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1.
目的:研究致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制。方法:应用佛波酯(PMA)诱导THP-1单核细胞分化为巨噬细胞进行实验研究。选用5-氨基酮戊酸(ALA)作为声敏剂,进行声动力治疗(SDT)。应用流式细胞术证实SDT显著促进了细胞凋亡;应用Rhod 2/AM实时监测线粒体Ca~(2+)水平;通过蛋白质免疫印迹对全细胞蛋白中的Bax、Cleaved-caspase3、电压依赖性阴离子通道1(VDAC1)和三磷酸肌醇Ⅲ型受体(IP3R-Ⅲ)进行检测;应用VDAC1抗体进行免疫共沉淀,检测VDAC1和IP3R-Ⅲ之间的相互作用;实时监测线粒体Ca~(2+)水平,检测VDAC抑制剂DIDS和IP3Rs抑制剂2-ABP对SDT效果的影响。结果:与对照组相比,仅SDT组出现了显著的细胞凋亡(P0.001)。与对照组相比,ALA对线粒体Ca~(2+)水平无明显影响,超声诱导了线粒体Ca~(2+)水平的明显升高,SDT诱导了线粒体Ca~(2+)水平快速且大幅度的升高,且去除超声后仍维持在较高水平。与对照组相比,超声对Bax、Cleaved-caspase3、VDAC1和IP3R-Ⅲ的表达无明显影响,ALA诱导了VDAC1(P0.01)和IP3R-Ⅲ表达量的增加(P0.05),SDT诱导了Bax(P0.001)、Cleaved-caspase3(P0.001)、VDAC1(P0.01)和IP3R-Ⅲ(P0.05)表达量的增加,VDAC1和IP3R-Ⅲ的增加幅度与ALA组接近;ALA和SDT均诱导了VDAC1和IP3R-Ⅲ之间相互作用的显著增强(P0.05)。DIDS和2-ABP均明显抑制了SDT诱导的线粒体Ca~(2+)增加。结论:在致凋亡的SDT作用于THP-1巨噬细胞的过程中,ALA诱导了线粒体外膜Ca~(2+)转运通道VDAC1和内质网重要Ca~(2+)转运通道IP3R-Ⅲ的表达量增加与二者间相互作用的增强,在内质网和线粒体之间建立了大量的Ca~(2+)转运通道,超声的作用则在于触发这些Ca~(2+)转运通道的开放,进而引发线粒体钙的迅速增加。这是后续线粒体凋亡通路启动的重要机制之一。  相似文献   

2.
Excessive Ca2+ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca2+-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca2+-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca2+ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca2+ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca2+ release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca2+ uptake into the mitochondria.  相似文献   

3.
VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca2 + across the OMM and because Ca2 + has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca2 + levels ([Ca2 +]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca2 + and induce VDAC1 over-expression. Direct elevation of [Ca2 +]i by the Ca2 +-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca2 +]i using the cell-permeable Ca2 +-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca2 +]i levels and apoptosis induction, as induced by H2O2 or As2O3, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca2 +-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca2 +]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca2 + promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau  相似文献   

4.
The role of the antiapoptotic protein Bcl-xL in regulating mitochondrial Ca2+ ([Ca2+]mito) handling was examined in wild-type (WT) and Bcl-xL knock-out (Bcl-xL-KO) mouse embryonic fibroblast cells. Inositol 1,4,5-trisphosphate-generating agonist evoked cytosolic Ca2+ transients that produced a larger [Ca2+]mito uptake in WT cells compared with Bcl-xL-KO. In permeabilized cells, stepping external [Ca2+] from 0 to 3 μm also produced a larger [Ca2+]mito uptake in WT; moreover, the [Ca2+]mito uptake capacity of Bcl-xL-KO cells was restored by re-expression of mitochondrially targeted Bcl-xL. Bcl-xL enhancement of [Ca2+]mito uptake persisted after dissipation of the mitochondrial membrane potential but was absent in mitoplasts lacking an outer mitochondrial membrane. The outer membrane-localized voltage-dependent anion channel (VDAC) is a known Ca2+ permeability pathway that directly interacts with Bcl-xL. Bcl-xL interacted with VDAC1 and -3 isoforms, and peptides based on the VDAC sequence disrupted Bcl-xL binding. Peptides reduced [Ca2+]mito uptake in WT but were without effect in Bcl-xL-KO cells. In addition, peptides reduced [Ca2+]mito uptake in VDAC1 and VDAC3 knock-out but not VDAC1 and -3 double knock-out mouse embryonic fibroblast cells, confirming that Bcl-xL interacts functionally with VDAC1 and -3 but not VDAC2. Thus, an interaction between Bcl-xL and VDAC promotes matrix Ca2+ accumulation by increasing Ca2+ transfer across the outer mitochondrial membrane.  相似文献   

5.
VDAC is the major permeability pathway in the mitochondrial outer membrane and can control the flow of metabolites and ions. Therefore Ca2+ flux across the outer membrane occurs mainly through VDAC. Since both Ca2+ fluxes and VDAC are involved in apoptosis, we examined whether Ca2+ is required for channel formation by VDAC isolated from rat liver. The voltage gating of VDAC does not require Ca2+ and it functions normally with or without Ca2+. Additionally, VDAC generally shows a higher permeability to Ca2+ in the closed states (states with lower permeability to metabolites) than that in the open state. Thus VDAC closure, which induces apoptosis, also favors Ca2+ flux into mitochondria, which can also lead to permeability transition and cell death. These results are consistent with the view that VDAC closure is a pro-apoptotic signal.  相似文献   

6.
Cardiac aging is associated with compromised myocardial function and morphology although the underlying mechanism remains elusive. Aldehyde dehydrogenase 2 (ALDH2), an essential mitochondrial enzyme governing cardiac function, displays polymorphism in humans. This study was designed to examine the role of ALDH2 in aging-induced myocardial anomalies. Myocardial mechanical and intracellular Ca2+ properties were examined in young (4–5 months) and old (26–28 months) wild-type and ALDH2 transgenic mice. Cardiac histology, mitochondrial integrity, O2 generation, apoptosis, and signaling cascades, including AMPK activation and Sirt1 level were evaluated. Myocardial function and intracellular Ca2+ handling were compromised with advanced aging; the effects were accentuated by ALDH2. Hematoxylin and eosin and Masson trichrome staining revealed cardiac hypertrophy and interstitial fibrosis associated with greater left-ventricular mass and wall thickness in aged mice. ALDH2 accentuated aging-induced cardiac hypertrophy but not fibrosis. Aging promoted O2 release, apoptosis, and mitochondrial injury (mitochondrial membrane potential, levels of UCP-2 and PGC-1α), and the effects were also exacerbated by ALDH2. Aging dampened AMPK phosphorylation and Sirt1, the effects of which were exaggerated by ALDH2. Treatment with the ALDH2 activator Alda-1 accentuated aging-induced O2 generation and mechanical dysfunction in cardiomyocytes, the effects of which were mitigated by cotreatment with activators of AMPK and Sirt1, AICAR, resveratrol, and SRT1720. Examination of human longevity revealed a positive correlation between life span and ALDH2 gene mutation. Taken together, our data revealed that ALDH2 enzyme may accentuate myocardial remodeling and contractile dysfunction in aging, possibly through AMPK/Sirt1-mediated mitochondrial injury.  相似文献   

7.
In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca2+ homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca2+and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca2+ homeostasis and mitochondria-mediated apoptosis will be discussed.  相似文献   

8.

Background

Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca2+ concentration ([Ca2+]i) elevation induced by infection can cause cell death, but [Ca2+]i changes and high [Ca2+]i-induced death of macrophages due to infection of Leptospira have not been previously reported.

Methodology/Principal Findings

We first used a Ca2+-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca2+]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca2+]i elevation was caused by both extracellular Ca2+ influx through the purinergic receptor, P2X7, and Ca2+ release from the endoplasmic reticulum, as seen by suppression of [Ca2+]i elevation when receptor-gated calcium channels were blocked or P2X7 was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3), which in turn induces intracellular Ca2+ release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S-1. Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca2+]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca2+]i. Moreover, PI-PLCs (PI-PLC-β3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca2+]i increases induced apoptosis and necrosis of macrophages, while mild [Ca2+]i elevation only caused apoptosis.

Conclusions/Significance

This study demonstrated that L. interrogans infection induced [Ca2+]i elevation through extracellular Ca2+ influx and intracellular Ca2+ release cause macrophage apoptosis and necrosis, and the LB361 gene product was shown to be a novel PI-PLC of L. interrogans responsible for the [Ca2+]i elevation.  相似文献   

9.
Obesity-related kidney diseases are becoming serious health problems worldwide, yet the mechanism by which obesity causes kidney injury is not fully understood. The purpose of current study was to investigate the role of Mas receptor in lipid-induced kidney injury. In mice fed with high-fat diet (HFD), the protein abundance of markers of autophagy, endoplasmic reticulum stress (ER stress) and apoptosis was dramatically increased in the kidney cortex, which was markedly prevented by Mas deletion (Mas−/−) or Mas receptor antagonist A779. Palmitic acid (PA) induced persistently increased autophagy, ER stress, and apoptosis as well as mitochondrial injuries in primary cultured proximal tubular cells from wild type, but not from Mas−/− mice. In human proximal tubular HK2 cells, PA-induced autophagy and ER stress was aggravated by Mas agonists Ang (1–7) or AVE0991, but attenuated by A779 or Mas knockdown. Stimulation of Mas resulted in elevated intracellular calcium levels [Ca2+]i in HK2 cells treated with PA, whereas inhibition or knockdown of Mas decreased [Ca2+]i. Mitochondrial outer membrane located voltage-dependent anion channel (VDAC1) was markedly upregulated in HK2 cells treated with PA, which was associated with impaired mitochondrial morphology and depolarization. These were enhanced by AVE0991 and suppressed by A779 or Mas knockdown. Mas knockdown in HK2 cells prevented impaired interactions among VDAC1, autophagy adaptor P62, and ubiquitin, induced by PA, leading to a potential ubiquitination of VDAC1. In conclusion, Mas receptor-mediated lipid-induced impaired autophagy and ER stress in the kidney, likely contributing to tubular injuries in obesity-related kidney diseases.Subject terms: Autophagy, Chronic kidney disease  相似文献   

10.

Background

Transglutaminase 2 (TG2) is a protein cross-linking enzyme known to be associated with the in vivo apoptosis program of T cells. However, its role in the T cell apoptosis program was not investigated yet.

Results

Here we report that timed overexpression of both the wild type (wt) and the cross-linking mutant of TG2 induced apoptosis in Jurkat T cells, the wt being more effective. Part of TG2 colocalised with mitochondria. WtTG2-induced apoptosis was characterized by enhanced mitochondrial Ca2+ uptake. Ca2+-activated wtTG2 cross-linked RAP1, GTP-GDP dissociation stimulator 1, an unusual guanine exchange factor acting on various small GTPases, to induce a yet uncharacterized signaling pathway that was able to promote the Ca2+ release from the endoplasmic reticulum via both Ins3P and ryanodine sensitive receptors leading to a consequently enhanced mitochondrial Ca2+uptake.

Conclusions

Our data indicate that TG2 might act as a Ca2+ sensor to amplify endoplasmic reticulum-derived Ca2+ signals to enhance mitochondria Ca2+ uptake. Since enhanced mitochondrial Ca2+ levels were previously shown to sensitize mitochondria for various apoptotic signals, our data demonstrate a novel mechanism through which TG2 can contribute to the induction of apoptosis in certain cell types. Since, as compared to knock out cells, physiological levels of TG2 affected Ca2+ signals in mouse embryonic fibroblasts similar to Jurkat cells, our data might indicate a more general role of TG2 in the regulation of mitochondrial Ca2+ homeostasis.  相似文献   

11.
12.
DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl channels. Some Cl channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca2+ dependence of PS scrambling, we explored whether inhibitors of Cl channels (DIDS, NPPB) or of Ca2+-activated Cl? channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca2+ concentration ([Ca2+]i) and activity of Ca2+-activated K+ (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 °C) with Cl? channels inhibitors decreased [Ca2+]i in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca2+]i was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl channel blockers further modified the alterations of [Ca2+]i following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca2+ ionophore ionomycin (1 μM). The ability of the Cl? channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca2+]i as TA and AO1 had a particularly strong decreasing effect on [Ca2+]i but at the same time enhanced PS exposure. In conclusion, Cl channel inhibitors affect Gardos channels, influence Ca2+ homeostasis and induce PS exposure of hRBCs by Ca2+-independent mechanisms.  相似文献   

13.
Ischemia-induced ionic imbalance leads to the activation of numerous events including mitochondrial dysfunction and eventual cell death. Dysregulation of mitochondrial Ca2+ (Ca2+m) plays a critical role in cell damage under pathological conditions including traumatic brain injury and stroke. High Ca2+m levels can induce the persistent opening of the mitochondrial permeability transition pore and trigger mitochondrial membrane depolarization, Ca2+ release, cessation of oxidative phosphorylation, matrix swelling and eventually outer membrane rupture with release of cytochrome c and other apoptogenic proteins. Thus, the dysregulation of mitochondrial Ca2+ homeostasis is now recognized to play a crucial role in triggering mitochondrial dysfunction and subsequent apoptosis. Recent studies show that some secondary active transport proteins, such as Na+-dependent chloride transporter and Na+/Ca2+ exchanger, contribute to ischemia-induced dissipation of ion homeostasis including Ca2+m.Key words: ischemia, intracellular Ca2+ dysregulation, changes of mitochondrial Ca2+, cytochrome c, apoptosis  相似文献   

14.
15.
The aim of this study was to assess the effect of the β‐amyloid fragment Aβ25–35 on mitochondrial structure and function and on the expression of proteins associated with the mitochondrial permeability transition pore (MPTP) in rat hippocampal neurons. Ninety clean‐grade Sprague–Dawley rats were randomly assigned to six groups (n = 15 per group). Aβ25–35 (1, 5, or 10 µg/rat) was injected into hippocampal area CA1. Normal saline was injected as a control. The effect of Aβ25–35 injection on hippocampal structure was assessed by transmission electron microscopy. Ca2+‐ATPase activity, [Ca2+]i, and mitochondrial membrane potential were measured. The expression of genes associated with the MPTP, including the voltage‐dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (Cyp‐D), were evaluated. Results showed that Aβ25–35 injection damaged the mitochondrial structure of hippocampal neurons, decreased Ca2+‐ATPase activity and mitochondrial membrane potential, and increased [Ca2+]i. The expression levels for VDAC, ANT, and Cyp‐D in all groups were significantly (P < 0.05) higher than those in the normal control group after Aβ25–35 injection. These results indicate that Aβ25–35 damages mitochondria in rat hippocampal neurons and effects mitochondrial dysfunction, as well as increasing the expression of genes associated with the MPTP. Mitochondrial dysfunction may result in increased MPTP gene expression, leading to neurodegenerative effects. J. Cell. Biochem. 112: 1450–1457, 2011. © 2011 Wiley‐Liss, Inc.  相似文献   

16.
Khz is a compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating JNK to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the translocation of regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca2+]i, which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca2+]i, JNK activation, and ROS generation via NADPH oxidase and mitochondria.  相似文献   

17.
Recent studies have suggested that mitochondria may play important roles in the Ca2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca2+ flux can regulate the generation of Ca2+ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca2+ (Cai 2+) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and CaWs were induced in the presence of high (4 mM) external Ca2+ (Cao 2+). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai 2+ levels even after depletion of SR Ca2+ in the absence of Cao 2+ , suggesting Ca2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m) or Ru360 (a mitochondrial Ca2+ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca2+ release and uptake exquisitely control the local Ca2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.  相似文献   

18.
《Free radical research》2013,47(3):338-346
Abstract

Polycystic ovary syndrome (PCOS) is a common inflammatory and oxidant disease with an uncertain pathogenesis. N-acetyl cysteine (NAC) decreases oxidative stress, intracellular free calcium ion [Ca2+]i, and apoptosis levels in human neutrophil. We aimed to investigate the effects of NAC on apoptosis, oxidative stress, and Ca2+ entry through transient receptor potential vanilloid 1 (TRPV1) and TRP melastatin 2 (TRPM2) channels in neutrophils from patients with PCOS. Neutrophils isolated from PCOS group were investigated in three settings: (1) after incubation with TRPV1 channel blocker capsazepine or TRPM2 channel blocker 2-aminoethyl diphenylborinate (2-APB), (2) after supplementation with NAC (for 6 weeks), and (3) with combination (capsazepine + 2-APB + NAC) exposure. The neutrophils in TRPM2 and TRPV1 experiments were stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 μM) and capsaicin (10 μM) as concentration agonists, respectively. Neutrophil lipid peroxidation and capsaicin-induced increase in [Ca2+]i concentrations were reduced by capsazepine and NAC treatments. However, the [Ca2+]i concentration did not change by fMLP stimulation. Neutrophil lipid peroxidation, apoptosis, caspase-3, caspase-9, cytosolic reactive oxygen species production, and mitochondrial membrane depolarization values were decreased by NAC treatment although neutrophil glutathione peroxidase and reduced glutathione levels were increased by the NAC treatment. Serum lipid peroxidation, luteinizing hormone, testosterone, insulin, interleukin-1 beta, and homocysteine levels were decreased by NAC treatment although serum vitamin A, beta-carotene, vitamin E, and total antioxidant status were increased by the NAC treatment. In conclusion, NAC reduced oxidative stress, apoptosis, cytokine levels, and Ca2+ entry through TRPV1 channel, which provide supportive evidence that oxidative stress and TRPV1 channel plays a key role in etiology of PCOS.  相似文献   

19.
Selenium (Se), as one of the essential trace elements, plays an anti-inflammatory, antioxidation, and immune-enhancing effect in the body. In addition, Se can also improve nervous system damage induced by various factors. Earlier studies have described the important role of mitochondrial dynamic imbalance in lipopolysaccharide (LPS)-induced nerve injury. The inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (GRP75)/voltage-dependent anion channel 1 (VDAC1) complex is considered to be the key to regulating mitochondrial dynamics. However, it is not clear whether Selenomethionine (SeMet) has any influence on the IP3R/GRP75/VDAC1 complex. Therefore, the aim of this investigation was to determine whether SeMet can alleviate LPS-induced brain damage and to elucidate the function of the IP3R/GRP75/VDAC1 complex in it. We established SeMet and/or LPS exposure models in vivo and in vitro using laying hens and primary chicken nerve cells. We noticed that SeMet reversed endoplasmic reticulum stress (ERS) and the imbalance in mitochondrial dynamics and significantly prevented the occurrence of neuronal apoptosis. We made this finding by morphological observation of the brain tissue of laying hens and the detection of related genes such as ERS, the IP3R/GRP75/VDAC1 complex, calcium signal (Ca2+), mitochondrial dynamics, and apoptosis. Other than that, we also discovered that the IP3R/GRP75/VDAC1 complex was crucial in controlling Ca2+ transport between the endoplasmic reticulum and the mitochondrion when SeMet functions as a neuroprotective agent. In summary, our results revealed the specific mechanism by which SeMet alleviated LPS-induced neuronal apoptosis for the first time. As a consequence, SeMet has great potential in the treatment and prevention of neurological illnesses (like neurodegenerative diseases).  相似文献   

20.
Panaxydol, a polyacetylenic compound derived from Panax ginseng roots, has been shown to inhibit the growth of cancer cells. In this study, we demonstrated that panaxydol induced apoptosis preferentially in transformed cells with a minimal effect on non-transformed cells. Furthermore, panaxydol was shown to induce apoptosis through an increase in intracellular Ca2+ concentration ([Ca2+]i), activation of JNK and p38 MAPK, and generation of reactive oxygen species (ROS) initially by NADPH oxidase and then by mitochondria. Panaxydol-induced apoptosis was caspase-dependent and occurred through a mitochondrial pathway. ROS generation by NADPH oxidase was critical for panaxydol-induced apoptosis. Mitochondrial ROS production was also required, however, it appeared to be secondary to the ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the membrane translocation of regulatory p47phox and p67phox subunits and shown to be necessary for ROS generation by panaxydol treatment. Panaxydol triggered a rapid and sustained increase of [Ca2+]i, which resulted in activation of JNK and p38 MAPK. JNK and p38 MAPK play a key role in activation of NADPH oxidase, since inhibition of their expression or activity abrogated membrane translocation of p47phox and p67phox subunits and ROS generation. In summary, these data indicate that panaxydol induces apoptosis preferentially in cancer cells, and the signaling mechanisms involve a [Ca2+]i increase, JNK and p38 MAPK activation, and ROS generation through NADPH oxidase and mitochondria.  相似文献   

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