Terminal dUTP Nick End Labeling Research Articles

Hippocampal Crhr1 conditional gene knockout ameliorated the depression-like behavior and pathological damage in male offspring mice caused by chronic stress during pregnancy

Numerous studies have demonstrated that chronic stress during pregnancy (CSDP) can induce depression and hippocampal damage in offspring. It has also been observed that high levels of corticotropin-releasing hormone (CRH) can damage hippocampal neurons, and intraperitoneal injection of a corticotropin releasing hormone receptor 1 (CRHR1) antagonist decreases depression-like behavior and hippocampal neuronal damage in a mouse depression model. However, whether CSDP causes hippocampal damage and depression in offspring through the interaction of CRH and hippocampal CRHR1 remains unknown and warrants further investigation. Therefore, hippocampal Crhr1 conditional gene knockout mice and C57/BL6J mice were used to study these questions. Depression-related indexs in male offspring mice were examined using the forced swim test (FST), sucrose preference test (SPT), tail suspension test (TST) and open field test (OFT). Serum CRH levels were measured by enzyme-linked immunosorbent assay (ELISA). Golgi-Cox staining was used to examine the morphological changes of hippocampal neuronal dendrites. Neuronal apoptosis in the hippocampal CA3 regions was detected by terminal deoxynucleotidy transferase dUTP nick end labeling (TUNEL) staining. The levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR) and protein kinase B (AKT) proteins were measured by Western blot analysis. This study showed that CSDP induces depression-like behavior, hippocampal neuronal dendrite damage and apoptosis in male offspring mice. Conditional gene knockout of hippocampal Crhr1 in mice reduced CSDP-induced depression-like behavior, hippocampal neuronal dendrite damage and apoptosis in male offspring, and counteracted the CSDP-induced decreased expression of p-Akt and mTOR activity in male offspring hippocampus. These findings demonstrated that CSDP might inhibit the Akt/mTOR pathway by increasing the levels of CRH, leading to increased CRH-mediated activation of hippocampal CRHR1, thereby inducing synaptic impairment and apoptosis in hippocampal neurons, which in turn leads to depression-like behavior in offspring.

P-070 The effect of surgical interventions in sperm DNA fragmentation: a systematic review and meta-analysis

Abstract Study question Do surgical interventions improve sperm DNA fragmentation? Summary answer Orchiectomy, bariatric surgery and varicocelectomy improve sperm DNA fragmentation. What is known already Sperm DNA fragmentation, which is known to be one of the functional alterations that harms men’s fertile potential, is caused by several factors, and one of the most important is oxidative stress, caused by accumulation of reactive oxygen species (ROS) beyond the physiological limits of the organism. Some examples of diseases that cause this increase in ROS are testicular tumors, obesity and varicocele. Once they all have surgical corrections, the aim of this study was to observe if orchiectomy, bariatric surgery and varicocelectomy had a positive impact in sperm DNA fragmentation. Study design, size, duration A systematic review was developed, including 33 articles: 1 about orchiectomy, 4 about bariatric surgery, and the remaining ones about varicocelectomy. After the review was finished, a meta-analysis was conducted with all the numeric information extracted from the articles. The study was registered on PROSPERO database and was performed based on PRISMA guideline. Participants/materials, setting, methods Data extraction was made by two reviewers independently, and all the information was tabulated in a systematic spreadsheet. 7 articles were excluded, because they did not meet the inclusion criteria: they either included analysis of adolescent spermatogenesis or did not include analysis of sperm DNA fragmentation, and 5 articles were excluded from statistical analysis due to the studies’ designs and lack of essential statistical data. The statistical analysis was conducted by dmetar, metafor e meta. Main results and the role of chance The studies were carried out with men whose seminal samples were analysed before and after surgical intervention. The type of intervention (orchiectomy, bariatric surgery or varicocelectomy) and the way sperm DNA fragmentation was analysed (Terminal deoxynucleotidyl transferase dUTP nick end labeling – TUNEL, Cometa Assay or The Sperm Chromatin Structure Assay – SCSA) were not moderators of the estimated effect. However, the effect estimated increased according to the duration of the follow-up. When duration of follow-up was short (up to six months), there was no effect of interventions on sperm DNA fragmentation. The effect was greater when the duration was average (between six and nine months) and long (more than nine months). There was a reduction in sperm DNA fragmentation with interventions (effect estimated [confidence range 95%] = -1.22 [-1.65; -0.78]; p < 0.001). Despite different techniques were used to analyse sperm DNA fragmentation, the present study showed an improvement in this functional alteration after surgical interventions. Limitations, reasons for caution The small number of participants included in each article analysed, the heterogeneity between the follow-up time and the lack of quality numeric information in literature are possible limitations of this study. Besides, the fact that varicocelectomy had a bigger weight in this study must be analysed critically. Wider implications of the findings Therefore, this study is considered an important source for consistent and reliable information about sperm DNA fragmentation and the effect of surgical interventions on it. It is necessary to expand the studies on this topic, mostly due to the lack of randomized clinical trials. Trial registration number not applicable

Injection of Collagen Binding Domain-Brain Derived Neurotrophic Factor Promotes SIRT1 Expression: Improving Neuroinflammation in Experimental Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease, often leading to neuroinflammation and neuronal damage. Activation of the Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is closely associated with post-SAH neuroinflammation, while activation of Nicotinamide Adenine Dinucleotide (NAD)-dependent deacetylase sirtuin-1 (SIRT1) has neuroprotective effects. This study aimed to investigate the impact of injectable Collagen Binding Domain-Brain Derived Neurotrophic Factor (CBD-BDNF) on neuroinflammation and neuronal damage following SAH. After establishing the SAH model, experimental animals were divided into three groups: sham surgery group (Sham), SAH group, and SAH+neuroregenerative scaffold (CBD-BDNF treatment) group. Behavioral performance was evaluated using neurofunctional deficit, beam balance, and Y-maze tests. Expression of inflammatory factors and essential proteins was quantitatively analyzed using Enzyme-Linked Immunosorbent Assay (ELISA) kits and immunoblotting. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) staining was used to assess cell apoptosis. To further investigate the mechanism of action of CBD-BDNF on SIRT1, the model animals were treated with EX527 (SIRT1 inhibitor) for comparative studies. Neurological deficit tests, CBD-BDNF improves functional outcomes after SAH. Compared to the SAH group, the SAH+neuroregenerative scaffold group showed significantly increased expression of SIRT1 protein and significantly decreased expression of NLRP3, Apoptosis-associated speck-like protein containing a CARD (ASC), and c-caspase-1. The inflammatory cytokines Interleukin-1 beta (IL-1β), IL-6, and IL-18 levels also significantly decreased in the SAH+neuroregenerative scaffold group. Additionally, animals in the SAH+neuroregenerative scaffold group showed better neurofunctional recovery in neurofunctional deficit and beam balance tests. The number of apoptotic cells significantly decreased in the SAH+neuroregenerative scaffold group compared to the SAH group. However, when SIRT1 was inhibited with EX527, the aforementioned neuroprotective effects were reversed, indicating the involvement of CBD-BDNF through SIRT1 activation. This study demonstrates that injectable CBD-BDNF can significantly alleviate neuroinflammation and neuronal damage resulting from SAH by blocking NLRP3 inflammasome activation and promoting SIRT1 expression. These findings provide a new therapeutic strategy for neuroprotection after SAH and reveal the mechanism of action of CBD-BDNF as a potential therapeutic agent. Future research will further explore the long-term efficacy and safety of CBD-BDNF.

Ganoderic acid a derivative induces apoptosis of cervical cancer cells by inhibiting JNK pathway

ObjectiveGanoderic acid A can inhibit the proliferation and promotes the apoptosis of cancer cells. Surprisingly, the molecular mechanisms underlying the anti-cancer effects of ganoderic acid A still remain poorly defined. Ganoderic acid A derivative (GaAD19) is an effective ingredient obtained by structural modification of ganoderic acid A. The purpose of this study was to evaluate the anti-proliferation effect of GaAD19 on cervical cancer cells. MethodsThrough the HeLa cervical cancer cell model, the drug target of GaAD19 was predicted using the SwissTargetPrediction database and molecular docking. Subsequently, computer analysis results were verified by a series of molecular biology experiments, such as flow cytometry, Western blot, immunocytochemical staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), quantitative real time polymerase chain reaction (qPCR), and so on. Then, pathway agonists and inhibitors were used to investigate the mechanism of GaAD19. Finally, the mouse model of cervical cancer was established to evaluate the inhibitory effect of GaAD19 on tumor growth in U14 cervical cancer mice. ResultsGaAD19 induced apoptosis and inhibited the growth of tumors. It also blocked the transition from the G1 to the S phase of the cell cycle. However, in the presence of a c-Jun N-terminal kinase (JNK)agonist, the effects of GaAD19 on the proliferation, apoptosis, and cell cycle transition of cancer cells were suppressed. ConclusionsThis study showed that GaAD19 can play an anti-cervical cancer role by inhibiting the JNK signaling pathway. These results will be helpful in further exploring the mechanism of GaAD19 in the treatment of cervical cancer.

Therapeutic Effect of Donepezil on Neuroinflammation and Cognitive Impairment after Moderate Traumatic Brain Injury.

Despite the important clinical issue of cognitive impairment after moderate traumatic brain injury (TBI), there is currently no suitable treatment. Here, we used in vitro and in vivo models to investigate the effect of Donepezil-an acetylcholinesterase (AChE) inhibitor-on cognitive impairment in the acute period following injury, while focusing on neuroinflammation and autophagy- and mitophagy-related markers. The purpose of the in vitro study was to investigate potential neuroprotective effects in TBI-induced cells after donepezil treatment, and the in vivo study, the purpose was to investigate therapeutic effects on cognitive impairment in the acute period after injury by analyzing neuroinflammation and autophagy- and mitophagy-related markers. The in vitro TBI model involved injuring SH-SY5Y cells using a cell-injury controller and then investigating the effect of donepezil at a concentration of 80 μM. The in vivo TBI model was made using a stereotaxic impactor for male C57BL/6J mice. Immuno-histochemical markers and cognitive functions were compared after 7 days of donepezil treatment (1 mg/kg/day). Mice were divided into four groups: sham operation with saline treatment, sham operation with donepezil treatment, TBI with saline treatment, and TBI with donepezil treatment (18 mice in each group). Donepezil treatment was administered within 4 h post-TBI. In vitro, donepezil was found to lead to increased cell viability and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), along with decreased reactive oxygen species (ROS), lactate-dehydrogenase (LDH), 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA)-positive cells, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. The mRNA and protein expressions of neuroinflammation (Cyclooxygenase-2, COX-2; NOD-like receptor protein 3, NLRP3; Caspase-1; and Interleukin-1 beta, IL-1β), as well as autophagy- and mitophagy-related markers (death-associated protein kinase 1, DAPK1; PTEN-induced kinase 1, PINK1; BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like, BNIP3L; Beclin-1, BECN1; BCL2-associated X protein, BAX; microtubule-associated protein 1A/1B-light chain 3B (LC3B); Sequestosome-1; and p62) were all found to decrease after donepezil treatment. The in vivo study also showed that donepezil treatment resulted in decreased levels of cortical tissue losses and brain swelling in TBI compared to the TBI group without donepezil treatment. Donepezil treatment was also shown to decrease the mRNA and Western blotting expressions of all markers, and especially COX-2 and BNIP3L, which showed the most significant decreases. Moreover, TBI mice showed an decreased escape latency, increased alteration rate, and improved preference index, altogether pointing to better cognitive performance after donepezil treatment. Donepezil treatment may be beneficial in improving cognitive impairment in the early phase of moderate traumatic brain injury by ameliorating neuroinflammation, as well as autophagy and mitophagy.

Synergistic Effects of 125I Seed Implantation Brachytherapy and Gemcitabine in Pancreatic Tumors.

Monotherapy consisting of radiotherapy or chemotherapy has limited efficacy in pancreatic tumors. This study aims to investigate whether the combination of 125I brachytherapy and gemcitabine (GEM) chemotherapy has a synergistic effect on pancreatic cancer (PC). In vitro, PANC-1 cells in the exponential phase were treated with 125I radioactive seeds (6 Gy) and GEM (30 nM). Cell proliferation, apoptosis, and mitochondrial membrane potential were measured using the Cell Counting Kit-8 (CCK-8) assay, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and flow cytometry, respectively. In vivo, we examined the inhibitory effect of three different treatment regimens on tumor growth in mice when combined with 125I brachytherapy and GEM. Next, we investigated the effects of the optimal scheme among the three on the tumor microenvironment, tumor tissue morphology, tumor cell apoptosis, systemic inflammatory response, and levels of apoptosis-related proteins in the tumor. Changes in the tumor microenvironment and levels of apoptosis-related proteins were measured by Western blot. The extent of damage to tumor tissue morphology was assessed by Hematoxylin and Eosin (HE) staining. Tumor cell apoptosis was measured by TUNEL staining. Changes in inflammation-related factors were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The results of in vitro cell experiments demonstrated that the combination of 125I radioactive seeds (6 Gy) and GEM (30 nM) had a stronger inhibitory effect on PANC-1 cells than either alone (p < 0.05). In vivo, data showed that the GEM (after 3 d) + 125I treatment group had the strongest tumor inhibition effect on PC (p < 0.05). Western blot analysis showed that the combined treatment of 125I brachytherapy and GEM caused changes in the expression of collagen and connexin in the tumor microenvironment, promoted tumor cell apoptosis, upregulated the expression of pro-apoptotic proteins, and helped to restore pancreatic function (p < 0.01). Our research results suggest that the strategy of 125I seed implantation surgery in mice after 3 days of GEM treatment has a more pronounced synergistic effect on the treatment of PC.

KLF13 Attenuates Lipopolysaccharide-Induced Alveolar Epithelial Cell Damage by Regulating Mitochondrial Quality Control via Binding PGC-1α.

Sepsis is a clinically life-threatening syndrome, and acute lung injury is the earliest and most serious complication. We aimed to assess the role of kruppel-like factor 13 (KLF13) in lipopolysaccharide (LPS)-induced human alveolar type II epithelial cell damage and to reveal the possible mechanism related to peroxisome proliferator-activated receptor-γ co-activator 1-α (PGC-1α). In LPS-treated A549 cells with or without KLF13 overexpression or PGC-1α knockdown, cell viability was measured by a cell counting kit-8 assay. Enzyme-linked immunosorbent assay kits detected the levels of inflammatory factors, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining measured cell apoptosis. Besides, mitochondrial reactive oxygen species (MitoSOX) and mitochondrial membrane potential were detected using MitoSOX red- and JC-1 staining. Expression of proteins related to mitochondrial quality control (MQC) was evaluated by western blot. Co-immunoprecipitation (Co-IP) assay was used to analyze the interaction between KLF13 and PGC-1α. Results indicated that KLF13 was highly expressed in LPS-treated A549 cells. KLF13 upregulation elevated the viability and reduced the levels of inflammatory factors in A549 cells exposed to LPS. Moreover, KLF13 gain-of-function inhibited LPS-induced apoptosis of A549 cells, accompanied by upregulated BCL2 expression and downregulated Bax and cleaved caspase3 expression. Furthermore, MQC was improved by KLF13 overexpression, as evidenced by decreased MitoSOX, JC-1 monomers and increased JC-1 aggregates, coupled with the changes of proteins related to MQC. In addition, Co-IP assay confirmed the interaction between KLF13 and PGC-1α. PGC-1α deficiency restored the impacts of KLF13 upregulation on the inflammation, apoptosis, and MQC in LPS-treated A549 cells. In conclusion, KLF13 attenuated LPS-induced alveolar epithelial cell inflammation and apoptosis by regulating MQC via binding PGC-1α.

OIP5-AS1/miR-455-3p/microfibril-associated protein 2 axis exacerbates the progression of thyroid carcinoma.

The long non-coding RNA (lncRNA) Opa interacting protein 5-antisense RNA 1 (OIP5-AS1) has been shown to participate in numerous biological and pathological processes, notably including oncogenesis. OIP5-AS1 modulates oncogenic or anti-tumor activities by controlling various microRNAs (miRs) in diverse cancer types. This study sought to examine the potential role of the lncRNA OIP5-AS1-mediated miR-455-3p/microfibril-associated protein 2 (MFAP2) axis and its influence on the progression of thyroid carcinoma. Cell proliferation, migration, and apoptosis were assessed through in vitro experimental measurements, which involved the use of Cell Counting Kit 8 (CCK8), transwell, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining techniques. The estimate algorithm was employed to examine the relationship between MFAP2 and the Stromal score, Immune score, and ESTIMATE score. OIP5-AS1 expression was significantly more elevated in the thyroid carcinoma tissues and cell lines than the corresponding normal non-tumor tissues and cell lines. Following transfection with short-hairpin (sh)-OIP5-AS1, the CAL62 and SW1736 cells upregulated miR-455-3p and downregulated the MFAP2 expression levels. The downregulation of OIP5-AS1 expedited cellular apoptosis and hindered cellular proliferation and migration in the CAL62 and SW1736 cells. The in vitro experiments showed that both the suppression of MFAP2 and the increased expression of miR-455-3p exerted significant anti-cancer effects. In addition, the overexpression of MFAP2 counteracted the in vitro antineoplastic effects of the sh-OIP5-AS1 and miR-455-3p mimics. The results suggest that lncRNA OIP5-AS1 plays a crucial role in the advancement of thyroid carcinoma by inhibiting miR-455-3p to activate MFAP2.

GOLPH3 knockdown alleviates the inflammation and apoptosis in lipopolysaccharide-induced acute lung injury by inhibiting Golgi stress mediated autophagy

Pneumonia, an acute inflammatory lesion of the lung, is the leading cause of death in children aged < 5 years. We aimed to study the function and mechanism of Golgi phosphoprotein 3 (GOLPH3) in infantile pneumonia. Lipopolysaccharide (LPS)-induced acute lung injury (ALI) mice and injury of MLE-12 cells were used as the pneumonia model in vitro. After GOLPH3 was knocked down, the histopathological changes of lung tissues were assessed by hematoxylin-eosin (H&E) staining. The Wet/Dry ratio of lung tissues was calculated. The enzyme-linked immunosorbent assay (ELISA) method was used to detecte the contents of inflammatory factors in bronchoalveolar lavage fluid (BALF). The damaged DNA in apoptotic cells in lung tissues was tested by Terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling (TUNEL) staining. Immunofluorescence staining analyzed LC3II and Golgi matrix protein 130 (GM130) expression in lung tissues and MLE-12 cells. The apoptosis of MLE-12 cells was measured by flow cytometry analysis. Additionally, the expression of proteins related to apoptosis, autophagy and Golgi stress was examined with immunoblotting. Results indicated that GOLPH3 knockdown alleviated lung tissue pathological changes in LPS-triggered ALI mice. LPS-induced inflammation and apoptosis in lung tissues and MLE-12 cells were remarkably alleviated by GOLPH3 deficiency. Besides, GOLPH3 depletion suppressed autophagy and Golgi stress in lung tissues and MLE-12 cells challenged with LPS. Moreover, Rapamycin (Rap), an autophagy inhibitor, counteracted inflammation and apoptosis inhibited by GOLPH3 silencing in LPS-induced MLE-12 cells. Furthermore, brefeldin A (BFA) pretreatment apparently abrogated the inhibitory effect of GOLPH3 knockdown on autophagy in MLE-12 cells exposed to LPS. To be concluded, GOLPH3 knockdown exerted lung protective effect against LPS-triggered inflammation and apoptosis by inhibiting Golgi stress mediated autophagy.

An alternative approach of TUNEL assay to specifically characterize DNA fragmentation in cell model systems

DNA damage is one of the most important effects induced by chemical agents. We report a comparative analysis of DNA fragmentation on three different cell lines using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, generally applied to detect apoptosis. Our approach combines cytogenetic techniques and investigation in detached cellular structures, recovered from the culture medium with the aim to compare the DNA fragmentation of three different cell line even beyond the cells adherent to substrate. Consequently, we detect any fragmentation points on single chromosomes, whole nuclei and other cellular structures. Cells were exposed to resveratrol (RSV) and doxorubicin (Doxo), in single and combined treatments. Control and treated astrocytes showed DNA damage in condensed nuclei and detached structures. Caco-2 cells showed fragmented DNA only after Doxo-treatment, while controls showed fragmented chromosomes, indicating DNA damage in replicating cells. MDA-MB-231 cells showed nuclear condensation and DNA fragmentation above all after RSV-treatment and related to detached structures. This model proved to perform a grading of genomic instability (GI). Astrocytes show a hybrid level of GI. Caco-2 cells showed fragmented metaphase chromosomes, proving that the DNA damage was transmitted to the daughter cells probably due to an absence of DNA repair mechanisms. Instead, MDA–MB-231 cells showed few or no fragmented metaphase, suggesting a probable activation of DNA repair mechanisms. By applying this alternative approach of TUNEL test, we obtained data that can more specifically characterize DNA fragmentation for a suitable application in various fields.

Protective Effects of Allium senescens Extract against 6-Hydroxydopamine in Neurons

Oxidative neurodegeneration causes various neuronal diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. This study aimed to demonstrate the protective effect of Allium senescens leaf extracts on 6-hydroxydopamine (6-OHDA)-stressed SH-SY5Y cells, which are known to be optimal for neurotoxic research. The levels of apoptotic markers were evaluated using quantitative polymerase chain reaction (qPCR) and flow cytometry. The localization of apoptotic cells in vivo was analyzed using whole-mount immunochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Additionally, the production of reactive oxygen species (ROS) was estimated using flow cytometry. 6-OHDA induced ROS production in neuroblasts and in vivo, but treatment with the extract protected against the 6-OHDA-induced increase in ROS levels. Under oxidative stress, the extract performs three protective functions: decreasing ROS production, preventing mitochondrial apoptosis, and protecting the central and ventral nervous systems. These results also suggest that the extract can be useful in the development of functional foods for the prevention of neural damage caused by oxidative stress.

Dexmedetomidine ameliorates x-ray-induced myocardial injury via alleviating cardiomyocyte apoptosis and autophagy

BackgroundRadiotherapy is a primary local treatment for tumors, yet it may lead to complications such as radiation-induced heart disease (RIHD). Currently, there is no standardized approach for preventing RIHD. Dexmedetomidine (Dex) is reported to have cardio-protection effects, while its role in radiation-induced myocardial injury is unknown. In the current study, we aimed to evaluate the radioprotective effect of dexmedetomidine in X-ray radiation-treated mice.Methods18 male mice were randomized into 3 groups: control, 16 Gy, and 16 Gy + Dex. The 16 Gy group received a single dose of 16 Gy X-ray radiation. The 16 Gy + Dex group was pretreated with dexmedetomidine (30 µg/kg, intraperitoneal injection) 30 min before X-ray radiation. The control group was treated with saline and did not receive X-ray radiation. Myocardial tissues were collected 16 weeks after X-ray radiation. Hematoxylin-eosin staining was performed for histopathological examination. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining was performed to assess the state of apoptotic cells. Immunohistochemistry staining was performed to examine the expression of CD34 molecule and von Willebrand factor. Besides, western blot assay was employed for the detection of apoptosis-related proteins (BCL2 apoptosis regulator and BCL2-associated X) as well as autophagy-related proteins (microtubule-associated protein 1 light chain 3, beclin 1, and sequestosome 1).ResultsThe findings demonstrated that 16 Gy X-ray radiation resulted in significant changes in myocardial tissues, increased myocardial apoptosis, and activated autophagy. Pretreatment with dexmedetomidine significantly protects mice against 16 Gy X-ray radiation-induced myocardial injury by inhibiting apoptosis and autophagy.ConclusionIn summary, our study confirmed the radioprotective effect of dexmedetomidine in mitigating cardiomyocyte apoptosis and autophagy induced by 16 Gy X-ray radiation.

Yishen Jiangzhuo decoction attenuates cisplatin‑induced acute kidney injury by inhibiting inflammation, oxidative stress and apoptosis through the TNF signal pathway.

The present study aimed to investigate the therapeutic effects and mechanisms of Yishen Jiangzhuo decoction (YSJZD) in a mouse model of cisplatin-induced acute kidney injury (AKI). The mice were divided into the NC, cisplatin and cisplatin + YSJZD groups. A concentration-dependent effect of YSJZD on cisplatin-induced AKI was observed and the optimal concentration for intervention was calculated. Changes in blood urea nitrogen and serum creatinine levels combined with hematoxylin and eosin and periodic acid-Schiff staining and transmission electron microscopy observations indicated that YSJZD enhanced renal function, reduced pathological injury and protected renal tubular epithelial cells in cisplatin-induced AKI mice. The results of the transcriptomic and enrichment analyses showed that the mechanisms of YSJZD may be associated with inflammation, oxidation, apoptosis and the TNF signal pathway. Immunofluorescence, oxidative stress index, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and western blotting revealed that YSJZD downregulated apoptosis in the renal tissues of AKI mice and further decreased the expression levels of p-p65, p-p38 MAPK, TNF-α, cleaved-caspase-3 and malondialdehyde, while increasing the levels of NAD-dependent protein deacetylase sirtuin-3, glutathione and superoxide dismutase. Overall, the results showed that YSJZD could effectively abrogate cisplatin-induced AKI in mice through mechanisms primarily related to its anti-inflammatory, antioxidative and antiapoptotic effects by inhibited the TNF signal pathway. YSJZD warrants further investigation as a clinical empirical prescription.

JP4-039, a Mitochondria-Targeted Nitroxide, Mitigates the Effect of Apoptosis and Inflammatory Cell Migration in the Irradiated Mouse Retina.

We hypothesize that the injection of JP4-039, a mitochondria-targeted nitroxide, prior to irradiation of the mouse retina may decrease apoptosis and reduce neutrophil and macrophage migration into the retina. In our study, we aimed to examine the effects of JP4-039 in the mouse retina using fluorescent microscopy, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and flow cytometry. Forty-five mice and one eye per mouse were used. In Group 1, fluorescent microscopy was used to determine retinal uptake of 10 µL (0.004 mg/µL) of intravitreally injected BODIPY-labeled JP4-039 at 0, 15, and 60 min after injection. In Group 2, the TUNEL assay was performed to investigate the rate of apoptosis after irradiation in addition to JP4-039 injection, compared to controls. In Group 3, flow cytometry was used to determine the extent of inflammatory cell migration into the retina after irradiation in addition to JP4-039 injection, compared to controls. Maximal retinal uptake of JP4-039 was 15 min after intravitreal injection (p < 0.0001). JP4-039-treated eyes had lower levels of retinal apoptosis (35.8 ± 2.5%) than irradiated controls (49.0 ± 2.7%; p = 0.0066) and demonstrated reduced migration of N1 cells (30.7 ± 11.7% vs. 77.7 ± 5.3% controls; p = 0.004) and M1 cells (76.6 ± 4.2 vs. 88.1 ± 3.7% controls, p = 0.04). Pretreatment with intravitreally injected JP4-039 reduced apoptosis and inflammatory cell migration in the irradiated mouse retina, marking the first confirmed effect of this molecule in retinal tissue. Further studies may allow for safety profiling and potential use for patients with radiation retinopathy.

Idebenone Antagonizes P53-Mediated Neuronal Oxidative Stress Injury by Regulating CD38-SIRT3 Protein Level.

Idebenone, an antioxidant used in treating oxidative damage-related diseases, has unclear neuroprotective mechanisms. Oxidative stress affects cell and mitochondrial membranes, altering Adp-ribosyl cyclase (CD38) and Silent message regulator 3 (SIRT3) protein expression and possibly impacting SIRT3's ability to deacetylate Tumor protein p53 (P53). This study explores the relationship between CD38, SIRT3, and P53 in H2O2-injured HT22 cells treated with Idebenone. Apoptosis was detected using flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining after determining appropriate H2O2 and Idebenone concentrations.In this study, Idebenone was found to reduce apoptosis and decrease P53 and Caspase3 expression in H2O2-injured HT22 cells by detecting apoptosis-related protein expression. Through bioinformatics methods, CD38 was identified as the target of Idebenone, and it further demonstrated that Idebenone decreased the expression of CD38 and increased the level of SIRT3. An increased NAD+/NADH ratio was detected, suggesting Idebenone induces SIRT3 expression and protects HT22 cells by decreasing apoptosis-related proteins. Knocking down SIRT3 downregulated acetylated P53 (P53Ac), indicating SIRT3's importance in P53 deacetylation.These results supported that CD38 was used as a target of Idebenone to up-regulate SIRT3 to deacetylate activated P53, thereby protecting HT22 cells from oxidative stress injury. Thus, Idebenone is a drug that may show great potential in protecting against reactive oxygen species (ROS) induced diseases such as Parkinson's disease, and Alzheimer's disease. And it might be able to compensate for some of the defects associated with CD38-related diseases.

Integrated transcriptomic and metabolomic analysis provides insight into the pollen development of CMS-D1 rice

BackgroundCytoplasmic male sterility (CMS) has greatly improved the utilization of heterosis in crops due to the absence of functional male gametophyte. The newly developed sporophytic D1 type CMS (CMS-D1) rice exhibits unique characteristics compared to the well-known sporophytic CMS-WA line, making it a valuable resource for rice breeding.ResultsIn this research, a novel CMS-D1 line named Xingye A (XYA) was established, characterized by small, transparent, and shriveled anthers. Histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays conducted on anthers from XYA and its maintainer line XYB revealed that male sterility in XYA is a result of delayed degradation of tapetal cells and abnormal programmed cell death (PCD) of microspores. Transcriptome analysis of young panicles revealed that differentially expressed genes (DEGs) in XYA, compared to XYB, were significantly enriched in processes related to chromatin structure and nucleosomes during the microspore mother cell (MMC) stage. Conversely, processes associated with sporopollenin biosynthesis, pollen exine formation, chitinase activity, and pollen wall assembly were enriched during the meiosis stage. Metabolome analysis identified 176 specific differentially accumulated metabolites (DAMs) during the meiosis stage, enriched in pathways such as α-linoleic acid metabolism, flavone and flavonol biosynthesis, and linolenic acid metabolism. Integration of transcriptomic and metabolomic data underscored the jasmonic acid (JA) biosynthesis pathway was significant enriched in XYA during the meiosis stage compared to XYB. Furthermore, levels of JA, MeJA, OPC4, OPDA, and JA-Ile were all higher in XYA than in XYB at the meiosis stage.ConclusionsThese findings emphasize the involvement of the JA biosynthetic pathway in pollen development in the CMS-D1 line, providing a foundation for further exploration of the molecular mechanisms involved in CMS-D1 sterility.

Phenethyl isothiocyanate inhibits the carcinogenic properties of hepatocellular carcinoma Huh7.5.1 cells by activating MAPK/PI3K-Akt/p53 signaling pathways.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with limited effective treatment options. Phenethyl isothiocyanate (PEITC) is a bioactive substance present primarily in the cruciferous vegetables. PEITC has exhibited anti-cancer properties in various cancers, including lung, bile duct, and prostate cancers. It has been demonstrated that PEITC can inhibit the proliferation, invasion, and metastasis of SK-Hep1 cells, while effectively inducing apoptosis and cell cycle arrest in HepG2 cells. However, knowledge of its anti-carcinogenic effects on Huh7.5.1 cells and its underlying mechanism remains elusive. In the present study, we aim to evaluate the anti-carcinogenic effects of PEITC on human HCC Huh7.5.1 cells. MTT assay and colony formation assay was performed to investigate the anti-proliferative effects of PEITC against Huh7.5.1 cells. The pro-apoptosis effects of PEITC were determined by Annexin V-FITC/PI double staining assay by flow cytometry (FCM), mitochondrial transmembrane potential (MMP) measurement, and Caspase-3 activity detection. A DAPI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was conducted to estimate the DNA damage in Huh7.5.1 cells induced by PEITC. Cell cycle progression was determined by FCM. Transwell invasion assay and wound healing migration assay were performed to investigate the impact of PEITC on the migration and invasion of Huh7.5.1 cells. In addition, transcriptome sequencing and gene set enrichment analysis (GSEA) were used to explore the potential molecular mechanisms of the inhibitory effects of PEITC on HCC. Quantitative real-time PCR (qRT-PCR) analysis was performed to verify the transcriptome data. MTT assay showed that treatment of Huh7.5.1 cells with PEITC resulted in a dose-dependent decrease in viability, and colony formation assay further confirmed its anti-proliferative effect. Furthermore, we found that PEITC could induce mitochondrial-related apoptotic responses, including a decrease of mitochondrial transmembrane potential, activation of Caspase-3 activity, and generation of intracellular reactive oxygen species. It was also observed that PEITC caused DNA damage and cell cycle arrest in the S-phase in Huh7.5.1 cells. In addition, the inhibitory effect of PEITC on the migration and invasion ability of Huh7.5.1 cells was assessed. Transcriptome sequencing analysis further suggested that PEITC could activate the typical MAPK, PI3K-Akt, and p53 signaling pathways, revealing the potential mechanism of PEITC in inhibiting the carcinogenic properties of Huh7.5.1 cells. PEITC exhibits anti-carcinogenic activities against human HCC Huh7.5.1 cells by activating MAPK/PI3K-Akt/p53 signaling pathways. Our results suggest that PEITC may be useful for the anti-HCC treatment.

Hederagenol improves multiple sclerosis by modulating Th17 cell differentiation.

Multiple sclerosis (MS) is a common autoimmune illness that is difficult to treat. The upregulation of Th17 cells is critical in the pathological process of MS. Hederagenol (Hed) has been shown to lower IL-17 levels, although its role in MS pathophysiology is uncertain. In this study, we explore whether Hed could ameliorate MS by modulating Th17 cell differentiation, with the goal of identifying new treatment targets for MS. The experimental autoimmune encephalomyelitis (EAE) mouse model was conducted and Hed was intraperitoneally injected into mice. The weight was recorded and the clinical symptom grade was assessed. Hematoxylin-eosin staining was carried out to determine the extent of inflammation in the spinal cord and liver. The luxol Fast Blue staining was performed to detect the pathological changes in the myelin sheath. Nerve damage was detected using NeuN immunofluorescence staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. Immunohistology approaches were used to study alterations in immune cells in the spinal cord. The proportions of T cell subsets in the spleens were analyzed by flow cytometry. RORγt levels were measured using quantitative real-time PCR or Western blot. The activity of the RORγt promoter was analyzed by Chromatin immunoprecipitation. Hed administration reduced the clinical symptom grade of EAE mice, as well as the inflammatory infiltration, demyelination, and cell disorder of the spinal cord, while having no discernible effect on the mouse weight. In addition, Hed treatment significantly reduced the number of T cells, particularly Th17 cells in the spinal cord and spleen-isolated CD4+ T cells. Hed lowered the RORγt levels in spleens and CD4+ T cells and overexpression of RORγt reversed the inhibitory effect of Hed on Th17 differentiation. Hed decreased nerve injury by modulating Th17 differentiation through the RORγt promoter. Hed regulates Th17 differentiation by reducing RORγt promoter activity, which reduces nerve injury and alleviates EAE.

Circadian rhythm disruption-mediated downregulation of Bmal1 exacerbates DSS-induced colitis by impairing intestinal barrier.

Circadian rhythm disruption (CRD) is thought to increase the risk of inflammatory bowel disease. The deletion of Bmal1, a core transcription factor, leads to a complete loss of the circadian rhythm and exacerbates the severity of dextran sodium sulfate (DSS)-induced colitis in mice. However, the underlying mechanisms by which CRD and Bmal1 mediate IBD are still unclear. We used a CRD mouse model, a mouse colitis model, and an in vitro model of colonic epithelial cell monolayers. We also knocked down and overexpressed Bmal1 in Caco-2 cells by transfecting lentivirus in vitro. The collected colon tissue and treated cells were assessed and analyzed using immunohistochemistry, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction, western blot, flow cytometry, transmission electron microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling staining. We found that CRD mice with downregulated Bmal1 expression were more sensitive to DSS-induced colitis and had more severely impaired intestinal barrier function than wild-type mice. Bmal1-/- mice exhibited more severe colitis, accompanied by decreased tight junction protein levels and increased apoptosis of intestinal epithelial cells compared with wild-type mice, which were alleviated by using the autophagy agonist rapamycin. Bmal1 overexpression attenuated Lipopolysaccharide-induced apoptosis of intestinal epithelial cells and impaired intestinal epithelial cells barrier function in vitro, while inhibition of autophagy reversed this protective effect. This study suggests that CRD leads to the downregulation of Bmal1 expression in the colon, which may exacerbate DSS-induced colitis in mice, and that Bmal1 may serve as a novel target for treating inflammatory bowel disease.

Effects of Attractive Pickering emulsion gels loaded with oxaliplatin and lactate dehydrogenase inhibitors in hepatocellular carcinoma.

e16235 Background: Oxaliplatin (OXA) can be used as a palliative treatment for advanced hepatocellular carcinoma (HCC). While most patients still have rapid disease progression after OXA due to the drug resistance. The lactate dehydrogenase A (LDHA) inhibitors can reduce the inflammation-induced effects, metastasis, and proliferation potential of cancer cells. The combination of OXA and LDHA inhibitor may be an effective treatment for HCC. Methods: We adopt the water-in-oil attractive Pickering emulsion gel (APEG) to deliver OXA and LDHA inhibitor, GSK2837808A (GSK). OXA is dissolved in water and GSK is dissolved in iodized oil. We injected OXA+GSK@gel into the peritumoral tissues of subcutaneously tumorigenic HCC model mice, then observe the tumor size. Next, we apply immunohistochemical staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay on the dissected tumors to explore the cell proliferation and apoptosis. Meanwhile, flow analysis on the dissected tumors is applied to investigate the tumor immune microenvironment in HCC. Results: This drugs-loaded APEG has good biocompatibility and can release OXA and GSK slowly. OXA+GSK@gel has significant anti-tumor effect on HCC model, which can effectively inhibit tumor cell proliferation and promote tumor cell apoptosis. Meanwhile, flow analysis confirm that it could activate the tumor immune microenvironment in HCC. The infiltration of CD8+ T cells was increased, thereby providing better anti-tumor effect. Conclusions: The results suggest that the APEGs loaded with OXA and GSK can effectively improve the delivery efficiency and enhance the anti-tumor therapy.