Effect Of Heat Shock Response Research Articles

Quercetin counteracts the pro‐viral effect of heat shock response in grass carp cells with its therapeutic potential against aquareovirus

We have previously shown that heat shock protein (Hsp70) is significantly up-regulated by grass carp reovirus (GCRV) infection, and Hsp70 plays a positive role in facilitating GCRV infection in vitro through unknown mechanisms. Considering the fact that Hsp70 is the most highly induced heat shock protein in cells undergoing the conserved heat shock response (HSR), we determined here that both aquareovirus infection and heat shock induced expression of Hsp70 in grass carp ctenopharyngodon idella kidney cell line (CIK). The pro-viral function of heat shock response was associated with the involvement of Hsp70 in the replication and transcription complex (RTC) of GCRV104 as shown by co-localization of Hsp70 with viral double-stranded RNA (dsRNA). Furthermore, we evaluated the potential of grass carp Hsp70 as a novel fish antiviral target through its down-regulation by plant-derived quercetin, a major dietary flavonoid and known mammalian transcriptional repressor for heat shock factor 1 (HSF-1). In the presence of 50 µM quercetin, grass carp Hsp70 expression was significantly suppressed in cells stressed with either GCRV-104 infection or heat shock. Reduced viral protein expression and descendent virions correlated with the down-regulation of Hsp70 by quercetin in GCRV104-infected grass carp cells, and the pro-viral effect of heat shock could be counteracted by quercetin, which highlighted the involvement of heat shock response protein Hsp70 as a quercetin-mediated antiviral target. Antiviral effects of quercetin in a dose-dependent manner were also observed for GCRV JX-01, a different genotype of GCRV, which predicted a broad-spectrum nature of quercetin in suppressing aquareovirus replication. Overall, the data here indicated that quercetin acted as a specific suppressor for grass carp Hsp70 expression in cells stressed with either aquareovirus or heat shock, and the suppressing effect of quercetin on aquareovirus infection implied its potential as an environmental-friendly and broad-spectrum antiviral drug through targeting fish heat shock response protein.

Heat Shock Response Inhibits NF-κB Activation and Cytokine Production in Murine Kupffer Cells

Kupffer cells play a crucial role in the pathogenesis of sepsis through production of proinflammatory mediators and control of systemic endotoxemia. The anti-inflammatory effects of heat shock response (HSP) have been well documented. However, the role of HSP in lipopolysaccharide (LPS) induced Kupffer cell activation has not been fully investigated. In this study, we investigated the effects of HSP on LPS induced Kupffer cell NF-kappaB activation and cytokine production. Kupffer cells were isolated from mice by collagenase digestion and HSP was induced by culturing Kupffer cells with sodium arsenite. Kupffer cells were stimulated in vitro by LPS. Heat shock protein (HSP)-70 expression and cytoplasmic IkappaBalpha protein was determined by Western blot. Supernatant tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-10 levels were measured by ELISA. NF-kappaB activation was analyzed by electrophoresis mobility shift assay. Cytokine and IkappaBalpha mRNA expression were determined by RT-PCR. Toll-like receptor 4 expression on Kupffer cells was determined by flow cytometry. HSP pre-conditioning significantly inhibited LPS-induced cytokine TNF-alpha and IL-6 production and mRNA expression. NF-kappaB activation and IkappaBalpha degradation induced by LPS were attenuated by HSP. HSP up-regulated expression of IkappaBalpha mRNA. No effect of HSP on cell surface expression of TLR4 was observed. Increased IkappaBalpha stability and up-regulation of IkappaBalpha gene expression may be one of the mechanisms of the inhibition of LPS induced Kupffer cell activation by HSP. HSP also inhibited expression of the anti-inflammatory cytokine IL-10, and the mechanism and biological significance of this effect merit further investigation.

HEAT SHOCK RESPONSE INHIBITS RELEASE OF HIGH MOBILITY GROUP BOX 1 PROTEIN INDUCED BY ENDOTOXIN IN MURINE MACROPHAGES

The purpose of this study was to evaluate the kinetic changes and the localization of high-mobility group box 1 protein (HMGB1) and to observe the effect of heat shock response (HSR) on the expression and release of HMGB1 in lipopolysaccharide (LPS)-activated murine macrophage-like RAW 264.7 cells. Reverse transcriptase (RT)-PCR and Western blot were used to examine HMGB1 expression after LPS treatment. The intracellular localization of HMGB1 in normal or LPS-activated cells was investigated by immunocytochemical analysis and HMGB1 released from cultured macrophages by Western blot. HSR was performed by incubating RAW 264.7 cells at 42.5 degrees C for 1 h then recovery at 37 degrees C for 12 h. The effect of HSR on expression and release of HMGB1 was observed. The results showed that a decrease of HMGB1 mRNA expression was observed at 18 h after LPS (500 ng/mL) treatment, although the total intracellular HMGB1 protein levels were not affected. A visible translocation of HMGB1 from the nuclear to the cytoplasm was observed at 20 h after stimulation with LPS (500 ng/mL). Furthermore, HMGB1 was released into the medium by LPS-activated RAW 264.7 cells in a time- and dose-dependent manner. Heat shock pretreatment significantly inhibited LPS-induced release of HMGB1 and the translocation of HMGB1 from the nucleus to the cytoplasm in RAW 264.7 cells. These findings suggest that the release of HMGB1 by LPS-activated macrophages is a late event in the pathogenesis of sepsis and that HSR could inhibit the release and translocation of HMGB1 induced by LPS.

Inhibition of cyclooxygenase-2 gene expression by the heat shock response in J774 murine macrophages

The heat shock response is a highly conserved mechanism of protection elicited in the cell by various kinds of stimuli, such as heat, sodium arsenite, oxidants and inflammation. Among the mechanisms potentially involved in mediating the protective effects of hsp, one of the most investigated is the inhibition of pro-inflammatory gene expression such as inducible nitric oxide synthase (iNOS) and inflammatory cytokines. Nevertheless, data about the effects of heat shock response on cyclooxygenase-2 expression in activated macrophages are so far not available in literature. The aim of this study was to investigate the changes in cyclooxygenase-2 expression following lipopolysaccharide stimulation of heat shocked J774 murine macrophages. We found, by Western blotting analysis and reverse transcription–polymerase chain reaction analysis (RT-PCR), that the lipopolysaccharide-induced cyclooxygenase-2 gene expression was reduced in heat shocked cells. Such a reduction was associated to activation of heat shock factor, increased levels of heat shock protein 72 and inhibition of lipopolysaccharide-induced nuclear factor-κB binding activity. These data suggest that the heat shock response inhibits cyclooxygenase-2 gene expression at transcriptional level, i.e. by preventing the activation of nuclear factor-κB, and provide additional information about mechanism(s) underlying the anti-inflammatory effect of the heat shock proteins.

Protective effects of heat shock response on circulatory collapse induced by hyperthermia

To investigate the protective effects and mechanism of heat shock response (HSR) on circulatory collapse induced by hyperthermia. Two experiments were carried out: (1) Protective effects of HSR. Rats were divided into 2 groups: heat shock (HS) group, sham control (SC) group. After HS group was pretreated with heat shock and recovered for 20 h at room temperature, both groups were exposed to heat till death, and blood pressure, electrocardiogram were measured continuously during exposure. Mean arterial pressure (MAP), survival time etc were acquired through Chart software. (2) Mechanism of effects. Rats were divided into 3 groups: HS group, SC group and normal control (NC) group. The treatment in HS and SC groups was identical with that in the first experiment, but it would be terminated at 73 min after heat exposure. Systolic pressure (Ps), diastolic pressure (Pd) etc were recorded and content of NO and HSP70 in myocardium were measured. (1) The survival time in HS group [(102.3 +/- 11.4) min] was longer than that in SC group [(87.9 +/- 7.7) min] and shock revealed later (P < 0.01); (2) During early heat exposure MAP in HS group was not different from that in SC group, but after 60 min MAP in HS group were higher than that in SC group; (3) MAP, Ps, Pd, HR and HSP70 in HS group were significantly higher but content of NO was lower than those in SC group (P < 0.01, P < 0.05). HSR may induce upregulation of HSP70 and inhibit excessive production of NO in myocardium, thus result in relief of circulatory collapse induced by hyperthermia.

Differential Regulation of Interleukin-12 and Interleukin-10 by Heat Shock Response in Murine Peritoneal Macrophages

Heat shock response is a conserved stress response and has been shown to have anti-inflammatory effects. We investigated the effect of heat shock response on LPS-induced production of IL-12 and IL-10, which are two important cytokines playing contradictory roles in regulation of immune response, by murine peritoneal macrophages. The data showed that induction of heat shock response strongly suppressed LPS-induced production of IL-12 while augmented that of IL-10, suggesting the pleiotropic effects of heat shock response on immune regulatory gene expression. Also, the novel observation on up-regulation of IL-10 by heat shock response adds to the mechanism by which heat shock response exerts its anti-inflammatory effects.

The Effect of Heat Shock Response on the Tumor Necrosis Factor-alpha-induced Acute Lung Injury in Rats

Background : Heat-treated cells are known to be protected from lysis by TNF, which is considered to play a central role in the pathogenesis of sepsis-induced acute lung injury. The objective of the study was to investigate the effect of heat shock response by heat-pretreatment on the acute lung injury of the rats induced by intratracheally administered TNF-, Methods : We intratracheally instilled either saline or TNF (R&D, 500ng) with and without heat pretreatment in Sprague-Dawley rats weighing 250~350 g. The heated rats were raised their rectal temperature to and was maintained thereafter for 13 minutes at 18 h before intratracheal administration of saline or TNF. After 5 h of intratracheal treatment, lung leak, lung myeloperoxidase activity (MPO) and heat shock proteins were measured in rats. Lung leak index was defined as counts per minute of in the right lung divided by counts per minutes of in 1.0 ml of blood. All data are expressed as means SE. Results : There is no difference in acute lung leak index ( vs ) among the rats given saline intratracheally with and without heat pretreatment, but MPO activity showed a decreased tendency in heat-pretreated rats () compared with heat-unpretreated rats () (P=0.064). Rats administered TNF intratracheally with heat-pretreatment had decreased lung leak index () and lung MPO activity () compared with those of heat-unpretreated and TNF-administered rats (, ) (P