Attenuation of sepsis-induced apoptosis by heat shock pretreatment in rats

Abstract

Apoptosis is a process by which cells undergo a form of non-necrotic cellular suicide. Although it is a programmed process, apoptosis can be induced by various stressors. During sepsis, apoptosis has been regarded as an important cause of cell death in the immune system, leading to unresponsiveness to treatment. This study was designed to investigate how prior heat shock induction can influence the rate of apoptosis in animals that have experienced sepsis. Sprague-Dawley rats were used, and experimental sepsis was induced by cecal ligation and puncture (CLP). Animals in the heated group were anesthetized and received heat shock by whole-body hyperthermia. They were sacrificed 9 h and 18 h after CLP as early and late sepsis, respectively. Apoptosis was evaluated by "DNA ladder" detection in agarose electrophoresis and Tdt-mediated dUTP nick end-labeling (TUNEL) assay. Hsp72 was detected by Western blot analysis. The results showed that the DNA ladder was detected most clearly in the thymus at the late phase of sepsis with time course dependence, while it showed less clearly in heat shock treated animals. Histopathological study by TUNEL assay obtained similar results in the thymus, where the cortex was more susceptible to apoptosis than the medulla. The Western blot analysis showed that the heat shock induced Hsp72 concomitant with an increase in Bcl-2:Bax ratio. In conclusion, heat shock pretreatment prevents rats from sepsis-induced apoptosis that may account for the better outcome of experimental sepsis. An increase in the Bcl-2:Bax ratio may in part explain the molecular mechanism of the effect of heat shock pretreatment.

Fig 1.

Analysis of DNA fragmentation in various organs of late sepsis by agarose gel electrophoresis. Organs were obtained from rats 18 h after cecal ligation and puncture (CLP) operation. DNA was prepared by chloroform/phenol method. Equal amount (8 μg) of DNA was applied to each lane. Li: liver, K: kidney, Lu: lung, T: thymus, S: spleen, P: Peyer's patch, Ly: lymphocytes, M: DNA ladder marker

Fig 2.

Effect of hyperthermia pretreatment on sepsis-inducedapoptosis in the thymus by DNA ladder detection. Hyperthermia pretreatment was performed 24 h before the cecal ligation and puncture (CLP) operation. Equal amounts (8 μg) of DNA were loaded in each lane. S: sham operation, E: sham heated early stage of CLP-induced sepsis, L: sham heated late stage of CLP-induced sepsis, HL: late stage of CLP-induced sepsis with hyperthermia pretreatment, M: DNA ladder marker

Fig 3.

Immunohistochemical detection of apoptotic cells in the thymus during cecal ligation and puncture (CLP)-induced sepsis by TUNEL assay. Apoptotic cells were visualized by fluorescence stain and appeared as bright green spots under fluoromicroscopy. Panels A, B, C, and D indicate the specimens obtained from rats of sham operation, sham heated early stage, sham heated late stage of CLP-induced sepsis, and late stage of CLP-induced sepsis with hyperthermia pretreatment (100×). Panel E indicates high-power magnification of apoptotic cells from sham heated late sepsis (1000×), while panel F is the negative control

Fig 4.

Detection of Hsp72 in the thymus by Western blotting and immunochemical study. Equal amount of cellular extract was loaded to each lane. Bands of Hsp72 and β-tubulin, acting as the internal standard, were quantified using a densitometer. Samples from preheated rats were statistically compared with those of nonheated rats. (A) Immunochemical study. (B) Statistical analysis of relative content of Hsp72 (ratio of ODHsp72 to tubulin). S: sham operation, E: sham heated early stage of CLP-induced sepsis, L: sham heated late stage of CLP-induced sepsis, HL: late stage of CLP-induced sepsis with hyperthermia pretreatment. Number of rats is shown in parentheses in each column. * = p < 0.05

Fig 5.

Detection of Bcl-2 and Bax in the thymus by Western blotting and immunochemical study. Equal amounts of cellular extract were loaded in each lane. Immunochemical detection of Bcl-2 and Bax was carried out simultaneously on the same nitrocellulose membrane, and the bands were quantified by a densitometer. The ratio was calculated and analyzed statistically. (A) Immunochemical study. (B) Statistical analysis of Bcl-2:Bax ratio. S: sham operation, E: sham heated early stage of CLP-induced sepsis, L: sham heated late stage of CLP-induced sepsis, HL: late stage of CLP-induced sepsis with hyperthermia pretreatment. Number of rats is shown in parentheses in each column. * = p < 0.05