Breakdown of intestinal mucosa via accelerated apoptosis increases intestinal permeability in experimental severe acute pancreatitis

BACKGROUND: Bacterial translocation plays an important role for infectious complications in severe acute pancreatitis (SAP). Breakdown of intestinal mucosal integrity may increase intestinal permeability and may be implicated in bacterial translocation. It is suggested that increase in intestinal permeability is correlated with the changes of tight junction and/or apoptosis in intestinal epithelial cells. The aim of this study was to investigate the changes of intestinal mucosa and its permeability in SAP. METHODS: SAP was induced by injection of 3% sodium deoxycholate into the biliopancreatic ducts in rats. Permeability of intestinal wall was assayed ex vivo by measuring the leaked amount of FITC-dextran from the ileum pouch. Alteration of tight junction proteins such as zonula occludens (ZO)-1 and Occludin was evaluated by Western blotting and immunofluorescence staining. Apoptotic change of intestinal mucosa was detected by TUNEL staining and DNA fragmentation ELISA. In vitro, apoptosis-inducing effect of pancreatitis-associated ascitic fluid (PAAF) was examined using T84 cells. Integrity of monolayer cells was assessed by transepithelial electric resistance (TEER). RESULTS: Permeability of ileum was significantly increased 6 h after induction of SAP. Blood endotoxin level was significantly elevated and bacterial translocation occurred 18 h after induction of SAP. Six hours after induction of SAP, expressions of ZO-1 and Occludin were not altered, but apoptosis of ileum mucosa was significantly accelerated. Addition of PAAF to T84 cells did not affect expressions of ZO-1 or Occludin, but significantly increased the apoptosis and significantly decreased TEER. CONCLUSIONS: These results suggest that breakdown of intestinal mucosa via accelerated apoptosis may increase in intestinal permeability in SAP and that PAAF contains factor(s) that accelerates the apoptosis of intestinal epithelial cells.     

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.     

Administration of nitric oxide with caspase inhibitors minimizes bacterial translocation in experimental intestinal transplantation

BACKGROUND: Bacterial translocation (BT) has been suggested to be responsible for the high incidence of infections occurring after small-bowel transplantation (Trp). Nitric oxide (NO) and apoptosis could affect cell demise. The aim of this study was to asses whether supplementation of University of Wisconsin (UW) solution with NO donors and apoptosis inhibitors can abolish BT in Trp. METHODS: The following experimental groups were studied: sham, Trp, intestinal transplantation, Trp+spermine NONOate (NONOs), and Trp+NONOs+caspase inhibitor Z-Val-Ala-Asp(Ome)-fluoromethylketone(Z-VAD-fmk). Histologic analysis, caspase-3 activity, DNA fragmentation, and BT from graft to mesenteric lymph nodes, liver, and spleen were measured in tissue samples after transplantation. RESULTS: During intestinal transplantation, apoptosis and necrosis were increased, showing graft injury and high levels of BT. The rats treated with NONOs showed a histologic protection of transplanted graft and a decrease in BT despite caspase-3 and DNA fragmentation-inducing effects. Administration of caspase inhibitor Z-VAD to NONOs-treated rats reversed the NO apoptosis-inducing effects and showed the lowest levels of BT in all tissues. CONCLUSIONS: Exogenous administration of NO associated with the inhibition of apoptosis maintains the graft in optimal conditions in terms of BT and improves the histology of the graft after intestinal transplantation in rats.     

Hypoxia/re-oxygenation injury induces apoptosis of LLC-PK1 cells by activation of caspase-2

Hypoxia/re-oxygenation injury induces apoptosis in renal tubule cells, but its underlying molecular pathways are not fully elucidated. Activation of caspase-2 has recently been proposed as a novel mechanism of apoptosis in fibroblasts. In this study we examined whether hypoxia/re-oxygenation injury induces apoptosis in proximal tubule cells by activation of caspase-2. Porcine proximal tubule (LLC-PK1) cells were subjected to hypoxia/re-oxygenation injury in the presence or absence of caspase inhibitors. Apoptosis was detected by DNA laddering, flow cytometry, and immunocytochemistry for Bax and cytochrome c. The activity of caspases-2, 8 and 9 was measured. Apoptosis was evident after hypoxia/re-oxygenation and was best prevented by pretreatment with caspase-2 inhibitor. Hypoxia/re-oxygenation resulted in a dramatic increase in caspase-2 activity (32-fold, in comparison with a 16-fold increase in caspase-8 activity and a tenfold increase in caspase-9 activity). Immunocytochemistry revealed Bax activation and translocation to mitochondria and cytochrome c release into the cytosol following hypoxia/re-oxygenation, both of which were significantly suppressed by pretreatment with caspase-2 inhibitor. These results indicate that hypoxia/re-oxygenation injury in cultured proximal tubule cells induced apoptosis by activation of caspase-2, which is required for the mitochondrial translocation of Bax.     

表皮生长因子对重症胰腺炎肠外营养大鼠肠道细菌易位的影响

探讨表皮生长因子（ＥＧＦ）对重症胰腺炎（ＳＡＰ）全肠外营养（ＴＰＮ）支持大鼠肠道细菌易位的影响。方法：４８只ＳＤ大鼠随机分为ＳＡＰ组、ＳＡＰ＋ＴＰＮ组及ＳＡＰ＋ＴＰＮ＋ＥＧＦ组。分别测定肠系膜淋巴结、肝脏及脾脏细菌易位率,利用图像分析仪对小肠粘膜进行检测,并测定回肠粘膜厌氧菌与需氧菌的比率。结果：ＳＡＰ＋ＴＰＮ＋ＥＧＦ组肠系膜淋巴结、肝脏及脾脏细菌易位率明显降低,小肠粘膜绒毛面积、高度、隐窝深度增加,回肠厌氧菌与需氧菌的比率显著提高。结论：ＥＧＦ具有保护重症胰腺炎全肠外营养大鼠肠粘膜机械屏障、生物屏障和降低肠道细菌易位率的作用。     

Prevention of mucosal atrophy: Role of glutamine and caspases in apoptosis in intestinal epithelial cells

Glutamine starvation induces apoptosis in enterocytes; therefore glutamine is important in the maintenance of gut mucosal homeostasis. However, the molecular mechanisms are unknown. The caspase family of proteases constitutes the molecular machinery that drives apoptosis. Caspases are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were to (1) identify specific caspases activated by glutamine starvation and (2) determine whether a general caspase inhibitor blocks glutamine starvation-induced apoptosis in intestinal epithelial cells. Rat intestinal epithelial (RIE-I) cells were deprived of glutamine. Specific caspase activation was measured using fluorogenic substrate assay. Apoptosis was quantified by DNA fragmentation and Hoechst nuclear staining. Glutamine starvation of RIE-1 cells resulted in the time-dependent activation of caspases 3 (10 hours) and 2 (18 hours), and the induction of DNA fragmentation (12 hours). Caspases 1 and 8 remained inactive. ZVAD-fluoromethyl ketone, a general caspase inhibitor, completely blocked glutamine starvation-induced caspase activation, DNA fragmentation, and nuclear condensation. These results indicate that glutamine starvation selectively activates specific caspases, which leads to the induction of apoptosis in PIE-1 cells. Furthermore, inhibition of caspase activity blocked the induction of apoptosis, suggesting that caspases are potential molecular targets to attenuate apoptotic responses in the gut. Supported by National Institutes of Health grants F32 DK09867, KO8 CA64191, PO1 DK35608, and EY 08547; the American Cancer Society, Texas Division, Inc.; the Lions Eye Bank Fotmdation; and the Walls Medical Research Foundation. Dr. Papaconstantinou is a Visiting Scientist from the University of Cincinnati Medical Center, Cincinnati, Ohio, and is the recipient of a Clinical Oncology Fellowship from the American Cancer Society, Texas Division, Inc. Presented at the Fortieth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Fla., May 16–19, 1999, and published as an abstract in Gastroenterology 116:A1325, 1999.     

Intracellular mechanisms of cyclosporin A-induced tubular cell apoptosis

Tubular cell apoptosis contributes to the pathogenesis of renal injury. However, the intracellular pathways that are active in tubular epithelium are poorly understood. The lethal pathways activated by cyclosporin A (CsA), a nephrotoxin that induces caspase-dependent apoptosis in tubular epithelium, were explored. Fas expression, caspase activation, and mitochondrial injury were assessed by Western blot, flow cytometry, and microscopy in cultured murine tubular epithelial cells exposed to CsA. The influence of FasL antagonists, Bax antisense oligodeoxynucleotides, and caspase inhibitors on cell survival was explored. Tubular cells constitutively express FasL. CsA increased the expression of Fas. However, Fas had no role in CsA-induced apoptosis, as CsA did not sensitize to FasL-induced apoptosis, caspase-8 activity was not increased, and neither blocking anti-FasL antibodies nor caspase-8 inhibition prevented CsA-induced apoptosis. Apoptosis induced by CsA is associated with the translocation of Bax to the mitochondria and Bax antisense oligodeoxynucleotides protected from CsA-induced apoptosis. CsA promoted a caspase-independent release of cytochrome c and Smac/Diablo from mitochondria. CsA also led to a caspase-dependent loss of mitochondrial membrane potential. Caspase-2, caspase-3, and caspase-9 were activated, and specific caspase inhibitor prevented apoptosis and increased long-term survival. Evidence for endoplasmic reticulum stress, such as induction of GADD153, was also uncovered. However, endoplasmic reticulum-specific caspase-12 was not activated. CsA induces changes in several apoptotic pathways. However, the main lethal apoptotic pathway in CsA-exposed tubular epithelial cells involves mitochondrial injury.     

Bile salts induce resistance to apoptosis through NF-kappaB-mediated XIAP expression

Apoptosis plays a critical role in intestinal mucosal homeostasis. We previously showed that the bile salt taurodeoxycholate has a beneficial effect on the intestinal mucosa through an increase in resistance to apoptosis mediated by nuclear factor (NF)-kappaB. The current study further characterizes the effect of bile salts on intestinal epithelial cell susceptibility to apoptosis and determines if the X-linked inhibitor of apoptosis protein (XIAP) regulates bile salt-induced resistance to apoptosis. Exposure of normal intestinal epithelial cells (IEC-6) to the conjugated bile salts taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) resulted in an increase in resistance to tumor necrosis factor (TNF)-alpha and cycloheximide (CHX)-induced apoptosis, and NF-kappaB activation. Treatment with TDCA and TCDCA resulted in an increase in XIAP expression. Specific inhibition of NF-kappaB by infection with an adenoviral vector that expresses the IkappaBalpha super-repressor (IkappaBSR) prevented the induction of XIAP expression and the bile salt-mediated resistance to apoptosis. Treatment with the specific XIAP inhibitor Smac also overcame this increase in enterocyte resistance to apoptosis. Bile salts inhibited formation of the active caspase-3 from its precursor procaspase-3. Smac prevented the inhibitory effect of bile salts on caspase-3 activation. These results indicate that bile salts increase intestinal epithelial cell resistance to apoptosis through NF-kappaB-mediated XIAP expression. Bile salt-induced XIAP mediates resistance to TNF-alpha/CHX-induced apoptosis, at least partially, through inhibition of caspase-3 activity. These data support an important beneficial role of bile salts in regulation of mucosal integrity. Decreased enterocyte exposure to luminal bile salts, as occurs during starvation and parenteral nutrition, may have a detrimental effect on mucosal integrity.     

Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation), non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study, we demonstrate that nitrogen-containing bisphosphonates (risedronate, alendronate, pamidronate, and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts, human osteoclastoma-derived osteoclasts, and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes, loss of mitochondrial membrane potential, and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed, dying osteoclasts and osteoclast-like cells using a cell-permeable, fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation, because apoptosis resulting from alendronate, clodronate, or zoledronic acid treatment was suppressed by zVAD-fmk, a broad-range caspase inhibitor, or by SB-281277, a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore, caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts, whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3, therefore, appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins, because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, whereas FTI-277, a specific inhibitor of protein farnesylation, had no effect on apoptosis or caspase activity.     

Chondrocyte apoptosis induced by hydrogen peroxide requires caspase activation but not mitochondrial pore transition.

The primary objective of this study was to test the hypothesis that inhibition of mitochondrial permeability transition and/or inhibition of caspase family enzymes can block chondrocyte apoptosis induced by H2O2. Primary human chondrocytes were isolated from normal cartilage by enzymatic digestion. Apoptosis was induced by exposure to H2O2. Chondrocyte apoptosis was quantified using an ELISA for nucleosome formation. Independent confirmation of apoptosis was obtained by TUNEL analysis. H2O2 induced apoptosis in primary human chondrocytes in a time and dose dependent manner. The effects of candidate apoptosis inhibitors were then tested. Chondrocytes were pre-treated with inhibitors of mitochondrial permeability transition, or one of three different caspase inhibitors, and then incubated with H2O2. Apoptosis was then measured after 16 h of exposure to H2O2. Pre-treatment with inhibitors of mitochondrial permeability transition did not block apoptosis induced by H2O2. A non-selective caspase inhibitor, a caspase 3-selective inhibitor, and a caspase 1-selective inhibitor, all blocked chondrocyte apoptosis induced by H2O2. These results show that H2O2 triggers chondrocyte apoptosis through caspase activation, independent of mitochondrial membrane permeability transition.     

The nitric oxide donor molsidomine improves survival and reduces hepatocyte apoptosis in cholestasis and endotoxemia

BACKGROUND: Cholestasis and endotoxemia have been demonstrated to cause hepatocyte apoptosis through caspase-mediated pathways. In vitro nitric oxide (NO) donors reduce hepatocyte apoptosis and caspase activation in several models. The nitric oxide donor molsidomine improves survival in an in vivo model of endotoxemia. We tested the effect of molsidomine on survival and hepatocyte apoptosis in a model of obstructive jaundice and endotoxemia. STUDY DESIGN: Sprague-Dawley rats underwent common bile duct ligation on day 1. On day 3, animals were given either 100 mg/kg of molsidomine or an equivalent volume of saline, and 30 minutes later they were given endotoxin 3 mg/kg or 10 mg/kg intravenously. Animals were sacrificed 4 or 16 hours after endotoxin injection. Serum samples were analyzed for alanine aminotransferase and frozen liver samples were analyzed for caspase 3 activity. Paraffin-embedded liver sections were assayed for apoptosis using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Survival was measured in a separate experiment in which animals underwent the same protocol, but were given three different doses of endotoxin and were observed for 72 hours before sacrifice. RESULTS: At endotoxin 3 mg/kg, the 72-hour survival in saline-treated animals was 92%, which decreased to 45% at 10 mg/kg and to 29% at 15 mg/kg. All of the molsidomine-treated animals survived all endotoxin doses. Alanine aminotransferase was reduced in molsidomine-treated animals compared with those treated with saline. Apoptosis was attenuated in molsidomine-treated animals. Caspase 3 activity was decreased in molsidomine-treated animals compared with those given saline. CONCLUSIONS: Molsidomine attenuates caspase activation and hepatocyte apoptosis and improves survival after cholestatic endotoxic injury.     

Dual roles for NF-kappaB activation in osteoblastic cells by serum deprivation: osteoblastic apoptosis and cell-cycle arrest

To clarify the mechanisms of osteoblastic cell death, we examined whether serum deprivation would cause activation of the apoptotic signal cascade and arrest of the cell cycle in mouse osteoblastic MC3T3-E1 cells. Serum withdrawal from osteoblastic cell cultures resulted in growth arrest and cell-cycle arrest at G0/G1, which actions were accompanied by transient and potent activation of NF-kappaB, caspase-8, caspase-2, caspase-3, and caspase-9 in this order. Apoptosis, but not necrosis, in serum-deprived cells could be detected by FACS using Annexin-V/propidium iodine double staining. Serum deprivation also resulted in transient activation of the 20S proteasome, which is an important component for regulation of the cell cycle by the ubiquitin-proteasome system. The 20S proteasome inhibitor (PSI) but not NF-kappaB inhibitor SN50 suppressed the activation of proteasomes in serum-deprived cells. Although caspase inhibitors could not prevent the G0/G1 arrest in the serum-deprived cells, SN50 and the 20S proteasome inhibitor could block it. Since SN50, 20S proteasome inhibitor and caspase inhibitor could rescue cells from serum deprivation-induced apoptosis, the pathway for NF-kappaB/caspase activation is independent of the NF-kappaB/cell-cycle pathway, and the events downstream of the NF-kappaB/caspase-9 cascade lead to apoptosis. Taken together, our present results identify a novel role for NF-kappaB in cell-cycle and apoptosis regulation and underscore the significance of each independent signal cascade in serum-deprived osteoblastic cells.     

STAT3在急性重症胰腺炎大鼠肠黏膜屏障功能障碍中的作用

目的　研究重症胰腺炎(SAP)时大鼠肠上皮细胞凋亡、氧化损伤及凋亡相关蛋白的激活情况。方法　Wistar大鼠4 8只,分为S(假手术)组、C(对照)组、A(急性胰腺炎)组、N(抗氧化剂)组,A组、N组采用胆胰管内逆行注入5 %牛磺胆酸钠诱导大鼠SAP模型,C组注入生理盐水。术后3h、2 4h分批处死大鼠,取胰腺组织行HE染色明确胰腺炎程度;留取血浆检测D 乳酸水平;刮取小肠黏膜检测丙二醛(MDA)、黄嘌呤氧化酶(XO)水平;小肠组织增殖细胞核抗原(PCNA)免疫组化染色;小肠组织TUNEL染色及黏膜DNA琼脂糖凝胶电泳;提取小肠黏膜蛋白行Westernblot检测Bcl 2、Bax、p STAT3表达水平。结果　A组术后3h血浆D 乳酸含量、肠上皮细胞凋亡增加,2 4h最显著(P <0 . 0 1) ,D 乳酸含量、凋亡指数分别为(3 6. 1±0 . 98) μg/ml、(4 5. 3±1. 95 ) % ,二者正相关(r =0 . 5 74 ,P <0 . 0 1) ;A组术后3h氧化应激水平高于其他各组,小肠黏膜MDA含量及XO活力分别为(4 .85±1 .0 5 )nmol/mg、(18. 1±3 .0 3)U/g ;A组术后3h肠黏膜Bax、p STAT3表达增加,2 4h最明显,二者存在相关性(r=0 . 5 91,P <0 .0 1)。结论　急性重症胰腺炎大鼠肠上皮氧化应激导致的细胞凋亡是肠黏膜通透性增加的机制之一;氧化应激可能通过激活STAT3信号传导通路调控凋亡相关蛋白的     

The role of caspases in methotrexate-induced gastrointestinal toxicity.

BACKGROUND: Enterocolitis is the major toxicity of methotrexate-based cancer chemotherapy, which limits its clinical applications. Methotrexate induces gut mucosal apoptosis in vivo; however, little is known about the molecular mechanism involved. The effectors of apoptosis include the caspase family of proteases, which are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were (1) to establish an in vitro model of methotrexate-induced gut apoptosis and (2) to determine the role of caspases in methotrexate-induced apoptosis in intestinal epithelial cells. METHODS: Rat intestinal epithelial cells (RIE-1) were treated with methotrexate in the absence or presence of ZVAD-fluoromethyl ketone, a general caspase inhibitor. Apoptosis was quantified by means of deoxyribonucleic acid (DNA) fragmentation assays and Hoechst nuclear staining. Caspase activation was measured with the use of fluorogenic substrates. RESULTS: Methotrexate induced apoptosis and decreased cell number in RIE-1 cells. DNA fragmentation was preceded by the sequential activation of caspases 9, 2, and 3, whereas caspases 1 and 8 remained inactive. ZVAD-fluoromethyl ketone inhibited methotrexate-induced caspase activation, DNA fragmentation, and nuclear condensation. CONCLUSIONS: These results indicate that methotrexate activates specific caspases and induces apoptosis in RIE-1 cells. Furthermore, caspases may play an important role in methotrexate-induced apoptosis in RIE-1 cells and may be potential therapeutic targets to attenuate methotrexate-induced enterocolitis.     

Multiple caspases mediate acute renal cell apoptosis induced by bacterial cell wall components

The stimulus for caspase-mediated renal cell apoptosis in septic acute renal failure (ARF) is unclear. To demonstrate the nephrotoxic effects of bacterial cell wall components, the anti-cellular activity of bacterial muropeptides (muramyl dipeptides), peptidoglycans, and lipopolysaccharides was investigated in rabbit kidney cells. Changes in the cell membrane (APOPercentage? dye uptake), caspase activities, and DNA degradation were quantified colorimetrically and using densitometric assays and their inhibition by caspase-specific and pan-caspase inhibitors was determined. The onset and levels of APOPercentage? dye-positive rabbit kidney cells, caspase activities, and DNA degradation were closely associated. Specific caspase-1, -2, -3, -4, -8, -10, and -12 inhibitors reduced caspase-3 activity by ≥40%, but only caspase-3 and -8-specific inhibitors reduced apoptotic DNA levels. Pan-caspase inhibitor Q-VD-OPh was 10-fold more effective at inhibiting rabbit kidney cell death, caspase activation, and DNA degradation than caspase-family inhibitor Z-VAD-FMK. Apoptosis was inhibited effectively by both pan-caspase inhibitors when applied early during the stimulus-to-response period. Multiple initiator and effector caspases were activated suggesting extrinsic, intrinsic, and endoplasmic reticulum/stress apoptotic pathway stimulation in rabbit kidney cells treated with bacterial cell wall components. The results provide in vitro support for bacterial cell wall-induced apoptosis as a pathogenic mechanism of renal cell death in septic ARF and support the potential prophylactic use of pan-caspase inhibitors to suppress septic ARF.     

补充双歧杆菌可促进烫伤大鼠肠道分泌型sIgA合成与分泌

目的　探讨严重烫伤后补充双歧杆菌与肠道sIgA合成、分泌的关系。　 方法　Wistar大鼠随机分为烫伤对照组 (BC组 ,30只 )、烫伤治疗组 (BT组 ,30只 )、假伤组 (NC组 ,10只 )。BT组大鼠烫伤后灌胃双歧杆菌悬液 (5 0× 10 9CFU/ml) 1 5ml,2次 /d。观测大鼠细菌移位、肠黏膜菌群双歧杆菌量、肠道sIgA分泌和表达情况等。　 结果　 (1)伤后 3d ,BC组与BT组大鼠脏器细菌移位率分别为 4 2 %和 16 % (P =0 0 0 4 ) ;伤后 5d分别为 30 %和 8% (P =0 0 0 2 )。 (2 )伤后大鼠肠黏膜菌群中双歧杆菌减少 10～ 6 0倍 ,应用悬液后双歧杆菌明显增多。 (3)BC组大鼠肠黏液sIgA平均减少 30 % ,伤后 3d达最低 ;BT组 3d后基本恢复正常。伤后大鼠肠道sIgA表达减弱 ,补充双歧杆菌后sIgA表达显著增强。 (4)肠膜菌群中双歧杆菌量与肠黏液sIgA浓度呈显著正相关。　 结论　大鼠严重烫伤后肠道sIgA产生明显受抑制 ,补充外源性双歧杆菌可促进肠道sIgA合成与分泌。     

不同营养方式对肠道缺血再灌注大鼠肠屏障功能的影响

目的探讨不同营养物质及支持途径对肠道缺血再灌注大鼠肠屏障功能和细菌易位的影响。方法60只雄性SD大鼠建立肠道缺血再灌注模型,随机分成普通肠外营养组(PN),富含谷氨酰胺的肠外营养组(G-PN),普通肠内营养组(EN)及免疫增强型肠内营养组(IEN)。从术后第1天起连续营养支持7d,各组等氮、等热卡。观察肠道形态学、肠道黏膜通透性、肠道细菌易位情况和血浆内毒素水平及肠道免疫功能检测。结果PN组肠黏膜明显萎缩,其绒毛高度、黏膜厚度、隐窝深度及绒毛表面积均显著低于其他各组(P<0.05);其肠黏膜通透性及内毒素值显著高于其他各组(P<0.05),细菌易位率(100%)明显高于其他各组(G-PN组60.0%,EN组33.3%,IEN组20.0%)。PN组CD4 T淋巴细胞和IgA 浆细胞分布显著低于其他各组(P<0.01)。结论EN在维护肠黏膜屏障功能、防止细菌及内毒素易位方面优于PN。免疫增强型EN在维护肠黏膜屏障、改善肠道免疫功能、防止细菌易位方面作用优于普通EN。     

Role of bacterial adherence and the mucus barrier on bacterial translocation: effects of protein malnutrition and endotoxin in rats.

OBJECTIVE: The purpose of the study was to investigate the potential relations between mucosal bacterial adherence, intestinal mucus and mucin content, and bacterial translocation. SUMMARY BACKGROUND DATA: The attachment of bacteria to mucosal surfaces is the initial event in the pathogenesis of most bacterial infections that originate at mucosal surfaces, such as the gut. The intestinal mucus layer appears to function as a defensive barrier limiting micro-organisms present in the intestinal lumen from colonizing enterocytes. Consequently, studies focusing on the biology of bacterial adherence to the intestinal mucosa likely are to be important in clarifying the pathogenesis of gut origin sepsis. METHODS: To explore the relations between intestinal bacterial adherence, mucus bacterial binding, and bacterial translocation, two models were used. One (protein malnutrition) in which profound alterations in intestinal morphology occurs in the absence of significant translocation and one (endotoxin challenge) in which bacterial translocation occurs and intestinal morphology is relatively normal. RESULTS: Protein malnutrition was not associated with bacterial translocation and measurement of enteroadherent, mucosally associated bacterial population levels documented that the total number of gram-negative enteric bacilli adherent to the ileum and cecum was less in the protein-malnourished rats than in the normally nourished animals (p < 0.01). Furthermore, there was an inverse relation between the duration of protein malnutrition and bacterial adherence to the intestinal mucosa (r = 0.62, p < 0.002). In contrast, after endotoxin challenge, the level of enteroadherent bacteria was increased and bacterial translocation was observed. The binding of Escherichia coli to immobilized ileal mucus in vitro was decreased significantly in protein-malnourished rats, whereas E. coli binding to insoluble ileal mucus was increased in the rats receiving endotoxin. CONCLUSIONS: This study indicates that the adherence of bacteria to the intestinal mucosal surface is an important factor in bacterial translocation, that intestinal mucus modulates bacterial adherence, and that increased levels of mucosally associated bacteria are associated with a loss intestinal barrier function to bacteria.     

Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro

BACKGROUND: Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. METHODS: Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. RESULTS: Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. CONCLUSION: Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.     

谷氨酰胺对腹部手术后肠通透性及细菌易位影响的临床研究

目的观察外源性谷氨酰胺（Gln）对腹部手术后病人肠道通透性的影响,继而探讨其与肠细菌易位的关系。方法将暨南大学附属第一医院2004年5月至2007年5月收治的76例腹部大中型手术病人随机分成Gln治疗组（n=38）和对照组（n=38）。两组均于术后第2d开始给予肠外营养支持,持续7d。Gln治疗组于肠外营养中加入Gln。两组分别于术前、术后检测血谷氨酰胺水平、尿乳果糖/甘露醇（L/M）比值、外周血肠源性细菌DNA。结果术后7d治疗组Gln与术前相比差异无统计学意义［(61614±4213) μmol/L vs (611354±3981)μmol/L］,P﹥005。与对照组比较Gln显著升高,差异具有统计学意义, P﹤001。术后L/M比值对照组较术前显著升高,P﹤005;治疗组与术前比较无升高,P﹥005。两组术后相比较对照组高于治疗组,差异具有统计学意义,P﹤001。外周血细菌DNA行PCR扩增后阳性率为1053%（8/76）,PCR阳性组Gln水平较PCR阴性组显著降低,P﹤005;L/M则显著增高,P﹤001。结论外源性Gln的补充可以有效的提高腹部手术后病人Gln水平,保护肠黏膜屏障功能,减少细菌易位的发生。