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 < 0.05), an important indicator of apoptosis. During heat-induced apoptosis, more AKTs were activated, showing increased phosphorylation. An increase of BAD phosphorylation, which is an inhibitory modification, ensued. In rat IEC-6 cell line, a significant higher level of AKT phosphorylation was observed at 2 h after heat exposure. This coincided with a marked reduction of apoptosis.

Conclusion: Together, these results suggest that heat stress caused damages to rat jejunum and induced apoptosis to a greater degree. HSPs and pro-survival factors were involved in response to heat stress. Among them, AKT played a key role in inhibiting heat-induced apoptosis.

Figure 1

Heat stress induced serious affect on physiology. The weight of rats decreased significantly after heat stress, and rectal temperature significantly increased. Values represent the mean±SD, n=6 rats for each group. *p<0.05 control versus heat stress.

Figure 2

Changes of glucocorticoid concentrations between control and heat-stressed group. In the stressed group, the serum corticosterone increased significantly. Values represent the mean ±SD, n=6 rats for each group. *p<0.05 heat-stressed versus control.

Figure 3

Photomicrographs of hematoxylin and eosin-stained sections of control (A) and heat-treated (B) stressed groups. After treated by hyperthermia, the integrity of small intestine was damaged (jejunum), with desquamation at the top of the intestinal villi and exposure of the lamina propria(indicated by arrows).

Figure 4

Expression of rat HSP genes which were detected by RT-PCR. The expression of HSP genes increased significantly after heat exposure. Values represent the mean ± SD, n = 6 rats for each group. *p <0.05 heat-stressed versus control.

Figure 5

Apoptosis in jejunum of non-treated and heat-treated rats. The apoptotic cells of stressed group (B,D) was increased after stress compared with control (A,C) (indicated by arrows).(A,B 40×; C,D 200×).

Figure 6

Ratio of Bax/Bcl-2 before and after heat stress detected by RT-PCR. The ratio Bax/Bcl-2 was significantly higher in heat-treated group than in control. Values represent the mean ± SD, n = 6 rats for each group. *p <0.05 heat-stressed versus control.

Figure 7

Effect of AKT on heat-induced apoptosis. (A) Phosphorylation of AKT and BAD in rat small intestine was significantly increased after heat exposure. (B) AKT and Bad proteins extracted from rat IEC-6 cells. Cells were treated with heat for 15 min, 30 min, 1 h, 2 h, 4 h and 8 h, respectively. The phosphorylation of AKT and BAD were significantly higher at 2 h of heat exposure than at other time points. (C) was observed after heat exposure. Compared to control, heat stress caused damages to cell morphology. The damage was more serious at 4 h (b,d) of heat exposure than at 2 h(a,c)(a,b 200×; c,d 400×). *p <0.05, ** p <0.01 heat-stressed versus control.