAT1-overexpressed groups (all P>0.05). Conclusion Endo-genous SO2 inhibited the hypoxic HPAECs apoptosis stimulated by the treatment of CoCl2. Key words: Sulfur dioxide; Aspartate aminotransferase 1; Cobalt chloride; Human pulmonary arterial endothelial cells; Apoptosis

MiR-125b blocks Bax/Cytochrome C/Caspase-3 apoptotic signaling pathway in rat models of cerebral ischemia-reperfusion injury by targeting p53

ABSTRACTObjective: To explore the potential effect of miR-125b on p53-mediated regulation of Bax/Cytochrome C/Caspase-3 apoptotic signaling pathway in rats with cerebral ischemia-reperfusion (CIR) injury.Methods: Sprague-Dawley (SD) rats were used to conduct CIR injury and injected with miR-125b mimic/inhibitor or p53 inhibitor (Pifithrin-α, PFT-α). Dual-luciferase reporter gene assay was used to analyze the targeting relationship between miR-125b and p53. Longa scoring and Triphenyl tetrazolinm chloride (TTC) staining were used to test the neurologic function and determine infarct size, respectively. Hematoxylin-eosin (HE) and Nissl’s stainings were conducted to observe the morphology of cortical neurons. Neuronal nuclei (NeuN) expression was detected by immunohistochemical staining. QRT-PCR was performed to detect the expressions of miR-125b and p53. TUNEL staining and Western blotting was used to determine neuronal apoptosis and expressions of Bax/Cytochrome C/Caspase-3 signaling pathway-related proteins, respectively.Results: Our results showed that miR-125b could directly target p53. As observed, overexpression of miR-125b could obviously reduce the neurological score, infarct size, and brain water content after CIR in rats, which also improved the morphology of cortical neurons, increased the number of neurons, reduced neuronal apoptosis, and inhibited the expressions of Bax/Cytochrome C/Caspase-3 pathway. Moreover,the similar results were observed in rats with CIR after injected with PFT-α. But no significant differences in each index were found in CIR group and CIR + anti-miR-125b + PFT-α group.Conclusion: MiR-125b exerts protective effects on CIR injury through inhibition of Bax/Cytochrome C/Caspase-3signaling pathway via targeting p53, which is likely to be a promising treatment for CIR.Abbreviations: 3’–UTR: 3–untranslated region; CIR: cerebral ischemia–reperfusion; CIS: cerebral ischemic stroke; PFT–α: Pifithrin–α; PVDF: polyvinylidene fluoride; SD: Sprague–Dawley; TBST: tris buffered saline with tween. TTC staining: Triphenyl tetrazolinm chloride staining; TUNEL: Terminal deoxynucleotidyl transferase–mediated dUTP–biotin nick end labeling.

Curcumin ameliorates cisplatin-induced cystopathy via activating NRF2 pathway.

The present work evaluated preventive effect of curcumin on cisplatin-induced bladder cystopathy. Fifteen female rats were divided into (i) Control group administered with physiological saline solution for 5 days; (ii) Cis-P group injected with cisplatin (6 mg/kg); and (iii) Cis-Cur group given cisplatin (6 mg/kg) with curcumin for 5 consecutive days. The function of bladder was measured by means of urodynamic analysis. Furthermore, hematoxylin-eosin staining and Masson trichrome staining were performed for morphological analysis. The cell apoptosis was evaluated through terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry. The expression of nerve growth factor (NGF), NF-E2-related factor 2, and hemeoxygenase-1 (HO-1) levels were measured through Western blotting. Urodynamic assay and histopathological manifestations revealed that curcumin ameliorated the bladder dysfunction induced by cisplatin. The level of cisplatin-induced apoptosis in the bladder decreased following curcumin treatment. Also, the increased protein expression of NGF indicated that the curcumin could offer neuroprotection for bladder against cisplatin. Curcumin also activated NRF2, and elevated the expression of HO-1, but curcumin could not rescue cisplatin-induced apoptosis in the cell lines with knockdown of NRF2. Taken together, the results of this paper showed that curcumin could ameliorate cisplatin-induced cystopathy and inhibit the apoptosis of bladder cell in cisplatin-treated rats. This may be attributed to curcumin's broad biological functions, particularly antioxidant effect, and to its ability to activate the NRF2 protein.

The effect of NF-κB on NLRP3 of lung tissue in rats with acute lung injury induced by phosgene

Objective To observe the effect of signal transduction pathway of nuclear factor-kappa B (NF-κB) on Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and pyroptosis in rats with acute lung injury induced (ALI) by phosgene. Methods The rats were randomly(random number) divided into 3 groups: air exposure control group, phosgene exposure group and PDTC group with phosgene exposure after 100 mg/kg pyrrolidine dithiocarbamate (PDTC) administration. The specimens of serum, bronchoalveolar lavage fluid (BALF) and lung tissue were collected 6 h after exposure. Morphological changes were observed by HE staining. The expression of NLRP3 in the lung of three groups was detected by immunohistochemistry. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expressions of NF-κB p65,NLRP3, ASC and caspase-1 mRNA in the lung tissue. NF-κB p65, NLRP3, ASC and caspase-1 protein levels in the lung tissue were quantified by Western blot. The concentrations of IL-1β, IL-18 and IL-33 in the serum and BALF were measured by enzyme-linked immunosorbent assay (ELISA). Pyroptosis was observed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). Results The model of phosgene-induced ALI was successfully established in rats. Morphological changes with inflammatory cell infiltration were observed in the lung tissues of phosgene group, in which NLRP3 positive cells also could be observed by immunohistochemical staining. The mRNA expressions and protein levels of NF-κB p65, NLRP3 and caspase-1 in lung tissues were significantly increased (P<0.05) in phosgene group, compared with air control group. The mRNA expressions and protein levels of NF-κB p65, NLRP3 and caspase-1 in lung tissues were significantly decreased (P<0.05) in PDTC group, compared with phosgene group. The IL-1β, IL-18 and IL-33 protein levels in serum and BALF were significantly increased (P<0.05) in phosgene group, compared with air control group. The IL-1β, IL-18 and IL-33 protein levels in serum and BALF were significantly decreased (P<0.05) in PDTC group, compared with phosgene group. TUNEL results showed that pyroptosis in the lung tissue obviously increased in phosgene group, while decreased in PDTC group. Conclusions NLRP3 inflammasome and lung cell pyroptosis were induced through NF-κB signal transduction pathway in rats with acute lung injury caused by phosgene inhalation. Blockade of NF-κB can alleviate acute lung injury by down-regulating the expression of NLRP3 inflammasome to inhibit pyroptosis. Key words: Nuclear factor-kappa B (NF-κB); Phosgene; acute lung injury; Pyrin domain-containing 3 (NLRP3) innammasome; Inhalation; pyroptosis; Inflammation; Pyrrolidinedithiocarbamic acid (PDTC)

A DNA Vaccine Expressing Consensus Hemagglutinin-Esterase Fusion Protein Protected Guinea Pigs from Infection by Two Lineages of Influenza D Virus.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax (n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 105 50% tissue culture infective dose(s) (TCID50)] D/OK (n = 6) or D/600 (n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine (n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 105 TCID50) with IDV D/OK (n = 6) or D/660 (n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model.IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections.

Busulfan-mediated oxidative stress and genotoxicity decrease in sperm of Satureja Khuzestanica essential oil-administered mice

ABSTRACTHere, we studied the protective effects of Satureja Khuzestanica essential oil (SKEO) as a potent anti-oxidant, against damage caused by chemotherapy with busulfan in testis and epididymal sperm of adult mice. The NMRI adult mice were assigned: G1: control, G2: was treated with busulfan (4 days, 3.2 mg/kg), G3: was treated with busulfan (4 days, 3.2 mg/kg) and SKEO (28 days, 225 mg/kg) at the same time, and G4: was pre-treated with SKEO (7 days, 225 mg/kg) and subsequently co-treated with busulfan (4 days, 3.2 mg/kg) and SKEO (28 days, 225 mg/kg). Apoptosis and Bcl-2 family gene expression were evaluated in sperm by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and Real-Time PCR, respectively. The level of oxidative stress was studied in sperm and testis by Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx) assays. Lactate Dehydrogenase (LDH), and Thiobarbituric assays were used for analyzing cytotoxicity and lipid peroxidation in testis and sperm of mice, (TBA) respectively. The results showed a significant decrease in the percentage of apoptotic sperm in G4 versus G2 and G3 (p < 0.05). SKEO pre-treatment potentially increased Bcl-2 expression and decreased BAX expression in sperm of G4 compared with G2 and G3. The activities of SOD and GPx were increased, also, LDH and TBA decreased significantly in testis and sperm of G4 compared with G2 and G3 (p < 0.05). SKEO pre-treatment had a notable role in reducing oxidative stress, apoptosis, cytotoxicity, and genotoxicity in sperm of busulfan-treated mice. In addition, cytotoxicity and oxidative stress were decreased significantly in testes of this group. Thereby, SKEO may inhibit busulfan-mediated apoptosis in sperm via decreasing oxidative stress and regulating Bcl-2 family genes expression. In conclusion, the beneficial properties of SKEO pre-treatment and co-treatment by its herbal potent anti-oxidants may reduce adverse effects of chemotherapy in the reproductive system in a rodent system.Abbreviations: SKEO: Satureja Khuzestanica essential oil; SOD: superoxide dismutase; GPx: glutathione peroxidase; LDH: lactate dehydrogenase; GC–MS: gas chromatography/mass spectrometry; TdT: terminal deoxynucleotidyl transferase; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; ROS: reactive oxygen species

Lead exposure reduces sperm quality and DNA integrity in mice.

Toxicity of lead on male reproductive functions has raised wide public concern as environmental lead contamination remains common worldwide. Conflicting and controversial data are available regarding effects of lead on male fertility. More importantly, our knowledge on effects of lead on sperm DNA integrity is significantly limited. Thus, further studies should focus on this issue. In the current study, adult male mice were exposed to a series of lead acetate concentrations in drinking water for six weeks. Following administration, lead levels in blood, testicles, and epididymis were measured, and potential changes in morphology of testis and epididymis due to lead exposure were identified. We also analyzed sperm parameters, including sperm density, viability, motility, and morphology, to evaluate quality of sperm collected from epididymis. Especially, hypothetical influence of lead on sperm DNA integrity was also evaluated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, alkaline comet assay, and sperm chromatin structure assay. Lead exposure possibly exerted no effect on growth of mice because these animals acquired similar body weight gain during the experimental period. However, high lead concentrations (0.5% and 1%) in drinking water affected sperm motility and increased percentage of spermatozoa with abnormal morphology. In groups treated with 0.25%, 0.5%, and 1% lead acetate, percentages of sperm cells showing DNA breaks and chromatin structure damage significantly increased. Altogether, lead exposure not only exhibits adverse effects on sperm physiological parameters, but also impairs DNA structure and integrity. These effects may lead to significant decline in male fertility.

Cognitive-enhancing effect of polysaccharides from Flammulina velutipes on Alzheimer's disease by compatibilizing with ginsenosides

This study was performed to evaluate the cognitive-enhancing effect Flammulina velutipes polysaccharides (FVP) by compatibilizing with ginsenosides on D-galactose-induced Alzheimer's disease (AD) rats. Results of the Morris water maze test suggested that the cognitive ability of AD rats was significantly elevated after combined treatment with FVP and ginsenosides. Moreover, the anti-oxidant effect was also elevated as reflected by the increased levels of superoxide dismutase, catalase, and glutathione peroxidase and decreased levels of malondialdehyde in the combination group. Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling (TUNEL), immunohistochemistry, and Western blot analysis showed that the TUNEL apoptosis cell rate, Bax, cytochrome C, caspase-3, caspase-9, and apoptosis-inducing factor expression levels were obviously decreased, and the Bcl-2 expression levels were obviously increased in the hippocampi of rats treated with FVP and ginsenosides. The results suggested that the beneficial effect of FVP against AD can be significantly elevated by compatibilizing with ginsenosides. These findings provide scientific evidence supporting the use of FVP as a safe and effective drug to prevent and treat AD.

Impact of Nicotine Exposure on Hair Cell Toxicity and Embryotoxicity During Zebrafish Development.

Objectives Nicotine has various adverse effects including negative impacts associated with maternal exposure. In the current study, we examined nicotine-induced damage of hair cells and embryotoxicity during zebrafish development.Methods Zebrafish embryos were exposed to nicotine at several concentrations (5, 10, 20, and 40 μM) and embryotoxicity were evaluated at 72 hours, including hatching rate, mortality, teratogenicity rate, and heart rate. Hair cells within the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) neuromasts were identified at 120 hours. Apoptosis and mitochondrial damage of hair cells were analyzed using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) and DASPEI (2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide) assays, respectively, and changes of ultrastructure were observed by scanning electron microscopy.Results The control group without nicotine appeared normal with overall mortality and teratogenicity rate <5%. The hatching rate and mortality rate was not significantly different according to nicotine concentration (n=400 each). The abnormal morphology rate (n=400) increased and heart rate (n=150) decreased with increasing nicotine concentration (P<0.05). Nicotine-induced hair cell damage significantly increased as nicotine concentration increased. A significantly greater number of TUNEL-positive cells (P<0.01) and markedly smaller DASPEI area (P<0.01) were shown as nicotine concentration increased.Conclusion The current results suggest that nicotine induces dose-dependent hair cell toxicity in embryos by promoting apoptosis and mitochondrial and structural damage.

Combination of mild therapeutic hypothermia and adipose-derived stem cells for ischemic brain injury.

Mild therapeutic hypothermia has been shown to mitigate cerebral ischemia, reduce cerebral edema, and improve the prognosis of patients with cerebral ischemia. Adipose-derived stem cell-based therapy can decrease neuronal death and infiltration of inflammatory cells, exerting a neuroprotective effect. We hypothesized that the combination of mild therapeutic hypothermia and adipose-derived stem cells would be neuroprotective for treatment of stroke. A rat model of transient middle cerebral artery occlusion was established using the nylon monofilament method. Mild therapeutic hypothermia (33°C) was induced after 2 hours of ischemia. Adipose-derived stem cells were administered through the femoral vein during reperfusion. The severity of neurological dysfunction was measured by a modified Neurological Severity Score Scaling System. The area of the infarct lesion was determined by 2,3,5-triphenyltetrazolium chloride staining. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The regeneration of microvessels and changes in the glial scar were detected by immunofluorescence staining. The inflammatory responses after ischemic brain injury were evaluated by in situ staining using markers of inflammatory cells. The expression of inflammatory cytokines was measured by reverse transcription-polymerase chain reaction. Compared with mild therapeutic hypothermia or adipose-derived stem cell treatment alone, their combination substantially improved neurological deficits and decreased infarct size. They synergistically reduced the number of TUNEL-positive cells and glial fibrillary acidic protein expression, increased vascular endothelial growth factor levels, effectively reduced inflammatory cell infiltration and down-regulated the mRNA expression of the proinflammatory cytokines interleukin-1β, tumor necrosis factor-α and interleukin-6. Our findings indicate that combined treatment is a better approach for treating stroke compared with mild therapeutic hypothermia or adipose-derived stem cells alone.

MicroRNA-27a Suppresses Detrusor Fibrosis in Streptozotocin-Induced Diabetic Rats by Targeting PRKAA2 Through the TGF-β1/Smad3 Signaling Pathway

We examined the effects of microRNA-27a (miR-27a) on detrusor fibrosis in streptozotocin (STZ)-induced diabetic rats. Eighty healthy Sprague-Dawley (SD) rats were randomly allocated into control, diabetic, miR-27a mimics, mimics control, miR-27a inhibitors, inhibitors control, siRNA-PRKAA2 (siPRKAA2) and inhibitors + siPRKAA2 groups (the latter 7 groups were established as STZ-induced diabetic rat models and treated in different manners). Detrusor cell apoptosis in bladder tissues was determined through terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining. Detrusor cells were assigned to the blank, miR-27a mimics, mimics control, miR-27a inhibitors, inhibitors control, siPRKAA2 and inhibitors + siPRKAA2 groups. Flow cytometry determined the cell cycle stage and apoptosis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting (WB) were used to assess the expression of miR-27a, PRKAA2, TGF-β1, Smad3, p-Smad3, fibronectin (FN), connective tissue growth (CTGF), and collagen-I (COL-I) in tissues and cells. Compared with the control group, the diabetic, miR-27a mimics, and siPRKAA2 groups showed reduced weight and PRKAA2 expression, but elevated blood glucose, serum creatinine (sCr), blood urea nitrogen (BUN), cell apoptosis, and expression of TGF-β1, Smad3, FN, COL-I, CTGF, and p-Smad3. The opposite trend was observed in the miR-27a inhibitors group. PRKAA2 is a target gene of miR-27a. Compared to the blank group, the miR-27a mimics and siPRKAA2 groups indicated markedly increased TGF-β1, Smad3, FN, COL-I, CTGF and p-Smad3 expression; decreased PRKAA2 expression; and increased cell apoptosis. The miR-27a inhibitors group showed the opposite trend. These results indicate that miR-27a may contribute to detrusor fibrosis in STZ-induced diabetic rats by targeting PRKAA2 via the TGF-β1/Smad3 signaling pathway.