Effects of Batroxobin on Spatial Learning and Memory Disorder of Rats with Temporal Ischemia and the Expression of HSP32 and HSP70

The effect of Batroxobin on spatial memory disorder of left temporal ischemic rats and the expression of HSP32 and HSP70 were investigated with Morri`s water maze and immunohistochemistry methods. The results showed that the mean reaction time and distance of temporal ischemic rats in searching a goal were significantly longer than those of the sham-operated rats and at the same time HSP32 and HSP70 expression of left temporal ischemic region in rats was significantly increased as compared with the sham-operated rats. However, the mean reaction time and distance of the Batroxobin-treated rats were shorter and they used normal strategies more often and earlier than those of ischemic rats. The number of HSP32 and HSP70 immune reactive cells of Batroxobin-treated rats was also less than that of the ischemic group. In conclusion, Batroxobin can improve spatial memory disorder of temporal ischemic rats; and the down-regulation of the expression of HSP32 and HSP70 is probably related to the attenuation of ischemic injury.

Batroxobin Against Anoxic Damage of Rat Hippocampal Neurons in Culture:Morphological Changes and Hsp70 Expression

Batroxobin,the thrombin-like enzyme,is used for therapeutic defibrination. We have found that batroxobin has good therapeutic effect in ischemic reperfusion rats and clinical practices in vivo. But we have not studied the neuroprotective effect of batroxobin on anoxic hippocampal neurons in vitro. The purpose of this study was to obtain further information on the mechanism of the batroxobin-induced neuroprotection and examine the neuroprotective effect on neurons exposed to anoxia. The effect of batroxobin on anoxic damages in cultured hippocampal neurons of neonatal rats was investigated by using morphological changes and heat shock protein 70Kd (Hsp70) immunoreactive expression as indicators. The results indicate that batroxobin, besides its defibrination, may have a direct neuroprotective effect on anoxic damage of hippocampal neurons.

Effects of heat shock on change of HSC70/HSP68,acid and alkaline phosphatases before and after rat partial hepatectomy

INTRODUCTIONOnly the liver has the great capability ofregeneration in mammal.Few hepatocytes are inthe phase of division in the normal liver of an adultmammal (including human beings),but theremaining hepatocytes can be induced to proliferatequickly by partial hepatectomy (PH),and,to somedegree,they stop dividing and re-differentiate intocells functioning as hepatocytes.This shows

Purification of heat shock protein 70-associated tumor peptides and their antitumor immunity to hepatoma in mice

AIM:To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect.METHODS:By ConA-sepharose affinity chromatography,ADP-agarose affinity chromatography, and DEAE anion exchange chromatography, we were able to purify HSP70-associated peptides from mouse hepatoma (HCaF) cells treated in heat shock at 42℃. Specific active immunization and adoptive cellular immunization assay were adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes isolated from HcaF.RESULTS: The finally purified HSP-associated peptides had a very high purity and specificity found by SDS-PAGE and Western blot. Mice immunized with HSP70-associated peptide complexes purified from HCaF cells were protected from HCaF living cell challenge. This effect was dose dependent.Adoptive immunization of immune spleen cells of mice immunized with HSP70-associated peptide complexes could elicit immunity against HCaF challenge, and the tumor-free mice could resist repeated challenges. This effect could be continuously enhanced by repeated challenge with HCaF living cells. The tumor-free mice could tolerate the challenge for as high as 1×10^7 HCaF cells. The mice immunized once with spleen cells pulsed with HSP70-associated peptide complexes in vitro could also result in a certain adoptive immunity against HCaF.CONCLUSION:High purity and specificity of HSP70-associated peptides could be achieved from tumor cells by the low-pressure affinity chromatography method used in this study. HSP70-associated peptide complexes derived from the HCaF can elicit non-MHC-I molecule restrictive immunoprotective effect against HCaF.This effect can be transferred by adoptive immunization to mice and enhanced by repeated challenge with HCaF live cells.

Sleep deprivation increase the expression of inducible heat shock protein 70 in rat gastric mucosa

AIM: To investigate if sleep deprivation is able to increase the expression of inducible heat shock protein 70 in gastric mucosa and its possible role in mucosal defense. METHODS: Rats for sleep disruption were placed inside a computerized rotating drum, gastric mucosa was taken from rats with 1, 3 and 7d sleep deprivation. RT-PCR, immunohistochemistry and Western blotting were used to determine the expression of heat shock protein 70. Ethanol (500mL.L(-1), i.g.) was used to induce gastric mucosa damage. RESULTS: RT-PCR, Western blotting and immunostaining confirmed that the sleep deprivation as a stress resulted in significantly greater expression of inducible heat shock protein 70 in gastric mucosa of rats. After the 500mL.L(-1) ethanol challenge, the ulcer area found in the rats with 7d sleep deprivation (19.15 +/- 4.2)mm(2) was significantly lower (P【0.01) than the corresponding control (53.7 +/- 8.1) mm(2). CONCLUSION: Sleep deprivation as a stress, in addition to lowering the gastric mucosal barrier, is able to stimulate the expression of inducible heat shock protein 70 in gastric mucosa of rats, the heat shock protein 70 may play an important role in gastric mucosal protection.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Detection of the expression of heat shock protein 70 using quantum dots as fluorescence probe in human renal carcinoma

Objective： To detect the expression of heat shock protein 70 （HSP70） in human renal carcinoma tissues and cultured ACHN cells by using quantum dots-tagged fluorescence technology and its significance. Methods： Using the fluorescence property of quantum dots, indirect immunofluorescence method and immunocytochemical method were used to detect the expression of HSP70 tagged by quantum dots in renal carcinoma tissues and ACHN cells cultured in vitro. Results： Confocal fluorescence microscopy showed that HSP70 were significantly expressed in renal carcinoma tissues and ACHN cells cultured in vitro characterized by homogeneous distribution of intensive salmon pink fluorescence. Compared with FITC tagging, quantum dots tagged fluorescence had good specificity and signal to background. There was no notable quenching after excitation by quantum dots for 30 rain. Conclusion： Quantum dots can be used to label subcellular proteins and have obvious advantages compared with the traditional fluorescence methods. The quantum dots-tagged fluorescence could be applied as a new method for clinical labeling detection.

RNA干扰技术沉默热休克蛋白70对喉癌Hep-2细胞增殖的影响

喉癌是上呼吸道常见的恶性肿瘤,严重影响着人类的健康。研究表明热休克蛋白70(heat shock protein 70,HSP70)在正常组织中多不表达,但在包括头颈鳞状细胞癌在内的多种肿瘤组织却有异常表达。肿瘤细胞中高表达的HSP70,对细胞的增殖、侵袭及转移、机体对肿瘤治疗耐受性的发生及肿瘤预后等方面均有很重要的作用[1]。RNA干扰(RNA interference,RNAi)技术是近年来发展起来的一项新的基因技术。

Expression of Hsp70 and Caspase-3 in rabbits after se- vere traumatic brain injury

Objective： To investigate the expres- sion of Caspase-3 and Hsp70 in rabbits after severe trau- matic brain injury （TBI） and to explore the feasibility of its application in estimation of injury time in forensic medicine. Methods： Arabbit model of heavy TBI was developed by high velocity impact on the parietal bone with an iron stick. Totally 8 healthy adult New Zealand white rabbits were randomly divided into control group （n=2） and injury group （n=6）. Four hours after injury, tissue specimens from the parietal lobe, temporal lobe, occipital lobe, cerebellum and brainstem were harvested to detect the expression of Hsp70 and Caspase-3 by immunohistochemistry. Besides, the gray values of cells positive for HspT0 and Caspase-3 were analyzed with an image analyzer. Results： Immunohistochemistry staining demonstrated a low level of Caspase-3 and Hsp70 expression in normal control group. While in injury group, both the Caspase-3 and Hsp70 expression was significantly elevated （P〈0.05）. Positive cells gathered around the lesion focus. Occipital lobe and cerebellum had fewer positive cells while temporal and brainstem had the fewest. Conclusion： The expression of Caspase-3 and HspT0 at an early stage following severe TBI is characteristic and can be applied to estimate the time of injury.

Effect of moxibustion on expressions of HSP70 mRNA and protein in gastric cancer-bearing rats

Objective:To observe the effect of moxibustion on the mRNA and protein expressions of heat-shock protein 70(HSP70)in gastric cancer-bearing rats.Methods:A total of 40 healthy Sprague-Dawley(SD)rats were adaptively fed for one week.The gastric cancer model was prepared by Walker-256 cancer tissue transplantation.After 7 d,10 rats were randomly selected to verify the successful modeling,and the remaining 30 rats were divided into a model group,a moxibustion group and an infrared group by the random number table method,with 10 rats in each group.After enrollment,the moxibustion group received suspended moxibustion at Zhongwan(CV 12),Guan yuan(CV 4)and bilateral Zusanli(ST 36),(the first group of acupoints)on the 1st day,and suspended moxibustion at bilateral Pishu(BL 20)and Weishu(BL 21),(the second group of acupoints)on the 2nd day,20 min each time,once a day.Moxibustion was alternately performed every other day at the two groups of acupoints for 21 d.From the day of en roll me nt,rats in the infrared group were irradiated with the infrared radiatio n at the stomach area on the 1st day,and at the T12-Ti3 interspinous region on the 2nd day,20 min each time,once a day,and the two locations were alternately irradiated every other day for 21 d.During the treatment,rats in the model group were intervened by grasping and fixation without treatment.At the end of the treatment,blood was collected from the inner eye orbit,and the HSP70 expression in peripheral blood was determined by enzyme linked immunosorbent assay(ELISA).Rats were sacrificed,the tumor volume and growth inhibition rate were measured.The position and changes of HSP70 in gastric cancer were observed by streptavidin-perosidase(SP);HSP70 protein expression was determined by ELISA;HSP70 mRNA expression in cancer tissues was determined by reverse transcription-polymerase chain reaction(R「PCR)assay.Results:In comparison of the model group,the volume growth of the gastric cancer in the moxibustion group was significantly restricted(P<0.01);the volume growth inhibition rate in the moxibustion group was 37.93%;the HSP70 expressi on in peripheral blood and the cancer tissues was sign ifica ntly in creased(both P<0.01);the expressio n of HSP70 mRNA and HSP70 content in gastric tumor were both obviously in creased in the moxibustion group(P<0.01);and a large amount of HSP70 was released to the outside of cancer cells in the moxibustion group.In comparison of the model group,the volume growth of the gastric cancer in the infrared group was slightly restricted(P<0.05)with a volume growth inhibition rate of 15.89%;the HSP70 expression in the infrared group was increased significantly in peripheral blood(P<0.01)and in the gastric cancer tissues(P<0.05);more HSP70 was released outside of the cancer cells in the infrared group.In comparison of the infrared group,the volume growth of gastric cancer was more restricted in the moxibustion group(P<0.05)z and the HSP70 expression in the gastric cancer tissues was also higher(P<0.05);more HSP70 was released outside of the cancer cells in the moxibustion group.Conclusion:Moxibusti on and in fra red treatme nt inhibit the gastric can cer growth in the gastric cancer-bearing rats,up-regulate the HSP70 expression in gastric cancer tissues,and promote the production and extracellular release of HSP70,and the effect of moxibustion is more obvious.

Prokaryotic expression and purification of fusion protein HSP70-EGFP and its application in the study of dendritic cells internalization antigen

To study the endocytic activity of dendritic cells(DCs)by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood.Fusion protein HSP70-EGFP was prokaryotically expressed,isolated and purified.DCs were isolated and cultured from human peripheral blood.The DCs were divided into 3 groups in the endocytic experiment.There were 106 DCs in each group.Group 1 and 2 were respectively incubated for 30 min.with HSP70-EGFP and EGFP.Group 3 was incubated with HSP70 for 30 min,and then incubated for 30 min.with HSP70-EGFP.Subsequently,3 groups were placed in an incubator at 37℃for 0.5,1,2 and 24 h.Flow cytometry(FCM)was adopted to detect the amount of DCs with EGFP inside.IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12.There were 5 types in the experiment:LPS,inactive LPS,HSP70-EGFP,EGFP and no antigen.Fusion protein HSP70-EGFP was successfully obtained and its molecular weight was 97000.It accounted for 35.32%of the total protein.Under irradiation of an ultraviolet lamp,the protein solution sent out viridescent fluorescence.The result detected by FCM indicated that after incubation for 0.5 h at 37℃,the positive rate in group 1 was 63%,while the other 2 groups were negative.After incubation for 1,2 and 24 h at 37℃,the positive rates in the 3 groups were above 80%.The IL-12 Eli-spot examination shows that with HSP70-EGFP being loaded,the amount of DCs secreting IL-12 was 134.09±31.78/10^(5)cells,a little lower than that of DCs with LPS loaded(with the average point of 156.36±15.73).There was no significant difference between the 2 groups(P<0.01).By contrast,both of them were significantly higher than inactive LPS-(33.78±1.40)/10^(5)cells and EGFP-loaded(23.13±4.57)/105 cells DC groups in the amount of DCs secreting IL-12(P<0.01).The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs internalizing HSP70-EGFP.With increasing incubation time,pinocytosis begins to dominate.HSP70-EGFP may promote DCs to secret cell factor IL-12.

Effect of Moxibustion on Expression of HSP70 and Apoptosisrelated Factors in Rats with Acute Gastric Mucosal Damage

Objective： To observe the effect of preventively used moxibustion on the expression of apoptosis-related factors in rats with acute gastric mucosal damage （GMD）, and analyze the relationship between those factors and heat shock protein 70 （HSP70）, for discovering the mechanism of moxibustion for protecting gastric mucosa from the aspect of mitochondria signal transduction of apoptosis. Methods： Thirty-two Wistar rats were randomly divided into binding, model, acupoint-moxibustion and non-acupoint-moxibustion groups, 8 in each group. Moxibustion was applied preventively to acupoint-moxibustion and non-acupointmoxibustion groups respectively for 8 d. Rats in binding and model groups were served with restraint without moxibustion. The GMD models were induced in groups except for A group, and the expression of cytochrome c （Cyt-c） in gastric mucosa was measured by Western-blot analysis, the expression of HSP70, cell apoptosis index （AI）, apoptotic protease activating factor 1 （Apaf-1）, Caspase-9, Caspase-3, Bcl-2 and Bax in gastric mucosal cells were studied by immunohistochemical detection. Results： Compared with binding group, the expressions of HSP70, AI, Cyt-c, Apaf-1, Caspase-9, Caspase-3, Bcl-2 and Bax of gastric mucosa in model group were significantly higher （P〈0.05 or P〈0.01）. Compared with model group, the expressions of HSP70 and Bcl-2 were significantly higher （P〈0.01）, while AI, Cyt-c, Apaf-1, Caspase-9, Caspase-3, Bax were significantly lower （P〈0.01） in acupoint-moxibustion group.Compared with the acupoint-moxibustion group, the expressions of HSP70 and Bcl-2 were significantly lower （P〈0.01）, while AI, Cyt-c, Apaf-1, Caspase-9, Caspase-3, Bax were significantly higher （P〈0.05 or P〈0.01） in non-acupoint-moxibustion group. Conclusion： Moxibustion inhibits the apoptosis of rats＇ gastric mucosal cells and protect them from damage, through up-regulating the expression of HSP70, and modulating the expressions of Cyt-c, Apaf-1, Caspase-9, Caspase-3, Bcl-2 and Bax which were related to the mitochondria signal transduction of apoptosis.

RADIX SALVIAE MILTIORRHIZAE PROTECTS RAT HIPPOCAMPAL NEURON IN CULTURE FROM ANOXIC DAMAGE

Radix Salviae Miltiorrhizae (RSM), a well-known traditional Chinese medicinal herb, has been used to improve blood circulation and resolve blood stasis. We have previously found that RSM has neuroprotective effect on ischemia and/ or ischemia-reperfusion rats. The purpose of this study was to obtain further information on the mechanism of the RSM-in-duced neuroprotection and to examine the neuroprotective effect on neurons exposed to anoxia. The effect of RSM on anoxic damage in cultured hippocampal neurons of neonatal rat was investigated by using morphological changes and heat shock protein 70kD (HSP70) expression as indicators. RSM given 0.5h before 2h-anoxia followed by 48 hours reoxygenation could significantly increase survival rate of hippocampal neurons and number of HSP70 positive cells. The results suggest that RSM has a direct neuroprotective effects on anoxic damage in hippocampal neurons.

Zinc is a potent heat shock protein inducer during liver cold preservation in rats

OBJECTIVE: A simple liver cold preservation model was established to study the synthesis of heat shock protein 70 (HSP70) induced by zinc (ZnSO(4), i.p.) and its protection during liver cold preservation in rat. METHODS: Male Wistar rats were divided into 5 groups (n = 6). In control group rat received no pretreatment; in Zn-1 group, Zn-2 group, and Zn-3 group rats were pretreated with zinc sulfate at a dose of 5 mg/kg, 10 mg/kg, 15 mg/kg respectively; and in H group rat received heat shock preconditioning (42.5 degrees C x 15 min). Livers were preserved in UW solution for 6, 12 and 24 h, respectively. HSP70 was analyzed by Western blot. Aspartate transaminase (AST) and lactate dehydrogenase (LDH) values of the perfusion solution and the histology of the liver were evaluated. RESULTS: HSP70 expression was markedly elevated after pretreatment with zinc and heat shock. AST and LDH values in the Zn-1, Zn-2 and H groups were significantly lower than those in the control group, respectively (P 0.05), whereas the AST and LDH values in the Zn-3 group were much higher than those in the control group. Histology results showed that liver injury in the Zn-1, Zn-2 and H groups were minimal, while it was severe in the Zn-3 group. CONCLUSIONS: Zn(2+) is a potent and feasible inducer of HSP expression and is able to protect liver from cold preservation injury. The proper inducing dosage of Zn(2+) ranged from 5 mg/kg to 10 mg/kg. The dosage of 15 mg/kg for Zn(2+) as a HSP inducer is not indicated for its severe toxicity to the liver.

A combined dynamic inhalation device designed for moxa-smoking toxicity testing

OBJECTIVE： To design a combined dynamic inhalation device for testing the toxicity induced by moxa smoking. METHODS： The new apparatus （Patent No. 201120101911.5） includes air renewal and recycling systems, a gas generating device, a gas control unit, and a device to measure and control tem- perature and humidity. Sprague-dawley rats were tested for acute and sub-chronic toxicity after exposure to moxa-burning smoke.METHODS： The new apparatus （Patent No. 201120101911.5） includes air renewal and recycling systems, a gas generating device, a gas control unit, and a device to measure and control tem- perature and humidity. Sprague-dawley rats were tested for acute and sub-chronic toxicity after exposure to moxa-burning smoke.RESULTS： We found an LQ0 of 1.2× 10^4 mg/m^3 in the acute toxicity assays. In sub-chronic toxicity tests the organ coefficients studied showed no sig-nificant differences within rats groups of the same gender after treatment with moxa smoke or a month of recovery. However, mean gray degree of lung 70 heat shock protein （HSP70） was significantly elevated in the high dose group in comparison with the low dose group （P 〈 0.05）, mean gray degree, mean optical density, gross area of HSP70 in other organs and caspase-9 parameters showed no significant differences between groups.CONCLUSION： These results suggest that moxa smoke had no overt toxicity in rats. This work pro- vides evidence and reference for the design of dy- namic inhalation exposure systems.

Expression of endoplasmic reticulum molecular chaperon GRP94 in human lung cancer tissues and its clinical significance

OBJECTIVE: To investigate the relationship between the expression of glucose regulated protein 94 (GRP94) at the level of mRNA and protein in vivo and in human lung cancer. METHODS: RT-PCR, immunohistochemistry and/or Western blot were used in 54 cases of lung cancer tissues and corresponding normal lung tissues. RESULTS: There was a significant overexpression of GRP94 mRNA and protein in lung cancer tissues as compared with lung normal tissues. In lung cancer tissue, the relative level of GRP94 mRNA as evaluated by RT-PCR was 3.48 +/- 2.06, the level of GRP94 protein as evaluated by immunohistochemistry was + + to + + +, and by Western blot was 256.7 +/- 80.6. In lung normal tissue, the relative level of GRP94 mRNA was 2.01 +/- 1.83, the level of GRP94 protein was + to + + and 108.1 +/- 42.3. The differences in expression of GRP94 between the two tissues were significant (P

Effects of Acupuncture Pretreatment on Ischemic Cardiac Muscle Cell Apoptosis and Gene Expression in Ischemia-reperfusion Rats

Objective： To investigate the protective effects of acuptmcture pretreatment on ischemic myocardium, the protective mechanism of acupuncture pretreatment on ischemic myocardium was explored by observing the cardiac muscle cell apoptosis and the expression of HSP70 mRNA of ischemia-reperfusion injury rats treated with acupuncture pretreatment. Methods： Sixty-four Wistar rats were randomly divided into eight groups： control group, sham surgery group, ischemia-reperfusion group, ischemia pretreatment group, manual acupuncture pretreatment group （once a day）, electroacupuncture pretreatment group （once a day）, manual acupuncture pretreatment group （twice a day）, and electroacupuncture pretreatment group （twice a day）. The reperfusion model of rat myocardial ischemia was made. Expression of HSP70 mRNA was assayed by in situ hybridization, and cell apoptosis by TUNEL. Results： Compared with those in the control group and the sham surgery group, the apoptosis and the expression of HSP70 mRNA were increased in the ischemia-reperfusion group. Compared with those in the ischemia-reperfusion group, the cardiac muscle cell apoptosis was decreased and the HSP70 mRNA was increased in the rats treated with acupuncture pretreatment; meanwhile, acupuncture pretreatment twice a day had stronger effects than acupuncture pretreatment once a day and ischemia pretreatrnent. Conclusion： Acupuncture pretreatment can inhibit the cardiac muscle cell apoptosis, and up-regulate the expression of HSP70 mRNA in ischemia-reperfusion rats. Acupuncture pretreatment twice a day has stronger effects than pretreatment once a day.