The complement anaphylatoxin C5a induces apoptosis in adrenomedullary cells during experimental sepsis

Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis.     

Indigestible material attenuated changes in apoptosis in the fasted rat jejunal mucosa

We have previously demonstrated that fasting induced apoptosis and decreased cell proliferation in the rat intestinal mucosa. The aim was to investigate the effect of expanded polystyrene as indigestible material on apoptosis and cell proliferation in rat small intestinal mucosa during fasting. Male SD rats were divided into 3 groups. The first group was fed with chow and water ad libitum. The second group fasted for 72 hrs. The third group was fasted for 24 hrs and was fed expanded polystyrene. Intestinal apoptosis was evaluated by percent fragmented DNA assay, terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end-labeling (TUNEL) staining, and caspase-3 assay. Cell proliferation was analyzed by 5-bromo-2'-deoxyuridine (5-BrdU) uptake. Truncal vagotomy was performed to evaluate a role of the central nervous system. In the 72-hr fasted rat, mucosal height of the rat jejunum was decreased to 73% of that in rats fed ad libitum, and this decrease was partly restored to 90% in rats fed expanded polystyrene. The fragmented DNA was increased in fasted rats (28.0%) when compared with that in rats fed ad libitum (2.6%). The increase in fragmented DNA in fasted rats was recovered by feeding them expanded polystyrene (8.3%). TUNEL staining confirmed this result. The effect of polystyrene on apoptosis was decreased by truncal vagotomy. Expression of cleaved caspase-3 was increased in fasted rats, which was then decreased by feeding of expanded polystyrene. In contrast to apoptosis, feeding of expanded polystyrene had no reconstructive effect on 5-BrdU uptake in the intestinal epithelium, which was decreased by fasting to 60% of that in rats fed ad libitum. In conclusion, feeding of indigestible material partly restored the decrease in intestinal mucosal length in the fasted rats through the apoptotic pathway without any influence on BrdU uptake. Further exploration focused on the mechanism of this effect of indigestible material is required.     

Plasma membrane phospholipid asymmetry precedes DNA fragmentation in different apoptotic cell models

 Biochemical alterations occurring in many cell types during apoptosis include the loss of plasma membrane phospholipid asymmetry and nuclear DNA fragmentation. Annexin V staining detects phosphatidylserine translocation into the outer plasma membrane layer occurring during cell death, while the in situ tailing (IST or TUNEL) reaction labels the DNA strand breaks typical of apoptosis. To compare the time course of these processes we investigated methylprednisolone-induced apoptosis of rat thymocytes, topoisomerase inhibitor-induced apoptosis in the human histiocytic lymphoma cell line U937, and serum deprivation-induced apoptosis in the rat pheochromocytoma cell line, PC12. At all time points, FACS analysis and quantitative fluorescence light microscopy showed a higher proportion of annexin V-positive than IST-positive cells, with significantly different time courses in the apoptotic cell models investigated (Anova test). Results were confirmed by confocal microscopy. Our data indicate that the exposure of phosphatidylserine, a potential phagocyte recognition signal on the cell surface of apoptotic cells in vivo, precedes DNA strand breaks during apoptosis in different cell types. Accepted: 29 June 1998     

Choline deficiency induces apoptosis in primary cultures of fetal neurons.

Treatment of rats with choline during brain development results in long-lasting enhancement of spatial memory whereas choline deficiency has the opposite effect. Changes in rates of apoptosis may be responsible. We previously demonstrated that choline deficiency induced apoptosis in PC12 cells and suggested that interruption of cell cycling due to a decrease in membrane phosphatidylcholine concentration was the critical mechanism. We now examine whether choline deprivation induces apoptosis in nondividing primary neuronal cultures of fetal rat cortex and hippocampus. Choline deficiency induced widespread apoptosis in primary neuronal cells, indicating that cells do not have to be dividing to be sensitive to choline deficiency. When switched to a choline-deficient medium, both types of cells became depleted of choline, phosphocholine and phosphatidylcholine, and in primary neurons neurite outgrowth was dramatically attenuated. Primary cells could be rescued from apoptosis by treatment with phosphocholine or lysophosphatidylcholine. As described previously for PC12 cells, an increase in ceramide (Cer) was associated with choline deficiency-induced apoptosis in primary neurons. The primary neuronal culture appears to be an excellent model to explore the mechanism whereby maternal dietary choline intake modulates apoptosis in the fetal brain.     

Involvement of mitochondrial dysfunction in human islet amyloid polypeptide-induced apoptosis in INS-1E pancreatic beta cells: An effect attenuated by phycocyanin

Misfolded human islet amyloid polypeptide (hIAPP) in pancreatic islets is associated with the loss of insulin-secreting beta cells in type 2 diabetes. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. Currently, there is little information about the effect of hIAPP on mitochondrial function. In this study, we used INS-1E rat insulinoma beta cells as a model to investigate the role of mitochondria in hIAPP-induced apoptosis and the protective effects of phycocyanin (PC). We demonstrated that hIAPP induced apoptosis in INS-1E cells was associated with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (ΔΨ(m)). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS-1E cells was effectively restored by co-treatment of PC. Moreover, there was crosstalk between the extrinsic and intrinsic apoptotic pathways as demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. Taken together, we demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis. Attenuation of mitochondrial dysfunction provides a mechanism for the protective effects of PC.     

Nerve growth factor withdrawal-mediated apoptosis in naive and differentiated PC12 cells through p53/caspase-3-dependent and -independent pathways

Programmed cell death is regulated in response to a variety of stimuli, including the tumor suppressor protein p53, that can mediate cell cycle arrest through p21/Waf1 and apoptosis through the Bcl-2/Bax equilibrium and caspases. Neuronal cell apoptosis has been reported to require p53, whereas other data suggest that neuronal cell death may be independent of p53. Comparison of wild type PC12 to a temperature-sensitive PC12 cell line that depresses the normal function of p53 has permitted investigation of the importance of p53 in a variety of cell functions. This study examined the role of p53 in trophic factor withdrawal-mediated apoptosis in both na?ve and differentiated PC12 cells. Our data show that as PC12 cells differentiate they are more poised to undergo apoptosis than their undifferentiated counterparts. Survival assays with XTT (sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 h for na?ve and 36 h for differentiated cells. Apoptosis involved caspases 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53, concomitant with the delayed apoptosis. When the expression of caspase 3 was silenced with interference RNA, wild type PC12 cells revealed a morphology and biochemistry similar to PC12[p53ts] cells, indicating that caspase 3 accounts for the observed delay in apoptosis in p53 dysfunction. These results suggest that p53 is important, but not essential, in factor withdrawal-mediated apoptosis. Parallel pathways of caspase-mediated apoptosis are activated later in the absence of functional p53.     

Numb/Notch Signaling Plays an Important Role in Cerebral Ischemia-induced Apoptosis

Numb has been shown to play diverse roles in the central nervous system of adult mammals, and accumulating evidence indicates a role for Numb in apoptosis. In this study, we characterize the role of Numb in ischemia-induced apoptosis, and investigate the underlying pathway involved in this process. In vivo, exposure of pheochromocytoma (PC12) cells to glucose deprivation (GD) resulted in caspase-3-dependent apoptosis. Numb expression was upregulated by GD in a time-dependent manner, while Notch expression was down regulated. Knocking down endogenous Numb expression via siRNA protected PC12 cells from GD-induced apoptosis, whereas Numb overexpression sensitized PC12 cells to GD-induced apoptosis. In vivo, significantly increased Numb expression levels, together with activation of apoptosis, can be observed in the ischemic penumbra following cerebral ischemia. Taken together, our data show that Numb promotes ischemia-induced apoptosis. Based on these results, we conclude that inhibition of Numb could be a novel therapeutic approach for inhibiting apoptosis in the ischemic penumbra.     

Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway

Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.     

Prostaglandin E1 transported into cells blocks the apoptotic signals induced by nerve growth factor deprivation

Neuronal apoptosis in rat pheochromocytoma PC12 cells, which was confirmed by TUNEL (terminal transferase-mediated dUTP-biotin nick end-labeling) staining and detection of chromatin condensation, appeared within 8 h after nerve growth factor (NGF) deprivation. Prostaglandin (PG) E1 (10(-7)-10(6) M) reduced the incidence of apoptotic cell death in PC12 cells. The genes encoding PG transporter specific to prostaglandins such as PGE2 or PGF2alpha were expressed in the cell lines as shown by RT-PCR. Bromcresol green, an inhibitor of PG transporter, reversed the antiapoptotic effect of PGE1. Moreover, treatment of PC12 cells with an antisense oligonucleotide corresponding to PG transporter cDNA also blocked the inhibitory effects of PGE1 on apoptotic cell death. In addition, PGE1 counteracted the increased activities of stress-activated protein kinase/cJun N-terminal kinase within 1-2 h after NGF deprivation in PC12 cells. These results indicated that the antiapoptotic effect of PGE1 in NGF-deprived PC12 cells was achieved by inhibitory signals following uptake into neurons through the PG transporter.     

miR-96-5p靶向FOXO1调节脑缺血再灌注损伤机制

摘要 目的：探究miR-96-5p在脑缺血再灌注损伤（CIRI）中的作用及机制。方法：通过qRT-PCR检测36例确诊的缺血性脑卒中患者（IS患者组）和30例健康体检者（健康组）的血清miR-96-5p水平。将PC12细胞分为5组：对照组、NC-ag组、miR-96-5p-ag组、NC-an组、miR-96-5p-an组。采用Lipofectamine 2000对PC12细胞进行转染,通过qRT-PCR验证转染效率。将PC12细胞分为6组：对照组、氧糖剥夺/复氧复糖（OGD/R）组、OGD/R+NC-ag组、OGD/R+miR-ag组、OGD/R+NC-an组、OGD/R+miR-an组。根据分组对PC12细胞进行OGD/R处理和转染。通过MTT法检测PC12细胞活力,TUNEL法检测PC12细胞凋亡。采用改良Longa法建立大鼠CIRI模型,然后将大鼠分为假手术组、CIRI组、CIRI+NC-an组和CIRI+miR-an组。假手术组和CIRI组大鼠尾静脉注射生理盐水,CIRI+NC-an组和CIRI+miR-an组大鼠分别尾静脉注射NC-antagomir和miR-96-5p-antagomir。然后检测各组大鼠的神经功能评分、脑梗死体积和脑组织细胞凋亡情况。按照试剂盒说明书测定PC12细胞和大鼠脑组织中MDA、SOD和GSH-Px的含量。通过qRT-PCR检测PC12细胞和大鼠脑组织miR-96-5p和Forkhead box O1（FOXO1） mRNA水平,通过Western blot检测FOXO1、Ac-FOXO1、Bax和Bcl-2的蛋白表达水平。结果：与Health组比较,IS组患者的血清miR-96-5p水平显著升高（P<0.001）。与对照组和NC-ag组比较,miR-96-5p-ag组的miR-96-5p水平升高,FOXO1的mRNA和蛋白表达水平均降低,FOXO1的乙酰化水平升高（P<0.05）。与对照组和NC-an组比较,miR-96-5p-an组的miR-96-5p水平降低,FOXO1的mRNA和蛋白表达水平均升高,FOXO1的乙酰化水平降低（P<0.05）。与OGD/R组和OGD/R+NC-an组比较,OGD/R+miR-an组的相对细胞活力升高,TUNEL阳性率降低,Bax的蛋白相对表达量降低,Bcl-2的蛋白相对表达量升高,MDA水平降低,SOD和GSH-Px水平升高,miR-96-5p水平降低,FOXO1的mRNA和蛋白表达水平升高,FOXO1的乙酰化水平降低（P<0.05）。与CIRI组和CIRI+NC-an组比较,CIRI+miR-an组大鼠的神经功能评分和脑梗死体积降低,TUNEL阳性率降低,Bax的蛋白相对表达量降低,Bcl-2的蛋白相对表达量升高,MDA水平降低,SOD和GSH-Px水平升高,miR-96-5p水平降低,FOXO1的mRNA和蛋白表达水平升高,FOXO1的乙酰化水平降低（P<0.05）。结论：miR-96-5p在CIRI发生过程中表达上调,而下调miR-96-5p表达可能通过负调控FOXO1以减轻CIRI程度。     

Formation of retinoic acid from retinol in the rat

1. The formation in vivo of retinoic acid from microgram quantities of intrajugularly administered [15-(14)C]retinol was demonstrated in the rat. 2. Endogenously formed retinoic acid (about 0.1mug./rat) was found in liver, and to a much smaller extent in intestine, 12hr. after retinol administration. 3. Excretion of some of the endogenously formed retinoic acid occurred in the bile of bile-duct-cannulated rats. 4. Excretion of unaltered retinoic acid in the urine of intact rats did not occur even after the intrajugular administration of preformed retinoic acid.     

Intracerebroventricular administration of leptin-induced apoptosis in the rat small intestinal mucosa

The localization of leptin and leptin receptors in the stomach and small intestine has been reported. Their function is still unknown, although leptin is a hormone that regulates appetite and fat-related metabolism. The small intestine is one of the important organs for regulating metabolism. The purpose of the present study was to investigate whether leptin regulates apoptosis in the small intestinal mucosa. Intestinal apoptosis was evaluated by percent fragmented DNA, electrophoresis, TUNEL staining, and western blotting analysis of caspase-3. Mucosal apoptosis in the rat jejunum and ileum was evaluated at 0, 3, 6, 12, and 24 hrs after injection. Rats were tested after ad libitum feeding and 24-hr fasting to exclude the anorectic effect of leptin. Leptin was injected intraperitoneally (ip) at a dose of 200 microg/rat and infused into the rat third cerebroventricle (icv) at a dose of 8 microg/rat. Leptin at a dose of 8 microg/rat significantly induced intestinal apoptosis in the small intestine at 3 and 6 hrs after icv administration in both ad libitum feeding and 24-hr fasted rats. This increase in apoptosis was not attenuated by vagotomy. Intestinal apoptosis increased 12 and 24 hrs after ip injection of leptin at a dose of 200 microg/rat. The peak of the increase in apoptosis in icv rats appeared earlier than that in ip rats. Leptin induced jejunal and ileal mucosal apoptosis in the rat, indicating that leptin might control intestinal function through the regulation of intestinal apoptosis.     

Arachidonic acid release by cPLA2 may be causally related to NO-induced apoptosis in vascular smooth muscle cells

Apoptosis has been shown to occur in vascular smooth muscle cells during the development of atherosclerosis. In order to investigate the possible role of arachidonic acid during apoptosis in vascular smooth muscle, we induced apoptosis in cultured rat aortal smooth muscle cells (SMCs) by treatment with either UV (ultraviolet) radiation, tumor necrosis factor-alpha (TNF-alpha) or NO donor drugs (sodium nitroprusside, or S-nitroso-N-acetyl-D-penicillamine, SNAP). Apoptosis was detected by either DNA fragmentation analysis or by TUNEL analysis. UV radiation, TNF-alpha and NO were observed to stimulate apoptosis in the cells as well as to stimulate arachidonate release from the cells. NO also increased levels of cPLA2 in the cells, which is an enzyme that is frequently activated in cells that release arachidonate. These agents stimulated arachidonate release somewhat earlier than they stimulated apoptosis in the cells. The inhibition of cPLA2 by arachidonyl trifluoromethyl ketone (AACOCF3) also led to the inhibition of arachidonate release from the cells as well as the inhibition of nitroprusside stimulated apoptosis. Arachidonic acid itself could induce apoptosis in the cultured cells. These observations provide evidence that arachidonate may be involved in apoptosis in vascular smooth muscle.     

Regulated expression of inhibitor of apoptosis protein 3 in the rat corpus luteum

We sought to investigate the role inhibitor of apoptosis proteins (IAPs) play in the life cycle of the corpus luteum (CL) of the rat. We isolated two clones with amino acid homology to rat IAP2 (BIRC 3) and three to rat IAP3 (rIAP3; BIRC 4). The expression of rIAP3 mRNA was examined in the rat CL during and after pregnancy, in Day 8 pregnant rats after 24-h treatment of gonadotropin-releasing hormone-agonist (GnRH-Ag), and in a CL organ culture model of spontaneous apoptosis in the absence of tropic support with and without superoxide dismutase. We used real-time RT-PCR to quantitate rIAP3 mRNA expression. Interestingly, a significant reduction in rIAP3 levels was seen at the time of CL regression in the course of natural pregnancy and the GnRH-Ag model. Surprisingly, rIAP3 mRNA levels in the CL organ culture model of spontaneous apoptosis failed to show significant changes, although TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) reaction showed 30%-40% of the cells undergoing DNA fragmentation after 2 h in culture. In situ hybridization revealed that rIAP3 expression was localized to the cytoplasm of luteal and granulosa cells. These data clearly demonstrate both the presence of IAPs in the rat CL and the regulation of rIAP3 during in vivo apoptotic cell death, indicating a role for IAPs in the maintenance of CL function and demise.     

The Role of HSPA12B in Regulating Neuronal Apoptosis

Heat shock protein A12B (HSPA12B) is the newest member of a recently defined subfamily of proteins distantly related to the 70-kDa family of heat shock proteins (HSP70) family. HSP70s play a crucial role in protecting cells, tissues, organs and animals from various noxious conditions. Here we studied the dynamic expression changes and localization of HSPA12B after middle cerebral artery occlusion (MCAO) with reperfusion induced ischemic insult processes in adult rats. Apoptosis, as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, was also increased in the peri-ischemic cortex compared to non-ischemic hemisphere. The expression of HSPA12B was strongly induced in the ischemic hemisphere of MCAO reperfusion rats in vivo. In vitro studies indicated that the up-regulation of HSPA12B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knockdown of HSPA12B in cultured differentiated PC12 cells by siRNA showed that HSPA12B inhibited the expression of active caspase-3. Collectively, these results suggested that HSPA12B may be required for protecting neurons from ischemic insults.     

Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.