Apoptosis-related Proteins Caspase-3 Research Articles

Glutamine supplementation provides neuroprotection by inhibiting endoplasmic reticulum stress response after traumatic brain injury in rats

Objective To investigate the effects of glutamine (Gln) supplementation on neurologica severity score, brain edema, neuron apoptosis, and endoplasmic reticulum stress (ERS) response after traumatic brain injury (TBI) in rats. Methods TBI rat models were established using modified Feeney's method. Eighty Sprague-Dawley rats were divided into 4 groups with a random number table: sham operation group (Sham group), TBI group, Gln supplementation group (TBI+ Gln group) and ERS inducer 2-deoxy-D-glucose group (TBI+ Gln+ 2-DG group). We measured the rats' neurobehavioral outcomes by modified neurologic severity score (mNSS) on day 1, 3, 7 and 14 after TBI. Neuron apoptosis was detected using TUNEL staining. Brain water content was measured with wet-dry weight method.The apoptosis-related protein (caspase-12, caspase-3, and Bcl-2) and ERS-related cytokines [inositol-requiring enzyme 1(IRE-1), C/EBP homologous protein (CHOP)] expressions in TBI cerebral cortex were determined by immunohistochemistry staining and Western blot. Results Compared with the Sham group, the levels of brain edema, mNSS, apoptosis-related protein (caspase-12, caspase-3, Bcl-2) and ERS-related proteins (IRE-1, CHOP) were significantly increased in the other three groups(all P=0.00). Compared with the TBl group, the TBI+ Gln group showed significant lower brain water content[3 d: (81.39±0.59)% vs.(83.54±0.52)%, P=0.04; 7 d: (74.86±0.38)% vs. (77.32±0.66)%, P=0.03], improved mNSS(8.63±0.22 vs. 10.37±0.29, P=0.03), suppressed expressions of apoptosis- and ERS-related proteins (caspase-12, caspase-3, IRE-1, and CHOP)(P=0.01, P<0.01), and increased expression of anti-apoptotic protein Bcl-2 (P=0.02). Compared with the TBI+ Gln group, the expression of ERS-related factors (IRE-1 and CHOP), brain edema level, and neurological severity were increased in the TBI+ Glu+ 2-DG group. Conclusion Glutamine supplementation may have neuroprotection function, demonstrated as reducing brain edema and neuron apoptosis, and improving neurobehaviroal outcomes after TBI, possibly mediated by inhibiting TBI-induced ERS response. Key words: Traumatic brain injury; Glutamine; Endoplasmic reticulum stress; Neuroprotection

Thioridazine Sensitizes Apoptotic Effect of TRAIL in Human Lung Cancer PC9 Cells Through ER Stress Mediated Up-regulation of DR5

背景与目的肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducting ligand, TRAIL）可诱导肿瘤细胞发生凋亡，然而相当数量的肿瘤细胞可耐受TRAIL诱导的凋亡而得以存活。本实验观察硫利达嗪（thioridazine, THZ）通过诱导内质网应激（endoplasmic reticulum stress, ER stress）介导的死亡受体5（death receptor 5, DR5）表达上调，继而增敏TRAIL对肺腺癌细胞PC9的生长抑制及凋亡诱导效应，探讨其机制。方法不同浓度硫利达嗪及TRAIL单独或联合处理PC9细胞，MTT法检测细胞活性变化，流式细胞术检测细胞表面DR5表达及细胞凋亡率，Western blotting检测内质网应激相关蛋白GRP78（glucose regulated protein 78）、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白（C/EBP homologous protein, CHOP）、p-PERK（PKR-like ER kinase）、p-eIF2α（eukaryotic initiation factor-2α, eIF2α）、ATF4（activating transcription factor 4, ATF4）及凋亡相关蛋白Caspase-3、Caspase-9、Caspase-8、PARP、DR5表达变化。结果硫利达嗪对PC9细胞的增殖抑制效应呈浓度依赖性（P < 0.05），硫利达嗪可增加TRAIL对PC9细胞的抑制作用及凋亡诱导作用且可上调PC9细胞表面DR5表达水平，流式细胞术结果显示：TRAIL联合硫利达嗪组细胞凋亡率较单独TRAIL组显著增加（P < 0.05），Western blotting结果显示：TRAIL联合硫利达嗪组细胞Cleaved-caspase-8、Cleaved-PARP、DR5表达水平较单独TRAIL组明显上调。DR5表达上调及促凋亡效应是通过诱导内质网应激发生，并伴随着GRP78及CHOP表达上调发生的，且该效应可被4-苯基丁酸（4-phenylbutyric acid, 4-PBA）可抑制（P < 0.05）。结论硫利达嗪增敏TRAIL对PC9细胞的增殖抑制效应显著，其机制可能与硫利达嗪内质网应激介导的DR5上调有关。

Daphnetin-mediated Nrf2 antioxidant signaling pathways ameliorate tert-butyl hydroperoxide (t-BHP)-induced mitochondrial dysfunction and cell death

Daphnetin (Daph), a natural coumarin derivative isolated from plants of the Genus Daphne, possesses abundant biological activities, such as anti-inflammatory, antioxidant and anticancer properties. In the present study, we focused on investigating the protective effect of Daph against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage, mitochondrial dysfunction and the involvement of underlying molecular mechanisms. Our findings indicated that Daph effectively inhibited t-BHP-stimulated cytotoxicity, cell apoptosis, and mitochondrial dysfunction, which are associated with suppressed reactive oxygen species (ROS) generation, decreased malondialdehyde (MDA) formation, increased superoxide dismutase (SOD) levels and glutathione (GSH)/GSSG (oxidized GSH) ratio. Further investigation indicated that Daph significantly suppressed cytochrome c release and NLRP3 inflammasome activation and modulated apoptosis-related protein Bcl-2, Bax, and caspase-3 expression. Moreover, Daph dramatically induced the expression of the glutamate-cysteine ligase modifier (GCLM) subunit and the glutamate-cysteine ligase catalytic (GCLC) subunit, heme oxygenase-1 (HO-1), and NAD (P) H: quinone oxidoreductase (NQO1), which is largely dependent on upregulating the nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation, reducing the Keap1 protein expression, and strengthening the antioxidant response element (ARE) promoter activity. Additionally, Daph remarkably activated a c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation, but ERK and JNK inhibitor pretreatment exhibited an evident decrease of the level of Daph-enhanced Nrf2 nuclear translocation. Furthermore, Daph exposure suppressed t-BHP-induced cytotoxicity and ROS overproduction, which are mostly blocked in Nrf2 knockout RAW 264.7 cells and peritoneal macrophages. Accordingly, Daph exhibited protective roles against t-BHP-triggered oxidative damage and mitochondrial dysfunction by the upregulation of Nrf2 antioxidant signaling pathways, which may be involved in the activation of JNK and ERK.

Anti-proliferation and anti-metastasis effect of barbaloin in non-small cell lung cancer via inactivating p38MAPK/Cdc25B/Hsp27 pathway.

Non-small cell lung carcinoma (NSCLC) is the most common lung cancer with high morbidity and mortality. The traditional treatment for NSCLC is particularly liable to relapse with many side-effects. Barbaloin is a natural compound with anticancer efficacy. The present study aimed to investigate the anticancer potential of barbaloin in NSCLC. The results displayed that barbaloin inhibited the viability of A549 cells by decreasing cell growth and the expression level of Ki-67 and proliferating cell nuclear antigen (PCNA), especially at high concentrations (50 and 100µM). Besides, barbaloin increased the apoptosis rate of A549 cells and induced an accumulation of G2/M phase. Increased expression of apoptosis-related proteins (caspase-3, -8 and -9) and the changed levels of cell cycle checkpoint proteins (p27, p53 and cyclinA) further convinced of the anti-viability effect of barbaloin in A549 cells. On the other hand, barbaloin significantly suppressed the invasion and migration of A549 cells, and restrained the expression of tumor metastasis-related proteins. We further explored the activation of pro-survival or pro-metastasis signaling pathways, including AKT, nuclear factor kappa B (NF-κB), mitogen-actived protein kinase (MAPK) and β-catenin. The results revealed that barbaloin inactivated the p38MAPK/Cdc25B/Hsp27 pathway by inhibiting p38 nucleus translocation, while no significant influence was observed among other pathways. Finally, barbaloin restrained the growth and hepatic metastases of A549 cells invivo. Taken together, our research indicated that barbaloin inhibited the proliferation and metastasis of NSCLC cells invivo and invitro. This may provide safer and more effective aspects for the treatment of NSCLC.

Role of miR-15a in intervertebral disc degeneration through targeting MAP3K9.

Accumulating evidence indicates that microRNAs are involved in various cellular processes, including cell proliferation, differentiation, apoptosis and metastasis. miR-15a is an important regulator of immune responses and angiogenesis, endogenous controls as well as potential targets and hallmarks of cancer. However, the role of miR-15a in intervertebral disc degeneration (IDD) has not been elucidated. Total RNA was extracted from degenerative nucleus pulposus (NP) tissues of 20 patients with IDD and NP cells, respectively. The expression levels of miR-15a were examined by quantitative real-time PCR. The stable overexpress or silence miR-15a expression cell lines and control cell lines were constructed by lentivirus infection. Subsequently, 3-(4,5-dimethylthia zol-2-yl)-2,5-diphenylte trazolium bromide (MTT) assay, flow cytometry test, TdT-mediated dUTP Nick-End Labeling (TUNEL) experiment, colony formation assay and western blot analysis were performed to detect the biological functions of miR-15a. Moreover, a luciferase reporter assay was conducted to confirm its target associations. Herein, the results found that miR-15a was dramatically up-regulated in degenerative NP tissues and NP cells compared with the controls. Overexpression of miR-15a promoted NP cells proliferation and induced apoptosis. Moreover, apoptosis-related protein caspase-3 was significantly up-regulated and bcl-2 was observably down-regulated when NP cells were transfected with miR-15a mimics, while bax and caspase-3 were significantly down-regulated as well as bcl-2 was observably up-regulated when NP cells were transfected with miR-15a inhibitors. Further, luciferase reporter assay showed that MAP3K9, an upstream activator of MAPK kinase, was putative target of miR-15a. There was a negatively relationship between miR-15a and MAP3K9 expression in NP cells. In addition, knockdown MAP3K9 inhibited NP cells proliferation and promoted apoptosis, which further inhibited the activation of p38 and ERK MAPK pathway. This present study revealed that miR-15a might be considered as a novel therapeutic target for IDD treatment.

Hyaluronic acid-coated cationic nanostructured lipid carriers for oral vincristine sulfate delivery

The goal of this research is to structure a hyaluronic acid modified nanostructured lipid carrier (HA-NLCs) for vincristine sulfate (VCR) delivery, and detect its efficiency to improve the oral bioavailability. Emulsion solvent evaporation method was used to prepare the HA-NLCs nanoparticles. The particle size, zeta potential and entrapment efficiency of VCR-NLCs and HA-NLCs were 187 ± 3.52 nm, −8.61 ± 1.29 mV, 33.12 ± 1.16% and 192 ± 4.41 nm, −32.82 ± 2.64 mV, 34.41 ± 2.21%, respectively. HA-NLCs could significantly improve the cellular uptake efficiency and cytotoxicity in MCF-7 cells than other VCR formulations. The expressions of apoptosis related protein Caspase-3, Caspase-9, Bax and Bcl-2 were estimated by western blot assay in MCF-7 cells, and HA-NLCs exhibited the strongest effect in promoting cell apoptosis. The pharmacokinetics of HA-NLCs was evaluated in Sprague–Dawley male rats and the relative oral bioavailability of HA-NLCs and VCR-NLCs was improved about 1.8-fold and two-fold compared with VCR solution, respectively. Therefore, these results indicated that HA-NLCs could significantly improve the oral bioavailability and was a promising vehicle for the oral delivery of VCR.

Celastrol alleviates arthritis by modulating the inflammatory activities of neutrophils

ObjectiveNeutrophils play an important role in the pathogenesis of rheumatoid arthritis (RA). In this study, we used the adjuvant-induced arthritis murine model to evaluate the efficacy of celastrol on neutrophil-mediated inflammation in RA. MethodsFreund's complete adjuvant-induced arthritis was used as the murine model of RA. Celastrol was intraperitoneally administrated daily after onset of the disease. The joint diameter and inflammatory score were evaluated daily during the treatment period. Myeloperoxidase (MPO) and neutrophil elastase (NE) activities were evaluated by immunohistochemical analyses. Quantitative PCR and enzyme-linked immunoabsorbent assay were used to quantify the expression of cytokines. The expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 were evaluated by western blot. ResultsCelastrol suppressed inflammation in joints of arthritic mice and diminished the expression of MPO and NE in the joint tissue. Celastrol significantly inhibited the expression of TNFα and IL-6 induced by LPS in neutrophils in a dose-dependent manner in vitro. Moreover, celastrol induced apoptosis of LPS-stimulated neutrophils by increasing the expression of Bax and cleaved caspase-3 while decreasing Bcl-2 expression. ConclusionOur findings show that celastrol significantly alleviates murine arthritis by modulating the inflammatory activities of neutrophils. These results indicate that celastrol could serve as an alternative or adjunct modality for the treatment of RA.

STAT3 signaling pathway regulates glioma stem cells induced host macrophage malignance

Background: Continuous activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway contributes to cellular proliferation, angiogenesis and activation of anti-apoptosis pathways to promote tumor progression. Besides, STAT3 activation plays an important role in the polarisation of M2 macrophages and immune suppression. Our previous study found that glioma stem cells (SU3) could induce host macrophage malignance in the immune microenvironment, and a cancerous cell line SU3-induced host celiac tumor cells (SU3-ihCTCs) was obtained. We also found STAT3 was activated in SU3-ihCTCs. Methods: The ratio of F4/80 positive cells in SU3-ihCTCs was tested by flow cytometry, and the expression of p-STAT3 and macrophage markers were analyzed with RT-PCR or Western Blotting, respectively. SU3-ihCTCs were treated with 1–6 μmol/L WP1066 for 24 hours, then the expression of STAT3, p-STAT3, Bcl-2 and Cleaved caspase-3 were detected with Western Blotting, and the ratio of apoptosis were tested by flow cytometry. BalB/C nude mice received subcutaneous implants of SU3-ihCTCs, the tumor size was measured every other day, and the weight of tumor was measured. The expression of STAT3, p-STAT3, Bcl-2 and Cleaved caspase3 of the transplanted tumor were tested by Western Blotting and immunohistochemical analysis. Results: SU3-ihCTCs expressed higher of p-STAT3 and M2 macrophage makers. When SU3-ihCTCs were treated with the STAT3 inhibitor WP1066, the expressions of M2 makers, p-STAT3 and Bcl-2 of the cells were decreased, and the expression of cleaved caspase-3 was increased. At the same time, the proliferation of SU3-ihCTCs was inhibited and apoptosis was enhanced. In vivo studies had shown that WP1066 treatment significantly decreased the volume and weight of SU3-ihCTCs xenografts in nude mice, and immunohistochemical and Western blotting analyses showed that the expression levels of p-STAT3 and Bcl-2 decreased and those of the apoptosis-related protein caspase-3 were upregulated. Conclusions: We suggest that glioma stem cells induce host macrophage malignance in tumor microenvironment (TME), STAT3 pathway play an important role in the transformation process. These results present new evidence that macrophages in TME promote tumor proliferation. STAT3 signaling pathway may present a therapeutic target to suppress the tumor development.

Sequential Induction of Cell Cycle Arrest and Mitotic Catastrophe Followed By Apoptosis Via Activation of PTEN By GL-V9 in Human T-Cell Malignancies

GL-V9, a newly synthetic flavonoid derivative, is an active cytotoxic component and exhibits anticancer activities. Here, we demonstrated the sequence of death reponses to different concentration of GL-V9 in human T-cell malignancies cell Jurkat and HuT78. Responses included transient accumulation of cells at the G2/M border followed by also transient rise in several mitotic markers and mitotic attempts were largely abnormal, resulting in numerous micronucleated and multinucleated cells in low drug concentration (2 μM), as shown by flow cytometre analyses and Giemsa staining. These events, indicative of mitotic catastrophe, were not associated with immediate cell death. However, MTT assay showed that high concentration of GL-V9 (4, 8, 16μM) causes near complete cell growth inhibition at 12 h. Also, the classical manifestations of apoptosis (including phosphatidylserine externalization, mitochondrial dysfunction, and caspase activation) were marginal at 24 h, as shown by Annexin V-PI staining, JC-1, and western blot assay. The mechanism research reveals that low dose of GL-V9 induced mitotic catastrophe via decreasing EEA1, which is required for cytokinesis, and increasing the expression of PTEN. Besides, high concentration of GL-V9 induced cell apoptosis by up-regulating the expression of apoptosis-related proteins caspase 3 and Bim in lymphoid tumor cell lines and primary ALL cells. Collectively, these findings show that different concentrations of GL-V9 can induce mitotic catastrophe and ultimate cell death event of human T-cell malignancies respectively, which would be a potential therapeutical compound for treating human lymphoid tumor. DisclosuresNo relevant conflicts of interest to declare.

Cordycepin Induces Apoptosis and Inhibits Proliferation of Human Lung Cancer Cell Line H1975 via Inhibiting the Phosphorylation of EGFR.

Cordycepin is an active component of the traditional Chinese medicine Cordyceps sinensis and Cordyceps militaris with notable anticancer activity. Though the prominent inhibitory activity was reported in different kinds of cancer cell lines, the concrete mechanisms remain elusive. It was reported that cordycepin could be converted into tri-phosphates in vivo to confuse a number of enzymes and interfere the normal cell function. For the inhibitory mechanism of EGFR inhibitors and the structure similarity of ATP and tri-phosphated cordycepin, human lung cancer cell line H1975 was employed to investigate the inhibitory effect of cordycepin. The results showed that cordycepin could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Cell cycle analysis revealed that H1975 cells could be arrested at the G0/G1 phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives.

MicroRNA-184 Promotes Proliferation and Inhibits Apoptosis in HaCaT Cells: An In Vitro Study.

BackgroundThis study aimed to investigate the role of miR-184 in the proliferation and apoptosis of keratinocyte (HaCaT cells).Material/MethodsHaCaT cells were cultured in a growth medium. The miR-184 was transfected with siRNA, then cell viability and apoptosis were assayed by MTT and flow cytometry, respectively. The colony-forming efficacy of HaCaT cells were detected as well. mRNA expressions of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-β1 were measured with RT-PCR. The expressions of apoptosis-related proteins caspase-3 and Bcl-x in HaCaT cells were determined by Western blot.ResultsAfter miR-184 was transfected with siRNA, cell viability and colony forming ability decreased significantly, and apoptosis was significantly increased. The expressions of growth factors TGF-β1 and bFGF mRNAs, as well as apoptosis-related proteins Bcl-x, in HaCaT cells declined significantly after miR-184 was transfected with siRNA. In addition, the expression of pro-apoptotic protein caspase-3 increased significantly.ConclusionsOur results suggest distinct roles of miR-184 during the growth, proliferation, and apoptosis of keratinocytes.

Crosstalk between Beclin-1-dependent autophagy and caspase‑dependent apoptosis induced by tanshinoneIIA in human osteosarcoma MG-63 cells.

The aim of the present study was to ascertain whether or not autophagy is induced by tanshinone IIA (TanIIA), and to explore the crosstalk between autophagy and apoptosis in regards to the antitumor effects of TanIIA on MG-63 cells and the potential mechanism. MG-63 cells were cultured in vitro with various concentrations of TanIIA (0, 2.5, 5, 10 and 20 mg/l) for 0, 24, 48 and 72 h, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to evaluate the inhibition of the proliferation of osteosarcoma MG-63 cells by TanIIA or in the presence/absence of chloroquine (CQ). Autophagic vacuoles and characteristic autophagosomes were observed by transmission electron microscopy (TEM). TanIIA-induced autophagy in MG-63 cells was confirmed by GFP-LC3 punctate fluorescence. The expression levels of apoptosis-related proteins caspase-3, caspase-8, caspase-9 and cleaved-PARP and autophagy-related proteins LC3II/LC3I and Beclin-1 were detected by western blotting. FITC-Annexin V/propidium iodide (PI) staining, flow cytometry and Hoechst 33258 staining were used to analyze the apoptotic rate. Fluorescence intensity of reactive oxygen species (ROS) was examined under a fluorescence microscope using an analysis software system. Cell proliferation was obviously inhibited by TanIIA in a dose- and time-dependent manner. Generation of autophagy was triggered by TanIIA (0–20 mg/l) treatment, and in a Beclin-1-dependent manner. Compared with the control group, the apoptosis ratio following treatment with 2.5 mg/l TanIIA failed to achieve statistical significance. Expression of caspase-3, -8 and -9, and cleaved-PARP in the other groups was gradually enhanced in dose-dependent manner. Our analysis also suggested that the influence of autophagy on TanIIA cytotoxicity had a phase effect; with low-dose drugs and shorter treatment periods, autophagy functioned as a damage repair mechanism. In conrast, when the cells were treated with higher doses of TanIIA for longer treatment periods, autophagic cell death contributed to apoptosis. Furthermore, generation of ROS occurred in a dose-dependent manner and pretreatment with NAC, a selective ROS scavenger, blocked the coexistence of Beclin-1 autophagy and caspase-dependent apoptosis. In conclusion, our findings provide strong evidence that TanIIA may be a potential therapeutic drug against osteosarcoma. Moreover, its cytotoxity can be enhanced with ROS agonists.

Acid-sensing ion channel 1a regulates the survival of nucleus pulposus cells in the acidic environment of degenerated intervertebral discs.

Activation of acid-sensing ion channel 1a (ASIC1a) is responsible for tissue injury caused by acidosis in nervous systems. But its physiological and pathological roles in nucleus pulposus cells (NPCs) are unclear. The aim of this study is to investigate whether ASIC1a regulates the survival of NPCs in the acidic environment of degenerated discs. NPCs were isolated and cultured followed by immunofluorescent staining and Western-blot analysis for ASIC1a. Intracellular calcium ([Ca2+]i) was determined by Ca2+-imaging using Fura-2-AM. Cell necrosis, apoptosis, and senescence following acid exposure were determined using lactate dehydrogenase (LDH) release assay, annexin V-fluorescein isothiocyanate/propidium iodide dual-staining and cell cycle analysis, respectively, followed by Western-blot analysis for apoptosis-related proteins (Bax, Bcl-2, and caspase-3) and senescence-related proteins (p53, p21, and p16). Effects of treatment with psalmotoxin-1 (PcTX1, blocker of ASIC1a) on [Ca2+]i and cell survival were investigated. ASIC1a was detected in healthy NPCs, and its expression was significantly higher in degenerated cells. When NPCs were treated with PcTX1, acid-induced increases in [Ca2+]i were significantly inhibited. PcTX1 treatment also resulted in decreased LDH release, cell apoptosis and cell cycle arrest in acid condition. Acid exposure decreased the expression of Bcl-2 and increased the expression of Bax, cleaved caspase-3 and senescence-related proteins (p53, p21, and p16), which was inhibited by PcTX1. The present findings suggest that further understanding of ASIC1a functionality may provide not only a novel insight into intervertebral disc biology but also a novel therapeutic target for intervertebral disc degeneration.

Regulation of the Ku70 and apoptosis-related proteins in experimental diabetic nephropathy

BackgroundApoptosis contributes nephropathy pathogenesis in diabetes. However, its mechanisms still remain unclear. We examined the extent to which the angiotensin-II type 1 receptor blocker (AT1RB) irbesartan and the angiotensin converting enzyme inhibitor (ACEI) perindopril affected the apoptosis-related proteins Bcl-2, Bax, caspase-3, cytochrome-c and Ku70 in streptozotocin (STZ)-diabetic rats. Materials and methodsAnimals were divided into five groups of eight each, four of which received STZ (60mg/kg in a single dose, i.p.) to induce diabetes. The groups were performed as untreated diabetic; non-diabetic control; daily irbesartan (15mg/kg/day) or perindopril (6mg/kg/day) and also combined irbesartan and perindopril (respectively, 5mg/kg/day, 3mg/kg/day) were applied by gavage for 30days to STZ-diabetic rats. The kidney tissue analysis was performed by using immunohistochemical staining with Bcl-2, Bax, caspase-3, cytochrome-c and Ku70 antibodies and by using Western blot analysis with caspase-3 and cytochrome-c antibodies. ResultsImmunoreactivity of Bax, caspase-3, cytochrome-c and Ku70 was increased in the tubuli and glomeruli of the untreated diabetic group, but decreased in all treated diabetic groups. In the irbesartan and perindopril treated diabetic groups Bcl-2 immunoreactivity was higher than that of the untreated diabetic group. Caspase-3 and cytoplasmic cytochrome-c protein levels increased in the untreated diabetic group. ConclusionsWe conclude that the increased expression of Bax and caspase-3, and the increased level of cytoplasmic cytochrome-c relate to renal tissue injury. This case is also seen in the early stages of diabetes as a result of the damage caused by local increased expression of renin angiotensin system (RAS) in the renal tissue, which is induced by hyperglycemia. The increase of the cytosolic cytochrome-c, caspase-3 and Ku70 expression in the tubuli is suggestive of apoptosis. Overall, our results show that treatments of irbesartan and perindopril are effective and efficient in preventing renal tissue injury and apoptosis by blocking the RAS in experimental diabetic nephropathy and reducing the expression of proteins associated with apoptosis.

Maternal DHA supplementation protects rat offspring against impairment of learning and memory following prenatal exposure to valproic acid

Docosahexaenoic acid (22:6n-3; DHA) is known to play a critical role in postnatal brain development. However, there have been no studies investigating the preventive effect of DHA on prenatal valproic acid (VPA)-induced behavioral and molecular alterations in offspring. The present study was to evaluate the neuroprotective effects in offspring using maternal feeding of DHA to rats exposed to VPA in pregnancy. In the present study, rats were exposed to VPA on day 12.5 of pregnancy; DHA was administered at the dosages of 100, 300 and 500 mg/kg/day for 3 weeks from day 1 to 21 of pregnancy. The results showed that maternal feeding of DHA to the prenatal exposed to VPA (1) prevented VPA-induced learning and memory impairment but did not change social-related behavior, (2) increased total DHA content in offspring plasma and hippocampus, (3) rescued VPA-induced neuronal loss and apoptosis of pyramidal cells in hippocampal CA1, (4) influenced the content of malondialdehyde and glutathione and the activities of superoxide dismutase and glutathione in the hippocampus, (5) altered levels of apoptosis-related proteins (Bcl-2, Bax and caspase-3) and inhibited the activity of caspase-3 in offspring hippocampus and (6) enhanced relative levels of p-CaMKII and p-CREB proteins in the hippocampus. These findings suggest that maternal feeding with DHA may prevent prenatal VPA-induced impairment of learning and memory, normalize several different molecules associated with oxidative stress and apoptosis in the hippocampus of offspring, and exert preventive effects on prenatal VPA-induced brain dysfunction.

Sarcolemmal ATP-sensitive potassium channel protects cardiac myocytes against lipopolysaccharide-induced apoptosis.

The sarcolemmal ATP-sensitive K+ (sarcKATP) channel plays a cardioprotective role during stress. However, the role of the sarcKATP channel in the apoptosis of cardiomyocytes and association with mitochondrial calcium remains unclear. For this purpose, we developed a model of LPS-induced sepsis in neonatal rat cardiomyocytes (NRCs). The TUNEL assay was performed in order to detect the apoptosis of cardiac myocytes and the MTT assay was performed to determine cellular viability. Exposure to LPS significantly decreased the viability of the NRCs as well as the expression of Bcl-2, whereas it enhanced the activity and expression of the apoptosis-related proteins caspase-3 and Bax, respectively. The sarcKATP channel blocker, HMR-1098, increased the apoptosis of NRCs, whereas the specific sarcKATP channel opener, P-1075, reduced the apoptosis of NRCs. The mitochondrial calcium uniporter inhibitor ruthenium red (RR) partially inhibited the pro-apoptotic effect of HMR-1098. In order to confirm the role of the sarcKATP channel, we constructed a recombinant adenovirus vector carrying the sarcKATP channel mutant subunit Kir6.2AAA to inhibit the channel activity. Kir6.2AAA adenovirus infection in NRCs significantly aggravated the apoptosis of myocytes induced by LPS. Elucidating the regulatory mechanisms of the sarcKATP channel in apoptosis may facilitate the development of novel therapeutic targets and strategies for the management of sepsis and cardiac dysfunction.

Combined cytotoxic effects of pesticide mixtures present in the Chinese diet on human hepatocarcinoma cell line

Consumers might be simultaneously exposed to several pesticide residues contained in their food. Based on the results of previous studies, 20 pesticides were selected due to their high exposure levels to which the Chinese population is likely exposed through the diet. The purpose of this study was to measure the cytotoxicity of these pesticides in HepG2 cells in vitro, as an alternative approach to assess the toxicity of chemicals. Then, the pesticides and some of the mixtures with comparatively high cell-proliferating inhibitory activities were selected to test the cellular ROS level and apoptosis-related protein Caspase-3/7 content in HepG2 cells. The combined effects of these pesticide mixtures with the prediction was based on a combination index (CI)-isobologram equation and the pesticide combinations exhibited various types of interactions (synergism, antagonism, and additivity). Two individuals, one binary combinations, and three uniform design (UD) mixtures of the pesticides were found to have significant cytotoxic effects, along with significant time- and dose-dependent induction of caspase-3/7 activity in vitro, indicating that cytotoxicity caused by these pesticides might be attributed to the pro-oxidative and apoptosis induced potential.

Gene Expression Profile of Apoptosis in Leukemia Cells Induced by Hsp90 Selective inhibitor 17-AAG

To investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism. CCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays. The inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment. The 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.

Heme oxygenase 1 induction protects myocardiac cells against hypoxia/reoxygenation-induced apoptosis

Although recent studies have found that heme oxygenase (HO)-1 plays an important role in myocardiac cell survival, the precise mechanisms occurring during hypoxia/reoxygenation (H/R) injury remain unclear. Therefore, the aim of this study was to investigate the cytoprotective mechanisms of HO-1 against H/R-induced myocardiac cell apoptosis and to explore whether the Akt signaling pathway contributed to the protection provided by HO-1. Cobalt protoporphyrin (CoPP, apharmacologic inducer of HO-1) was employed to induce the over-expression of HO-1 before H/R in H9c2 cells. Hoechst staining and flow cytometry were used to examine the extent of apoptosis. Furthermore, the effect of HO-1 on Akt, JNK, and the expression of apoptosis-related proteins (c-JUN and Caspase-3) was determined by Western blotting. The results showed that over-expressed HO-1 could significantly protect myocardiac cells against H/R-induced apoptosis in H9c2 cells. Furthermore, the protein expression of p‑Akt increased and of p‑JNK decreased in the H/R injury H9c2 cells when treated with CoPP. The apoptosis-related proteins c‑Jun and caspase-3 were both inhibited by over-expression of HO-1. At the same time, retreatment with zinc protoporphyrin (ZnPP, aspecific inhibitor of HO-1 enzymatic activity) or LY294002 (an inhibitor of Akt1) reversed the HO-1-induced changes. In summary, our results suggest that HO-1 can decrease H/R-induced myocardiac cell apoptosis; the mechanism may be related to the activation of the Akt signaling pathway and, furthermore, to the inhibition of the JNK/c-Jun/caspase-3 signaling pathway.

Alpha-ketoglutarate enhances milk protein synthesis by porcine mammary epithelial cells.

Alpha-ketoglutarate (AKG), a key intermediate in the Krebs cycle, has been reported to promote protein synthesis through activating mechanistic targeting of rapamycin (mTOR) in enterocytes. The study tested the hypothesis that AKG may enhance growth and milk protein synthesis in porcine mammary epithelial cells (PMECs). PMECs were cultured for 96h in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing prolactin (2µg/ml) and AKG (0 or 1.5mM). At the end of 96-h culture, the abundance of apoptosis-related proteins (caspase-3, caspase-9), milk-specific proteins (α-lactalbumin and β-casein), mTOR signaling proteins (mTOR, p-mTOR, PERK, p-PERK, eIF2a, P70S6K and p-P70S6K), and endoplasmic reticulum stress (ERS)-associated proteins (BiP and CHOP) in PMEC were determined. Addition of AKG dose-dependently enhanced cell viability in the absence or presence of prolactin, with optimal concentrations of AKG being at 1.0 and 1.5mM, respectively. In the presence of prolactin, addition of 1.5mM AKG: (1) decreased (P<0.05) the abundance of caspase-3 and caspase-9 by 21 and 39%; (2) enhanced (P<0.05) the phosphorylation of p-mTOR and p-P70S6K by 39 and 89%, respectively; (3) increased (P<0.05) the production of β-casein and α-lactalbumin by 16 and 20%, respectively; (4) attenuated (P<0.05) the expression of CHOP by 34% but promoted (P<0.05) the expression of BiP by 46%; (5) increased (P<0.05) the secretion of lactose by 15%, when compared to the 0mM AKG group. Rapamycin (50nM; an inhibitor of mTOR) attenuated (P<0.05) the stimulatory effect of AKG on mTOR signaling and syntheses of milk protein and lactose, while relieving (P<0.05) an inhibitory effect of AKG on expression of proteins related to ERS. Collectively, our results indicate that AKG enhances milk proteinproduction by modulating mTOR and ERS signaling pathways in PMECs.