Expression of hypoxia-inducible factor 1α in human gingival tissues with chronic periodontitis

AIM：To observe the expression of hypoxia-inducible factor 1α (HIF-1α) in human gingival tissues with chronic periodontitis. METHODS：A total of 55 volunteers, including 15 healthy controls, 20 cases of moderate chronic periodontitis and 20 cases of severe chronic periodontitis, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining, and the expression of HIF-1α in gingival tissues was detected by immunohistochemical staining. RESULTS： The proportion of HIF-1α positive cells in gingival tissues was significantly higher in chronic periodontitis groups than that in healthy control group (P<0.01), and that in severe chronic periodontitis group was significantly higher than that in moderate chronic periodontitis group (P<0.05). There was a significantly positive correlation between the severity of chronic periodontitis and the proportion of HIF-1α positive cells in gingival tissues. CONCLUSION：The expression of HIF-1α in human gingival tissues is increased with the severity of chronic periodontitis, suggesting that hypoxia may play an important role in chronic periodontitis.     

Expression of hypoxia-inducible factor 1α in pulmonary tissues from patients with chronic obstructive pulmonary disease

AIM：To study the expression of hypoxia-inducible factor 1α (HIF-1α) in pulmonary tissues from patients with chronic obstructive pulmonary disease (COPD) μg/L and its effects on the pathogenesis of COPD. METHODS：Pulmonary tissues were obtained from 32 subjects (16 patients with COPD and 16 without COPD as controls) who were undergoing single or bilateral lobectomy or wedge resection for lung cancer. The specimens were obtained as far away from the cancer foci (≥8 cm) as possible. The expression of HIF-1α protein in pulmonary tissues was measured by Western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS：The expression of HIF-1α protein in pulmonary tissues from controls and COPD patients was as follows: (0.96±0.43) μg/L and (0.16±0.07) μg/L (ELISA, P<0.05); 0.71±0.22 and 0.53±0.15 (Western blotting, P<0.05). Furthermore, the level of HIF-1α protein in pulmonary tissues from mild and moderate COPD patients was obviously higher than that from severe COPD patients (P<0.05). CONCLUSION：HIF-1α may play an important role in the progress of COPD.     

Improving effects of ginsenoside Rb1 on glucose metabolism in cardiomyocytes under hypoxia by hypoxia-inducible factor 1α

AIM: To elucidate the effect of ginsenoside Rb1 (Gs-Rb1) on the glucose metabolism to improve the viability of the cardiomyocytes under hypoxia, and whether hypoxia-inducible factor 1α (HIF-1α) and/or AMPKα are involved in the process.METHODS: The neonatal rat cardiomyocytes were cultured, and randomly divided into control group, hypoxia (1% O₂, 94% N₂ and 5% CO₂) group, Gs-Rb1 (200 μmol/L) group, Ara-A (500 μmol/L) group, Gs-Rb1+Ara-A group, YC-1 (5 μmol/L) group, Gs-Rb1+YC-1 group, Ara-A+YC-1 group and Gs-Rb1+YC-1+Ara-A group. After the intervention for 8 h, the cell viability was analyzed by MTT assay. The protein levels of AMPK, HIF-1α and glucose transporter-4 (GLUT-4) were determined by Western blot. The activities of heterophosphatase (HK), phosphofructokinase (PFK) and lactic dehydrogenase (LDH) were measured by ELISA.RESULTS: Gs-Rb1 significantly improved the viability of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 and Ara-A. In addition, YC-1 and Ara-A had a synergistic effect. Gs-Rb1 increased the protein levels of AMPK and HIF-1α in the hypoxic cardiomyocytes, which was significantly inhibited by Ara-A and YC-1. Gs-Rb1 significantly increased the expression of GLUT-4 on the cytomembrane of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 or Ara-A, especially Ara-A+YC-1. Gs-Rb1 significantly increased the activities of HK, PFK and LDH, all those were significantly inhibited by YC-1 or Ara-A. Besides, YC-1 and Ara-A had a synergistic effect.CONCLUSION: Gs-Rb1 improves the viability of hypoxic cardiomyocytes, which may be related to the regulation of glucose uptake and enhancement of glycolysis by synergy of both HIF-1α and AMPK.     

Regulatory pathway of hypoxia-inducible factor and its role in intestinal diseases

Intestinal tract is the largest organ of the human body. It plays an important role in the absorption of nutrients and the resistance to pathogens. At the same time, it is also regulated by oxygen supply. Hypoxia-inducible factor (HIF) is a key factor in the regulation of hypoxia and plays a key role in maintaining the homeostasis of the intestinal environment. In the development of inflammatory bowel disease, intestinal tumor, ischemia-perfusion injury and intestinal flora imbalance, HIF responds to inflammation and hypoxia, and causes an important adaptive response in intestinal mucosa, thus maintaining the function of the intestinal epithelium or promoting the development of intestinal tumors. In this review, the recent advances in HIF structure, function and regulatory pathways, and its role in intestinal diseases are reviewed.     

Effect of psychological stress on development of periodontitis and expression of periodontal hypoxia-inducible factor 1α in rats

AIM: To investigate the effect of psychological stress on the development of periodontitis and the expression of periodontal hypoxia-inducible factor 1α(HIF-1α) in rats.METHODS: Forty-eight male Wistar rats(SPF grade) were randomly divided into 4 groups:(1) normal control group, i.e. naive rats;(2) experimental periodontitis group: the periodontitis model was induced by wrapping 3-0 silk ligature inoculated with putative periodontopathic bacteria around the left maxillary second molar of the rats;(3) stress group: the rats were treated with stress alone;(4) periodontits with stress group, the periodontitis model was induced as above,and the rats were treated with stress. The rats were sacrificed at week 1, 4, 6 and 8 after the ligature. The attachment losses(AL) were measured by home-made probe. The histological changes of periodontal tissues stained with hematoxylin and eosin(HE) were observed under microscope. The HIF-1α expression level in the periodontal epithelium was determined by immunohistochemistry that was used to evaluate the severity of hypoxia by measuring the average rate of HIF-1α-positive cells.RESULTS: No significant difference of AL between stress group and normal control group was observed(P>0.05).The AL and the average rate of HIF-1α-positive cells in periodontitis with stress group were significantly higher than those in experimental periodontitis group at time points of week 4,6 and 8 after ligature(both P<0.01).CONCLUSION: Psychological stress is one of the periodontitis inducing factors in the animal model. Psychological stress may aggravate periodontitis by decreasing tissue oxygenation in rats.     

Expression and significance of hypoxia-inducible factor-1α on synovium from collagen-induced arthritis

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α) in the pathogenesis of rheumatoid arthritis (RA).METHODS: The collagen-induced arthritis (CIA) model was set up in male Wistar rats.The arthritis activity indexes,pathological changes and expression of HIF-1α on synovium at different time were observed through HE and immunohistochemistry staining.The correlations of HIF-1α expression with arthritis activity indexes and pathological scores were analyzed.RESULTS: Cytoplasmic and nucleic HIF-1α expressions were found on both lining and sublining area of synovium.HIF-1α expression in synovium increased gradually with the prolonged disease course.Synovial HIF-1α expression correlated significantly with arthritis activity indexes,total pathological score,synovial hyperplasia score and angiogenesis score,but not with inflammation score.CONCLUSION: HIF-1α may play important roles in the pathogenesis of RA through inducing synovial hyperplasia and angiogenesis.     

Expression of HIF-1α in rat's radiation-induced oral mucositis

AIM: To detect the mRNA and protein level of HIF-1 alpha in the tissues of rat's radiated mucosa. METHODS: The left buccal mucosa was irradiated and excised. The right buccal mucosa was excised to serve as own control tissue. The mRNA of HIF-1 alpha was determined by using the semi quantitative RT-PCR. SABC method was employed to immunostain and to elucidate the localization, intensity and distribution of HIF-1 alpha protein.RESULTS: A Sprague-Dawley rat's model of radiation-induced oral mucositis (ROM) was successfully established. The results of RT-PCR indicated that the left buccal mucosa expressed HIF-1 alpha mRNA while the right buccal mucosa did not or seldom expressed it. Immunohistochemical analysis of HIF-1 alpha demonstrated that the left side mucosa expressed HIF-1 alpha protein.CONCLUSION: The mucosa of ROM expresses the mRNA and protein of HIF-1 alpha. The expressions of HIF-1 alpha are correlated with the severity of ROM.     

Effect of hypoxia-inducible factor-1α knockdown by siRNA on viability and CEACAM1 expression in oral squamous cell carcinoma

AIM: To investigate the relationship between hypoxia-inducible factor-1α (HIF-1α) and viability and apoptosis of oral squamous cell carcinoma and its mechanism. METHODS: The expression of HIF-1α and carcinoembryonic antigen-related cell adhesion molecular 1 (CEACAM1) at mRNA and protein levels in oral squamous cell carcinoma cell lines Tca8113 and CAL27 and normal epithelial cell line NOK was determined by RT-PCR and Western blot. The expression of HIF-1α in CAL27 cells was silenced by RNA interference (RNAi) technique. The cells were divided into blank control group, non-sense control group and siRNA-HIF-1α group. The viability of CAL27 cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry. The protein levels of HIF-1α, P21, vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined by Western blot. RESULTS: The expression of HIF-1α and CEACAM1 in oral squamous cell carcinoma cells was significantly higher than that in normal cells (P<0.05), and the expression of HIF-1α and CEACAM1 was positively correlated. The protein expression of HIF-1α in siRNA-HIF-1α group was significantly lower than that in blank control group (P<0.05). Knockdown of HIF-1α significantly inhibited CAL27 cell viability (P<0.05), promoted apoptosis (P<0.05), increased the protein levels of P21 and Bax (P<0.05), and significantly decreased the levels of VEGF and Bcl-2 (P<0.05). CONCLUSION: HIF-1α is over-expressed in oral squamous cell carcinoma. Knockdown of HIF-1α significantly inhibits cell viability and promotes apoptosis possibly through regulating the expression of HIF-1α downstream target genes and tumor angiogenesis.     

Influence of hypoxia-inducible factor-1 alpha on lung cancer cell A549 growth in vitro and in vivo

AIM: To study the role of hypoxia-inducible factor-1alpha(HIF-1α) on lung cancer cells A549 growth in vitro and in vivo. METHODS: To observe the growth rate of A549 cells after HIF-1α transfected, A549 cells (1×10⁶/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group (group A, n=10), the HIF-1α transfected group(group B, n=10). The weights of subcutaneous tumor were detected. The resected specimens were made into paraffin-embedded sections. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemistry(ISH). The expressions of HIF-1α、 apoptosis-related protein survivin and bcl-2 were analyzed by Western blot. RESULTS: The growth rates of the HIF-1α transfected lung cancer cells A549 were significantly increased, and more importantly, the HIF-1α transfected lung cancer cells A549 was able to enhance lung cancer growth in nude mice(P＜0.05). The PCNA were increased significantly in group B, compared with group A. The expressions of HIF-1α, survivin and bcl-2 in group B were increased significantly than that of group A. CONCLUSIONS: HIF-1α increases lung cancer cells A549 growth in vitro and in vivo and its mechanism may be due to promotion of proliferation and inhibition of apoptosis.     

Effects of isorhapontigenin on cell cycle arrest and down-regulation of cyclin D1 expression in bladder cancer cells

AIM：To explore the inhibitory mechanism of isorhapontigenin (ISO) on the proliferation, migration and invasion of UMUC3 bladder cancer cells. METHODS：Human UMUC3 bladder cancer cells were pretreated with ISO, and the proliferation of the cells was observed under phase-contrast microscope and by ATPase assay. The expression of cyclin D1 was determined by RT-PCR and Western blotting. The cell cycle alteration was detected by flow cytometry, and the cell migration was examined by wound-healing assay. RESULTS：Over 20 μmol/L of ISO significantly inhibited the proliferation of UMUC3 cells with the IC50 of (22.5±2.8) μmol/L. The mRNA and protein levels of cyclin D1 in UMUC3 cells were markedly decreased after treatment with ISO. Exposure of UMUC3 cells to low dose (5 μmol/L) of ISO led to significant induction of G0/G1 growth arrest at both 12 h (58.82%) and 24 h (63.94%), compared with the negative control cells (47.33%) without inducing obvious apoptosis. ISO at dose of 5 μmol/L also markedly inhibited the cell migration. CONCLUSION：ISO significantly exhibits inhibitory effects on the proliferation and migration of human bladder cancer cells by down-regulation of cyclin D1 expression accompanying with G0/G1 cell cycle arrest.     

Expression of zinc-finger transcription factor Snail 1 in the kidney of diabetic rats

AIM: To explore the expression of Snail 1 in renal tissues of diabetic rats, and to investigate its contribution to the progression of diabetic nephropathy. METHODS: Streptozotocin-induced diabetic rats were randomly divided into 2, 4, 8, 12, 16, 20, 24 weeks groups and 16 week A, 20 week A and 24 week A groups. A groups were treated with insulin to control blood glucose to normal level from the 13th week. Control groups were set up in age-matched time points. Blood glucose, 24 h urine protein, serum creatinine (Scr) and kidney index of rats were measured. Periodic acid-silver (PAS) staining was used to observe the renal pathological changes. The mRNA and protein expressions of Snail 1 and FN in renal cortex were detected by RT-PCR and immunohistochemical staining, respectively. Western blotting was employed to detect the expression of Snail 1 protein in the renal cortex. RESULTS: The levels of blood glucose, Scr, kidney weight index were increased remarkably in diabetic rats as compared with those in control groups (P<0.05, P<0.01), and decreased remarkably in the insulin-treated rats as compared with those in the diabetic rats (P<0.05, P<0.01). The Snail 1 protein was not detected by immunohistochemical staining in normal renal tissues. However, strongly positive staining was observed in renal tubules of diabetic rats. A time-dependent loss of Snail 1 expression was detected in the kidney in insulin-treated rats. The Snail 1 protein and mRNA of Snail 1 and FN were significantly up-regulated in the diabetic rats as compared with those in controls (P<0.01), while down-regulated in the insulin-treated diabetic rats (P<0.01). A close positive relationship existed between the mRNA expression of Snail 1 and FN (r=0.800, P<0.01). The level of Snail 1 protein expression was positively correlated with blood glucose, urine protein, Scr, kidney index (r=0.877, 0.694, 0.522, 0.875, P<0.01).CONCLUSION: These findings suggest that Snail 1 gene and protein expression are up-regulated in the kidney of rats with diabetes and may be involved in the pathogenesis of diabetic nephropathy.     

苹果属小金海棠转录因子MxMYB1基因的克隆及其原核表达

 MxMYB1是苹果属小金海棠MYB类转录因子。MxMYB1含1个重复序列及MYB蛋白特有的氨基酸组成。酶切、PCR扩增及测序分析表明构建的原核表达载体结构正确, 未出现碱基突变及移码现象。1 mmol/L IPTG诱导2 h后, 在预期的蛋白分子量38 kD处出现1条表达加强的蛋白条带, 而未经诱导的转化子没有此蛋白条带。为进一步目的蛋白的纯化和鉴定提供试验基础。     

Changes of plasma endothelin-1 and von Willebrand factor in patients undergoing cerebral digital substration angiography

AIM: To investigate the effects of cerebral digital substration angiography (DSA) on the expression of endothelin-1(ET-1) and von Willebrand factor(vWF) in vascular endothelial cells from the patients with ischemic cerebrovascular disease. METHODS: The plasma levels of ET-1 and vWF were measured by enzyme-linked immunosorbent assay in 108 patients before, immediately and 24 h after cerebral DSA. RESULTS: The plasma level of endothelin-1 significantly increased immediately and 24 h after cerebral DSA as compared with that before the procedure (P<0.05). The level of vWF was not changed before and after operation. CONCLUSION: The marked and sustained increase in endothelin-1 plasma level after cerebral DSA is probably related to mechanical endothelial injury by the procedure of cerebral DSA.     

Effect of BDNF expression in cerebral cortex and hippocampus on ability of learning and memory in APP/PS1 transgenic mice

AIM: To investigate the expression changes of brain-derived neurotrophic factor (BDNF) in the cerebral cortex and hippocampus and their effects on the ability of learning and memory in the wild-type (WT) mice and APP/PS1 transgenic mice. METHODS: WT mice and APP/PS1 transgenic mice were selected as study subjects. Aβ plaques, apoptosis rate and BDNF expression in the cerebral cortex and hippocampus of WT mice and APP/PS1 transgenic mice were detected by the methods of Congo red staining, TUNEL, immunofluorescence and Western blot. The abilities of learning and memory were determined by Morris water maze test. RESULTS: The Aβ plaques appeared in the cerebral cortex and hippocampus of APP/PS1 transgenic mice, and the number of Aβ plaques in 12-month-old mice was larger than that in 6-month-old mice (P<0.05). The number of apoptotic neurons in the cerebral cortex and hippocampus of 12-month-old APP/PS1 transgenic mice was larger than that of WT mice (P<0.01). The expression level of BDNF in the cerebral cortex and hippocampus of WT mice was higher than that of APP/PS1 transgenic mice (P<0.01). The Morris water maze test showed that the escape latency in APP/PS1 transgenic mice was longer than that in WT mice, and the times across the platform quadrant in 60 s was less than that in WT mice (P<0.01). The swim-tracking path of APP/PS1 transgenic mice was disordered and irregular. CONCLUSION: The expression of BDNF in the cerebral cortex and hippocampus of APP/PS1 transgenic mice was lower than that of WT mice, accompanied by increased neuronal apoptosis and decreased spatial learning and memory ability. The decrease in learning and memory ability may be related to decreased BDNF expression in the cerebral cortex and hippocampus of APP/PS1 transgenic mice, leading to increased neuronal apoptosis, which may be one of the pathological mechanisms of Alzheimer disease.     

Expression and histogenesis of matrix metalloproteinase-9 and transforming growth factor-beta 1 in acute cerebral ischemia model of rats

AIM:To observe the expression and tissue localization of matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta 1 (TGF-β1) in the rat acute cerebral ischemia model. METHODS:Male Wistar rats were used to establish acute cerebral ischemia model by a suturing method. The rats were divided into normal control group, sham group and ischemia 6 h, 12 h, 1 d, 2 d, 6 d and 14 d groups. The rat cerebral cortex and hippocampus of the brain were collected at different time points.The mRNA and protein levels of MMP-9 and TGF-β1 in the brain tissues were detected by real-time PCR and in situhistochemistry staining, respectively. The levels of MMP-9 and TGF-β1 in the plasma were also measured by ELISA. RESULTS:The results of real-time PCR showed that the mRNA levels of MMP-9 began to increase 6 h after acute ischemia and reached to a peak 2 d after acute ischemia. Similarly, the mRNA level of TGF-β1began to rise 12 h after acute ischemia and reached to the highest level 6 d after acute ischemia. Compared with the sham rats, the mRNA levels of MMP-9 and TGF-β1 in the rat brains that collected at ischemic time of 12 h, 1 d, 2 d, 6 d and 14 d were significantly increased. Moreover, results of in situhistochemical staining showed that the expression of MMP-9 was detected at cerebral cortex and hippocampus 1 d after acute cerebral ischemia.Further studies showed that MMP-9 dyeing of the rat cerebral cortex was most obvious 2 d after the acute cerebral ischemia. Similarly, the rat cortex and hippocampus began to express TGF-β1 2 d after acute ischemia and TGF-β1 staining at rat cerebral cortex was most obvious 6 d after the acute cerebral ischemia. In addition, ELISA showed that the increase in MMP-9 and TGF-β1 was detected in the plasma 12 h after ischemia. Compared with the sham rats, the level of these 2 factors significantly upregulated since 1 d after ischemia. CONCLUSION: The brain tissue itself contributes to the upregulation of MMP-9 and TGF-β1 post acute cerebral ischemia, which shed light on the related research in the field.     

茎瘤芥AP2/EREBP 转录因子基因BjABR1 的克隆和表达分析

 以茎瘤芥（Brassica juncea var. tumida Tsen et Lee）‘永安’为材料,通过RACE 和RT-PCR 技术得到1 个AP2/EREBP 转录因子基因的cDNA 全长序列和基因组DNA（gDNA）序列,该基因与拟南 芥AtABR1 基因的氨基酸序列相似性高达72%,因此命名为BjABR1（GenBank 登录号：JQ713825.1）。BjABR1 基因gDNA 序列含1 个内含子;cDNA 序列全长1 514 bp,含有1 个1 146 bp 的开放阅读框（ORF）,编 码381 个氨基酸;其推定编码的蛋白分子量为41.674 kD,等电点为9.11,具有14 个磷酸化位点,含1 个典型的AP2 DNA 结合域和1 个CMX-1 基序。洋葱表皮细胞的瞬时表达显示,BjABR1 蛋白定位于细 胞核。荧光定量PCR 分析结果表明,该基因在茎瘤芥不同发育时期的根、茎、叶中均有表达,但在根中 表达量极高;在对茎瘤芥组培幼苗进行高盐、渗透压和低温3 种非生物胁迫处理后发现,3 种胁迫均能诱 导该基因表达,但其对高盐的响应更为迅速。