Triphosphate (dUTP) Nick End Labeling Research Articles

Ameliorative effect of pedunculoside on sepsis-induced acute lung injury, inflammation and pulmonary fibrosis in mice model via suppressing AKT/NF-κB pathway.

Sepsis-induced acute lung injury (ALI) is the typical complications of sepsis with a high global incidence and mortality. Inhibition of inflammatory response is a crucial and effective strategy for sepsis-induced ALI. Pedunculoside (PE) has been shown to have an anti-inflammatory effect on various diseases. However, the effect and mechanism of PE on sepsis-induced ALI remain unknown. A mice model of sepsis-induced ALI was constructed by cecal ligation and puncture (CLP). The effect of PE on the CLP-induced mice were assessed using pathological staining, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), reverse transcription quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot assays. PE reduced pathological symptoms and scores, apoptosis and the W/D ratio of lung tissues in CLP-induced mice. Besides, PE decreased the level of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α), pulmonary fibrosis and the expression of fibrosis markers. Mechanically, PE inhibited AKT/NF-κB signaling in CLP-induced mice. Activation of AKT/NF-κB pathway abolished the ameliorative effect of PE on the pathological symptoms, the release of inflammatory factors and pulmonary fibrosis of CLP-induced mice. PE improved inflammation and pulmonary fibrosis by inhibiting AKT/NF-κB pathway in CLP-induced mice.

Protein inhibitor of activated STAT1 (PIAS1) alleviates cerebral infarction and inflammation after cerebral ischemia in rats

BackgroundIschemic stroke is a severe disorder with high incidence, disability rate and mortality. Multiple pathogenesis mechanisms are involved in ischemic stroke, such as inflammation and neuronal cell apoptosis. Protein inhibitor of activated signal transducer and activators of transcription 1 (PIAS1) plays a crucial role in various biological processes, including inflammation. PIAS1 is also downregulated in ischemia-reperfusion injury and involved in the disease processes. However, the role of PIAS1 in cerebral ischemia is unclear. MethodsSprague-Dawley (SD) rats were induced with middle cerebral artery occlusion (MCAO). The role and mechanisms of PIAS1 in ischemic cerebral infarction were explored by Longa test, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Morris water maze (MWM) test, hematoxylin-eosin (HE) staining, quantification of brain water content, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), Western blot and immunofluorescence assays. ResultsThe expression of PIAS1 in MCAO-induced rat was declined compared to sham rats. Overexpression of PIAS1 reduced the Longa neurological scores, the percent of infarction area, the pathological abnormality, the escape latency of swimming and the percent of brain water content, and increased the number of platform crossings and time in the target quadrant in the MCAO-induced rats. Besides, overexpression of PIAS1 decreased the MCAO-induced the contents of IL-1β, IL-6 and TNF-α, but further elevated the concentrations of IL-10 in both sera and brain tissues. Moreover, overexpression of PIAS1 reversed the MCAO-induced apoptosis rate and the relative protein level of Bax, cleaved caspase3 and Bcl-2. Overexpression of PIAS1 also reversed the level of proteins involved in NF-κB pathway. ConclusionPIAS1 reduced inflammation and apoptosis, thereby alleviating ischemic cerebral infarction in MCAO-induced rats through regulation NF-κB pathway.

Zhi Zhu Ma Ren pill relieves constipation in mice through endoplasmic reticulum stress-mediated apoptosis.

This study explores the detailed effects and mechanisms of Zhi Zhu Ma Ren Pill (ZZMRP) on constipation. Mouse constipation was induced by using loperamide (Lop). The effects and mechanisms of ZZMRP on constipation were addressed by various methods including charcoal meals, hematoxylin and eosin (H&E) staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL), transmission electron microscopy, and western blot experiments. Lop-treated mice exhibited delayed transit and reduced ink progradation rates after charcoal meal administration. H&E staining confirmed severe pathologic symptoms in these mice. Additionally, a decline in interstitial Cajal cells (ICCs) was observed in Lop-treated mice, accompanied by reduced concentrations of 5-hydroxytryptamine (5-HT), acetylcholinesterase (AchE), substance P (SP), and vasoactive intestinal peptide (VIP), coupled with an elevated concentration of nitric oxide synthase (NOS). However, ZZMRP treatment markedly ameliorated these changes. In addition, ZZMRP introduction significantly reversed the Lop-induced enhancement in apoptosis rate, endoplasmic reticulum (ER) stress, and levels of proapoptotic proteins and ER stress proteins, and the decrease in the expression of antiapoptotic proteins. These effects were further confirmed with the use of 4-phenylbutyrate (4-PBA), which also reversed the changes in apoptosis rate and protein levels. ZZMRP alleviates constipation primarily through modulating ER stress-mediated apoptosis in Lop-treated mice, suggesting its use as a therapeutic agent for constipation.

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.

Cucurbitacin B suppresses hepatocellular carcinoma progression through inducing DNA damage-dependent cell cycle arrest

BackgroundThe mortality rate of liver cancer ranks third in the world, and hepatocellular carcinoma (HCC) is a malignant tumor of the digestive tract. Cucurbitacin B (CuB), a natural compound extracted from Cucurbitaceae spp., is the main active component of Chinese patent medicine the Cucurbitacin Tablet, which has been widely used in the treatment of various malignant tumors in clinics, especially HCC. PurposeThis study explored the role and mechanism of CuB in the suppression of liver cancer progression. MethodsCell Counting Kit-8 (CCK-8) and colony formation assays were used to detect the inhibitory function of CuB in Huh7, Hep3B, and Hepa1/6 hepatoma cells. Calcein-AM/propidium iodide (PI) staining and lactate dehydrogenase (LDH) measurement assays were performed to determine cell death. Mitochondrial membrane potential (Δψm) was measured, and flow cytometry was performed to evaluate cell apoptosis and cell cycle. Several techniques, such as proteomics, Western blotting (WB), and ribonucleic acid (RNA) interference, were utilized to explore the potential mechanism. The animal experiment was performed to verify the results of in vitro experiments. ResultsCuB significantly inhibited the growth of Huh7, Hep3B, and Hepa1/6 cells and triggered the cell cycle arrest in G2/M phage without leading to cell death, especially apoptosis. Knockdown of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a target of CuB, did not reverse CuB elicited cell cycle arrest. CuB enhanced phosphorylated ataxia telangiectasia mutated (p-ATM) and phosphorylated H2A histone family member X (γ-H2AX) levels. Moreover, CuB increased p53 and p21 levels and decreased cyclin-dependent kinase 1 (CDK1) expression, accompanied by improving phosphorylated checkpoint kinase 1 (p-CHK1) level and suppressing cell division cycle 25C (CDC25C) protein level. Interestingly, these phenomena were partly abolished by a deoxyribonucleic acid (DNA) protector methylproamine (MPA). Animal studies showed that CuB also significantly suppressed tumor growth in BALB/c mice bearing Hepa1/6 cells. In tumor tissues, CuB reduced the expression levels of proliferating cell nuclear antigen (PCNA) and γ-H2AX but did not change the terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) level. ConclusionThis study demonstrated for the first time that CuB could effectively impede HCC progression by inducing DNA damage-dependent cell cycle arrest without directly triggering cell death, such as necrosis and apoptosis. The effect was achieved through ataxia telangiectasia mutated (ATM)-dependent p53-p21-CDK1 and checkpoint kinase 1 (CHK1)-CDC25C signaling pathways. These findings indicate that CuB may be used as an anti-HCC drug, when the current findings are confirmed by independent studies and after many more clinical phase 1, 2, 3, and 4 testings have been done.

Gentiopicroside inhibits retinoblastoma cell proliferation, invasion, and tumorigenesis in nude mice by suppressing the PI3K/AKT pathway.

Retinoblastoma is a prevalent pediatric intraocular tumor. The suppressive effect of gentiopicroside (GPS) has been reported on various tumors. This study sought to determine the effect of GPS on retinoblastoma cell proliferation, apoptosis, invasion, and epithelial-mesenchymal transition (EMT), and tumorigenesis in nude mice. The effect and mechanism of GPS on growth, apoptosis, invasion, and EMT were determined by cell counting kit-8 (CCK-8), western blot, flow cytometry, and transwell assays in retinoblastoma cells. Y79 cells were injected into the vitreous cavity of BALB/c‑nude mice to construct a retinoblastoma mouse model. Tumor growth and mouse weight were monitored for sequential 5weeks. The effect of GPS in vivo was assessed by immunohistochemistry (IHC), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and western blot assays. GPS decreased the cell viability of both Y79 and Weri-Rb1 cells with the IC50 of 18.85μM and 27.57μM, respectively. Besides, GPS reduced the relative expression of proteins involved in proliferation and EMT, and the number of invading cells, while increased the apoptosis rate and the relative expressions of apoptosis proteins in retinoblastoma cells. Mechanically, GPS decreased the relative protein level of PI3K/AKT pathway, which was then recovered after 740 Y-P was applied. Correspondingly, 740 Y-P reversed the inhibitory effect of GPS on growth, invasion, and EMT, and the increased effect of GPS on apoptosis. Additionally, GPS decreased tumor volume and weight as well as the relative level of Ki-67, VEGF, p-PI3K/PI3K, and p-AKT/AKT, while increased the apoptosis rate in vivo. GPS inhibited retinoblastoma cell proliferation and invasion via deactivating the PI3K/AKT pathway in both cell and animal models.

Arctigenin hinders the invasion and metastasis of cervical cancer cells via the FAK/paxillin pathway

ContextCervical cancer is the most common gynecological pernicious tumor with high morbidity and mortality worldwide, especially in developing countries. Arctigenin (ARG), a nature-derived component, has exhibited anti-tumor activity in various tumors. ObjectiveTo explore the effect of ARG on cervical cancer. Materials and methodsThe effect and mechanism of ARG on cervical cancer cells were explored by cell counting kit-8 (CCK-8), flow cytometry, transwell and Western blot assays. Additionally, in vivo experiment was conducted in xenografted mice by immunohistochemistry (IHC), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) and Western blot assays. ResultsARG treatment induced both concentration-dependent and time-dependent reductions in the cell viability of SiHa and HeLa cells with a IC50 value of 9.34 μM and 14.45 μM, respectively. ARG increased the apoptosis rate and the protein levels of cleaved-caspase 3 and E-cadherin, but decreased the invaded cell numbers and the protein levels of Vimentin and N-cadherin in vitro. Mechanically, ARG inhibited the expression of focal adhesion kinase (FAK)/paxillin pathway, which was confirmed by the overexpression of FAK in SiHa cells. The inhibitory role of overexpression of FAK in proliferation and invasion, as well as its promoted role in apoptosis were reversed with ARG treatment. Meanwhile, ARG suppressed growth and metastasis, and enhanced apoptosis in vivo. Consistently, ARG administration reduced the relative protein level of p-FAK/FAK and p-paxillin/paxillin in tumor tissues of xenografted mice. ConclusionARG inhibited proliferation, invasion and metastasis, but enhanced apoptosis of cervical cancer via the FAK/paxillin axis.

Levistolide A and periplogenin inhibit the growth of gastric cancer cells in vitro and in vivo

Aim: In the present study, the natural products levistolide A (LA) and periplogenin (PPG) were studied for their growth inhibitory effects on the development of gastric cancer cells in vitro and, more critically, in vivo, alone or in combination with the therapeutic medication 5-fluorouracil (5-FU). Methods: Methyl thiazolyl tetrazolium (MTT), also known as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays were used for the cell viability study. Apoptosis was detected by western blot to detect the cleavage of caspase substrate poly (ADP-ribose) polymerase (PARP) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assays. The nude mice bearing gastric cancer cells were used for the anti-cancer activity detection of LA and its combinational treatment effect with 5-FU. Results: The results in the present study shown that the two compounds were able to inhibit the viability of the cancer cells in a dose- and time-dependent manner by MTT method. They could trigger apoptosis when used alone, and more potently, in combination with 5-FU detected by TUNEL positivity and the cleavage of caspase substrate PARP. In nude mice bearing gastric cancer cells, injection (i.p.) of LA or PPG alone inhibited the growth of the cancer cells. The treatment using one of the compounds in combination with 5-FU inhibited the cancer cell growth at a higher level than the treatment by a compound alone. Conclusions: LA and PPG could inhibit the growth of the cancer cells, alone or in combination with 5-FU, in vitro and in vivo, suggesting that they are promising investigational drugs for therapeutic development.

Cordycepin suppresses vascular inflammation, apoptosis and oxidative stress of arterial smooth muscle cell in thoracic aortic aneurysm with VEGF inhibition

BackgroundThoracic aortic aneurysm (TAA) is a type of common and serious vascular disease, in which inflammation, apoptosis and oxidative stress are strongly involved in the progression. Cordycepin, a bioactive compound from Cordyceps militaris, exhibits anti-inflammatory and anti-oxidative activities. This study aimed to address the role and mechanism of cordycepin in TAA. MethodsThe thoracic aortas were perivascularly administrated with calcium chloride (CaCl2), and human aortic smooth muscle cells (HASMCs) were incubated with angiotensin II (Ang II) to simulate the TAA model in vivo and in vitro, respectively. The effect and mechanism of cordycepin in TAA were explored by hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), immunofluorescence (IF), western blot, biochemical test, cell counting kit-8 (CCK-8), and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assays. ResultsCordycepin improved the CaCl2-induced the aneurysmal alteration and disappearance of normal wavy elastic structures of the aorta tissues, TAA incidence and thoracic aortic diameter in rats, and Ang II-induced the cell viability of HASMCs. Cordycepin reversed the CaCl2-induced the relative protein expression of cleaved caspase 9, cleaved caspase 3, interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1β, and the relative levels of glutathione (GSH), malonaldehyde (MDA) and reactive oxygen species (ROS) in vivo, or Ang II-induced these changes in vitro. Mechanically, cordycepin reduced the relative protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), cluster of differentiation 31 (CD31) and endothelial nitric oxide synthase (eNOS) in the Ang II-induced HASMCs. Correspondingly, overexpression of VEGF increased the levels of the indicators involved in apoptosis, inflammation and oxidative stress, which were antagonized with the cordycepin incubation in the Ang II-induced HASMCs. ConclusionCordycepin inhibited apoptosis, inflammation and oxidative stress of TAA through the inhibition of VEGF.

Differential Expression of NME4 in Trophoblast Stem-Like Cells and Peripheral Blood Mononuclear Cells of Normal Pregnancy and Preeclampsia.

Preeclampsia (PE) is known to arise from insufficient trophoblast invasion as uterine spiral arteries lack remodeling. A significant reduction in placental perfusion induces an ischemic placental microenvironment due to reduced oxygen delivery to the placenta and fetus, leading to oxidative stress. Mitochondria are involved in the regulation of cellular metabolism and the production of reactive oxygen species (ROS). NME/NM23 nuceloside diphosphate kinase 4 (NME4) gene is known to have the ability to supply nucleotide triphosphate and deoxynucleotide triphosphate for replication and transcription of mitochondria. Our study aimed to investigate changes in NME4 expression in PE using trophoblast stem-like cells (TSLCs) from induced pluripotent stem cells (iPSCs) as a model of early pregnancy and peripheral blood mononuclear cells (PBMNCs) as a model of late preterm pregnancy. Transcriptome analysis using TSLCs was performed to identify the candidate gene associated with the possible pathophysiology of PE. Then, the expression of NME4 associated with mitochondrial function, p53 associated with cell death, and thioredoxin (TRX) linked to ROS were investigated through qRT-PCR, western blotting and deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay. In patients with PE, NME4 was significantly downregulated in TSLCs but upregulated in PBMNCs. p53 was shown to be upregulated in TSLCs and PBMNCs of PE. In addition, western blot analysis confirmed that TRX expression had the tendency to increase in TSLCs of PE. Similarly, TUNEL analysis confirmed that the dead cells were higher in PE than in normal pregnancy. Our study showed that the expression of the NME4 differed between models of early and late preterm pregnancy of PE, and suggests that this expression pattern may be a potential biomarker for early diagnosis of PE.

Hispidulin protective impact on sepsis induced acute kidney injury is mediated by regulation of AKT and NF-κB pathway

Sepsis-induced acute kidney injury is associated with inflammatory dysregulations within the kidney. This study aimed to explore the renal protective effect of hispidulin on suppressing the apoptosis rate, and inhibiting reactive oxygen species production and inflammatory response after cecal puncture (CLP) operation. In order to gain a deeper understanding of the relationship between sepsis and acute kidney injury, the CLP induced kidney injury animal model was established. The automated biochemical analyzer was used to measure the kidney function related biomarkers including serum cystatin C (ScysC), blood urea nitrogen (BUN), and serum creatinine (Scr). The pathological changes of damaged kidney tissues were detected by hematoxylin and eosin (H&E) staining. The expression of inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) were detected by their corresponding test kits and Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). The level of reactive oxygen species production-related protein including myeloperoxidase (MPO), glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in kidney tissue from each group were quantized using Enzyme-linked immunosorbent assay (ELISA). The protein expression was measured using western blot and the apoptotic rate of kidney tissue was measured by terminal deoxynucleotidyl transferase Deoxyuridine Triphosphate (dUTP) nick end labeling (TUNEL) assay. Our results revealed that hispidulin has the protective ability in sepsis-induced acute kidney injury. The potential mechanism of hispidulin on sepsis-induced cell apoptosis, oxidative stress and inflammatory response was also investigated. Finally, our results highlighted that hispidulin exerted a protective effects on CLP-induced acute kidney injury by suppressing the protein kinase B (AKT) and Nuclear factor kappa B (NF-κB) signaling pathways. In summary, the current study provided a piece of novel evidence, that hispidulin can be explored as a potential drug in CLP-induced acute kidney injury by examining its effects on suppressed the oxidative stress, inflammatory responses, and apoptosis in kidney tissue.

The Mitigatory Effect of Shen-Qi Compound on the Diabetic Thoracic Aortic Complications through Inhibiting the Inflammatory Microenvironment by miR-223-3p/RBP-J/IRF8 Axis.

Background Disruption of the vascular immunological inflammatory microenvironment is linked to metabolic memory impairment. Even though it has been proven that the Shen-Qi compound (SQC) can efficiently halt metabolic memory and preserve vascular endothelial cells, extensive studies need to be done to investigate if it can also change the vascular immune-inflammatory microenvironment by regulating the immune system. This will help figure out the role of stopping metabolic memory. Methods After 4 weeks on a high-fat diet (HFD), GK rats were used to create a model for diabetic thoracic aortic problems. The effect and mechanisms of SQC on diabetic thoracic aortic complications were assessed by hematoxylin-eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), biochemical analysis, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), reverse transcription, real-time polymerase chain reaction (RT-qPCR), immunofluorescence (IF), western blot, and luciferase reporter assays. Results SQC treatment ameliorates the HFD-induced pathological symptoms as well as the HFD-induced increased concentrations of fasting blood glucose (FBG), fasting insulin (FINS), total cholesterol (TC), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C) and decreased concentrations of high-density lipoprotein cholesterol (HDL-C). Besides, SQC counteracted the HFD-induced average fluorescence intensity of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), as well as the concentrations of endothelin-1 (ET-1) and monocyte chemoattractant protein-1 (MCP-1), while rescuing the HFD-induced concentrations of nitric oxide (NO) and nitric oxide synthetase (NOS). Also, SQC decreases apoptosis and oxidative stress in rats with diabetic thoracic aortic complications. In addition, SQC facilitated the polarization of macrophages, stimulated the activation of dendritic cells, and regulated the inflammatory milieu in rats with diabetic thoracic aortic complications. Furthermore, SQC also modulated the miR-223-3p/RBP-J/IRF8 axis in the macrophages of rats with diabetic thoracic aortic complications. Conclusion SQC ameliorated diabetic thoracic aortic complications through the regulation of apoptosis, oxidative stress, and inflammatory microenvironment mediating by the miR-223-3p/RBP-J/IRF8 axis.

Mitoapocynin Attenuates Organic Dust Exposure-Induced Neuroinflammation and Sensory-Motor Deficits in a Mouse Model.

Increased incidences of neuro-inflammatory diseases in the mid-western United States of America (USA) have been linked to exposure to agriculture contaminants. Organic dust (OD) is a major contaminant in the animal production industry and is central to the respiratory symptoms in the exposed individuals. However, the exposure effects on the brain remain largely unknown. OD exposure is known to induce a pro-inflammatory phenotype in microglial cells. Further, blocking cytoplasmic NOX-2 using mitoapocynin (MA) partially curtail the OD exposure effects. Therefore, using a mouse model, we tested a hypothesis that inhaled OD induces neuroinflammation and sensory-motor deficits. Mice were administered with either saline, fluorescent lipopolysaccharides (LPSs), or OD extract intranasally daily for 5 days a week for 5 weeks. The saline or OD extract-exposed mice received either a vehicle or MA (3 mg/kg) orally for 3 days/week for 5 weeks. We quantified inflammatory changes in the upper respiratory tract and brain, assessed sensory-motor changes using rotarod, open-field, and olfactory test, and quantified neurochemicals in the brain. Inhaled fluorescent LPS (FL-LPS) was detected in the nasal turbinates and olfactory bulbs. OD extract exposure induced atrophy of the olfactory epithelium with reduction in the number of nerve bundles in the nasopharyngeal meatus, loss of cilia in the upper respiratory epithelium with an increase in the number of goblet cells, and increase in the thickness of the nasal epithelium. Interestingly, OD exposure increased the expression of HMGB1, 3- nitrotyrosine (NT), IBA1, glial fibrillary acidic protein (GFAP), hyperphosphorylated Tau (p-Tau), and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-positive cells in the brain. Further, OD exposure decreased time to fall (rotarod), total distance traveled (open-field test), and olfactory ability (novel scent test). Oral MA partially rescued olfactory epithelial changes and gross congestion of the brain tissue. MA treatment also decreased the expression of HMGB1, 3-NT, IBA1, GFAP, and p-Tau, and significantly reversed exposure induced sensory-motor deficits. Neurochemical analysis provided an early indication of depressive behavior. Collectively, our results demonstrate that inhalation exposure to OD can cause sustained neuroinflammation and behavior deficits through lung-brain axis and that MA treatment can dampen the OD-induced inflammatory response at the level of lung and brain.

Modulation of SIRT1 expression improves erectile function in aged rats.

Silent information regulator 2-related enzyme 1 (SIRT1) is an aging-related protein activated with aging. Herein, we evaluated the role of SIRT1 in aging-related erectile dysfunction. The expression of SIRT1 was modulated in aged Sprague-Dawley rats following intragastric administration of resveratrol (Res; 5 mg kg-1), niacinamide (NAM; 500 mg kg-1) or Res (5 mg kg-1) + tadalafil (Tad; phosphodiesterase-5 [PDE5] inhibitor; 5 mg kg-1) for 8 weeks. Then, we determined erectile function by the ratio of intracavernosal pressure (ICP)/mean systemic arterial pressure (MAP). Cavernosal tissues were extracted to evaluate histological changes, cell apoptosis, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP), the superoxide dismutase (SOD)/3,4-methylenedioxyamphetamine (MDA) level, and the expression of SIRT1, p53, and forkhead box O3 (FOXO3a) using immunohistochemistry, terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL), enzyme-linked immunosorbent assays, and western blot analysis. Compared with the control, Res treatment significantly improved erectile function, reflected by an increased content of smooth muscle and endothelium, NO/cGMP and SOD activity, and reduced cell apoptosis and MDA levels. The effect of Res was improved by adding Tad. In addition, the protein expression of SIRT1 was increased in the Res group, accompanied by decreased p53 and FOXO3a levels. In addition, inhibition of SIRT1 by NAM treatment resulted in adverse results compared with Res treatment. SIRT1 activation ameliorated aging-related erectile dysfunction, supporting the potential of SIRT1 as a target for erectile dysfunction treatment.

Endoplasmic reticulum stress is involved in retinal injury induced by repeated transient spikes of intraocular pressure.

Clinically, a large proportion of glaucoma patients undergo repeated intraocular pressure (IOP) spike (Spike IOP) attacks during their sleep, which may facilitate retinopathy. In this study, we established a mouse model of repeated transient Spike IOP to investigate the direct damage to the retina following Spike IOP attacks, and elucidated the underlying molecular mechanism. We analyzed the changes in the number of retinal ganglion cells (RGCs) via immunofluorescence. Thereafter, we detected retinal cell apoptosis via terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining, and performed RNA sequencing (RNA-seq) to reveal the underlying molecular mechanism. Finally, we validated the expression of key molecules in the endoplasmic reticulum (ER) stress pathway using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Results revealed a time-dependent RGC loss in Spike IOP, evidenced by a reduction in the number of Brn3a-positive RGCs in experimental eyes following a 7-d continuous treatment with Spike IOP. In addition, TUNEL staining indicated that apoptosis of retinal cells started in the outer nuclear layer (ONL), and then spread to the ganglion cell layer (GCL) with time. RNA-seq analysis revealed that ER stress might be involved in Spike IOP-induced retinal injury. This result was corroborated by western blot, which revealed upregulation of ER stress-related proteins including binding immunoglobulin protein/glucose-regulated protein 78 (BiP/GRP78), phosphorylated inositol-requiring enzyme 1 (p-IRE1), unspliced X-box-binding protein 1 (XBP1-u), spliced X-box-binding protein 1 (XBP1-s), phosphorylated c-Jun N-terminal kinase (p-JNK), C/EBP-homologous protein (CHOP), and B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax). These findings indicate that repeated IOP transients are detrimental to the retina, while ER stress plays an important role in retinal cell apoptosis in this situation. Notably, repeated Spike IOP among glaucoma patients is a crucial factor for progressive retinopathy.

AFB1-induced mice liver injury involves mitochondrial dysfunction mediated by mitochondrial biogenesis inhibition

Aflatoxin B1 (AFB1) pollutes foodstuffs and feeds, causing a food safety problem and seriously endangering human and animal health. Liver is the principal organ for AFB1 accumulation and biotransformation, during which AFB1 can cause acute and chronic liver damage, however, the specific mechanism is not completely clear. Mitochondria are the primary organelle of cellular bio-oxidation, providing 95% energy for liver to execute its multiple functions. Therefore, we speculated that mitochondrial dysfunction is involved in AFB1-induced liver injury. To verify the hypothesis, a total of eighty healthy male mice were randomly divided into four groups on average, and exposed with 0, 0.375, 0.75 and 1.5 mg/kg body weight AFB1 by intragastric administration for 30 d. The results displayed that AFB1 triggered liver injury accompanied by oxidative stress. AFB1 exposure also damaged mitochondria structure, decreased mitochondrial membrane potential (MMP), as well as increased cytoplasmic cytochrome c (Cyt-c) protein expression, Bax, p53, Caspase-3/9 protein and/or mRNA expression levels and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine-5'-triphosphate (dUTP) nick end labeling (TUNEL) staining positive cells in mice liver. Meanwhile, AFB1 exposure elevated pyruvate content, inhibited tricarboxylic acid (TCA) cycle rate-limiting enzymes and electron transport chain (ETC) complexes I-V activities, disturbed ETC complexes I-V subunits mRNA expression levels and reduced adenosine triphosphate (ATP) level in mice liver. These results indicated that AFB1 destroyed mitochondrial structure, activated mitochondrion-dependent apoptosis and induced mitochondrial dysfunction. In addition, AFB1 disrupted mitochondrial biogenesis, presented as the abnormalities of protein and/or gene expression levels of voltage dependent anion channel protein 1 (VDAC1), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (Nrf1) and mitochondrial transcription factor A (Tfam). This may contribute to hepatic and mitochondrial lesions induced by AFB1. These results provide a new perspective for elucidating the mechanisms of AFB1 hepatotoxicity.

The role of neuronal apoptosis in Valproic Acid brain-related teratogenesis: a histochemical and immunohistochemical study in BALB/c mice.

Objectives: The purpose of this study was to examine the teratogenic effects of Valproic Acid (VPA) and to investigate the role of apoptosis in neural tissue development. Although an apoptotic activity due to VPA has been reported, a direct connection of VPA-induced apoptosis with embryonic brain and/or spine malformations and teratogenesis has not yet been established. Materials and Methods: VPA was administered to BALB/c mice, from the 7th to the 10th gestational days. Macroscopical congenital anomalies were registered under a stereomicroscope and were further histologically studied. Immunohistochemistry was performed with terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining. Results: Birth defects were described and an increase of the apoptotic activity in the brain was immunohistochemically identified. Conclusions: Considering the increased and very intense TUNEL expression of the neural cells of treated animals’ fetuses, it is suggested that VPA triggers a pathological increase of apoptosis resulting in an imbalance between cell proliferation and cell death, the final result of which is malformation.

S-oxiracetam Facilitates Cognitive Restoration after Ischemic Stroke by Activating α7nAChR and the PI3K-Mediated Pathway.

S-oxiracetam (S-ORC), a nootropic drug, was used to protect against ischemic stroke by lessening the blood brain barrier dysfunction and inhibiting neuronal apoptosis. However, the potential effects of S-ORC in the recovery of cognitive functions after ischemic stroke and the underlying mechanisms remains unclear. In this study, middle cerebral artery occlusion/reperfusion (MCAO/R) in rats was used as the animal model. By using Y-maze test, Morris water maze, triphenyl tetrazolium chloride (TTC)staining, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate(dUTp) nick end labeling(TUNEL) assay, hematoxylin and eosin, immunohistochemical staining and western blot to evaluate the protective effect of S-ORC on cognitive recovery, we were able to confirm that S-ORC ameliorated spatial learning impairment, tissue loss, and hippocampal neuronal apoptosis and injury induced by MCAO/R in rats. These cognitive effects were achieved by restoring the normal function of synaptophysin and increasing PSD95 expression in the hippocampus. Furthermore, we found that methyllycaconitine, the antagonist of α7 nicotinic acetylcholine receptor (α7nAChR), and LY294002, the inhibitor of phosphoinositide 3-kinase(PI3K), were able to block the cognitive effects of S-ORC after MCAO/R in rats. In conclusion, α7nAChR and PI3K are key molecules that mediated the signaling pathway leading to S-ORC-induced cognitive restoration after MCAO/R.

The Role of SHIP1 on Apoptosis and Autophagy in the Adipose Tissue of Obese Mice.

Obesity-induced adipocyte apoptosis promotes inflammation and insulin resistance. Src homology domain-containing inositol 5′-phosphatase 1 (SHIP1) is a key factor of apoptosis and inflammation. However, the role of SHIP1 in obesity-induced adipocyte apoptosis and autophagy is unclear. We found that diet-induced obesity (DIO) mice have significantly greater crown-like structures and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)-positive cells than ob/ob or control mice. Using RNA sequencing (RNA-seq) analysis, we identified that the apoptosis- and inflammation-related gene Ship1 is upregulated in DIO and ob/ob mice compared with control mice. In particular, DIO mice had more SHIP1-positive macrophages and lysosomal-associated membrane protein 1 (LAMP1) as well as a higher B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio compared with ob/ob or control mice. Furthermore, caloric restriction attenuated adipose tissue inflammation, apoptosis, and autophagy by reversing increases in SHIP1-associated macrophages, Bax/Bcl2-ratio, and autophagy in DIO and ob/ob mice. These results demonstrate that DIO, not ob/ob, aggravates adipocyte inflammation, apoptosis, and autophagy due to differential SHIP1 expression. The evidence of decreased SHIP1-mediated inflammation, apoptosis, and autophagy indicates new therapeutic approaches for obesity-induced chronic inflammatory diseases.

The possible mechanisms of simvastatin on apoptosis of lung adenocarcinoma cells

Objective: To explore the possible mechanisms of simvastatin-induced apoptosis in lung adenocarcinoma cells. Methods: The experiment was divided into control group (vehicle treated A549 cells), different concentrations (10, 20, 40, 80 mg/L) simvastatin group (simvastatin treated with different concentrations of A549 cells), aspartate specific proteinase (caspase) inhibitor (Z-VAD-FMK) group (50 μmol/L Z-VAD-FMK treated A549 cells), 40 mg/L simvastatin combined with Z-VAD-FMK group (40 mg/L simvastatin combined with 50 μmol/L Z-VAD-FMK co-treated A549 cells), interleukin-6 (IL-6) group (IL-6 acts on A549 cells) and different concentrations (10, 20, 40 mg/L) simvastatin combined with IL-6 group (simvastatin combined with IL-6 act on A549 cells). Cell counting kit-8 (CCK8) method was used to detect the effect on survival rate of lung adenocarcinoma A549 cells; Flow cytometry was used to detect the effect of simvastatin on A549 cell cycle; Mitochondrial membrane potential-1 (JC-1) fluorescent probe was wsed to detect the effect of simvastatin on mitochondrial membrane potential (MMP); Flow-type phosphatidl serine protein antibody Annexin V/propidium iodide (Annexin V-FITC/PI) double staining method was used to detect the effect of simvastatin on A549 cell apoptosis; CCK8 method was used to detect the effect of Z-VAD-FMK on the survival rate of A549 cells; TdT-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling (TUNEL) method was used to detect the effect of Z-VAD-FMK on simvastatin-induced apoptosis in A549 cells; Western blot method was used to detect the effect of simvastatin on the expression levels of Janus kinase 2 and activation of signal transducers and activators of transcription 3 (JAK2/STAT3) pathway-related proteins phosphorylated JAK2 (p-JAK2), JAK2, phosphorylated STAT3 (p-STAT3), and STAT3 before and after the activator IL-6 of JAK2/STAT3 pathway acted on A549 cells. Results: The survival rates of A549 cells in the 20-80 mg/L simvastatin-treated groups were significantly lower than that in the control group (all P<0.05), and gradually decreased with the increase of the concentration of the simvastatin and the extension of the action time. The cells in the G(0)/G(1) phase of the simvastatin group were significantly higher than those in the control group, and the cells in the G(2)/M phase were significantly lower than those in the control group (all P<0.01). The MMP of the treatment group with different concentrations of simvastatin was significantly lower than that of the control group (all P<0.05). The apoptosis rate of the 20 mg/L and 40 mg/L simvastatin-treated group was significantly higher than that of the control group (both P<0.01). The cell survival rate of the 40 mg/L simvastatin group and the 40 mg/L simvastatin combined with Z-VAD-FMK group were (52.2±2.7)% and (57.5±3.8)%, respectively, were lower than that of the control group (100.0±2.7)% (both P<0.01). But the difference between 40 mg/L simvastatin group and the simvastatin combined with Z-VAD-FMK group was not statistically significant (P>0.05). The cell numbers with positive fluorescent staining in the 40 mg/L simvastatin group were significantly more than those in the control group, but the cell numbers with positive fluorescent staining in the 40 mg/L simvastatin combined with Z-VAD-FMK group had no statistical significance compared with the simvastatin group (P>0.05). The specific value of p-JAK2/JAK2 and p-STAT3/STAT3 protein relative expressions in the simvastatin-treated group (20, 40 mg/L) were significantly lower than that in the control group, respectively (both P<0.05). The specific value of p-JAK2/JAK2 and p-STAT3/STAT3 protein relative expressions in IL-6 group were significantly higher than those in control group (both P<0.05), the specific value of p-JAK2/JAK2 and p-STAT3/STAT3 protein relative expressions in simvastatin (20, 40 mg/L) combined with IL-6 groups were lower than those in IL-6 group (all P<0.05), respectively. Conclusion: Simvastatin can induce the apoptosis of A549 cells through a non-caspase-dependent mitochondrial apoptosis pathway, which may be achieved by inhibiting the JAK2/STAT3 pathway.