Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus)

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets (Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3'-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets (n=14) and also in ovariectomized marmosets (n=13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E+P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)--a proliferation marker and steroid receptors, ERalpha and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E+P group as compared to other groups of ovariectomized animals (P<0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERalpha and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E+P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.     

VEGF, bFGF, and their receptors in the endometrium of rhesus monkey during menstrual cycle and early pregnancy

A number of cytokines and growth factors are known to modulate proliferation and differentiation of human endometrium. In this study, the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and VEGF receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing region (KDR), and bFGF receptor 1 (Flg) were examined in the endometrium of rhesus monkey on Day 5, 10, 16, 20, 25 of menstrual cycle and on Day 19 of early pregnancy. Western blot analysis showed the specificity of the anti-human antibodies with the monkey tissue. The expression of mRNA and protein of VEGF was correlated with that of its receptor KDR, which was detected in epithelial, vascular, and myometrial cells. The localization of bFGF and its receptor Flg was similar to that of VEGF, except that the Flg was absent in the endothelial cells. Strong expression of VEGF and bFGF in the glandular epithelial cells was observed in the proliferative phase, declined in the secretory phase during the cycle. Stronger staining of these factors was also observed in the decidual cells of the pregnant uterus, as compared with the stromal cells of cycling uterus. No expression of Flt1 was detected in the tissue examined in this study. These data suggest that VEGF, bFGF, and their receptors play important roles in epithelial and stromal development, angiogenesis, and blood vessel function in the endometrium during the menstrual cycle and early pregnancy of the rhesus monkey.     

Alveolar epithelial type II cell apoptosis in vivo during resolution of keratinocyte growth factor-induced hyperplasia in the rat

Keratinocyte growth factor (KGF) induces rapid and transient hyperplasia of alveolar epithelial type II cells. We sought to determine components of the apoptotic process involved in the resolution of this hyperplasia and the fate of the apoptotic cells. Rats received intrabronchial instillation of 5 mg KGF/kg body weight or diluent. Lungs were fixed 1, 2, 3, 5, and 7 days later. Apoptosis was identified by TdT-mediated dUTP nick-end labeling (TUNEL), double-labeling for TUNEL and the type II cell marker MNF116, and electron microscopy. Fas, FasL, Bax, Bcl-2, and pro- and active caspase-3 were studied by immunohistochemistry. Changes were quantified by stereology. Cell type specificity was investigated by immunofluorescence double staining. Type II cells exhibited Fas, FasL, Bcl-2, and procaspase-3 irrespective of treatment and time. Immunoelectron microscopy revealed Fas at the apical type II cell membrane. Bax staining was prominent in controls (45-95% of type II cell surface fraction), markedly decreased during hyperplasia at days 2 (20-40%) and 3 (0-10%), and reappeared at day 7 (25-45%) when apoptosis was prominent. Remnants of apoptotic type II cells were incorporated in membrane-bound vacuoles of type II cell neighbors as well as alveolar macrophages. The results indicate that type II cells can enter the Fas/FasL/caspase-3 pathway regulated by Bax and Bcl-2. High Bcl-2:Bax levels favor type II cell survival and a low rate of apoptosis during hyperplasia. Low Bcl-2:Bax levels favor type II cell apoptosis during resolution. Because of time-dependent changes that occur within a short time, the KGF-treated rat lung provides a useful in vivo model to investigate apoptosis in the context of tissue remodeling and repair.     

Endomorphins, endogenous opioid peptides, induce apoptosis in human leukemia HL-60 cells

Opioids play a role in the apoptosis machinery. We studied the induction of apoptosis in endomorphin 1 (EM1) and endomorphin 2 (EM2), 2 newly isolated endogenous mu-opioid receptor agonists. These endomorphins were able to reduce the viability of cultured HL-60 cells. The antiproliferative properties of endomorphins appeared to be attributable to their induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 indicated down-regulation, but the Bax, Fas, and FasL expression showed up-regulation as compared with the untreated control cells. These data support the idea that endomorphins induce apoptosis in HL-60 cells through the activation of the Bcl-2-Bax and the Fas-FasL pathway. We suggest that endomorphins may play an important role in the regulation of tumor cell death.     

Lung injury after ischemia-reperfusion of small intestine in rats involves apoptosis of type II alveolar epithelial cells mediated by TNF-α and activation of Bid pathway

Although ischemia-reperfusion (I/R) of small intestine is known to induce lung cell apoptosis, there is little information on intracellular and extracellular molecular mechanisms. Here, we investigated the mechanisms of apoptosis including the expression of Fas, Fas ligand (FasL), Bid, Bax, Bcl-2, cytochrome c, and activated caspase-3 in the rat lung at various time-points (0–24 h) of reperfusion after 1-h ischemia of small intestine. As assessed by TUNEL, the number of apoptotic epithelial cells, which were subsequently identified as type II alveolar epithelial cells by electron microscopy and immunohistochemical double-staining, increased at 3 h of reperfusion in the lung. However, intravenous injections of anti-TNF-α antibody decreased the number of TUNEL-positive cells, indicating involvement of tumor necrosis factor-α (TNF-α) in the induction of lung cell apoptosis. Western blotting and/or immunohistochemistry revealed a marked up-regulation of Fas, FasL, Bid, Bax, cytochrome c and activated caspase-3 and down-regulation of Bcl-2 in lung epithelial and stromal cells at 3 h of reperfusion. Our results indicate that I/R of small intestine results in apoptosis of rat alveolar type II cells through a series of events including systemic TNF-α, activation of two apoptotic signaling pathways and mitochondrial translocation of Bid.     

Selenium as a modulator of membrane stability parameters and surface changes during the initiation phase of 1,2-dimethylhydrazine induced colorectal carcinogenesis

Epithelial ovarian cancer (EOC) represents the most challenging of gynecological malignancies. Defective apoptosis is a major causative factor in the development and progression of cancer. The two important pathways of apoptosis are extrinsic death receptor pathway (Fas family) and intrinsic mitochondrial pathway (Bcl-2 family). In this study, differential protein expression of the major Fas family members (Fas, FasL, and FAP-1) and Bcl-2 family members (Bax, Bcl-2, and Bcl-X(L)) in benign versus malignant surface epithelial ovarian tumors was evaluated at the protein level by immunohistochemistry. The expression of these molecules was compared in 30 benign versus 35 malignant surface epithelial ovarian tumors. The findings of the present study showed that there was no significant difference in the expression of the Fas family members in benign and malignant ovarian tumors. However, benign tumors showed higher levels of anti-apoptotic Bcl-2 protein levels (p < 0.009), whereas malignant tumors showed higher levels of pro-apoptotic Bax (p < 0.001). In general, there was no significant difference in Bcl-X(L) protein levels. The observations made in the present study suggest that alterations in expression of the Fas family and the Bcl-2 family members occur and play a key role in the deregulated growth of epithelial ovarian cancer.     

Dexamethasone induces apoptosis in human T cell clones expressing low levels of Bcl-2

Previous results of ours have demonstrated that the same clonotype can express both a sensitive and a resistant phenotype to Dex-mediated PCD induction depending on its cell cycle phase. In particular, we demonstrated that human T lymphocytes, arrested in the G0/G1 phase of the cell cycle, are susceptible, while proliferating T cells are resistant to Dex-mediated apoptosis. In this paper, we have further characterized the sensitive and resistant phenotypes and investigated whether a different expression of the apoptotic genes Fas, FasL, Bcl-2, Bcl-x and Bax is involved in the regulation of Dex-mediated apoptosis. The results show that the amount of Bcl-2 expression, that changes during cell cycle phases, determines susceptibility or resistance to apoptosis induced by Dex. In fact, undetectable expression of Bcl-2 in sensitive cells favors Dex-mediated apoptosis while high expression of Bcl-2 in proliferating cells counterbalances apoptosis induction. Moreover, the addition of exogenous IL-2, in the presence of Dex, fails to up-regulate Bcl-2 expression and to revert Dex-mediated apoptotic phenomena.     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.     

Expression of apoptosis-related genes Fas/FasL, Bax/Bcl-2 and Caspase-3 in rat corpus luteum during luteal regression

Corpus luteum (CL) is a transient endocrine organ formed from the ovulated follicle. CL produces progesterone and estrogen that are important in preparing the uterine environment for implantation and maintaining gestation. Pregnancy maintains the CL function; otherwise, CL re-gresses rapidly. Cycling formation and regression of CL is essential for initiation of new follicular growth and differentiation, and subsequently ovulation and luteinization[1]. Luteal regression could be divided int…     

Expression of Bcl—2 inhibited Fas—mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Apoptosis plays an important role in embryonic development,tissue remodeling,immune regulation and tumor regression.Two groups of molecules(Bcl-2 family and “Death factor” family) are involved in regulating apoptosis.In order to know about the effect of Bcl-2 on apoptosis induced by Fas,a typical member of “Death factor” family,the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells,a human hepatocellular carcinoma cell line which expresses endogenous Fas,but not FasL and Bcl-2.The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells.However,the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis.Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis.These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.     

Identical changes in Bax expression, but not Fas ligand expression, occur in structural luteolysis in gonadotropin releasing hormone agonist- and prolactin-treated superovulated rats

Structural luteolysis induced by gonadotropin releasing hormone agonist (GnRHa) or prolactin (PRL) is defined as histological involution of the corpus luteum. We reported that one of the mechanisms of structural luteolysis induced by PRL was tissue remodeling by matrix metalloproteinase (MMP) and also apoptosis in superovulated rats. We also reported that GnRHa induced structural luteolysis with elevation of MMP. In this study, we investigated whether GnRHa caused apoptosis in mature corpus luteum of superovulated rats and also examined the expression of apoptosis-related molecules (Fas, Fas ligand (FasL), Bcl-2, Bax). We gave 4-day GnRHa treatment 5 days after hCG injection to immature female rats treated with pregnant mare surum gonadotrophin (PMSG) and hCG to induce structural involution of mature corpus luteum. PMSG-hCG-treated rats without GnRHa treatment, rats treated with bromocryptine (Brom) to induce functional luteolysis and rats treated with Brom followed by PRL (Brom+PRL) to mimic the PRL surge to induce structural luteolysis as we previously reported were used for comparison. GnRHa treatment caused structural luteolysis characterized by structural involution, a decrease in the serum progestin level, and apoptotic bodies as well as structural luteolysis induced by Brom+PRL. FasL expression in corpora lutea was elevated after Brom treatment, but there was no elevation of FasL after GnRHa treatment started. FasL expression decreased and Bax expression increased in structural luteolysis induced by GnRHa as well as Brom+PRL treatment, although Fas and Bcl-2 expression did not change throughout the luteal phase. In summary, both GnRHa and Brom+PRL caused structural luteolysis, one of whose mechanisms was apoptosis with an increase in Bax expression, but not with an identical change in FasL expression. It is speculated that the significance in alteration of FasL may involve some mechanism other than apoptosis.     

Expression of apoptosis-related genes Fas/FasL,Bax/Bcl-2 and Caspase-3 in rat corpus luteum during luteal regression

Using immunohistochemistry, in situ hybridization, Western blot and TUNEL methods, we have studied the expression of Fas/FasL, Bcl-2/Bax and caspase-3 in the corpora lutea (CL) at various stages of pseudopregnant rat induced by injection of PMSF/hCG. The results showed that no apoptotic signal could be observed until Day 14 after hCG injection. Fas weakly expressed in the CL at all the stages increased when luteolysis took place. FasL signal increased dramatically on Day 14 and reached the maximum level on Day 21. The expression of Bcl-2 and Bax was detected in a time-dependent manner. At the early stage of CL development, Bcl-2 expression was stronger, while Bax was low. The expression of Bcl-2 and Bax in the CL was completely reversed. Caspase-3 antigen could be detected throughout all the phases of CL development in a time-dependent fashion, low on Day 2 and reaching the maximum on Day 21. These results suggest that luteal regression at the late phases may be related to cell apoptosis.     

Immunohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland epithelium

The expression of the apoptosis-related proteins Bcl-2 and Bax was investigated by immunohistochemistry in the normal non-lactating human mammary gland in relation to cell proliferation and apoptosis. In order to characterize individual Bax/Bcl-2-immunoreactive cells, the epithelial markers cytokeratin 14 and 19 and the macrophage marker CD 68 were used. Secretory-like differentiation of epithelial cells was characterized by histochemistry and lectin staining of surface glycoconjugates. Cell proliferation was exclusively found in glandular epithelial cells with broad contact to the ductular lumen, whereas nuclei with apoptosis-related DNA fragmentation were seen predominantly in basally located glandular epithelial cells and in myoepithelial cells. Weak immunoreactivity for Bcl-2 and Bax was present throughout all epithelia, suggesting a balance between pro- and antiapoptotic effects in the majority of epithelial cells. However, specific cells showed a strong staining for Bax or Bcl-2. The strongly Bcl-2-immunoreactive epithelial cells were not identical with proliferating cells, but they resembled them in configuration and in the luminal intraepithelial position. In contrast, the strongly Bax-positive epithelial cells had no or only a narrow contact to the ductular lumen. The different patterns of Bax/Bcl-2 immunoreactivity in specific glandular epithelial cells suggest that there are also different grades of susceptibility towards apoptotic stimuli in individual glandular epithelial cells. We conclude that specific Bax/Bcl-2 expression patterns could reflect particular cell differentiation states, and that the strongly Bcl-2-positive cells in part could represent epithelial stem cells.     

重组荞麦胰蛋白酶抑制剂对人肝癌细胞的凋亡及半胱氨酸天冬酶活性的影响

先前的研究表明,基因重组荞麦胰蛋白酶抑制剂 (rBTI) 具有诱导不同肿瘤细胞凋亡的作用.为了揭示其诱导肿瘤细胞凋亡的可能机理,从基因水平上探讨与凋亡有关的分子事件,本研究用不同浓度的 rBTI 体外作用于人肝癌细胞 HepG2 后,采用 MTT 比色法检测抑制剂对epG2 细胞的抑制率,用 DNA 凝胶电泳和细胞核的形态学观察检测 HepG2 细胞的凋亡.结果表明,rBTI 在体外能够明显抑制 HepG2 细胞的增长,并诱导细胞凋亡.另外,细胞凋亡与Bcl-2/Bax mRNA 水平有关.通过 RT-PCR 检测发现,细胞经过rBTI处理后,抗凋亡基因Bcl-2 mRNA 水平下调,促凋亡基因 Bax mRNA 有所上调,而对照 GAPDH 无变化.对 HepG2细胞中 Fas/Fas 配体及半胱氨酸天冬酶（caspase）的研究证明,细胞经过 rBTI 处理后,对死亡受体 Fas mRNA没有影响; rBTI 可明显激活caspase-3 和 caspase-9 酶活性, 对caspase-8 活性几乎无影响.上述结果表明,rBTI 对HepG2 细胞具有明显的诱导凋亡作用,其诱导细胞凋亡的机制与 caspase-3 依赖性凋亡调节信号通路有关,未涉及 Fas/Fas 配体途径.     

Reciprocal control of apoptosis and proliferation in cultured rat hepatoma arl-6 cells: Roles of nutrient supply, serum, and oxidative stress

Summary In order to understand how cancer cells accumulate, rat hepatoma ARL-6 cells were cultured for 8 d to identify factors involved in spontaneous cell proliferation and apoptosis. With increasing time in culture, the proportion of cells in the proliferative phases of the cell cycle and the rate of deoxyribonucleic acid (DNA) synthesis decreased. The waning of proliferation was associated with a gradual reduction of cell viability, and this was temporally related to the appearance of typical apoptotic morphology and DNA laddering. Medium replacement or supplementation with fetal calf serum (FCS) suppressed apoptosis, while medium change, but not fetal calf serum alone, enhanced cell proliferation. Apoptosis was also suppressed by dimethyl sulfoxide (DMSO), but supplementary glutathione was without effect. Expression of poly(adenosine diphosphate[ADP]-ribose)polymerase peaked on days 3–4 of culture, and was followed by a progressive decrease thereafter, consistent with proteolytic cleavage. This decrease was prevented to varying extents by complete medium replacement, FCS and DMSO, indicating a close temporal relationship between poly(ADP-ribose)polymerase activation and apoptosis. Expression of Fas and Bcl-2 did not change appreciably over the 8-d culture, but there was a gradual increase in Bax expression; medium change, FCS and DMSO all partly inhibited Bax expression. These data indicate that spontaneous apoptosis in cultured ARL-6 cells is inversely related to cell proliferation, and that nutrient supply, and to a lesser extent, serum-derived factors and oxidative stress modulate apoptosis in this system. Proteolytic cleavage of poly(ADP-ribose)polymerase and expression of Bax are likely to be mechanistically involved with the control of spontaneous apoptosis in ARL-6 cells, whereas changes in the levels of Fas and Bcl-2 do not play a role.     

Phenylbutyrate-induced apoptosis and differential expression of Bcl-2, Bax, p53 and Fas in human prostate cancer cell lines

OBJECTIVE: To assess the mechanisms of action of phenylbutyrate (PB), an investigational chemotherapeutic agent for prostate cancer (PCa), in apoptosis induction in PCa cell lines in vitro. STUDY DESIGN: We analyzed the differential expression of different apoptosis modulators, Bcl-2, Bax, p53 and Fas, for their potential role in PB-induced apoptosis using relative quantitative flow cytometry (FCM). Both androgen-dependent (LNCaP) and androgen-independent (C-4-2, PC-3-PF and DU145) human PCa cell lines were examined. RESULTS: PB induced apoptosis in PCa cell lines in a dose-dependent manner. Fifty percent apoptosis could be induced by 5-10 mM PB. Bcl-2 was down-regulated 30-75% and the Bax:Bcl-2 ratio elevated in apoptotic PCa cell lines regardless of their androgen dependency or p53 status. FCM revealed a heterogeneous stimulatory effect on the expression of Bax and Bcl-2 in PC3-PF cells at 0.5-2.5 mM PB. In a p53-positive cell line (DU145), p53 was repressed by 70% and Fas elevated sixfold with 10 mM PB. CONCLUSION: Our data show that PB-induced PCa apoptosis is associated with the relative repression of Bcl-2 and with up-regulation of Bax and Fas proteins and that this PB-induced apoptosis is independent of p53 and androgen-dependency status of PCa cell lines.     

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

Induction of cell death in rat small intestine by ischemia reperfusion: differential roles of Fas/Fas ligand and Bcl-2/Bax systems depending upon cell types

Although ischemia reperfusion (I/R) induces apoptotic damage of mammalian small intestine, the molecular mechanism is largely unknown. We investigated the appearance of apoptosis at various time-points (0–24 h) of reperfusion after 1-h ischemia and the expression of various apoptosis-related proteins, such as Bcl-2, Bax, Fas, Fas ligand (FasL), activated caspase-3, and cytochrome c, immunohistochemically in rat small intestine. As assessed by TUNEL and electron microscopy, apoptotic cells were increased at 3 h of reperfusion in all intestinal parts (villous epithelium, crypt epithelium, and stroma of intestine). Moreover, the TUNEL-positive cells in the stroma were later identified as T cells. The expression of Fas and FasL as well as activated caspase-3 was markedly increased at 3 h of reperfusion in the stroma. In the villous epithelium, a transient decrease in Bcl-2 expression was found while in the crypt epithelium, Fas expression was induced. Finally, intraperitoneal injection of leupeptin (an SH-protease inhibitor) after I/R resulted in a significant inhibition of the induction of apoptosis in the stroma and crypt epithelium. Our results indicate that the triggering molecules of apoptosis in the I/R rat small intestine may vary depending on cell type and that the use of a broad-spectrum protease inhibitor may reduce intestinal damage.