JC-1 Assay Research Articles

Protective mechanism of gold nanoparticles on human neural stem cells injured by β-amyloid protein through miR-21–5p/SOCS6 pathway

Alzheimer’s disease (AD) is a neurodegenerative disorder with progressive memory loss in dementia. Gold nanoparticles (AuNPs) were reported beneficial for human neural stem cells (hNSCs) treated with Amyloid-beta (Aβ), but the neuroprotective mechanisms still are unknown. First, the hNSCs induced by Aβ to construct AD cell model in vitro and AuNPs was performed to assess the therapeutic effect of Aβ-targeted AD treatment. Then, we investigated the effects of AuNPs on hNSCs viability and proinflammatory factors (interleukin 6 and tumor necrosis factor-alpha) by Cell Counting Kit-8 (CCK-8) and enzyme-linked immunosorbent (ELISA). FACS was carried out to determinate Tuj-1 and glial fibrillary acidic protein (GFAP). Reactive oxygen species (ROS) generation and mitochondrial membrane potential was evaluated by ROS and JC-1 assay kit. In addition, miRNA array was used to systematically detect the differential miRNAs. Dual-luciferase reporter assay was applied to verify the targeting relationship between miR-21–5p and the suppressor of cytokine signalling 6(SOCS6). Quantitative PCR (qPCR) and Western blot assessments were also used to detect related gene expression intracellularly or in the supernatant. The results demonstrate that AuNPs co-treatment repressed the high expression of total tau (T-tau), phosphorylated tau (P-tau), and Aβ protein, and reduced apoptosis rate of hNSCs. Aβ-induced decreased mitochondrial membrane potential and mitochondria in the hNSCs were damaged, while AuNPs co-treatment showed a protective effect on mitochondrial membrane potential. Co-treatment with AuNPs significantly increased dynamin-related protein 1 (DRP1), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (TFAM) mRNA levels. AuNPs may improve mitochondrial function impairment due to Aβ by elevating mitochondrial membrane potential, upregulating regulators of mitochondrial biogenesis, and inhibiting ROS production. hNSCs transfected with miR-21–5p inhibitor reversed AuNPs mediated cytoprotection induced by Aβ. AuNPs upregulation of miR-21–5p expression and exert a mitochondrial protective function. Overexpression of miR-21–5p contributes to enhancing the effect of cytoprotection of AuNPs. MiR-21–5p direct targeting SOCS6 and overexpression SOCS6 exerted opposite effects on hNSCs compared with miR-21–5p mimic group. In conclusion, AuNPs can protect hNSCs from Aβ injury and decrease mitochondrial damage by regulating the miR-21–5p/SOCS6 pathway.

PINK1-mediated mitophagy contributes to glucocorticoid-induced cathepsin K production in osteocytes.

Glucocorticoid (GC) is one of frequently used anti-inflammatory agents, but its administration is unfortunately accompanied with bone loss. Although sporadic studies indicated that osteocytes are subject to a series of pathological changes under GC stress, including overexpression of cathepsin K, the definite role of osteocytes in GC-induced bone loss remains largely unclear. Gene expression of Ctsk and protein levels of cathepsin K were assessed in MLO-Y4 cell lines exposed to dexamethasone (Dex) of different time (0, 12, 24 hours) and dose (0, 10-8 and 10-6 M) courses by RT-qPCR and western blotting, respectively. Confocal imaging and immunostaining were then performed to evaluate the effects of osteocyte-derived cathepsin K on type I collagen in a primary osteocyte ex vivo culture system. MitoTracker Red was used to stain mitochondria for mitochondria morphology assessment and JC-1 assay was employed to evaluate the mitochondria membrane potential in MLO-Y4 cells following Dex treatment. Activation of PINK1-mediated mitophagy was evaluated by immunostaining of the PINK1 protein and CytoID assay. Mdivi-1 was used to inhibit mitophagy and siRNAs were used for the inhibition of Pink1 and Atg5. GC triggered osteocytes to produce excessive cathepsin K which in turn led to the degradation of type I collagen in the extracellular matrix in a primary osteocyte ex vivo culture system. Meanwhile, GC administration increased mitochondrial fission and membrane depolarization in osteocytes. Further, the activation of PINK1-mediated mitophagy was demonstrated to be responsible for the diminishment of dysfunctional mitochondria in osteocytes. Examination of relationship between mitophagy and cathepsin K production revealed that inhibition of mitophagy via knocking down Pink1 gene abolished the GC-triggered cathepsin K production. Interestingly, GC's activation effect towards cathepsin K via mitophagy was found to be independent on the canonical autophagy as this effect was not impeded when inhibiting the canonical autophagy via Atg5 suppression. GC-induced PINK1-mediated mitophagy substantially modulates the production of cathepsin K in osteocytes, which could be an underlying mechanism by which osteocytes contribute to the extracellular matrix degradation during bone loss. Findings of the current study indicate a possible role of osteocyte mitophagy in GC-induced bone loss, which provides a potential therapeutic approach to alleviate GC-induced osteoporosis by targeting PINK1-mediated osteocytic mitophagy.

The effects of cycloastragenol on bovine embryo development, implantation potential and telomerase activity.

Telomerase reverse transcriptase is a key factor responsible for structural and cellular alterations in aged oocytes and changes in the structure of the zona pellucida and mitochondria. Telomerase expression is reduced in aged cumulus oocyte complexes, and its activation or enhanced expression would be beneficial for in vitro oocyte maturation and in vitro embryo development. This study aimed to investigate telomerase activation by cycloastragenol and its effect on bovine oocyte in vitro maturation, fertilisation, and early embryo development. We used qPCR, Western blot, immunofluorescence, reactive oxygen species (ROS) assay,TUNEL assay, JC-1 assay, and invasion assay to analyse the affect of cycloastragenol (CAG) on bovine oocyte maturation, embryo development, embryo quality and implantation potential. Cycloastragenol treatment of oocytes in in vitro maturation (IVM) media significantly (P <0.05) improved oocyte IVM (90.87%), embryo cleavage (90.78%), blastocyst hatching (27.04%), and embryo implantation potential. Telomerase also interacts with mitochondria, and JC-1 staining results showed significantly (P <0.05) higher mitochondrial membrane potential (ΔΨ m) in the CAG-treated group. Furthermore, the inner cell mass (OCT4 and SOX2) and trophoblasts (CDX2) of the control and CAG groups were examined. Moreover, CAG treatment to primary cultured bovine cumulus cells substantially enhanced telomerase activity. Telomerase activation via cycloastragenol is beneficial for bovine oocyte IVM and for the production of high-quality bovine embryos. Cycloastragenol is a natural telomerase activator, and could be useful as a permanent component of oocyte maturation media.

Curcumin Attenuates Damage to Rooster Spermatozoa Exposed to Selected Uropathogens.

Artificial insemination, as an essential pillar of the modern poultry industry, primarily depends on the quality of semen collected from stud roosters. Since the collection and storage of ejaculates is not a sterile process, antimicrobial agents have become essential supplements to semen extenders. While the use of traditional antibiotics has been challenged because of rising bacterial resistance, natural biomolecules represent an appealing alternative because of their antibacterial and antioxidant properties. As such, this study strived to compare the effects of 50 μmol/L curcumin (CUR) with 31.2 µg/mL kanamycin (KAN) as a conventional antibiotic on rooster sperm quality in the presence of Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa. Changes in sperm structural integrity and functional activity were monitored at 2 and 24 h of culture. Computer-assisted semen analysis revealed significant sperm motility preservation following treatment with KAN, particularly in the case of Salmonella enterica and Pseudomonas aeruginosa (p &lt; 0.001) after 24 h. On the other hand, CUR was more effective in opposing ROS overproduction by all bacteria (p &lt; 0.05), as determined by luminol-based luminometry, and maintained sperm mitochondrial activity (p &lt; 0.001 in the case of Salmonella enterica; p &lt; 0.05 with respect to Escherichia coli and Pseudomonas aeruginosa), as assessed by the fluorometric JC-1 assay. The TUNEL assay revealed that CUR readily preserved the DNA integrity of rooster sperm exposed to Salmonella enterica (p &lt; 0.01) and Escherichia coli (p &lt; 0.001). The bacteriological analysis showed higher efficiency of KAN in preventing the growth of all selected bacterial species (p &lt; 0.0001) as opposed to CUR. In conclusion, CUR provided protection to rooster spermatozoa against alterations caused by uropathogens, most likely through its antioxidant activity. Hence, CUR supplementation to poultry semen extenders in combination with properly selected antibacterial substances may become an interesting strategy in the management of bacterial contamination during semen storage.

Spirulina platensis Mitigates the Inhibition of Selected miRNAs that Promote Inflammation in HAART-Treated HepG2 Cells.

The introduction of highly active antiretroviral therapy (HAART) in the treatment of HIV/AIDS has recently gained popularity. In addition, the significant role of microRNA expression in HIV pathogenesis cannot be overlooked; hence the need to explore the mechanisms of microRNA expression in the presence of HAART and Spirulina platensis (SP) in HepG2 cells. This study investigates the biochemical mechanisms of microRNA expression in HepG2 cells in the presence of HAART, SP, and the potential synergistic effect of HAART−SP. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability following SP treatment. The cellular redox status was assessed using the quantification of intracellular reactive oxygen species (ROS), lipid peroxidation, and a lactate dehydrogenase (LDH) assay. The fluorometric JC-1 assay was used to determine mitochondrial polarisation. The quantitative polymerase chain reaction (qPCR) was also employed for micro-RNA and gene expressions. The results show that MiR-146a (p < 0.0001) and miR-155 (p < 0.0001) levels increased in SP-treated cells. However, only miR-146a (p < 0.0001) in HAART−SP indicated an increase, while miR-155 (p < 0.0001) in HAART−SP treatment indicated a significant decreased expression. Further inflammation analysis revealed that Cox-1 mRNA expression was reduced in SP-treated cells (p = 0.4129). However, Cox-1 expression was significantly increased in HAART−SP-treated cells (p < 0.0001). The investigation revealed that HepG2 cells exposed to HAART−SP treatment showed a significant decrease in Cox-2 (p < 0.0001) expression. mRNA expression also decreased in SP-treated cells (p < 0.0001); therefore, SP potentially controls inflammation by regulating microRNA expressions. Moreover, the positive synergistic effect is indicated by normalised intracellular ROS levels (p < 0.0001) in the HAART−SP treatment. We hereby recommend further investigation on the synergistic roles of SP and HAART in the expression of microRNA with more focus on inflammatory and oxidative pathways.

Mitochondrial Dysfunction Involved in the Cytotoxicity of Tramadol in Human Endometrial Carcinoma Cells

Tramadol is a common anesthetic used to treat cancer pain, including endometrial cancer, but its function in endometrial cancer remains unclear. The purpose of this study was to elucidate the antitumor effects of tramadol on human endometrial cancer cells. Colony formation, BrdU, cell cycle profiles, apoptosis, ROS, and Western blot analyses were used to study the response of endometrial cancer cells to tramadol. JC-1 and seahorse metabolic flux assays were used to detect the effect of tramadol on mitochondria in endometrial cancer cells. Combination index was used to detect the interaction of tramadol with chemotherapy drugs in endometrial cancer cells. In this study, we found that tramadol was able to inhibit proliferation and induce cell cycle arrest, ROS generation, and apoptosis in two types of endometrial cancer cells. In addition, tramadol treatment also induced mitochondrial dysfunction in endometrial cancer cells by causing a loss of mitochondrial membrane potential and a decreased oxygen consumption rate. More importantly, the synergetic effect of tramadol with doxorubicin or cisplatin was further confirmed in endometrial cancer cells by the results of the combination index and apoptosis assay. In summary, our findings indicate that tramadol has an antitumor effect on endometrial cancer cells, which might serve as a potential adjuvant therapy strategy for endometrial cancer.

Neuroprotective effects of a lead compound from coral via modulation of the orphan nuclear receptor Nurr1.

To screen coral-derived compounds with neuroprotective activity and clarify the potential mechanism of lead compounds. The lead compounds with neuroprotective effects were screened by H2 O2 and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP+ )-induced cell damage models in SH-SY5Y cells. CCK8 and LDH assays were used to detect cell viability. The anti-apoptosis of lead compounds was evaluated by flow cytometry. JC-1 and MitoSox assays were performed to examine the changes in mitochondrial membrane potential and mitochondrial ROS level. Survival of primary cortical and dopaminergic midbrain neurons was measured by MAP2 and TH immunoreactivities. The Caenorhabditis elegans (C. elegans) model was established to determine the effect of lead compounds on dopaminergic neurons and behavior changes. Three compounds (No. 63, 68, and 74), derived from marine corals, could markedly alleviate the cell damage and notably reverse the loss of worm dopaminergic neurons. Further investigation indicated that compound 63 could promote the expression of Nurr1 and inhibit neuronal apoptosis signaling pathways. Lead compounds from marine corals exerted significant neuroprotective effects， which indicated that coral might be a new and potential resource for screening and isolating novel natural compounds with neuroprotective effects. Furthermore, this study also provided a new strategy for the clinical treatment of neurodegenerative diseases such as Parkinson's disease.

Response of Skin-Derived and Metastatic Human Malignant Melanoma Cell Lines to Thymoquinone and Thymoquinone-Loaded Liposomes.

Thymoquinone has been proved to be effective against neoplasms, including skin cancer. Its high lipophilicity, however, may limit its potential use as a drug. Melanoma remains the deadliest of all skin cancers worldwide, due to its high heterogeneity, depending on the stage of the disease. Our goal was to compare the anti-cancer activity of free thymoquinone and thymoquinone-loaded liposomes on two melanoma cell lines that originated from different stages of this cancer: skin-derived A375 and metastatic WM9. We evaluated the proapoptotic effects of free thymoquinone by flow cytometry and Western blot, and its mitotoxicity by means of JC-1 assay. Additionally, we compared the cytotoxicity of free thymoquinone and thymoquinone in liposomes by WST-1 assay. Our results revealed a higher antiproliferative effect of TQ in WM9 cells, whereas its higher proapoptotic activity was observed in the A375 cell line. Moreover, the thymoquinone-loaded liposome was proved to exert stronger cytotoxic effect on both cell lines studied than free thymoquinone. Differences in the response of melanoma cells derived from different stages of the disease to thymoquinone, as well as their different responses to free and carrier-delivered thymoquinone, are essential for the development of new anti-melanoma therapies. However, further research is required to fully understand them.

Exploring oxidative stress and endothelial dysfunction as a mechanism linking bisphenol S exposure to vascular disease in human umbilical vein endothelial cells and a mouse model of postnatal exposure

BackgroundStructural analogues used to replace bisphenol A (BPA) since the introduction of new regulatory restrictions are considered emerging environmental toxicants and remain understudied with respect to their biological actions and health effects. Studies reveal a link between BPA exposure and vascular disease in human populations, whereas the vascular effects of BPA substitutes remain largely unknown. ObjectivesTo determine the effect of BPS, a commonly used BPA substitute, on redox balance, nitric oxide (NO) availability and microvascular NO-dependent dilation. MethodsIn human umbilical vein endothelial cells (HUVEC), production of reactive oxygen species (ROS) and NO after exposure to BPS was measured using fluorescent probes for DCFDA and DAF-FM diacetate, respectively. The contribution of endothelial NO synthase (eNOS) uncoupling to ROS generation was determined by measuring ROS in the presence or absence of an eNOS inhibitor (L-NAME) or eNOS co-factor, BH4, while the contribution of mitochondria-derived ROS was determined by treating cells with mitochondria-specific antioxidants prior to BPS exposure. Bioenergetic profiles were assessed using Seahorse extracellular flux analysis and mitochondria membrane polarization was measured with TMRE and JC-1 assays. In a mouse model of low dose BPS exposure, NO-mediated endothelial function was assessed in pressurized microvessels by inducing endothelium-dependent dilation in the presence or absence of L-NAME. ResultsBPS exposure (≥25 nM) reduced NO and increased ROS production in HUVEC, the latter corrected by treating cells with L-NAME or BH4. BPS exposure led to a loss of mitochondria membrane potential but had no impact on bioenergetic parameters except for a decrease in the spare respiratory capacity. Treatment of HUVEC with mitochondria-specific antioxidants abolished the effect of BPS on NO and ROS. NO-mediated vasodilation was impaired in male mice exposed to BPS. DiscussionExposure to BPS may promote cardiovascular disease by perturbing NO-mediated vascular homeostasis through the induction of oxidative stress.

Neurexin 2 p.G849D variant, implicated in Parkinson's disease, increases reactive oxygen species, and reduces cell viability and mitochondrial membrane potential in SH-SY5Y cells.

Parkinson's disease (PD) is a neurodegenerative movement disorder, affecting 1-2% of the human population over 65. A previous study by our group identified a p.G849D variant in neurexin 2α (NRXN2) co-segregating with PD, prompting validation of its role using experimental methods. This novel variant had been found in a South African family with autosomal dominant PD. NRXN2α is an essential synaptic maintenance protein with multiple functional roles at the synaptic cleft. The aim of the present study was to investigate the potential role of the translated protein NRXN2α and the observed mutant in PD by performing functional studies in an in vitro model. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells to assess the effect of the mutant on cell viability and apoptosis [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay; ApoTox-Glo™ Triplex Assay)], mitochondrial membrane potential (MMP; MitoProbe™ JC-1 Assay), mitochondrial network analysis (MitoTracker®) and reactive oxygen species (ROS; ROS-Glo™ H2O2 Assay). Cells transfected with the mutant NRXN2α plasmid showed decreased cell viability and MMP. They also exhibited increased ROS production. However, these cells showed no changes in mitochondrial fragmentation. Our findings led us to speculate that the p.G849D variant may be involved in a toxic feedback loop leading to neuronal death in PD. Mitochondrial dysfunction and synaptic dysfunction have been linked to PD. Therefore, findings from this exploratory study are in line with previous studies connecting these two processes and warrants further investigation into the role of this variant in other cellular and animal models.

Infectious spleen and kidney necrosis virus induces the reactive oxidative species/Nrf2-mediated oxidative stress response for the regulation of mitochondrion-mediated Bax/Bak cell death signals in GF-1 cells.

Infectious spleen and kidney necrosis virus (ISKNV) infections can trigger host cell death and are correlated with viral replication; however, they have rarely been considered in terms of the host organelle involvement. In the present study, we demonstrated that ISKNV triggered an oxidative stress signal in the Nrf2-mediated oxidative stress response and induced stress signals for Bax/Bak-mediated host cell death in fish GF-1 cells. The results showed that after ISKNV infection, the levels of reactive oxidative species (ROS) increased by 60–80% from day 3 to day 5, as assessed by an H2DCFDA assay for tracing hydrogen peroxide (H2O2), which was correlated with up to a one-fold change in the fish GF-1 cells. Furthermore, we found that ISKNV infection induced Nrf2-mediated ROS stress signals from D1 to D5, which were correlated with the upregulation of antioxidant enzymes, such as catalase, SOD1, and SOD2; these effects were blocked by the antioxidants GSH and NAC. By analyzing Nrf2-mediated ROS stress signals for cell death regulation via an apoptotic assay, we found that treatment with antioxidants reduced annexin-V-positive signals by 10% (GSH) to 15% (NAC); moreover, necrotic-positive signals were reduced by 6% (GSH) and 32% (NAC) at day 5 (D5) in GF-1 cells, as indicated by PI staining. Furthermore, we found that Nrf2-mediated ROS stress regulated mitochondrion-mediated Bax/Bak death signals at D3 and D5; this was effectively blocked by antioxidant treatment in the GF-1 cells, as demonstrated by a JC1 assay (ΔΨm) and western blot analysis. In addition, we found that downstream signals for caspase-9 and -3 activation were apparently blocked by antioxidant treatment at D3 and D5. Finally, we found that treatment with GSH and NAC reduced major capsid protein (MCP) expression and virus titer (TCID50%) by up to 15-fold at D5 in GF-1 cells. Thus, our data suggest that ISKNV can induce ROS production, which triggers Nrf2-mediated stress signals. Then, these stress signals can regulate mitochondrion-mediated Bax/Bak apoptotic signaling, which is connected to downstream caspase-9 and -3 activation. If ISKNV-induced Nrf2-mediated stress signaling is blocked, then the antioxidants GSH and NAC can also suppress apoptotic signals or reduce viral replication. These findings may provide insights into the control and treatment of double-stranded DNA viruses.

Chitosan-poly(ethylene oxide) nanofibrous mat as a vaginal platform for tenofovir disoproxyl fumarate – The effect of vaginal pH on drug carrier performance

In the present work, a solution blow spun nanofibrous mat comprised of chitosan (CS) and poly(ethylene oxide) (PEO) was obtained as vaginal platform for tenofovir disoproxil fumarate (TDF) to prevent sexually transmitted infections. Apart from physicochemical and mechanical analysis, the specific steps involved studies on nanofibrous mat mucoadhesive and swelling characteristics upon pH fluctuations over the physiological range. Physicochemical analysis showed uniform drug distribution within the CS/PEO mat volume and pointed toward physical interactions between the drug and polymers. TDF-loaded CS/PEO nanofibrous mat was shown potentially safe when evaluated by the MTT metabolic activity and JC-1 assays in human vaginal epithelial cells VK2-E6/E7. In vitro antiviral studies indicated inhibition efficacy of TDF-CS/PEO nanofibrous mat toward HSV-2 virus and proved the SBS process does not change the microbicidal activity of drug molecule. Fluctuations in the physiological vaginal pH range of 3.8 to 5.0 substantially affected mucoadhesive and swelling behavior of chitosan which in turn impacted drug dissolution rate from polymer carrier. The rate of permeation and accumulation of TDF in vaginal tissue differed in response to vaginal pH. Faster drug permeation assessed at pH 5.0 suggests that an increase in vaginal pH could improve TDF bioavailability at earlier time points.

Artemisia argyi extract induces apoptosis in human gemcitabine-resistant lung cancer cells via the PI3K/MAPK signaling pathway

Ethnopharmacological relevance: Artemisia argyi H. Lév. & Vaniot (Asteraceae), also called “Chinese mugwort”, is frequently used as a herbal medicine in China, Japan, Korea, and eastern parts of Russia. It is known as “ai ye” in China and “Gaiyou” in Japan. In ancient China, the buds and leaves of A. argyi were commonly consumed before and after Tomb-sweeping Day. It is used to treat malaria, hepatitis, cancer, inflammatory diseases, asthma, irregular menstrual cycle, sinusitis, and pathologic conditions of the kidney and liver. Although A. argyi extract (AAE) has shown anti-tumor activity against various cancers, the therapeutic effect and molecular mechanism of AAE remains to be further studied in lung cancer.Aim of the study: This study aimed to demonstrate the anti-tumor effect of AAE and its associated biological mechanisms in CL1-0 parent and gemcitabine-resistant (CL1-0-GR) lung cancer cells.Experimental procedure: Human lung cancer cells CL1-0 and CL1-0-GR cells were treated with AAE. Cell viability was assessed using the MTT, colony, and spheroid formation assays. Migration, invasion, and immunofluorescence staining were used to determine the extent of epithelial– mesenchymal transition (EMT). JC-1 and MitoSOX fluorescent assays were performed to investigate the effect of AAE on mitochondria. Apoptosis was detected using the TUNEL assay and flow cytometry with Annexin V staining.Result: We found that A. argyi significantly decreased cell viability and induced apoptosis, accompanied by mitochondrial membrane depolarization and increased ROS levels in both parent cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0-GR). AAE-induced apoptosis is regulated via the PI3K/AKT and MAPK signaling pathways. It also prevents CL1-0 and CL1-0-GR cancer cell invasion, migration, EMT, colony formation, and spheroid formation. In addition, AAE acts cooperative with commercial chemotherapy drugs to enhance tumor spheroid shrinkage.Conclusion: Our study provides the first evidence that A. argyi treatment suppresses both parent and gemcitabine-resistant lung cancer cells by inducing ROS, mitochondrial membrane depolarization, and apoptosis, and reducing EMT. Our finding provides insights into the anti-cancer activity of A. argyi and suggests that A. argyi may serve as a chemotherapy adjuvant that potentiates the efficacy of chemotherapeutic agents.

Benzoylaconine improves mitochondrial function in oxygen-glucose deprivation and reperfusion-induced cardiomyocyte injury by activation of the AMPK/PGC-1 axis.

Heart failure (HF) has become one of the severe public health problems. The detailed role of mitochondrial function in HF was still unclear. Benzoylaconine (BAC) is a traditional Chinese medicine, but its role in HF still needs to be explored. In this study, oxygen-glucose deprivation and reperfusion (OGD/R) was executed to mimic the injury of H9C2 cells in HF. The viability of H9C2 cells was assessed via MTT assay. OGD/R treatment markedly decreased the viability of H9C2 cells, but BAC treatment evidently increased the viability of OGD/R-treated H9C2 cells. The apoptosis of H9C2 was enhanced by OGD/R treatment but suppressed by BAC treatment. The mitochondrial membrane potential was evaluated via JC-1 assay. BAC improved the mitochondrial function and suppressed oxidative stress in OGD/R-treated H9C2 cells. Moreover, Western blot analysis revealed that the protein expression of p-AMPK and PGC-1α were reduced in OGD/R-treated H9C2 cells, which was reversed by BAC. Rescue assays indicated that AMPK attenuation reversed the BAC-mediated protective effect on OGD/R-treated cardiomyocytes. Moreover, BAC alleviated myocardial injury in vivo. In a word, BAC modulated the mitochondrial function in OGD/R-induced cardiomyocyte injury by activation of the AMPK/PGC-1 axis. The findings might provide support for the application of BAC in the treatment of HF.

Extract of Codiaeum luzonicum Merr. overcomes multidrug resistance in human colon cancer cells by modulating P-glycoprotein

Objective: To investigate anti-multidrug resistance (MDR) activity and safety of the bioactive fraction (CL11) from Codiaeum luzonicum crude leaf extract. Methods: Cytotoxic activity of CL11 against MDR and non- resistant colon cancer cells was assessed using MTT assay. Mode of cell death was investigated by annexin V-propidium iodide staining, TUNEL, and JC-1 assays. To examine mechanism of action, the effect on the expression and function of the MDR-implicated protein P-glycoprotein was tested using Western blotting and calcein assay, respectively. Results: CL11 had an EC50 of 0.18, 1.03 and 38.52 μg/mL against HCT-15, HCT-15/Dox and HCT116, respectively. Cytotoxicity was mediated by inhibition of P-glycoprotein function and expression. The mode of cell death involved mitochondrial membrane depolarization and was mostly non-apoptotic at EC50 concentrations against HCT-15 and HCT-15/Dox. Conclusions: Fraction CL11 of Codiaeum luzonicum induces non- apoptotic cell death in MDR cancer cells by overcoming MDR through inhibition of P-glycoprotein expression and function.

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men.

This study focused on the identification of bacterial profiles of semen in normozoospermic men and their possible involvement in changes to the sperm structural integrity and functional activity. Furthermore, we studied possible fluctuations of selected cytokines, oxidative markers, and antibacterial proteins as a result of bacterial presence in the ejaculate. Sperm motility was assessed with computer-assisted sperm analysis, while sperm apoptosis, necrosis and acrosome integrity were examined with fluorescent methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL protocol and chromatin-dispersion test, while the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cytokine levels were quantified with the biochip assay, whilst selected antibacterial proteins were quantified using the ELISA method. The predominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were Staphylococcus hominis, Staphylococcus capitis and Micrococcus luteus. The results revealed that the sperm quality decreased proportionally to the increasing bacterial load and occurrence of conditionally pathogenic bacteria, including Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains to ampicillin, vancomycin, tobramycin, and tetracycline. Furthermore, an increased bacterial quantity in semen was accompanied by elevated levels of pro-inflammatory cytokines, including interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor alpha as well as ROS overproduction and lipid peroxidation of the sperm membranes. Our results suggest that semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia may be associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for the development of subfertility, even in normozoospermic males.

Palmitic acid, a common long-chain saturated fatty acid, demonstrates anti-proliferative and anti-tumorigenic effects in pre-clinical models of endometroid endometrial cancer (262)

<b>Objectives:</b> The consumption of saturated free fatty acids, such as palmitic acid (PA), has been implicated in diseases related to lipotoxicity (i.e., obesity and diabetes), including cancer development. Although some studies have shown PA to have pro-tumorigenic effects, emerging evidence suggests that PA may also have antitumorigenic activity for some cancer types, including hepatocellular and breast cancer. The impact of PA on endometrial cancer (EC) development and progression has yet to be explored, despite EC being a highly obesity-driven disease. Thus, we aimed to delineate the effects of PA on cell proliferation and tumor growth in EC cell lines and a transgenic mouse model of EC. <b>Methods:</b> The human endometrioid EC cell lines ECC-1 and Ishikawa were used. Cell proliferation was assessed by MTT assay. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was measured by DCFH-DA and JC1 assay. Adhesion was assessed by laminin-1 assay, whereas migration was determined by wound healing assay. Western immunoblotting was performed to determine the effects of PA on BCL-2, MCL-1, PERK, Bip, and PD1. The LKB1^fl/flp53^fl/fl mouse model of endometrioid EC was used in this study to determine the antitumorigenic activity of PA. Mice were treated with PA (10 mg/kg, oral gavage, daily) or control for four weeks. Immunohistochemistry of the endometrial tumors was performed to assess the effects of PA on cell proliferation as measured by Ki-67 staining. <b>Results:</b> PA potently inhibited cell proliferation in a dose-dependent manner in both cell lines with a median IC50 of 166µM and 356µM after 72 hours of treatment in ECC-1 and Ishikawa, respectively. PA (250µM) increased the activity of cleaved caspase-3 by 8.2-fold in ECC-1 and 2.9-fold in Ishikawa (p<0.05). Treatment with PA significantly increased ROS and JC1 production (p<0.05), and induced PERK, Bip, and PD1 expression in both cell lines. Cellular adhesion (decreased by 14% in ECC-1 and 14% in Ishikawa, p<0.05) and wound healing (decreased by 53% in ECC-1 and 55% in Ishikawa, p<0.05) were significantly impaired after treatment with PA in both cell lines. In LKB1^fl/flp53^fl/fl mice, PA decreased tumor weight by 44% (p<0.05) as compared to control mice, along with a corresponding decrease in the expression of Ki-67 in the endometrial tumor tissues (p<0.05). <b>Conclusions:</b> We found that PA, the most common saturated free fatty acid, had antiproliferative and antitumorigenic effects in endometrioid EC. Further studies are needed to assess if PA would be a novel dietary intervention in the treatment of EC.

Ruellia tuberosa Ethyl Acetate Leaf Extract Induces Apoptosis and Cell Cycle Arrest in Human Breast Cancer Cell Line, MCF-7

Ruellia tuberosa L. has been previously shown to possess antioxidant and antiproliferative activities on cancer cells but its underlying mechanisms are largely unknown. This study aimed to elucidate the mode of action underlying this inhibitory effect on MCF-7 using ethyl acetate extract obtained after liquid-liquid partition of methanol crude extract. Antiproliferative effect of R. tuberosa ethyl acetate leaf extract (RTEAL) was evaluated using MTT assay. Its ability to induce apoptosis was assessed by DNA ladder formation, JC-1, Annexin V, and methylene blue staining assays. Perturbation of cell cycle progression was determined using flow cytometry. RTEAL was found to selectively inhibit the proliferation of MCF-7 cells with the IC50 value of 28 µg/mL. Morphological changes such as nuclear fragmentation and chromatin condensation were observed although DNA laddering was undetected in agarose gel. RTEAL-induced apoptotic pathways by inhibiting the expression of anti-apoptotic BCL-2 while upregulating pro-apoptotic BAX, caspase 7 and caspase 8. RTEAL also caused cell cycle arrests at the S and G2/M phase and dysregulation of cell cycle regulators. These findings collectively demonstrate that RTEAL extract inhibited cell growth by inducing apoptosis and cell cycle arrest, suggesting its therapeutic potential against breast cancer.

Dapagliflozin Protects Methamphetamine-Induced Cardiomyopathy by Alleviating Mitochondrial Damage and Reducing Cardiac Function Decline in a Mouse Model.

Background: Methamphetamine (METH)-induced cardiovascular toxicity has been attributed to its destructive effect on mitochondrial function at least to some extent. Previous studies highlighted the benefits of dapagliflozin (DAPA) on the cardiovascular system, but the response of METH-induced cardiomyopathy to DAPA is never addressed before. The present study aimed to investigate the potential ability of DAPA in preventing METH-induced cardiomyopathy. Materials and Methods: C57BL/6 mice were randomly divided into control group (n = 24), METH group (n = 24), and METH + DAPA group (n = 24). The METH-induced cardiomyopathy group received intraperitoneal METH injections at gradually increasing doses thrice weekly for 14 weeks. Mice in the METH + DAPA group were simultaneously treated with DAPA 1 mg/kg/day by intragastric administration. Echocardiography was performed to assess cardiac function. Reactive oxygen species (ROS), JC-1, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were performed to evaluate oxidative stress, mitochondrial damage, and apoptosis, respectively. Mitochondrial and apoptosis-related protein expression was measured by western blotting. Results: Mice exposed to METH exhibited reduced cardiac function (left ventricular ejection fraction [LVEF]: 56.51 ± 6.49 vs. 73.62 ± 1.42, p < 0.01), fibrotic remodeling, and mitochondrial dysfunction, leading to apoptosis (apoptotic cells%: 7.4 ± 1.3 vs. 1.3 ± 0.5, p < 0.01). DAPA significantly reduced mitochondrial dynamics and function, ROS, apoptosis (apoptotic cells%: 2.4 ± 0.8 vs. 7.4 ± 1.3, p < 0.01), cardiac function decline (LVEF: 70.99 ± 4.936 vs. 56.51 ± 6.49, p < 0.01), and fibrotic remodeling. These results indicated that DAPA could be considered as an effective therapeutic agent in the protection against METH-associated cardiomyopathy. Conclusion: DAPA protects against METH-induced cardiomyopathy in mice by decreasing mitochondrial damage and apoptosis.

Isobavachalcone induces cell death through multiple pathways in human breast cancer MCF-7 cells

To explore the effects of isobavachalcone (IBC) on cell death of human breast cancer MCF-7 cells and explore the possible mechanism. MCF-7 cells were treated with different concentrations of IBC, and the changes in cell proliferation were assessed using MTT assay. Apoptosis of MCF-7 cells following treatment with 10, 20, and 40 μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy, and the expressions of apoptosis- and autophagy-related proteins (Bax, Bcl-2, Akt, p-Akt, p62, and LC3) were detected with Western blotting. Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40 μmol/L IBC. JC-1 assay kit, ATP assay kit, and reactive oxygen species (ROS) kit were used to determine the effect of IBC on mitochondrial function of the cells. MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration- and time-dependent manner, with IC50 values of 38.46, 31.31, and 28.26 μmol/L at 24, 48, and 72 h, respectively. IBC also concentration-dependently induced apoptosis of MCF-7 cells. IBC-induced cell death was inhibited by z-VAD-fmk, a caspase inhibitor (P < 0.05), but not by the necroptosis inhibitor necrostatin-1 (Nec-1). Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2, Akt and p-Akt-473 (all P < 0.05). With the increase of IBC concentration, the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively. Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells. IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells (P < 0.05). IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells, suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.