Vascular endothelial growth factor and nitric oxide in rat liver regeneration

In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65+/-10.70; PH-Control: 88.13+/-1.60(); Sh-SNP: 90.35+/-3.11(); PH-SNP: 119.5+/-12.10()(#); Sh-AG: 33.27+/-5.23, PH-AG: 36.80+/-3.40(#)) (p<0.05 vs Sh-Control; (#)p<0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats.     

硝普钠促肺成纤维细胞增殖和凋亡的作用

用MTT测定、琼脂糖凝胶电泳和流式细胞术等方法,观察了一氧化氮供体硝普钠（sodium nitroprusside,SNP）对体外培养的肺成纤维细胞增殖和凋亡以及Bcl-2、Bax和p53蛋白含量的影响。结果发现：MTT吸光度、细胞数和增殖指数（proliferation index,PI）均较对照增加;凋亡细胞数也增加,但尚不足以出现明显的“梯形”凋亡电泳条带;同时,细胞内Bcl-2蛋白下调和Bax蛋白上调;而细胞内p53蛋白含量无明显变化。结果表明,外源性NO有增强肺成纤维细胞增殖和凋亡的双重作用,但以促细胞增殖为主;此作用的分子机制与Bcl-2蛋白下调和Bax蛋白上调有关。     

Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis

This study examined the role of nitric oxide (NO) in cytokine-induced apoptosis in adult cardiac fibroblasts (CFbs). In cultured adult rat CFbs, IL-1beta (5 ng/ml), but not interferon-gamma (10 ng/ml) or tumor necrosis factor-alpha (10 ng/ml), induced inducible NO synthase (iNOS) expression and NO production that was associated with an increase in caspase-3 activity and apoptotic cell death. Apoptotic frequency was reduced by the iNOS inhibitor S-methylisothiourea (3 x 10(-5) M). Apoptosis in response to IL-1beta was attenuated by the caspase-3 inhibitor [Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DVED-FMK)] but not by inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). IL-1beta-induced CFb apoptosis was associated with an increase in p53 and Bax protein expression with no changes in Bcl-2 or Bcl-x(L). Nuclear condensation and fragmentation occurred when isolated nuclei were exposed to an NO donor [Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate) 10(-5) M], an effect that was not blocked by the peroxynitrite scavenger Mn(III)tetrakis(4-benzoic acid) porphyrin chloride. Moreover, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride attenuated but did not eliminate IL-1beta-induced CFb apoptosis, indicating that the proapoptotic effect of NO can occur independently of its conversion to peroxynitrite. Our results demonstrate that IL-1beta-induced iNOS expression can trigger NO-dependent apoptosis in adult CFbs, which appears to result from DNA damage and may be mediated by a p53-dependent apoptotic pathway.     

Nitric oxide-mediated inhibition of follicular apoptosis is associated with HSP70 induction and Bax suppression

Nitric oxide (NO) has recently emerged as a potential regulator of follicular development because of its involvement in the regulation of several physiological functions of the ovary. NO influences apoptotic cell death of follicular cells as a follicle survival factor. The present study was conducted (1) to investigate the mechanism involved in the protective effect of NO on spontaneously induced follicular apoptosis in serum-free condition and (2) to determine the role of NO on the expression of mRNAs and proteins for HSP70 and Bax. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 hr in serum-free medium with or without sodium nitroprusside (SNP), a NO generator. Granulosa cells within follicles incubated in medium alone for 24 hr exhibited extensive apoptosis. Treatment of SNP in the culture medium blocked this onset of apoptosis. Both mRNA and protein levels of HSP70 were highly increased with SNP than those of control group. On the contrary, those of Bax were suppressed with SNP treatment. Results of the present study suggest that NO prevents rat preovulatory follicular apoptosis in vitro by stimulating HSP70 and suppressing Bax expression.     

LPS/IFN-γ-Induced RAW 264.7 Apoptosis is Regulated by Both Nitric Oxide–Dependent and –Independent Pathways Involving JNK and the Bcl-2 Family

Lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) stimulate macrophages to produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) and activate stress signaling cascades including the c-jun-N-terminal kinase (JNK) pathway. These events trigger an apoptotic cascade that ultimately results in death. Since JNK regulates pro-apoptotic and anti-apoptotic Bcl-2 family members, the role of NO in LPS/IFN-γ-induced activation of JNK and its effects on the Bcl-2 family was examined in RAW 264.7 macrophage-like cells. Inhibition of JNK by siRNA verified a role for JNK in LPS/IFN-γ-induced apoptosis. Suppression of NO production by a pharmacologic agent, i.e. iNOS inhibitor L-NIL, altered the kinetics of JNK activation by LPS/IFN-γ. Examination of mitochondrial and nuclear compartments of RAW 264.7 cells demonstrated NO-dependent activation of mitochondrial JNK by LPS/IFN-γ, but NO-independent, cytokine-induced phosphorylation of Bim. NO did not affect phosphorylation, but did inhibit Bax phosphorylation. These results suggest a novel mechanism of LPS/IFN-γ-induced apoptosis in macrophages involving NO-independent phosphorylation of Bim and NO-dependent dephosphorylation of Bax.     

Regulation of apoptosis by somatostatin and substance P in peritoneal macrophages

Recent studies have shown that somatostatin (SOM) inhibits interleukin 6 (IL-6) and interferon gamma (IFNgamma) production by lymphocytes and peritoneal macrophages, whereas substance P (SP) enhances these cytokines production. To define the mechanism of the cytokine production enhancements and inhibitions by SOM and SP, we examined the expression of apoptosis modulator, p53, Bcl-2, Bax, inducible nitric oxide synthase (iNOS), Fas, caspase-8 and nitric oxide (NO) in thioglycolate-elicited peritoneal macrophages. SOM caused up-regulation of p53, Bcl-2, Fas and caspase-8 activities, and down-regulation of iNOS expression and NO production. On the other hand, SP slightly induces p53 and highly induces Bcl-2, iNOS expression and NO production. These data suggest that apoptosis by SOM may occur by a Bax- and NO-independent p53 accumulation, and through Fas and caspase-8 activation pathways, and that the inducible expression of Bcl-2 and NO production by SP may contribute to prevent the signals of apoptosis by Bax, and via Fas and caspase-8 activation.     

Protein SV-IV promotes nitric oxide production not associated with apoptosis in murine macrophages

SV-IV (seminal vesicle protein no. 4) is a potent immunomodulatory and anti-inflammatory secretory protein (Mr 9758) produced in large amounts by the rat seminal vesicle epithelium. Here we show that this protein possesses the ability to upregulate in J774 macrophages the expression of the gene coding for the inducible nitric oxide synthase (iNOS). The increase in NO production consequent on the marked enhancement of iNOS activity was not associated with apoptotic damage of the SV-IV-treated cells. In the same experimental model, however, LPS induced upregulation of iNOS coupled with an increase in NO production and marked apoptotic death. Differences in the ability of SV-IV and LPS to control the life/ death signal balance in target cells via trans-membrane activation of apoptotic (mediated by TNF-alpha and NO/iNOS system) and anti-apoptotic (mediated by bcl-2, c-myc, etc.) pathways are suggested to be the basis of the apoptotic fate of the experimentally treated cells. In addition, considering the important role played by NO in the process of mammalian reproduction, SV-IV may be involved in the fine tuning of NO concentration in the female genital tract mucosa via an SV-IV-mediated control of iNOS gene expression in local macrophages.     

Penehyclidine hydrochloride attenuates LPS-induced iNOS production by inhibiting p38 MAPK activation in endothelial cells

The aim of this study was to investigate the inhibitory effect of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production in human endothelial cell. Cultured endothelial cells were pretreated with PHC, followed by LPS treatment. NO activity were determined. iNOS expression and p38 mitogen-activated protein kinase (p38 MAPK) protein expression were measured by Western blot analysis. LPS treatment significantly induced p38 MAPK activation, iNOS expression, and NO production, which could be attenuated by 2 μg/ml PHC pretreatment. Furthermore, our study showed LPS-induced NO production and iNOS expression were suppressed by p38 MAPK inhibitor SB203580 pretreatment. We concluded that PHC attenuates NO production and iNOS expression by suppressing the activation of p38 MAPK pathway, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced endothelial cell injury.     

Modulation of balance between apoptosis and proliferation by lipid peroxidation (LPO) during rat liver regeneration

BACKGROUND: This work aims to investigate the role of lipid peroxidation (LPO) at early stages of liver regeneration and to evaluate the balance between apoptosis and cell proliferation during this process. METHODS: Sham and partial hepatectomized (PH) male Wistar rats were randomized in seven groups: Control (untreated), E-Control (injected with vitamin E-vehicle), C-Control (injected with vitamin C-vehicle), E1 (vitamin E 100 mg/kg body weight), E2 (vitamin E 600 mg/kg body weight), C1 (vitamin C 30 mg/kg body weight), C2 (vitamin C 100 mg/kg body weight). RESULTS: Vitamin treatments attenuated the increase of LPO level observed in total homogenate and microsomes at 3 and 5 hr after PH. Both antioxidant vitamins attenuated the increase in Bax pro-apoptotic protein and augmented Bcl-xL antiapoptotic protein levels (35%) at 3 and 5 hr post-PH; Bcl-xL/Bax ratio was, therefore, increased. A direct linear relationship between LPO levels and Bax mitochondrial protein levels was seen. Vitamin-treatments diminished the apoptosis index with respect to PH-Control values, so that this parameter showed a linear relationship with LPO levels. At 24 hr after PH, the vitamin treatments increased the peak of [(3) H]-thymidine incorporation into DNA and the proliferative index (PI), measured as PCNA expression; an inverse relationship between PI and LPO levels could be demonstrated. CONCLUSION: Our data show that the diminution of LPO levels by vitamin-treatment post-PH produces both an attenuation of cellular apoptosis and a marked increase in the proliferation process, suggesting that the modulation of LPO has a role in liver regeneration process.     

P53-dependent miRNAs mediate nitric oxide-induced apoptosis in colonic carcinogenesis

Both miRNAs and nitric oxide (NO) play important roles in colonic inflammation and tumorigenesis. Resistance of colonic epithelial cells to apoptosis may contribute to tumor development. We hypothesized that some miRNAs could increase the resistance of colonic cancer cells to nitric oxide-induced apoptotic cell death. Here we show that NO induced apoptosis and stimulated expression of some miRNAs. Loss of p53 not only blocked NO-induced apoptosis but also dramatically inhibited the expression of NO-related miRNAs, such as miR-34, miR-203, and miR-1301. In addition, blockage of p53-dependent miRNAs significantly reduced NO-induced apoptosis. Furthermore, forced expression of these miRNAs rendered HT-29 cells, which are resistant to apoptosis with mutant p53, more sensitive to NO-induced apoptotic cell death. Most interestingly, in a colitis-associated colon cancer mouse model, the level of miRNAs dropped significantly, accompanied by downregulation of p21, which is a key target gene of p53. In human colorectal cancer samples, the expression of miR-34 significantly correlated with the level of inducible nitric oxide synthase (iNOS). We contend that increased NO production may select cells with low levels of p53-dependent miRNAs which contributes to human colonic carcinogenesis and tumor progression.     

Functional proteomics of resveratrol-induced colon cancer cell apoptosis: caspase-6-mediated cleavage of lamin A is a major signaling loop

The study investigated the molecular basis of resveratrol (RSV)-evoked apoptosis in four (Bax+/-, Bax-/-, p53+/+, and p53-/-) HCT116 colon cancer cell lines. RSV induced apoptosis in all the cells in a dose-dependent manner; however, Bax+/- and p53+/+ cells were more susceptible than their knockout counterparts (Bax-/- and p53-/-, respectively). Using Bax+/- cells as a model, proteomic analysis revealed four RSV-responsive events: fragmentation of lamin A/C protein; increase in concentration of a more basic isoelectric variant of the ribosomal protein P0; and decrease in concentration of dUTPase as well as stathmin 1. Lamin A cleavage in response to RSV treatment was confirmed using Western blot analysis. Caspase-6 was activated, which was evidenced by cleavage and accumulation in active form of caspase-6 as well as upregulation of the protease activity. RSV-elicited lamin A cleavage and apoptosis were entirely abrogated by the peptide inhibitors of caspase-6. Likewise, partial knockdown of caspase-6 expression using small interfering RNA resulted in significant inhibition of RSV-elicited lamin A cleavage and apoptosis. Furthermore, the lower apoptosis sensitivity of the knockout cells (Bax-/- and p53-/-) correlated with the relatively reduced processing of caspase-6 and lamin A cleavage. Taken together, these data highlight the critical role of caspase-6 and its cleavage of lamin A in apoptotic signaling triggered by RSV in the colon carcinoma cells, which can be activated in the absence of Bax or p53.     

Nitric oxide-induced cell death of cerebrocortical murine astrocytes is mediated through p53- and Bax-dependent pathways

We have investigated the mechanism by which nitric oxide (NO) induces the death of mouse astrocytes. We show that NO (from donor diethylenetriamine-NO adduct) induces death with several features of apoptosis, including chromatin condensation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, Bax translocation to the mitochondria and cytochrome c release, but no caspase activation or nuclear fragmentation is observed. Nitric oxide also elevates p53 expression, causing a concomitant increase in p53 serine 18 phosphorylation and p53 translocation from the cytoplasm to the nucleus. Activation of Bax and p53 is important for NO-induced apoptosis-like cell death because Bax- or p53-deficient astrocytes are much more resistant than wild-type cells to the same NO treatment. We further demonstrate that LY294002-sensitive kinases are responsible for controlling serine 18 phosphorylation of p53, thereby regulating the pro-apoptotic activity of p53 in astrocytes. While apoptosis is suppressed in the presence of LY294002, however, death by necrosis is increased, suggesting that LY294002-sensitive kinases additionally suppress a latent necrotic response to NO. We conclude that NO-induced death in astrocytes is mediated by p53- and Bax-dependent mechanisms, although full manifestation of apoptosis is aborted by concomitant inhibition of caspase activation. More generally, our data suggest that apoptotic mediators should be evaluated as the cause of cell death even in cases where a full apoptotic phenotype is lacking.     

UVC radiation-induced effect on human primary thyroid cell proliferation and HLA-DR expression

The aim of this study was to examine how UVC irradiation will affect normal human thyroid cell proliferation and HLA-DR expression. Primary human thyroid cells were exposed to UVC (254 nm wavelength) irradiation. In some experiments 0.5 mM buthionine sulfoximine (BSO) was added. Apoptosis was detected measuring annexin V, proteins involved in apoptotic process (p53, Bax, Bcl-2, caspase 3, and 9) by immunoblot analysis and HLA-DR expression by FACS. UVC induced a cell cycle arrest in G0/G1 phase in the first 24 h, accumulation of cells in the S phase 72 h after treatment, and an increase of apoptotic cells. BSO pretreatment showed an earlier appearance and a higher percentage of apoptosis. p53, caspase 3 and 9 were increased, while Bax and Bcl-2 were decreased. We also observed a transient significant increase in HLA-DR expression. UVC inhibited cell proliferation and induced apoptosis in normal human primary thyroid cells. An inhibitor of glutathione synthesis induced an earlier appearance and higher percentage of apoptosis suggesting that oxidative stress may play a role. Apoptotis involved components of the intrinsic mitochondrial pathway. A transient increase in HLA-DR expression after UVC irradiation could play a role in inducing AITD.     

Nitric oxide augments oridonin-induced efferocytosis by human histocytic lymphoma U937 cells via autophagy and the NF-κB-COX-2-IL-1β pathway

Abstract We previously demonstrated that oridonin-induced autophagy enhanced efferocytosis (phagocytosis of apoptotic cells) by macrophage-like U937 cells through activation of the inflammatory pathways. In this study, exposure of U937 cells to 2.5 μM oridonin caused up-regulation of inducible nitric oxide synthase (iNOS) expression and continuous endogenous generation of nitric oxide (NO), which was reversed by pre-treatment with the inhibitors of nitric oxide synthase 1400 W (dihydrochloride) or L-NAME (hydrochloride). NO donor sodium nitroprusside (SNP) and efferocytosis irritant lipopolysaccharide (LPS) could also exert NO generation and iNOS expression. Moreover, oridonin-induced stimulation of efferocytosis was significantly suppressed by 1400 W or L-NAME. In addition, 1400 W or L-NAME impaired oridonin-induced autophagy. Inhibition of autophagy with 3-methyladenine (3MA) or Beclin-1 siRNA attenuated the uptake of apoptotic cells with a slight increase in the production of NO. The pro-inflammatory cytokine interleukin-1β (IL-1β) has been reported to be involved in oridonin-induced efferocytosis in U937 cells and interact with NO to contribute to inflammatory responses. 1400 W or L-NAME blocked the secretion of IL-1β and the activation of NF-κB and COX-2. Provision of SNP or LPS in place of oridonin resulted in the similar enhancement of efferocytosis, autophagy, the release of IL-1β and the expression of signal protein. NO augmented the oridonin-induced efferocytosis by mediating autophagy and activating the NF-κB-COX-2-IL-1β pathway. Inhibition of NF-κB or COX-2 in turn decreased the production of NO and the expression of iNOS. There exists a positive feedback loop between NO generation and NF-κB-COX-2-IL-1β pathway.     

Neuroprotective and Anti-Apoptotic Propensity of Bacopa monniera Extract Against Sodium Nitroprusside Induced Activation of iNOS,Heat Shock Proteins and Apoptotic Markers in PC12 Cells

Sodium nitroprusside (SNP) is a widely used nitric oxide (NO) donor, known to exert nitrative stress by up-regulation of inducible nitric oxide synthase (iNOS). Nω-nitro-l-arginine-methyl esther (L-NAME) is a NO inhibitor, which inhibits iNOS expression, is used as positive control. The present study was designed to assess neuroprotective propensity of Bacopa monniera extract (BME) in SNP-induced neuronal damage and oxido-nitrative stress in PC12 cells via modulation of iNOS, heat shock proteins and apoptotic markers. Our results elucidate that pre-treatment of PC12 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP (200 μM) as evidenced by MTT and LDH assays. BME pre-treatment inhibited NO generation by down regulating iNOS expression. BME replenished the depleted antioxidant status induced by SNP treatment. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic protein biomarkers such as Bax, Bcl-2, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. Q-PCR results further elucidated up-regulation of neuronal cell stress markers like HO-1 and iNOS and down-regulation of BDNF upon SNP exposure was attenuated by BME pre-treatment. By considering all these findings, we report that BME protects PC12 cells against SNP-induced toxicity via its free radical scavenging and neuroprotective mechanism.     

Hck Promotes Neuronal Apoptosis Following Intracerebral Hemorrhage

The hematopoietic cell kinase (Hck) is a member of the Src family protein kinases which regulates many signal transduction pathways including cell growth, proliferation, differentiation, migration, and apoptosis. However, the expression and function of Hck after intracerebral hemorrhage (ICH) are unknown. Western blot, immunohistochemistry, and immunofluorescence showed that Hck was obviously up-regulation in neurons adjacent to the hematoma after ICH. In addition, the temporary raise of Hck expression was paralleled with the expression of p53, Bax, and active caspase-3, suggesting that Hck was involved in neuronal apoptosis. Hck siRNA dramatically decrease hemin-induced expression of p53, Bax, and active caspase-3 as well as the amount of apoptotic SH-SY5Y cells in vitro. Furthermore, Hck interacted with p53. Hence, Hck might promote neuronal apoptosis via p53 signaling pathway after ICH.     

The Effect of Selenium on the Cd-Induced Apoptosis via NO-Mediated Mitochondrial Apoptosis Pathway in Chicken Liver

Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na₂SeO₃, 2 mg/kg), Cd (CdCl₂, 150 mg/kg), or Se + Cd (150 mg/kg of CdCl₂ and 2 mg/kg of Na₂SeO₃) for 90 days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.