Increased hsp70 of glucocorticoid receptor complex induced by scald and heat stress and its possible effect on the affinity of glucocorticoid receptor

Background Glucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro. Methods The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting.Results Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols.Conclusions The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity.     

Increased hsp70 of glucocorticoid receptor complex induced by scald and heat stress and its possible effect on the affinity of glucocorticoid receptor

Background Glucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro. Methods The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting.Results Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols.Conclusions The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity.     

人参茎叶皂苷对失血性休克大鼠糖皮质激素受体的影响

目的 观察人参茎叶皂昔(ginsenosides，GSS)对失血性休克大鼠糖皮质激素受体(GR)的影响，并分析其作用机制，为研制及时抢救失血性休克患者的天然药物制剂提供实验依据。方法 雄性SD大鼠随机分为失血性休克组和对照组，失血性休克组分别每日ig200，100，50mg／kg GSS水溶液，对照组和模型组ig蒸馏水2mL，共10d。以[^3H]地塞米松为配体，用一点分析法测脑和肝胞液GR结合活性(Rs)、半定量RT—PCR方法测肝胞液GR mRNA水平、放免法测血浆促肾上腺皮质激素(ACTH)和皮质酮(GC)浓度。结果 GSS组大鼠脑和肝胞液的GR结合活性高于单纯失血性休克组，其中以中剂量组最明显(P＜0．01)；GSS组大鼠肝胞液GR mRNA表达水平高于单纯失血性休克组；GSS组大鼠血浆ACTH和GC浓度和单纯失血性休克组没有明显差别。结论 GSS可减轻失血性休克大鼠GR结合活性的下降幅度，作用机制可能与其促进了GR mRNA表达有关，并可能存在一个最佳剂量。     

HSP70在严重烧伤大鼠心肌中的表达及其保护作用的研究

目的 研究热休克蛋白70(HSP70)在严重烧伤大鼠心肌中的表达变化规律，并观察诱导热休克反应对烧伤后心肌损害的保护作用。方法 72只Wistar大鼠随机分为单纯烫伤组(B组)、亚砷酸钠预处理组(SA组)，于致伤前及30％TBSAⅢ度烫伤后3、6、12、24、48h检测血清心肌钙蛋白Ⅰ含量变化，Western blot检测心肌组织HSP70蛋白表达情况。结果 大鼠严重烧伤后3h心肌组织HSP70蛋白表达显著升高，伤后48h达高峰。大鼠烫伤后3h血清心肌钙蛋白Ⅰ(cTnⅠ)含量即开始明显升高，伤后12h达高峰。亚砷酸钠预处理可显著诱导机体的热休克反应，SA组伤后多数时相点血清cTnⅠ含量显著低于B组，且波动幅度较小。结论 严重烧伤早期即可引起心肌组织HSP70蛋白表达显著增强，HSP70蛋白可能在烧伤后早期心肌损害中发挥其内源性抗损伤机制。     

复合辅酶对烫伤休克后大鼠脏器功能的保护作用

目的研究复合辅酶对烫伤休克后大鼠脏器功能的保护作用。方法通过制作大鼠烫伤休克/复苏模型,将40只大鼠随机分为4组：对照组、单纯烫伤组、复合辅酶治疗组、谷胱甘肽治疗组,每组10只。测定大鼠肝功能、心肌酶谱。并采用光镜、透射电镜观察心脏、肝的组织形态及细胞的超微结构。结果单纯烫伤组大鼠肝功能、心肌酶谱指标明显高于其他各组（P〈0.05或P〈0.01）。烫伤大鼠经复合辅酶和谷胱甘肽治疗后各项指标明显改善,复合辅酶组中ALT、CK-MB低于谷胱甘肽组（P〈0.05）。光镜观察复合辅酶治疗组细胞形态基本完整,排列尚规则,炎细胞浸润不明显,细胞损伤较单纯烫伤组、谷胱甘肽治疗组轻。结论复合辅酶对烫伤休克后大鼠的心脏、肝具有保护作用。     

The glucocorticoid resistance in burn and shock

The changes of glucocorticoid receptor (GR) and the reactivity of target cells toglucocorticoids (GC) were studied experimentally.The results showed that GC resistance,which wascaused mainly by the decrease of GR,developed in burned and shocked rats and dogs.Thesechanges may exacerbate the shock state,and even lead to the development of multiple organfailuue.The protection of GC against intestinal ischemic shock of dogs was demonstrated after theconcentration of dexamethasone (Dex) in plasma was elevated to 10~(-6) mol/L by iv injection of 5mgDex/kg body weight.     

人参茎叶皂苷对热损伤大鼠不同脏器糖皮质激素受体的影响

目的:观察人参茎叶皂苷(ginsenosides,GSS)对热损伤大鼠不同脏器糖皮质激素受体(glucocorticoidreceptor,GR)的影响,并探讨其作用机制。方法:雄性SD大鼠随机分为:(1)正常对照组,室温下饲养,蒸馏水灌胃;(2)GSS治疗组,室温下饲养,GSS灌胃;(3)热损伤模型组,蒸馏水灌胃,制作热损伤模型;(4)热损伤模型GSS治疗组,GSS灌胃,制作热损伤模型。采用放射配体结合法检测大鼠脑、胸腺、肺和肝细胞液GR结合活性;逆转录聚合酶链反应法测定脑、肝细胞液GRmRNA的水平;放射免疫法测定血浆促肾上腺皮质激素(adrenocorticotropin,ACTH)和皮质酮(corticosterone,CS)的浓度。结果:热损伤模型GSS治疗组大鼠脑、肺和肝细胞液GR结合活性以及脑和肝细胞液GRmRNA表达水平均明显高于单纯热损伤模型组(P<0.05或P<0.01);热损伤模型GSS治疗组大鼠血浆ACTH和CS浓度与单纯热损伤模型组比较则无明显差异。结论:GSS可缓解热损伤大鼠不同脏器GR结合活性的下降幅度,其作用机制可能与促进GRmRNA的表达有关。     

Effects of HSP70 antisense oligonucleotide on the proliferation and apoptosis of human hepatocellular carcinoma cells

The study investigated the effects of heat shock protein 70(HSP70) antisense oligonucleotide(ASODN) on the proliferation and apoptosis of a human hepatocellular carcinoma cell line(SMMC-7721 cells) in vitro.HSP70 oligonucleotide was transfected into SMMC-7721 cells by the mediation of SofastTM transfection reagent.Inhibition rate of SMMC-7721 cells was determined by using MTT method.Apoptosis rate and cell cycle distribution were measured by flow cytometry.Immunocytochemistry staining was used to observe th...     

热疗作用于肝癌细胞株SMMC-7721后几种凋亡相关基因的表达

目的　探讨热疗前后人肝癌SMMC -772 1细胞几种凋亡相关基因的表达情况 ,解释热疗促进肿瘤细胞凋亡的分子机制。方法　采用免疫组化S- P法检测热疗前后肝癌细胞中P5 3、HSP 70、c Myc蛋白的表达情况。结果　各个热处理组与对照相比 ,HSP-70、P5 3、c Myc蛋白的表达均增高 ,有统计学差异 (P <0 .0 1 ) ;42℃加热 1h和2h与加热 3 0min比较HSP 70蛋白的表达增多 ,有统计学意义 (P <0 .0 5 ) ;而P5 3和c Myc蛋白在各个热处理组间表达没有差异。结论　加热可以诱导HSP70蛋白的大量产生 ,可以激活P5 3基因诱导肝癌细胞凋亡 ,上调c- Myc基因 ,通过调节细胞凋亡来提高细胞的热敏感性。     

生脉散组分配伍对热损伤大鼠糖皮质激素受体的影响

目的：研究生脉散组分配伍对热损伤大鼠糖皮质激素受体（glucocorticoid receptor,GR）的影响。 方法：雄性SD大鼠32只随机分为正常对照组、模型组、人参总皂苷组和生脉散组分配伍组（简称组分配伍组）,每组8只,连续灌胃1周进行预处理。除正常对照组外,其余各组大鼠于末次给药30min后复制大昂热损伤模型。随后立即断头处死动物,收集血清和肝、肺、肾脏组织。酶联免疫吸附测定法（enzyme-linked immkinosorbent assay,ELISA）检测血清皮质酮（corticosterone,CS）的浓度,放射免疫检测法（radioimmunoassay,RIA）检测血清促肾上腺皮质激素（adrenocorticotropic hormone,ACTH）的浓度;放射性配体受体结合法（radioligand receptor binding assay,RRA）检测肝、肺、肾细胞液GR结合容量。 结果：模型组肝、肺、肾胞液GR明显低于正常对照组（P〈0.01）。组分配伍组肝和肺胞液GR结合容量明显高于模型组（P〈0．01）,肾胞液GR结合容量与模型组比较,差异无统计学意义。组分配伍组肝胞液GR明显高于人参总皂苷组（P〈0．01）,肺和肾胞液GR结合容量与人参总皂苷组比较有上升的趋势．但差异无统计学意义。正常大鼠CS及ACTH水平与模型组、组分配伍组和人参总皂苷组比较,差异有统计学意义（P〈0．01）,模型组CS及ACTH水平与各给药组比较,差异无统计学意义。 结论：生脉散组分配伍可增强人参总皂苷上调热损伤大鼠GR水平的作用。     

NMDA受体对烫伤应激大鼠海马糖皮质激素受体基因表达的影响

目的：观察烫伤应激后海马糖皮质激素受体（GR）基因表达的变化，探讨N－甲基－D－天冬氨酸（N－methyl-D-aspartate,NMDA）受体在这种变化中的作用。方法：利用RT－PCR技术，检测烫伤前腹腔注射NMDA受体拮抗剂MK－801及激动剂NMDA对烫伤应激后2h GR mRNA水平的影响。结果：烫伤应激后2h海马GR mRNA水平明显降低（P＜0．01）；应激前腹腔注射MK－801使烫伤后降低的GR mRNA水平明显恢复（P＜0．01），注射NMDA使其进一步降低（P＜0．05），注射生理盐水无明显变化。结论：NMDA受体介导了烫伤应激导致的海马GR转录水平基因表达的下降。     

烫伤后大鼠肝糖皮质激素受体减少的机理——肾上腺部分切除动物的研究

部分去除肾上腺的大鼠烫伤后,血清糖皮质激素无明显升高,而肝胞液糖皮质激素受体(GR)却有升高的趋势,其中烫伤后6 h组和对照组的相差有非常显著的意义。进一步用打点杂交进行的研究表明,部分去腺大鼠烫伤后6h肝GR mRNA也有增加。这些结果提示,完整动物烫伤时肝胞液GR的减少依赖于血中糖皮质激素的升高。     

小鼠急性肺损伤后糖皮质激素受体的活性改变与热休克蛋白的关系

目的 研究急性肺损伤(ALI)后机体内糖皮质激素受体(GR)的配体结合活性显著下降与其分子伴侣热休克蛋白(Hsps)改变的关系.方法 以小鼠油酸急性肺损伤为模型,用Western blotting和受体的放射配体结合试验动态观察伤后GR、Hsp90、Hsp70和GR的结合容量和亲和力变化.结果 伤后小鼠肺组织水肿、肺体指数和肺含水量显著升高;GR在伤后1 h升高,然后显著下降,在12 h达最低点;Hsp90和Hsp70在伤后的表达逐渐升高,在12 h达最高点.配体结合试验显示在伤后GR Bmax逐渐下降,Kd值升高,与Hsp90/GR的变化趋势一致.结论 急性肺损伤后的GR配体结合活性的改变可能与Hsp90的变化有关.     

人hsp90β基因的诱导表达与染色质活化的关系

目的 研究人ｈｓｐ９０β基因的热诱导表达与染色质活化的关系。方法 采用有机汞亲和层析柱分离热诱导前后Ｊｕｒｋａｔ细胞内的活性和非活性染色质。然后采用狭缝杂交技术比较热诱地前后人ｈｓｐ９０β基因在活性和非活性染色质中的分布,并用Ｎｏｒｔｈｅｒｎ印迹杂交的方法研究热休克诱导下的ｈｓｐ９０βｍＲＮＡ表达。结果 发现热休克诱导使细胞内人ｈｓｐ９０β基因在活性染色质中的含量明显增加,并与内源性的ｈｓｐ９０β     

羟基红花黄色素A对烫伤大鼠休克期血管通透性的影响

目的:通过羟基红花黄色素A对烧伤后休克期大鼠的抗渗作用进行实验观察,探讨羟基红花黄色素A在烧伤休克期的应用价值.方法:制作大鼠烫伤模型,将烫伤大鼠随机分为A、B、C三组.A:空白对照组;B:烫伤补液组;C:烫伤补液+羟基红花黄色素A组.用改良伊文思蓝渗出法及称取组织干湿重法测定各组大鼠创周和空肠组织血管通透性、组织含水量.结果:烫伤大鼠血管通透性比较,C组显著低于A和B组(P＜0.05);组织含水量比较,C组显著低于B组.(P＜0.05).结论:羟基红花黄色素A能降低烫伤大鼠血管通透性和组织含水量,减轻组织水肿.     

丙酮酸钠液对烫伤休克大鼠肾血管通透性的影响

目的观察丙酮酸钠对烫伤休克大鼠肾通透性的影响。方法采用50%TBSAⅢ度烫伤大鼠模型,大鼠随机分为假烫组、烫伤组、氯化钠组和丙酮酸钠组,每组6只。应用伊文斯蓝法测定伤后4h肾组织通透性及含水量的变化,检测血肌酐（Cr）及血细胞压积（HCT）变化。结果烫伤后4h单纯烫伤组,氯化钠组肾伊文斯兰含量、Cr、HCT及含水量较假烫组明显增加（P〈0.05）,丙酮酸钠组伊文斯兰含量、Cr、HCT及含水量较单纯烫伤组、氯化钠组明显降低（P〈0.05）,而与假烫组无明显统计学差异。结论丙酮酸钠能明显减轻烧伤早期血管通透性的增加,有助于烧伤后休克及并发症的防治。     

Experimental study on protective effect of chinese herbal medicine on glucocorticoid receptor

Objective: To probe the relationship of glucocorticoid receptor and some Chinese medicinal herbs.Methods: The model of Qi-Yang exhaustion and Qi-Yin exhaustion were made with hemorrhagic rats and heat-stressed rats respectively. The effect of Shenfu decoction (SFD) and Shengmai powder (SMP) on plasma glucocorticoid (GC) and its receptor (GcR) in hepatic cytosol of the models were measured respectively.Results: The activity of GcR decreased in both models, while their blood level of GC increased markedly. SFD and SMP showed no regulating effect on blood GC, but displayed obvious up-regulation on GcR level in both models.Conclusion: SFD and SMP could up-regulate the activity of GcR in Qi-Yang and Qi-Ying exhaustion models. This study was supported by National Natural Science Foundation (No. 30670882)     

外源性p16基因导入对人原发性肝癌细胞迁移能力和VEGF及nm23表达的影响

目的 探讨外源性p16基因导入对人原发性肝癌细胞迁移能力和VEGF及nm23表达的影响。方法 细胞划痕法检测外源性p16基因导入对人肝癌细胞SMMC-7721迁移能力的影响；免疫细胞化学法分析外源性p16基因导入对VEGF表达的影响；Western blotting法分析外源性p16基因导入对nm23表达的影响。结果 外源性p16基因导入人肝癌细胞SMMC-7721后，转染组细胞迁移能力较空载体对照组和空白对照组SMMC-7721细胞明显下降。外源性p16基因导入人肝癌细胞SMMC-7721后，转染组VEGF表达较空载体对照组和空白对照组SMMC-7721细胞减弱。外源性p16基因导入人肝癌细胞SMMC-7721后，转染组nm23表达较空载体对照组和空白对照组SMMC-7721细胞增强。结论 外源性p16基因导入能降低人肝癌细胞SMMC-7721的侵袭和迁移能力。     

Effects of Dexamethasone on glucocorticoid receptor expression in a human ovarian carcinoma cell line 3AO

Ourpreviousworkhasshownthatdexamethasone (Dex) ,asyntheticglucocorticoid ,inhibitshumancarcinomacell(3AO)proliferationandinducesdifferentiationinatime andconcentration dependentmanner 1　Butthemolecularmechanismbehindtheseobservationswasunclear Thepresentstudyinvestigatestheexpressionofglucocorticoidreceptor (GR)in 3AOcellsbyDexaswellastheDex inducedcellulareffectsthatweremediatedbyGR METHODSMaterialsRPMI 16 40mediumwasobtainedfromNissuiPharmaceuticalCo ,Ltd (Tokyo ,Japan) 1,2 ,4 …