Protein kinase C-dependent activation of P44／42 mitogen-activated protein kinase and heat shock protein 70 in signal transduction during hepatocyte ischemic preconditioning

AIM: To investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (MAPKs) and heat shock protein (HSP)70 signal transduction during hepatocyte ischemic preconditioning. METHODS: In this study we used an in vitro ischemic preconditioning (IP) model for hepatocytes and an in vivo model for rat liver to investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (P44/42 MAPKs) and heat shock protein 70 (HSP70) signal transduction in IP. Through a normal liver cell hypoxic preconditioning (HP) model in which cultured normal liver cells were subjected to 3 cycles of 5 rain of incubation under hypoxic conditions followed by 5 rain of reoxygenation and subsequently exposed to hypoxia and reoxygenation for 6 h and 9 h respectively. PKC inhibitor, activator and MEK inhibitor were utilized to analyze the phosphorylation of PKC, the expression of P44/42 MAPKs and HSP70. Viability and cellular ultrastructure were also observed. By using rat liver as an in vivo model of liver preconditioning (3 cycles of 10-min occlusion and 10-min reperfusion), in vivo phosphorylation of PKC and P44/42MAPKs, HSP70 expression were further analyzed. AST/ALT concentration, cellular structure and ultrastruture were also observed. All the data were statistically analyzed. RESULTS: Similar results were obtained in both in vivo and in vitro IP models. Compared with the control withouts IP (or HP), the phosphorylation of PKC and P44/42 MAPKs and the expression of HSP70 were obviously increased in IP (or HP) treated model in which cytoprotection could be found. The effects of preconditioning were mimicked by stimulating PKC with 4β phorobol-12-myristate 13-acetate (PMA). Conversely, inhibiting PKC with chelerythrine abolished the protection given by preconditioning. PD98059, inhibitor of MEK (the upstream kinase of P44/42MAPKs), also reverted the cytoprotection exerted by preconditioning. CONCLUSION: The results demonstrate that preconditioning induces a rapid activation of P44/421VlAPKs and PKC activation plays a pivotal role in the activation of P44/42 MAPKs pathway that participates in the preservation of liver cells. HSP expression is regulated by signals in PKC dependent P44/42 MAPKs pathway.     

Effect of acetyl-L-carnitine on hypersensitivity in acute recurrent caerulein-induced pancreatitis and microglial activation along the brain’s pain circuitry

BACKGROUND Acute pancreatitis(AP)and recurring AP are serious health care problems causing excruciating pain and potentially lethal outcomes due to sepsis.The validated caerulein-(CAE)induced mouse model of acute/recurring AP produces secondary persistent hypersensitivity and anxiety-like behavioral changes for study.AIM To determine efficacy of acetyl-L-carnitine(ALC)to reduce pain-related behaviors and brain microglial activation along the pain circuitry in CAE-pancreatitis.METHODS Pancreatitis was induced with 6 hly intraperitoneal(i.p.)injections of CAE(50μg/kg),3 d a week for 6 wk in male C57BL/6J mice.Starting in week 4,mice received either vehicle or ALC until experiment’s end.Mechanical hypersensitivity was assessed with von Frey filaments.Heat hypersensitivity was determined with the hotplate test.Anxiety-like behavior was tested in week 6 using elevated plus maze and open field tests.Microglial activation in brain was quantified histologically by immunostaining for ionized calcium-binding adaptor molecule 1(Iba1).RESULTS Mice with CAE-induced pancreatitis had significantly reduced mechanical withdrawal thresholds and heat response latencies,indicating ongoing pain.Treatment with ALC attenuated inflammation-induced hypersensitivity,but hypersensitivity due to abdominal wall injury caused by repeated intraperitoneal injections persisted.Animals with pancreatitis displayed spontaneous anxiety-like behavior in the elevated plus maze compared to controls.Treatment with ALC resulted in increased numbers of rearing activity events,but time spent in“safety”was not changed.After all the abdominal injections,pancreata were translucent if excised at experiment’s end and opaque if excised on the subsequent day,indicative of spontaneous healing.Post mortem histopathological analysis performed on pancreas sections stained with Sirius Red and Fast Green identified wide-spread fibrosis and acinar cell atrophy in sections from mice with CAE-induced pancreatitis that was not rescued by treatment with ALC.Microglial Iba1 immunostaining was significantly increased in hippocampus,thalamus(intralaminar nuclei),hypothalamus,and amygdala of mice with CAE-induced pancreatitis compared to na?ve controls but unchanged in the primary somatosensory cortex compared to na?ves.CONCLUSION CAE-induced pancreatitis caused increased pain-related behaviors,pancreatic fibrosis,and brain microglial changes.ALC alleviated CAE-induced mechanical and heat hypersensitivity but not abdominal wall injury-induced hypersensitivity caused by the repeated injections.     

Heat shock proteins and autophagy in rats with cerulein-induced acute pancreatitis

Background/Aims

Heat shock proteins (HSPs) protect rats from cerulein-induced acute pancreatitis (AP) by preventing the subcellular redistribution of cathepsin B and the activation of trypsinogen. Autophagy plays a critical role in the secretion of digestive enzymes and triggering of cerulein-induced AP via the colocalization of trypsinogen and lysosomes. Therefore, using a rat cerulein-induced AP model, we investigated whether HSPs prevent AP by regulating autophagy.

Methods

Twelve hours after fed standard laboratory chow and water, the experimental groups (cerulein, water-immersion [WI]-cerulein and heat-shock [HS]-cerulein) and the control groups (control, WI, and HS) received one intraperitoneal injection of cerulein (50 µg/kg) or saline, respectively. All of the rats were sacrificed at 6 hours after injection. The severity of the AP was assessed based on the serum amylase level and the histological and electron microscopy findings. Western blotting was also performed for HSP60/70 and LC3B-II.

Results

WI and HS induced HSP60 and HSP70, respectively. The induced HSP60/70 effectively prevented the development of cerulein-induced AP. Autophagy developed in the rats with cerulein-induced AP and was documented by the expression of LC3-II and electron microscopy findings. The WI-stressed rats and HS-treated rats did not develop cerulein-induced autophagy.

Conclusions

HSPs exert protective effects against cerulein-induced AP in rats by inhibiting autophagy.     

热激蛋白70(HSP70)及血吸虫HSP70研究进展

热激蛋白(heat shock proteins,HSP)是细胞或生物体受到外界各种刺激时产生的一种具有重要生物学功能和具有高度保守性的多肽类蛋白质分子家族,其中HSP70是最受关注且研究较为深入的一种.该文阐述了HSP及血吸虫HSP70的研究进展,并对其研究意义作一综述.     

肺结核患者血浆中热休克蛋白70的表达及意义

目的通过检测肺结核患者外周血中热休克蛋白70(HSP70)的表达情况,旨在探讨HSP70与肺结核的关系。方法采用双抗体夹心ELISA法检测32例活动性肺结核组患者外周血中HSP70蛋白的表达情况,并与25例稳定性肺结核组及30例健康对照组进行对照分析。结果活动性肺结核患者血浆中HSP70蛋白表达水平为(7.5±1.3)ng/ml,显著高于稳定性肺结核组(5.5±1.7)ng/ml及健康对照组(5.3±0.8)ng/m(P<0.01);稳定性肺结核组与健康对照组比较差异无显著性(P>0.05)。结论活动性肺结核患者存在HSP70的过度表达,提示HSP70可能通过某些未明机制参与活动性肺结核的发病,并可辅助肺结核活动性的判断。     

2型糖尿病患者热休克蛋白70基因多态性的变化

目的探讨热休克蛋白70（HSP70）基因多态性与2型糖尿病的关系。方法应用聚合酶链反应-限制性片段长度多态性（PCR．RFLP）方法,检测HSP70基因型在112例2型糖尿病患者和110例对照组的分布情况。结果112例2型糖尿病患者HSP70—1A／A、A／B和B／B3种基因型的分布频率分别为73．2％、23．2％和3．6％,HSP70．2A／A、A／B和B／B3种基因型的分布频率分别是55．4％、34．8％和9．8％,HSP70-hom A／A、A／B和B／B3种基因型频率分别是58．9％、37．5％和3．6％;110例对照组HSP70—1A／A、A／B、B／B基因型分布频率分别是54．5％、40．9％和4．6％,HSP70-2A／A、A／B、B／B3种基因型频率分别是52．7％,36．4％和10．9％,HSP70-hom A／A、A／B、B／B3种基因型频率分别是47．3％、47．3％和5．4％。结论112例2型糖尿病患者HSP70-1A／A、A／B和B／B3种基因型的分布与110例对照组比较差异有统计学意义（P〈0．05）,而HSP70-2和HSP70-homA／A、A／B和B／B3种基因型的分布与对照组比较差异无统计学意义（P〉0．05）。     

热休克蛋白70对供心细胞Bcl-2、Bax蛋白表达的影响

目的探讨热休克蛋白70(HSP70)对大鼠供心心肌细胞凋亡相关基因Bcl-2、Bax蛋白表达的影响。方法Wistar大鼠18只,分为两组:对照组(C,n=9),腹腔注射生理盐水0.5ml,24h后取离体心脏灌注HTK心脏保护液,4℃保存3h后建立Langendorff离体心脏灌注模型,灌注KH液2h;实验组(E,n=9)腹腔注射重酒石酸去甲肾上腺素(溶于生理盐水中)3.1μmol/kg(0.53mg/kg),腹腔注射24h后取离体心脏,处理方法同C组。运用免疫组化SP法测定心肌HSP70含量、Bcl-2、Bax蛋白的含量以及相关生化指标并进行统计学处理。结果HSP70含量E组(17.78±1.82)较C组(5.22±1.05)明显增高(P<0.01),Bcl-2的表达E组(41.88±5.09)较C组(31.36±3.27)明显增多(P<0.01),Bax的表达E组(22.61±3.49)较C组(40.52±4.17)明显减少(P<0.01),Bcl-2/Bax比值E组(1.86±0.11)较C组(0.77±0.01)明显增高。生化指标E组明显优于C组。结论心肌HSP70高表达对供心具有明显的保护效应,其促进心肌抗凋亡蛋白Bcl-2的表达,减少促凋亡蛋白Bax表达,增加Bcl-2/Bax比率,以此抑制心肌细胞凋亡,从而发挥其对供心心肌细胞保护作用。     

兔动脉粥样硬化易损斑块中高表达热激蛋白70细胞与细胞凋亡的关系

目的:探讨热激蛋白(HSP)的高表达和细胞凋亡在兔动脉粥样硬化斑块中的作用。方法:取兔动脉粥样硬化易损斑块28例,镜下将斑块分为4个区,分别为纤维帽区、脂核区、肩部区和中膜平滑肌区;采用免疫组化技术检测各区不同细胞HSP70的高表达情况、原位末端标记法(TUNEL)检测各区不同细胞凋亡情况。结果:纤维帽区与肩部区HSP70的表达阳性炎性细胞数均显著高于脂核区、中膜平滑区,纤维帽区平滑肌细胞HSP70的阳性表达显著高于其他3个区。脂核区TUNEL阳性细胞数明显高于其他3个区。纤维帽区与肩部中表达HSP70的阳性细胞数大于TUNEL阳性细胞数(P<0.05,P<0.01);脂核区内TUNEL阳性细胞数明显大于表达HSP70的阳性细胞数(P<0.01)。结论:易损斑块内HSP70的高表达主要在纤维帽区及肩部区,而细胞凋亡主要在斑块脂核周围。     

HSP70,HSP90在结肠癌中表达及其和生物学行为的相关性

目的:探讨HSP70,HSP90在人结肠癌中的表达情况及其和结肠癌细胞生物学行为的相关性.方法:结肠癌患者手术标本40例,分别以癌组织、癌旁2cm组织和远离癌灶的正常黏膜组织作为病例组和对照组.用亲和免疫组织化学技术方法检测HSP70和HSP90,并收集该40例患者的临床Duke's分期,分析HSP70和HSP90蛋白的阳性程度和结肠癌临床分期是否存在相关性.结果:癌组织HSP70和HSP90的表达明显增高,癌组织、癌旁组织和正常黏膜组织三组之间HSP70和HSP90表达率有明显差异(HSP70:82.5%vs52.5%vs25%,χ2=26.58,P=0.000;72.5%vs42.5%vs22.5%,χ2=20.41,P=0.000);HSP70和HSP90蛋白的阳性程度和结肠癌临床分期存在相关性(HSP70:tau_b=0.392,P=0.006;HSP90:tau_b=0.396,P=0.006).结论:结肠癌细胞存在HSP70和HSP90蛋白的过度表达,HSP70,HSP90和结肠癌细胞的生物学行为存在相关性.     

热休克蛋白70对应激性胃溃疡黏膜的保护作用

目的:探讨热休克蛋白70(HSP70)表达是否随应激性胃渍疡病程的进展而变化.方法:收集30例应激性胃溃疡出血患者及10例术前患者的胃液及外周血,采用TRIzol一步法提取胃液脱落细胞及外周血白细胞总RNA,采用半定量RT-PCR检测HSP70的表达;同时,采用免疫组化技术检测外周血白细胞HSP70的表达.结果:RT-PCR检测结果提示应激性溃疡各期的HSP70表达明显高于对照组的表达(血标本:1期0.6289±0.1839,2期1.1322±0.3683,3期0.6855±0.1923,对照组0.3868±0.2071;胃液标本:1期0.7741±0.2442,2期1.2385±0.4558,3期0.8790±0.2722,对照组0.4626±0.2416:均P＜0.05),应激性溃疡2期HSP70表达显著高于1期和3期(P＜0.05),而1期和3期之间无统计学差异(P＞0.05).免疫组化结果为应激性溃疡患者外周血白细胞HSP70的表达水平在不同病期有所不同(1期40%,2期80%,3期30%,对照组10%,χ2=10.8383,P＜0.05),以2期表达水平最高,对照组表达最低.结论:应激性溃疡时患者外周血细胞和胃液脱落细胞均可检测到HSP70的表达,应激状态可诱导HSP70的表达,HSP70对胃黏膜具有保护作用,并可能促进应激性溃疡的愈合.     

超速起搏预适应的延迟保护与热休克蛋白70的表达

目的观察在超速心室起搏（ventricular overdrive pacing,VOP）预适应延迟保护阶段热休克蛋白70（HSP70）的表达水平。方法新西兰兔24只,随机分为3组,单纯结扎组,起搏组,起搏+放线菌素D组。制作超速起搏预适应和缺血/再灌注的动物模型,检测CK和CK-MB的变化,动态描记再灌注时心电图,免疫组织化染色检测HSP70抗原。结果缺血后起搏组心肌酶的水平在再灌注时均低于单纯结扎组和起搏+放线菌素D组（P<0.01）;单纯结扎组中在再灌注过程中共有5只发生心律失常,起搏+放线菌素组也有4只,而起搏组无心律失常发生。起搏组和单纯结扎组之间有显著性差别（P<0.05）。起搏组HSP70的阳性表达的程度明显高于其他两组。结论超速起搏预适应可以模拟缺血预适应,其延迟保护作用可能与HSP70表达增加密切相关。