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Sigma 1 receptor (S1R) is expressed in retinal ganglion cells (RGCs) and astrocytes, and its activation is neuroprotective. We evaluated the contribution of S1R within optic nerve head astrocytes (ONHAs) to growth and survival of RGCs in vitro.Wild-type (WT) RGCs and WT or S1R knockout (S1R KO) ONHAs were cocultured for 2, 4, or 7 days. Total and maximal neurite length, neurite root, and extremity counts were measured. Cell death was measured using a TUNEL assay. Signal transducer and activator of transcription 3 phosphorylation levels were evaluated in ONHA-derived lysates by immunoblotting.The coculture of WT RGCs with WT or S1R KO ONHAs increased the total and maximal neurite length. Neurite root and extremity counts increased at 4 and 7 days when WT RGCs were cocultured with WT or S1R KO ONHAs. At all timepoints, the total and maximal neurite length decreased for WT RGCs in coculture with S1R KO ONHAs compared with WT ONHAs. Root and extremity counts decreased for WT RGCs in coculture with S1R KO ONHAs compared with WT ONHAs at 2 and 7, but not 4 days. RGC apoptosis increased in S1R KO ONHA coculture and S1R KO-conditioned medium, compared with WT ONHA coculture or WT-conditioned medium. S1R KO ONHA-derived lysates showed decreased phosphorylated signal transducer and activator of transcription 3 levels compared with WT ONHA-derived lysates.The absence of S1R within ONHAs has a deleterious effect on RGC neurite growth and RGC survival, reflected in analysis of WT RGC + S1R KO ONHA indirect cocultures. The data suggest that S1R may enhance ganglion cell survival via glia-mediated mechanisms.

Retinal pigment epithelium (RPE) cell apoptosis arising from all-trans-retinal (atRAL) is in close contact with the etiology of dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its underlying mechanisms remain elusive. In this study, we reported that c-Jun N-terminal kinase (JNK) activation facilitated atRAL-induced apoptosis of RPE cells. Reactive oxygen species production and endoplasmic reticulum stress were identified as two of major upstream events responsible for activating JNK signaling in atRAL-loaded RPE cells. Inhibiting JNK signaling rescued RPE cells from apoptosis induced by atRAL through attenuating caspase-3 activation leading to poly-ADP-ribose polymerase (PARP) cleavage, and DNA damage response. Abca4

We question whether, in men with an abnormal rate of sperm DNA fragmentation, the magnetic-activated cell sorting (MACS) could select spermatozoa with lower rates of DNA fragmentation as well as spermatozoa with unbalanced chromosome content. Cryopreserved spermatozoa from six males were separated into nonapoptotic and apoptotic populations. We determined the percentages of spermatozoa with (i) externalization of phosphatidylserine (EPS) by annexin V-Fluorescein isothiocyanate (FITC) labeling, (ii) DNA fragmentation by TdT-mediated-dUTP nick-end labeling (TUNEL), and (iii) numerical abnormalities for chromosomes X, Y, 13, 18, and 21 by fluorescence in situ hybridization (FISH), on the whole ejaculate and selected spermatozoa in the same patient. Compared to the nonapoptotic fraction, the apoptotic fraction statistically showed a higher number of spermatozoa with EPS, with DNA fragmentation, and with numerical chromosomal abnormalities. Compared to the whole ejaculate, we found a significant decrease in the percentage of spermatozoa with EPS and decrease tendencies of the DNA fragmentation rate and the sum of disomy levels in the nonapoptotic fraction. Conversely, we observed statistically significant higher rates of these three parameters in the apoptotic fraction. MACS may help to select spermatozoa with lower rates of DNA fragmentation and unbalanced chromosome content in men with abnormal rates of sperm DNA fragmentation.

During inflammatory processes of the central nervous system, helper T cells have the capacity to cross the blood-brain barrier and injure or kill neural cells through cytotoxic mechanisms. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is part of the astrocyte cytoskeleton that can become fragmented in neuroinflammatory conditions. The mechanism of action by which helper T cells with cytotoxic properties injure astrocytes is not completely understood. Primary human astrocytes were obtained from fetal brain tissue. Human helper (CD4+ ) T cells were isolated from peripheral blood mononuclear cells and activated with the superantigen staphylococcal enterotoxin E (SEE). Granzyme B was detected by enzyme linked immunosorbent assay and intracellular flow cytometry. GFAP fragmentation was monitored by western blotting. Cell death was monitored by lactic acid dehydrogenase release and terminal biotin-dUTP nick labeling (TUNEL). Astrocyte migration was monitored by scratch assay. Adult human oligodendrocytes were cultured with sublethally injured astrocytes to determine support function. Helper T cells activated with SEE expressed granzyme B but not perforin. Helper T cells released granzyme B upon contact with astrocytes and caused GFAP fragmentation in a caspase-dependent, MHCII-independent manner. Sublethally injured astrocytes were not apoptotic; however, their processes were thin and elongated, their migration was attenuated, and their ability to support oligodendrocytes was reduced in vitro. Helper T cells can release granzyme B causing sublethal injury to astrocytes, which compromises the supportive functions of astrocytes. Blocking these pathways may lead to improved resolution of neuroinflammatory lesions.

Embryonic development is a critical period wherein brain neurons are generated and organized. Maternal dietary folate, a cofactor in one-carbon metabolism, modulates neurogenesis and apoptosis in foetal brain neurons. We hypothesized that aberrant neuronal apoptosis may affect the development of the central nervous system during maternal folic acid deficiency, with evident effects because maternal folic acid deficiency modulates the microRNA-34a associated with Bcl-2 pathway during embryonic development. Four-week-old female Sprague-Dawley rats were divided randomly into two groups (10 rats per group): a folate-deficient diet group and a folate-normal diet group. The diets were administered to the rats 60 d before mating, which was continued for the pregnant dams until parturition. Maternal folic acid deficiency increased neuronal apoptosis in the hippocampus and the cortex in the offspring. Furthermore, maternal folic acid deficiency increased the ratio of cleaved caspase-3/caspase-3, followed by an increase in caspase-3 activity. Moreover, maternal folic acid deficiency downregulated Bcl-2 and upregulated Bax, and this effect associate with maternal folic acid deficient increases expression of microRNA-34a. Together, the present results indicate that maternal folic acid deficiency stimulates neuronal apoptosis via microRNA-34a associated with Bcl-2 signalling in rat offspring.

To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. Here we show that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. We show that Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells., Organ regeneration by stem cells is required to compensate for tissue damage during aging, although how stem cells are maintained in adulthood is poorly understood. Here, the authors show in Drosophila that Shavenbaby interacts with Yorkie, a mediator of Hippo signalling, to ensure adult stem cell survival.

Background The microbiome of the gastrointestinal tract is a vast collection of microorganisms implicated in numerous aspects of normal physiology and disease pathogenesis. The use of gnotobiotic mouse models, with single or specific communities of microbes comprising the microbiome, can enhance our understanding of the microbiome-host relationship. We hypothesized that gnotobiotic mice would exhibit differences in mucosal homeostasis when compared with mice with conventional flora (CF). Materials and methods Single-organism gnotobiotic mice were generated containing Escherichia coli MG1655, Akkermansia muciniphila, Bacteroides eggerthii, and Clostridium symbiosum, representing four of the major phyla present in the gastrointestinal tract. Distal ileal segments were harvested from adult mice, and histologic sections were H&E stained and used to measure villus height and crypt depth. Immunohistochemistry was performed with Ki67 and TUNEL as markers of proliferation and apoptosis, respectively. Results When compared to the ileum from CF mice, the ileum from all groups of gnotobiotic mice had significant increases in nearly all measured parameters. In addition, significant differences were seen among certain gnotobiotic groups for villus height, crypt depth, and apoptosis. Conclusions Single-organism gnotobiotic mice demonstrate enhanced morphometric parameters compared with mice with CF and show differences in growth patterns among bacterial species. These findings suggest unique interactions between individual bacteria and the host animal which hold potential for future therapeutic strategies aimed at mucosal restoration. The mechanisms involved in this process therefore warrant further study.

Nuclear co-localization labels are critical to ocular research. Among these, the TUNEL assay has been established as a gold standard of cell death and apoptosis. While several validated computer-based methods exist to quantitate these markers, including ImageJ Retina Analysis (RA) Toolkit and ImagePro, none verify the count with the nuclear counter stain to confirm nuclear co-localization. We established a new ImageJ-based automated multichannel thresholding (MCT) method to quantitate nuclear co-localized labeling. The MCT method was validated by comparing it with the two published TUNEL analysis in TUNEL-positive photoreceptors in an experimental retinal detachment (RD) model. RDs were induced in murine eyes and cross-sectional images of TUNEL and DAPI counter stain were obtained. Images were classified as “typical” or high density ”hotspot” TUNEL regions (n = 10/group). Images were analyzed and compared between the MCT method and the published techniques including both ”standard” and ”high” settings of the RA Toolkit for detecting lower or higher TUNEL densities, respectively. Additional testing of the MCT method with built-in ImageJ thresholding algorithms was performed to produce fully automated measurements. All images were compared with Bland-Altman mean difference plots to assess the difference in counts and linear regression plots to assess correlation. Comparison between the MCT method and the ImagePro method were found to be well correlated (typical: R(2) = 0.8972, hotspot: R(2) = 0.9000) with minor to non-significant differences. The RA Toolkit settings were found to be mostly well correlated as well (standard/typical: R(2) = 0.8036, standard/hotspot: R(2) = 0.4309, high/typical: R(2) = 0.7895, high/hotspot: R(2) = 0.8738) but were often found to have significantly higher counts than the MCT. In conclusion, the MCT method compared favorably with validated computer-based methods of nuclear marker immunofluorescence quantitation and avoids staining artifacts through the incorporation of the nuclear counter stain to confirm positive cells.

Objective: To investigate the protective activity of tanshinone in a rat model of myocardial ischemia-reperfusion and determine its effect on the expression of Bcl-2 and Bax in cardiomyocytes. Methods: We established a rat model of myocardial ischemia-reperfusion. Rats were randomly divided into blank (no surgery); saline; and low-dose (2 mg/ml), medium-dose (15 mg/ml), and high-dose (50 mg/ml) tanshinone groups. We measured heart rate and troponin (cTnI) levels, performed TUNEL to detect cardiomyocyte apoptosis, and detected LDH and CK-MB activities in serum by ELISA. We performed RT-qPCR and western blot to detect the expression of Bcl-2 and Bax mRNA and protein in cardiomyocytes. Results: Rats treated with tanshinone experienced more stable heart rate after ischemia-reperfusion compared with those in the saline control group. cTnI decreased after ischemia-reperfusion in mice injected with tanshinone, while cTnI in saline-treated mice increased significantly compared with that in the blank control group. TUNEL staining showed that there were greater apoptotic cardiomyocytes in the saline group, but the tanshinone groups showed fewer apoptotic cardiomyocytes. LDH and CK-MB activities were significantly increased after reperfusion in the saline group (p0.05). The expression levels of Bcl-2 mRNA and protein in cardiomyocytes of rats were higher in the three tanshinone groups in a dose-sensitive manner than those in the blank and saline groups (p

In recent years, the detrimental effects of high iodine on intelligence are gaining tons of attention, but the relationship between high iodine and neurotoxicity is controversial. This study aimed to explore whether high iodine intake may impair intelligence and the roles of apoptosis and autophagy in high iodine-induced neurotoxicity. The results showed that high iodine exposure reduced brain coefficient and intelligence of rats, and caused histopathological abnormalities in hippocampus. Moreover, high iodine increased hippocampal apoptosis, as confirmed by elevation of apoptotic proteins and TUNEL-positive incidence. Further study showed that high iodine impaired mitochondrial ultrastructure and caused elevation of Bax, cytochrome c and decline of Bcl2, indicating the participation of mitochondrial apoptotic pathway. Simultaneously, high iodine also increased the number of autophagosomes. Intriguingly, the expression of autophagosomes formation protein Atg7, Beclin1 and autophagic substrate p62 were elevated, suggesting that the accumulated autophagosomes is not only due to the enhancement of formation but also the decline of clearance. These, together with the numerous damaged organelles observed in hippocampal ultrastructure, reveal the crucial role of disordered autophagy flux in high iodine-elicited neurotoxicity. Collectively, these findings suggest that excessive apoptosis and disordered autophagy flux contribute to high iodine-elicited neurotoxicity.

Aims The bioactivities of Santolina corsica Jord. & Fourr. n-hexane (EHS) and methanol (EMS) extracts were evaluated in relation to their chemical profile. Main methods EHS and EMS were analysed by gas chromatography-mass spectrometry () and high performance liquid chromatography-diode array detection (HPLC-DAD), respectively. Antioxidant activity was determined by β-carotene bleaching, Ferric Reducing Activity Power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) tests. Nitric oxide (NO) production was assessed in LPS-stimulated RAW 264.7 cells. Anti-proliferative activity was evaluated by MTT assay on A549, HeLa, PC3, MCF-7, MDA-MB-231 cancer cells, and non-tumorigenic MCF10 A cells. Cell motility, migration and invasion were assessed by wound-healing scratch, migration and invasion assays, respectively. DNA fragmentation was tested by TUNEL assay. Cells morphology was studied by phase-contrast microscopy. Procaspase-8, -9, poly (ADP-ribose) polymerase and COX-2 expression levels were evaluated by immunoblotting analysis. Key findings Kaempferol-3-O-glucoside (5878.67 mg/100 g of extract), chlorogenic acid (746.11 mg/100 g), and rosmarinic acid (550.16 mg/100 g) were the dominant EMS constituents. EHS showed myrcene (18.86%) as the main compound, followed by palmitic acid methyl and ethyl esters (9.35 and 9.16%, respectively), β-phellandrene (8.48%), and ar-curcumene (5.63%). Both extracts showed promising anti-proliferative activity on all tested cancer cells, without inducing cytotoxicity in non-tumorigenic cells MCF-10 A. Moreover, extracts inhibited motility, migration, and invasion of MDA-MB-231 cells, inducing apoptosis. EHS decreased NO production, showing anti-inflammatory activity. Significance S. corsica extracts might be potentially useful in cancer treatment, since reduce invasive and migratory potential of MDA-MB-231 cells triggering apoptosis.

Inflammatory bowel disease (IBD), characterized by sustained inflammation, is a latent risk factor of colon tumorigenesis. Silibinin has been reported to be anti-inflammatory and antineoplastic, but its efficacy on colitis-associated cancer (CAC) has not been reported. Interlukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) is the key signaling pathway involved in CAC. We evaluated the chemopreventive effect of silibinin on a CAC mouse model and determined its impact on IL-6/STAT3 signaling. Intestinal tumor cells (IMCE and HCT-116 cell lines) were also treated by graded concentration of silibinin, and cellular viability was determined. Silibinin (750 mg/kg/day) was administered to an azoxymethane/dextran sulfate sodium (AOM/DSS) C57BL/6 mouse model for 10 weeks by gavage. Body weight, colon length, and the amount and diameter of colon tumors were documented, respectively. Specimens were subjected to H&E staining for colitis and tumor scoring, immunohistochemical staining and terminal deoxynucleotidyl transferase dUTP nick end labeling for proliferation assessment, and immunofluorescent staining for intestinal mucosa barrier assessment. Production of inflammatory cytokines was determined by real-time PCR. IL-6/STAT3 pathway activation was evaluated through immunohistochemical staining and western blot. In the current study, silibinin significantly inhibited the viability of intestinal tumor cells. The production of inflammatory cytokines and the phosphorylation of STAT3 were both inhibited in intestinal tumor cells. Meanwhile, silibinin decreased the amount and size of tumors in AOM/DSS mice. Colitis and tumor scores were decreased accompanying with inhibition of colonic tumor cell proliferation and promotion of cellular apoptosis. Additionally, silibinin could reduce the production of inflammatory cytokines and attenuate the impairment of colonic mucosal barrier. Furthermore, STAT3 phosphorylation was significantly suppressed by silibinin. In conclusion, silibinin could protect against colitis-associated tumorigenesis in mice via inhibiting IL-6/STAT3, which showed promising chemopreventive potential of CAC.

The ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of many neurodegenerative diseases. Endoplasmic reticulum (ER) stress is shown to play a pathological role in the development of diabetes and its complications. Hence, the current study is aimed to investigate the role of UPS and ER stress in the cerebral cortex of diabetic rats and examine the therapeutic effect of 4-phenylbutyric acid (4-PBA), an ER stress inhibitor. Male Sprague-Dawley rats were divided into three groups: control, diabetes, and diabetes plus 4-PBA treatment group. Diabetes was induced by single intraperitoneal streptozotocin injection (37 mg/kg body weight [bw]), and 4-PBA was administered (40 mg/kg bw/d, intraperitoneal) for 2 months, starting from 2 months of diabetes induction. At the end of 4 months, cerebral cortex was collected for analysis. Declined proteasome activity and ubiquitin C-terminal hydrolase (UCH)-L1 expression, increased ubiquitinated proteins, and apoptosis were observed in the diabetic rats. The expression of the ubiquitin-activating enzyme E1, UCHL5, and ER stress markers (ATF6, pPERK, and CHOP) was markedly elevated, whereas the expression of ER-associated protein degradation (ERAD) components was downregulated in the diabetic rats. 4-PBA intervention attenuated ER stress, alterations in UPS, and ERAD components in diabetic rats. Importantly, neuronal apoptosis was lowered in 4-PBA-treated diabetic rats. Our observations demonstrate that altered UPS could be one of the underlying mechanisms of neuronal apoptosis in diabetes and chemical chaperones such as 4-PBA could be potential candidates for preventing these alterations under hyperglycemic conditions.

Survivin, a member of the inhibitors of apoptosis protein gene family, inhibits the activity of caspase, leading to a halt of the apoptotic process. Our study focused on the neuroprotective effect of survivin after transient middle cerebral artery occlusion (MCAO) with intraparenchymal administration of an adeno-associated virus (AAV) vector. His-tagged survivin was cloned and packaged into the AAV-rh10 vector. Four-week-old Sprague-Dawley rats were injected with 4 × 109 vg of AAV-GFP or AAV-His-survivin into the right striatum and were treated 3 weeks later with transient MCAO for 90 min. Twenty-four hours after MCAO, functional and histological analyses of the rats were performed. The result showed that rats that had been treated with AAV-green fluorescent protein (GFP) and those that had been treated with AAV-His-survivin did not show a significant difference in neurological scores 24 hr after MCAO, however, infarction volume was significantly reduced in the AAV-His-survivin group compared to that in the AAV-GFP group. Although the neutrophil marker myeloperoxidase did not show a significant difference in the ischemic boundary zone, cells positive for active caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling were significantly decreased in the AAV-His-survivin group. In conclusion, survivin overexpression in the ischemic boundary zone induced by using an AAV vector has the potential for amelioration of ischemic damage via an antiapoptotic mechanism.

This study aimed to investigate the expression of miRNA-21 during intestinal barrier dysfunction induced by intestinal ischemia reperfusion. Forty SPF SD rats were divided into 5 groups randomly. Intestinal ischemia-reperfusion injury (IRI) was induced by mesenteric artery occlusion for 1 h and reperfusion for 1 h, and the rats were sacrificed at 1, 3, 6 and 12 h after reperfusion. Fresh intestine tissues were immediately isolated for the measurement of transepithelial electrical resistance (TER). The levels of cytokines, ICAM-1, DAO, iFABP and MPO in serum were determined by ELISA. Intestinal tight junction proteins occludin and claudin-1 were detected by immunofluorescence analysis and Western blot analysis. miR-21 expression in intestinal tissues was measured by RT-PCR. Compared with sham group, the levels of pro-inflammatory cytokines TNF-α and IL-6 and ICAM-1, DAO, iFABP and MPO increased while IL-10 level decreased in intestinal ischemia-reperfusion group. In addition, the levels of intestinal tight junction proteins occludin and claudin-1 decreased while miR-21 level increased in intestinal ischemia-reperfusion group, compared with sham group. In conclusion, miR-21 expression is upregulated during intestinal barrier dysfunction induced by IRI. miR-21 may play an important role in the regulation of intestinal barrier function. Keywords: Intestinal epithelial barrier, MicroRNA-21, Ischemia-reperfusion injury, Tight junction

Chemo-PTT, which combines chemotherapy with photothermal therapy, offers a viable approach for the complete tumor eradication but would likely fail in drug-resistant situations if conventional chemotherapeutic agents are used. Here we show that a type of copper (Cu)-palladium (Pd) alloy tetrapod nanoparticles (TNP-1) presents an ideal solution to the chemo-PTT challenges. TNP-1 exhibit superior near-infrared photothermal conversion efficiency, thanks to their special sharp-tip structure, and induce pro-survival autophagy in a shape- and composition-dependent manner. Inhibition of autophagy with 3-methyl adenine or chloroquine has a remarkable synergistic effect on TNP-1-mediated PTT in triple-negative (4T1), drug-resistant (MCF7/MDR) and patient-derived breast cancer models, achieving a level of efficacy unattainable with TNP-2, the identically-shaped CuPd nanoparticles that have a higher photothermal conversion efficiency but no autophagy-inducing activity. Our results provide a proof-of-concept for a chemo-PTT strategy, which utilizes autophagy inhibitors instead of traditional chemotherapeutic agents and is particularly useful for eradicating drug-resistant cancer., “Conventional chemotherapy-photothermal therapy combination has limited efficacy in drug resistant cancers. Here they develop Copper-palladium tetrapod nanoparticles to overcome these challenges and show them to work in synergy with autophagy inhibitors to treat drug resistant cancers”

Leiomyomas (myomas) are the most common benign smooth muscle cell tumor of the myometrium. Resveratrol, a stilbene, has been used as an anti-inflammatory and antitumor agent. In the current study, we investigated the inhibitory effect of resveratrol on the proliferation of primary human myoma cell cultures. Resveratrol arrested cell proliferation via integrin αvβ3. It also inhibited integrin αvβ3 expression and protein accumulation. Concurrently, constitutive AKT phosphorylation in myoma cells was inhibited by resveratrol. Expressions of proapoptotic genes, such as cyclooxygenase (COX)-2, p21 and CDKN2, were induced by resveratrol in myoma cells. On the other hand, expressions of proliferative (anti-apoptotic) genes were either inhibited, as in BCL2, or unchanged, as in cyclin D1 and proliferating cell nuclear antigen (PCNA). The accumulation of insulin-like growth factor (IGF)-1 receptor (IGF-1R) was inhibited by resveratrol in primary myoma cells. IGF-1-induced cell proliferation was inhibited by co-incubation with resveratrol. Therefore, growth modulation of myoma cells occurs via mechanisms dependent on cross-talk between integrin αvβ3 and IGF-1R. Our findings suggest that resveratrol can be considered an alternative therapeutic agent for myomas.

Background Although studies have reported an increased risk for mood disorders in Hashimoto’s thyroiditis (HT) patients even in the euthyroid state, the mechanisms involved remain unclear. Neuroinflammation may play a key role in the etiology of mood disorders in humans and behavioral disturbances in rodents. Therefore, this study established a euthyroid HT model in mice and investigated whether HT itself was capable of triggering neuroinflammation accompanied by emotional alterations. Methods Experimental HT was induced by immunizing NOD mice with thyroglobulin and adjuvant twice. Four weeks after the last challenge, mice were tested for anxiety-like behavior in the open field and elevated plus maze tests and depression-like behavior in the forced swimming and tail suspension tests. Then, animals were sacrificed for thyroid-related parameter measure as well as detection of cellular and molecular events associated with neuroinflammation. The changes in components of central serotonin signaling were also investigated. Results HT mice showed intrathyroidal monocyte infiltration and rising serum thyroid autoantibody levels accompanied by normal thyroid function, which defines euthyroid HT in humans. These mice displayed more anxiety- and depressive-like behaviors than controls. HT mice further showed microglia and astrocyte activation in the frontal cortex detected by immunohistochemistry, real-time RT-PCR, and transmission electron microscopy (TEM). These observations were also accompanied by enhanced gene expression of proinflammatory cytokines IL-1β and TNF-α in the frontal cortex. Despite this inflammatory response, no signs of neuronal apoptosis were visible by the TUNEL staining and TEM in the frontal cortex of HT mice. Additionally, IDO1 and SERT, key serotonin-system-related genes activated by proinflammatory cytokines, were upregulated in HT mice, accompanied by reduced frontal cortex serotonin levels. Conclusions Our results are the first to suggest that HT induces neuroinflammation and alters related serotonin signaling in the euthyroid state, which may underlie the deleterious effects of HT itself on emotional function.

Subarachnoid hemorrhage (SAH) is a neurologically destructive stroke in which early brain injury (EBI) plays a pivotal role in poor patient outcomes. Expanding upon our previous work, multiple techniques and methods were used in this preclinical study to further elucidate the mechanisms underlying the beneficial effects of apolipoprotein E (ApoE) against EBI after SAH in murine apolipoprotein E gene-knockout mice (Apoe−/−, KO) and wild-type mice (WT) on a C57BL/6J background. We reported that Apoe deficiency resulted in a more extensive EBI at 48 h after SAH in mice demonstrated by MRI scanning and immunohistochemical staining and exhibited more extensive white matter injury and neuronal apoptosis than WT mice. These changes were associated with an increase in NADPH oxidase 2 (NOX2) expression, an important regulator of both oxidative stress and inflammatory cytokines. Furthermore, immunohistochemical analysis revealed that NOX2 was abundantly expressed in activated M1 microglia. The JAK2/STAT3 signaling pathway, an upstream regulator of NOX2, was increased in WT mice and activated to an even greater extent in Apoe−/− mice; whereas, the JAK2-specific inhibitor, AG490, reduced NOX2 expression, oxidative stress, and inflammation in Apoe-deficient mice. Also, apoE-mimetic peptide COG1410 suppressed the JAK2/STAT3 signaling pathway and significantly reduced M1 microglia activation with subsequent attenuation of oxidative stress and inflammation after SAH. Taken together, apoE and apoE-mimetic peptide have whole-brain protective effects that may reduce EBI after SAH via M1 microglial quiescence through the attenuation of the JAK2/STAT3/NOX2 signaling pathway axis.

The present study was conducted to demonstrate cytotoxicity, apoptosis and hepatic damage induced by gemcitabine in laboratory mice. Animals were treated with a single dose of gemcitabine (415 mg/kg body wt), equivalent to a human therapeutic dose, and sacrificed after 1, 2 and 3 weeks. A significant decrease in mean body weight and absolute liver weight was registered. The levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased as a result of this induced stress. Various structural changes were observed in the liver tissue of treated mice, as evident in the histological sections. Specifically, gemcitabine exposure was able to induce apoptosis in liver cells, and the incidence of TUNEL positive liver cells was increased compared to the control group. DNA fragmentation appeared on agarose gel and flow cytometry analysis confirmed the induction of apoptosis. These findings in gemcitabine-treated animal tissues suggest that inhibition or disruption of cells' DNA synthesis may be the mechanism by which this drug induces toxicity in the animal body.

Cisplatin is the first-line chemotherapeutic agent, but its systemic toxicity and side effects severely limit its clinical use. We report a microneedle technique to mediate the transdermal delivery of lipid-coated cisplatin nanoparticles (LCC-NPs) for efficient and safe cancer therapy. Cisplatin was encapsulated by tumor-targeting pH-responsive lipid nanoparticles with a high loading rate of 80%, and the encapsulation substantially increased the solubility of cisplatin and enhanced its antitumor efficiency in vitro. The LCC-NPs were embedded in dissolvable microneedles, and released from the microneedles after inserting into the skin. This enabled the nanoparticles to pass the stratum corneum for safe local delivery. An in vivo study with a xenograft tumor animal model demonstrated that microneedle arrays loaded with cisplatin nanoparticles significantly increased cytotoxicity and apoptosis in cancer cells with an apoptotic index of 58.6%, resulting in significantly reduced tumor volume and weight. Moreover, serum platinum, pulmonary toxicity, hepatotoxicity, and nephrotoxicity were not detected in vivo, indicating that this technique is biosafe. The cisplatin-nanoparticle microneedle system developed in this study may offer promising opportunities in cancer therapy for enhancing antitumor effects and reducing systemic toxicity and side effects.

Signaling abnormalities play important roles during podocyte injury and have been indicated as crucial events for triggering many glomerular diseases. There is emerging evidence demonstrating significant improvements in preventing renal injury and restoring podocytes after islet transplantation. However, whether signaling abnormalities affect the therapeutic efficacy of islet transplantation remain unclear. This study was established to investigate the impact of Notch-1 signaling activation on renal injury and podocyte restoration after islet transplantation. Experiments were performed in vivo and in vitro under conditions of diabetic nephropathy and high-glucose medium, respectively. Podocyte injury in vitro was induced by high-glucose concentration, and expression levels of genes associated with the Notch-1 pathway were also regulated by Jagged-1/FC and N-[N-(3,5-Difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT). Podocytes were co-cultured with islets to investigate the protective effect of islets in high-glucose conditions. Histopathological staining and transmission electron microscopy were performed to assess pathological changes in podocytes in glomeruli. The results from this study showed that Notch-1 signaling in podocytes was significantly decreased by functional islet cells in vivo and in vitro. Compared with the co-cultured group and transplanted group, highly activated Notch-1 signaling significantly moderated the effect of islets in affecting podocyte restoration and renal injury. Renal damage and podocyte injury were alleviated after DAPT treatment. Furthermore, the balance between apoptosis and autophagy was diverse under different treatments. All the data in this study showed that highly activated Notch-1 signaling could affect the therapeutic efficacy of islet transplantation on renal injury and podocyte restoration in high-glucose conditions. The balance between apoptosis and autophagy was also closely associated with the degree of podocyte restoration. This finding may suggest that the in vivo microenvironment plays a critical role in podocyte restoration after islet transplantation, which provides a promising and individual assessment and targeting treatment for different diabetic nephropathy patients after islet transplantation into the future.

Tumor growth is modulated by crosstalk between cancer cells and the tumor microenvironment. Recent advances have shown that miRNA dysfunction in tumor cells can modulate the tumor microenvironment to indirectly determine their progression. However, this process is poorly understood in testicular germ cell tumors (TGCTs). We reported here that miR-125b was repressed in TGCT samples by epigenetic modifications rather than genetic alternations. Furthermore, miR-125b overexpression significantly alleviated the tumor growth in two NCCIT human embryonic carcinoma xenograft models in vivo, whereas miR-125b did not stimulate autonomous tumor cell growth in vitro. Notably, forced expression of miR-125b in NCCIT embryonic carcinoma cells decreased the abundance of host tumor-associated macrophages (TAMs) within tumor microenvironment. Selective deletion of host macrophages by clodronate abolished the anti-tumoral ability of miR-125b in xenograft models. By RNA profiling, Western blot and luciferase reporter assay, we further observed that miR-125b directly regulated tumor cell-derived chemokine CSF1 and CX3CL1, which are known to control the recruitment of TAMs to tumor sites. Lastly, we found that one set of miRNAs, which are under the regulation of miR-125b, might convergently target CSF1/CX3CL1 in NCCIT cells using miRNA profiling. These findings uncover the anticancer effect of miR-125b via mediating tumor-stroma crosstalk in xenograft models of TGCTs and raise the possibility of targeting miR-125b as miRNA therapeutics.

The aim of this study was to explore the role of micro ribonucleic acid (miR)-133 in the apoptosis of human placental trophoblasts through the Ras homolog gene family (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway.The plasma samples were collected from 30 patients with pre-eclampsia (PE) undergoing treatment and 30 healthy subjects (control group) who received physical examination in our hospital. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was utilized to measure the expression of miR-133 in PE patients and healthy people. Meanwhile, blood pressure, urine protein content, liver function, and kidney function were detected in patients of both groups as well. Subsequently, the placental trophoblasts were extracted and transfected with inhibitors and miRNA mimics to suppress and overexpress miR-133, respectively. The transfection efficiency was determined by RT-PCR. The levels of interleukin-6 (IL-6), IL-1, and tumor necrosis factor-alpha (TNF-α) were measured in both groups. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was performed to determine the apoptosis of trophoblasts. Next, the RT-PCR and Western blotting were carried out to detect the expressions of the Rho/ROCK pathway. Furthermore, the influence of miR-133 on the apoptosis of trophoblasts in human placenta tissues through Rho/ROCK was comprehensively observed.In vivo experiments demonstrated that the urinary protein content, miR-133 level, systolic blood pressure, diastolic blood pressure, and liver function and renal function indexes were significantly elevated in pre-eclampsia (PE) patients in comparison with normal subjects (p0.05). After transfection of mimics and inhibitors, the expression of miR-133 was remarkably up- and down-regulated, respectively. The content of the inflammatory factors in miR-133 mimics group was overtly higher than the other two groups. The TUNEL staining results showed that the number of apoptotic cells significantly increased and decreased in the miR-133 mimics group and miR-133 inhibitors group, respectively. Subsequent experiments indicated that the expressions of apoptosis gene Caspase3, pathway gene, and protein ROCKI were notably up-regulated in miR-133 mimics group. However, they were evidently down-regulated in miR-133 inhibitors group than in the control group. In addition, a consistent trend was observed in the protein expression level.MiR-133 participates in the development and progression of PE through the Rho/ROCK signaling pathway, which may affect the apoptosis of trophoblasts in the placenta tissues.

The toxic RGD peptide, rLj-RGD4, characterized by 4 (Arg-Gly-Asp) (RGD) motifs, is a novel mutant of rLj-RGD3 from the salivary gland of Lampetra japonica. Our previous study showd that rLj-RGD3 exerts a protective effect against cerebral ischemia injury in rats. Through the induction of middle cerebral artery occlusion/reperfusion (MCAO) injuries in rats, the present study investigated the effects and the mechanism through which rLj-RGD4 protects against ischemic stroke. In rats, treatment with rLj-RGD4 2 h after MCAO enhanced survival rate, improved movement and ameliorated the severity of brain infarction and apoptosis by diminishing pathological changes. This study demonstrated that rLj-RGD4 can reverse the downregulation of Bcl2, and the upregulation of Caspase-3. Mechanistic studies showed that rLj-RGD4 upregulated the expression levels of FAK, p-FAK, PI3K and p-Akt. In contrast, caspase-3 expression was inhibited. These results showed that rLj-RGD4 may reduce cerebral ischemia-reperfusion injury.

Surfactant protein B (SP-B) is essential for life and plays critical roles in host defense and lowering alveolar surface tension. A single-nucleotide polymorphism (SNP rs1130866) of human SP-B (hSP-B) alters the N-linked glycosylation, thus presumably affecting SP-B function. This study has investigated the regulatory roles of hSP-B genetic variants on lung injury in pneumonia-induced sepsis.Wild-type (WT) FVB/NJ and humanized transgenic SP-B-T and SP-B-C mice (expressing either hSP-B C or T allele without mouse SP-B gene) were infected intratracheally with 50 μL (4 × 10 colony-forming units [CFUs]/mouse) Pseudomonas aeruginosa Xen5 or saline, and then killed 24 or 48 h after infection. Bacterial dynamic growths were monitored from 0 to 48 h postinfection by in vivo imaging. Histopathological, cellular, and molecular changes of lung tissues and bronchoalveolar lavage fluid (BALF) were analyzed. Surface tension of surfactants was determined with constrained drop surfactometry.SP-B-C mice showed higher bioluminescence and CFUs, increased inflammation and mortality, the higher score of lung injury, and reduced numbers of lamellar bodies in type II cells compared with SP-B-T or WT (P 0.05). Minimum surface tension increased dramatically in infected mice (P 0.01) with the order of SP-B-CSP-B-TWT. Levels of multiple cytokines in the lung of infected SP-B-C were higher than those of SP-B-T and WT (P 0.01). Furthermore, compared with SP-B-T or WT, SP-B-C exhibited lower SP-B, higher NF-κB and NLRP3 inflammasome activation, and higher activated caspase-3.hSP-B variants differentially regulate susceptibility through modulating the surface activity of surfactant, cell death, and inflammatory signaling in sepsis.

A high-fat diet (HFD) is correlated with a higher risk of metabolic syndrome. The effect of HFD on neuroinflammation and apoptosis in acute stage after traumatic brain injury (TBI) in rats is not well known. Five-week-old male Sprague-Dawley (SD) rats were fed a HFD or normal diet (ND) for 8 weeks. Anesthetized male SD rats were divided into two subgroups: sham-operated and TBI. Motor function was measured using an inclined plane. The numbers of apoptotic neurons (markers Neu-N, TUNEL, caspase-3), activated astrocytes (marker GFAP) and microglia (marker OX42), and TNF-α expression in microglia and astrocytes in the ischemic cortex were investigated using an immunofluorescence assay. All of the parameters were measured on the 3rd day after TBI. Rats fed a HFD for 8 weeks had higher body weight, serum cholesterol, and triglycerides. TBI-induced motor deficits, neuronal decrease, neuronal apoptosis, astrocyte and microglia activation, and TNF-α expression in microglia and astrocytes in the ischemia cortex were significantly increased in ND and HFD rats compared to sham rats. However, these parameters were not significantly different between the ND and HFD TBI groups, except motor deficit. HFD has no significant effects on neuronal apoptosis or neuroinflammation in acute stage compared with ND for 8 weeks after moderate TBI in experimental rats.

Oxidative stress has a causative role in ischemic reperfusion-induced cell death. Evidence has shown that metformin is capable to reduce ischemic reperfusion injuries. The current study investigated the effect of metformin on ischemia/reperfusion-induced apoptosis in PC12 cells by evaluation of Bcl-2 family proteins expression. Cells were exposed to a time-dependent in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) injury and then treated with metformin. The intracellular reactive oxygen species (ROS) levels were measured. Western blotting was used to examine the expression of anti- and pro-apoptotic proteins. Moreover, the number of apoptotic cell death was evaluated by TUNEL assay. Our results showed that metformin attenuated ROS generation, downregulated pro-apoptotic BAX expression, and upregulated expression of the Bcl-2 protein in the PC12 cells. Moreover, metformin reduced cell death under OGD/R condition which was confirmed by lower apoptotic cell death in the TUNEL assay. These findings suggest that neuroprotective effect of metformin on OGD/R-induced cell death is possibly mediated by inhibition of ROS-induced apoptosis pathway.

Summary We assessed whether nicorandil, an adenosine triphosphate–sensitive K + channel opener, reduces burn wound progression in a rat comb burn model. A total of 24 rats were used. Following thermal injury, one dose of nicorandil (10 or 30 mg/kg) was administered intragastrically twice daily for 3 days. At days 1 and 3 after injury, skin was harvested for histopathological examination and protein isolation. Rats treated with the 10-mg/kg and 30-mg/kg doses of nicorandil exhibited significantly increased tissue survival in the zone of stasis at days 1 and 3 after injury. The 10-mg/kg and 30-mg/kg nicorandil doses also significantly increased skin perfusion in the zone of stasis at days 1 and 3 after injury. At 30 mg/kg, nicorandil significantly reduced hypoxia-inducible factor-1α expression in the zone of stasis at day 1 after injury and reduced inflammatory responses in the zone of stasis. The latter effect included decreased polymorphonuclear neutrophil leukocyte infiltration and interleukin-1β release at day 1 after injury. At 30 mg/kg, nicorandil also significantly reduced expression of nuclear factor-κB p65, akey transcriptional factor in the regulation of inflammatory mediators, in the zone of stasis at day 1 after injury. Our study demonstrates that a 30 mg/kg dosing schedule of nicorandil increases tissue survival in the zone of stasis by attenuating ischemia–reperfusion injury. This effect is mediated by the enhancement of skin blood flow and reduction in the inflammatory response. Therefore, our findings suggest that nicorandil has potential clinical applications for patients with burns.

In an effort to reduce smoking-related diseases, alternative products such as e-cigarettes have been proposed. However, despite their growing popularity, the potential toxicity of e-cigarettes remains largely unknown. In this study, human gingival fibroblasts were repeatedly exposed to cigarette smoke condensate (CSC) and to nicotine-rich (NR) or nicotine-free (NF) e-vapor condensates for 60 min once a day for various time periods. They were then used to perform different analyses. Results indicate that cells exposed to CSC or NR condensates showed an altered morphology and a reduced proliferation rate, as ascertained by MTT and BrdU assays. Fibroblast cultures exposed to either CSC or e-vapor condensates also showed increased levels of TUNEL-positive apoptotic cells, compared to that recorded in the control. Furthermore, the cell scratch test revealed that repeated exposures to CSC or to e-vapor condensates delayed both fibroblast migration and wound healing. It should be noted that CSC was much more damageable to gingival fibroblasts than were the NR and NF e-vapor condensates. The representative chain of damage thus translates to CSC > NR e-vapor condensate > NF e-vapor condensate.

Inflammation is implicated in epileptogenesis. Activated microglia and macrophages (MG/MΦ) are found in the brains of patients with epilepsy-related diseases and animal models of epilepsy. It is not yet known how the MG/MΦ activation phenotype affects pathological changes in the brain after a single seizure. In this study, we had 2 main purposes: first, to characterize post-status epilepticus (SE) inflammation by tracking MG/MΦ polarization, and, second, to explore the role of an innate immune receptor adaptor protein, namely, myeloid differentiation primary response gene 88 (MyD88), in the induction of SE in a mouse model. A lithium–pilocarpine model of seizure conditions was generated in C57BL/6 mice. The intensity and distribution of MG/MΦ polarization were tracked by fluorescent immunohistochemistry and Western blotting for the polarization markers inducible nitrogen oxygenized synthase, arginase-1, CD163, and mannose receptor. We observed steadily increasing M1 MG/MΦ along with MyD88 signal upregulation after SE in the hippocampi of mice, whereas the M2 marker arginase-1 was localized mainly in astrocytes rather than in MG/MΦ. Inhibition or gene knockout of MyD88 reduced M1 MG/MΦ and gliosis although increasing M2 MG/MΦ in the hippocampi of SE mice. MyD88 inhibition also augmented glutamate transporter 1 expression and reduced N-methyl-D-aspartate receptor NR1 subunit expression in the hippocampus to protect pyramidal neurons from apoptosis. These data suggest that MG/MΦ polarization after SE impacts the pathological outcome of the hippocampus via MyD88 signaling and point to MyD88 as a potential neuroprotective target for epilepsy therapy. Electronic supplementary material The online version of this article (10.1007/s13311-018-0653-0) contains supplementary material, which is available to authorized users.

The fovea is an anatomical specialization of the central retina containing closely packed cone-photoreceptors providing an area of high acuity vision in humans and primates. Despite its key role in the clarity of vision, little is known about the molecular and cellular basis of foveal development, due to the absence of a foveal structure in commonly used laboratory animal models. Of the amniotes the retina in birds of prey and some reptiles do exhibit a typical foveal structure, but they have not been studied in the context of foveal development due to lack of availability of embryonic tissue, lack of captive breeding programs, and limited genomic information. However, the genome for the diurnal bifoveate reptile species Anolis carolinensis (green anole) was recently published and it is possible to collect embryos from this species in captivity. Here, we tested the feasibility of using the anole as a model to study foveal development. Eyes were collected at various stages of development for histological analysis, immunofluorescence, and apoptosis. We show that at embryonic stage (ES) 10 there is peak ganglion cell density at the incipient central foveal region and a single row of cone photoreceptor nuclei. At ES17 the foveal pit begins to form and at this stage there are 3-4 rows of cone nuclei. Post-hatching a further increase in cone density and lengthening of inner and outer segments is observed. A yellowish pigment was seen in the adult central foveal region, but not in the temporal fovea. At ES14 Pax6 was localized across the entire retina, but was more prominent in the ganglion cell layer (GCL) and the part of the inner nuclear layer (INL) containing amacrine cell bodies. However, at ES17 Pax6 expression in the ganglion cells of the central retina was markedly reduced. Bioinformatic analysis revealed that 86% of human candidate foveal hypoplasia genes had an orthologous gene or DNA sequence in the green anole. These findings provide the first insight into foveal morphogenesis in the green anole and suggest that it could be a very useful model for investigating the molecular signals driving foveal development, and thus inform on human foveal development and disease.

Background Subcutaneous injection of insulin can lead to problems such as hypoglycemia and edema. Objective The purpose of this research was to evaluate the effect of oral insulin-coated trimethyl chitosan nanoparticles on control of glycemic status, IGF-1 and IGF-2 levels, and apoptosis in the hippocampus of rats with diabetes mellitus. Methods Insulin-coated trimethyl chitosan nanoparticles were prepared by the polyelectrolyte complex method (PEC) method. Insulin loading content, loading efficiency, quantity and quality of particle size were evaluated. In vivo study was performed in different treatment groups of male Wistar rats with diabetes mellitus by insulin-coated trimethyl chitosan nanoparticles or subcutaneous injection of trade insulin. The duration of diabetes was eight weeks and the treatment was started after that time and continued for another two weeks. Body weight, fasting blood glucose (FBS), hippocampal apoptosis, and immunohistochemical (IHC) protein levels of IGF-1 and IGF-2 were assessed at the end of the experiments. Results The size and polydispersity indexes were 533 nanometers and 0.533, respectively. Insulin coated trimethyl chitosan nanoparticles showed high loading efficiency (97.67% ) and loading content (48.83% ). The spherical shape of nanoparticle was confirmed by transmission electron microscopic (TEM). The amine, amide, ether and aliphatic groups were evaluated using FT-IR spectrophotometer which represented the correctness of the insulin coated trimethyl chitosan nanoparticles. Although the apoptotic index was not changed either by insulin-coated nano-particles or commercial insulin, in vivo results showed the efficacy of insulin-coated nanoparticles as well as commercial insulin in compensated weight loss, FBS and protein levels of IGF-1 and IGF-2. Conclusions The present study showed the efficacy of insulin coated nanoparticle in oral route manner that can be tested in Phase I- III clinical trials. However, a behavioral study could reveal the efficacy of insulin-loaded nanoparticles in the improvement of cognitive changes through the modulation of IGF-1 and IGF-2 levels in the hippocampus.

Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic "DNA ladder" pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.

Demir, Selim/0000-0002-1863-6280 WOS: 000446534300004 PubMed: 30009691 The effect of the electromagnetic field (EMF) established when cell phones are in use on human health, and particularly the head, has been the subject of major scientific research. Phones are usually carried near the lumbar region when not in use, and the kidneys will also inevitably be affected by such fields. We investigated the effects on the kidneys of female rats exposed to a continuous 900-megahertz (MHz) EMF for 1 h daily in mid-late adolescence. Control, sham, and EMF groups were established. The EMF was applied to the application group rats daily on postnatal days 35-59. A pseudo-megahertz effect was applied to sham group rats. All animals were euthanized on postnatal day 60. Right kidney tissues were subjected to routine procedures. Malondialdehyde, total antioxidant status, and total oxidant status (TOS) were investigated in left kidneys, and the oxidative stress index (OSI) was also calculated from these. Histopathological analysis revealed no pathology in either the control or sham groups. However, findings including hemorrhage in glomerulus, vacuolization and irregularity in the proximal and distal tubular epithelium, diffuse glomerular degeneration and edema, occasional degeneration in Bowman capsules, hemorrhage in the medullary region, disturbed nucleus location and morphology, and tubular edema in the cortex were observed in the EMF groups. TOS and OSI values were lower in the EMF group (9.4316 +/- 1.0211 and 0.8461 +/- 0.0826, respectively) and the sham group (8.2171 +/- 0.6437 and 0.7358 +/- 0.0545, respectively) than in the control group (11.1522 +/- 1.3389 and 1.0085 +/- 0.1174, respectively) (p < 0.05). In conclusion, exposure to a continuous 900-MHz EMF for 1 h daily during middle and late adolescence may cause various changes in the female rat kidney at postnatal day 60.

HIV-associated neurocognitive disorders (HAND) are prevalent despite combined antiretroviral therapy, affecting nearly half of HIV-infected patients worldwide. During HIV infection of macrophages secretion of the lysosomal protein, cathepsin B, is increased. Secreted cathepsin B has been shown to induce neurotoxicity. Oxidative stress is increased in HIV-infected patients, while antioxidants are decreased in monocytes from patients with HIV-associated dementia (HAD). Dimethyl fumarate (DMF), an antioxidant, has been reported to decrease HIV replication and neurotoxicity mediated by HIV-infected macrophages. Thus, we hypothesized that DMF will decrease cathepsin B release from HIV-infected macrophages by preventing oxidative stress and enhancing lysosomal function. Monocyte-derived macrophages (MDM) were isolated from healthy donors, inoculated with HIV-1ADA, and treated with DMF following virus removal. After 12 days post-infection, HIV-1 p24 and total cathepsin B levels were measured from HIV-infected MDM supernatants using ELISA; intracellular reactive oxygen and nitrogen species (ROS/RNS) were measured from MDM lysates, and functional lysosomes were assessed using a pH-dependent lysosomal dye. Neurons were incubated with serum-free conditioned media from DMF-treated MDM and neurotoxicity was determined using TUNEL assay. Results indicate that DMF reduced HIV-1 replication and cathepsin B secretion from HIV-infected macrophages in a dose-dependent manner. Also, DMF decreased intracellular ROS/RNS levels, and prevented HIV-induced lysosomal dysfunction and neuronal apoptosis. In conclusion, the improvement in lysosomal function with DMF treatment may represent the possible mechanism to reduce HIV-1 replication and cathepsin B secretion. DMF represents a potential therapeutic strategy against HAND.

Background:Conversion to full-thickness tear in partial-thickness rotator cuff tears (PTRCTs) is based on the quality and thickness of the normal-looking untorn rotator cuff layer. However, whether the untorn tendon is a healthy tissue remains to be elucidated.Purpose:To compare the apoptotic gene expression of the untorn articular layer with the torn bursal layer in PTRCTs.Study Design:Controlled laboratory study.Methods:Tendon tissues were harvested from 20 patients undergoing arthroscopic surgery for partial-thickness rotator cuff repair. As a control group, the tissues were harvested during intramedullary nail fixation in 10 proximal humeral fractures. In the experimental group, the samples were harvested from 2 sites: the torn bursal-sided tendon and the untorn articular-sided tendon. Hematoxylin and eosin (H&E) staining was conducted for basic histological evaluation, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to detect apoptosis of tissue cells. The expression of caspase 3, 8, and 9 was confirmed immunohistochemically. Western blot analysis was used to assay the caspase activities.Results:In H&E staining, the direction of collagen bundles in untorn tendon was disoriented when compared with those of control tendon. However, the shape of the nuclei was not different, although the nuclei of the untorn tendon showed apoptosis in the TUNEL staining similar to those of the torn tendon. The immunohistochemical staining of caspase 3, 8, and 9 was increased concomitantly in untorn and torn tendons. All of the caspase activities in the untorn articular layer and torn bursal layer were significantly higher than in controls ( P < .05). However, no significant differences were found between the two layers ( P > .05).Conclusion:The study demonstrates that apoptotic gene expression is increased not only in the torn bursal layer but also in the untorn articular layer of PTRCTs.Clinical Relevance:The untorn articular layer of PTRCTs is abnormal, which triggers postoperative pain and further rotator cuff tears. Therefore, treatment of the abnormal untorn articular layer is essential in bursal-sided PTRCTs.

Purpose: Soft-tissue reconstruction is complicated by ischemia and reperfusion injury. Animal trials have documented the independent healing benefits of hyperbaric oxygen preconditioning (HBOP) and stem cell delivery in cutaneous flaps. We explored the role of HBOP and stem cell delivery in flap preconditioning and survival. Methods: We designed a randomized controlled trial to assess the effects of hyperbaric oxygen preconditioning and stromal vascular fraction (SVF) delivery on flap survival. Of the first 24 guinea pigs, six received neither HBOP nor injections, and six underwent HBOP without injections. Of the remaining 12 animals, six received SVF or saline injections in the absence of HBOP. The final six animals received autologous SVF injections or saline injections followed by four HBOP treatments. To enhance clinical relevance, a group of 6 animals underwent HBOP prior to SVF or saline injections. Thereafter, an unfavorably designed cutaneous flap was elevated and assessed via study-blinded observer, as well as by quantification of TUNEL-positive cells. Results: Distal necrosis of the tissue flap was more ex- tensive in the no-intervention group (45% of flap). Flaps treated with HBOP only and those treated with SVF injections demonstrated only 38.2% and 27.1% distal necrosis. The most significant difference occurred in the combination HBOP and SVF group, where distal necrosis was only 21.1% of the flap (p ≤ 0.05). SVF delivery immediately prior to flap elevation further minimized distal necrosis of the flap to 15.6%. These findings were mirrored by the TUNEL assay. Conclusions: Combining HBOP and SVF improves flap viability.

Objectives The present work evaluated preventive effect of curcumin on cisplatin-induced bladder cystopathy. Methods 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. Results 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. Conclusions 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.

Cdc42, a Rho family small GTPase, regulates cytoskeleton organization, vesicle trafficking, and other cellular processes in development and homeostasis. However, Cdc42’s roles in prenatal tooth development remain elusive. Here, we investigated Cdc42 functions in mouse enamel organ. Cdc42 showed highly dynamic temporospatial patterns in the developing enamel organ, with robust expression in the outer enamel epithelium, stellate reticulum (SR), and stratum intermedium layers. Strikingly, epithelium-specific Cdc42 deletion resulted in cystic lesions in the enamel organ. Cystic lesions were first noted at embryonic day 15.5 and progressively enlarged during gestation. At birth, cystic lesions occupied the bulk of the entire enamel organ, with intracystic erythrocyte accumulation. Ameloblast differentiation was retarded upon epithelial Cdc42 deletion. Apoptosis occurred in the Cdc42 mutant enamel organ prior to and synchronously with cystogenesis. Transmission electron microscopy examination showed disrupted actin assemblies, aberrant desmosomes, and significantly fewer cell junctions in the SR cells of Cdc42 mutants than littermate controls. Autophagosomes were present in the SR cells of Cdc42 mutants relative to the virtual absence of autophagosome in the SR cells of littermate controls. Epithelium-specific Cdc42 deletion attenuated Wnt/β-catenin and Shh signaling in dental epithelium and induced aberrant Sox2 expression in the secondary enamel knot. These findings suggest that excessive cell death and disrupted cell-cell connections may be among multiple factors responsible for the observed cystic lesions in Cdc42 mutant enamel organs. Taken together, Cdc42 exerts multidimensional and pivotal roles in enamel organ development and is particularly required for cell survival and tooth morphogenesis.

Tooth enamel is manufactured by the inner enamel epithelium of the multilayered enamel organ. Msx2 loss-of-function mutation in a mouse model causes an abnormal accumulation of epithelial cells in the enamel organ, but the underlying mechanism by which Msx2 regulates amelogenesis is poorly understood. We therefore performed detailed histological and molecular analyses of Msx2 null mice. Msx2 null ameloblasts and stratum intermedium (SI) cells differentiated normally in the early stages of amelogenesis. However, during subsequent developmental stages, the outer enamel epithelium (OEE) became highly proliferative and transformed into a keratinized stratified squamous epithelium that ectopically expressed stratified squamous epithelium markers, including Heat shock protein 25, Loricrin, and Keratin 10. Moreover, expression of hair follicle–specific keratin genes such as Keratin 26 and Keratin 73 was upregulated in the enamel organ of Msx2 mutants. With the accumulation of keratin in the stellate reticulum (SR) region and subsequent odontogenic cyst formation, SI cells gradually lost the ability to differentiate, and the expression of Sox2 and Notch1 was downregulated, leading to ameloblast depolarization. As a consequence, the organization of the Msx2 mutant enamel organ became disturbed and enamel failed to form in the normal location. Instead, there was ectopic mineralization that likely occurred within the SR. In summary, we show that during amelogenesis, Msx2 executes a bipartite function, repressing the transformation of OEE into a keratinized stratified squamous epithelium while simultaneously promoting the development of a properly differentiated enamel organ competent for enamel formation.

Cartilage‑derived morphogenetic protein‑1 (CDMP1) is a polypeptide growth factor with specific cartilage inducibility, which is predominantly expressed in the developmental long bone cartilage core and in the pre‑cartilage matrix in the embryonic stage. The aim of the present study was to investigate the roles and the mechanisms of CDMP1 overexpression on the apoptosis of rat dorsal root ganglia (DRG) neurons that were induced by inflammatory cytokines. Cell counting Kit‑8 assay, flow cytometry and TdT‑mediated dUTP nick‑end labeling assay were performed to examine cell viability and apoptosis. ELISA, hematoxylin and eosin staining and immunohistochemistry assays were performed to examine the levels of several factors in DRG tissues. Western blot analysis and reverse transcription‑quantitative polymerase chain reaction assays were used to determine the mRNA and protein expression levels, respectively. The results demonstrated that CDMP1 expression was downregulated, while inflammatory cytokine expression was upregulated in DRG tissues derived from lumbar disc herniation (LDH) model rats. In addition, DRG cells from LDH rats exhibited increased apoptosis compared with control rats. CDMP1 overexpression enhanced the cell viability of inflammatory cytokine‑induced DRG cells, and suppressed the apoptosis of inflammatory cytokine‑induced DRG cells via regulating the expression levels of Caspase‑3/8/9, BCL2 apoptosis regulator, and BCL2 associated X. Furthermore, CDMP1 overexpression was demonstrated to affect the Wnt/β‑Catenin pathway in the inflammatory cytokine‑induced DRG cells. In conclusion, the present findings suggested that CDMP1 overexpression mediated inflammatory cytokine‑induced apoptosis via Wnt/β‑Catenin signaling in rat DRG cells.

The present study assessed the antitumor effect of artesunate (ART) in vitro and in vivo, as well as its underlying mechanism of action in HCT116 colon cancer cells. An MTT assay, DAPI staining, flow cytometry, western blotting, immunohistochemistry, transmission electron microscopy and TUNEL assay were performed to study the molecular mechanism underlying the antitumor effects of ART in HCT116 colon cancer cells. ART was observed to inhibit proliferation by inducing the apoptosis of HCT116 cells both in vitro and in vivo. Flow cytometry analysis demonstrated that treatment with 2 and 4 µg/ml ART for 48 h induced early apoptosis in 22.7 and 33.8% of cells, respectively. In the xenograft tumors of BALB/c nude mice, TUNEL‑positive cells increased in the ART group compared with that in the normal saline group. Furthermore,the associated mitochondrial cleaved‑caspase 3, poly‑ADP ribose polymerase (PARP), caspase 9 and Bcl‑2‑associated X protein levels increased while B‑cell lymphoma‑2 (Bcl‑2) decreased both in the cell and animal ART‑treated group. ART‑treated cells also exhibited autophagy induction, as evidenced by increased protein expression levels of light chain 3 (LC3) and beclin‑1, and the presence of autophagosomes. Notably, pharmacological blockade of autophagy activation using hydroxychloroquine markedly enhanced ART‑induced apoptosis and increased the protein levels of cleaved caspase 3 and PARP, while decreasing the levels of LC3 and beclin‑1. These findings suggested that the ART‑induced autophagy may have a cytoprotective effect by suppressing apoptosis. In conclusion, ART may be a potentially clinically useful anticancer drug for human colon cancer. In addition, co‑treatment with ART and an autophagy inhibitor may be an effective anticancer therapy.

Purpose To investigate the protective effects of miR-320 on cerebral ischemia/reperfusion (I/R) injury in mice and PC12 cells. Methods miR-320 normal control (NC), inhibitor fragment and overexpression fragments were injected into mice lateral ventricles. Thereafter, the middle cerebral artery occlusion (MCAO) for left cerebrum method was employed in order to establish a cerebral I/R injury mice model. Apoptosis in the peripheral region of cerebral infarction and the volume of cerebral infarction and brain edema were tested as well. For simulating MCAO in vitro, the PC12 cells were subjected to oxygen-glucose deprivation. The lentivirus transfection technique was used to overexpress or inhibit miR-320. Furthermore, the effects of miR-320 on the survival and apoptosis of PC12 were monitored by MTT and TUNEL detection. Results A cerebral I/R injury mice model was successfully established. MiR-320 enhanced cell apoptosis of the injury side cortical infarcted peripheral zone and increased brain infarction volume and edema volume in MCAO/R mice. In addition, Insulin growth factor-1 (IGF-1) mRNA and protein expressions in MCAO/R mice were inhibited by miR-320 as well. Moreover, the results of cell studies were consist with the animal studies. Conclusions miR-320 may be involved in the regulation of cerebral I/R for brain parenchyma injury by inhibition of IGF-1 pathway.

Aims Ovarian cancer (OC) has the highest mortality rate of all gynecological cancers. Currently, the first-line OC treatment consists of cytoreductive surgery and platinum-based chemotherapy. However, most patients develop chemoresistance after the first-line treatment limits the success of treatment. Therefore, there is an urgent need to identify effective therapeutic agents. Main methods Cell viabilities were detected by MTS assay; Annexin V-FITC/PI assay and western blotting assay were performed to analyze the apoptotic cells in vitro; An immunofluorescence assay was performed to analyze the TUNEL+ apoptotic cells in vivo; Patient-derived xenografts were established to test the in vivo antitumor effects; The key proteins of p53, caspase-mediated apoptotic pathway and Akt/mTOR pathway were detected by Western blotting. Key findings Icaritin, a prenylflavonoid derivative from Epimedium Genus, inhibited the proliferation of drug-sensitive OC cells (OV2008 and C13*) and cisplatin resistant OC cells A2780cp. Icaritin induced OC cell apoptosis in vitro, as indicated by the increase of Annexin V+/PI+ apoptotic cells analyzed with flow cytometry, and the cleavage of caspase 9, caspase 3 and poly-ADP-ribose polymerase (PARP) detected with western blotting. Icaritin also inhibited tumor growth and induced OC cells apoptosis in patient-derived xenografts, as indicated by the tumor growth delay and increase of TUNEL-positive cells in tumor tissues. The icaritin-induced OC cell apoptosis may be associated with the activation of p53 and the suppression of Akt/mTOR pathway. Significance This study sheds light on the underlying mechanisms of antitumor effect of icaritin, and warrants clinical trial for treatment of OC.

Objective Ciliary neurotrophic factor (CNTF) has been evaluated as a candidate therapeutic agent for diabetes and its neural complications. However, its role in diabetic retinopathy has not been fully elucidated. Methods This is a randomized unblinded animal experiment. Wistar rats with streptozocin (STZ)-induced diabetes were regularly injected with CNTF or vehicle control in their vitreous bodies beginning at 2 weeks after STZ injection. A total of five injections were used. In diabetic rats, the levels of CNTF and neurotrophin-3 (NT-3) were evaluated by enzyme-linked immunosorbent assays (ELISA) and real-time PCR. The abundance of tyrosine hydroxylase (TH) and β-III tubulin was detected by western blot. Transferase-mediated dUTP nick-end labeling staining (TUNEL) was used to detect cell apoptosis in the retinal tissue. The activation of caspase-3 was also measured. Results The protein and mRNA levels of CNTF in diabetic rat retinas were reduced compared to control rats. In addition, retinal ganglion cells (RGCs) and dopaminergic amacrine cells appeared to undergo degeneration in diabetic rat retinas, as revealed by transferase-mediated dUTP nick-end labeling staining (TUNEL). Tyrosine hydroxylase (TH) and β-III tubulin protein levels also decreased significantly. Intraocular administration of CNTF rescued RGCs and dopaminergic amacrine cells from neurodegeneration and counteracted the downregulation of β-III tubulin and TH expression, thus demonstrating its therapeutic potential. Conclusion Our study suggests that early diabetic retinal neuropathy involves the reduced expression of CNTF and can be ameliorated by an exogenous supply of this neurotrophin.