| Summary: | 博士 === 高雄醫學大學 === 醫學研究所博士班 === 95 === From our previous study results, it is getting more and more clear that failure in the induction of heat shock protein (Hsps) is highly associated with the poor outcome of sepsis, and their over-expression significantly do benefit to subjects facing a severe infection. Moreover, the Hsps synthesis is inhibited during sepsis, which might be a crucial factor leading to a poor prognosis of the disease. However the mechanism is still obscure. The expression of Hsps is regulated by a multi-step manner. Heat shock factor 1 (HSF-1) is recognized as a central component of the hsp gene expression. Recently, glutamine is attracted by researchers and believed to play a beneficial effect in many tissues of sepsis by inducing Hsps, although the molecular mechanism is still not clear. Unfortunately, majority of the reported data show that the liver, a central organ of metabolism of the body, seems less responsive to the treatment. Accordingly, the aims of this study were:
1) to investigate the mechanism of transcription failure of Hsp72 in sepsis, focusing on the HSF-1 turn-activation, and the possible effect of heat shock pretreatment was discussed
2) to explore the effect of glutamine in Hsp72 expression in experimental septic livers
3) to evaluate the effect of glutamine-induced activation of hsp72 gene preconditioned with heat shock treatment
Experimental sepsis was induced by cecal ligation and puncture (CLP) in adult male Sprague-Dawley (SD) rats. Heat shock treatment was applied to the rats’ whole bodily using an electric heating pad. Glutamine was administered 1 hr after initiation of sepsis via tail vein injection. To investigate the effect of glutamine in Hsp70 gene expression in heat-shock pretreated rats, glutamine was administered via tail vein injection 24 hours after heat shock treatment. Quercetin, an inhibitor of Hsps expression, was treated 6 hours before heat shock treatment.
First, the results showed that both non-phosphorylated HSF-1 and phosphorylated HSF-1 were detectable in the cytosolic protein, while non-phosphorylated HSF-1 was only detectable in nuclear protein of early and late phase of sepsis. HSF1-HSE binding activity was detectable but neither the hsp mRNA nor Hsp72 was expressed in early or late stage of sepsis. The expression of PKC?? was not different in cytosolic protein during all phases, but that in the nucleus was significantly different between non-heat and heat group. These results suggest that, in sepsis, HSF-1 was activated and translocated into the nucleus, and then bound with HSE on the promoter of hsp gene, but the subsequent transcription was inhibited. The nuclear PKC?? might be the critical role in influencing the expression of Hsp72 during sepsis.
Secondary, in the study of the effect of glutamine administration in experimental septic livers, the results showed that Hsp72 content noticeably increased in the livers of preheated rats supplied by glutamine 1 hr after sepsis. However, no further synthesis of Hsp72 was found in septic livers or sham glutamine-treated livers. Hsp72, which was induced by preheat, decreased with time while a large amount of Hsp72 could be detected by glutamine administration. RT-PCR data indicated that Hsp72 mRNA could only be detected in the group treated with preheat and glutamine administration. However, only the preheated group showed the phosphorylation in HSF-1. With the administration of glutamine, the nuclear accumulation of phosphorylated HSF-1 was observed to decline significantly 9 and 18 hr after CLP when the Hsp72 mRNA became detectable. These results demonstrated that Hsp72 could be induced by glutamine in septic liver only if the liver has been preconditioned by heat shock response. The selective facilitating effect might depend on decreasing the accumulation of intranuclear phosphorylated HSF-1 caused by previous heat shock treatment.
Finally, to evaluate the effect of glutamine-induced activation of hsp72 gene preconditioned with heat shock treatment, the results showed that glutamine administration enhanced Hsp72 induction significantly only in rats’ livers preconditioned by heat shock. Further evaluation of hsp72 mRNA synthesis and HSF-1 activation showed that glutamine induced hsp72 mRNA expression through the pathway of HSF-1 activation and that the reactivation of hsp72 mRNA expression was correlated to the amount of intracellular Hsp72 in a dose-dependent manner. In conclusion, glutamine reactivated the Hsp72 gene expression in liver preconditioned by heat shock treatment through the pathway of HSF-1 activation.
In conclusion, HSF-1 phosphorylation was failure during sepsis and heat shock treatment maintained phosphorylated HSF-1 in nucleus. Hsp72 could only be induced by glutamine in septic liver preconditioned by heat shock treatment. The effect might depend on decreasing the accumulation of intranuclear phosphorylated HSF-1 caused by previous heat shock treatment. Glutamine-induced reactivation of hepatic hsp70 gene expression is Hsp72 dependent and the activation might be through the pathway of HSF-1 activation. The present studies provide the fundamental information for exploring the therapeutic potential of glutamine-induced Hsps in sepsis, especially the protection of liver.
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