Cytosolic Cytochrome Research Articles

Crohn's Disease Pathobiont Adherent-Invasive E coli Disrupts Epithelial Mitochondrial Networks With Implications for Gut Permeability.

Background & AimsAdherent-invasive Escherichia coli are implicated in inflammatory bowel disease, and mitochondrial dysfunction has been observed in biopsy specimens from patients with inflammatory bowel disease. As a novel aspect of adherent-invasive E coli–epithelial interaction, we hypothesized that E coli (strain LF82) would elicit substantial disruption of epithelial mitochondrial form and function.MethodsMonolayers of human colon-derived epithelial cell lines were exposed to E coli–LF82 or commensal E coli and RNA sequence analysis, mitochondrial function (adenosine triphosphate synthesis) and dynamics (mitochondrial network imaging, immunoblotting for fission and fusion proteins), and epithelial permeability (transepithelial resistance, flux of fluorescein isothiocyanate–dextran and bacteria) were assessed.ResultsE coli–LF82 significantly affected epithelial expression of ∼8600 genes, many relating to mitochondrial function. E coli–LF82–infected epithelia showed swollen mitochondria, reduced mitochondrial membrane potential and adenosine triphosphate, and fragmentation of the mitochondrial network: events not observed with dead E coli–LF82, medium from bacterial cultures, or control E coli. Treatment with Mitochondrial Division Inhibitor 1 (Mdivi1, inhibits dynamin-related peptide 1, guanosine triphosphatase principally responsible for mitochondrial fission) or P110 (prevents dynamin-related peptide 1 binding to mitochondrial fission 1 protein) partially reduced E coli–LF82–induced mitochondrial fragmentation in the short term. E coli–LF82–infected epithelia showed loss of the long isoform of optic atrophy factor 1, which mediates mitochondrial fusion. Mitochondrial Division Inhibitor 1 reduced the magnitude of E coli–LF82–induced increased transepithelial flux of fluorescein isothiocyanate dextran. By 8 hours after infection, increased cytosolic cytochrome C and DNA fragmentation were apparent without evidence of caspase-3 or apoptosis inducing factor activation.ConclusionsEpithelial mitochondrial fragmentation caused by E coli–LF82 could be targeted to maintain cellular homeostasis and mitigate infection-induced loss of epithelial barrier function. Data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession numbers GSE154121 and GSE154122 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154121).

16-Hydroxycleroda-3,13-Dien-15,16-Olide Induces Apoptosis in Human Bladder Cancer Cells through Cell Cycle Arrest, Mitochondria ROS Overproduction, and Inactivation of EGFR-Related Signalling Pathways

A clerodane diterpene compound 16-hydroxycleroda-3,13-dien-15,16-olide (CD) is considered a therapeutic agent with pharmacological activities. The present study investigated the mechanisms of CD-induced apoptosis in T24 human bladder cancer cells. CD inhibited cell proliferation in a concentration and time-dependent manner. CD-induced overproduction of reactive oxygen species and reduced mitochondrial membrane potential, associated with reduced expression of Bcl-2 and increased levels of cytosolic cytochrome c, cleaved PARP-1 and caspase-3. In addition, CD treatment led to cell cycle arrest at the G0/G1 phase and inhibited expression of cyclin D1 and cyclin-dependent kinases 2 and 4 and led to increased levels of p21, p27Kip1 and p53. All of these events were accompanied with a reduction of pEGFR, pMEK1/2, pERK1/2, pAkt, pmTOR, pP70S6K1, HIF-1α, c-Myc and VEGF. RNAseq-based analysis revealed that CD-induced cell death was characterised by an increased expression of stress and apoptotic-related genes as well as inhibition of the cell cycle-related genes. In summary, CD induces apoptosis in T24 bladder cancer cells through targeting multiple intracellular signaling pathways as a result of oxidative stress and cell cycle arrest.

MCLR induces dysregulation of calcium homeostasis and endoplasmic reticulum stress resulting in apoptosis in Sertoli cells

Microcystins-LR (MCLR) is a potent reproductive system toxin. We have previously shown that MCLR induced endoplasmic reticulum (ER) stress and apoptosis in testis. ER is the main calcium storage site in cells, and its calcium homeostasis plays an important role in the regulation of apoptosis. Hence, in the present study, we have investigated the role of calcium (Ca2+) in inducing apoptosis and how it affect the mitochondria and endoplasmic reticulum in TM4 cells. Our study found that MCLR induced an increase in Ca2+ concentration in TM4 cells. Compared to the controls, MCLR induced phosphorylation of calmodulin-dependent protein kinase II (CaMKII) which was involved in MAPKs activation, resulting in the induction of mitochondrial apoptosis pathways. Ca2+ chelator Bapta-AM partially reversed MCLR-induced apoptosis, confirming the possible involvement of calcium homeostasis disruption after MCLR exposure. Meanwhile, MCLR activated unfolded protein response and activated the ER apoptotic pathway by activating caspase-12. In addition, exposure to MCLR causes mitochondrial defects and increased apoptosis by up-regulating caspase 3 and cytosol cytochrome c expression. Collectively, these results demonstrated that MCLR disturbed calcium homeostasis, which caused ER-mitochondria dysfunction, ultimately promoted cell apoptosis in Sertoli cells.

Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria

Cancer cells have developed chemoresistance and have improved their survival through the upregulation of autophagic mechanisms that protect mitochondrial function. Here, we report that the traditional Chinese anticancer agent tubeimoside I (Tub), which is a potent inhibitor of autophagy, can promote mitochondria-associated apoptosis in lung cancer cells. We found that Tub disrupted both mitochondrial and lysosomal pathways. One of its mechanisms was the induction of DRP1-mediated mitochondrial fragmentation. Another mechanism was the blocking of late-stage autophagic flux via impairment of lysosomal acidification through V-ATPase inhibition; this blocks the removal of dysfunctional mitochondria and results in reactive oxygen species (ROS) accumulation. Excessive ROS accumulation causes damage to lysosomal membranes and increases lysosomal membrane permeability, which leads to the leakage of cathepsin B. Finally, cathepsin B upregulates Bax-mediated mitochondrial outer membrane permeability and, subsequently, cytosolic cytochrome C-mediated caspase-dependent apoptosis. Thus, the cancer cell killing effect of Tub is enhanced through the formation of a positive feedback loop. The killing effect of Tub on lung cancer cells was verified in xenografted mice. In summary, Tub exerts a dual anticancer effect that involves the disruption of mitochondrial and lysosomal pathways and their interaction and, thereby, has a specific and enhanced killing effect on lung cancer cells.

The insect molting hormone 20-hydroxyecdysone protects dopaminergic neurons against MPTP-induced neurotoxicity in a mouse model of Parkinson's disease

20-hydroxyecdysone (20E), a steroidal prohormone, is secreted from the prothoracic glands. While 20E has been shown to have neuroprotective effects in Parkinson's disease (PD) models in vitro, its effects have not yet been examined in vivo. We sought to assess the behavioral and mechanistic effects of 20E on MPTP-induced toxicity in mice. To this end, we used behavioral tests, stereological analyses of dopaminergic neurons by tyrosine hydroxylase immunohistochemistry, and assessments of apoptotic mechanisms, focusing on Nrf2 signaling through Western blotting and ELISA assays. A 20E treatment protected against MPTP-induced motor incoordination, postural imbalance, and bradykinesia, and significantly reduced dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc) and the striatum (ST). It also attenuated dopamine deficiency in the ST, modulated levels of antioxidative enzymes superoxide dismutase, catalase, and glutathione in the SNpc, increased the Bcl-2/Bax ratio, and inhibited cytosolic cytochrome c release and caspase-9, -7, and -3 activity in the SNpc. These results indicated that 20E inhibited the apoptotic cascade. Furthermore, the attenuation of MPTP neurotoxicity was associated with inhibited cleaved-caspase signaling pathways, along with upregulated Nrf2 pathways in the SNpc, suggesting that 20E mitigates MPTP-induced neurotoxicity via mitochondria-mediated apoptosis by modulating anti-oxidative activities. Our results suggest that 20E can inhibit MPTP-induced behavioral and neurotoxic effects in mice. This lays the foundation for further research on 20E as a potential target for therapeutic use.

Melatonin against acute ischaemic stroke dependently via suppressing both inflammatory and oxidative stress downstream signallings.

This study tested the hypothesis that melatonin (Mel) therapy preserved the brain architectural and functional integrity against ischaemic stroke (IS) dependently through suppressing the inflammatory/oxidative stress downstream signalling pathways. Adult male B6 (n = 6 per each B6 group) and TLR4 knockout (ie TLR4−/−) (n = 6 per each TLR4−/− group) mice were categorized into sham control (SCB6), SCTLR4−/−, ISB6, ISTLR4−/−, ISB6 + Mel (i.p. daily administration) and ISTLR4−/− + Mel (i.p. daily administration). By day 28 after IS, the protein expressions of inflammatory (HMBG1/TLR2/TLR4/MAL/MyD88/RAM TRIF/TRAF6/IKK‐α/p‐NF‐κB/nuclear‐NF‐κB/nuclear‐IRF‐3&7/IL‐1β/IL‐6/TNF‐α/IFN‐γ) and oxidative stress (NOX‐1/NOX‐2/ASK1/p‐MKK4&7/p‐JNK/p‐c‐JUN) downstream pathways as well as mitochondrial‐damaged markers (cytosolic cytochrome C/cyclophilin D/SRP1/autophagy) were highest in group ISB6, lowest in groups SCB6 and SCTLR4−/−, lower in group ISTLR4−/− + Mel than in groups ISTLR4−/− and ISB6 + Mel and lower in group ISB6 + Mel than in group ISTLR4−/− (all P < .0001). The brain infarct volume, brain infarct area and the number of inflammatory cells in brain (CD14/F4‐88) and in circulation (MPO+//Ly6C+/CD11b+//Ly6G+/CD11b+) exhibited an identical pattern, whereas the neurological function displayed an opposite pattern of inflammatory protein expression among the six groups (all P < .0001). In conclusion, TLR inflammatory and oxidative stress signallings played crucial roles for brain damage and impaired neurological function after IS that were significantly reversed by Mel therapy.

Ultrafine silicon dioxide nanoparticles cause lung epithelial cells apoptosis via oxidative stress-activated PI3K/Akt-mediated mitochondria- and endoplasmic reticulum stress-dependent signaling pathways

Silicon dioxide nanoparticles (SiO2NPs) are widely applied in industry, chemical, and cosmetics. SiO2NPs is known to induce pulmonary toxicity. In this study, we investigated the molecular mechanisms of SiO2NPs on pulmonary toxicity using a lung alveolar epithelial cell (L2) model. SiO2NPs, which primary particle size was 12 nm, caused the accumulation of intracellular Si, the decrease in cell viability, and the decrease in mRNAs expression of surfactant, including surfactant protein (SP)-A, SP-B, SP-C, and SP-D. SiO2NPs induced the L2 cell apoptosis. The increases in annexin V fluorescence, caspase-3 activity, and protein expression of cleaved-poly (ADP-ribose) polymerase (PARP), cleaved-caspase-9, and cleaved-caspase-7 were observed. The SiO2NPs induced caspase-3 activity was reversed by pretreatment of caspase-3 inhibitor Z-DEVD-FMK. SiO2NPs exposure increased reactive oxygen species (ROS) production, decreased mitochondrial transmembrane potential, and decreased protein and mRNA expression of Bcl-2 in L2 cells. SiO2NPs increased protein expression of cytosolic cytochrome c and Bax, and mRNAs expression of Bid, Bak, and Bax. SiO2NPs could induce the endoplasmic reticulum (ER) stress-related signals, including the increase in CHOP, XBP-1, and phospho-eIF2α protein expressions, and the decrease in pro-caspase-12 protein expression. SiO2NPs increased phosphoinositide 3-kinase (PI3K) activity and AKT phosphorylation. Both ROS inhibitor N-acetyl-l-cysteine (NAC) and PI3K inhibitor LY294002 reversed SiO2NPs-induced signals described above. However, the LY294002 could not inhibit SiO2NPs-induced ROS generation. These findings demonstrated first time that SiO2NPs induced L2 cell apoptosis through ROS-regulated PI3K/AKT signaling and its downstream mitochondria- and ER stress-dependent signaling pathways.

Protective effect of L-carnitine on platelet apoptosis during storage of platelet concentrate.

Platelet apoptosis is considered as one of the important factors involved in platelet storage lesion (PSL) and affect the quality of platelets during storage. The beneficial effect of L-carnitine (LC) on platelet apoptosis during platelet concentrates (PCs) storage has not been fully investigated. The aim of this study was to evaluate the effects of LC on platelets of PC regarding their apoptosis markers during storage. Ten PCs from healthy donors were investigated in this study. PCs were prepared by platelet rich plasma (PRP) method and stored at 22±2°C with gentle agitation during storage. The effects of LC (15mM) on the platelet apoptosis were assessed by analyzing different indicative presence or absence of LC. Sampling was performed to evaluate apoptosis markers during platelet storage. The results indicated significantly higher mitochondrial membrane potential for LC-treated platelets than the untreated on the days 2 and 5 of storage (Pday2=0.001, Pday5=0.001). Phosphatidylserine (PS) exposure significantly increased on the untreated compared with LC-treated platelets on the second and third days of storage (Pday2=0.014, Pday3=0.012). Also, active caspase 3 was lower in the LC- treated platelets than the control group on the day 5 of storage (Pday5=0.004). Cytosolic cytochrome C was so significantly lower in LC-treated compared to the untreated platelets during storage time (Pday2=0.002, Pday3=0.001, Pday5=0.001). The results of this study indicate that the use of LC as an additive solution in platelets may be useful to reduce PSL by decreasing platelet apoptosis via mitochondrial pathway and increase platelet quality during storage.

(-)-Syringaresinol-4-O-β-D-glucopyranoside from Cortex Albizziae inhibits corticosterone-induced PC12 cell apoptosis and relieves the associated dysfunction

The neuroprotective effects and potential mechanisms of (-)-Syringaresinol-4-O-β-D-glucopyranoside (SRG), a natural lignan glycoside extracted from Cortex Albizziae, were investigated using corticosterone (CORT)-induced PC12 cells as an in vitro anxiety model. PC12 cells were treated with 100 μM CORT and 5, 10, or 20 μM SRG for 48 h. Cell viability and lactate dehydrogenase (LDH) leakage were measured. Apoptosis were detected using FITC-coupled Annexin V (AV) and propidium iodide (PI) staining flow cytometric analyses and TUNEL assays. Rhodamine 123 and Fluo-3-AM staining flow cytometric analyses were used to detect mitochondrial membrane potential (ΔΨm) and intracellular calcium concentration ([Ca2+]i), respectively. Western blot was used to detect brain-derived neurotrophic factor (BDNF), Bax, Bcl-2, cAMP-response element binding protein (CREB), cytosolic cytochrome c (Cyt c), caspase-3, and cleaved caspase-3. Experimental data showed that SRG promoted cell proliferation, reduced LDH release, inhibited apoptosis, improved ΔΨm values, decreased [Ca2+]i, up-regulated CREB, BDNF, and Bcl-2, down-regulated Bax and Cyt c protein expression levels, and reduced caspase-3 activity. This suggests that SRG has neuroprotective and antiapoptotic effects in the pathogenesis of anxiety disorders, and its mechanisms are partly connecte to inhibition of the mitochondrial apoptotic pathway and activation of pathways involving CREB and BDNF.

Taurine attenuates isoproterenol-induced H9c2 cardiomyocytes hypertrophy by improving antioxidative ability and inhibiting calpain-1-mediated apoptosis.

Pathological cardiac hypertrophy is ultimately accompanied by cardiomyocyte apoptosis. Apoptosis mainly related to calpain-1-mediated apoptotic pathways. Studies had proved that taurine can maintain heart health through antioxidation and antiapoptotic functions, but the effect of taurine on cardiachypertrophy is still unclear. This study aimed to determine whether taurine could inhibit calpain-1-mediated mitochondria-dependent apoptotic pathways in isoproterenol (ISO)-induced hypertrophic cardiomyocytes. We found that taurine could inhibit the increase in cell surface area and reduce the protein expression levels of the hypertrophic markers atrial natriuretic peptide, brain natriuretic polypeptide, and β-myosin heavy chain. Taurine also reduced ROS, intracellular Ca2+ overload and mitochondrial membrane potential. Moreover, taurine inhibited cardiomyocyte apoptosis by decreasing the protein expression of calpain-1, Bax, t-Bid, cytosolic cytochrome c, Apaf-1, cleaved caspase-9 and cleaved caspase-3 and by enhancing calpastatin and Bcl-2 protein expression. Calpain-1 small interfering RNA transfection results showed similar antiapoptotic effects as the taurine prevention group. However, compared with the two treatments, taurine inhibited the expression of cleaved caspase-9 more significantly. Therefore, we believe that taurine prevents ISO-induced H9c2 cardiomyocyte hypertrophy by inhibiting oxidative stress, intracellular Ca2+ overload, the calpain-1-mediated mitochondria-dependent apoptotic pathway and cleaved caspase-9 levels.

Targeting mitochondria to protect the heart: a matter of balance?

Mitochondria are dynamic, undergoing both fission and fusion. Evidence indicates that a balance between these two processes is necessary to maintain a healthy state. With ischemia/reperfusion (I/R) of the heart, fission is enhanced and is associated with mitochondrial swelling, depolarization, and production of reactive oxygen species (ROS), as well as apoptosis. Blocking fission is effective in reducing I/R-induced tissue damage and contractile dysfunction. In a groundbreaking study appearing in Clinical Science, Maneechote et al. assessed whether correcting the imbalance in mitochondrial dynamics with I/R by enhancing fusion would also be protective. Using a rat model, they investigated the efficacy of pharmacological intervention with mitochondrial fusion promoter-M1 (M1) given before ischemia, during ischemia, or at the onset of reperfusion. With pretreatment being the most effective, they found that M1 attenuated the incidence of arrhythmias, reduced infarct size, preserved cardiac function, and decreased mortality. M1 reduced I/R-induced increases in cytosolic cytochrome c, cleaved caspase 3, and apoptosis. All M1 groups exhibited modestly attenuated I/R-induced mitochondrial ROS levels and swelling, and preserved mitochondrial membrane potential. M1 also prevented a decrease in complex V levels with I/R. However, exactly how M1 stimulates mitochondrial fusion is unclear and other nonfusion-related actions of this phenylhydrazone compound should be considered, such as anti-oxidant actions, preconditioning signaling, or effects on putative mitochondrial connexin 43.

Involvement of p53-dependent apoptosis signal in antitumor effect of Colchicine on human papilloma virus (HPV)-positive human cervical cancer cells.

Colchicine, a plant-derived alkaloid with relatively low toxicity on normal human epidermal keratinocytes (HEKn), has selective inhibitory effect on the growth of CaSki (HPV16-positive) and HeLa (HPV18-positive) human cervical cancer cell lines via the induction of apoptosis. Colchicine (2.5, 5.0 and 10.0 ng/ml) significantly reduced the expression of human papilloma virus (HPV) 16 E6/E7 mRNA and protein in CaSki and HeLa cells. Moreover, reduced expression of E6 and E7 induced by Colchicine resulted in the up-regulation of tumor suppressor proteins, p53 and Rb, as well as down-regulation of phospho Rb (pRb) protein. In addition, Bax, cytosolic cytochrome c and cleaved caspase-3 protein were increased while Bcl-2 protein was decreased significantly by 48 h of Colchicine treatment. These results implied that Colchicine could be explored as a potent candidate agent for the treatment and prevention of HPV-associated cervical cancer without deleterious effects.

PM2.5 induces apoptosis, oxidative stress injury and melanin metabolic disorder in human melanocytes.

Recent growing evidence suggested that particulate matter 2.5 (PM2.5) has strong toxic effects on skin systems. However, the possible effects and the mechanisms of PM2.5 on vitiligo remain poorly understood. Therefore, the present study aimed to further investigate the effects and possible mechanisms of PM2.5 on vitiligo. Human keratinocytes (HaCaT cells) and human melanocytes (PIG1 cells and PIG3V cells) were exposed to PM2.5 (0-200 µg/ml) for 24 h. The cell viability of the three cell lines was measured by a Cell Counting Kit-8 assay. The secretions of stem cell factor (SCF) and basic fibroblast growth factor (bFGF) in HaCaT cells were evaluated by ELISA. The melanin contents, cellular tyrosinase activity, apoptosis, cell migration, malondialdehyde (MDA) contents, superoxide dismutase (SOD) levels, glutathione peroxidase (GSH-Px) levels and related protein expressions in PIG1 cells and PIG3V cells were evaluated by a NaOH assay, DOPA assay, Annexin V-FITC/Propidium Iodide staining, MDA assay, SOD assay, GSH-Px assay and western blotting, respectively. It was demonstrated that PM2.5 exposure inhibited cell viability of all three cell lines (HaCaT, PIG1 and PIG3V cells). PM2.5 exposure attenuated the secretions of SCF and bFGF in HaCaT cells. Moreover, PM2.5 exposure attenuated the activation of tyrosinase and melanogenesis, inhibited cell migration, and induced apoptosis and oxidative stress injury in PIG1 cells and PIG3V cells. In addition, PM2.5 exposure caused upregulated cytosolic cytochrome C and activated caspase-3 in PIG1 cells and PIG3V cells. Furthermore, PM2.5 exposure activated the nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 signaling pathway. The present results suggested that PM2.5 exposure could inhibit the secretions of SCF and bFGF in keratinocytes, and cause oxidative stress injury and melanin metabolic disorder in melanocytes. Therefore, PM2.5 could be a new risk factor for vitiligo.

Total triterpenes from the fruits of Chaenomeles speciosa (Sweet) Nakai protects against indomethacin-induced gastric mucosal injury: involvement of TFF1-mediated EGF/EGFR and apoptotic pathways.

Our previous studies indicated that the triterpenes from the fruits of Chaenomeles speciosa (Sweet) Nakai (TCS) owned effectively therapeutic effects on gastric ulcer patients and animals, but its mechanisms have not been fully understood. The current study was to further investigate its protective effect on indomethacin (IND)-damaged RGM-1 cells and rats, as well as its mechanisms involved. The gastroprotection of TCS was evaluated with IND-induced gastric lesions model in RGM-1 cells and rats. In vitro, the proliferation, migration, mitochondrial viability and apoptosis were assessed. In vivo, ulcer index, ulcer inhibition rate, gastric juice acidity, gastric wall mucus (GWM) and histopathology of gastric mucosa were detected. The gastroprotective effects of TCS through the TFF1-mediated EGF/EGFR and apoptotic pathways were measured by qRT-PCR and Western blot assays. The results demonstrated that TCS had gastroprotective function, which was related to the amelioration in promoting IND-damaged RGM-1 cell proliferation and migration, hoisting gastric juice acidity and GWM, improving ulcer index and ulcer inhibition rate, attenuating the haemorrhage, oedema, epithelial cell loss and inflammatory cell infiltration of gastric mucosa, upregulating PCNA, Bcl-2, Bcl-xl mRNA and TFF1, EGF, p-EGFR, p-Src, pro-caspase-3, pro-caspase-9 protein expressions, mitochondrial viability, mitochondrial cytochrome c concentration and p-EGFR/EGFR, p-Src/Src, Bcl-2/Bax, Bcl-xl/Bad ratioes, downregulating Bax, Bad, Apaf-1 mRNA and cleaved-caspase-3, cleaved-caspase-9, cleaved PARP-1 protein expressions and cytosol cytochrome c concentration. Our present study demonstrated that TCS's gastroprotective effect was closely connected with boosting TFF1 expression, activating TFF1-mediated EGF/EGFR pathway, thus restraining mitochondrial-dependent apoptosis, which provided new insights into interpreting its underlying mechanism and promised to act as a candidate drug to treat gastric mucosal injury.

Taurine prevents cardiomyocyte apoptosis by inhibiting the calpain-1/cytochrome c pathway during RVH in broilers

The calpain-1-activated apoptotic pathway plays a key role in right ventricular hypertrophy (RVH). Taurine has been shown to attenuate apoptosis by inhibiting calpain activity. This experiment aimed to determine whether taurine could prevent RVH by inhibiting the calpain-1/cytochrome c apoptotic pathway. The broilers were given 1% taurine dissolved in drinking water and were raised at 10°C ~ 12°C from day 21 to day 42. At 21 d, 28 d, 35 d and 42 d, the right ventricular (RV) tissues were collected. Increased RVH index, angiotensin II, norepinephrine and atrial natriuretic peptide mRNA expression were reduced by taurine in the broiler RVs. Taurine obviously inhibited cardiomyocyte apoptosis via maintaining the mitochondrial membrane potential and decreased the activation of caspase-9 and caspase-3 in the broiler RVs. The antioxidant assay demonstrated that taurine enhanced the activities of superoxide dismutase, total antioxidant capacity and glutathione peroxidase and the glutathione/glutathione disulfide ratio. Western blot results revealed that taurine also downregulated the expression of calpain-1 and cytosolic cytochrome c while upregulating the expression of Bcl-2/Bax and mitochondrial cytochrome c in broiler cardiomyocytes during RVH. In summary, we found that taurine could enhance cardiomyocyte antioxidant ability and further prevented cardiomyocyte apoptosis by inhibiting the calpain-1/cytochrome c pathway during RVH in broilers.

Copper Induces Oxidative Stress and Apoptosis in the Mouse Liver

Copper (Cu) is an essential trace element involved in the normal physiological processes of animals. However, excessive exposure to Cu can produce numerous detrimental impacts. The aim of this study was to investigate the effects of Cu on oxidative stress and apoptosis as well as their relationship in the mouse liver. Four-week-old ICR mice (n = 240) were randomly assigned to different Cu (Cu2+-CuSO4) treatment groups (0, 4, 8, and 16 mg/kg) for periods of 21 and 42 days. The high doses of Cu exposure could induce oxidative stress, by increasing the levels of reactive oxygen species (ROS) and protein carbonyls (PC) and decreasing the activities of antisuperoxide anion (ASA) and antihydroxyl radical (AHR) and content of glutathione (GSH), as well as activities and mRNA expression levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Moreover, high doses of Cu exposure induced hepatic apoptosis via the mitochondrial apoptotic pathway, as characterized by the depolarization of mitochondrial membrane potential (MMP); significantly increased mRNA and protein expression levels of cytosolic cytochrome (Cyt c), apoptosis-inducing factor (AIF), endonuclease G (Endo G), apoptosis protease-activating factor-1 (Apaf-1), cleaved caspase-9, cleaved caspase-3, cleaved PARP, Bcl-2 antagonist killer (Bak), Bcl-2-associated X protein (Bax), and Bcl-2-interacting mediator of cell death (Bim); and decreased mRNA and protein expression levels of B-cell lymphoma-2 (Bcl-2) and Bcl-extra-large (Bcl-xL). Furthermore, the activation of the tumor necrosis factor receptor-1 (TNF-R1) signaling pathway was involved in Cu-induced apoptosis, as characterized by the significantly increased mRNA and protein expression levels of TNF-R1, Fas-associated death domain (FADD), TNFR-associated death domain (TRADD), and cleaved caspase-8. These results indicated that exposure to excess Cu could cause oxidative stress triggered by ROS overproduction and diminished antioxidant function, which in turn promoted hepatic apoptosis via mitochondrial apoptosis and that the TNF-R1 signaling pathway was also involved in the Cu-induced apoptosis.

Effects of Immunosuppressive Medications on Mitochondrial Function

BackgroundImmunosuppressive medications are widely used for the prevention of allograft rejection in transplantation and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Despite their clinical utility, these medications are accompanied by multiple off-target effects, some of which may be mediated by their effects on mitochondria. MethodsWe examined the effect of commonly used immunosuppressive reagents, mycophenolate mofetil (MMF), cyclosporine A (CsA), rapamycin, and tacrolimus on mitochondrial function in human T-cells. T-cells were cultured in the presence of immunosuppressive medications in a range of therapeutic doses. After incubation, mitochondrial membrane potential, reactive oxygen species (ROS) production, and apoptotic cell death were measured by flow cytometry after staining with DiOC6, MitoSOX Red, and Annexin V and 7-AAD, respectively. Increases in cytosolic cytochrome c were demonstrated by Western blot. T-cell basal oxygen consumption rates were measured using a Seahorse bioanalyzer. ResultsT-cells demonstrated significant levels of mitochondrial depolarization after treatment with therapeutic levels of MMF but not after treatment with CsA, tacrolimus, or rapamycin. Only MMF induced T-cell ROS production and induced significant levels of apoptotic cell death that were associated with increased levels of cytosolic cytochrome c. MMF decreased T-cell basal oxygen consumption within its therapeutic range, and CsA demonstrated a trend toward this result. ConclusionsThe impairment of mitochondrial function by commonly used immunosuppressive reagents may impair T-cell differentiation and function by decreasing energy production, producing toxic ROS, and inducing apoptotic cell death.

Repetitively hypoxic preconditioning attenuates ischemia/reperfusion-induced liver dysfunction through upregulation of hypoxia-induced factor-1 alpha-dependent mitochondrial Bcl-xl in rat.

Repetitive hypoxic preconditioning (HP) enforces protective effects to subsequently severe hypoxic/ischemic stress. We hypothesized that HP may provide protection against ischemia/reperfusion (I/R) injury in rat livers via hypoxia-induced factor-1 alpha (HIF-1α)/reactive oxygen species (ROS)-dependent defensive mechanisms. Female Wistar rats were exposed to hypoxia (15 h/day) in a hypobaric hypoxic chamber (5500 m) for HP induction, whereas the others were kept in sea level. These rats were subjected to 45 min of hepatic ischemia by portal vein occlusion followed by 6 h of reperfusion. We evaluated HIF-1α in nuclear extracts, MnSOD, CuZnSOD, catalase, Bad/Bcl-xL/caspase 3/poly-(ADP-ribose)-polymerase (PARP), mitochondrial Bcl-xL, and cytosolic cytochrome C expression with Western blot and nitroblue tetrazolium/3-nitrotyrosine stain. Kupffer cell infiltration and terminal deoxynucleotidyl transferase-mediated nick-end labeling method apoptosis were determined by immunocytochemistry. The ROS value from liver surface and bile was detected by an ultrasensitive chemiluminescence-amplification method. Hepatic function was assessed with plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. HP increased nuclear translocation of HIF-1α and enhanced Bcl-xL, MnSOD, CuZnSOD, and catalase protein expression in a time-dependent manner. The response of HP enhanced hepatic HIF-1α, and Bcl-xL expression was abrogated by a HIF-1α inhibitor YC-1. Hepatic I/R increased ROS levels, myeloperoxidase activity, Kupffer cell infiltration, ALT and AST levels associated with the enhancement of cytosolic Bad translocation to mitochondria, release of cytochrome C to cytosol, and activation of caspase 3/PARP-mediated apoptosis. HP significantly ameliorated hepatic I/R-enhanced oxidative stress, apoptosis, and mitochondrial and hepatic dysfunction. In summary, HP enhances HIF-1α/ROS-dependent cascades to upregulate mitochondrial Bcl-xL protein expression and to confer protection against I/R injury in the livers.

Sulforaphane improves voiding function via the preserving mitochondrial function in diabetic rats

Hyperglycemia evoked oxidative stress contributing to diabetes (DM)-induced voiding dysfunction. We explored whether antioxidant sulforaphane,a NF-E2-related nuclear factor erythroid-2 (Nrf-2) activator, may ameliorate DM-induced bladder dysfunction. DM was induced by streptozotocin andsulforaphanewas administered before DM induction.Bladder reactive oxygen species (ROS) were determined by an ultrasensitive chemiluminescence analyzer. Mitochondrial function index mitochondrial Bax and cytosolic cytochrome c, antioxidant defense Nrf-2/HO-1, endoplasmic reticulum stress marker ATF-6/CHOP, and caspase 3/PARP were evaluated by Western blot. DM increased Keap1 and reduced Nrf-2 expression, associated with increase of bladder ROS, mitochondrial Bax translocation, cytosolic cytochrome c release, ATF-6/CHOP, caspase-3/PARP in bladders which resulted in voiding dysfunction by increased intercontraction intervals and micturition duration. However,sulforaphanesignificantly increased nuclear Nrf-2/HO-1axis expression, decreased bladder ROS amount, mitochondrial Bax translocation, cytochrome c release, ATF-6/CHOP and caspase 3/PARP/apoptosis, thereby improved the voiding function by the shortened intercontraction intervals and micturition duration. We suggest that sulforaphanevia activating Nrf-2/HO-1 signaling preserved mitochondrial function and suppressed DM-induced ROS, endoplasmic reticulum stress, apoptosis and voiding dysfunction.

In vitro apoptotic effect on human lymphatic filarial parasite by piperidine derivatives and thymidine reversal study.

A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.