Transferase-mediated Deoxyuridine Triphosphate Nick End Research Articles

Non-Invasive Radiofrequency Hyperthermia Attenuates HMGB1/TLR4/NF-κB Inflammatory Axis in a Chronic Prostatitis/Chronic Pelvic Pain Syndrome Rat Model.

The primary goal of this study is to evaluate the effect of the non-invasive radiofrequency hyperthermia (RFHT) device on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) rat model and investigate the underlying mechanism. In this study, Sprague-Dawley rats were randomly distributed into three groups: (1) normal control group, (2) CP/CPPS group, and (3) RFHT group. CP/CPPS rat models were induced by 17β-estradiol and dihydrotestosterone for 4 weeks and RFHT was administered for 5 weeks after model establishment. During RFHT administration, core body temperatures were continuously monitored with a rectal probe. After administering RFHT, we assessed pain index for all groups and collected prostate tissues for Western blot analysis, immunofluorescence, and immunohistochemistry. We also collected adjacent organs to the prostate including urinary bladder, testes, and rectum for safety assessment via H&E staining along with a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. After administering RFHT, pain in rats was significantly alleviated compared to the CP/CPPS group. RFHT reduced high-mobility group box 1 (HMGB1) expression and improved inflammation by downregulating subsequent proinflammatory cytokines through inhibition of the toll-like receptor 4 (TLR4)-nuclear factor kappa B (NF-κB) pathway. In prostate-adjacent organs, no significant histological alteration or inflammatory infiltration was detected. The area of cell death also did not increase significantly after RFHT. In conclusion, RFHT demonstrated anti-inflammatory effects by inhibiting the HMGB1-TLR4-NF-κB pathway in CP/CPPS rat models. This suggests that RFHT could serve as a safe and promising therapeutic strategy for CP/CPPS.

Vasopressin Analog [V4Q5]dDAVP Exerts Cooperative Anticancer Effects in Combination With Low-Dose 5-Fluorouracil on Aggressive Colorectal Cancer Models.

Colorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide. Despite being an essential component of systemic chemotherapy for advanced CRC, 5-fluorouracil (5-FU) clinical use has severe limitations, such as high toxicity, low selectivity and drug resistance. [V4Q5]dDAVP (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) is a peptide vasopressin analog and a selective agonist of the arginine vasopressin type 2 membrane receptor (AVPR2), expressed in microvascular and tumor tissue. This synthetic compound has well-proven antitumor and antimetastatic activity in different tumor types, including metastatic CRC. The objective of this work was to assess the potential combinational benefits in preclinical CRC models after [V4Q5]dDAVP addition to 5-FU. Effects on cellular viability, cell cycle progression, apoptosis and molecular mechanisms associated to [V4Q5]dDAVP treatment in combination with 5-FU were evaluated in murine CT-26 and human COLO-205 cell lines. In vivo, impact of dual therapy was explored on CRC tumor growth and metastatic spread. In CRC cells, [V4Q5]dDAVP (1 µM) addition to sub-IC50 5-FU concentrations resulted in the enhancement of cytostatic effects induced by chemotherapy. Reduction of cell viability after combined treatment was associated with cell cycle arrest in the G0/G1 phase, induction of apoptosis and increased gene expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the tumor suppressor p53 (TP53) in malignant cells, as assessed by flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. In vivo, intravenous administration of [V4Q5]dDAVP (0.3 µg/kg) in combination with safe low doses of 5-FU (50 or 80 mg/kg for CT-26 or COLO-205 tumor models, respectively) effectively abrogated CRC growth, reducing aggressiveness of primary lesions and increasing survival of tumor-bearing mice. In addition, concomitant administration of [V4Q5]dDAVP and 5-FU inhibited pulmonary metastasis formation by CT-26 cells in immunocompetent mice, especially reducing macrometastatic disease. [V4Q5]dDAVP seems to enhance the efficacy of 5-FU-based chemotherapy in CRC by modulating tumor progression, as well as metastatic dissemination, suggesting its potential role as a safe and cost-effective co-adjuvant agent for the management of advanced CRC.

An in vivo and in vitro model on the protective effect of cilnidipine on contrast-induced nephropathy via regulation of apoptosis and CaMKⅡ/mPTP pathway.

Contrast-induced nephropathy (CIN) is an acute kidney injury (AKI) observed after the administration of contrast media. Calcium channel blockers (CCBs) have been reported to exert a renal protective effect. This study aims to investigate the role of cilnidipine, a novel CCBs, on CIN by regulating the calcium/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)/mitochondrial permeability transition pore(mPTP) pathway. Here, iohexol, a representative contrast media, was used to establish CIN model. KN-93 (CaMKⅡ inhibitor) and atractyloside (mPTP opener) were administered in rats, and CaMKⅡ overexpression was used in Human proximal tubular epithelial cells. Markers of renal injury (serum creatinine, blood urea nitrogen, and urinary NAGL), hematoxylin-eosin stain, oxidative stress (ROS, superoxide dismutase[SOD], and malondialdehyde [MDA] levels), cell death (MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling[TUNEL]), mitochondrial function (mPTP, mitochondrial membrane potential[MMP], and ATP) were assessed. Western blots were used to measure the expression levels of Bax/Bcl-2, caspase-3, CaMKⅡ/mPTP signaling pathways. Results showed that cilnidipine markedly improved kidney function, and alleviated tubular cell apoptosis, oxidative stress and mitochondrial damage induced by iohexol in vitro and in vivo. The underlying mechanism may be that cilnidipine relieved CaMKⅡ activation and mPTP opening induced by iohexol. All of these protective effects of cilnidipine were attenuated by CaMKⅡ overexpression and atractyloside (mPTP opener) pretreatment. Moreover, KN-93 (CaMKⅡ inhibitor) treatment showed a similar renal protective effect with cilnidipine, while the protective effect of cilnidipine on kidney in CIN rats was not further suppressed by KN-93 cotreatment. These in vitro and in vivo results point towardthe fact that cilnidipine might be a novel therapeutic drug against contrast-induced nephrotoxicity in a CaMKⅡ-dependent manner.

Proliferative and apoptotic evaluations of renal preventive effects of coenzyme Q10 in radioiodine-131 induced renal damage

The aim of this study was to investigated anti-proliferative and anti-apoptotic effects of coenzyme Q10 (CoQ10) in the prevention of radioiodine-131 (RAI) (I131) induced kidney damage. A total of 24 Wistar albino rats were separated into equal three groups (n = 8/group): Group 1 (control): untreated group; Group 2 (RAI): 3 mCi/kg RAI oral route; Group 3 (RAI+CoQ10): 3 mCi/kg RAI oral route and intraperitoneally 30 mg/kg/day CoQ10. CoQ10 treatment was started two days before RAI administration and was continued five days once daily after RAI. Pathomorphological parameters of kidneys were measured using hematoxylin–eosin and Masson’s trichrome staining. Immunohistochemically; proliferating cell nuclear antigen (PCNA), caspase 8, caspase 9 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) were used to determine proliferation and apoptosis. With the exception of the control group, varying degrees of inflammation, degeneration, necrosis, and interstitial/perivascular fibrosis were detected in the kidneys of all rats. This histopathological damage was found to be significantly less in CoQ10 group versus RAI group (P<0.05). The all immunohistochemical examinations demonstrated that administration of CoQ10 had reduced proliferation and apoptosis (P<0.05). The results of kidney histopathology and immunohistochemistry demonstrated that administration of CoQ10 had reduced inflammation, proliferation, and apoptosis. These findings show CoQ10 can play an important role in the radioprotection of kidneys against RAI-induced damage

Sevoflurane attenuates hepatic ischemia reperfusion injury by the miR-122/Nrf2 pathway

BackgroundSevoflurane can protect organs from ischemia-reperfusion (IR) injury, but the mechanism is still unclear. MicroRNA-122 (miR-122) is a liver-specific microRNA (miRNA) and regulates liver function. Therefore, this study aims to elucidate the relationship between the protective effect of sevoflurane and miR-122 in liver IR injury.MethodsWistar rats were divided into the following groups: sham, IR, IR + sevoflurane, IR + miR-122 antagomir, and IR + miR-122 antagomir + sevoflurane. Hematoxylin and eosin (H&E) staining and Suzuki score were used to evaluate the pathological damage of the liver. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 in the serum and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO) in the liver homogenate supernatant were detected by using the corresponding kit. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and flow cytometry was applied to evaluate the apoptosis of liver tissues. The expression of nuclear factor E2-related factor 2 (Nrf2), miR-122, p53, and HO-1 in liver tissue was evaluated by using immunohistochemistry, qRT-PCR, and western blot as needed.ResultsCompared to the IR group, the sevoflurane post-treatment or miR-122 antagomir groups showed improved liver injury, decreased Suzuki score, inhibited the levels of AST, ALT, LDH, MDA, NO, TNF-α, IL-1β, and IL-6, increased levels of SOD, IL-10, and inhibited hepatocyte apoptosis. Regarding the molecular mechanism, sevoflurane post-treatment fostered the expression of HO-1, promoted the transport of Nrf2 from cytoplasm to the nucleus, and decreased the expression of miR-122 and p53. The combined use of miR-122 antagomir and sevoflurane enhanced the protective effect of miR-122 antagomir in liver injury in IR rats.ConclusionsSevoflurane protected the liver from IR damage by regulating the miR-122/Nrf2/HO-1 pathway.

Reduced Bone Mass and Increased Osteocyte Tartrate-Resistant Acid Phosphatase (TRAP) Activity, But Not Low Mineralized Matrix Around Osteocyte Lacunae, Are Restored After Recovery From Exogenous Hyperthyroidism in Male Mice.

Hyperthyroidism causes secondary osteoporosis through favoring bone resorption over bone formation, leading to bone loss with elevated bone fragility. Osteocytes that reside within lacunae inside the mineralized bone matrix orchestrate the process of bone remodeling and can themselves actively resorb bone upon certain stimuli. Nevertheless, the interaction between thyroid hormones and osteocytes and the impact of hyperthyroidism on osteocyte cell function are still unknown. In a preliminary study, we analyzed bones from male C57BL/6 mice with drug-induced hyperthyroidism, which led to mild osteocytic osteolysis with 1.14-fold larger osteocyte lacunae and by 108.33% higher tartrate-resistant acid phosphatase (TRAP) activity in osteocytes of hyperthyroid mice compared to euthyroid mice. To test whether hyperthyroidism-induced bone changes are reversible, we rendered male mice hyperthyroid by adding levothyroxine into their drinking water for 4 weeks, followed by a weaning period of 4 weeks with access to normal drinking water. Hyperthyroid mice displayed cortical and trabecular bone loss due to high bone turnover, which recovered with weaning. Although canalicular number and osteocyte lacunar area were similar in euthyroid, hyperthyroid and weaned mice, the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive osteocytes was 100% lower in the weaning group compared to euthyroid mice and the osteocytic TRAP activity was eightfold higher in hyperthyroid animals. The latter, along with a 3.75% lower average mineralization around the osteocyte lacunae in trabecular bone, suggests osteocytic osteolysis activity that, however, did not result in significantly enlarged osteocyte lacunae. In conclusion, we show a recovery of bone microarchitecture and turnover after reversal of hyperthyroidism to a euthyroid state. In contrast, osteocytic osteolysis was initiated in hyperthyroidism, but its effects were not reversed after 4 weeks of weaning. Due to the vast number of osteocytes in bone, we speculate that even minor individual cell functions might contribute to altered bone quality and mineral homeostasis in the setting of hyperthyroidism-induced bone disease. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Neuroprotective effects of verbascoside against Alzheimer\u2019s disease via the relief of endoplasmic reticulum stress in A\u03b2-exposed U251 cells and APP/PS1 mice

BackgroundEndoplasmic reticulum (ER) stress is involved in the progression of Alzheimer’s disease (AD). Verbascoside (VB), an active phenylethanoid glycoside that was first isolated from Verbascum sinuatum (the wavyleaf mullein), possesses anti-inflammatory, antioxidative, and anti-apoptotic effects. The purpose of this study was to elucidate the beneficial effects of VB in amyloid β (Aβ)1–42-damaged human glioma (U251) cells and in APPswe/PSEN1dE9 transgenic (APP/PS1) mice.MethodsU251 cells were co-incubated with 10 μM of Aβ1-42 and treated with VB. The protective effects of VB were investigated by using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay, flow cytometry, fluorescence staining, and transmission electron microscopy. APP/PS1 transgenic mice were treated for 6 weeks with VB. Learning and memory were evaluated using a Morris water maze test. Immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, thioflavin-S staining, and proteomics analysis were performed to study the potential neuroprotective mechanism. Enzyme-linked immunosorbent assays and western blot were performed to analyze altered protein levels of brain lysates in APP/PS1 mice and/or Aβ1-42-damaged U251 cells.ResultsIn Aβ1-42-damaged U251 cells, VB significantly improved cell viability, inhibited apoptosis, reduced calcium accumulation and the intracellular concentrations of reactive oxygen species, and improved the morphology of mitochondria and ER. In APP/PS1 mice, 6-week administration of VB significantly improved memory and cognition. VB inhibited apoptosis, reduced the deposition of Aβ, reduced the formation of neurofibrillary tangles formed by hyperphosphorylated tau protein, and downregulated the expression levels of 4-hydroxynonenal and mesencephalic astrocyte-derived neurotrophic factor in the brains of APP/PS1 mice. Proteomics analysis of mouse hippocampus suggested that the neuroprotective effect of VB may be related to the reduction of ER stress. This was indicated by the fact that VB inhibited the three branches of the unfolded protein response, thereby attenuating ER stress and preventing apoptosis.ConclusionsThe results confirmed that VB possesses significant neuroprotective effects, which are related to the reduction of ER stress. These findings support the status of VB as a potentially effective treatment for AD and warrant further research.

Hsa_circNFXL1_009 modulates apoptosis, proliferation, migration, and potassium channel activation in pulmonary hypertension

In this study, we explored the circular RNA (circRNA) profile in pulmonary arterial hypertension (PAH) patients caused by chronic obstructive pulmonary disease (COPD) and the effects of hsa_circNFXL1_009 on abnormal proliferation, apoptosis, and migration of human pulmonary arterial smooth muscle cells (hPASMCs) driven by hypoxia. Using microarrays, we screened the circRNA profile in whole-blood samples from three pairs of subjects and found 158 dysregulated circRNAs in patients with PAH-COPD. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis further validated that hsa_circNFXL1_009 was dramatically downregulated with the highest area under a receiver operating characteristic curve (ROC) in 21 pairs of subjects. Consistently, exposure to hypoxia markedly reduced the hsa_circNFXL1_009 level in cultured hPASMCs. Delivery of exogenous hsa_circNFXL1_009 attenuated hypoxia-induced proliferation, apoptotic resistance, and migration of hPASMCs, as evidenced by immunocytochemistry, 5-ethynyl-2′-deoxyuridine incorporation, wound healing, and a TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) assay. A luciferase assay showed that hsa_circNFXL1_009 directly sponged hsa-miR-29b-2-5p (miR-29b) and positively regulated the expression of voltage-gated potassium (K+) channel subfamily B member 1 (KCNB1) at the mRNA level. Using patch-clamp electrophysiology, we proved that overexpression of hsa_circNFXL1_009 promoted a whole-cell K+ current in hPASMCs. Taken together, these studies identify hsa_circNFXL1_009 as a key regulator of PAH, and it may be used as a potential therapeutic target for the treatment of PAH.

PD08-03 EFFECTS OF DIFFERENT RADIOFREQUENCY ABLATION MODES ON NORMAL RENAL TISSUE AROUND THE ABLATION ZONE

You have accessJournal of UrologyKidney Cancer: Localized: Ablative Therapy (PD08)1 Apr 2020PD08-03 EFFECTS OF DIFFERENT RADIOFREQUENCY ABLATION MODES ON NORMAL RENAL TISSUE AROUND THE ABLATION ZONE Linfang Yao*, Wenmin Cao, Wei Wang, Wei Chen, Jiong Shi, Xingqun Zhao, and Hongqian Guo Linfang Yao*Linfang Yao* More articles by this author , Wenmin CaoWenmin Cao More articles by this author , Wei WangWei Wang More articles by this author , Wei ChenWei Chen More articles by this author , Jiong ShiJiong Shi More articles by this author , Xingqun ZhaoXingqun Zhao More articles by this author , and Hongqian GuoHongqian Guo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000835.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To explore the effects of radiofrequency ablation "slow" or "fast" mode on normal renal tissue around the ablation zone. METHODS: The animal model of rabbit renal VX2 tumor was established. Radiofrequency ablation of rabbit renal VX2 tumors was performed in a "slow" or "fast" mode. The thermocouple temperature measurement system(NATIONAL INSTRUMENTS Company, USA) monitored the temperature changes of the ablation zone and the renal tissue around the ablation zone during the radiofrequency ablation. The formation and range of radiofrequency ablation were monitored in real time by B-ultrasound imaging system (Flex Focus 800 (BK Medical).Conventional pathological hematoxylin-eosin staining was used to observe the necrosis. Cell necrosis and apoptosis were detected by trypan blue staining and TUNEL(terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling). The change of cell microstructure was examined under a transmission electron microscope. RESULTS: Radiofrequency ablation of rabbit renal VX2 tumors in either "slow" or "fast" mode formed an ideal range of ablation zone to cover the tumor.The average temperature within the ablation area reached 80 degree centigrade and a significant ablation zone could be formed. But the "fast" mode of radiofrequency ablation often leaded to a temperature of more than 60 degree centigrade in normal kidney tissue around the ablation zone. While under a "slow" mode, the temperature of normal surrounding kidney tissue was about 40 degree centigrade. The "fast" mode of radiofrequency ablation could easily lead to damage to normal renal tissue around the ablation zone followed with degeneration and necrosis. Trypan blue staining was positive in the damaged tissue cells. Apoptosis index and mitochondrial injury were increased. While the "slow" mode of radiofrequency ablation did not cause significant damage to normal renal tissue surrounding the ablation zone. CONCLUSIONS: Radiofrequency ablation of renal tumors should be performed in a "slow" mode to minimize damage to normal renal tissue around the ablation zone. Source of Funding: This work was supported by the National Natural Science Foundation of China (Grant No. 81972388, 81772710) , Nanjing health science and technology development special fund project (YKK18076), Jiangsu postdoctoral research fund (1701023B), the Project of Invigorating Health Care through Science, Technology and Education, Jiangsu Provincial Key Medical Discipline (Laboratory) (ZDXKB2016014). © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e168-e168 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Linfang Yao* More articles by this author Wenmin Cao More articles by this author Wei Wang More articles by this author Wei Chen More articles by this author Jiong Shi More articles by this author Xingqun Zhao More articles by this author Hongqian Guo More articles by this author Expand All Advertisement PDF downloadLoading ...

Responses of Immune Organs after Cerebral Ischemic Stroke.

Stroke is a leading cause of death and disability worldwide. Recently, secondary damage to the brain has been hypothesized as a key aggravating element in the ischemic cascade. However, the interaction between cerebral infarction and immune organs is not fully understood. In this study, we investigated changes in rat brain, spleen, thymus, mesenteric lymph node, and liver at 3, 7, and 13 days after transient middle cerebral artery occlusion (tMCAO) by immunohistochemistry. Rat models of stroke were made by tMCAO. Functional assessment was performed at 3 h and 1, 3, 5, 7, 9, 11, and 13 days after MCAO. Rat organs were harvested for 2,3,5-triphenyltetrazolium chloride staining and immunohistochemistry. The number of CD8α+ T cells decreased in spleen, thymus, mesenteric lymph node, and liver and increased in brain. Numbers of Iba1+ and CD68+ macrophages decreased in spleen, thymus, and mesenteric lymph node and increased in brain and liver. Ki67+ cells exhibited the same characteristics as macrophages, and increased numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) -positive apoptotic cells were present in spleen, mesenteric lymph node, liver, and brain. The present results indicate that stroke is a systemic disease that, in addition to affecting the brain, also induces responses in immune organs. These results suggest that systemic treatment might be a good strategy for clinical stroke care.

Long-term effect of low-level diode laser irradiation on proliferation of stem cells from human exfoliated deciduous teeth after cryopreservation protocol

Low-level laser irradiation (LLLI) could be useful for the stimulation of stem-cell proliferation. LLLI has important physiological properties which increase the mitochondrial activity of cells. This study aimed to evaluate the biological effect of LLLI on cryopreserved stem cell from human exfoliated deciduous teeth (SHEDs). Cryopreserved and characterised SHEDs from a previous study were used for this study. The SHEDs were exposed to GaAlAs diode laser with energy densities of 1.2 (Group 2) and 2 J cm−2 (Group 3). For the control group (Group 1), SHEDs were cultured in culture medium including Dulbecco’s modified Eagle’s Medium supplemented with 15% foetal bovine serum, 1% L-glutamine, 1% penicillin-streptomycin, 1% gentamycin, and amphotericin-B. The cells in the experimental groups were irradiated as previously indicated, and after the irradiation were incubated for 0, 24, 48, and 72 h at 37 °C in 5% CO2. Proliferation of SHEDs was analysed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay at incubation periods of 0, 24, 48, and 72 h after the single-laser irradiation. The effects of laser irradiation on apoptosis of SHEDs at the last incubation period (72 h) were detected with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method. Group 2 showed increased TUNEL-positive cells compared to Groups 1 and 3, but no significant differences between the three groups were observed (p > 0.05). Comparing Groups 2 and 3 to Group 1, the proliferation of SHEDs significantly increased after 24, 48, and 72 h (p < 0.05), but no significant differences were observed between the groups at 0 h (p > 0.05). In addition to this, at 72 h, Group 3 showed significantly higher proliferative effect on SHEDs compared to Group 2 (p > 0.05). In terms of clinical usage of long-term cryopreserved SHEDs, we can recommend to clinicians and researchers that prior to their use, LLLI may be used to increase the proliferation rate.

G protein-coupled estrogen receptor activation improves contractile and diastolic functions in rat renal interlobular artery to protect against renal ischemia reperfusion injury.

This work aimed to investigate whether G protein-coupled estrogen receptor (GPER) can improve the renal interlobular artery vascular function by increasing the NO content, thereby protecting against renal ischemia-reperfusion (IR) injury. This study classified ovariectomised (OVX) female Sprague-Dawley rats into OVX, OVX + IR, OVX + IR + G1 (the GPER agonist G1), OVX + IR + G1+G15 (GPER blocker) and OVX + IR + G1+L-NAME (eNOS blocker) groups. Enzyme-linked immunosorbent assay was performed to detect the estrogen levels in the body and eliminate interference from endogenous estrogens. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and HE staining, renal function test and Paller scoring were performed to identify the successful model and detect the degree of renal and renal interlobular arteries injury. The in vitro microvascular pressure diameter measurement technique was used to detect the contraction and diastolic activities of the renal interlobular arteries in each group. Immunofluorescence technique was used to observe the localisation and expression levels of GPER and eNOS in renal interlobular arteries. The GPER and eNOS protein expression levels in each group were detected by Western blot. The NO content in the serum of each group was detected by the nitrate reductase method. After OVX, the estrogen level in the body decreased significantly (P < 0.01), and TUNEL staining showed a significant increase in the degree of renal tubular epithelial cell apoptosis in the IR group. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were significantly increased in the IR group (P < 0.01), and the Paller score showed significantly increased kidney damage. When performing drug treatment, the G1 intervention group significantly decreased serum BUN and SCr levels after IR injury (P < 0.01). The Paller score showed significantly decreased the degree of renal injury (P < 0.01). After IR, the renal interlobular artery contraction rate and systolic velocity of blood vessels were significantly decreased (P < 0.01). The G1 intervention group significantly restored contraction rate and systolic velocity of blood vessels (P < 0.01), and G15 and L-NAME partially reversed this effect (P < 0.01). Immunofluorescence technique showed that GPER was expressed in renal interlobular artery smooth muscle and endothelial cells. After IR injury, the GPER protein expression increased, and the eNOS protein expression decreased significantly (P < 0.01). Western blot showed that after IR injury, the GPER protein expression increased, and the eNOS protein expression decreased significantly. After G1 intervention, the GPER content did not change, and the eNOS content increased significantly (P < 0.01). After ischemia and reperfusion, the serum NO content decreased significantly, but it increased after G1 intervention. G15 and L-NAME reversed the effects of G1 to varying degrees (both at P < 0.01). GPER may improve the renal interlobular artery vascular function by increasing the NO content, thereby protecting against renal IR injury.

Evaluation of low-level diode laser irradiation and various irrigant solutions on the biological response of stem cells from exfoliated deciduous teeth

This study aimed to evaluate cytotoxic effects and the apoptosis of Gallium-Aluminum-Arsenide (GaAlAs) diode laser irradiation, sodium hypochlorite (NaOCl), ozonated water and ethylene diamine tetraacetic acid (EDTA) on stem cells from human exfoliated deciduous teeth (SHEDs). Cells were exposed to EDTA (5%, 8.5%, 17%), NaOCl (1%, 2.5%, 5%) ozonated water (5, 10, 20 μg/ml) and GaAlAs diode laser irradiation (energy densities of 0.5, 1, 1.5 j/cm2). Culture medium included D-MEM, supplemented with 15% foetal bovine serum, 1% l-glutamine, 1% penicillin-streptomycin, 1% gentamycin, amphotericin-B and served as control group. The prepared irrigants were added to the relevant wells and incubated with the cells at 37 °C for 5, 10 and 15 min. The cells in the laser group were also incubated at 37 °C for 5, 10 and 15 min after the laser application. Cell viability and proliferation were analysed with the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The percentage of cell viability showed a significant reduction in all concentrations of the EDTA and NaOCl groups when compared to the control group, diode laser irradiation and ozonated water groups at 5th, 10th and 15th minutes respectively but high cytotoxic effects of all EDTA and NaOCl groups with decreased over 50% of cell viability were observed at the 15th minute. Also EDTA group with 17% concentration (17%E) presented the lowest survival rate on SHEDs with mean of 21.67% ± 6.101 at this time interval. The lowest toxic effects were observed at the 5th minutes compared to other time periods at experimental groups. For detection of apoptotic cells, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method was performed. According to the MTT results, doses showed the highest toxicity (cell survival decreased over 50%) in each group were selected for TUNEL assay (17% EDTA; 1% NaOCl; 10 μg/ml Ozonated water; 1.5 j/cm2 diode laser irradiation). The significantly lowest percentages of TUNEL-positive cells were detected in ozonated water (10.67% ± 2.93) and diode laser irradiation (13.24% ± 7.61) compared to EDTA (39.89% ± 11.54) and NaOCl (31.15% ± 10.64) respectively. Also the difference between percentage of TUNEL-positive cells in EDTA and NaOCl groups was not significant. Synergistic combination of ozonated water and diode laser irradiation may be used in the disinfection step of necrotic root canals.

Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-β1).

ObjectivesThe role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.MethodsIn this study, TGF-β1 from osteoclasts and knee joints were analyzed using a co-cultured cell model and an OA rat model, respectively. Five patients with a femoral neck fracture (four female and one male, mean 73.4 years (68 to 79)) were recruited between January 2015 and December 2015. Results showed that TGF-β1 was significantly upregulated in osteoclasts by cyclic loading in a time- and dose-dependent mode. The osteoclasts were subjected to cyclic loading before being co-cultured with chondrocytes for 24 hours.ResultsA significant decrease in the survival rate of co-cultured chondrocytes was found. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay demonstrated that mechanical stress-induced apoptosis occurred significantly in co-cultured chondrocytes but administration of the TGF-β1 receptor inhibitor, SB-505124, can significantly reverse these effects. Abdominal administration of SB-505124 can attenuate markedly articular cartilage degradation in OA rats.ConclusionMechanical stress-induced overexpression of TGF-β1 from osteoclasts is responsible for chondrocyte apoptosis and cartilage degeneration in OA. Administration of a TGF-β1 inhibitor can inhibit articular cartilage degradation.Cite this article: R-K. Zhang, G-W. Li, C. Zeng, C-X. Lin, L-S. Huang, G-X. Huang, C. Zhao, S-Y. Feng, H. Fang. Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-β1). Bone Joint Res 2018;7:587–594. DOI: 10.1302/2046-3758.711.BJR-2018-0057.R1.

Survivin overexpression via adeno-associated virus vector Rh10 ameliorates ischemic damage after middle cerebral artery occlusion in rats.

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 3weeks later with transient MCAO for 90min. 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 24hr 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.

What should be done for men with sperm DNA fragmentation?

In an age when a small quantity of sperm can lead to pregnancy through in vitro fertilization or intracytoplasmic sperm injection, selecting healthy sperm is important. Sperm DNA fragmentation (SDF) is known to be higher in infertile men. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and the alkaline comet test are SDF tests that directly measure DNA damage and have shown closer correlations with assisted reproduction results than indirect tools such as the sperm chromatin structure assay or the sperm chromatic dispersion test. It is difficult; however, to endorse a single test as the best test overall; instead, it is best to select a testing method based on each patient's clinical condition and goals. In a couple struggling with infertility, if the male partner has a high level of SDF, he should aim to decrease SDF through lifestyle modifications, antioxidant treatment, and ensuring an appropriate duration of abstinence, and physicians need to treat the underlying diseases of such patients. If sperm DNA damage continues despite the patient's and physician's efforts, other methods, such as micromanipulation-based sperm selection or testicular sperm extraction, should be used to select healthy sperm with nuclear DNA integrity.

Integration of sperm DNA damage assessment into OECD test guidelines for genotoxicity testing using the MutaMouse model

The Organisation for Economic Co-operation and Development (OECD) endorses test guidelines (TG) for identifying chemicals that are genotoxic, such as the transgenic rodent gene mutation assay (TG 488). Current OECD TG do not include assays for sperm DNA damage resulting in a critical testing gap. We evaluated the performance of the Sperm Chromatin Structure Assay (SCSA) and the Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick end Labeling (TUNEL) assay to detect sperm DNA damage within the recommended TG 488 protocol. MutaMouse males received 0, 0.5, 1, or 2 mg/kg/day triethylenemelamine (TEM), a multifunctional alkylating agent, for 28 days orally and tissues were collected two (blood) and three (sperm and bone marrow) days later. TEM significantly increased the frequency of lacZ mutants in bone marrow, and of micronuclei (MN) in both reticulocytes (%MN-RET) and normochromatic erythrocytes (%MN-NCE) in a dose-dependent manner (P < 0.05). The percentage of DNA fragmentation index (%DFI) and %TUNEL positive cells demonstrated dose-related increases in sperm (P < 0.05), and the two assay results were strongly correlated (R = 0.9298). Within the same animal, a good correlation was observed between %MN-NCE and %DFI (R = 0.7189). Finally, benchmark dose modelling (BMD) showed comparable BMD10 values among the somatic and germ cell assays. Our results suggest that sperm DNA damage assays can be easily integrated into standard OECD designs investigating genotoxicity in somatic tissues to provide key information on whether a chemical is genotoxic in germ cells and impact its risk assessment.

Ketamine induces reactive oxygen species and enhances autophagy in SV-HUC-1 human uroepithelial cells.

This study was aimed at exploring the underlying mechanisms of ketamine in the SV-40 immortalized human ureteral epithelial (SV-HUC-1) cells. The viability and apoptosis of SV-HUC-1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit-8(CCK-8) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis-related proteins (B-cell lymphoma 2 [Bcl-2] and Bax) and autophagy-associated proteins (light chain 3-I [LC3-I] and LC3-II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3-methyladenine (3-MA) or N-acetyl-l-cysteine (NAC), the biological functions of SV-HUC-1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK-8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV-HUC-1 cells and accelerated apoptosis of SV-HUC-1 cells through regulating the expression level of IKBα (phospho), nuclear factorкB (P65), Bcl-2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine-treated SV-HUC-1 cells treated with ketamine. Ketamine-induced autophagosomes in SV-HUC-1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV-HUC-1 cells in a dose-dependent and time-dependent manner. Additionally, cotreatment of NAC with 3-MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV-HUC-1 cell autophagy and impaired the cell viability of SV-HUC-1 cells by inducing ROS.

P38 Mitogen-Activated Protein Kinase is Involved in the Pathogenesis of Endometriosis by Modulating Inflammation, but not Cell Survival.

Local pro-inflammatory environment and enhanced cell survival contribute to the endometriosis development. A serine/threonine kinase p38 mitogen-activated protein kinase (MAPK) mediates intracellular signaling of cytokine production, cell proliferation, and apoptosis in different cell types. The current study compares p38 MAPK activity in normal endometrium and endometriosis, and assesses role(s) of p38 MAPK on cytokine production and cell survival in endometriosis. Immunohistochemical levels of total and phosphorylated (active) p38 MAPK as well as its correlation with interleukin 8 (IL-8) expression, and cell proliferation and apoptosis were compared in normal human endometrium and endometriosis. The action of p38 MAPK on pro-inflammatory cytokine-induced IL-8 and monocyte chemotactic protein (MCP)-1 expression in endometriotic cells were assessed by enzyme-linked immunosorbent assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival, 5-bromo-2'-deoxyuridine incorporation, and Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assays were used to determine the function of p38 MAPK in cultured human endometriotic stromal cell proliferation and apoptosis. p38 MAPK activity was significantly higher in both eutopic and ectopic endometria compared to normal endometria during late proliferative and early secretory phases ( P < .05). Increased p38 MAPK activity in endometriotic cells correlated with IL-8 expression (Pearson correlation coefficient r = 0.83, P < .01), but not with apoptosis in vivo. The pro-inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α induced activation of p38 MAPK. Inhibition of p38 MAPK activity blocked IL-1β and TNF-α-induced IL-8 and MCP-1 secretion in cultured endometriotic stromal cells ( P < .05), but did not impact on endometriotic cell survival. These results suggest that rather than modulating cell survival, increased p38 MAPK activity in endometriotic cells contributes to the pathogenesis of endometriosis by promoting the local inflammatory milieu.

Sperm DNA and detection of DNA fragmentations in sperm.

The questionable effectiveness of routine sperm parameters in determining male factor infertility problems and increasing the success rates of assisted reproductive techniques have led to the investigation of more detailed sperm parameters that could affect the male fertility and reproduction. Thus, the effects of different sperm parameters such as sperm DNA integrity was started to be investigated thanks to the previously described methods such as single cell gel electrophoresis (COMET) assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), terminal deoxynucleotidyl transferase-mediated deoxyuridine (TdT) triphosphate (dUTP) nick end labeling (TUNEL) assay and sperm chromatin dispersion (SCD) test. However, studying on sperm DNA might be very complex because the sperm DNA differs from the somatic cell DNA with its unique structure. Also, the sperm DNA undergoes many changes during spermatogenesis and it is condensed by being packaged tightly with different types and numbers of protamines in different species. Despite all these difficulties, these methods provide important information about the reasons and consequences of DNA damages in sperm and the effects of these damages on reproduction.