Modified mild heat shock modality attenuates hepatic ischemia/reperfusion injury.

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury is a pathologic process caused by hepatic surgery and transplantation, and still remains a severe clinical problem. It was shown that preconditioning by hyperthermia might protect tissues against I/R injury. But hyperthermia could be laborious and time-consuming. Alternatively, the application of mild electrical stimulation (MES) has been reported to have positive effects in clinical settings on several medical ailments. Thus, we modified the preconditioning approach by combining short-term mild heat shock (HS) and MES, and evaluated the effect of HS+MES pretreatment on hepatic injury induced by I/R. MATERIALS AND METHODS: C57BL/6J mice were sham treated or treated three times with HS (42 degrees C) and/or MES (12V) for 20min, carried out every other d within 1 wk. After the last treatment, mice were subjected to hepatic ischemia for 30 or 60min and reperfusion for 6h. Liver injury was assessed by evaluating the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The expressions of pro-inflammatory cytokines and heat shock protein (Hsp) 72 in liver tissues were also assessed by real-time PCR and Western blotting analyses, respectively. RESULTS: HS+MES pretreatment suppressed the hepatic I/R-induced release of serum AST and ALT and the mRNA levels of some pro-inflammatory cytokines. In addition, HS+MES up-regulated the expression of Hsp72 in mice liver. CONCLUSIONS: HS+MES preconditioning ameliorated hepatic I/R injury possibly through Hsp72 induction, and suppressed pro-inflammatory cytokine expression in mice liver.

A novel combination of mild electrical stimulation and hyperthermia: general concepts and applications.

This review discusses the basic concepts, effects and applications of hyperthermia and mild electrical stimulation (MES) using low-intensity direct current. It also proposes a novel combinatorial use of MES and hyperthermia, and briefly outlines the rationale and the effects of MES and hyperthermia combination treatment on certain diseases (diabetes, hepatic ischaemia/reperfusion injury and gastric ulcer). The integrated modalities of MES and hyperthermia might find therapeutic applications to stress-induced diseases and intractable diseases of dysregulated signalling pathways.

IFN-gamma down-regulates Hsp27 and enhances hyperthermia-induced tumor cell death in vitro and tumor suppression in vivo.

Hyperthermia is used as one of the treatment modalities for various types of cancer, but the acquisition of thermotolerance in cancer cells, through the induction of heat shock proteins (Hsps), renders hyperthermia less effective. Among the Hsp family members, Hsp27 is frequently associated with thermotolerance and chemoresistance. Thus, down-regulation of Hsp27 expression during hyperthermic or chemotherapeutic applications is a promising approach to efficient tumor treatment. In the present study, we found that the cytokine interferon-gamma (IFN-gamma) suppresses the basal, the heat shock-induced and the cisplatin-induced expression of Hsp27 in HSC-2 (oral squamous carcinoma) and A549 (lung cancer) cells but not in 16HBE14o- (normal bronchial epithelial cells). Neither IFN-alpha nor IFN-beta affected Hsp27 expression, suggesting the specificity of IFN-gamma. We also demonstrate here that IFN-gamma suppresses Hsp27 basal transcription and promoter activity, and this is mediated specifically through one of the two Sp1 sites in the proximal region of the Hsp27 promoter. More importantly, pre-treatment of cells with IFN-gamma enhanced the induction of cell death by hyperthermia and cisplatin treatments in the tumor cell lines, HSC-2 and A549, but has no effect in 16HBE14o-, indicating a tumor cell-specific effect of IFN-gamma. Furthermore, the combination treatment of hyperthermia and IFN-gamma suppressed tumor growth in vivo more effectively than hyperthermia treatment alone. Together, our findings propose that IFN-gamma could be a useful potentiator of hyperthermia and cisplatin in cancer therapy.

Promoter hypomethylation of Toll-like receptor-2 gene is associated with increased proinflammatory response toward bacterial peptidoglycan in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) is the most common lethal inherited disorder caused by mutation in the gene encoding CF transmembrane regulator (CFTR). The clinical course of CF is characterized by recurrent pulmonary infections and chronic inflammation. Here, we show that toll-like receptor-2 (TLR2) expression and response were strongly enhanced in the human CF bronchial epithelial cell line, CFBE41o-. Treatment of the cells with 5-azacytidine decreased the promoter methylation within TLR2 proximal promoter and increased endogenous expression of TLR2 in non-CF 16HBE14o- cells, suggesting that TLR2 expression is epigenetically regulated by CpG methylation. Moreover, bisulfite sequence analysis revealed that TLR2 promoters were highly demethylated in CFBE41o- cells, implying that decreased methylation of the TLR2 promoter is responsible for CF-related up-regulation of TLR2. Finally, stable expression of WT-CFTR in CFBE41o- cells (CFBE41o-/WT-CFTR cells) reduced TLR2 expression and the response to its ligand peptidoglycan (PGN), implying a causal relationship between CFTR dysfunction and TLR2 up-regulation. Consistent with reduced expression of TLR2 in CFBE41o-/WT-CFTR cells, CpG methylation was increased in CFBE41o-/WT-CFTR cells. Taken together, our results demonstrate that TLR2 expression is epigenetically up-regulated in CF bronchial epithelial cells and suggest that TLR2 overexpression or prolonged activation of TLR2 signaling might be critical in CF pathogenesis.

Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells.

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling. Here, we found that membrane-anchored CD14 is required for LPS-induced downregulation of TLR4 and MD-2 in CHO cells. Moreover, pretreatment of the cells with sterol-binding agent filipin reduced LPS-induced TLR4 downregulation, suggesting the involvement of caveolae-mediated endocytosis pathway. Involvement of caveolae in LPS-induced TLR4 endocytosis was further confirmed by immunoprecipitation. Thus, our data indicate that caveolae-dependent endocytosis pathway is involved in LPS-induced TLR4 downregulation and that this is dependent on membrane-anchored CD14 expression.