TdT-mediated dUTP-biotin Nick End Labeling Research Articles

Effect of Acridine Orange and Zoledronic Acid on Bone Metastasis in Renal Cell Carcinoma.

The increasing incidence of renal cell carcinoma (RCC) and its associated bone metastasis pose challenges in surgical interventions, warranting the exploration of novel therapeutic approaches. Therefore, this study aimed to assess the impact of hematogenously administering acridine orange (AO) alone and in combination with zoledronic acid (ZA) on bone metastasis in RCC. RENCA cells (1.0×106 cells/10 μl) were directly injected into the right femur of male BALB/c mice. The mice were categorized into four groups based on the applied therapeutic intervention and were euthanized after five weeks. Micro-computed tomography was performed to quantify the extent of periosteal reaction, indicative of bone metastasis, along the entire length of the femur. Tumor weight and volume were measured at euthanization. Hematoxylin and eosin staining was used to examine the extent of tumor development in the bone. Apoptotic cell, osteoclast, and vascular endothelial growth factor (VEGF)-positive cell counts were assessed using TdT-mediated dUTP-biotin nick end labeling, tartrate-resistant acid phosphatase staining, and VEGF staining, respectively. The periosteal reaction was significantly reduced in the intervention groups compared to the control group (p<0.05). The apoptotic cell numbers in the intervention groups surpassed that in the control group (p<0.05), whereas those of osteoclasts and VEGF-positive cells in the intervention groups were lower than those in the control group (p<0.05). AO hinders bone metastasis progression in RCC, and combination therapy with ZA may be more effective than AO administration alone.

Lijing-Anfei Decoction Attenuates COPD in a Mouse Model: Network Pharmacology Analysis, Molecular Docking, and Experimental Validation

Background Today, network pharmacology and molecular docking were utilized for investigation and prediction of possible mechanisms in fields such as pharmacognosy and traditional Chinese medicine formulas when treating disease. Purpose Chronic obstructive pulmonary disease (COPD) is caused by exposure to toxic particles and leads to chronic bronchitis and lung dysfunction. Lijing-Anfei decoction (LAD) is a traditional Chinese herbal decoction used to cure COPD. This study explored the effects of this decoction and its intrinsic mechanism in a COPD mouse model. Materials and methods Possible targets were retrieved from several databases and combined using Veny. Protein-protein interaction (PPI) networks were constructed via STRING database. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed via the Metascape database. The COPD model was established by exposing the mice to cigarette smoke for 12 weeks. After the model was established, LAD was administered for four weeks. Enzyme-linked immunosorbent assay (ELISA) was performed, and body weights and lung wet/dry ratios were measured. Lung morphology was observed by hematoxylin-eosin staining. Results After the screening, 32 overlapping targets were used to generate PPI networks and the following hub genes were identified: JUN, IL6, RELA, IL1B, HIF1A, MAPK1, PPARG, PTGS2, MAPK14, and NOS2. The KEGG analysis indicated that hypoxia-inducible factor-1 (HIF-1) and other signaling pathways are possible signaling pathways. The molecular docking results demonstrated tight links between major compounds and their targets. In animal experiments, LAD attenuated body weight loss, downregulated inflammatory cell numbers, and inhibited the enhancement of Fe2+ levels. The mean linear intercept index changes indicated that LAD ameliorated the lung structural damage. ELISA revealed that it downregulated pro-inflammatory cytokine levels and inhibited excessive oxidant production, TdT-mediated dUTP-biotin nick end labeling assay demonstrated that it alleviated cell death in the lungs, and immunohistochemical staining revealed that it inhibited HIF-1α and PTGS2. ML228—an agonist of HIF-1—blocked the effect of LAD, validating the role of HIF-1-mediated ferroptosis in the pharmacological effects of this decoction. Conclusion This study revealed that LAD was successful in treating COPD.

Isocucurbitacin B inhibits glioma growth through PI3K/AKT pathways and increases glioma sensitivity to TMZ by inhibiting hsa-mir-1286a.

Aim: Glioma accounts for 81% of all cancers of the nervous system cancers and presents one of the most drug-resistant malignancies, resulting in a relatively high mortality rate. Despite extensive efforts, the complete treatment options for glioma remain elusive. The effect of isocucurbitacin B (isocuB), a natural compound extracted from melon pedicels, on glioma has not been investigated. This study aims to investigate the inhibitory effect of isocuB on glioma and elucidate its underlying mechanisms, with the objective of developing it as a potential therapeutic agent for glioma. Methods: We used network pharmacology and bioinformatics analysis to predict potential targets and associated pathways of isocuB in glioma. Subsequently, the inhibitory effect of isocuB on glioma and its related mechanisms were assessed through Counting Kit-8 (CCK-8), wound healing, transwell, Western blot (WB), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and other in vitro experiments, alongside tumor formation experiments in nude mice. Results: Based on this investigation, it suggested that isocuB might inhibit the growth of gliomas through the PI3K-AKT and MAPK pathways. Additionally, we proposed that isocuB may enhance glioma drug sensitivity to temozolomide (TMZ) via modulation of hsa-mir-1286a. The CCK-8 assay revealed that isocuB exhibited inhibitory effects on U251 and U87 proliferation and outperformed TMZ. Wound healing and transwell experiments showed that isocuB inhibited the invasion and migration of U251 cells by suppressing the activity of MMP-2/9, N-cadherin, and Vimentin. The TdT-mediated dUTP-biotin nick end labeling (TUNEL) and flow cytometry (FCM) assays revealed that isocuB induced cell apoptosis through inhibition of BCL-2. Subsequently, we conducted RT-qPCR and WB experiments, which revealed that PI3K/AKT and MAPK pathways might be involved in the mechanism of the inhibition isocuB on glioma. Additionally, isocuB promoted the sensitivity of glioma U251 to TMZ by inhibiting hsa-mir-1286a. Furthermore, we constructed TMZ-resistant U251 strains and demonstrated effective inhibition by isocuB against these resistant strains. Finally, we confirmed that isocuB can inhibit tumor growth in vivo through experiments on tumors in nude mice. Conclusion: IsocuB may protect against glioma by acting on the PI3K/AKT and MAPK pathways and promote the sensitivity of glioma U251 to TMZ by inhibiting hsa-mir-1286a.

Micro-Osteoperforations Accelerate Tooth Movement without Exacerbating the Progression of Root Resorption in Rats.

A recent study reported that micro-osteoperforations (MOPs) accelerated tooth movement by activating alveolar bone remodeling. However, very little is known about the relationship between MOPs and external apical root resorption during orthodontic treatment. In this study, in order to investigate the mechanism through which MOPs accelerate tooth movement without exacerbating the progression of root resorption, we measured the volume of the resorbed root, and performed the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method on exposed MOPs during experimental tooth movements in rats. Male Wistar rats (11 weeks old) were divided into three groups: 10 g orthodontic force (optimal force) applied to the maxillary first molar (optimal force: OF group), 50 g orthodontic force application (heavy force: HF group), and 10 g force application plus three small perforations of the cortical plate (OF + MOPs group). On days 1, 4, 7, 10, and 14 after force application, the tooth movement and root volume were investigated by micro-computed tomography. Furthermore, the number of apoptotic cells in the pressured sides of the periodontal ligament (PDL) and surrounding hard tissues were determined by TUNEL staining. The OF + MOPs group exhibited a 1.8-fold increase in tooth movement on days 7, 10, and 14 compared with the OF group. On days 14, the HF group had a higher volume of root loss than the OF and OF + MOPs groups. On the same day, the number of TUNEL-positive cells in the HF group increased at the root (cementum) site whereas that in the OF group increased at the alveolar bone site. Furthermore, the number of TUNEL-positive cells in the OF + MOPs group increased at the alveolar bone site compared with the OF group. These results suggest that MOPs accelerate orthodontic tooth movement without exacerbating the progression of root resorption.

Development of injectable in situ hydrogels based on hyaluronic acid via Diels-Alder reaction for their antitumor activities studies

The objective of this study was to develop an injectable hydrogel based on furfuryl amine-conjugated hyaluronic acid (FA-conj-HA) and evaluate the in vivo anti-4 T1 tumor activity of doxorubicin-loaded hydrogel (DOX@FA-conj-HAgel). The cargo-free hydrogel (FA-conj-HAgel) was fabricated through a Diels-Alder reaction at 37 °C with FA-conj-HA as a gel material and four armed poly(ethylene glycol)2000-maleimide (4-arm-PEG2000-Mal) as a cross-linker. The bio-safety of FA-conj-HAgel were assessed, and the in vivo antitumor activity of DOX@FA-conj-HAgel was also investigated. Many 3D network structures were observed from scanning electron microscope (SEM) photograph, confirming the successful preparation of FA-conj-HAgel. The absence of cytotoxicity from FA-conj-HAgel was proved by the high viability of 4 T1 cells. In vivo bio-safety studies suggested that the obtained FA-conj-HAgel did not induce acute toxicity or other lesions in treated mice, confirming its high bio-safety. The reduced tumor volumes, hematoxylin-eosin staining (H&E), and TdT-mediated dUTP-biotin nick end labeling (TUNEL) analysis indicated the potent in vivo anti-4 T1 tumor effects of DOX@FA-conj-HAgel. In conclusion, the favorable bio-safety and potent antitumor activity of DOX@FA-conj-HAgel highlighted its potential application in oncological therapy.

Empagliflozin ameliorates diabetic cardiomyopathy probably via activating AMPK/PGC-1α and inhibiting the RhoA/ROCK pathway.

Diabetic cardiomyopathy (DCM) increases the risk of hospitalization for heart failure (HF) and mortality in patients with diabetes mellitus. However, no specific therapy to delay the progression of DCM has been identified. Mitochondrial dysfunction, oxidative stress, inflammation, and calcium handling imbalance play a crucial role in the pathological processes of DCM, ultimately leading to cardiomyocyte apoptosis and cardiac dysfunctions. Empagliflozin, a novel glucose-lowering agent, has been confirmed to reduce the risk of hospitalization for HF in diabetic patients. Nevertheless, the molecular mechanisms by which this agent provides cardioprotection remain unclear. To investigate the effects of empagliflozin on high glucose (HG)-induced oxidative stress and cardiomyocyte apoptosis and the underlying molecular mechanism. Twelve-week-old db/db mice and primary cardiomyocytes from neonatal rats stimulated with HG (30 mmol/L) were separately employed as in vivo and in vitro models. Echocardiography was used to evaluate cardiac function. Flow cytometry and TdT-mediated dUTP-biotin nick end labeling staining were used to assess apoptosis in myocardial cells. Mitochondrial function was assessed by cellular ATP levels and changes in mitochondrial membrane potential. Furthermore, intracellular reactive oxygen species production and superoxide dismutase activity were analyzed. Real-time quantitative PCR was used to analyze Bax and Bcl-2 mRNA expression. Western blot analysis was used to measure the phosphorylation of AMP-activated protein kinase (AMPK) and myosin phosphatase target subunit 1 (MYPT1), as well as the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and active caspase-3 protein levels. In the in vivo experiment, db/db mice developed DCM. However, the treatment of db/db mice with empagliflozin (10 mg/kg/d) for 8 wk substantially enhanced cardiac function and significantly reduced myocardial apoptosis, accompanied by an increase in the phosphorylation of AMPK and PGC-1α protein levels, as well as a decrease in the phosphorylation of MYPT1 in the heart. In the in vitro experiment, the findings indicate that treatment of cardiomyocytes with empagliflozin (10 μM) or fasudil (FA) (a ROCK inhibitor, 100 μM) or overexpression of PGC-1α significantly attenuated HG-induced mitochondrial injury, oxidative stress, and cardiomyocyte apoptosis. However, the above effects were partly reversed by the addition of compound C (CC). In cells exposed to HG, empagliflozin treatment increased the protein levels of p-AMPK and PGC-1α protein while decreasing phosphorylated MYPT1 levels, and these changes were mitigated by the addition of CC. Adding FA and overexpressing PGC-1α in cells exposed to HG substantially increased PGC-1α protein levels. In addition, no sodium-glucose cotransporter (SGLT)2 protein expression was detected in cardiomyocytes. Empagliflozin partially achieves anti-oxidative stress and anti-apoptotic effects on cardiomyocytes under HG conditions by activating AMPK/PGC-1α and suppressing of the RhoA/ROCK pathway independent of SGLT2.

Environmental enrichment enhanced neurogenesis and behavioral recovery after stroke in aged rats.

Age is identified as a significant prognostic factor for poorer outcome after stroke. However, environmental enrichment (EE) has been reported to promote functional recovery after ischemic stroke. The purpose of this study was to investigate whether environmental enrichment was beneficial to ischemic stroke in aged rats. Aged rats were randomly assigned as control rats, rats subjected to cerebral ischemia, and rats with cerebral ischemia treated with EE for 30 days. Focal cortical ischemia was induced by intracranial injection of endothelin-1 (ET-1). EE housing began one day after focal ischemia and was maintained for the whole experimental period. We used immunofluorescence staining to analyze the neurogenesis in the subventricular zone (SVZ) and TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay to evaluate apoptosis. The expression of neuronal nuclei, glial fibrillary acidic protein (GFAP) and Iba-1 around the infarcted area were also measured by double immunohistochemistry. EE enhanced the proliferation of newborn neurons in the SVZ, as well as increased the long-term survival of newborn neurons. EE also exerted effects on inflammation after stroke. Furthermore, EE suppressed apoptosis and improved the motor functions after stroke in the aged rats. EE improved post-stroke recovery on the basis of enhancing neurogenesis in aged rats.

Kaempferol induces apoptosis through the MAPK pathway and regulates JNK-mediated autophagy in MC-3 cells.

This study sought to determine the anticancer effect of kaempferol, a glycone-type flavonoid glycoside with various pharmacological benefits, on human oral cancer MC-3 cells. In vitro studies comprised a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, annexin V and propidium iodide staining, western blotting analysis, and acridine orange staining, while the in vivo studies entailed a xenograft model, hematoxylin and eosin staining, and TdT-mediated dUTP-biotin nick end labelling. In vitro, kaempferol reduced the rate of survival of MC-3 cells, mediated intrinsic apoptosis, increased the number of acidic vesicular organelles, and altered the expression of autophagy-related proteins. Further, treatment with the autophagy inhibitors revealed that the induced autophagy had a cytoprotective effect on apoptosis in kaempferol-treated MC-3 cells. Kaempferol also decreased the expression of phosphorylated extracellular signal-regulated kinase and increased that of phosphorylated c-Jun N-terminal kinase (p-JNK) and phosphorylated p38 kinase in MC-3 cells, suggesting the occurrence of mitogen-activated protein kinase-mediated apoptosis and JNK-mediated autophagy. In vivo, kaempferol reduced tumor growth inducing apoptosis and autophagy. These results showed that kaempferol has the potential use as an adjunctive agent in treating oral cancer.

Yi-Shen-Xie-Zhuo formula alleviates cisplatin-induced AKI by regulating inflammation and apoptosis via the cGAS/STING pathway.

Yi-Shen-Xie-Zhuo formula (YSXZF) is a traditional Chinese medicine prescription developed from the classic prescription Mulizexie powder documented in the book of Golden Chamber Synopsis and the Buyanghuanwu Decoction recorded in the book of Correction of Errors in Medical Classics. According to our years of clinical experience, YSXZF can effectively improve qi deficiency and blood stasis in kidney disease. However, its mechanisms need further clarification. Apoptosis and inflammation play key roles in acute kidney disease (AKI). The Yi-Shen-Xie-Zhuo formula, consisting of four herbs, is commonly used for treating renal disease. However, the underlying mechanism and bioactive components remain unexplored. This study aimed to investigate the protective effects of YSXZF against apoptosis and inflammation in a cisplatin-treated mouse model, and identify the main bioactive components of YSXZF. C57BL/6 mice were administered cisplatin (15mg/kg) with or without YSXZF (11.375 or 22.75g/kg/d). HKC-8cells were treated with cisplatin (20μM) with or without YSXZF (5% or 10%) for 24h. Renal function, morphology, and cell damage were evaluated. UHPLC-MS was used to analyze the herbal components and metabolites in the YSXZF-containing serum. Blood urea nitrogen (BUN), serum creatinine, serum and urine neutrophil gelatinase-associated lipocalin (NGAL) levels were clearly increased in the cisplatin-treated group. Administration of YSXZF reversed these changes; it improved renal histology, downregulated kidney injury molecule 1 (KIM-1) expression, and lowered the number of TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells. YSXZF significantly downregulated cleaved caspase-3 and BAX, and upregulated BCL-2 proteins in renal tissues. YSXZF suppressed increase in cGAS/STING activation and inflammation. In vitro treatment with YSXZF markedly reduced cisplatin-induced HKC-8cell apoptosis, relieved cGAS/STING activation and inflammation, improved mitochondrial membrane potential (MMP), and lowered reactive oxygen species (ROS) overgeneration. Small RNA interference (siRNA)-mediated silencing of cGAS or STING inhibited the protective effects of YSXZF. Twenty-three bioactive constituents from the YSXZF-containing serum were identified as key components. This is the first study to demonstrate that YSXZF protects against AKI by suppressing inflammation and apoptosis via the cGAS/STING signaling pathway.

Protective role and mechanism of tubastatin A on renal and intestinal injuries after cardiopulmonary resuscitation in swine

To investigate the protective effect and potential mechanism of tubastatin A (TubA), a specific inhibitor of histone deacetylase 6 (HDAC6), on renal and intestinal injuries after cardiopulmonary resuscitation (CPR) in swine. Twenty-five healthy male white swine were divided into Sham group (n = 6), CPR model group (n = 10) and TubA intervention group (n = 9) using a random number table. The porcine model of CPR was reproduced by 9-minute cardiac arrest induced by electrical stimulation via right ventricle followed by 6-minute CPR. The animals in the Sham group only underwent the regular operation including endotracheal intubation, catheterization, and anesthetic monitoring. At 5 minutes after successful resuscitation, a dose of 4.5 mg/kg of TubA was infused via the femoral vein within 1 hour in the TubA intervention group. The same volume of normal saline was infused in the Sham and CPR model groups. Venous samples were collected before modeling and 1, 2, 4, 24 hours after resuscitation, and the levels of serum creatinine (SCr), blood urea nitrogen (BUN), intestinal fatty acid binding protein (I-FABP) and diamine oxidase (DAO) in serum were determined by enzyme-linked immunoadsordent assay (ELISA). At 24 hours after resuscitation, the upper pole of left kidney and terminal ileum were harvested to detect cell apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) were detected by Western blotting. After resuscitation, renal dysfunction and intestinal mucous injury were observed in the CPR model and TubA intervention groups when compared with the Sham group, which was indicated by significantly increased levels of SCr, BUN, I-FABP and DAO in serum. However, the serum levels of SCr and DAO starting 1 hour after resuscitation, the serum levels of BUN starting 2 hours after resuscitation, and the serum levels of I-FABP starting 4 hours after resuscitation were significantly decreased in the TubA intervention group when compared with the CPR model group [1-hour SCr (μmol/L): 87±6 vs. 122±7, 1-hour DAO (kU/L): 8.1±1.2 vs. 10.3±0.8, 2-hour BUN (mmol/L): 12.3±1.2 vs. 14.7±1.3, 4-hour I-FABP (ng/L): 661±39 vs. 751±38, all P < 0.05]. The detection of tissue samples indicated that cell apoptosis and necroptosis in the kidney and intestine at 24 hours after resuscitation were significantly greater in the CPR model and TubA intervention groups when compared with the Sham group, which were indicated by significantly increased apoptotic index and markedly elevated expression levels of RIP3 and MLKL. Nevertheless, compared with the CPR model group, renal and intestinal apoptotic indexes at 24 hours after resuscitation in the TubA intervention group were significantly decreased [renal apoptosis index: (21.4±4.6)% vs. (55.2±9.5)%, intestinal apoptosis index: (21.3±4.5)% vs. (50.9±7.0)%, both P < 0.05], and the expression levels of RIP3 and MLKL were significantly reduced [renal tissue: RIP3 protein (RIP3/GAPDH) was 1.11±0.07 vs. 1.39±0.17, MLKL protein (MLKL/GAPDH) was 1.20±0.14 vs. 1.51±0.26; intestinal tissue: RIP3 protein (RIP3/GAPDH) was 1.24±0.18 vs. 1.69±0.28, MLKL protein (MLKL/GAPDH) was 1.38±0.15 vs. 1.80±0.26, all P < 0.05]. TubA has the protective effect on alleviating post-resuscitation renal dysfunction and intestinal mucous injury, and its mechanism may be related to inhibition of cell apoptosis and necroptosis.

Effects of Iron on Efficacy of Photodynamic Therapy Using Photolon in a Mouse Model of CT26 Colon Cancer.

Photodynamic therapy (PDT) -a minimally invasive anti-cancer therapy-is undergoing experimental studies to increase its anti-cancer effects. This study investigated the influence of iron on the anti-cancer effects of PDT. PDT was performed in a cancer-bearing mouse model, which was created by using a murine colon carcinoma (CT26) cell line after administration of Photolon and iron. Tumor volume and the results of TdT-mediated dUTP-biotin nick end labeling (TUNEL), 8-OHdG, and TBARS assays were used to measure anti-cancer effect. On day 14, tumor volume had increased by 49% in the PDT group and decreased by 72% in the iron+PDT group. The percentage of TUNEL-positive cells in tumor tissues was 45% in the PDT group and 69% in the iron+PDT group, suggesting that the proportion of TUNEL-positive cells had increased in the iron+PDT group. The 8-OHdG content in tumor tissues was 33% higher in the iron+PDT group than in the PDT group. The TBARS content in tumor tissues was 46% higher in the iron+PDT group than in the PDT group. Iron enhances the anti-cancer effect of PDT using Photolon, most likely by increasing oxidative damage.

Immunohistochemical analysis with apoptosis and autophagy markers in periodontitis and peri-implantitis: Clinical comparative study.

Recently, the terms autophagy and apoptosis have been studied on implants, especially in cell culture and in vitro studies, but in vivo evaluations are limited. The aim of this study was to compare the differences in apoptosis and autophagy intensity at the molecular and cellular level in periodontal and peri-implant diseases. Sixty-four biopsy samples were obtained from 52 patients, 36 female and 16 male, whose mean age was between 18 and 75, and were included in the study. The periodontitis group was defined as PG (n:30 sample) and the peri-implantitis group as IG (n:34 samples). Granulation tissues as biopsy materials were collected, and immunohistochemical analysis was performed with hematoxylin-eosin, Masson's trichrome, anti-MAP1LC3A, anti-beclin, and anti-active caspase-3 antibodies and terminal TdT-mediated dUTP-biotin nick end labeling (TUNEL) methods. The histological slide images were evaluated with the ImageJ software program. Inflammatory cell density in epithelial tissue, inflammatory cell density in connective tissue, the density of necrotic tissue debris, and collagen density in connective tissue were scored between 0 and 3 (0: none, 1: minimal, 2: moderate, 3: severe by hematoxylin-eosin and Masson's trichrome). The antibody binding reaction areas were evaluated per unit area (mm2 ) in connective tissue by immunohistochemical examination. As histochemical evaluations, there was no statistically significant differences the mean inflammatory cell density value in the epithelial tissue, inflammatory cell density value in the connective tissue, density value of necrotic tissue debris, collagen density value in the connective tissue between the groups. There was no statistically significant difference on immunohistochemical staining with LC3, caspase-3, Beclin-1 and TUNEL between the two groups (p > .05). A higher rate of inflammatory accumulation was shown on peri-implantitis, but no difference was found between periodontitis and peri-implantitis according to autophagy and apoptosis markers. Studies with high sample sizes with different markers are needed.

Interaction between Porcine Alveolar Macrophage-Tang Cells and Streptococcus suis Strains of Different Virulence: Phagocytosis and Apoptosis

Streptococcus suis is an important swine bacterial pathogen that activates macrophages to secrete inflammatory cytokines. Primary porcine alveolar macrophages (PAMs) are inconvenient to obtain, but it is unknown whether immortalized PAM-Tang cells can replace them as a better cell model for the study of the interaction between S. suis and macrophages. In this study, the phagocytic integrity, polarization, and pro-inflammatory cytokine secretion of PAM-Tang cells were confirmed by live-cell imaging, electron microscopy, confocal microscopy, and ELISA. Interestingly, the S. suis serotype 9 avirulent strain W7119 induced higher levels of adhesion and pro-inflammatory cytokines in PAM-Tang cells than the S. suis serotype 2 virulent strain 700794. Prolonged incubation with S. suis caused more cytotoxic cell damage, and the virulent strain induced higher levels of cytotoxicity to PAM-Tang cells. The virulent strain also induced higher levels of apoptosis in PAM-Tang cells, as shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay. In addition, it is the first report of virulent and avirulent S. suis inducing PAM-Tang polarization towards pro-inflammatory M1 macrophages and p53- and caspase-dependent apoptosis in PAMs. Taken together, this study contributes to a better understand of interactions between macrophages and S. suis isolates of different virulence, and confirms that PAM-Tang cells provide a long-term, renewable resource for investigating macrophage infections with bacteria.

Neuroprotective effects of Chrysanthemum morifolium on cerebral ischemia- reperfusion injury contributes to the oxidative stress suppression and related Keap1/Nrf2 pathway

ABSTRACT Background Ischemic stroke, the cause of death and disability worldwide, is closely related to oxidative stress damage. Chrysanthemum has profound antiantioxidant activity. We aimed to verify whether Chrysanthemum morifolium extract (CME) influences brain injury in cerebral ischemia-reperfusion injury (CR/RI) model. Methods In vitro, rat hippocampal H19-7 neurons were pretreated with CME, CR/RI was simulated with oxygen glucose deprivation/reoxygenation (OGD/R). The cell viability, apoptosis, lactate dehydrogenase release, reactive oxygen species (ROS) generation, malonaldehyde (MDA) content and superoxide dismutase（SOD） activity were detected. In vivo, middle cerebral artery occlusion (MCAO) model rats were pre-administered with CME, and then behavioral test, triphenyltetrazolium chloride (TTC), hematoxylin-eosin staining (HE), terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), ROS immunofluorescence, MDA and SOD activity were tested. Furthermore, Keap1/Nrf2 signaling of CME in CI/RI was investigated. Results In OGD/R induced in H19-7 cells, CME increased OGD/R-induced cell viability and reduced cell apoptosis, which was reversed by siNrf2 transfection . In MCAO rats, CME improved the neurological deficits and alleviated brain injury. However, co-treatment with MLK385 counteracted these neuroprotective effects of CME on MCAO rats. Conclusion CME could significantly reduce oxidative stress and nerve injury in vitro and in vivo models of CI/RI by regulating the Keap1/Nrf2 pathway.

Electroacupuncture inhibits hippocampal neuronal apoptosis and improves cognitive dysfunction in mice with vascular dementia via the JNK signaling pathway.

Electroacupuncture (EA) has been shown to reduce cognitive impairment in vascular dementia (VaD) patients. However, the mechanism of action remains unknown. The c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in apoptosis. Herein, we focused on whether EA can inhibit apoptosis and alleviate cognitive impairment by regulating the JNK signaling pathway using a mouse model of VaD induced by modified bilateral common carotid artery occlusion (BCCAo). In experiment I, 60 mice were randomly divided into a Sham group, BCCAo group, BCCAo + EA group, BCCAo + Sham-EA group, BCCAo + SP group (receiving the selective JNK inhibitor SP600125) and BCCAo + SP + EA group. Morris water maze tests, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining and flow cytometry were used to evaluate the effect of the EA intervention on VaD. In experiment II, 30 mice were randomly divided into a Sham group, BCCAo group, BCCAo + EA group, BCCAo + SP group and BCCAo + SP + EA group. Western blotting and real-time reverse transcription polymerase chain reaction were used to detect protein and mRNA expression of key factors in the JNK signaling pathway in the hippocampus. EA, SP600125 and EA + SP600125 significantly inhibited hippocampal apoptosis and improved cognitive impairment in VaD model mice. There were no significant differences between the BCCAo group and the BCCAo + Sham-EA group. EA, EA + SP600125 and SP600125 inhibited the phosphorylation of JNK and caspase-3. EA and EA + SP600125 promoted protein and mRNA expression of B-cell lymphoma 2 (Bcl-2) in the hippocampus of VaD mice and inhibited protein and mRNA expression of activator protein (AP)-1, p53 and Bax. EA can reverse cognitive deficits and inhibit hippocampal neuronal apoptosis in VaD model mice, at least partially through inhibition of the JNK signaling pathway and regulation of apoptosis signals.

Bloodletting Acupuncture at Jing-Well Points Alleviates Myocardial Injury in Acute Altitude Hypoxic Rats by Activating HIF-1α/BNIP3 Signaling-Mediated Mitochondrial Autophagy and Decreasing Oxidative Stress.

To explore the protective effect and possible mechanisms of bloodletting acupuncture at Jing-well points (BAJP) pre-treatment on acute hypobaric hypoxia (AHH)-induced myocardium injury rat. Seventy-five rats were randomly divided into 5 groups by a random number table: a control group (n=15), a model group (n=15), a BAJP group (n=15), a BAJP+3-methyladenine (3-MA) group (n=15), and a BANA (bloodletting at nonacupoint; tail bleeding, n=15) group. Except for the control group, the AHH rat model was established in the other groups, and the corresponding treatment methods were adopted. Enzyme-linked immunosorbent assay (ELISA) was used to detect creatine kinase isoenzyme MB (CK-MB) and cardiac troponins I (CTnI) levels in serum and superoxide dismutase (SOD) and malondialdehyde (MDA) levels in myocardial tissue. Hematoxylin-eosin (HE) staining was used to observe myocardial injury, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to observe cell apoptosis. Transmission electron microscopy detection was used to observe mitochondrial damage and autophagosomes in the myocardium. The mitochondrial membrane potential of the myocardium was analyzed with the fluorescent dye JC-1. Mitochondrial respiratory chain complex (complex I, III, and IV) activities and ATPase in the myocardium were detected by mitochondrial respiratory chain complex assay kits. Western blot analysis was used to detect the autophagy index and hypoxia inducible factor-1α (HIF-1α)/Bcl-2 and adenovirus E1B 19k Da-interacting protein 3 (BNIP3) signaling. BAJP reduced myocardial injury and inhibited myocardial cell apoptosis in AHH rats. BAJP pretreatment decreased MDA levels and increased SOD levels in AHH rats (all P<0.01). Moreover, BAJP pretreatment increased the mitochondrial membrane potential (P<0.01), mitochondrial respiratory chain complex (complexes I, III, and IV) activities (P<0.01), and mitochondrial ATPase activity in AHH rats (P<0.05). The results from electron microscopy demonstrated that BAJP pretreatment improved mitochondrial swelling and increased the autophagosome number in the myocardium of AHH rats. In addition, BAJP pretreatment activated the HIF-1α/BNIP3 pathway and autophagy. Finally, the results of using 3-MA to inhibit autophagy in BAJP-treated AHH rats showed that suppression of autophagy attenuated the treatment effects of BAJP in AHH rats, further proving that autophagy constitutes a potential target for BAJP treatment of AHH. BAJP is an effective treatment for AHH-induced myocardial injury, and the mechanism might involve increasing HIF-1α/BNIP3 signaling-mediated autophagy and decreasing oxidative stress.

Comparison of Cytotoxic and Ototoxic Effects of Lipoplatin and Cisplatin in Neuroblastoma In Vivo Tumor Model.

Background:This study aimed to compare the cytotoxic, cytostatic, and ototoxic effects of lipoplatin compared to cisplatin application in the subcutaneous xenograft nude mouse neuroblastoma tumor model.Methods:In this study, C1300 neuroblastoma cells were administered subcutaneously to 21 male nude mice. When the tumor reached 150 mm3 diameter, mice were randomized into 3 groups. Saline, cisplatin, and lipoplatin were given intraperitoneally. The auditory function tests were performed before administration and 72 hours after administration. Mice were sacrificed and the tumor and cochlea were removed after 72 hours. Histopathologic evaluation of necrosis and apoptosis was determined by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Cyclooxygenase 2, superoxide dismutase 2, and inducible nitric oxide synthase levels were determined by immunohistochemistry in tissue samples.Results:Apoptosis and necrosis rates were higher in lipoplatin group than in cisplatin group (P = .035 and P = .010, respectively) in tumor tissue. In the spiral ganglion, apoptosis and necrosis were lower in the lipoplatin group than in cisplatin group (P = .002 and P = .002, respectively). Cyclooxygenase 2 pattern in the cochlea was positive in both control and lipoplatin group and negative in cisplatin group (P = .001). Superoxide dismutase 2 and inducible nitric oxide synthase 2 protein expressions showed no difference between groups. The auditory functions were similar to baseline values and had a better threshold value in lipoplatin group than cisplatin group.Conclusion:For the treatment of neuroblastoma, the use of lipoplatin seems to be beneficial in reducing side effects of cisplatin. We recommend that the mechanism of these properties of lipoplatin should be evaluated in further studies.

Dynorphin/KOR inhibits neuronal autophagy by activating mTOR signaling pathway to prevent acute seizure epilepsy.

In previous studies, we found that dynorphin exerts antiepileptic effect by activating the kappa opioid receptor (KOR). However, the role of neuronal autophagy in dynorphin/KOR-mediated antiepileptic is still unclear. This study aimed to investigate the molecular mechanism of dynorphin's antiepileptic effect by inhibiting autophagy and reducing neuronal apoptosis. Here, a pilocarpine-induced rat model of epilepsy was established and hippocampal neurons were treated with Mg2+ -free exposed for epileptiform activity induction. The real-time polymerase chain reaction and Western blot analysis were used to evaluate messengerRNA and protein expression. The TdT-mediated dUTP-biotin nick end labeling staining and flow cytometry were used to analyze cell apoptosis in vivo and in vitro. Neuron cells viability was detected by Cell Counting Kit-8 assay. Immunofluorescent staining and green fluorescent protein-light chain 3 immunofluorescence were used to measure autophagy in vivo and in vitro. Results showed that overexpression ofprodynorphinalleviated neuronal apoptosis, activated the mammalian target of rapamycin (mTOR) signaling pathway, and inhibited neuronal autophagy in epileptic rats. Dynorphin inhibited Mg2+ -free-induced seizure-like neuron apoptosis, partially reversing the effect of Mg2+ -free on the mTOR signaling pathway and seizure-like neuron autophagy. Further, using rapamycin, we found that dynorphin inhibited Mg2+ -free-induced seizure-like neuron autophagy and apoptosis by activating the mTOR signaling pathway. In conclusion, dynorphin inhibits autophagy by activating the mTOR signaling pathway and has a protective effect on epilepsy acute seizure and epilepsy-induced brain injury.

Transcriptome analysis revealed gene expression feminization of testis after exogenous tetrodotoxin administration in pufferfish Takifugu flavidus

Tetrodotoxin (TTX) is a deadly neurotoxin and usually accumulates in large amounts in the ovaries but is non-toxic or low toxic in the testis of pufferfish. The molecular mechanism underlying sexual dimorphism accumulation of TTX in ovary and testis, and the relationship between TTX accumulation with sex related genes expression remain largely unknown. The present study investigated the effects of exogenous TTX treatment on Takifugu flavidus. The results demonstrated that exogenous TTX administration significantly incresed level of TTX concentration in kidney, cholecyst, skin, liver, heart, muscle, ovary and testis of the treatment group (TG) than that of the control group (CG). Transcriptome sequencing and analysis were performed to study differential expression profiles of mRNA and piRNA after TTX administration of the ovary and testis. The results showed that compared with female control group (FCG) and male control group (MCG), TTX administration resulted in 80 and 23 piRNAs, 126 and 223 genes up and down regulated expression in female TTX-treated group (FTG), meanwhile, 286 and 223 piRNAs, 2 and 443 genes up and down regulated expression in male TTX-treated group (MTG). The female dominant genes cyp19a1, gdf9 and foxl2 were found to be up-regulated in MTG. The cyp19a1, whose corresponding target piRNA uniq_554482 was identified as down-regulated in the MTG, indicating the gene expression feminization in testis after exogenous TTX administration. The KEGG enrichment analysis revealed that differentially expressed genes (DEGs) and piRNAs (DEpiRNAs) in MTG vs MCG group were more enriched in metabolism pathways, indicating that the testis produced more metabolic pathways in response to exogenous TTX, which might be a reason for the sexual dimorphism of TTX distribution in gonads. In addition, TdT-mediated dUTP-biotin nick end labeling staining showed that significant apoptosis was detected in the MTG testis, and the role of the cell apoptotic pathways was further confirmed. Overall, our research revealed that the response of the ovary and testis to TTX administration was largely different, the ovary is more tolerant whereas the testis is more sensitive to TTX. These data will deepen our understanding on the accumulation of TTX sexual dimorphism in Takifugu.

Activation of the Nrf2/GPX4 Signaling by Pratensein From Trifolium pretense Mitigates Ferroptosis in OGD/R-Insulted H9c2 Cardiomyocytes

Background: Pratensein (PTS) is a type of flavonoid that has been identified in various plants, such as Trifolium pretense L., with a considerable cytoprotective effect against exogenous stimuli. However, the biological function of PTS in cardiomyocytes in response to ischemia-reperfusion (I/R) conditions is unclear. Purpose: In our study, we examined the function of PTS in the progression of myocardial infarction (MI). Methods: In this study, we established an oxygen-glucose deprivation/reoxygenation (OGD/R) model in H9c2 cells. The Cell Counting Kit-8 assay was used to assess the viability of H9c2 cells. The TdT-mediated dUTP-biotin nick end labeling and flow cytometry assays confirmed apoptosis of H9c2 cells. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) content, and Fe2+ level were evaluated. Western blotting was used to detect relative protein expression. Results: We firstly found that PTS reduced apoptosis of H9c2 cells in response to OGD/R stimulation. PTS attenuates the increase in ROS and MDA production and the decrease in GSH content caused by OGD/R. The increased Fe2+ level in OGD/R-treated H9c2 cells was also restrained by PTS. For mechanism studies, we found that the decreased expression levels of Nrf2 and GPX4 in OGD/R-treated H9c2 cells were significantly elevated after PTS treatment. Knockdown of Nrf2 in H9c2 cells reversed the protective effect of PTS on ferroptosis in H9c2 cells induced by OGD/R, indicated by reduced cell viability, increased apoptotic cells and oxidation markers, and increased Fe2+ level. Conclusion: Based on these findings, we speculated that PTS may protect H9c2 cells from OGD/R-caused ferroptosis by modulating the Nrf2/GPX4 signaling.