Deoxynucleotidyl Transferase Deoxyuridine Triphosphate Nick Research Articles

ANGPTL2 knockdown induces autophagy to relieve alveolar macrophage pyroptosis by reducing LILRB2-mediated inhibition of TREM2.

Acute lung injury (ALI) is featured with a robust inflammatory response. Angiopoietin-like protein 2 (ANGPTL2), a pro-inflammatory protein, is complicated with various disorders. However, the role of ANGPTL2 in ALI remains to be further explored. The mice and MH-S cells were administrated with lipopolysaccharide (LPS) to evoke the lung injury invivo and invitro. The role and mechanism of ANGPTL was investigated by haematoxylin-eosin, measurement of wet/dry ratio, cell count, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling, reverse transcription quantitative polymerase chain reaction, immunofluorescence, enzyme-linked immunosorbent assay, detection of autophagic flux and western blot assays. The level of ANGPTL2 was upregulated in lung injury. Knockout of ANGPTL2 alleviated LPS-induced pathological symptoms, reduced pulmonary wet/dry weight ratio, the numbers of total cells and neutrophils in BALF, apoptosis rate and the release of pro-inflammatory mediators, and modulated polarization of alveolar macrophages in mice. Knockdown of ANGPTL2 downregulated the level of pyroptosis indicators, and elevated the level of autophagy in LPS-induced MH-S cells. Besides, downregulation of ANGPTL2 reversed the LPS-induced the expression of leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) and triggering receptor expressed on myeloid cells 2 (TREM2), which was reversed by the overexpression of LILRB2. Importantly, knockdown of TREM2 reversed the levels of autophagy- and pyroptosis-involved proteins, and the contents of pro-inflammatory factors in LPS-induced MH-S cells transfected with si ANGPTL2, which was further inverted with the treatment of rapamycin. Therefore, ANGPTL2 silencing enhanced autophagy to alleviate alveolar macrophage pyroptosis via reducing LILRB2-mediated inhibition of TREM2.

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.

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.

Monotropein alleviates sepsis-elicited acute lung injury via the NF-κB pathway.

To address the effect and mechanism of Monotropein (Mon) on sepsis-induced acute lung injury (ALI). ALI model was established by lipopolysaccharide (LPS)-stimulated mouse lung epithelial cell lines (MLE-12) and cecal ligation and puncture (CLP)-treated mice, respectively. The function of Mon was examined by cell counting kit-8 (CCK-8), pathological staining, the pulmonary function examination, flow cytometry, enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labellingand western blot. Mon increased the LPS-reduced viability but decreased the LPS-evoked apoptosis rate in MLE-12 cells. Mon suppressed the concentrations and protein expressions of proinflammatory factors, and the expressions of fibrosis-related proteins in LPS-challenged MLE-12 cells compared with LPS treatment alone. Mechanically, Mon downregulated the levels of NF-κB pathway, which was confirmed with the application of the receptor activator of nuclear factor-κB ligand (RANKL). Correspondingly, RANKL reversed the ameliorative effect of Mon on the proliferation, apoptosis, inflammation and fibrosis. Moreover, Mon improved the pathological manifestations, apoptosis, the W/D ratio and pulmonary function indicators in CLP-treated mice. Consistently, Mon attenuated inflammation, fibrosis and NF-κB pathway in CLP-treated mice. Mon inhibited apoptosis, inflammation and fibrosis to alleviate sepsis-evoked ALI via the NF-κB pathway.

FTZ promotes islet β-cell regeneration in T1DM mice via the regulation of nuclear proliferation factors

Ethnopharmacological relevanceFufang-Zhenzhu-Tiaozhi capsule (FTZ), a Traditional Chinese Medicine (TCM) patent prescription commonly used in clinical practice, has a significant curative effect on hyperglycemia and hyperlipidemia. Previous studies have shown that FTZ can treat diabetes, but the effect of FTZ on β-cell regeneration needs to be further explored in T1DM mice. Aim of the studyThe aim is to investigate the role of FTZ in promoting β-cell regeneration in T1DM mice, and to further explore its mechanism. Materials and methodsC57BL/6 mice were used as control. NOD/LtJ mice were divided into the Model group and FTZ group. Oral glucose tolerance, fasting blood glucose, and fasting insulin level were measured. Immunofluorescence staining was used to detect the level of β-cell regeneration and the composition of α-cells and β-cells in islets. Hematoxylin and eosin staining was used to detect the infiltration degree of inflammatory cells. The apoptosis of islet cells was detected by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. Western blotting was used to detect the expression levels of Pancreas/duodenum homeobox protein 1 (PDX-1), V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), and Neurogenin-3 (NGN3). ResultsFTZ could increase insulin levels and reduce the glucose level of T1DM mice and promote β-cell regeneration. FTZ also inhibited the invasion of inflammatory cells and the islet cell apoptosis, and maintained the normal composition of islet cells, thus preserving the quantity and quality of β-cells. Furthermore, FTZ promoting β-cell regeneration was accompanied by increasing the expression of PDX-1, MAFA, and NGN3. ConclusionFTZ can restore the insulin-secreting function of the impaired pancreatic islet, improve blood glucose level, possibly via the enhancing β cell regeneration via upregulation of PDX-1, MAFA, and NGN3 in T1DM mice, and may be a potential therapeutic drug for T1DM.

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.

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.

Neuroprotective effects of chlorogenic acid by controlling the Bcl-2 family protein in a stroke animal model.

Ischemic stroke causes severe brain damage and high mortality. Chlorogenic acid is a phenolic compound that has neuroprotective properties. B-cell lymphoma-2 (Bcl-2) family proteins are important for apoptosis regulation. Bcl-2 and Bcl-xL are proteins that inhibit apoptosis, and Bax and Bad induce apoptosis. In this study, we investigated whether chlorogenic acid exerts a neuroprotective effect against ischemic stroke damage by regulating Bcl-2 family proteins. We performed middle cerebral artery occlusion (MCAO) to induce ischemic stroke in adult male rats. The animals were intraperitoneally injected with normal saline as a vehicle or chlorogenic acid (30 mg/kg) 2 hr after MCAO. Cerebral cortex tissue was collected 24 hr after MCAO damage. MCAO damage caused histopathological changes and increased the number of terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling-positive cells, while chlorogenic acid attenuated these changes. RT-qPCR and Western blot results showed decreases in Bcl-2 and Bcl-xL expression and an increase in Bax and Bad expression in MCAO animals. However, chlorogenic acid treatment attenuated these changes due to MCAO damage. The interaction of Bax with Bcl-2 or Bcl-xL decreased in MCAO animals, and the binding of Bad with Bcl-2 or Bcl-xL increased. However, chlorogenic acid treatment reduced these changes. Chlorogenic acid also prevented MCAO-induced increases in caspase-3 and caspase-9 expression. This study provides evidence that chlorogenic acid has neuroprotective effects against MCAO damage by modulating Bcl-2 family proteins including Bcl-2, Bcl-xL, Bax, and Bad. Furthermore, chlorogenic acid regulates the interaction between Bcl-2 family proteins. In conclusion, chlorogenic acid contributes to neuroprotection against ischemic stroke damage by controlling Bcl-2 family proteins.

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.

Polyunsaturated fatty acids ameliorate renal stone-induced renal tubular damage via miR-93-5p/Pknox1 axis

ObjectivesPolyunsaturated fatty acids (PUFAs) can decrease the risk of calcium oxalate stone formation, which accounts for 80% of all renal stones. This study aimed to investigate the protective mechanisms of PUFAs against renal stones. MethodsUrine samples of patients with renal stones and biopsy tissue samples from patients with nephrocalcinosis were tested for miR-93-5p expression. A renal stone mouse model was established with intraperitoneal injection of glyoxylic acid, during which mice were treated with PUFAs and/or an miR-93-5p inhibitor adenovirus. Periodic acid–Schiff staining, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining, oil red O staining, triacylglycerol assay, and colorimetry testing were performed to assess glycogen deposition, apoptosis, lipid accumulation, blood urea nitrogen, and serum creatinine levels, respectively. Renal proximal tubular epithelial cells (human kidney 2 [HK-2]) were subjected to gain- and loss-of-function assays before calcium-oxalate monohydrate (COM) induction and PUFA treatment. Cell counting kit 8, flow cytometry, and lactate dehydrogenase activity assays were used to examine cell viability, apoptosis, and damage. A luciferase reporter gene assay verified the interaction between miR-93-5p and Pknox1, and miR-93-5p and Pknox1 levels were assessed using a reverse transcription-quantitative polymerase chain reaction and Western blot analysis. ResultsmiR-93-5p was downregulated in clinical samples with renal stones and negatively targeted Pknox1. PUFAs increased miR-93-5p expression and reduced apoptosis, glycogen deposition, and lipid accumulation in mice with renal stones, which were annulled by miR-93-5p downregulation. PUFAs increased proliferation and diminished apoptosis, lipid accumulation, and lactate dehydrogenase activity in COM-induced HK-2 cells, which were negated by miR-93-5p inhibition. Pknox1 overexpression reversed the effect of miR-93-5p upregulation on COM-induced HK-2 cells. ConclusionsPUFAs repressed renal stone-induced renal tubular damage via the miR-93-5p/Pknox1 axis.

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.

Beta-Boswellic Acid Protects Against Cerebral Ischemia/Reperfusion Injury via the Protein Kinase C Epsilon/Nuclear Factor Erythroid 2-like 2/Heme Oxygenase-1 Pathway.

Ischemic strokes are associated with a high rate of disability and death globally. Cerebral ischemia/reperfusion (I/R) injury is a type of brain damage associated with oxidative stress after an ischemic stroke. Beta-boswellic acid (β-BA) reportedly exerts antioxidant and neuroprotective effects, but its role in cerebral I/R injury is unclear. The aim of this research was to investigate the neuroprotective effects, as well as the mechanisms of β-BA in cerebral I/R injury. In vivo experiments were conducted using a rat middle cerebral artery occlusion and reperfusion (MCAO/R) model, and in vitro experiments were performed using a rat neuronal oxygen-glucose deprivation and reoxygenation (OGD/R) model. Triphenyltetrazolium chloride staining, neurological function scores, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, hematoxylin and eosin staining, and antioxidant levels in the brain were used to assess the effects of β-BA. Flow cytometry was used to detect reactive oxygen species and apoptotic cells. Western blotting and immunofluorescence staining were used to measure protein levels. The results showed that β-BA markedly improved neurological deficits and decreased infarct volume and necrotic neurons in rats. The in vitro results showed that β-BA protected neurons against OGD/R-induced injury. Additionally, β-BA significantly increased the phosphorylation of protein kinase C epsilon (PRKCE) at S729, the translocation of nuclear factor erythroid 2-like 2 (NFE2L2), and expression of heme oxygenase-1 (HMOX1). This study demonstrates that β-BA exerts neuroprotective effects against cerebral I/R via the activation of the PRKCE/NFE2L2/HMOX1 pathway and is a potential therapeutic candidate for ischemic stroke.

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.

Inhibition of Methamphetamine-Induced Cytotoxicity in the U87-Cell Line by Atorvastatin-Conjugated Carbon Nanotubes.

In biological systems, carbon nanotubes can enhance the biological effects of drugs and reduce their side effects. Methamphetamine (METH) is a stimulant drug that induces cell death in various cell types, primarily neural cells. On the other hand, specific doses of atorvastatin (ATO) can stimulate cell growth and inhibit cell death in different cell lines. This study aimed to investigate the improvement effect of ATO@single-walled carbon nanotube (SWCNT) on METH-induced cell cytotoxicity in the U87 glioblastoma cell line. In this study, cells were cultured in 10mM of METH during the cell treatment with 0-10nM of ATO and ATO@SWCNT. The conjugated drugs to SWCNT as Van der Waals were detected using field emission scanning electron microscopy, Fourier transform-infrared spectroscopy, and other analyses. Then, the in vitro proliferating of ATO@SWCNT was explored against glioblastoma cells compared to pure ATO. This examine was performed using methyl thiazole tetrazolium approach, terminal deoxynucleotidyl transferase deoxy uridine-triphosphate nick end labeling assay, caspase-3 method, lactate dehydrogenase assay, and RH-123 assay with 10mM METH. The results obtained from transmission electron microscopy analysis showed the average size of 50nm for ATO@SWCNT. This study indicated that U87 cells, which were exposed to METH and suffered cell death, were severely reduced in the presence of ATO, especially ATO@SWCNT (for its anti-apoptotic effect), but they survived. This study suggests that ATO, which was primarily used to reduce blood lipids, can significantly reduce brain cell death. The findings of this study indicate that by using SWCNT, more drugs can reach the target cells. This method reduces the total amount of required medication and shows a more beneficial therapeutic effect.

N-3-oxododecanoyl homoserine lactone exacerbates endothelial cell death by inducing receptor-interacting protein kinase 1-dependent apoptosis.

Endothelial dysfunction is associated with the initiation of sepsis-associated organ failure. Bacterial quorum-sensing molecules act as pathogen-associated molecular patterns; however, the effects of quorum-sensing molecules on endothelial cells remain less understood. This study investigated the molecular mechanisms of quorum-sensing molecule-induced cell death and their interaction with lipopolysaccharide (LPS) in human umbilical vein endothelial cells. Endothelial cells were treated with N-3-oxododecanoyl homoserine lactone (3OC12-HSL) and LPS derived from Pseudomonas aeruginosa. Treatment with 3OC12-HSL reduced cell viability in a dose-dependent manner, and cotreatment with 3OC12-HSL and LPS enhanced cell death. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay revealed an increase in apoptotic cell death following 3OC12-HSL treatment; furthermore, cotreatment with 3OC12-HSL and LPS enhanced apoptosis. Western blotting revealed that treatment with 3OC12-HSL activated the receptor-interacting protein kinase 1 (RIPK1) pathway, leading to an increase in the levels of cleaved caspase 8 and 3. In addition, we found that treatment with necrostatin-1, an RIPK1 inhibitor, reduced cell death and ameliorated the activation of the RIPK1-dependent apoptotic pathway in 3OC12-HSL-treated cells. In conclusion, 3OC12-HSL induced endothelial cell apoptosis via the activation of the RIPK1 pathway, independent of LPS toxicity. Inhibition of RIPK1 may act as a therapeutic option for preserving endothelial cell integrity in patients with sepsis by disrupting the mechanism by which quorum-sensing molecules mediate their toxicity.

Activation of the M3AChR and Notch1/HSF1 Signaling Pathway by Choline Alleviates Angiotensin II-Induced Cardiomyocyte Apoptosis.

Angiotensin II- (Ang II-) induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure. Choline exerts cardioprotective effects; however, its effects on Ang II-induced cardiomyocyte apoptosis are unclear. In this study, the role and underlying mechanism of choline in regulating Ang II-induced cardiomyocyte apoptosis were investigated using a model of cardiomyocyte apoptosis, which was induced by exposing neonatal rat cardiomyocytes to Ang II (10−6 M, 48 h). Choline promoted heat shock transcription factor 1 (HSF1) nuclear translocation and the intracellular domain of Notch1 (NICD) expression. Consequently, choline attenuated Ang II-induced increases in mitochondrial reactive oxygen species (mtROS) and promotion of proapoptotic protein release from mitochondria, including cytochrome c, Omi/high-temperature requirement protein A2, and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low P. The reversion of these events attenuated Ang II-induced increases in cardiomyocyte size and numbers of terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling-positive cells, presumably via type 3 muscarinic acetylcholine receptor (M3AChR). Indeed, downregulation of M3AChR or Notch1 blocked choline-mediated upregulation of NICD and nuclear HSF1 expression, as well as inhibited mitochondrial apoptosis pathway and cardiomyocyte apoptosis, indicating that M3AChR and Notch1/HSF1 activation confer the protective effects of choline. In vivo studies were performed in parallel, in which rats were infused with Ang II for 4 weeks to induce cardiac apoptosis. The results showed that choline alleviated cardiac remodeling and apoptosis of Ang II-infused rats in a manner related to activation of the Notch1/HSF1 pathway, consistent with the in vitro findings. Taken together, our results reveal that choline impedes oxidative damage and cardiomyocyte apoptosis by activating M3AChR and Notch1/HSF1 antioxidant signaling, and suggest a novel role for the Notch1/HSF1 signaling pathway in the modulation of cardiomyocyte apoptosis.

Anti-Inflammatory Dipeptide, a Metabolite from Ambioba Secretion, Protects Cerebral Ischemia Injury by Blocking Apoptosis Via p-JNK/Bax Pathway

MQ (l-methionyl-l-glutamic acid), anti-inflammatory dipeptide, is one of the metabolites of monocyte locomotion inhibitory factor, a thermostable pentapeptide secreted by Entamoeba histolytica. Monocyte locomotion inhibitory factor injection has been approved as an investigational drug for the potential neural protection in acute ischemic stroke. This study further investigated the neuroprotective effect of MQ in ischemic brain damage. Ischemia-reperfusion injury of the brain was induced in the rat model by middle cerebral artery occlusion. 2,3,5-triphenyltetrazolium chloride staining assay was used to measure cerebral infarction areas in rats. Laser Doppler measurement instrument was used to detect blood flow changes in the rat model. Nissl staining and NeuN staining were utilized to observe the numbers and structures of neuron cells, and the pathological changes in the brain tissues were examined by hematoxylin–eosin staining. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) staining was used to assess cell apoptosis. The changes in oxidative stress indexes, superoxide dismutase and malondialdehyde (MDA), were measured in serum. Methyl thiazolyl tetrazolium was used to measure the survival rates of PC12 cells. Flow cytometry assessed the apoptosis rates and the levels of reactive oxygen species. Real-time PCR was used to evaluate the mRNA expression levels, and Western blotting was used to analyze the changes in protein levels of p-JNK, Bax, cleaved Caspase3. We revealed that MQ improved neurobehavior, decreased cerebral infarction areas, altered blood flow volume, and the morphology of the cortex and hippocampus. On the other hand, it decreased the apoptosis of cortical neurons and the levels of MDA, and increased the levels of superoxide dismutase. In vitro studies demonstrated that MQ enhanced the cell survival rates and decreased the levels of reactive oxygen species. Compared to the oxygen-glucose deprivation/reperfusion group, the protein and mRNA expressions of p-JNK, Bax, cleaved Caspase3 was decreased significantly. These findings suggested that MQ exerts a neuroprotective effect in cerebral ischemia by blocking apoptosis via the p-JNK/Bax pathway.

Cancer-Preventive Role of Bone Marrow-Derived Mesenchymal Stem Cells on Colitis-Associated Colorectal Cancer: Roles of Gut Microbiota Involved.

BackgroundMesenchymal stem cells (MSCs) treatment showed promising results in inflammatory bowel disease in both rodent models and patients. Nevertheless, previous studies conducted conflicting results on preclinical tumor models treated with MSCs concerning their influence on tumor initiation and progression. This study is designed to demonstrate the role of bone marrow-derived MSCs and the potential mechanism in the colitis-associated colon cancer (CAC) model.MethodsBone marrow-derived MSCs were isolated from green fluorescent protein-transgenic mice, cultured, and identified by flow cytometry. Azoxymethane and dextran sulfate sodium were administrated to establish the CAC mouse model, and MSCs were infused intraperitoneally once per week. The mice were weighed weekly, and colon length, tumor number, and average tumor size were assessed after the mice were killed. MSC localization was detected by immunofluorescence staining; tumor cell proliferation and apoptosis were measured by immunohistochemistry staining of Ki-67 and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay, respectively. The colonic tumor tissues were isolated for RNA-seq, and fecal samples were collected for 16S ribosomal RNA sequencing of the microbiome.ResultsAfter injection intraperitoneally, MSCs migrated to the intestine and inhibited the initiation of colitis-associated colorectal cancer. This inhibition effect was marked by less weight loss, longer colon length, and reduced tumor numbers. Moreover, MSCs reduced tumor cell proliferation and induced tumor cell apoptosis. Furthermore, MSCs could inhibit chronic inflammation assessed by RNA-sequencing and promote gut microbiome normalization detected by 16S ribosomal RNA sequencing.ConclusionThe results proved that MSCs could migrate to the colon, inhibit chronic inflammation, and regulate gut microbiome dysbiosis to suppress the development of CAC.

Comparison of different energy response for lipolysis using a 1,060-nm laser: An animal study of three pigs.

Non-invasive body-sculpting procedures are becoming increasingly popular. The application of 1,060nm of laser energy transcutaneously to hyperthermically induce the disruption of fat cells in the abdomen is a type of non-invasive procedure. The purpose of this study was to compare the treatment results from two parameters of the same system, each with different energy output levels, in an in vivo porcine model to determine the most effective application. Female pigs (n=3) were used in this study. We examined the effects of the treatment using photography, ultrasonography, gross and microscopic pathology, and histological examination in order to determine the mechanism of action, efficacy, and safety of the procedure. Blood chemistry analysis was performed before each session to check lipid levels and to monitor for any adverse changes in markers that may indicate liver damage. Biopsies were taken and routinely processed with hematoxylin and eosin and Oil Red O stains to examine for tissue damage at baseline and after each treatment. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assays were performed to check for apoptotic-related DNA damage. Ultrasonic imaging of the same area before and after the application of 1,060nm of laser energy at outputs of 0.9 and 1.4W/cm2 showed that the density of the fat layer changed immediately after irradiation due to the transient heat transfer in the fat layer. Preclinical evaluation was performed to obtain comparison data on the safety and efficacy of subcutaneous fat reduction after applying the different energy outputs of 0.9 and 1.4W/cm2 . Based on our findings, we suggest that long-term histologic changes through the use of these devices suggest a comparative effectiveness of the treatment energy.

Ephedrine causes liver toxicity in SD rats via oxidative stress and inflammatory responses.

Ephedrine abuse has spread in many parts of the world and severely threatens human health. The mechanism of ephedrine-induced toxicity still remains unclear. This study was performed to investigate the effects of ephedrine treatment on the liver and explore the underlying mechanisms. Sprague Dawley rats were divided into saline and ephedrine groups. Rats were treated with ephedrine at 20 mg/kg or 40 mg/kg (n = 10) by oral gavage daily for 7 days. Pathological changes were examined by hematoxylin and eosin staining and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay. Enzyme-linked immunosorbent assays were used to measure the liver functional markers, oxidative stress markers, and inflammatory cytokines. Real-time polymerase chain reaction and Western blot were used to measure gene and protein expression, respectively. Our data showed that ephedrine treatment increased hepatocellular cell apoptosis and impaired liver function. Moreover, ephedrine treatment increased oxidative stress and inflammatory responses, which may be due to the increase of transforming growth factor β (TGF-β)/Smad3 expression. Our study demonstrated that short-term treatment of ephedrine caused liver toxicity in rats through regulating TGF-β/Smad pathway.