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Inhibition of heat-induced apoptosis in rat small intestine and IEC-6 cells through the AKT signaling pathway.

ABSTRACT: BACKGROUND: As the world warms up, heat stress is becoming a major cause of economic loss in the livestock industry. Long-time exposure of animals to hyperthermia causes extensive cell apoptosis, which is harmful to them. AKT and AKT-related serine-threonine kinases are known to be involved in signaling cascades that regulate cell survival, but the mechanism remains elusive. In the present study, we demonstrate that phosphoinositide 3-kinase (PI3K) /AKT signal pathway provides protection against apoptosis induced by heat stress to ascertain the key point for treatment. RESULTS: Under heat stress, rats showed increased shedding of intestinal epithelial cells. These rats also had elevated levels of serum cortisol and improved expression of heat shock proteins (Hsp27, Hsp70 and Hsp90) in response to heat stress. Apoptosis analysis by TUNEL assay revealed a higher number of villi epithelial cells that were undergoing apoptosis in heat-treated rats than in the normal control. This is supported by gene expression analysis, which showed an increased ratio of Bax/Bcl-2 (p?

SUBMITTER: Gao Z

PROVIDER: S-EPMC4220846 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Inhibition of heat-induced apoptosis in rat small intestine and IEC-6 cells through the AKT signaling pathway.

Gao Zhimin Z Liu Fenghua F Yin Peng P Wan Changrong C He Shasha S Liu Xiaoxi X Zhao Hong H Liu Tao T Xu Jianqin J Guo Shining S

BMC veterinary research 20131202

<h4>Background</h4>As the world warms up, heat stress is becoming a major cause of economic loss in the livestock industry. Long-time exposure of animals to hyperthermia causes extensive cell apoptosis, which is harmful to them. AKT and AKT-related serine-threonine kinases are known to be involved in signaling cascades that regulate cell survival, but the mechanism remains elusive. In the present study, we demonstrate that phosphoinositide 3-kinase (PI3K) /AKT signal pathway provides protection ...[more]

PMID: 24295139

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Project description:Intestinal epithelial (IEC-6) cells are resistant to apoptosis following the inhibition of ODC (ornithine decarboxylase) and subsequent polyamine depletion. The depletion of polyamines rapidly activates NF-kappaB (nuclear factor kappaB) and STAT3 (signal transducer and activator of transcription 3), which is responsible for the observed decrease in apoptosis. Since both NF-kappaB and STAT3 signalling pathways can be activated by Src kinase, we examined its role in the antiapoptotic response. Inhibition of ODC by DFMO (alpha-difluoromethylornithine) increased the activity of Src and ERK1/2 (extracellular-signal-regulated kinase 1/2) within 30 min, which was prevented by exogenous polyamines added to the DFMO-containing medium. Conversely, epidermal growth factor-mediated Src and ERK1/2 activation was not prevented by the addition of polyamines. Inhibition of Src with PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} and a DN-Src (dominant-negative Src) construct prevented the activation of Akt, JAK (Janus kinase) and STAT3. Spontaneous apoptosis was increased in DN-Src-expressing cells and the protective effect of polyamine depletion was lost. Polyamine depletion by DFMO increased integrin beta3 Tyr785 phosphorylation. Cells plated on fibronectin had significantly higher beta3 phosphorylation and Src activation compared with plastic. Exogenous polyamines added to the fibronectin matrix prevented Src activation. Arg-Gly-Asp-Ser inhibited beta3, Src and Akt phosphorylation and sensitized polyamine-depleted cells to tumour necrosis factor alpha/cycloheximide-mediated apoptosis. Fibronectin activated Src and subsequently protected cells from apoptosis. Together, these results suggest that the inhibition of ODC rapidly removes a small pool of available polyamines triggering the activation of beta3 integrin, which in turn activates Src. The subsequent Akt and JAK activation is accompanied by translocation of NF-kappaB and STAT3 to the nucleus and the synthesis of antiapoptotic proteins.

Project description:Background and Purpose: Novobiocin (NOVO), an ABC transporter inhibitor, decreases intestinal wall permeability of capsaicin (CAP), an ABC transporter substrate. However, the mechanism of this effect is not consistent with the action of NOVO as an ABC transporter inhibitor. We previously found that CAP can also be transported via TRPV1, which was site-specific in the permeability of CAP across the intestine. We explored the regulation by NOVO of TRPV1 in the present study. Methods: Rats and transfected IEC-6 cells were used as the models to assess intestinal permeability and expression of TRPV1. Ussing chamber and intracellular accumulation were used to evaluate the influence of NOVO on the transport of CAP in vitro. The expression of TRPV1 was detected after administration of NOVO by qRT-PCR, western blot and immunofluorescent imaging. In addition, MTT and lactate dehydrogenase (LDH) were used to evaluate the cytotoxicity of NOVO in both rat and cell models. Finally, the effect of NOVO on the absorption of CAP in vivo was studied by LC-MS/MS. Results: In vitro data showed that there existed a dose-dependent relationship in the range of concentration between 5 and 50 μM, and even 5 μM NOVO could decrease intestinal permeability of CAP across the intestine. Meanwhile, cytosolic accumulation of CAP decreased when NOVO was used simultaneously or 24 h in advance. NOVO exhibited an inhibition level similar to that of ruthenium red (RR) or SB-705498, a TRPV1-specific inhibitor. NOVO down-regulated TRPV1 expression in the intestine and in transfected cells in a concentration-dependent fashion, hinting that its inhibition of the permeability of CAP is due to its inhibition of TRPV1 expression. Immunofluorescent imaging data showed that the fluorescence intensity of TRPV1 was reduced after pre-treatment with NOVO and SB-705498. In vivo data further demonstrated that oral co-administration of NOVO decreased Cmax and AUC of CAP in dosage-dependent ways, consistent with its role as a TRPV1 inhibitor. Conclusion: NOVO could be a potential TRPV1 inhibitor by attenuating the expression of TRPV1 and may be used to attenuate permeability of TRPV1 substrates.

Dataset Information

Inhibition of heat-induced apoptosis in rat small intestine and IEC-6 cells through the AKT signaling pathway.

Publications

Inhibition of heat-induced apoptosis in rat small intestine and IEC-6 cells through the AKT signaling pathway.

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