Transferase-mediated Deoxyuridine Triphosphate Nick-end Labeling Research Articles

Effect of Ginsenoside Rb2 on a Myocardial Cell Model of Coronary Heart Disease through Nrf2/HO-1 Signaling Pathway.

The ginsenoside Rbs are the primary active compounds of Panax ginseng and ginsenoside Rb2 is a renowned component among the Rbs. This study aimed to investigate the potential effects of ginsenoside Rb2 on coronary heart disease (CHD). H9c2 cells were exposed to H2O2 to establish CHD model in vitro. Gene expression was determined by quantitative realtime PCR (qPCR) and Western blot. Cellular functions were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays. We found that Ginsenoside Rb2 promoted cell proliferation while suppressed oxidative stress and apoptosis of H9c2 cells induced by H2O2 exposure. Mechanistically, Ginsenodise Rb2 involves in the regulation of nuclear factor, erythroid 2 like 2 (Nrf2)/heme oxygenase (HO)-1 signaling pathway. Inactivation of Nrf2/HO-1 signaling pathway reversed the effects of ginsenoside Rb2 on H9c2 cells. Taken together, ginsenoside Rb2 exhibited a cardioprotective effect in vitro. The underlying mechanism of ginsenoside Rb2 in H9c2 cells could be standardized to Nrf2/HO-1 signaling pathway, inhibiting cell apoptosis and regaining cell proliferation. The present study has proposed a novel mechanism of ginsenoside Rb2 in the cardioprotective effect.

Redundant Functions of ERK1 and ERK2 Maintain Mouse Liver Homeostasis Through Down-Regulation of Bile Acid Synthesis.

Activation of extracellular signal–regulated kinase (ERK) 1/2 promotes hepatocyte proliferation in response to growth stimuli, but whether constitutive hepatocyte ERK1/2 signaling functions in liver physiology is unknown. To examine the role of ERK1/2 in hepatic homeostasis, the effects of a knockout of Erk1 and/or Erk2 in mouse liver were examined. The livers of mice with a global Erk1 knockout or a tamoxifen‐inducible, hepatocyte‐specific Erk2 knockout were normal. In contrast, Erk1/2 double‐knockout mice developed hepatomegaly and hepatitis by serum transaminases, histology, terminal deoxynucleotide transferase‐mediated deoxyuridine triphosphate nick end‐labeling, and assays of hepatic inflammation. Liver injury was associated with biochemical evidence of cholestasis with increased serum and hepatic bile acids and led to hepatic fibrosis and mortality. RNA sequencing and polymerase chain reaction analysis of double‐knockout mouse livers revealed that the rate‐limiting bile acid synthesis gene Cyp7a1 (cholesterol 7α‐hydroxylase) was up‐regulated in concert with decreased expression of the transcriptional repressor short heterodimer partner. Elevated bile acids were the mechanism of liver injury, as bile acid reduction by SC‐435, an inhibitor of the ileal apical sodium–dependent bile acid transporter, prevented liver injury. Conclusion: Constitutive ERK1 and ERK2 signaling has a redundant but critical physiological function in the down‐regulation of hepatic bile acid synthesis to maintain normal liver homeostasis.

Pirfenidone attenuates acetaminophen-induced liver injury via suppressing c-Jun N-terminal kinase phosphorylation

Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in Western countries. Pirfenidone (PFD), an orally bioavailable pyridone derivative, is clinically used for idiopathic pulmonary fibrosis treatment and has antifibrotic, anti-inflammatory, and antioxidant effects. Here we examined the PFD effect on APAP-induced liver injury.In a murine model, APAP caused serum alanine aminotransferase elevation attenuated by PFD treatment. We performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and vital propidium iodide (PI) stainings simultaneously. APAP induced TUNEL-positive/PI-negative necrosis around the central vein and subsequent TUNEL-negative/PI-positive oncotic necrosis with hemorrhage and caused the upregulation of hypercoagulation- and hypoxia-associated gene expressions. PFD treatment suppressed these findings. Western blotting revealed PFD suppressed APAP-induced c-Jun N-terminal kinase (JNK) phosphorylation despite no effect on JNK phosphatase expressions.In conclusion, simultaneous TUNEL and vital PI staining is useful for discriminating APAP-induced necrosis from typical oncotic necrosis. Our results indicated that PFD attenuated APAP-induced liver injury by suppressing TUNEL-positive necrosis by directly blocking JNK phosphorylation. PFD is promising as a new option to prevent APAP-induced liver injury.

Curcumin represses lipid accumulation through inhibiting ERK1/2-PPAR-γ signaling pathway and triggering apoptosis in porcine subcutaneous preadipocytes.

ObjectiveExcessive lipid accumulation in adipocytes results in prevalence of obesity and metabolic syndrome. Curcumin (CUR), a naturally phenolic active ingredient, has been shown to have lipid-lowering effects. However, its underlying mechanisms have remained largely unknown. Therefore, the study aims to determine the effect of CUR on cellular lipid accumulation in porcine subcutaneous preadipocytes (PSPA) and to clarify novel mechanisms.MethodsThe PSPA were cultured and treated with or without CUR. Both cell counting Kit-8 and lactate dehydrogenase release assays were used to examine cytotoxicity. Intracellular lipid contents were measured by oil-red-o staining extraction and triglyceride quantification. Apoptosis was determined by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling assay. Adipogenic and apoptosis genes were analyzed by quantitative polymerase chain reaction and Western blot.ResultsThe CUR dose-dependently reduced the proliferation and lipid accumulation of PSPA. Noncytotoxic doses of CUR (10 to 20 μM) significantly inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and expression of adipogenic genes peroxisome proliferation-activity receptor-γ (PPAR-γ), CCAAT/enhancer binding protein-α, sterol regulatory element-binding protein-1c, adipocyte protein-2, glucose transporter-4 as well as key lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase, while ERK1/2 activation significantly reversed CUR-reduced lipid accumulation by increasing PPAR-γ. Furthermore, compared with differentiation induced media treated cells, higher dose of CUR (30 μM) significantly decreased the expression of AKT and B-cell lymphoma-2 (BCL-2), while increased the expression of BCL-2-associated X (BAX) and the BAX/BCL-2 expression ratio, suggesting triggered apoptosis by inactivating AKT and increasing BAX/BCL-2 ratio and Caspase-3 expression. Moreover, AKT activation significantly rescued CUR inhibiting lipid accumulation via repressing apoptosis.ConclusionThese results demonstrate that CUR is capable of suppressing differentiation by inhibiting ERK1/2-PPAR-γ signaling pathway and triggering apoptosis via decreasing AKT and subsequently increasing BAX/BCL-2 ratio and Caspase-3, suggesting that CUR provides an important method for the reduction of porcine body fat, as well as the prevention and treatment of human obesity.

Fraxetin suppresses reactive oxygen species-dependent autophagy by the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells.

Isoflurane, a common volatile anesthetic, has been widely used to provide general anesthesia in operations. However, exposure to isoflurane may cause widespread neurotoxicity in the developing animal brain. Fraxetin, a natural coumarin derivative extracted from the bark of Fraxinus rhynchophylla, possesses versatile pharmacological properties including anti-oxidative, anti-inflammatory, and neuroprotective effects. However, the effect and action mechanism of fraxetin on neurotoxicity induced by isoflurane are unknown. Reactive oxygen species (ROS) generation, cell viability, lactate dehydrogenase (LDH) release, and apoptosis were estimated by 2',7'-dichlorofluorescin-diacetate (DCFH-DA) staining, MTT, LDH release, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining assays, respectively. The protein levels of light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were detected by western blot analysis. Isoflurane induced ROS, LDH release, apoptosis, and autophagy, but inhibited the viability in HT22 cells, which were overturned by fraxetin or ROS scavenger N-acetyl-L-cysteine. Fraxetin suppressed isoflurane-induced PI3K/Akt inactivation in HT22 cells. PI3K/Akt inactivation by LY294002 resisted the effects of fraxetin on isoflurane-induced autophagy and autophagy-modulated neurotoxicity in HT22 cells. In conclusion, fraxetin suppressed ROS-dependent autophagy by activating the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells.

Clinical Guidelines for Sperm DNA Fragmentation (SDF) is Associated with Male Infertility from Reactive Oxygen Species (ROS)

Sperm DNA fragmentation is common in infertile male. Besides, sperm DNA integrity is essential for fertilization and healthy offspring development. Numerous genetic and environmental elements are associated with impacting sperm DNA integrity negatively. Such as lifestyle, ageing, industrial toxins, and infection. The mechanisms behind SDF are many, but apoptosis and reactive oxygen species are considered the main SDF mechanisms. The management of male infertility has led to the desire for more advanced SDF diagnostic tools to diagnose sperm DNA. Numerous sperm DNA damage assays such as terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) assay and in situ nick translation (ISNT) are available to enhance SDF diagnosing and ultimately to better SDF management. Clinical SDF can lead to a low pregnancy rate, defects in embryo development and impaired offspring health. Moreover, SDF can impact the effectiveness of assisted reproductive technology through transfer genetics impartment to the embryo b in vitro fertilization or intracytoplasmic sperm injection. SDF can be mange through lifestyle changing, treating existing infection in the male reproductive tract and reactive oxygen species.

Butyric acid alleviated chronic intermittent hypoxia-induced lipid formation and inflammation through up-regulating HuR expression and inactivating AMPK pathways.

To investigate whether butyric acid could alleviate chronic intermittent hypoxia (CIH)-induced lipid formation in human preadipocytes-subcutaneous (HPA-s) through accumulation of human antigen R (HuR) and inactivation of AMP-activated protein kinase (AMPK) pathway, HPA-s were obtained and divided into three groups: Control group: cells were cultured under normal conditions; CIH group: cells were cultured in a three-gas incubator (10% O2); Butyric acid group: 10 mmol/l butyric acid added into cell culture medium. HuR-siRNA was futher transfected into CIH group for verification the function of HuR. Oil Red O was implemented for observation of lipid droplets within cells. Cell Counting Kit-8 (CCK8) assay was used for detecting cell viability. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) assay as well as flow cytometry analysis was employed for determining cell apoptosis. Western blotting was used for measurement of protein expression levels. RT-qPCR analysis was used for detecting mRNA expression. CIH treatment increased adipocytes proliferation, while butyric acid inhibited cell proliferation and promoted cell apoptosis. The treatment of butyric acid in CIH group down-regulated expression of inflammatory factors and increased cell apoptotic rate. Butyric acid treatment increased HuR expression in both cytoplasm and nucleus and decreased the level of p-AMPK and p-ACC, while transfection of AMPK activator or HuR-siRNA would down-regulate HuR expression. Moreover, butyric acid alleviated CIH-induced cell proliferation, lipid formation and inflammatory status and promoted cell apoptosis through regulating related genes including p21, PPARγ, C/EBPa, IL-1β, IL-6, TLR4, caspase-8 and caspase-3. In conclusion, butyric acid could alleviate CIH-induced inflammation, cell proliferation and lipid formation through accumulation of HuR and inactivation of AMPK pathway.

Cell death of hippocampal CA1 astrocytes during early epileptogenesis.

Growing evidence suggests that dysfunctional astrocytes are crucial players in the development of mesial temporal lobe epilepsy (MTLE). Using a mouse model closely recapitulating key alterations of chronic human MTLE with hippocampal sclerosis, here we asked whether death of astrocytes contributes to the initiation of the disease and investigated potential underlying molecular mechanisms. Antibody staining was combined with confocal imaging and semiquantitative real-time polymerase chain reaction analysis to identify markers of different cellular death mechanisms between 4h and 3days after epilepsy induction. Four hours after kainate-mediated induction of status epilepticus (SE), we found a significant reduction in the density of astrocytes in the CA1 stratum radiatum (SR) of the ipsilateral hippocampus. This reduction was transient, as within the next 3days, astrocyte cell numbers recovered to the initial values, which was accompanied by enhanced proliferation. Four hours after SE induction, a small proportion of astrocytes in the ipsilateral CA1 SR expressed autophagy-related genes and proteins, whereas we did not find astrocytes positive for cleaved caspase 3 or terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling, ruling out apoptosis-related astrocytic death. Importantly, at the same early time point post-SE, many astrocytes in the ipsilateral CA1 SR showed strong expression of genes encoding pro-necroptosis factors, including receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Phosphorylation of MLKL (pMLKL), formation of necrosome complexes composed of RIPK3 and pMLKL, and translocation of pMLKL to the nucleus and to the plasma membrane were often observed in astrocytes of the ipsilateral hippocampus 4h post-SE. The present study revealed that astrocytes die shortly after induction of SE. Our expression data and immunohistochemistry suggest that necroptosis and autophagy contribute to astrocytic death. These findings help to better understand how dysfunctional and pathological remodeling of astrocytes contributes to the initiation of temporal lobe epilepsy.

Protective effects of curcumin on chromatin quality, sperm parameters, and apoptosis following testicular torsion-detorsion in mice.

ObjectiveThe chief outcome of testicular torsion in clinical and experimental contexts is testicular ischemia. Curcumin, a compound with anti-inflammatory and antioxidant properties, has fascinated researchers and clinicians for its promise in the treatment of fertility diseases. MethodsThirty-five fully grown male mice were randomly classified into five groups: control, sham, testicular torsion, treatment group 1 (testicular torsion+short-term curcumin), and treatment group 2 (testicular torsion+long-term curcumin). Thirty-five days later, spermatozoa from the right cauda epididymis were analyzed with regard to count and motility. Toluidine blue (TB), aniline blue (AB), and chromomycin A3 (CMA3) staining assays were used to evaluate the sperm chromatin integrity. In addition, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) test was used to assess apoptosis. ResultsTreatment group 1 exhibited a remarkably elevated sperm count compared to the testicular torsion group. Additionally, notably lower sperm motility was found in the testicular torsion group compared to the control, treatment 1, and treatment 2 groups. Staining (CMA3, AB, and TB) and the TUNEL test indicated significantly greater testicular torsion in the torsion group compared to the control group (p<0.05). The data also revealed notably lower results of all sperm chromatin assays and lower apoptosis in both treatment groups relative to the testicular torsion group (p<0.05). Significantly elevated (p<0.05) AB and TB results were noted in treatment group 1 compared to treatment group 2. ConclusionCurcumin can compensate for the harmful effects of testicular ischemia and improve sperm chromatin quality in mice.

A Novel Therapeutic Approach to Corneal Alkaline Burn Model by Targeting Fidgetin-Like 2, a Microtubule Regulator.

PurposeThe purpose of this study was to determine the efficacy of nanoparticle-encapsulated Fidgetin-like 2 (FL2) siRNA (FL2-NPsi), a novel therapeutic agent targeting the FL2 gene, for the treatment of corneal alkaline chemical injury.MethodsEighty 12-week-old, male Sprague-Dawley rats were divided evenly into 8 treatment groups: prednisolone, empty nanoparticles, control-NPsi (1 µM, 10 µM, and 20 µM) and FL2-NPsi (1 µM, 10 µM, and 20 µM). An alkaline burn was induced onto the cornea of each rat, which was then treated for 14 days according to group assignment. Clinical, histopathologic, and immunohistochemical analyses were conducted to assess for wound healing. FL2-NPsi-mediated knockdown of FL2 was confirmed by in vitro quantitative polymerase chain reaction (qPCR). Toxicity assays were performed to assess for apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL] assay) and nerve damage (whole mount immunochemical staining). Statistical analyses were performed using Student's t-test and ANOVA.ResultsCompared with controls, FL2-NPsi-treated groups demonstrated enhanced corneal wound healing, with the 10 and 20 µM FL2-NPsi-treated groups demonstrating maximum rates of corneal re-epithelialization as assessed by ImageJ software, enhanced corneal transparency, and improved stromal organization on histology. Immunohistochemical analysis of vascular endothelial cells, macrophages, and neutrophils did not show significant differences between treatment groups. FL2-NPsi was not found to be toxic to nerves or induce apoptosis (p = 0.917).ConclusionsDose-response studies found both 10 and 20 µM FL2-NPsi to be efficacious in this rat model. FL2-NPsi may offer a novel treatment for corneal alkaline chemical injuries.Translational RelevanceBasic cell biology findings about the microtubule cytoskeleton were used to design a therapeutic to enhance corneal cell migration, highlighting the promise of targeting microtubules to regulate corneal wound healing.

High-Dose Vitamin C Administration Inhibits the Invasion and Proliferation of Melanoma Cells in Mice Ovary.

Several studies have been conducted to explore the anticancer effects of vitamin C (VC). However, the effect of high-dose VC administration on melanoma is still unknown. Therefore, in this study, we investigated the effects of high-dose VC (4 g/kg) on the invasion and proliferation of melanoma cells in various organs of mice. B16 melanoma cells (1 × 106 cells/100 µL) were intravenously injected into the tails of female mice, and VC solution (4 g/kg) was orally administered once a day for 14 d. On the 15th day, samples from the liver, lungs, jejunum, and ovaries were collected and analyzed for invasion and proliferation of melanoma cells. Oral VC administration decreased the number of dihydroxyphenylalanine (DOPA)-positive cells and gp100-positive melanoma cells in the ovaries and suppressed the invasion and proliferation of melanoma. Compared to melanoma-administered mice, macrophage inflammatory protein-2 levels and number of neutrophils were increased in the VC + melanoma-administered mice. Furthermore, the concentrations of VC, iron, and hydrogen peroxide, and the number of terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were significantly increased in the ovaries of VC + melanoma-administered mice compared to those of melanoma-administered mice. These results suggest that VC can reduce the invasion and proliferation of melanoma cells in the ovaries, and neutrophils in the ovaries play an important role in achieving this melanoma-suppressive effect.

Hepatocyte-Derived Igκ Exerts a Protective Effect against ConA-Induced Acute Liver Injury.

Immunoglobulin (Igκ) has been reported to be expressed in sorted liver epithelial cells of μMT mice, and the sequence characteristics of hepatocyte-derived Igκ were different from those of classical B-cell-derived Igκ. However, the physiological function of hepatocyte-derived Igκ is still unclear. The expression of Igκ was firstly identified in primary hepatocytes and normal liver cell line (NCTC1469), and hepatocyte-derived Igκ expression was elevated and displayed unique localization in hepatocytes of concanavalin A (ConA)-induced hepatitis model. Moreover, Igκ knockout mice were more sensitive to ConA-induced hepatitis and had higher serum aspartate aminotransferase (AST) levels, more severe histological injury and a greater number of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells as compared with littermate controls. Furthermore, knockdown of Igκ in primary hepatocytes and NCTC1469 cells led to accelerated activation of the mitochondrial death pathway and caspase-3 cleavage in vitro, which might be related to inhibition of NF-κB signaling pathway and activation of JNK via the cytoskeleton dynamics. Taken together, these results indicate that hepatocyte-derived Igκ mediates cellular resistance to ConA-induced liver injury by inhibiting activation of caspase-3 and the mitochondrial death pathway, suggesting that Igκ plays an important role in hepatocyte survival and exerts a protective effect against ConA-induced liver injury in mice.

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).

Liver-specific Mettl3 ablation delays liver regeneration in mice

This study investigated the role of N6-methyladenosine RNA methylation in liver regeneration following partial hepatectomy in mice. We created a liver-specific knockout mouse model by the deletion of Mettl3, a key component of the N6-methyladenosine methyltransferase complex, using the albumin-Cre system. Mettl3 liver-specific knockout mice and their wild-type littermates were subjected to 2/3 partial hepatectomy. Transcriptomic changes in liver tissue at 48 h after partial hepatectomy were detected by RNA-seq. Immunohistochemistry and immunofluorescence were used to determine protein expression levels of Ki67, hepatocyte nuclear factor 4 alpha, and cytokeratin 19. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling was also performed. Liver weight/body weight ratios after partial hepatectomy were significantly lower in Mettl3 liver-specific knockout mice than in wild-type mice at 48 h after 2/3 partial hepatectomy (3.1% ± 0.11% vs. 2.7% ± 0.03%). Compared with wild-type littermates, Mettl3 liver-specific knockout mice showed reduced bromodeoxyuridine staining and reduced Ki-67 expression at 48 h after 2/3 partial hepatectomy. RNA-seq analysis showed that Mettl3 liver-specific knockout delayed the cell cycle progression in murine liver by downregulating the expression levels of genes encoding cyclins D1, A2, B1, and B2. Loss of Mettl3-mediated N6-methyladenosine function led to attenuated liver regeneration by altering the mRNA decay of suppressor of cytokine signaling 6, thereby inhibiting the phosphorylation of signal transducer and activator of transcription 3 during early liver regeneration. These results demonstrated the importance of N6-methyladenosine mRNA modification in liver regeneration and suggest that Mettl3 targeting might facilitate liver regeneration.

A decrease in the tear secretion volume in a mouse model with ulcerative colitis

Purpose Dry eye syndrome is known to develop from several systemic inflammatory diseases. Although dry eye may develop due to extraintestinal complications of ulcerative colitis (UC), the pathogenesis is not well-known. This study aimed to investigate whether there was decrease in the tear secretion volume in a mice model with UC; the difference between the control and dextran sodium sulphate (DSS)-treated group was also determined. Materials and methods This study included a mice model with UC induced by the oral administration of 5.0% DSS for 7 days. Following the DSS treatment, the tear volume was measured using the Schirmer’s test. The colon and ocular tissues, including the lacrimal gland, were evaluated using histological and protein analyses. Additionally, tumour necrosis factor (TNF)-α and interleukin (IL)-6 in the plasma were determined. Differences between groups (DSS-treated versus control mice) were determined using Student’s t-test. Results The tear volume in DSS-treated mice was decreased compared to that in the control mice. Plasma levels of TNF-α and IL-6 in DSS-treated mice was higher than that of control. Morphological change was observed with the invasion of the inflammatory cell in the lacrimal gland of DSS-treated mice. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL)-positive cells were increased in the lacrimal glands of DSS-treated mice compared with control group. The distribution of aquaporin-5 expressed in the lacrimal gland of DSS-treated mice was decreased compared to that in the control group. Conclusions These findings suggest that a decrease in the tear volume in UC was associated with a functional decline in the inflamed lacrimal gland. This result therefore provides useful information that could contribute to the development of treatment approaches for dry eye associated with UC.

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.

The role of the globular heads of the C1q receptor in TcdA-induced human colonic epithelial cell apoptosis via a mitochondria-dependent pathway

BackgroundClostridioides (formerly Clostridium) difficile infection is the leading cause of antibiotic-associated colitis. Studies have demonstrated that C. difficile toxin A (TcdA) can cause apoptosis of many human cell types. The purpose of this study was to investigate the relationships among exposure to TcdA, the role of the receptor for the globular heads of C1q (gC1qR) gene and the underlying intracellular apoptotic mechanism in human colonic epithelial cells (NCM 460). In this study, gC1qR expression was examined using real-time polymerase chain reaction (PCR), western blotting and immunohistochemical staining. Cell viability was assessed by the water-soluble tetrazolium salt (WST-1) assay, and cell apoptosis was assessed by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Mitochondrial function was assessed based on reactive oxygen species (ROS) generation, changes in the mitochondrial membrane potential (ΔΨm) and the content of ATP.ResultsOur study demonstrated that increasing the concentration of TcdA from 10 ng/ml to 20 ng/ml inhibited cell viability and induced cell apoptosis (p < 0.01). Moreover, the TcdA-induced gC1qR expression and enhanced expression of gC1qR caused mitochondrial dysfunction (including production of ROS and decreases in the ΔΨm and the content of ATP) and cell apoptosis. However, silencing of the gC1qR gene reversed TcdA-induced cell apoptosis and mitochondrial dysfunction.ConclusionThese data support a mechanism by which gC1qR plays a crucial role in TcdA-induced apoptosis of human colonic epithelial cells in a mitochondria-dependent manner.

Telmisartan Exacerbates Cisplatin-Induced Nephrotoxicity in a Mouse Model.

Cisplatin (CDDP; cis-diamine dichloroplatinum)-induced nephrotoxicity is the main reason for dose limitations, which can reduce the efficacy of cancer treatment. Lower blood pressure and administration of renin angiotensin system (RAS) inhibitors have been reported as factors that exacerbate CDDP-induced nephrotoxicity; however, the detailed mechanisms remain unknown and the results of previous studies are conflicting. In this study, we examined the influence of various hypotensive drugs, including RAS inhibitors and calcium channel blockers, on CDDP-induced nephrotoxicity in BALB/c mice. The mice were divided into nine groups: (1) CDDP group (15 mg/kg CDDP), (2) AML group (5 mg/kg amlodipine), (3) ENA group (2.5 mg/kg enalapril), (4) telmisartan (TEL) group (10 mg/kg telmisartan), (5) LOS group (10 mg/kg losartan), (6) CDDP + AML group, (7) CDDP + ENA group, (8) CDDP + TEL group, and (9) CDDP + LOS group. Nephrotoxicity was evaluated by measuring serum creatinine (CRE) and blood urea nitrogen (BUN) levels. In addition, the kidney sections were stained with Masson's trichrome and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) to assess the renal fibrosis area and apoptotic area. Serum CRE and BUN levels were increased in the CDDP + ENA, CDDP + LOS, and CDDP + TEL groups compared to those in the CDDP alone group, and the CDDP + AML group showed an increasing trend. However, there was no correlation between ∆CRE or ∆BUN levels and ∆ systolic blood pressure. The CDDP + TEL group showed a significant increase in the renal fibrosis area. These results suggest that exacerbation of CDDP-induced nephrotoxicity is not correlated with systolic blood pressure but is associated with administration of RAS inhibitors, particularly TEL.

Deficiency of miR-208a Exacerbates CCl4-Induced Acute Liver Injury in Mice by Activating Cell Death Pathways.

Acute liver injury (ALI) is associated with multiple cellular events such as necrosis, apoptosis, oxidative stress and inflammation, which can lead to liver failure. In this study, we demonstrate a new role of microRNA (miR)‐208a in ALI. ALI was induced in wild‐type (WT) and miR‐208a knockout (KO) mice by CCl4 administration. Increased alanine aminotransferase and decreased hepatic miR‐208a levels were found in WT mice after acute CCl4 treatment. Histopathological evaluations revealed increased necrosis and decreased inflammation in miR‐208a KO compared with WT mice after CCl4 treatment. CCl4 treatment induced a higher alanine aminotransferase elevation and increased numbers of circulating extracellular vesicles (exosomes and microvesicles) in miR‐208a KO compared with WT mice. We found increased CCl4‐induced nuclear factor kappa B activation and tumor necrosis factor‐α induction and decreased monocyte chemoattractant protein 1 levels in miR‐208a KO compared with WT mice. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling assay indicated aggravated hepatic apoptosis and necrosis in CCl4 ‐treated miR‐208a KO compared with WT mice. CCl4 treatment induced a greater increase in cleaved caspase‐8, p18, and caspase‐3 in miR‐208a KO compared with WT mice. p53 is involved in various cell death pathways, including necrosis and apoptosis. Our in silico analysis revealed p53 as a predicted miR‐208a target, and we found enhanced p53 and cyclophilin D protein expressions in miR‐208a KO mice after CCl4 treatment. Increased liver injury in miR‐208a KO mice was further associated with increased Bax (B cell lymphoma 2–associated X protein) and p21 expression. Our in vitro results indicated a role of miR‐208a in cell death. We found that CCl4‐induced cytotoxicity was partially rescued by miR‐208a overexpression in RAW macrophages. Altogether, our results revealed a role of miR‐208a in ALI in mice and suggest a role for miR‐208a in regulating cell death.

TUNEL assay—Standardized method for testing sperm DNA fragmentation

Sperm DNA integrity is important for normal functions such as fertilization, implantation, pregnancy and fetal development. Sperm DNA fragmentation (SDF) is more common in infertile men and may be responsible for poor reproductive function. Although there are a number of tests available to measure SDF, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling TUNEL) assay using flow cytometry is becoming more popular to measure the sperm DNA fragmentation. It is a direct test that measures both single- and double- DNA strand breaks. In this review, we describe the protocol, quality control and measurement of sperm DNA fragmentation using a benchtop flow cytometer. We also briefly discuss the factors that can affect the results, challenges and clinical implications of TUNEL in assessing male infertility.