Apoptosis inhibition plays a greater role than necrosis inhibition in decreasing bacterial translocation in experimental intestinal transplantation

BACKGROUND: During small-bowel transplantation, necrosis and apoptosis are involved in the destruction of intestinal epithelial cells. This study was conducted to assess which mode of cell death plays a greater role as a trigger of the bacterial translocation (BT) associated with intestinal transplantation. METHODS: The following experimental groups were studied: sham, Tx (intestinal transplantation), Tx + poly (ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB), and Tx + caspase inhibitor Z-VAD-fmk. Histological analysis, caspase-3 activity, DNA fragmentation, and BT were measured in tissue samples after transplantation. RESULTS: During intestinal transplantation, apoptosis and necrosis both increased, showing graft injury and high levels of BT. Rats treated with 3-AB showed histological protection of the transplanted graft and a tendency toward lower BT despite the existence of high apoptosis levels. The rats treated with Z-VAD showed histological protection of the transplanted graft and decreased levels of caspase-3 and DNA fragmentation. The Tx + Z-VAD group showed the lowest levels of BT in all tissues. CONCLUSIONS: In small intestinal transplantation, both apoptosis and cell necrosis give rise to histological injury and BT. Apoptosis inhibition and necrosis inhibition treatments protect intestinal grafts from ischemia/reperfusion injury; Z-VAD supplementation has a greater effect on BT prevention than does administration of the PARS inhibitor 3-AB.     

Protective effect of caspase inhibitor on intestinal integrity in experimental severe acute pancreatitis

BACKGROUND AND AIM: Endotoxin/bacterial translocation (E/BT) plays an important role in systemic complications in severe acute pancreatitis (SAP). The breakdown of intestinal integrity is considered to be implicated in E/BT. We recently demonstrated that accelerated apoptosis of intestinal mucosa may have a part in E/BT. On the other hand, caspase is believed to play a central role in apoptosis. The aim of this study was to investigate the efficacy of caspase inhibitor on intestinal integrity and E/BT in SAP. METHODS: SAP was induced by retrograde injection of 3% sodium deoxycholate into the biliopancreatic ducts in rats. At the same time, polycaspase inhibitor (Z-VAD-fmk) was administered intraperitoneally. Caspase activation in the intestine was evaluated by immunohistochemical staining and Western blotting. Apoptosis of intestinal mucosa was detected by TdT-mediated dUTP-biotin nick end labeling staining and DNA fragmentation enzyme-linked immunosolvent assay. Intestinal permeability was assayed ex vivo by measuring the leaked amount of FITC-dextran. Blood endotoxin level, bacterial culture of the ascites and mesenteric lymph nodes, and 24-h mortality rate were evaluated. RESULTS: Immunoreactivities for activated caspase-10, -9, and -3 were increased 2 h after induction of SAP. Apoptosis and permeability of ileum were significantly increased 6 h after induction of SAP. Caspase inhibitor significantly improved the increasing apoptosis and permeability. It did not prevent the bacterial translocation but improved the disorder of intestinal mucosa and elevation of blood endotoxin 18 h after induction of SAP. Moreover, caspase treatment significantly improved the 24-h mortality rate. Z-VAD-fmk indeed inhibited the caspase-3 activation in intestinal mucosa of SAP. CONCLUSIONS: These results suggest that caspase activation has a key role in the accelerated apoptosis of intestinal epithelial cells in SAP and that breakdown of intestinal mucosa via accelerated apoptosis causes the increase in intestinal permeability following endotoxin translocation in SAP.     

Role of nitric oxide in apoptosis and cell necrosis for intestinal transplantation

INTRODUCTION: Nitric oxide (NO) is an important mediator of both physiological and pathological responses. Its dual role in the ischemia-reperfusion syndrome is still a matter of controversy. The aim of this study was to analyze the effect of NO on apoptosis and cell necrosis associated with heterotopic small bowel transplant. METHODS: Sprague-Dawley rats underwent heterotopic small bowel transplants with 3 hours of cold ischemia and 5 hours of reperfusion. Animals were assigned to the following study groups: Sham; bowel transplant (Trp); bowel transplant + NO donor (Trp + NONOS); bowel transplant + NO synthesis inhibitor (Trp + L-NAME). We studied histological changes and bacterial translocation in mesenteric nodes, liver and spleen as parameters of cell necrosis and caspase-3 activity as a parameter of apoptosis. RESULTS: Histological changes and bacterial translocation showed that exogenous administration of NO protected the transplant. Simple bowel transplant, with or without inhibition of NO synthesis, did not display this protective effect. Significantly greater levels of apoptosis were observe in grafts among the group administered NO at pharmacological doses. CONCLUSIONS: In experimental bowel transplantation rats administered exogenous NO show less necrosis but at the same time stimulation of apoptosis.     

Caspase 3 inhibition improves survival and reduces early graft injury after ischemia and reperfusion in rat liver transplantation

BACKGROUND: Apoptosis plays a crucial role after ischemia-reperfusion in organ transplantation. It is executed by caspases and influenced by the rheostat of pro- and anti-apoptotic proteins of the bcl-2 family. This study investigated the effect of specific inhibition of caspases 3 and 7 on graft function, survival, and hepatic bcl-2 levels after liver transplantation. METHODS: Lewis rats underwent syngeneic orthotopic liver transplantation after 16 hr of cold graft storage (in University of Wisconsin solution). Livers of donor animals treated with D(OMe)E(OMe)VD(OMe)-fluoromethylketone (specific inhibitor of apoptosis executor caspases 3 and 7), and appropriate control groups, were investigated. Early graft injury was quantified by measurement of bile flow and determination of microvascular graft injury by using in vivo fluorescence microscopy. Apoptosis and its regulation were examined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining and Western blot analysis of cell death effectors, respectively. RESULTS: After specific in vivo caspase inhibition, Western blot analysis revealed inhibition of caspase-induced cleavage of poly-ADP-ribose-polymerase. Inhibition of caspases 3 and 7 resulted in a significantly decreased number of apoptotic endothelial cells and improved microvascular perfusion. A cell protective effect was also suggested by an increase of bcl-2 levels at 7 days. Most important, specific caspase blockade resulted in improved rat survival after liver transplantation. CONCLUSION: Specific inhibition of apoptosis executor caspases effectively reduces graft ischemia-reperfusion injury and improves survival in liver transplantation. Better tissue preservation after caspase inhibition correlates with reduced apoptosis execution, improved microvascular perfusion, and bcl-2 up-regulation. Therefore, specific caspase inhibition represents a promising regimen for clinical use in liver transplantation.     

Protection against concanavalin A-induced hepatocyte apoptosis by molsidomine is time-dependent

BACKGROUND: Viral hepatitis and autoimmune liver diseases cause hepatocyte apoptosis. Concanavalin A (Con A)-induced hepatitis resembles human viral hepatitis and autoimmune hepatitis. The role of nitric oxide (NO) in liver injury was controversial in different liver injury models. We hypothesize both endogenous and exogenous NO protect liver against Con A-induced liver injury. Molsidomine is metabolized into SIN-1 by the liver, and SIN-1 subsequently generates NO. So, molsidomine was used as a NO donor in this study. STUDY DESIGN: To study a protective role of endogenous NO in Con A-induced liver injury, mice were pretreated with a specific inducible nitric oxide synthase (iNOS) inhibitor, L-N(6)-(1-iminoethyl)-lysine (L-NIL), before Con A challenge. To study a time-dependent protection against Con A-induced liver injury, animals were either given molsidomine, a NO donor, before or after Con A administration. Serum alanine aminotranferase (ALT) was analyzed. Liver samples were subjected to DNA fragmentation assay, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling stain, Western blot analysis, and caspase activity assays. RESULTS: Animals pretreated with L-NIL had significantly increased serum ALT levels compared with those challenged with Con A alone; but pretreatment with molsidomine dramatically decreased ALT levels in L-NIL-pretreated animals or in animals that received Con A alone. Administration of molsidomine 30 minutes before or 1, 2, and 3 hours after Con A injection significantly reduced serum ALT levels and attenuated hepatocyte apoptosis from caspase inactivation. The ALT reduction was associated with inhibition of both caspase-3 and caspase-8 activation and reduction of hepatocyte apoptosis. CONCLUSIONS: Endogenous NO plays an important protective role against Con A-induced liver injury by reducing hepatocyte apoptosis. Administration of a NO donor early after Con A injection protects the liver from injury. This is the first study demonstrating a time-dependent inhibition of liver injury induced by Con A administration.     

Early inhibition of caspase-3 activity lessens the development of graft coronary artery disease.

BACKGROUND: The role of apoptosis in the development of graft coronary artery disease (GCAD) is poorly understood. We have previously shown that early overexpression of the anti-apoptotic protein Bcl-2 lessens the development of GCAD. We hypothesized that early inhibition of apoptosis with a caspase-3 inhibitor would also lessen the development of GCAD. METHODS: Heterotopic heart transplantation was performed in 4 groups of rats. Donor hearts were pretreated with 50 microg DEVD-CHO, a cell-permeable caspase-3 inhibitor, or vehicle. Recipient animals were pretreated with 1.7 mg/kg intraperitoneal DEVD-CHO or vehicle. Animals were treated with 7.5 mg/kg/d cyclosporine for 10 days to prevent acute rejection. On post-operative day 90, the animals were sacrificed and the transplanted hearts were assessed morphometrically for evidence of GCAD. RESULTS: At 90 days, intimal proliferation was significantly higher in vehicle treated animals than in inhibitor treated animals. Moreover, the percentage of vessels with high-grade occlusion (>50%) was also lower in inhibitor treated animals. CONCLUSIONS: Early inhibition of caspase-3 activity with cell-permeable DEVD-CHO lessens the development of GCAD. Caspase-3 inhibition may be a useful strategy for prevention of GCAD in clinical transplantation.     

A possible mechanism for prevention of intestinal programmed cell death after ischemia-reperfusion injury by hepatocyte growth factor pretreatment

Background/Purpose: Intestinal ischemia-reperfusion (IR) injury produces necrosis and apoptosis resulting in tissue loss. The authors have observed previously that pretreatment with hepatocyte growth factor (HGF) attenuates enterocyte apoptosis after IR. This study investigated the effects of HGF on tissue levels of caspase-8, an apoptosis initiator, and caspase-3, an apoptosis effector, in intestinal mucosa after IR. Methods: Thirty rats underwent placement of jugular venous catheters connected to osmotic pumps; 15 rats received vehicle, and 15 rats received HGF (150 [mu ]g/kg/d). After a 48-hour infusion, 5 rats from each group underwent either 35 minutes of superior mesenteric artery occlusion alone, or ischemia followed by 2 or 6 hours of reperfusion. Mucosal protein was analyzed for caspase-8 and caspase-3 activity. DNA fragmentation was used to measure the presence of apoptosis. Statistical significance was determined using analysis of variance. Results: After 6 hours of reperfusion, caspase-3 activity was increased significantly in control animals (P [lt ] .05). In HGF-pretreated animals, caspase-8 and caspase-3 activities were significantly reduced at 6 hours compared with control animals (P [lt ] .05). Conclusion: By preventing the activation of enterocyte caspase enzymes, and, thus, reducing apoptosis, HGF may enhance preservation of the intestine after IR injury.     

Cornea graft endothelial cells undergo apoptosis by way of an alternate (caspase-independent) pathway

PURPOSE: To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process. MATERIALS AND METHODS: Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry. RESULTS: Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells. CONCLUSION: During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.     

Caspase-3-independent internucleosomal DNA fragmentation in ischemic acute kidney injury

Background/Aims: Renal tubular cell death in ischemia-reperfusion does not follow the classical apoptosis or necrosis phenotype. We characterized the morphological and biochemical features of injured tubular epithelial cells in ischemic acute kidney injury (AKI). Methods: Ischemic AKI was induced in rats by 60 min of ischemia followed by 24 h of reperfusion. Light and electron microscopic TUNEL (LM-TUNEL and EM-TUNEL), gel electrophoresis of extracted DNA, and caspase-3 involvement were examined during the development of death. Results: Damaged tubular epithelial cells with condensed and LM-TUNEL-positive (+) nuclei were prominent at 12 and 18 h after reperfusion with DNA 'ladder' pattern on gel electrophoresis. EM-TUNEL+ cells were characterized by nuclei with condensed and clumping chromatin, whereas the cytoplasm showed irreversible necrosis. The protein levels and activity of caspase-3 did not increase in kidneys after reperfusion. In addition, caspase inhibitor (ZVAD-fmk) failed to inhibit DNA fragmentation and prevent tubular epithelial cell death in ischemic AKI. Conclusion: Caspase-3-independent internucleosomal DNA fragmentation occurs in injured tubular epithelial cells undergoing irreversible necrosis in ischemic AKI. The manner of this cell death may be identical to the cell death termed apoptotic necrosis, aponecrosis, or necrapoptosis. Ischemia-reperfusion injury activates caspase-3-independent endonuclease, which in turn induces irreversible damage of tubular epithelial cells, and may contribute to the initiation and development of AKI.     

Reassessment of caspase inhibition to augment grafted dopamine neuron survival

One experimental therapy for Parkinson's disease (PD) is the transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted neurons die, many via apoptosis. Activated caspases play a key role in execution of the apoptotic pathway; therefore, exposure to caspase inhibitors may provide an effective intervention strategy for protection against apoptotic cell death. In the present study we examined the efficacy of two different caspase inhibitors, caspase-1 inhibitor Ac-YVAD-CMK and caspase-3 inhibitor Ac-DEVD-CMK, to augment mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival in culture and following implantation into the denervated striatum of rats. We report that treatment with Ac-YVAD-CMK provided partial but nonsignificant protection for TH-ir neurons against serum withdrawal in mesencephalic cultures plated at low density, while neither caspase inhibitor promoted TH-ir neuron survival in higher density cultures, simulating graft density. We demonstrate that plating procedures (full well vs. microislands) and cell density directly affect the degree of insult experienced by TH-ir neurons following serum withdrawal. This varying degree of insult directly impacts whether caspase inhibition will augment TH-ir neuron survival. Our grafting experiments demonstrate that Ac-YVAD-CMK does not augment grafted TH-ir neuron survival when added to mesencephalic cell suspensions prior to grafting or to mesencephalic reaggregates for 3 days in vitro prior to transplantation. These experiments provide further evidence of the failure of these caspase inhibitors to augment TH-ir neuron survival. Furthermore, we suggest that cell culture paradigms used to model grafting paradigms must more closely approximate the cell densities of mesencephalic grafts to effectively screen potential augmentative treatments.     

Apoptosis and allograft rejection in the absence of CD8+ T cells.

BACKGROUND: The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells. METHODS: Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant. RESULTS: Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts. CONCLUSION: These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.     

Porcine islet graft function is affected by pretreatment with a caspase-3 inhibitor

During the isolation procedure and after transplantation islets are subjected to numerous variables associated with the induction of apoptosis. The present study investigated the effect of transient pretreatment with caspase inhibitors on function and survival of transplanted pig islets. Isolated porcine islets (3000 IEQ) were incubated overnight in 200 microM of the caspase-3 inhibitor DEVD-CMK prior to transplantation into diabetic nude mice. Glucose-stimulated insulin release of pretreated islets was assessed during static incubation. DEVD-CMK successfully prevented the expression of capase-3 and DFF as demonstrated in heat-shocked pig islets. Nevertheless, transient pretreatment of freshly isolated pig islets with DEVD-CMK resulted in a significantly decreased final graft function of 50.0% (n = 16) compared to 85.7% (n = 14) in control islets (p < 0.05). Glucose-stimulated insulin release of porcine islets (n = 6) was not significantly effected by overnight culture with DEVD-CMK. Morphological assessment revealed that this caspase-3 inhibitor significantly increased the percentage of necrosis to a small, but nevertheless significant, extent in comparison to control islets (p < 0.05). The study demonstrates that short-time pretreatment with the caspase-3 inhibitor DEVD-CMK reduces the capacity of transplanted porcine islets to restore normoglycemia in diabetic nude mice.     

皮质酮诱导的大鼠Leydig细胞凋亡中caspase-3活化途径的研究

目的我们新近的研究表明，超生理剂量的皮质酮（大鼠体内的糖皮质激素）可通过激活caspase-3诱导大鼠Leydig细胞凋亡。本研究拟评价在皮质酮诱导的大鼠Leydig细胞凋亡过程中，caspase-3的激活是否有其上游的caspase-8平和 caspase-9的参与。方法采用荧光分光光度法榆测经皮质酮处理的大鼠Leydig细胞中caspase-8活性，以DNA梯状电泳条带作为评价细胞凋亡的指标，观察caspase-8抑制剂是否能够抑制细胞凋亡，采用RTPCR枪测皮质酮诱导的大鼠Leydig细胞中caspase-9的mRNA水平。结果在皮质酮诱导的大鼠Leydig细胞中出现caspase-8活性增高，以12h最为址著，升高的caspase-8的活性可被caspase-8抑制剂抑制，并导致Leydig细胞的凋亡过程被阻断。Leydig细胞中caspase-9的mRNA水平在皮质酮作用下上升，同样以12h最为显著。结论皮质酮诱导的大鼠Leydig细胞调亡与caspase-8和caspase-9有关。     

Early assessment of apoptosis in isolated islets of Langerhans

BACKGROUND: There is substantial evidence to link early graft loss after islet transplantation to isolation-induced islet cell apoptosis. Measurement of caspase 3 activity and detection of the lost cell membrane asymmetry, revealed by annexin V binding, are newly available assays that allow the analysis of early events of apoptosis. METHODS: In this study, we compared these tests with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and analysis of DNA fragmentation after gel electrophoresis in freshly isolated islets obtained from rats, before and after treatment with interleukin-1 beta, interferon gamma, and tumor necrosis factor a, cytokines known to induce islet cell damage. RESULTS: A measurable level of apoptosis was observed the day after isolation when caspase 3 activity and annexin V binding were used as assays, although no substantial DNA fragmentation was detected with TUNEL assay and DNA gel electrophoresis. Baseline caspase 3 activity was 0.8+/-0.3 U/100 islet equivalents and it increased to 1.4+/-0.45 U/100 islet equivalents 3 hr after cytokine stimulation (P<0.05 vs. unstimulated islets). The baseline level of apoptosis, as detected by annexin V binding, was 21.1%+/-5.8%, and it increased to 27.5%+/-8.1% 6 hr after addition of the cytokine cocktail (P<0.01 vs. unstimulated islets). An increase in the number of TUNEL-positive nuclei was detected 24 hr after stimulation and peaked at 48 hr. DNA laddering was also evident 24 hr after cytokine treatment. CONCLUSION: These data suggest that measurement of caspase 3 activity and annexin V binding analysis might represent reliable markers of early events of islet cell apoptosis.     

Pitavastatin prevents bacterial translocation after nonpulsatile/low-pressure blood flow in early atherosclerotic rat: inhibition of small intestine inducible nitric oxide synthase

BACKGROUND: Cardiopulmonary bypass decreases intestinal mucosal blood flow because of nonpulsatile and low-pressure blood flow resulting in bacterial translocation (BT) and atherosclerosis also has peripheral blood flow deficiency. The risk of nonpulsatile and low-pressure blood flow for atherosclerotic animals and the effect of statin administration, which has pleiotropic effects, were studied. METHODS: Wistar rats were divided into four groups: group N (normal diet), group C (high-cholesterol diet), group S (group C plus pitavastatin therapy), and group I [group C plus inducible nitric oxide (iNOS) inhibitor therapy]. First of all, vascular responses were measured. Then the rats underwent nonpulsatile/low-pressure blood flow in the intestine, and the serum peptidoglycan concentration as a parameter of BT, the small intestinal PO(2) ratio (intestinal PO(2)/PaO(2)) as a parameter of mucosal blood flow, and NO concentrations were measured before surgery (T0), at the end of 90 min of stenosis (T1), and 90 min after the release of stenosis (T2). Immunostaining for nitrotyrosine was also performed at T2. RESULTS: Group C had vascular endothelial dysfunction without histological changes, which indicated early atherosclerosis. The serum peptidoglycan concentration increased significantly at T2 only in group C. The intestinal PO(2) ratio was decreased at T1 in all the groups, and retuned to baseline at T2 in group N and group S, but not in group C or group I. Jejunal NO only in group C was significantly higher at all time points and ileal NO production at T1 and T2. There tended to be a positive stain for nitrotyrosine along the mucosal epithelium in group C. CONCLUSION: In the setting of early atherosclerosis, intestinal blood flow does not only improve after nonpulsatile/low-pressure blood flow but causes BT because of a large amount of NO from high enzymatic intestinal iNOS activity, and pitavastatin treatment can prevent BT by improving both issues.     

Role of Nitric Oxide and Peroxynitrite in Gut Barrier Failure

Bacterial translocation (BT) may be a normal physiologic process that is important for mucosal antigen sampling in the gut. However, physiologic insults such as endotoxemia, hemorrhagic shock, or necrotizing enterocolitis (NEC) may lead to pathologic BT and thus contribute to the pathogenesis of nosocomial infection. The mechanism may involve accelerated enterocyte apoptosis at the intestinal villus apex resulting, at least transiently, in a "bare area" at the villus tip where bacteria can attach and traverse the epithelium. Evidence suggests that sustained upregulation of the inducible isoform of nitric oxide synthase (NOS-2) co-localizes with enterocyte apoptosis and immunoreactivity to 3-nitrotyrosine, the footprint of peroxynitrite (ONOO-), a potent oxidant formed by the reaction of nitric oxide (NO) with superoxide. We propose that the bare area at the villus apex is caused by apoptosis of enterocytes that have migrated from the base of the crypts to the villus apex and are shed into the intestinal lumen. These bare areas, and thus the degree of BT, may be the result of an imbalance between enterocyte proliferation and apoptosis. We postulate that normal enterocyte apoptosis is mediated by the caspase cascade, whereas enterocyte proliferation and differentiation in the crypt may be regulated by tyrosine kinase-dependent signaling pathways. Both of these cellular pathways may be influenced by overproduction of NO and its metabolite ONOO-. Therefore, sustained NO production and ONOO- formation occurring in inflammatory states may differentially accelerate apoptosis in the villus apex and/or inhibit proliferation at the base of the crypts resulting in expanded extrusion zones at the villus tip and accelerated BT.     

Effects of caspase inhibition on the progression of experimental glomerulonephritis

BACKGROUND: Caspase-3 has a central role in the execution of apoptosis. In a nephrotoxic nephritis (NTN) model, we previously demonstrated an up-regulation of caspase-3 that was associated with inappropriate renal apoptosis, inflammation, tubular atrophy, and renal scarring. METHODS: We applied a pan caspase inhibitor, Boc-Asp (OMe)-fluoro-methyl-ketone (B-D-FMK), directly to rat NTN kidney using an intrarenal cannula fed from an osmotic pump. Animals were treated either for the first 7 days (acutely) to determine the effects on renal inflammation (ED-1 staining) and apoptosis (in situ end labeling of fragmented DNA), or for 28 days commencing 15 days after NTN (chronically) to observe the effects on cell death and renal fibrosis. Changes of caspase-3 and caspase-1 activity were detected by fluorometric substrate cleavage assay. Changes in caspase-3 and caspase-1, interleukin-1 beta (IL-1 beta), and collagen I, III, and IV proteins and mRNA were detected by Western blotting and Northern blotting, respectively. RESULTS: In both treated groups, caspase-3 activity was inhibited, and 17 and 24 kD active caspase-3 proteins were reduced significantly. A compensatory increase of caspase-3 mRNA occurred in the acutely treated group, but decreased in the chronically treated group (P < 0.05). Although there were no significant changes in caspase-1 activity and its active protein, the observed decrease in its precursor in the chronic group was increased by treatment (P < 0.05). Further, IL-1 beta precursor and its mRNA were significantly reduced by treatment only in the chronically treated group. Apoptosis was decreased in the glomeruli of acutely treated rats, and in the tubules and interstitium of chronically treated animals (P < 0.05). Glomerular inflammation was decreased only in the acutely treated group, whereas tubulointerstitial inflammation was lowered in both treated groups (P < 0.05). Glomerulosclerosis was reduced in both inhibitor groups, with a reduction in tubulointerstitial fibrosis and collagen I, III, and IV mRNA restricted to chronically treated animals (P < 0.05). Proteinuria was significantly decreased with caspase inhibition in both treated groups, but not serum creatinine level. CONCLUSION: This study clearly indicates that caspase inhibition reduces renal apoptosis, ameliorates inflammation and fibrosis, and improves proteinuria in experimental glomerulonephritis, which may mainly be related to changes in the caspase enzymatic system.     

N-硝基-L-精氨酸甲酯对大鼠隐睾生殖细胞凋亡的保护作用

目的探讨一氧化氮合酶(NOS)抑制剂N-硝基-L-精氨酸甲酯(L-NAME)对大鼠隐睾生殖细胞凋亡的保护作用.方法用22 d SD雄性大鼠复制单侧隐睾模型.实验分假手术组、隐睾组、隐睾+L-NAME组[术后腹腔注射L-NAME,10 mg/(kg·d)],每组大鼠各10只.术后7 d,用生物素-dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡,用硝酸还原酶法测定睾丸组织中NO含量,用化学比色法测定睾丸组织中NOS活性.结果术后第7 d,与假手术组睾丸相比,隐睾组睾丸发生凋亡的生殖细胞数显著增加,隐睾+L-NAME组睾丸发生凋亡的生殖细胞数比隐睾组显著减少(P＜0.01),隐睾+L-NAME组睾丸组织中NO含量及NOS活性与隐睾组相比显著降低(P＜0.01).结论隐睾组织中NO和NOS升高是隐睾生殖细胞凋亡增加的病理机制之一,L-NAME通过抑制NOS活性、减少NO的产生来降低睾丸组织生殖细胞的凋亡发挥其保护作用.