臭氧氧化预处理对大鼠肾缺血再灌注损伤的热休克蛋白表达的影响

目的 探讨臭氧氧化预处理通过诱导热休克蛋白70(HSP70)的合成,保护大鼠肾脏缺血再灌注损伤的作用与机制.方法 建立原位大鼠单侧肾缺血再灌注动物模型,I/R前15 d经直肠吹入氧气和臭氧的混合气体5.0～5.5 ml(臭氧浓度50 mg/L,1 mg/kg体蕈,每日 1次).全自动生化分析仪检测尿素氮(BUN)、肌酐(Cr),比色法测定血清的脂质过氧化产物丙二醛(MDA)、超氧化物歧化酶(SOD).Western blot检测HSP70蛋白的含量;逆转录聚合酶链反应(RT-PCR)方法检测HSP70的表达.结果 肾缺血再灌注24 h后,血清中BUN、Cr、MDA明显增高,肾组织内HSPT0表达明显增强(P<0.05),经臭氧氧化预处理后,血清中的BUN、Cr、MDA均降低,SOD升高;HSP70表达升高更加明显(P<0.05).结论 臭氧氧化预处理可以诱导大鼠肾缺血再灌注组织中HSP70表达,减轻大鼠肾脏缺血再灌注损伤.     

臭氧氧化预处理对大鼠肾缺血再灌注损伤的热休克蛋白表达的影响

目的 探讨臭氧氧化预处理通过诱导热休克蛋白70(HSP70)的合成,保护大鼠肾脏缺血再灌注损伤的作用与机制.方法 建立原位大鼠单侧肾缺血再灌注动物模型,I/R前15 d经直肠吹人氧气和臭氧的混合气体5.0～5.5 ml(臭氧浓度50 mg/L,1 mg/kg体重,每日1次).全自动生化分析仪检测尿素氮(BUN)、肌酐(Cr),比色法测定血清的脂质过氧化产物丙二醛(MDA)、超氧化物歧化酶(SOD).Western blot检测HSP70蛋白的含量;逆转录-聚合酶链反应(RT-PCR)方法检测HSPT0的表达.结果 肾缺血再灌注24 h后,血清中BUN、Cr、MDA明显增高,肾组织内HSP70表达明显增强(P＜0.05),经臭氧氧化预处理后,血清中的BUN、Cr、MDA均降低,SOD升高;HSP70表达升高更加明显(P＜0.05).结论 臭氧氧化预处理可以诱导大鼠肾缺血再灌注组织中HSP70表达,减轻大鼠肾脏缺血再灌注损伤.     

胸腺瘤细胞中PCNA、HSP70的表达水平的研究及意义

目的 研究PCNA、HSP70在胸腺瘤细胞及正常胸腺组织中的表达水平及其临床意义。方法 用免疫组织化学链霉菌抗生物素蛋白－过氧化物酶染色法（S—P法）检测20例恶性胸腺瘤、18例良性胸腺瘤及12例正常胸腺组织细胞中PCNA、HSP70蛋白的表达水平。结果 20例恶性胸腺瘤中PCNA、HSP70蛋白阳性表达分别为80．0％（16／20）、75．0％（15／20）；18例良性胸腺瘤中PCNA、HSP70蛋白阳性表达分别为38．9％（7／18）、27．8％（5／18）；12例正常胸腺组织细胞中分别为8．3％（1／12）、0（0／12），PCNA蛋白及HSP70蛋白的表达在良、恶性胸腺瘤和正常胸腺组织三者之间差异都有显著性（P〈0．001），且在良、恶性胸腺瘤之间相比差异也均有显著性（P〈0．05）。但良性胸腺瘤和正常胸腺组织相比，PCNA及HSP70蛋白的表达差异均无显著性（P〉0.05）。结论 通过检测HSP70与PCNA在胸腺瘤的表达水平，可以判断胸腺瘤的良、恶性，为术后进一步治疗提供了有价值的参考指标。     

氨基酸对大鼠肾缺血再灌注损伤bcl-2和HSP70表达影响的研究

目的：探讨8种L—支链氨基酸合剂对急性肾缺血—再灌注损伤的保护作用及作用机制。方法：夹闭大鼠双侧肾蒂45min，再灌注24h制成肾缺血再灌注损伤动物模型，将24只SD大鼠随机分为假手术组(A组)、对照组(B组)、氨基酸治疗组(C组)，通过检测尿量、血尿素氮(BUN)、血肌酐(Cr)，肾组织苏木精—伊红染色及用免疫组织化学进行bcl—2和HSP70表达检测。结果：3组肾组织中bcl—2和HSP70均有表达，但免疫反应强度不同。与A组相比，B组尿量增加，血BUN、Cr升高，肾小管上皮细胞呈现缺血性改变，HSP70表达明显增强，两组间差异有显著性意义，而bcl—2表达差异无显著性意义。与B组相比，C组尿量明显增加，血BUN、Cr明显下降，且缺血损伤程度有减轻，bcl—2表达明显增强，两组间差异有显著性意义，而HSP70表达差异无显著性意义。结论：该氨基酸合剂对大鼠急性肾缺血再灌注损伤有明显保护作用，其作用机制可能与它影响bcl—2表达有关。     

大鼠全脑缺血再灌注模型方法的改进及其在热休克蛋白70方面的应用

目的　改进大鼠全脑缺血再灌注模型的方法。方法　采用 42只成年雄性SD大鼠 ,四动脉阻断法制作全脑缺血再灌注模型 ,术中乙醚麻醉 ,41/ 2号注射器针头破坏椎动脉 ,动脉夹夹闭颈总动脉 ,苏木素 伊红 (HE)染色观察海马区病理改变 ,免疫组织化学法检测海马CA1区热休克蛋白 70 (HSP70 )表达 ,苏木素在体染色大体观察脑血供的阻断情况。结果　全脑缺血模型成功率为 77.8% ,四动脉阻断的动物海马区神经元大量坏死 ,HSP70表达 ,苏木素在体染色阴性 ,单纯颈总动脉阻断组和假手术组未见上述表现。结论　改进后的四动脉阻断方法简便 ,效果可靠 ,利于推广。     

Effects of heat stimulation via microwave applicator on cartilage matrix gene and HSP70 expression in the rabbit knee joint

The objective of this study was to investigate the effects of heat stimulation on the expression of extracellular matrix genes and heat‐shock protein 70 (HSP70) in rabbit articular cartilage in vivo. Heat stimulation was applied to the knee joints of Japanese white rabbits for 20 min using a microwave (MW) applicator (2.45‐GHz, 0–80 W). After 8–72 h, the articular cartilage was removed from the knee joints and proteins and total RNA were extracted. As controls, knee joints without heat stimulation were analyzed. The expression of HSP70 was confirmed by real‐time PCR and Western blotting. The expression of proteoglycan core protein (PG) and type II collagen (Col II) was quantified using real‐time PCR to assess cartilage matrix metabolism. Compared to controls, HSP70 expression was higher with more than 40 W of heat stimulation. The expression of PG and Col II mRNA was higher, with more than 20 W of heat stimulation and peaked with 40 W. When quercetin was used to inhibit the induction of HSP70 expression, PG mRNA expression did not increase. External MW application stimulated HSP70 expression in the articular cartilage in vivo. The expression of extracellular matrix genes was increased by appropriate heat stimulation. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:34–41, 2008     

热休克蛋白70保护肝缺血再灌注损伤的研究进展

肝缺血再灌注（IR）损伤是引起肝脏损害的重要因素,已成为研究的热点和难点。近年来研究发现HSP70在组织、器官的IR损伤中起了重要的保护作用,笔者就其在肝IR损伤中的作用作一综述,并重点阐述其作用机制,如调控炎症因子、抗细胞凋亡以及抗氧化作用等。     

Chronic bladder ischemia and oxidative stress: New pharmacotherapeutic targets for lower urinary tract symptoms

Chronic bladder ischemia is potentially a common cause of lower urinary tract symptoms in the elderly. Epidemiological studies have shown a close association between lower urinary tract symptoms and vascular risk factors for atherosclerosis, and investigations using transrectal color Doppler ultrasonography have shown a negative correlation between decreased lower urinary tract perfusion and International Prostate Symptom Score in elderly patients with lower urinary tract symptoms. Bladder blood flow is also known to decrease in men with bladder outlet obstruction as a result of benign prostatic hyperplasia. Studies in animal models suggest that chronic bladder ischemia and repeated ischemia/reperfusion during a micturition cycle might produce oxidative stress, leading to denervation of the bladder and the expression of tissue‐damaging molecules in the bladder wall, which could be responsible for the development of bladder hyperactivity progressing to bladder underactivity. The effects of drugs with different mechanisms of action; for example, α₁‐adrenoceptor antagonists, phosphodiesterase type 5 inhibitors, free radical scavengers and β₃‐adrenoceptor agonist, have been studied in animal models of chronic bladder ischemia. The drugs, representing different treatment principles for increasing blood flow and decreasing oxidative stress, showed protective effects not only on urodynamic parameters, but also on negative effects on muscle contractility and on detrimental structural bladder wall changes. Improvement of lower urinary tract perfusion and control of oxidative stress can be considered new therapeutic strategies for treatment of bladder dysfunction induced by chronic ischemia.     

热休克蛋白70-2基因沉默对大鼠精子附睾成熟的影响

目的 观察热休克蛋白(HSP)70-2基因沉默对大鼠附睾中精子成熟的影响.方法 构建HSP70-2特异的短发夹RNA(shRNA)质粒载体,注入大鼠附睾中,1周后处死大鼠,无菌切取附睾,用逆转录-聚合酶链反应(RT-PCR)方法 检测附睾中HSP70-2 mRNA表达,Western blot观察HSP70-2蛋白表达,应用苏木素-伊红(HE)染色检测附睾管内精子密度.结果 空白对照组HSP70-2蛋白表达量为1.31±0.10,附睾管内成熟精子密度为(63.46±14.43)%.基因沉默组HSP70-2蛋白表达量为0.93±0.13,附睾管内成熟精子密度为(31.51±10.67)%,与对照组比较均显著减少,差异有统计学意义(P＜0.01).结论 HSP70-2基因沉默影响附睾中精子成熟.     

乙醇预处理与HSP70的诱导及对肝脏缺血再灌流损伤的影响

目的探讨乙醇预处理对肝脏缺血再灌流损伤的影响以及与热休克蛋白70诱导的关系。方法雄性成年Wistar大鼠232只,胃饲乙醇浓度40%,剂量为5 g/kg体重,随机分为5组:正常对照组(N组)、胃饲乙醇组(E组)、缺血组(IR组)、生理盐水预处理组(NPC组)、胃饲乙醇预处理组(EPC组)。动物手术采用门静脉转流下的肝脏缺血模式,肝门阻断时限为90 min,于再灌流0、13、、6、12、24、72 h活杀留取血液及肝脏标本。结果EPC组3、6 h血清ALT(1 230.88±132.50、888.88±126.67)IU/L、AST(1 866.38±61.77)(、1 433.88±42.74)IU/L均明显低于IR组及NPC组,肝脏病理改变较轻,而肝组织HSP70含量高于后者。结论乙醇预处理可以减轻大鼠肝脏90 min的缺血再灌流损伤,其肝脏保护作用与肝组织HSP70升高相一致,HSP70可能是其发挥肝脏保护的物质基础之一。     

Combined microwave irradiation and intraarticular glutamine administration‐induced HSP70 expression therapy prevents cartilage degradation in a rat osteoarthritis model

The objective of the present study was to investigate the effects of heat stimulation and glutamine (Gln) on the expression of extracellular matrix genes and heat shock protein 70 (HSP70) in rat articular cartilage in vivo and to determine whether HSP70 expression achieved with a combination of microwave (MW) and Gln suppresses osteoarthritis (OA) progression in a rat OA model. Stimulation at 40 W was assumed to be appropriate in the present study, and the effects of heat treatment at this intensity were evaluated. Articular cartilage was collected at 8 h after heat stimulation and/or intraarticular Gln administration, and total RNA was extracted. The expression of HSP70, aggrecan, and type II collagen was quantified using real‐time RT‐PCR. Cartilage samples from the OA model were subjected to hematoxylin and eosin (HE) and safranin O staining. HSP70 and aggrecan expression was greatest in a group receiving both MW and Gln. In the rat OA model, the severity of OA was significantly milder in a group receiving MW and Gln than in the control group. HSP70, stimulated by the combination of MW heat and Gln, may be involved in the suppression of OA progression. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:401–407, 2012     

Different duration of crowding and noise exposure effects on exploratory behavior,cellular immunity and HSP70 expression in rats

This study examined the effects of different duration of stress exposure on cellular immunity and heat shock protein (HSP) 70 expression in rats. The different durations of crowding and noise were used as different stress modes. Observation of exploratory behavior in an open‐field test was used to indicate stress level. The expression of HSP70 and T‐cell subsets in blood samples were measured in the different groups of stressed rats. The results showed that there were significant reductions in the percentages of CD3⁺ and CD4⁺ T cells in the stressed groups. The lowest point was at 2 weeks from stress. There were significant increases in the percentage of CD8⁺ in 2 day, 1 week and 2 week stress groups compared with the control group. The CD4⁺/CD8⁺ ratio was also remarkably lower in rats of the 2 day, 1 week and 2 week stress groups compared with the control group. HSP70 expression rose significantly after stress and reached a maximum after 2 weeks of stress. Open field test activity showed higher vertical movement scores at 2 weeks stress compared with the control group. Grooming scores at 2 days stress was obviously more than that of the control and there were higher grooming scores at 2 weeks and 3 weeks stress compared to the 2 day stress group. Those results showed that the different duration stress exposure affects both exploratory behavior and immune function. The results also showed that not only immune function but also HSP70 expression was mainly dependent on the duration of stress. Copyright © 2006 John Wiley & Sons, Ltd.     

Expression of 60-kDa and inducible 70-kDa stress proteins in gentamicin-induced acute renal failure

Background Heat shock proteins contribute to the survival of cells under stress conditions. We have previously observed the expression of HSP90 and constitutive HSP70 in rat kidneys during stress loading. This study was designed to determine whether HSP60 and HSP70i are induced in rat kidneys during exposure to gentamicin. Methods During the continuous administration of gentamicin (40 mg/kg per day) to rats for 30 days, the changes of HSP60 and HSP70i in these kidneys were quantitatively investigated by immunoblot analyses using specific antibodies. Results The indicators for renal failure,N-acetyl-β-d-glucosaminidase, blood urea nitrogen, and serum creatinine, increased, and reached the maximum levels at day 12. Thereafter, they spontaneously decreased, and were close to their normal levels at day 21, despite continued gentamicin exposure. The time course of HSP60 levels showed a unique 2-peak pattern exclusively in the cytosols of the cortex; the gentamicin target site. The second HSP60 peak occurred during the recovery phase, and coincided with published reports of a second peak of gentamicin accumulation in the kidney. The time course of HSP70i level showed a single peak at day 12 when the renal failure was most intense; the level was similar between the soluble and insoluble fractions in all portions of the kidney, except the inner medulla. Conclusion The increase in cytosolic HSP60 levels, as well as HSP70i levels, may be related to the spontaneous recovery from renal failure at the target site of the drug.     

The effect of oxidative stress and Raftlin levels on wound healing

Reactive oxygen species (ROS) are crucial in all wound‐healing processes. Raftlin also plays an important role in the induction of the autoimmune response and the vascular inflammatory response. Inflammatory mediators induce continuous synthesis and secretion. To the best of our knowledge, although there are studies in the literature on antioxidant enzyme levels (superoxide dismutase [SOD], catalase [CAT]) and oxidative stress markers, there are no studies on the comparison of these levels in wound patients with the activities of Raftlin, which is known to play a role in the vascular endothelial response. The aim of this study was to compare the levels of oxidative stress and antioxidant response between wound patients and a control group and to compare the levels of Raftlin between the two groups, which is a new biomarker in inflammatory diseases. Between January 2018 and September 2018, 30 healthy control patients and 30 patients with wounds were enrolled in the study as volunteers. Tissue samples were collected and were sent to the biochemistry laboratory to determine the levels of oxidative stress, antioxidant enzymes, and Raftlin, which play an important role in wound healing. The following were evaluated: SOD and CAT levels (as a measure of antioxidant enzymes); malondialdehyde (MDA) levels (as a measure of free oxygen radicals); and Raftlin, which is a lipid raft protein used in determining the level of inflammatory and autoimmune response. The analyses determined a statistically significant correlation between MDA, SOD, CAT, and Raftlin values in wound patients (p<0.05). Raftlin was a considerable parameter in determining the prognostic process of wound healing. The levels of tissue Raftlin were significantly higher in wounded patients. A significant increase in MDA, SOD, and CAT activities of the wounded patients also suggested that the oxidant and antioxidant effect was balanced and that external antioxidant supplementation was not required.     

shRNA介导的热休克蛋白70敲低对人结肠癌细胞在裸鼠体内生长的影响

目的:探讨热休克蛋白70(HSP70)敲低对结肠癌细胞在裸鼠体内生长的影响。方法:构建两种干扰HSP70的shRNA(HSP70 shRNA-1和HSP70 shRNA-2)质粒表达载体,分别转染到结肠癌HT29细胞,并以空质粒转染(阴性对照)和未转染的HT29细胞(空白对照)为对照,用RT-PCR和Western blot方法检测各组HT29细胞HSP70基因和蛋白的表达;将HSP70shRNA-2转染、空质粒转染及未转染的HT29细胞分别接种至裸鼠皮下建立移3组植瘤模型,观察肿瘤生长情况,3周后剥离瘤块,用HE染色、免疫组化和TUNEL法分别检测移植瘤组织形态学、增殖细胞核抗原(PCNA)的表达及细胞凋亡情况。结果:RT-PCR和Western blot结果显示HSP70 shRNA-1和HSP70 shRNA-2均能明显抑制HT29细胞HSP70基因和蛋白的表达,且HSP70 shRNA-2的抑制作用更为明显,空质粒转染对HSP70的表达无明显影响;与空白对照组比较,HSP70 shRNA-2转染组裸鼠移植瘤生长明显较明显减慢(P<0.01),瘤组织中心出现明显坏死,PCNA表达明显降低,细胞凋亡率明显增加(P<0.01),而空质粒转染组无明显上述改变(P>0.05)。结论:HSP70沉默能抑制结肠癌HT29细胞裸鼠移植瘤的生长,促进细胞凋亡,HSP70可能是治疗结肠癌有效靶点。     

Protective effect of Propolfenol® on induced oxidative stress in human spermatozoa

The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack. Oxidative stress can induce propagation of a lipid peroxidation (LPO) chain reaction because spermatozoa contain high concentration of unsaturated fatty acids. This study aimed at evaluating in vitro the possible toxicity and/or the antioxidant properties of Propolfenol^® in ejaculated human spermatozoa. A colorimetric assay determined the total flavonoid content by spectrophotometry and a high‐performance liquid chromatography–diode array detection analysis the quantity of galangin, pinocembrin and caffeic acid phenylethilic ester (CAPE). Sperm parameters such as motility, vitality and DNA integrity were assessed utilising optical microscopy. The antioxidant properties Propolfenol^® against LPO induced by tert‐Butyl Hydroperoxide were evaluated using the C11‐BODIPY581/591 probe. Chemical analysis of Propolfenol^® revealed low quantities of galangin, pinocembrin and CAPE; cyclic voltammetry experiments showed that Propolfenol^® may exert an antioxidant activity. A protective action of Propolfenol^® (20 and 100 μg/ml) on induced LPO in human spermatozoa was detected. Propolfenol^® may be proposed as the supplement in media for sperm preparation techniques or cryopreservation to counteract the increased presence of reactive oxygen species generated by these methods.     

急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70表达的影响

目的:探讨急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70(heat shock protein 70,HSP70)表达的影响。方法:将32只8周龄雄性小白鼠随机均分为4组,饲养7d后,进行热应激处理,温度控制在(39±0.5)℃,时间分别为0.5、1和3h。应激后立即采血,分离血清测定谷草转氨酶(GOT)含量。一侧附睾制备精子悬液,用于计算精子密度和顶体畸形率;另一侧附睾、睾丸、输精管用于免疫组化研究。结果:应激后,小鼠体重、睾丸系数、顶体畸形率变化不显著(P>0.05),附睾系数和精子密度有不同程度的下降,GOT含量急剧升高(P<0.01)。随着应激时间的延长,小鼠精子密度呈递减趋势,顶体畸形率呈上升趋势。应激时间最短的0.5h组小鼠体重、睾丸系数、附睾系数的降幅反而最大。免疫组化法观察发现,HSP70在性成熟小鼠睾丸、附睾、输精管中均有表达。正常状态下,HSP70在睾丸组织间质细胞中少量表达,应激后分布于间质细胞核,此外在精母细胞核与精子细胞核中也有大量分布;附睾中HSP70主要分布于主细胞质,基细胞和亮细胞中没有表达,应激后附睾体的纤毛细胞中也发现大量棕色颗粒;输精管中HSP70主要定位在基细胞质,主细胞中不表达。随着应激时间的延长,HSP70在睾丸、附睾中的表达量明显升高,而在输精管中的增幅不明显。结论:急性热应激对性成熟雄性小鼠的生殖系统造成了损伤;HSP70在睾丸、附睾、输精管中的表达与定位具有区域特异性和细胞特异性,提示其可能参与精子的发生与成熟;HSP70在应激状态下表达量大幅上升的作用可能在于保护细胞免受高热损伤。