Transferase dUTP Nick-end Labeling Assay Research Articles

Depilatory laser miniaturizes hair by inducing bystander dermal papilla cell necrosis through thermal diffusion.

Depilatory laser targeting melanin has been widely applied for the treatment of hypertrichosis. Both selective photothermolysis and thermal diffusion have been proposed for its effect, but the exact mechanism of permanent hair reduction remains unclear. In this study, we explore the role of thermal diffusion in depilatory laser-induced permanent hair loss and determine whether nonpigmented cells are injured by thermal diffusion. C57BL/6 mice in anagen and telogen were treated with alexandrite laser (wavelength 755 nm, pulse duration 3 milliseconds, fluence 12 J/cm2 , spot size 12 mm), respectively. Histological analysis, terminal deoxynucleotidyl transferase dUTP nick-end labelingassay, and transmission electron microscopic imaging were employed to evaluate the injury to hair follicle (HF)cells. The proliferation status of HF cells was examined by 5-bromo-2'-deoxyuridinepulse labeling. The number of HF stem cells was quantified by fluorescence-activated cell sorting. The size of the regenerated hair was determined by measuring its length and width. We found that irradiating C57BL/6 mice in anagen with alexandrite laser led to hair miniaturization in the next anagen. In addition to thermal disruption of melanin-containing cells in the precortex region, we also detected necrosis of the adjacent nonpigmented dermal papilla cells due to thermal diffusion. Dermal papilla cells decreased by 24% after laser injury, while the number of bulge stem cells remained unchanged. When the laser was delivered to telogen HFs where no melanin was present adjacent to the dermal papilla, thermal necrosis and cell reduction were not detected in the dermal papilla and no hair miniaturization was observed. Our results suggest that depilatory laser miniaturizes hair by inducing thermal necrosis of dermal papilla cells due to secondary thermal diffusion from melanin-containing precortex cells in the anagen hair bulbs.

MicroRNA-194-5p Attenuates Doxorubicin-Induced Cardiomyocyte Apoptosis and Endoplasmic Reticulum Stress by Targeting P21-Activated Kinase 2.

ObjectiveMany studies have reported that microRNAs (miRs) are involved in the regulation of doxorubicin (DOX)-induced cardiotoxicity. MiR-194-5p has been reported significantly upregulated in patients with myocardial infarction; however, its role in myocardial diseases is still unclear. Various stimuluses can trigger the endoplasmic reticulum (ER) stress and it may activate the apoptosis signals eventually. This study aims to explore the regulatory role of miR-194-5p in DOX-induced ER stress and cardiomyocyte apoptosis.MethodsH9c2 was treated with 2 μM DOX to induce apoptosis, which is to stimulate the DOX-induced cardiotoxicity model. The expression of miR-194-5p was detected by quantitative real-time PCR (qRT-PCR); the interaction between miR-194-5p and P21-activated kinase 2 (PAK2) was tested by dual luciferase reporter assay; terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and caspase-3/7 activity were used to assess apoptosis; trypan blue staining was applied to measure cell death; Western blotting was performed to detect protein expressions; and ER-related factors splicing X-box binding protein 1 (XBP1s) was detected by polyacrylamide gel electrophoresis and immunofluorescence to verify the activation of ER stress.ResultsMiR-194-5p was upregulated in cardiomyocytes and mouse heart tissue with DOX treatment, while the protein level of PAK2 was downregulated. PAK2 was predicted as the target of miR-194-5p; hence, dual luciferase reporter assay indicated that miR-194-5p directly interacted with PAK2 and inhibited its expression. TUNEL assay, caspase-3/7 activity test, and trypan blue stain results showed that either inhibition of miR-194-5p or overexpression of PAK2 reduced DOX-induced cardiomyocyte apoptosis. Silencing of miR-194-5p also improved DOX-induced cardiac dysfunction. In addition, DOX could induce ER stress in H9c2, which led to XBP1 and caspase-12 activation. The expression level of XBP1s with DOX treatment increased first then decreased. Overexpression of XBP1s suppressed DOX-induced caspase-3/7 activity elevation as well as the expression of cleaved caspase-12, which protected cardiomyocyte from apoptosis. Additionally, the activation of XBP1s was regulated by miR-194-5p and PAK2.ConclusionOur findings revealed that silencing miR-194-5p could alleviate DOX-induced cardiotoxicity via PAK2 and XBP1s in vitro and in vivo. Thus, the novel miR-194-5p/PAK2/XBP1s axis might be the potential prevention/treatment targets for cancer patients receiving DOX treatment.

Light-activatable multifunctional paclitaxel nanoprodrug for synergistic chemo-photodynamic therapy in liver cancer.

Paclitaxel (Ptx) is widely utilized to treat liver cancer, and the treatment benefit of reactive oxygen species (ROS)-responsive Ptx nanoprodrug is investigated in this study. The one-step nano-precipitation method was utilized to self-assembly DSPE-PEG2000 -thioketal linker (TK)-Ptx with pyropheophorbide acid nanoparticles (PPa NPs) to form PPa/Ptx NPs. Dynamic light scattering and transmission electron microscopy were used for characterization, and 2'-7'dichlorofluorescin diacetate staining was utilized for intracellular ROS detection. HepG2 cells viability and tumor growth rate of HepG2 bearing mice were assayed. Hematoxylin and eosin staining, proliferating cell nuclear antigen detection, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay were utilized for histology assessment. PPa/Ptx NPs incubation with light irradiation showed superior cytotoxicity to HepG2 cells with increased intracellular ROS production than PPa/Ptx NPs incubation without light irradiation or PPa NPs incubation with light irradiation. At the same time, PPa/Ptx NPs with light irradiation could significantly decrease the tumor growth in vivo as indicated by diminished tumor volume with the largest necrotic area, the highest rate of apoptotic cells, and the least proliferating cells. PPa/Ptx NPs show synergistic chemo-photodynamic characteristics, which could be considered as a promising treatment option for liver cancer.

CHD7 in oocytes is essential for female fertility.

BackgroundChromodomain helicase DNA-binding protein 7 (CHD7), which is associated with CHARGE (Coloboma, Heart defect, Atresia choanae, Restricted growth, Genital hypoplasia and Ear abnormality) syndrome is an important regulator in many vital developmental processes. However, its role during oocyte development remains unknown.MethodsWe screened the Gene Expression Omnibus (GEO) database for expression levels of CHD7 during folliculogenesis. We generated a conditional knockout (cKO) mouse strain with oocyte-specific deletion of CHD7 (Gdf9-Cre:Chd7f/f) using the Cre-loxP approach. Evaluation of follicle numbers and reproductive ability was then conducted. In addition, granulosa cell (GC) apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and cleaved caspase-3, using immunohistochemistry (IHC) and immunofluorescence (IF). GC proliferation was measured by Ki67 staining as evaluated by IHC.ResultsIn our study, we demonstrated that CHD7 has high expression throughout all developmental stages of the oocyte. We found that deletion of Chd7 in oocytes can cause infertility or sub-fertility in female mice and is associated with decreased follicle numbers at all stages. In addition, we found that GC apoptosis was significantly higher in cKO mice.ConclusionsTo our knowledge, our study has been the first to show that CHD7 plays a specific role during oogenesis. Our findings provide new insights into CHD7-related infertility.

Effect of pirfenidone protecting against cigarette smoke extract induced apoptosis

INTRODUCTIONApoptosis of lung structural cells is a significant upstream event involved in COPD pathogenesis. This study was designed to explore whether pirfenidone (PFD) was able to attenuate apoptosis induced by cigarette smoke extract (CSE).METHODSA method of intraperitoneal CSE injection to BALB/C mice was used to establish emphysema mouse model. Terminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL) assay was applied to evaluate apoptotic cell ratio in mouse lung tissue. The cell viability of HBECs exposed to different concentrations of PFD was measured by Cell Counting Kit-8 (CCK-8) assay. The apoptosis index (AI) of HBECs was tested by flow cytometry. Levels of apoptosis-related protein were determined by Western blotting.RESULTSPFD treatment significantly decreased the AI value in emphysema mouse lung tissue by TUNEL. In HBECs, flow cytometry showed that PFD could significantly reduce AI led by CSE. Both in vitro and in vivo, protein levels of Bax and Cleaved-caspase 3 in CSE group significantly increased in contrast with the control group; while Bcl-2 protein level in CSE group was significantly decreased; moreover, PFD significantly reversed protein level changes of Bcl-2, Bax, and Cleaved-caspase 3 led by CSE.CONCLUSIONSThis study reveals that PFD may potentially protect against CSE induced apoptosis.

Effects of Radiotherapy in Combination With Irinotecan and 17-AAG on Bcl-2 and Caspase 3 Gene Expression in Colorectal Cancer Cells.

Introduction: In this study, the cytotoxic and anti-cancer effects of Irinotecan as a conventional chemotherapeutic agent compared to 17-(allyl amino)-17-demethoxygeldanamycin (17-AAG) as possible radiosensitizers in the HCT-116 cell line were investigated. Methods: HCT-116 cells were treated with various concentrations of irinotecan and 17-AAG and also irradiated with a 2-Gy of X-ray radiation. Then, the cell viability was examined by a water-soluble tetrazolium-1 assay after 24 hours. For single therapies and double and triple combination cases, IC50, 0.5×IC50 and 0.25×IC50 concentrations of each drug were selected respectively for a terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay and other tests. In treated and untreated cells, the caspase 3 and Bcl-2 gene expression ratios were evaluated by the real-time PCR method. Likewise, caspase 3 activity was detected with a colorimetric assay. Results: In all combined treatments, including 17-AAG- radiation, irinotecan - radiation, irinotecan -17-AAG, and irinotecan-17-AAG-radiation, decreased cellular viability and increased TUNEL positive cells were presented versus the control group (P < 0.05). There were increased TUNEL positive cells in the triple combination, in concentrations of 0.25×IC50 of each drug, in comparison with single and double agent treatments. Moreover, in triple combination, the caspase 3 mRNA level and caspase 3 activity increased versus related single treatments. Likewise, in the irinotecan-17-AAG-radiation combined treatment and the 17-AAG-radiation double treatment, the Bcl-2 gene expression level decreased in comparison with single therapies. Conclusion: It can be indicated that the combination of chemo-radiotherapy versus single treatments has significant anti-cancer effects.

Applicability of advanced endoscopic balloon-type irreversible electroporation catheter on the esophagus: Preclinical animal study.

339 Background: Irreversible electroporation (IRE) is a new destructive technique that removes undesirable tissues using an electric field. Main mechanism of IRE is through the creation of permanent, nano-sized pores on the cell membranes, resulting in cell death. It poses, however, one technical challenge as contractions of smooth muscle of the gastrointestinal tract may prevent IRE from effectively electroporate the cell. The present study aims to demonstrate the applicability of this newly designed endoscopic balloon-type IRE catheter in destroying undesired tissues. Methods: The electrical field generated at 1500V with 40 pulses during ablation with the balloon-type catheter in esophagus were simulated using COMSOL Multiphysics. After a pig was anesthetized, a 0.035-inch Jagwire was inserted through the duodenum using a gastroscope. An IRE catheter was then manually advanced into the esophagus along with a guidewire. The fluoroscopy with contrast medium was employed to determine target lesions with clips. Following the sequential ablation along the esophagus, electrical parameters as well as the number of interruptions encountered were recorded. The pigs used in the experiments were sacrificed after 24 hours and tissue specimens obtained were evaluated using H&amp;E and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: Based on the COMSOL simulation, the heated portion, approximately 57 degrees of Celsius, was observed only at the edge of the electrodes. Fluoroscopy demonstrated the balloon-type catheter was not adjacent to the heart and tightly blocked the lumen of esophagus as it adheres to the mucosal layer during inflation. The IRE catheter was able to overcome muscle contractions during ablative process and deliver all electrical energy of the scheduled sequences. A total of 12 ablations were performed in three pigs, and the success rate of balloon catheter was 91.7% (11/12). Histological slides from H&amp;E staining and TUNEL assay showed nuclei, stained brown, which indicates apoptosis at the ablation site. Conclusions: The IRE balloon-type catheter demonstrated its applicability and effectiveness as an electroporation-based treatment in esophagus as validated in the experimental results. Further studies regarding electrodes improvement to heat-free condition and its safety related assessment would be needed.

Testis-enriched Asb12 is not required for spermatogenesis and fertility in mice.

BackgroundMembers of the ankyrin repeat and SOCS box (Asb) family are expressed abundantly in testes. Some Asb genes/proteins are required for spermatogenesis, but the function of Asb12 during spermatogenesis is not clear. We investigated the physiological role of Asb12 in murine testes.MethodsThe clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system was used to generate Asb12-knockout (KO) mice. Histology and immunostaining were done to assess the effects of Asb12 KO on mouse testes and epididymides. Semen quality was analyzed using a computer-assisted sperm analyzer. The terminal deoxynucleotidyl transferase-dUTP nick-end labeling assay was employed to examine testicular apoptosis. Real-time reverse transcription-quantitative polymerase chain reaction (PCR) was conducted to calculate gene transcription levels.ResultsAsb12 was expressed predominantly in murine testes. Immunostaining of Asb12 protein revealed that Asb12 was located specifically in the acrosome of elongated spermatids, which suggested a potential role of Asb12 during spermatogenesis. However, Asb12-KO mice had normal fertility, and no overt difference was detected in testicular morphology, semen quality, or apoptosis when comparing Asb12-KO and Asb12-wild type (WT) mice. Gene expression of several Asb family members was increased significantly in the testes of Asb12-KO mice when compared with that in Asb12-WT mice, which suggested functional compensation from paralogs for Asb12 loss.ConclusionsWe demonstrated that Asb12 is not essential for the spermatogenesis and fertility of mice. Our findings will assist researchers in avoiding redundant efforts, and provide a baseline resource for genetic studies on human fertility.

A novel nordihydroguaiaretic acid analog, compound 3a, alleviates acute lung injury by exerting antiapoptotic and antiinflammatory effects

Acute lung injury (ALI) is a continuum of pulmonary changes caused by various lung insults. Previously, we synthesized a series of nordihydroguaiaretic acid analogs; of these, compound 3a exhibited excellent antioxidant capacity in a murine model of middle cerebral artery occlusion. However, it remains unclear whether compound 3a can modulate lipopolysaccharide (LPS)-induced ALI. ALI was induced via tracheal LPS administration, and the pathological changes were assessed. The level of inflammation was verified by immunofluorescence and immunohistochemical analyses. Apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick-end labeling assays and Western blotting. Changes in the levels of mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) pathway proteins were assessed by immunofluorescence assays and Western blotting. In vitro, RAW 264.7 cells were treated with compound 3a prior to LPS challenge, and the intracellular level of inflammation was assessed by quantitative PCR (qPCR). Relevant proteins were detected via immunofluorescence assays and Western blotting. Mice developed extensive lung inflammation by 24 h after LPS challenge. Histological examination revealed signs typical of ALI. Preadministration of compound 3a markedly ameliorated the histopathological changes and reduced fluid exudation into the alveolar space. Compound 3a also greatly reduced the levels of inflammation and apoptosis both in vivo and in vitro. Moreover, compound 3a markedly reduced phosphorylation of MAPK/NF-κB pathway-related proteins and p65 translocation, consistent with the in vitro observations. In summary, administration of compound 3a prior to LPS suppressed ALI via inhibition of the MAPK/NF-κB pathway.

Canonical Thyroid Hormone Receptor β Action Stimulates Hepatocyte Proliferation in Male Mice.

3,5,3'-L-triiodothyronine (T3) is a potent inducer of hepatocyte proliferation via the Wnt/β-catenin signaling pathway. Previous studies suggested the involvement of rapid noncanonical thyroid hormone receptor (TR) β signaling, directly activating hepatic Wnt/β-catenin signaling independent from TRβ DNA binding. However, the mechanism by which T3 increases Wnt/β-catenin signaling in hepatocytes has not yet been determined. We aimed to determine whether DNA binding of TRβ is required for stimulation of hepatocyte proliferation by T3. Wild-type (WT) mice, TRβ knockout mice (TRβ KO), and TRβ mutant mice with either specifically abrogated DNA binding (TRβ GS) or abrogated direct phosphatidylinositol 3 kinase activation (TRβ 147F) were treated with T3 for 6 hours or 7 days. Hepatocyte proliferation was assessed by Kiel-67 (Ki67) staining and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Activation of β-catenin signaling was measured in primary murine hepatocytes. Gene expression was analyzed by microarray, gene set enrichment analysis (GSEA), and quantitative reverse transcription polymerase chain reaction. T3 induced hepatocyte proliferation with an increased number of Ki67-positive cells in WT and TRβ 147F mice (9.2% ± 6.5% and 10.1% ± 2.9%, respectively) compared to TRβ KO and TRβ GS mice (1.2% ± 1.1% and 1.5% ± 0.9%, respectively). Microarray analysis and GSEA showed that genes of the Wnt/β-catenin pathway-among them, Fzd8 (frizzled receptor 8) and Ctnnb1 (β-catenin)-were positively enriched only in T3-treated WT and TRβ 147F mice while B-cell translocation gene anti-proliferation factor 2 was repressed. Consequently, expression of Ccnd1 (CyclinD1) was induced. Instead of directly activating Wnt signaling, T3 and TRβ induce key genes of the Wnt/β-catenin pathway, ultimately stimulating hepatocyte proliferation via CyclinD1. Thus, canonical transcriptional TRβ action is necessary for T3-mediated stimulation of hepatocyte proliferation.

Effect of Preventive Acupunture Combined with CMNS6-1 Electroacupuncture Instrument on Promoting Apoptosis of 7,12-Dimethylbenzene (a) Anthracene-Induced Breast Cancer Model Rats

The purpose of this study was to evaluate the effect of preventive acupuncture combined with CMNS6-1 electroacupuncture on 7,12-dimethylbenzanthracene induced mammary tumors in Sprague-Dawley rats and to explore the effect of preventive acupuncture on mammary tumors from the perspective of apoptosis. 48 w old female Sprague-Dawley rats were randomly divided into control (10), model (10), tamoxifen (10) and preventive acupuncture (10) groups. The model group, tamoxifen group and preventive acupuncture group were administered once intragastrically with 100 mg/kg body weight of 7,12-dimethylbenzanthracene to induce spontaneous breast cancer. On d 1 after modeling, acupuncture plus electrical stimulation was administered to the preventive acupuncture group. Density wave was selected for electrical stimulation with a frequency of 2 Hz/100 Hz and an intensity of 1 mA and three acupoints, "Zu San Li", "San Yin Jiao" and "Tan Zhong", were stimulated for 20 min per treatment, once every other day, three times a week for 16 w. Tamoxifen (3 mg/kg body weight, intragastrically, every day) was started the day after 7,12-dimethylbenzanthracene administration and continued for 16 w, as positive control treatment. Apoptosis of breast tumor tissue cells was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and expression of caspase-3 and B-cell lymphoma-2 was assessed by immunohistochemistry. During the experiment, no tumors were detected in rats from the control+sham group, all the rats in the 7,12-dimethylbenzanthracene+sham group developed tumors and the cumulative incidence of tumors was 100 %, meanwhile the cumulative incidence of tumors in the 7,12-dimethylbenzanthracene+preventive acupuncture group and the 7,12-dimethylbenzanthracene+tamox group were 60 % and 50 %, respectively. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay showed that preventive acupuncture could significantly promote apoptosis in breast tumor cells in 7,12-dimethylbenzanthracene induced rat, while increased the expression levels of pro-apoptotic proteins such as caspase-3 and B-cell lymphoma-2 associated X. The expression level of antiapoptotic protein B-cell lymphoma-2 was down-regulated and the ratio of B-cell lymphoma-2/B-cell lymphoma-2 associated X was increased. Preventive acupuncture therapy combined with CMNS6-1 electroacupuncture instrument inhibited 7,12-dimethylbenzanthracene-induced mammary tumorigenesis in rats and the mechanism may be related to the regulation of apoptosis-related protein expression and promotion of tumor cell apoptosis.

Interrelationships between amphiregulin, kisspeptin, FSH and FSH receptor in promotion of human ovarian cell functions.

The aim of this study was to investigate: (1) the ability of granulosa cells to produce amphiregulin (AREG), kisspeptin (KISS) and FSH receptor (FSHR); (2) the role of AREG and KISS in the control of ovarian functions; (3) the effect of FSH and KISS on AREG; and (4) the ability of KISS to affect FSHR and to modify FSH action on AREG output by human ovarian granulosa cells. We examined: (1) time-dependent accumulation of AREG; (2) effects of AREG (0, 1, 10, 100ng/mL) and KISS (0, 1, 10, 100ng/mL) on granulosa cell functions; and (3) the effects of KISS (0, 1, 10, 100ng/mL), FSH (0, 1, 10, 100ng/mL), and their combinations on AREG release. Viability, markers of proliferation [accumulation ofproliferating cell nuclear antigen (PCNA) cyclin B1 and sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy6-nitro)benzene sulfonic acid hydrate (XTT formazan)] and apoptosis (accumulation of bax, caspase 3 and terminal deoxynucleotidyl transferase dUTP nick-end labelling), accumulation of KISS, FSHR and steroid hormones, and AREG release were analysed by Trypan blue exclusion test, quantitative immunocytochemistry, XTT, terminal deoxynucleotidyl transferase dUTP nick-end labelling assays and enzyme-linked immunosorbent assay. AREG promoted cell viability, proliferation and steroid hormone output, and inhibited apoptosis. KISS (1 and 10ng/mL) stimulated viability, proliferation, steroid hormone release and occurrence of FSHR and suppressed apoptosis and AREG output; KISS (100ng/mL) had the opposite effect. FSH stimulated AREG release, whilst addition of KISS reversed this FSH effect. FSH mimicked and promoted the inhibitory effect of KISS on AREG release. These results suggest an intra-ovarian production and a functional interrelationship between AREG, KISS, FSH and FSHR in direct regulation of basic ovarian cell functions.

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer.

BackgroundSomatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.ObjectivesThis study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.MethodsThe viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer.ResultsMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption.ConclusionsSow urine-derived cells could be used to produce SCNT embryos.

Effects of melatonin on both testicular regeneration and recovery of spermatogenesis in busulfan-treated rats

Testicular damage is one of the most hazardous effects of chemotherapy as it is frequently associated with oligozoospermia and azoospermia. This study aimed at evaluating the protective effect of melatonin in a rat model of busulfan-induced testicular injury. Rats were divided into four groups: control, melatonin, busulfan, busulfan plus melatonin. After 15 days, the semen was collected from the epididymis and testes were assessed. Sperm removed from cauda epididymis and analyzed for sperm count and viability. Testis tissues were also removed, fixed in formalin and were embedded in paraffin. Sections of testis tissue were stained with hematoxylin-eosin for histological examination and prepared for TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) assay to detect apoptosis and PCNA (proliferating cell nuclear antigenassay) to detect proliferation cells. Serum and testes supernatants were separated to detect testosteron level and oxidative stress parameters. In histological examination, degenerative changes in seminiferous tubules were observed in the experimental groups. In biochemical examination, the total oxidant status (TOS) levels in Busulfan group were significantly higher than in the control group while the total antioxidant status (TAS) levels of all the groups were similar. In conclusion, the beneficial properties of melatonin treatment by its potent anti-oxidants may reduce adverse effects of chemotherapy in the reproductive system in a rodent system.

Dexmedetomidine attenuates acute stress-induced liver injury in rats by regulating the miR-34a-5p/ROS/JNK/p38 signaling pathway.

Dexmedetomidine (DEX) protects against acute stress-induced liver injury, but what's less clear lies in the specific mechanism. To elucidate the specific mechanism underlying DEX on acute stress-induced liver injury, an in vivo model was constructed on rats with acute stress-induced liver injury by 15 min of exhaustive swimming and 3 hr of immobilization. DEX (30 μg/kg) or miR-34a-5p agomir was injected into model rats. Open field test was used to verify the establishment of the model. Liver injury was observed by hematoxylin-eosin (H&E) staining. Contents of norepinephrine (NE), alanine aminotransfease (ALT) and aspartate aminotransferase (AST) in serum of rats were detected by enzyme-linked immunosorbent assay (ELISA) and those of oxidative stress markers (reactive oxygen species (ROS), Malondialdehyde (MDA), Glutathione (GSH), Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPX)) were measured using commercial kits. Apoptosis of hepatocytes was detected by Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Western blot was performed to detect the expressions of SOD2, COX-2, cytochrome C, Cleaved caspase 3, Bax, Bcl-2, P-JNK, JNK, P-p38, p38 and c-AMP, p-PKA and PKA in liver tissues. As a result, liver injury in model rat was alleviated by DEX. DEX attenuated the increase in the levels of NE, ALT, AST, MDA, ROS, apoptosis, SOD2, COX-2, Cytochrome C, cleaved caspase 3, Bax, and P-JNK, P-p38, c-AMP, P-PKA and miR-34a-5p, and the decrease in the levels of SOD, GPX, GSH and Bcl-2 in model rats. Furthermore, miR-34a-5p overexpression could partly reverse the effects of DEX. Collectively, DEX could alleviate acute stress-induced liver injury through ROS/JNK/p38 signaling pathway via downregulation of miR-34a-5p.

Reposition of the anti-inflammatory drug diacerein in an in-vivo colorectal cancer model

Excessive interleukin (IL)-6 production is a driver for malignancy and drug resistance in colorectal cancer (CRC). Our study investigated a seven-week post-treatment with the anti-inflammatory drug, Diacerein (Diac), alone or in combination with 5-fluorouracil (5-FU), using a 1,2-dimethylhydrazine (DMH) rat model of CRC. Diac alone and 5-FU+Diac reduced serum levels of carcino-embryonic antigen (CEA), while all regimens decreased serum levels of colon cancer-specific antigen (CCSA), a more specific CRC biomarker. Additionally, Diac, 5-FU and their combination suppressed colonic content/gene expression of IL-6, its downstream oncogene, Kirsten rat sarcoma viral oncogene homolog (K-Ras), and consequently Notch intracellular domain and nuclear factor-kappa B (NF-κB) p65. In turn, NF-κB downstream factors, viz., matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), c-Myc, and B-cell lymphoma-2 (Bcl-2) were also downregulated, while E-cadherin was elevated. Additionally, the drugs reduced the immunoreactivity of CD31 to prove their anti-angiogenic effect, while the TUNEL assay confirmed the apoptotic effect. The apoptotic effect was confirmed by transferase dUTP nick-end labeling assay. Moreover, these drugs inhibited colon content of p-Akt, β-catenin, and cyclin D1 immunoreactivity. The drugs also activated the tumor suppressor glycogen synthase kinase 3- β (GSK3-β) and upregulated the expression of the Nur77 gene, which represents the second arm of IL-6 signaling. However, only 5-FU upregulated miR-200a, another K-Ras downstream factor. The in-vitro cytotoxic and migration/invasion assays verified the molecular trajectories. Accordingly, we evaluated the antineoplastic effect of Diac alone and its possible chemosensitization effect when added to 5-FU. This combination may target critical oncogenic pathways, including the IL-6/K-Ras/Notch/NF-κB p65 axis, p-Akt/GSK3-β/β-catenin/cyclin D-1 hub, and Nur77.

In Vitro Studies on the Influence of Meloxicam on Cytotoxic Activity Induced by Risedronate Sodium in Canine (D-17) and Human (U-2 OS) Osteosarcoma Cell Lines.

Simple SummaryThe aim of this in vitro study was to reveal the pharmacological interactions between meloxicam and risedronate sodium, used jointly to induce a cytotoxic effect in canine (D-17) and human (U-2 OS) osteosarcoma cell lines. Meloxicam, a non-steroidal anti-inflammatory drug, is capable of intensifying the cytotoxic activity of risedronate sodium routinely used in bone tissue metabolic diseases. The cell cultures were incubated, tested, and evaluated according to standard protocols. The study demonstrated a greater susceptibility of canine osteosarcoma cells in vitro to the investigated drug combination than the human. In both cases, meloxicam alone showed low cytotoxic activity against the tested cell lines, but the two compounds combined were synergic.The study describes the cytotoxic effect against human and canine osteosarcoma (U-2 OS and D-17) cell lines induced by risedronate sodium and meloxicam per se and in combination. Both cell lines were prepared according to standard procedures for cell cultures studies. The cell viability was estimated in both cell lines treated with chosen concentrations of risedronate sodium and meloxicam. The apoptosis assessment was carried out using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. EC50 values, computed for risedronate sodium and meloxicam cytotoxicity, showed comparable effects against the canine OS cell line in similar concentration of both drugs. In case of human OS, the stronger cytotoxic effect of risedronate sodium was proved. The EC50 values for meloxicam in both cell lines were, statistically, significantly different (* p < 0.05). Moreover, the cytotoxic effect of a combined administration of meloxicam and risedronate sodium in doses 100 µg/mL, compared with the negative control showed statistically significant differences. The human OS cell line was more resistant to both compounds than the canine OS cell line. The apoptotic effect in canine and human osteosarcoma triggered by risedronate sodium and meloxicam was statistically significant (p < 0.05). The cytotoxic effect induced with 100 µg/mL of risedronate sodium proved statistically significant differences between both tested cell lines compared to negative control. The results obtained with 10 and 100 µg/mL of meloxicam were not statistically significant. The study showed the synergic mechanism of action of risedronate sodium and meloxicam, but the concentrations used in vitro will not be possible to achieve in in vivo. Therefore, our results serve as basis only to design future studies on the tissue level.

Salidroside inhibits NLRP3 inflammasome activation and apoptosis in microglia induced by cerebral ischemia/reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway.

BackgroundThe NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an important mediator of neuroinflammatory responses that regulates inflammatory injury following cerebral ischemia and may be a potential target. Salidroside (Sal) has good anti-inflammatory effects; however, it remains unclear whether Sal can regulate NLRP3 inflammasome activation through the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway after cerebral ischemia to alleviate inflammatory injury.MethodsWe established an oxygen-glucose deprivation and reoxygenation (OGD/R) model of BV2 cells and a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model. Cell Counting Kit-8 (CCK-8), flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay were used to detect the viability and apoptosis of BV2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of inflammatory factors. 2,3,5-triphenyltetrazolium chloride (TTC) staining and modified Neurological Severity Score (mNSS) were used to detect cerebral infarction volume and neurological deficit in rats. Western blot, immunohistochemistry and immunofluorescence staining were used to detect the protein expression levels.ResultsOur results showed that Sal increased viability, inhibited lactate dehydrogenase (LDH) release, and reduced apoptosis in OGD/R-induced BV2 cells. Sal reduced the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-8. Following induction by OGD/R, BV2 cells exhibited NLRP3 inflammasome activation and increased protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, IL-1β, and IL-18. Protein levels of key TLR4 signaling pathway elements, such as TLR4, myeloid differentiation primary response 88 (MyD88), and phosphorylated nuclear factor kappa B p65 (p-NF-κB p65)/NF-κB p65 were upregulated. Interestingly, it was revealed that Sal could reverse these changes. In addition, TAK242, a specific inhibitor of TLR4, had the same effect as Sal treatment on BV2 cells following induction by OGD/R. In the MCAO/R rat model, Sal was also observed to inhibit NLRP3 inflammasome activation in microglia, reduce cerebral infarction volume, and inhibit apoptosis.ConclusionsIn summary, we found that Sal inhibited NLRP3 inflammasome activation and apoptosis in microglia induced by cerebral ischemia/reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway, thus playing a protective role. Therefore, Sal may be a promising drug for the clinical treatment of ischemic stroke.

Protection of Retinal Function by Nucleoside Reverse Transcriptase Inhibitors Following Retinal Ischemia/Reperfusion Injury.

Purpose: Retinal ischemia/reperfusion (I/R) injury is a common cause of visual impairment and blindness for which there remain limited treatment options. Nucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT), have been shown to block the NLRP3 inflammasome and prevent retinal degeneration in a mouse model of age-related macular degeneration. The NLRP3 inflammasome has also been shown to be triggered in I/R injury. Therefore, we studied the neuroprotective effects of AZT using a pressure-induced retinal ischemia mouse model. Methods: C57BL/6J mice were randomly assigned to 1 of 2 treatment groups: vehicle-treated retinal I/R injury (n = 6) or AZT-treated retinal I/R injury (n = 6). Vehicle (1% dimethyl sulfoxide [DMSO] in phosphate-buffered saline [PBS]) or AZT 50 mg/kg in 1% DMSO in PBS were injected intraperitoneally twice daily for 5 days. On day 2 of treatment, retinal ischemia was induced by transient elevation of intraocular pressure for 45 min. Scotopic electroretinography (ERG) was used to quantify retinal function before and 1 week after retinal ischemic insult. Retinal morphology was examined 1 week after ischemic insult. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and caspase 1 immunostaining was performed 24 h after retinal I/R injury. Results: Following I/R injury, ERG a- and b-wave amplitudes were significantly reduced in the vehicle-treated mice. AZT treatment significantly attenuated I/R-induced loss of retinal function as compared with vehicle-treated mice. Additionally, AZT-treated mice experienced significantly less inner retinal thinning as compared with vehicle-treated mice. TUNEL-positive cells were prevalent in the vehicle-treated I/R injury mouse retinas compared with the AZT-treated I/R injury mouse retinas. More caspase-1 immunoreactivity was detected in ganglion cell layer and inner nuclear layer (INL) in vehicle-treated I/R injury group than in AZT-treated I/R injury group. Conclusion: AZT treatment resulted in relative preservation of retinal structure and function following ischemic insult as compared with controls. This suggests AZT may have therapeutic value in the management of retinal ischemic diseases.

Exosomes Derived from Mouse Adipose-Derived Mesenchymal Stem Cells Alleviate Benzalkonium Chloride-Induced Mouse Dry Eye Model via Inhibiting NLRP3 Inflammasome

Purpose: The objective of the study was to investigate efficacy and mechanisms of mouse adipose-derived mesenchymal stem cell-derived exosomes (mADSC-Exos) in the benzalkonium chloride (BAC)-induced mouse dry eye model. Methods: Exosomes in the mADSC culture supernatant were isolated by ultracentrifugation. Western blotting, nanoparticle tracking analysis, and transmission electron microscopy were used to characterize mADSC-Exos. An experimental mouse model of dry eye was established by instillation of 0.2% BAC. mADSC-Exos were administered following BAC treatment. The positive control group was treated with commercial eye drops (0.1% pranoprofen). Corneal fluorescein staining, tear secretion, and tear film break-up time (BUT) were evaluated, and histologic analysis of the cornea and conjunctiva was performed by hematoxylin and eosin and periodic acid-Schiff staining. Apoptosis in the corneal epithelium was detected with the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and by Western blotting. Levels of pro-inflammatory cytokines in the cornea and conjunctiva were evaluated by flow cytometry, and mRNA and protein levels of NLR family pyrin domain-containing 3 (NLRP3) pathway components were assessed by quantitative real-time PCR and Western blotting, respectively. Results: mADSC-Exos were characterized as vesicles with a bilayer membrane. The particle size distribution peak was at 134 nm. mADSC-Exos specifically expressed cluster of differentiation (CD)9, CD63, and CD81. mADSC-Exos treatment repaired ocular surface damage. Additionally, mADSC-Exos inhibited cell apoptosis, decreased the levels of interleukin (IL)-1β, IL-6, IL-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, and increased levels of the anti-inflammatory cytokine IL-10. Meanwhile, NLRP3 inflammasome activation and upregulation of caspase-1, IL-1β, and IL-18 were reversed by mADSC-Exos. Conclusions: mADSC-Exos alleviate ocular surface inflammation, suggesting that it is a promising treatment for dry eye.