Heat shock response in noise-induced hearing loss: effects of alanyl-glutamine dipeptide supplementation on heat shock proteins status / Resposta de choque térmico em perda auditiva induzida por ruído: efeitos da suplementação com dipeptídeos de alanil-glutamina sobre o estado das proteínas de choque térmico

Abstract Introduction: The 72 kDa heat shock protein, HSP72, located intracellularly provides cochlear cytoprotective and anti-inflammatory roles in the inner ear during stressful noise challenges. The expression of intracellular HSP72 (iHSP72) can be potentiated by alanyl-glutamine dipeptide supplementation. Conversely, these proteins act as pro-inflammatory signals in the extracellular milieu (eHSP72). Objective: We explore whether noise-induced hearing loss promotes both intracellular and extracellular HSP72 heat shock response alterations, and if alanyl-glutamine dipeptide supplementation could modify heat shock response and prevent hearing loss. Methods: Female 90 day-old Wistar rats (n = 32) were randomly divided into four groups: control, noise-induced hearing loss, treated with alanyl-glutamine dipeptide and noise-induced hearing loss plus alanyl-glutamine dipeptide. Auditory brainstem responses were evaluated before noise exposure (124 dB SPL for 2 h) and 14 days after. Cochlea, nuclear cochlear complex and plasma samples were collected for the measurement of intracellular HSP72 and extracellular HSP72 by a high-sensitivity ELISA kit. Results: We found an increase in both iHSP72 and eHSP72 levels in the noise-induced hearing loss group, which was alleviated by alanyl-glutamine dipeptide treatment. Furthermore, H-index of HSP72 (plasma/cochlea eHSP72/iHSP72 ratio) was increased in the noise-induced hearing loss group, but prevented by alanyl-glutamine dipeptide treatment, although alanyl-glutamine dipeptide had no effect on auditory threshold. Conclusions: Our data indicates that cochlear damage induced by noise exposure is accompanied by local and systemic heat shock response markers. Also, alanyl-glutamine reduced stress markers even though it had no effect on noise-induced hearing loss. Finally, plasma levels of 72 kDa heat shock proteins can be used as a biomarker of auditory stress after noise exposure.

Resumo Introdução: A proteína de choque térmico de 72 kDa, HSP72 localizada intracelularmente, tem papéis citoprotetores e anti-inflamatórios cocleares na orelha interna durante situações de ruído estressantes. A expressão dessa proteína pode ser potencializada pela suplementação com dipeptídeo de alanil-glutamina. Por outro lado, essas proteínas atuam como sinais pró-inflamatórios no meio extracelular. Objetivo: Investigar se a perda auditiva induzida por ruído promove alterações tanto das proteínas HSP72 intracelulares quanto extracelulares na resposta de choque térmico e se a suplementação com alanil-glutamina pode modificar a resposta de choque térmico e evitar a perda auditiva. Método: Ratos Wistar fêmeas, com 90 dias de idade (n = 32), foram divididos aleatoriamente em quatro grupos: controle, perda auditiva induzida por ruído, tratados com alanil-glutamina e perda auditiva induzida por ruído mais alanil-glutamina. Os potenciais evocados auditivos do tronco encefálico foram avaliados antes da exposição ao ruído (124 dB NPS por 2 h) e 14 dias após. A cóclea, o complexo nuclear coclear e amostras de plasma foram coletadas para mensuração de HSP72 intra e extracelular com um kit Elisa de alta sensibilidade. Resultados: Houve um aumento nos níveis de HSP72 intra e extracelular no grupo perda auditiva induzida por ruído, que foi minimizado pelo tratamento com alanil-glutamina. Além disso, o índice H das HSP72 (razão HSP72 extracelular/HSP72intracelular plasma/cóclea) aumentou no grupo perda auditiva induzida por ruído, mas foi limitado pelo tratamento com alanil-glutamina, embora o alanil-glutamina não tenha efeito no limiar auditivo. Conclusões: Nossos dados indicam que o dano coclear induzido pela exposição ao ruído é acompanhado por marcadores da resposta de choque térmico locais e sistêmicos. Além disso, alanil-glutamina reduziu os marcadores de estresse, mesmo não tendo efeito sobre a perda auditiva induzida por ruído. Finalmente, os níveis plasmáticos de proteínas de choque térmico de 72 kDa podem ser usados como biomarcador do estresse auditivo, após a exposição ao ruído.

Effect of heat shock protein 72 on apoptosis of glomerulus endothelial cells in rats with hypoxic environment / 医学研究生学报

Objective When the body is stimulated by hypoxia , the expression of heat shock protein 72 ( HSP72 ) is in-creased to produce anti-apoptosis effects .The aim of this paper is to study the effect of heat shock protein on apoptosis of cultured rat glomerulus endothelial cells ( GENC) under hypoxic environment . Methods Hypoxia was induced by cobalt chloride ( CoCl2 ) and GENC were divided into 5 groups ( normoxia group , hypoxia group , hypoxia+DMSO group , hypoxia+HSP72 inhibitor group , and hy-poxia+HSP72 agonist group ) according to the different intervention methods .The cell apoptosis was detected by flow cytometry and the expression of HSP72 was detected by Western blot . Results Compared with the normoxia group [(2.21 ±3.80)% and (0.23 ± 0.09)], the apoptosis rate and the expression of HSP72 were in-creased in the hypoxia group , hypoxia +DMSO group , hypoxia +HSP72 inhibitor group , and hypoxia +HSP72 agonist group [(24.54 ±3.59)% and (0.82 ±0.15), (29.25 ±1.63)% and (0.80 ±0.17), (36.07 ±1.19)%and (0.43 ±0.08), (18.10 ±2.59)%and (1.05 ±0.07)] (P0.05). Conclusion Hypoxia can induce the increased GENC apoptosis accompanied with the prolonged hypoxia .The increase or decrease of HSP 72 expression may lead to the decrease or increase of apoptosis , which is an important factor affecting the apoptosis of GENC under hypoxia .

Expression of heat shock protein 72 in liver tissues of patients with biliary atresia / 中国医师杂志

Objective To investigate the relationship between heat shock protein 72 (HSP72) and liver fibrosis in pediatric patients with biliary atresia and the significance of its expression.Methods The clinical and pathological data of 30 patients with biliary atresia were selected from June 2013 to June 2015,and the expression of HSP72 in liver tissues was determined by streptomycin peroxide (SP) immunohistochemical method.Results The greater group of liver fibrosis degree with biliary atresia was less than the lighter group in the expression of HSP72 (Z =-4.16,P ＜ 0.01).There was negative correlation between HSP72 expression intensity and operation age (r =-0.704,P ＜ 0.01).Meanwhile,there was negative correlation between HSP72 expression intensity and postoperative cholangitis (r =-0.371,P ＜ 0.05).When the expression of HSP72 was higher,the autologous liver survival rate was higher (x2 =9.482,P ＜ 0.01).Conclusions The expression of HSP72 in liver tissues of patients with biliary atresia is closely related to the degree of liver tissue fibrosis.HSP72 expression may be one of the factors affecting the prognosis of patients with biliary atresia,and can be used as a reference index for evaluating prognosis.

Effect of Heat Shock Protein 72 Expression on Etoposide-induced Cell Death of Rat Retinal Ganglion Cells

PURPOSE: To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. METHODS: Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. RESULTS: Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. CONCLUSIONS: Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease.

Effects of heat treatment and UVB radiation alone or in combination on the expression of heat shock protein 72 in human epidermal melanocytes / 中华皮肤科杂志

ObjectiveTo explore the effects of heat treatment and ultraviolet B (UVB) radiation alone or in combination on the expression of heat shock protein (HSP) 72 in human epidermal melanocytes.Methods Melanocytes were obtained from human foreskin,and subjected to primary culture.After 3 to 5 passages,the melanocytes were classified into 4 groups:control group (receiving no treatment),heat treatment group (treated with heat at 42 ℃ for 1 hour every day for 3 days),UVB group(irradiated with UVB at 50 mJ/cm2 daily for 3days),combination group(treated with heat at 42 ℃ for 1 hour followed by irradiation with UVB at 50 mJ/cm2daily for 3 days).After another 2- to 6-hour culture following the last treatment,melanocytes were collected and subjected to real time PCR and Western blot for the detection of HSP72 mRNA and protein expression,respectively.ResultsThe mRNA and protein expressions of HSP72 were significantly higher in the heat treatment group and combination group than in the control group (mRNA:6.584 ± 0.871 and 7.269 ± 0.454 vs.0.975 ± 0.089,both P ＜ 0.001; protein:2.022 ± 0.058 and 2.080 ± 0.045 vs.0.532 ± 0.033,both P ＜ 0.001 ),but was similar between the UVB group and control group (mRNA:0.832 ± 0.084 vs.0.975 ± 0.089,P ＞ 0.05;protein:0.546±0.021 vs.0.532 ± 0.033,P ＞ 0.05).The ANOVA of factorial design showed that neither heat treatment nor UVB irradiation had interaction effect on the mRNA or protein expression of HSP72 (F =2.106,1.399 respectively,both P ＜ 0.05).ConclusionsHeat treatment can cause an increase in the expression of HSP72,which may enhance the function of melanocytes and protect melanocytes from UVB induced damage.

Influence of Exercise at High Temperature on Blood Biochemical Indexes and HSP72 Expression in Adult Males / 华中科技大学学报（医学）（英德文版）

Summary: The influence of exercise at high temperature on adult males' routine blood indexes and biochemical indexes and the expression of HSP72 in peripheral blood lymphocytes (PBLs) was studied in order to provide theoretical ground for health supervision of adults receiving exercise at high temperature. 180 adult males were selected and divided into exercise group and control group, in which the exercise group was subdivided into subgroup 1 and subgroup 2 receiving exercise at high temperature in the afternoon and in the morning, respectively. Peripheral venous blood was phlebotomized before and after the exercise to examine routine blood indexes and blood biochemical indexes. The expression levels of HSP72 in PBLs were detected by flow cytometry. The results showed that the routine blood indexes and biochemical indexes in each group were within the range of normal values of male adults. There was no significant difference between each exercise group and control group in indexes before exercise. After exercise, the expression levels of HSP72 in PBLs in exercise groups were higher than those before exercise, and HSP72 expression levels'in subgroup 1 were obviously higher than those in subgroup 2 and control group. The contents of ALT, urea, Na+, Cl-, Ca2+and K+ in subgroups 1 and 2 were lower than those in control group, but CK level was higher than in control group (P<0.05). The contents of Na+ and Cl- in subgroup 1 were relatively lower than those in subgroup 2 (P<0.05). It was concluded that while receiving exercise at high temperature, adult males' HSP72 levels in PBLs could be increased and the biochemical indexes changed. Attention should be paid to health supervision to avoid obvious body injuries at high temperature.

Localization of Heat Shock Protein 72 in Potassium-deprivated Rat Kidney / 대한해부학회지

Heat shock protein 72 (HSP72) appears to play an important role in cell survival in the hypertonic conditions of the renal medulla. The purpose of this study was to examine the effect of potassium deprivation on renal HSP72 expression. Male Sprague-Dawley rats were fed potassium deficient diet for 2 weeks. Kidney tissues were preserved by in vivo perfusion with paraformaldehyde-lysine-periodate (PLP) and processed for Western blot analysis and immunocytochemistry. Serum potassium concentration and urine osmolality decreased in potassium deprivated animals. In control kidneys, HSP72 immunostaining was observed mainly in the inner medulla in almost all cells including the inner medullary collecting duct and papillary surface epithelial cells. In potassium deprivated kidneys, HSP72 expression decreased dramatically in the inner medulla. However, strong HSP72 immunostaining remained in some inner medullary collecting duct and papillary surface epithelial cell. These results demonstrated that potassium deprivation induced down regulation of HSP72 in the renal medulla, at least in part, through cell-specific manner.

Effects of Repetitive Ischemic Preconditioning on the Phosphorylation of Akt and Expression of HSP72 and HSP90 in the Rat Tibialis Anterior and Soleus Muscles / 체질인류학회지

Akt, heat shock protein (HSP72)72, and HSP90 induced by ischemic preconditioning protect cells from the ischemic injury. The purpose of this study was to examine the alterations of the level of phospho-Akt, HSP72, and HSP90 in the rat tibialis anterior and soleus muscles after cyclic episodes of ischemic preconditioning. Sprague-Dawley rats aged 35 weeks were divided into control and ischemic preconditioning (IP) groups. The IP group was divided into 3 subgroups based on cycles of IP. Left common iliac artery was occluded 3, 6, and 10 times for 5 minutes, followed by 5 minutes reperfusion. The experimental animals were sacrificed at 0, 3, 6, 24, and 72 hours after reperfusion, and left tibialis anterior and soleus muscles were removed. The expression of phospho-Akt, HSP72, and HSP90 were examined with immunohistochemical methods and Western blot analysis. The results were as follows; 1. In the 3 and 6 times of IP groups, the expression of phospho-Akt (p-Akt) was increased at 0 and 3 hours after reperfusion, compared with control group. The expression of p-Akt in the 10 times of IP group was lower than that in 3 and 6 times of IP groups. At 72 hours after reperfusion, the expression of p-Akt showed no difference among the IP groups. The expression of p-Akt was higher in Soleus than that in Tibialis anterior. 2. The expression of HSP72 in 3 times of IP group increased at 0 and 3 hours after reperfusion, compared with 6 and 10 times of IP groups. The expression of HSP72 in the 10 times of IP group was lower than that in 3 and 6 times of IP groups. At 72 hours after reperfusion, the expression of HSP72 showed no difference among the IP groups. The expression of HSP72 was higher in Soleus than that in Tibialis anterior. 3. In the 3 and 6 times of IP groups, the expression of HSP90 increased at 0 and 3 hours after reperfusion, compared with control group. The expression of HSP90 in the 10 times of IP group was lower than that in 3 and 6 times of IP groups. At 24 hours after reperfusion, the expression of HSP90 showed no difference with increasing episode of IP. The expression level of HSP90 was higher in Soleus than that in Tibialis anterior. These findings suggest that ischemic preconditioning increases the expression of p-Akt, HSP72 and HSP90 at early phase after reperfusion in the rat tibialis anterior and soleus muscles. However, increased cycles of ischemic preconditioning may not induce the expression of them.

Changes in heat shock protein 72 of regenerating rat skeletal muscle / 体力科学

This study investigated the time course of changes in heat shock protein (HSP) 72, muscle weight and cross-sectional area of muscle fibers after injection of bupivacaine (BPVC) into the soleus muscle. Adult male Wistar rats (n=36) were anesthetized by pentobarvital sodium and 0.5 ml of BPVC was injected into the left soleus muscle, whereas the contralateral right soleus muscle was served as the control. Bilateral soleus muscles were dissected at 1, 2, or 4 days or 1, 2, or 4 weeks after the injection. Muscle fibers were disrupted at days 1-2 after BPVC injection and regenerated fibers that have centrally located nuclei in cytoplasm appeared 4 days after the injection. The cross-sectional area of regenerated fibers gradually increased during 4 days-4 weeks after the injection. The soleus muscle weight decreased until 1 week after the injection, then gradually increased during 1-4 weeks of recovery. The content of HSP 72 was drastically decreased in the soleus muscle 1 day after BPVC injection (p<0.01), then gradually increased during 2 days-4 weeks of recovery, and returned to the control level at 4 weeks of recovery. A similar time course of change was observed for the cross-sectional area of the regenerating fibers and HSP 72 expression. These results suggest that the BPVC injection induces disruption of muscle fibers and degradation of HSP 72, however, regeneration of muscle fibers indicated by increases in cross-sectional area occurs with increases in HSP 72 content during 4 weeks of recovery period.

Heat Shock Protein 72 Expression Following Odor Exposure in Olfactory System of Rat

BACKGROUND AND OBJECTIVES: Heat shock proteins (HSPs) are group of evolutionary conserved proteins whose synthesis are greatly enhanced in cells following exposure to various stressors and play an important role in cellular protection and survival. The purpose of this study was to determine whether olfactory stimulation induces the synthesis of HSP72 in olfactory system of the rat. MATERIALS AND METHODS: Animals were exposed to odorant stimuli using 2% propionic acid odorant stimuli and expression pattern of HSP72 in the olfactory system were detected by immunohistochemistry using anti-HSP72 antibody according to time course and by Western blotting. RESULTS: HSP72 immunopositive cells were expressed in the olfactory epithelium and in the olfactory bulb neurons and a 72 kD band was detected by Western blotting. CONCLUSION: These results suggest that expression of HSP72 in olfactory system of the rat following exposure to odor may serve as a marker for cellular stress and potential damage and may be involved in cellular protection against injuries.

Zinc translocation and heat shock protein induction in rat brains following kainate seizures / 대한해부학회지

Translocation of synaptic zinc may mediate neuronal death in pathological conditions. In this study, we examined the possible correlation between zinc translocation and heat shock protein (HSP)72 induction in rat brains following kainate seizures. Zinc accumulation, visualized by Timm's method, occurred in degenerating neurons in hippocampus, amygdala, and cortex 6~24 h after kainate injection. Immunohistochemistry with anti-HSP72 antibody revealed HSP induction largely in areas where zinc accumulation occurred. At the cellular level, however, most HSP72 immunoreac-tive neurons were found to be Timm (-) and morphologically intact. Present results suggest that intense zinc translocation may induce neuronal death before possible HSP induction. However, we could not rule out the possibility that sublethal zinc translocation, below the detection limit by Timm's method, may play a role in HSP72 induction.

Stress Protein Expression in Kainate-Induced Experimental Temporal Lobe Epilepsy in Rats

To investigate neuronal injury developed in an experimental model of temporal lobe epilepsy, the expression of c-FOS, c-JUN and HSP72 on the amygdala, hippocampus and temporal neocortex was studied. Epileptic seizure was induced in rats by microinjection of kainic acid(1microgrm/microl) dissolved in phosphate buffer(0.1 M, pH 7.4) into the left amygdala. Selective and delayed neuronal injuries appeared in the CA3 region of the hippocampus after 14 days and were characterized by swelling of cytoplasm and neurites, nuclear pyknosis and loss of neurons. Early induction of c-FOS and c-JUN was noted on the injection-side amygdala at 1-12h, and delayed expression developed at 7 days after the injection. HSP72 expression appeared continuously 3 hrs after the injection. Delayed induction of c-FOS, c-JUN and continuous expression of HSP72 were observed in the hippocampus and entorhinal cortex. In the hippocampus, c-FOS expression was relatively strong in the neurons of CA3 and dentate gyrus at 7~14 days after the injection. Similar findings were also noted in the neurons of the entorhinal cortex. Induction of HSP72 occurred slightly later than on that of c-FOS and c-JUN in the amygdala, with the prominent induction being noted in the neurons of amygdala, CA2, CA3, CA4 and dentate gyrus at 3 to 21 days after the injection. These results suggested that the delayed expressions of c-FOS and c-JUN in the hippocampus correlated well with impending clinical epileptic seizure.

A Study of the Expression of Heat Shock Protein 72 in Guinea PigCochlea after Cisplatin Injection / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Several studies have attempted to show the reasons for ototoxicity induced by cisplatin, but the cochlear ototoxicity remained poorly understood. Recently, it is considered that free radicals play an important role in cisplatin ototoxicity. Heat shock proteins (HSP) are consistently present at some level in normal tissues and increased synthesis following stress. They have been implicated in the role of cellular protection during sub-lethal stressors. Many studies have demonstrated the increased synthesis of HSP following hyperthermia, ischemia, surgical injury and noise exposure. Free radicals are also considered as important inducer of HSP. The purpose of this study was to demonstrate the increase of HSP after cisplatin injection. MATERIALS AND METHOD: Thirty six Albano guinea pigs were used in this study. The animals were injected with a dose of 8 mg/kg cisplatin intraperitoneally. Cochleae are harvested 1, 3, 5 or 6, 12, 24 hours after injection. Immunocytochemistry and surface preparation method were used to detect the expression of HSP 72 in the cochlear tissues. RESULTS: The level of HSP 72 immunoreactivity began increasing by 3 hours after injection and continued to increase thereafter to reach maximal levels at 6 hours. Twelve hours after injection, the level of HSP 72 seemed to decrease to its normal levels. The increase of HSP 72 was mainly detected in Deiters' cells. CONCLUSION: Cisplatin induces a HSP in the guinea pig cochlea, particularly in the organ of Corti. However, further studies including quantitative analysis should be followed.