GLUT1-mediated microglial proinflammatory activation contributes to the development of stress-induced spatial learning and memory dysfunction in mice.

BACKGROUND: Stress is a recognized risk factor for cognitive decline, which triggers neuroinflammation involving microglial activation. However, the specific mechanism for microglial activation under stress and affects learning and memory remains unclear. METHODS: The chronic stress mouse model was utilized to explore the relationship between microglial activation and spatial memory impairment. The effect of hippocampal hyperglycemia on microglial activation was evaluated through hippocampal glucose-infusion and the incubation of BV2 cells with high glucose. The gain-and loss-of-function experiments were conducted to investigate the role of GLUT1 in microglial proinflammatory activation. An adeno-associated virus (AAV) was employed to specifically knockdown of GLUT1 in hippocampal microglia to assess its impact on stressed-mice. RESULTS: Herein, we found that chronic stress induced remarkable hippocampal microglial proinflammatory activation and neuroinflammation, which were involved in the development of stress-related spatial learning and memory impairment. Mechanistically, elevated hippocampal glucose level post-stress was revealed to be a key regulator of proinflammatory microglial activation via specifically increasing the expression of microglial GLUT1. GLUT1 overexpression promoted microglial proinflammatory phenotype while inhibiting GLUT1 function mitigated this effect under high glucose. Furthermore, specific downregulation of hippocampal microglial GLUT1 in stressed-mice relieved microglial proinflammatory activation, neuroinflammation, and spatial learning and memory injury. Finally, the NF-κB signaling pathway was demonstrated to be involved in the regulatory effect of GLUT1 on microglia. CONCLUSIONS: We demonstrate that elevated glucose and GLUT1 expression induce microglia proinflammatory activation, contributing to stress-associated spatial memory dysfunction. These findings highlight significant interplay between metabolism and inflammation, presenting a possible therapeutic target for stress-related cognitive disorders.

Blood-brain barrier dysfunction mediated by the EZH2-Claudin-5 axis drives stress-induced TNF-α infiltration and depression-like behaviors.

Growing evidence suggests that neurovascular dysfunction characterized by blood-brain barrier (BBB) breakdown underlies the development of psychiatric disorders, such as major depressive disorder (MDD). Tight junction (TJ) proteins are critical modulators of homeostasis and BBB integrity. TJ protein Claudin-5 is the most dominant BBB component and is downregulated in numerous depression models; however, the underlying mechanisms remain elusive. Here, we demonstrate a molecular basis of BBB breakdown that links stress and depression. We implemented an animal model of depression, chronic unpredictable mild stress (CUMS) in male C57BL/6 mice, and showed that hippocampal BBB breakdown was closely associated with stress vulnerability. Concomitantly, we found that dysregulated Cldn5 level coupled with repression of the histone methylation signature at its promoter contributed to stress-induced BBB dysfunction and depression. Moreover, histone methyltransferase enhancer of zeste homolog 2 (EZH2) knockdown improved Cldn5 expression and alleviated depression-like behaviors by suppressing the tri-methylation of lysine 27 on histone 3 (H3K27me3) in chronically stressed mice. Furthermore, the stress-induced excessive transfer of peripheral cytokine tumor necrosis factor-α (TNF-α) into the hippocampus was prevented by Claudin-5 overexpression and EZH2 knockdown. Interestingly, antidepressant treatment could inhibit H3K27me3 deposition at the Cldn5 promoter, reversing the loss of the encoded protein and BBB damage. Considered together, these findings reveal the importance of the hippocampal EZH2-Claudin-5 axis in regulating neurovascular function and MDD development, providing potential therapeutic targets for this psychiatric illness.

Homocysteine-Induced Disturbances in DNA Methylation Contribute to Development of Stress-Associated Cognitive Decline in Rats.

Chronic stress is generally accepted as the main risk factor in the development of cognitive decline; however, the underlying mechanisms remain unclear. Previous data have demonstrated that the levels of homocysteine (Hcy) are significantly elevated in the plasma of stressed animals, which suggests that Hcy is associated with stress and cognitive decline. To test this hypothesis, we analyzed the cognitive function, plasma concentrations of Hcy, and brain-derived neurotropic factor (BDNF) levels in rats undergoing chronic unpredicted mild stress (CUMS). The results showed that decreased cognitive behavioral performance and decreased BDNF transcription and protein expression were correlated with hyperhomocysteinemia (HHcy) levels in stressed rats. Diet-induced HHcy mimicked the cognitive decline and BDNF downregulation in the same manner as CUMS, while Hcy reduction (by means of vitamin B complex supplements) alleviated the cognitive deficits and BDNF reduction in CUMS rats. Furthermore, we also found that both stress and HHcy disturbed the DNA methylation process in the brain and induced DNA hypermethylation in the BDNF promoter. In contrast, control of Hcy blocked BDNF promoter methylation and upregulated BDNF levels in the brain. These results imply the possibility of a causal role of Hcy in stress-induced cognitive decline. We also used ten-eleven translocation (TET1), an enzyme that induces DNA demethylation, to verify the involvement of Hcy and DNA methylation in the regulation of BDNF expression and the development of stress-related cognitive decline. The data showed that TET1-expressing viral injection into the hippocampus inhibited BDNF promoter methylation and significantly mitigated the cognitive decline in HHcy rats. Taken together, novel evidence from the present study suggests that Hcy is likely involved in chronic stress-induced BDNF reduction and related cognitive deficits. In addition, the negative side-effects of HHcy may be associated with Hcy-induced DNA hypermethylation in the BDNF promoter. The results also suggest the possibility of Hcy as a target for therapy and the potential value of vitamin B intake in preventing stress-induced cognitive decline.

Norepinephrine promotes glioma cell migration through up-regulating the expression of Twist1.

BACKGROUND: Glioma cells are characterized by high migration ability, resulting in aggressive growth of the tumors and poor prognosis of patients. It has been reported that the stress-induced hormone norepinephrine (NE) contributes to tumor progression through mediating a number of important biological processes in various cancers. However, the role of NE in the regulation of glioma migration is still unclear. Epithelial-to-mesenchymal transition (EMT) is one of the most important steps for tumor migration and metastasis. Twist1, as a key regulator of EMT, has been found to be elevated during glioma migration. But it is still unknown whether Twist1 is involved in the effect of NE on the migration of glioma cells. METHODS: Wound healing assay and transwell assay were conducted to evaluate the migration of glioma cells upon different treatments. The mesenchymal-like phenotype and the expression of Twist1 after NE treatment were assessed by cell diameters, real-time PCR, western blot and immunofluorescence staining. The gain-and loss-of-function experiments were carried out to investigate the biological function of Twist1 in the migration induced by NE. Finally, the clinical significance of Twist1 was explored among three public glioma datasets. RESULTS: In this study, our finding revealed a facilitative effect of NE on glioma cell migration in a ß-adrenergic receptor (ADRB)-dependent way. Mechanistically, NE induced mesenchymal-like phenotype and the expression of Twist1. Twist1 overexpression promoted glioma cells migration, while knockdown of Twist1 abolished the discrepancy in the migration ability between NE treated glioma cells and control cells. In addition, the clinical analysis demonstrated that Twist1 was up-regulated in malignant gliomas and recurrent gliomas, and predicted a poor prognosis of glioma patients. CONCLUSIONS: NE enhanced the migration ability of glioma cells through elevating the expression of Twist1. Our finding may provide potential therapeutic target for protecting patients with glioma from the detrimental effects of stress biology on the tumor progression.

Dynamic effects of chronic unpredictable mild stress on the hippocampal transcriptome in rats.

Stress causes extensive changes in hippocampal genomic expression, leading to changes in hippocampal structure and function. The dynamic changes in hippocampal gene expression caused by stress of different durations are still unknown. mRNA sequencing was used to analyze the hippocampal transcriptome of rats subjected to chronic unpredictable mild stress (CUMS) of different durations. Compared with the control, 501, 442 and 235 differentially expressed genes (DEGs) were detected in the hippocampus of rats subjected to CUMS for 3 days and 2 and 6 weeks, respectively. Gene Ontology (GO) analysis was used to determine the potential mechanism underlying the dynamic harmful effects of stress on the hippocampus; Certain GO terms of the down­regulated DEGs in CUMS (3 days) rats were also found in the up­regulated DEGs in CUMS (6 weeks) rats. These results showed opposing regulation patterns of DEGs between CUMS at 3 days and 6 weeks, which suggested a functional change from adaptation to damage in during the early and late stages of chronic stress. GO analysis for upregulated genes in rats subjected to CUMS for 3 days and 2 weeks suggested significant changes in 'extracellular matrix' and 'wound healing'. Upregulated genes in rats subjected to CUMS for 2 weeks were involved in changes associated with visual function. GO analysis of DEGs in rats subjected to CUMS for 6 weeks revealed increased expression of genes associated with 'apoptotic process' and 'aging' and decreased expression of those associated with inhibition of cell proliferation and cell structure. These results suggest that the early and middle stages of chronic stress primarily promote adaptive regulation and damage repair in the organism, while the late stage of chronic stress leads to damage in the hippocampus.

Inverse Correlation Between Distress and Performance in the Medical Rescuers Against COVID-19 in Wuhan.

Background: During the COVID-19 pandemic, the Chinese government had transferred many medical rescuers to Wuhan, which provided effective support in disease control. The high-intensity working and mental stress during rescue could induce distress and negatively impact the performance of rescuer afterward. Materials and Methods: To identify the characteristics of stress load and its possible effects on performance, the study surveyed 90 medical rescuers in Wuhan using a mobile phone-based self-rated questionnaire. Results: The results showed an existence of universal but mostly mild distress in rescuers. About 95.6% of the participants reported that they had at least one symptom of distress, whereas, the median scores were <30 (100 as max). Compared with civilian rescuers, a higher proportion of working with immediate virus contact was found in military medical rescuers (P = 0.008); however, no statistical differences of stress load were found between civilians and militaries. The rescuers with positive cognition or good psychological preparation were found having lower stress loads than other rescuers. An inverse correlation between the stress load and performance (R = -0.24, P = 0.023) and a positive correlation between social support and working performance (R = 0.349, P = 0.001) were found in our survey, suggesting the possible negative effects of stress and the beneficial effects of social support on performance. Conclusion: Our study indicated that more attention should be paid to the distress of medical rescuers against COVID-19. Positive cognitions, good psychological preparations, and sufficient social support would be necessary to reduce the distress and improve the performance in COVID-19 rescue.

Chronic stress promotes glioma cell proliferation via the PI3K/Akt signaling pathway.

High malignancy and high mortality of glioma render it urgent to elucidate the underlying mechanisms of glioma carcinogenesis and explore novel targets for therapy. Epidemiologic and clinical studies have revealed that chronic stress promotes the progression of various solid tumors and is correlated with poor prognosis; however, findings reporting the involvement of chronic stress in glioma are rare. In the present study, a chronic restraint animal model and a chronic stress cell model were established to explore the effects of chronic stress on glioma and its molecular mechanisms. The results revealed that chronic stress promoted glioma growth in vivo, and the serum levels of the stress hormones glucocorticoid (GC) and noradrenaline (NE) were significantly increased. In addition, GC and NE were verified to accelerate the proliferation of glioma cells in vitro. Mechanistically, the phosphatidylinositol 3­kinase (PI3K)/Akt signaling pathway was revealed to be activated under stress conditions, and inhibition of the expression of p­Akt could restrain the stress hormone­induced glioma cell proliferation. In addition, our data indicated that the GC receptor (GR) and ß­adrenergic receptors (ADRBs) were both required for the biological functions of GC and NE in glioma cells. In conclusion, these results indicated that chronic stress and the stress hormones GC and NE activated PI3K/Akt signaling through binding to GR and ADRBs, thereby promoting glioma cell growth. Our findings may provide potential therapeutic targets and pave the way for the development of new strategies to protect patients with glioma from the detrimental effects of stress on tumor progression.

Loss of nuclear ARC contributes to the development of cardiac hypertrophy in rats.

AIM: Cardiac hypertrophy and myocardial apoptosis are two major factors in heart failure. As a classical regulator of apoptosis, apoptosis repressor with caspase recruitment domain (ARC) has recently also been found to have a protective effect against hypertrophy. However, the mechanism underlying this effect is still not fully understood. METHODS: In the present study, we established animal and cellular models to monitor the changes in total and nuclear ARC during cardiac hypertrophic processes. The preventive effects of nuclear ARC in cellular hypertrophy were verified by ARC regulation and nuclear export inhibition. To further explore the mechanism for nuclear ARC superficially, we analysed proteins that interact with ARC in the nucleus via Co-IP and mass spectrometry. RESULTS: The expression of total ARC in hypertrophic myocardial tissue and H9C2 cells remained invariant, while the level of nuclear ARC decreased dramatically. By altering the content of ARC in H9C2 cells, we found that both nuclear ARC transfection and nuclear ARC export blockade attenuated norepinephrine or angiotensin II-induced hypertrophy, while ARC knockdown had an inverse effect. Co-IP data showed that ARC interacted with prohibitin (PHB) in the nucleus and might participate in maintaining the level of PHB in cells. CONCLUSIONS: These findings suggest a novel mechanism for ARC in cardiac hypertrophy prevention and also indicate that the anti-hypertrophic roles of ARC are probably associated with its localization in nucleus, which imply the nuclear ARC as a potential therapeutic target for cardiac hypertrophy.

Identification, characterization, and functional investigation of circular RNAs in subventricular zone of adult rat brain.

Adult neural stem cells (NSCs) are able to self-renew and generate new neural cells. Identifying regulators of NSCs is significant for the development of NSC-based therapies for neurodegenerative diseases and brain injuries. Recently, circular RNAs (circRNAs) have been characterized in various cell lines and brain tissues, and found to participate in multiple biological processes. However, the expression pattern of circRNAs in adult NSCs is still unknown. Here, the subventricular zone (SVZ) of the lateral ventricle was isolated as the niche of NSCs in adult rat brain for RNA sequencing and the characteristics of circRNAs profiling in both SVZ and cerebral cortex were also investigated. As a result, 29 049 and 31 975 circRNAs were identified in SVZ and cortex, respectively. Among them, 41 were SVZ-specific and 48 were cortex-specific. 467 circRNAs were also found to express predominately in SVZ, while the cortex had other 423 circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the SVZ-specific circRNAs have close relationship with the regulation of NSC expansion and NSC-niche interaction, while the other differentially expressed circRNAs might be involved in neural cellular construction and nerve system function. Furthermore, the interactions between circRNAs and microRNAs were also explored, and the result showed that one SVZ-specific circRNA was capable to competitively bind miR-138-5p as a potential derepressive regulator in NSCs proliferation. Hence, our work has laid the foundations to decipher regulation mechanisms of circRNAs in adult NSCs and to develop circRNAs as novel biomarkers for adult NSCs.

Diet-induced elevation of circulating HSP70 may trigger cell adhesion and promote the development of atherosclerosis in rats.

Although accumulating evidence indicates that heat shock protein 70 (HSP70) could be secreted into plasma and its levels have been found to have an ambiguous association with atherosclerosis, our knowledge for the exact role of circulating HSP70 in the development of atherosclerosis is still limited. In the present study, we report an adhesion-promoting effect of exogenous HSP70 and evaluate the potential involvement of elevated circulating HSP70 in the development of atherosclerosis. Time-dependent elevation of plasma HSP70 was found in diet-induced atherosclerotic rats, whose effect was investigated through further in vitro experiments. In rat aortic endothelial cell (RAEC) cultures, exogenous HSP70 incubation neither produced cell injuries by itself nor had protective effects on cell injuries caused by Ox-LDL or homocysteine. However, exogenous HSP70 administration could lead to a higher adhesion rate between rat peripheral blood monocytes (PBMCs) and RAECs. This adhesion-promoting effect appeared only when PBMCs, rather than RAECs, were pretreated with HSP70 incubation. PBMCs in an HSP70 environment released more IL-6 to supernatant, which subsequently up-regulated the expression of ICAM-1 in RAECs. These results indicate that the diet-induced elevation of circulating HSP70 could trigger cell adhesion with the help of IL-6 as a mediator, which provides a novel possible mechanism for understanding the role of circulating HSP70 in the pathogenesis of atherosclerosis.

The involvement of homocysteine in stress-induced Aß precursor protein misprocessing and related cognitive decline in rats.

Chronic stress is a risk factor in the development of cognitive decline and even Alzheimer's disease (AD), although its underlying mechanism is not fully understood. Our previous data demonstrated that the level of homocysteine (Hcy) was significantly elevated in the plasma of stressed animals, which suggests the possibility that Hcy is a link between stress and cognitive decline. To test this hypothesis, we compared the cognitive function, plasma concentrations of Hcy, and the brain beta-amyloid (Aß) level between rats with or without chronic unexpected mild stress (CUMS). A lower performance by rats in behavioral tests indicated that a significant cognitive decline was induced by CUMS. Stress also disturbed the normal processing of Aß precursor protein (APP) and resulted in the accumulation of Aß in the brains of rats, which showed a positive correlation with the hyperhomocysteinemia (HHcy) that appeared in stressed rats. Hcy-targeting intervention experiments were used to verify further the involvement of Hcy in stress-induced APP misprocessing and related cognitive decline. The results showed that diet-induced HHcy could mimic the cognitive impairment and APP misprocessing in the same manner as CUMS, while Hcy reduction by means of vitamin B complex supplements and betaine could alleviate the cognitive deficits and dysregulation of Aß metabolism in CUMS rats. Taken together, the novel evidence from our present study suggests that Hcy is likely to be involved in chronic stress-evoked APP misprocessing and related cognitive deficits. Our results also suggested the possibility of Hcy as a target for therapy and the potential value of vitamin B and betaine intake in the prevention of stress-induced cognitive decline.

[Detection of endotoxin activity in water environment and analysis of influence factors for TAL assay].

Endotoxins, derived from cell walls of most Gram-negative bacteria and some cyanobacteria, are common pyrogen and highly immunogenic molecules, and related to many diseases. In this paper, a detection method for endotoxin activity in water environment using kinetic-turbid assay of Tachypleus Amebocyte Lysate (TAL) was established, the influence of pH and salts on TAL assay was investigated. Results showed that it was favorable for TAL assay in the pH range of 6.0-8.4, at low pHs, inhibition results were observed and opposite results were obtained at high pHs. The pH should be adjusted by Tris-HCl (pH = 7.4) buffer before the endotoxin detection. No significant interference was shown in the detection of water containing NaCl, Na2SO4, CaCl2, MgCl2 and KCl with a concentration of less than 50 mg x L(-1), however, the inhibition occurred at the concentration up to 1000-10,000 mg x L(-1). Only 2. 5 mg x L(-1) of FeCl, Fe2(SO4)3, AlCl3 and Al2 (SO4)3 caused significant inhibition. Endotoxin activities of ultrapure water, tap water and recreational water were detected by TAL assay, and their endotoxin activities were < 0.06 EU x mL(-1), 0.46 EU x mL(-1) and 432. 68 EU x mL(-1), respectively.

[Stress and metabolic regulation].

The stress is generated in response to changes in internal and external environment of the body is characterized by systemic reactions to neuroendocrine activation of the immune network. More and more evidence suggests that highly activated under stress conditions neuroendocrine immune network will have a strong and sustained influence on the body's metabolism. The effect of stress on the metabolism is not only confined to the glucose metabolism, fat metabolism and protein metabolism, and important role in regulating the metabolism of trace elements. Sustained high-load stress on the metabolism of the negative impact beyond the body's compensatory capacity will lead to a disease such as diabetes, atherosclerosis and secondary diseases.

[Expression of heat shock protein 70 in lung and plasma of rats with pulmonary fibrosis induced by SiO2].

OBJECTIVE: To investigate the dynamic expression of Heat shock protein 70 (Hsp70) in the lungs and plasma of rats with pulmonary fibrosis induced by silicon dioxide (SiO2). METHODS: Forty-eight Wistar rats were randomly divided into the control group exposed to normal solution and group exposed to SiO2 (50 mg/ml) with intratracheal injection. Each group was divided into four subgroups. The animals of SiO2 group and control group were sacrificed and lungs were collected on the 7th, 14th and 28th days after exposure, respectively. The left lung tissues were examined with the histopathologic HE staining. The expression and localization of Hsp70 protein in the lung tissues were examined with western blot assay and immunohistochemistry, respectively. The expression levels of Hsp70 protein in the plasma were measured by ELISA. RESULTS: The expression of Hsp70 in lung tissues of SiO2 group increased on the 7th day and reached the peak value on the 14th day then decreased, but still was significantly higher than that of the control group, the expression of Hsp70 in plasma of SiO2 group still was significantly higher than that of the control group (P < 0.05). The maximum expression level of Hsp70 in plasma of SiO2 group on the 21st day after exposure was 0.216 ± 0.027 µg/ml. CONCLUSION: The expression levels of Hsp70 protein in the lung tissues and plasma of the group exposed to SiO2 significantly increased, which were associated with the process of pulmonary fibrosis. It was suggested that Hsp70 protein may play an important biological role in the pulmonary fibrosis induced by SiO2.

[Changes of NGFI-B subcellular location in cardiomyocytes of stressed rats and its biological effects].

OBJECTIVE: To investigate the changes of expression and subcellular location of nuclear growth-induced protein-B(NGFI-B) in cardiomyocytes of stressed rats and its biological effect and to provide scientific evidences for exploring the mechanism underlying myocardium injury induced by stress. METHODS: The cell model of stress-induced cardiomyocyte injury were established. Western blot method and confocal microscopy method were used to investigate the subcellular location of NGFI-B in cardiomyocytes under stress. The flow-cytometry was selected to detect the apoptotic rate in cardiomyocytes in vitro. Western blot method was used to determine the content of cytochrome C protein in mitochondria and cytoplasm respectively. RESULTS: Stress induced the increase of NGFI-B content in the mitochondria of cardiomyocytes and the translocation of NGFI-B from the nucleus to the mitochondria. The translocation of NGFI-B promoted the release of cytochrome C from the mitochondria and the cardiomyocyte apoptosis. Treatment of stressed cardiomyocytes with leptomycin B, a non-specific blocker of nuclear export, resulted in nuclear retention of NGFI-B and abrogated its ability to induce the release of cytochrome C from the mitochondria. CONCLUSION: Stress could induce NGFI-B translocation from the nucleus to the mitochondria in cardiomyocytes, which activated the mitochondrial pathway of cell apoptosis.

Heat shock protein 70 is secreted from endothelial cells by a non-classical pathway involving exosomes.

Emerging evidence suggests that a high level of circulating heat shock protein 70 (HSP70) correlates with a lower risk of vascular disease; however, the biological significance of this inverse relationship has not been explored. Herein, we report that oxidative low density lipoprotein (Ox-LDL) and homocysteine (Hcy) induce HSP70 release from endothelial cells. In rat endothelial cells, Ox-LDL and Hcy induced robust release of HSP70, independent of the classical route of endoplasmic reticulum/Golgi protein trafficking or the formation of lipid rafts. In contrast, Ox-LDL and Hcy significantly enhanced the exosomal secretory rate and increased the HSP70 content of exosomes. Exogenous HSP70 had no impact on LPS-, Ox-LDL- and Hcy-induced activation of endothelial cells, whereas HSP70 did activate monocytes alone, resulting in monocyte adhesion to endothelial cells. These results indicate that exosome-dependent secretion of HSP70 from endothelial cells provides a novel paracrine mechanism to regulate vascular endothelial functional integrity.

[Effects of human HSF1 on transcriptional regulation of prohibitin promoter].

AIM: To assay the transcriptional regulation effect of hHSF1 on prohibitin gene promoter. METHODS: The total length of hHSF1 exon was amplified by PCR method and cloned into pcDNA3. 1(+) vector. pcDNA3. 1(+)-hHSF1 and pGI3 prohibitin were co-transfected into HEK293 cells. The luciferase activity was detected by Dual-Luciferase Reporter Assay System to evaluate the transcriptional regulation effect of hHSF1 on prohibitin gene promoter. RESULTS: The pcDNA3.1(+)-hHSF1 vector was constructed successfully. The assay of luciferase activity showed that the transcription of pGL3-prohibitin was dramatically upregulated by hHSF1. CONCLUSION: hHSF1 can transcriptionally regulate prohibitin expression.

[Level and effect of anti-HSP70 antibody changed in the procession of rat atherosclerosis].

AIM: To explore the level of anti-HSP70 antibody in plasma during atherosclerosis procedure induced by high-fat diet in rat and the relationship of them. METHODS: Twenty eight rat were divided into high-fat diet group (H) and control group (C). The total cholesterol (TC), Glyceride (TG), low density lipoprotein cholesterol (LDL-C) in serum, pathology change of rat Arch of the aorta were determined, the level of anti-HSP70 antibody and their Phenotype were evaluated by ELISA. RESULTS: After two weeks, the serum concentrations of TC and LDL-C in rat supplemented by high-fat diet were significantly higher than those in control group (P < 0.01), the serum TG were much lower than those in control group (P < 0.01). Four weeks later the level of anti-HSP70 antibody, IgM, IgG phenotype were significantly higher than those in control group (P < 0.01). There were lipin deposition and mottling formation in rat Arch of the aorta in rat supplemented by high-fat diet in 12th week. CONCLUSION: Atherosclerosis could be induced by high-fat diet in rat. Accompany with the atherosclerosis procession, the level of anti-HSP70 antibody was continuously elevated, the level of anti-HSP70 antibody was related to atherosclerosis. The level of anti-HSP70 antibody was closely associated with atherosclerosis.

[Construction and effect identification of MiR RNAi eukaryotic expression vectors of prohibitin].

AIM: To construct the RNAi eukaryotic vector of inhibitory member of the prohibitin (PHB-1) gene and observe the interfering effect in HEK293 cell line after the vector transfection. METHODS: The specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank, complementary single-strand DNA oligonucleotides were designed and synthesized, and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides , cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. PHB-1 expression was detected by Western blotting. RESULTS: The DNA sequence of interest clone to the vector was constructed to generate an entry clone and an expression clone successfully, which were proved by sequence determination. Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1. CONCLUSION: A RNAi eukaryotic vector containing prohibitin gene was successfully constructed.