The effects of linking loop structure between guanine ( G) repeats on G-quadruplex formation were investigated. The results show that the unfavorable effects of long linking loops on G-quadruplex formation can be overcome by introducing double-stranded structures in linking loop regions. This finding provides a new way for sensor design. The activity of G-quadruplex DNAzyme can be tuned by utilizing target-mediated formation of double-stranded structures in loops. As an example, T-T mismatches are introduced in loops to destroy double-stranded structures. The stabilization of Hg2+ to T-T mismatches promotes the reformation of double-stranded structures. Correspondingly, the oligonucleotide folds into G-quadruplex, which binds with hemin to form peroxidase-like G-quadruplex DNAzyme. Hg2+ sensor is designed based on this principle. Using this method, Hg2+ quantitation is achieved in the concentration range of 10-700 nmol/L, with a detection limit of 8. 7 nmol/L. Cysteine will compete with T bases to bind with Hg2+, releasing Hg2+from T-Hg2+-T base pairs. Thus cysteine can also be quantified with this system in the concentration range of 20-700 nmol/L, with a detection limit of 14 nmol/L.

Objective:To investigate the role of melatonin in calcium overload-induced heart injury.Methods:Thirty-two rats were divided into 4 groups:a control group (Control),a melatonin control group (Mel),a calcium overload group (CaP),and a calcium overload plus melatonin group (Mel+CaP).Isolated Sprague Dawley male rat hearts underwent Langendorffperfusion.Left ventricular developed pressure (LVDP) was calculated to evaluate the myocardial performance.Triphenyltetrazolium chloride staining was used to measure the infarct size of myocardium.Lactate dehydrogenase (LDH) activity in the coronary flow was determined.The expressions of caspase-3 and cytochrome c were determined by Western blot.The pathological morphological changes in myocardial fiber were analyzed by HE staining.Results:Compared with the control group,calcium overload significantly induced an enlarged infarct size (P＜0.01),accompanied by the disordered arrangement of myocardial fiber,up-regulation of cytochrome c and caspase-3 (P＜0.01),and the increased activity of LDH (P＜0.01).T hese effects were significantly attenuated by 10 μmol/L melatonin (P＜0.01).Conclusion:Melatonin can alleviate calcium overload-induced heart injury.

Background Research showed that the morbidity rate of primary angle closure glaucoma (PACG) in Mongolian population is 3.02 times more than Han nationality population.To understand the cause and mechanism of PACG in Mongolia is of an important significance.Objective This study was to investigate the pathogenesis of Mongolian PACG.Methods Thirty-two eyes of 32 PACG patients in Mongolia and 40 eyes of 40 PACG patients of Han peoples were included in Inner Mongolia Autonomous Region People's Hospital according to the diagnosis criteria of glaucoma group of Chinese Medical Ophthalmology Association (version 1987),and 13 eyes of 13 normal Mongolia and 17 eyes of 17 normal Han peoples who suffered with ocular truma were recruited as controls.Intraocular pressure(IOP) was measured before surgery.The trabecular meshwork tissue was obtained from all the eyes during the operation.Annexinv-FITC/PI double staining was performed and the apoptosis rate of trabecula cells was tested with flow cytometry.Written informed consent was obtained initial of the study.Results The IOP value in Mongolia PACG group,Han PACG group,Mengolia normal group and Han normal group was (35.97±7.11)mmHg,(38.70± 6.82) mmHg,(14.69 ± 2.91) mmHg and (13.59 ± 2.91) mmHg,respectively,showing a significant difference among the 4 groups(F=106.144,P=0.000),and the IOP was significantly higher in the Mengolia PACG group and Han PACG group than the normal groups(P＜0.05).The apoptosis rate of the cells was (7.14±0.67)％,(5.40±0.69) ％,(5.86±0.91) ％ and(2.29±0.65) ％ in the Mongolia PACG group,Han PACG group,Mongolia normal group and Han normal group,respectively,with a significant difference among them (F =174.888,P =0.000),and apoptosis rate of the Mongolia PACG group was significantly higher than that of the Han PACG group and the Mongolia normal group (P＜0.05).No significant difference was found between the Mongolia PACG group and the Han PACG group or between the Mongolia normal group and Han normal group (P＞0.05).The cell apoptosis rate was increased with the elevation of IOP (b =0.990,F=10.209,P =0.009) with the regression equition Y =2.788 +0.092X.Conclusions The apoptosis rate of trabecula cells in Mongolian is higher than Han people.If these results are associated with the high incidence of Mengolia PACG is worth of study.

Objective To evaluate the changes in mRNA expressions of Toll-like receptor 4 (TLR4) and its down-stream cytokines IL-1β,IL-6 and TNF-a in incisional tissues from a rat with postoperative pain.Methods Incisional pain was induced in 74 male adult SD rats weighing 200-250 g.Paw mechanical withdrawal threshold (PMWT) around the wound on the operated and nonoperated sides was measured at 1 day before operation and at 0.5,1,2,6 and 12 hours as well as 1,2,3,5 and 7 days after operation.Skin incisional tissues were removed for determination of mRNA expressions of TLR4,IL-1β,IL-6 and TNF-a using real-time quantitative PCR at 1 day before operation and at 2 and 8 hours as well as 1,2,3,5 and 7 days after operation.Results Compared with the baseline value before operation,PMWT on the operated side was significantly decreased at 0.5 hours-5 days after operation,mRNA expression of TLR4 around the wounds on the operated side was down-regulated at 2 hours after operation followed by a gradual increase,mRNA expressions of IL-1β,IL-6 and TNF-α on the operative side were up-regulated at 2 and 8 hours as well as 1,2,3 and 5 days after operation (P ＜ 0.05),but no significant changes were found in PMWT and mRNA expressions of TLR4,IL-1β,IL-6 and TNF-α on the non-operated side(P ＞ 0.05).PMWT on the operated side was lowest at 6 hours after operation followed by the gradual increase,mRNA expression of TLR4 on the operated side peaked at 2 days after operation,and mRNA expressions of IL-1β,IL-6 and TNF-a respectively peaked at 2 hours,1 day and 3 days after operation (P ＜ 0.05).mRNA expressions of TLR4,IL-1β,IL-6 and TNF-a were negatively correlated with PMWT on the operative side (r =-0.501,-0.743,-0.893,-0.657,P ＜ 0.05),and mRNA expressions of IL-1 β,IL-6 and TNF-a were positively correlated with the level of TLR4 mRNA(r=0.764,0.283,0.667,P＜0.05).Conclusion mRNA expressions of TLR4 and its down-stream cytokines IL-1 β,IL-6 and TNF-a in skin incisional tissues are up-regulated,which may be involved in the development and maintenance of postoperative pain.

OBJECTIVETo compare the intervention effects of four traditional Chinese medicines (TCMs) with typical cold or hot property on body temperature and temperature-sensitive transient receptor potential ion channel proteins (TRPs) of rats with yeast-induced fever.

METHODThe pyrexia model was induced by injecting yeast suspension subcutaneously. Totally 108 male SD rats were randomly divided into the normal group, the model group, the Rhei Radix et Rhizoma treated group, the Coptidis Rhizoma treated group, the Euodiae Fructus treated group, and the Alpiniae Officinarum Rhizoma treated group, with 18 rats in each group. At the 4 h, 8 h and 12 h after injection of yeast, the rats were sacrificed to collect their hypothalamus and dorsal root ganglion. The expressions of TRPV1 and TRPM8 were detected by immunohistochemistry and Western blot method.

RESULTCompared with the normal group, after injection of yeast, the temperature of rats in the model group notably increased, and reached the peak at 8 h (P < 0.01). The TRPV1 level in hypothalamus and dorsal root ganglia (DRG) of the model group significantly increased, whereas the TRPM8 level significantly reduced. Compared with the model group, the Rhei Radix et Rhizoma group and the Coptidis Rhizoma group showed significant decrease in the high body temperature of rats caused by yeast, down-regulation in the expression of TRPV1, and up-regulation in the expression of TRPM8 (P < 0.05 or P < 0.01). Euodiae Fructus and Alpiniae Officinarum Rhizoma had no significant effect on either temperature or TRPs of fever rats.

CONCLUSIONRhei Radix et Rhizoma and Coptidis Rhizoma, both are TCMs with cold property, can reduce the temperature of fever rats induced by yeast, which may be related to their effective regulation of TRPV1 and TRPM8 in hypothalamus and DRG, while Euodiae Fructus and Alpiniae Officinarum Rhizoma had no relevant effect.

Animals ; Antipyretics ; administration & dosage ; chemistry ; Body Temperature Regulation ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Fever ; drug therapy ; immunology ; microbiology ; physiopathology ; Gene Expression Regulation ; drug effects ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Saccharomyces cerevisiae ; immunology ; TRPM Cation Channels ; genetics ; immunology ; TRPV Cation Channels ; genetics ; immunology

OBJECTIVETo observe the effect of intracerebroventricular injection of rAAV-HIF-1α on hippocampal neuronal apoptosis in a rat model of Alzheimer disease (AD).

METHODSThirty-two male SD rats (250-300 g) were randomized into 4 groups (n=8), including the normal control group without any treatment, AD model group with right intracerebroventricular injection of 2 µl Aβ25-35 (10 mg/m1), sham-operated group with right intracerebroventricular injection of 2 µl normal saline, and AD+ rAAV-HIF-1α group with right intracerebroventricular injection of 10 µl rAAV-HIF-1a (1×10¹² v.g./m1) one week after Aβ25-35 injection. The rats were sacrificed to detect the expression of HIF-1α and apoptosis of hippocampal neurons 5 weeks after Aβ25-35 or saline injection.

RESULTSWestern blotting showed that the expression of HIF-1α was significantly higher in AD+rAAV-HIF-1α group (451.59±34.39) than in normal control group (229.05±41.28) and sham-operated group (216.29±37.08) (P<0.05) without significant difference between the latter two groups. The apoptotic ratio of the hippocampal neurons was significantly higher in AD model group ([19.49±2.59]%) than in normal control group ([5.41±0.75]%) and sham-operated group ([5.28±0.66]%) in (P<0.05), and intracerebroventricular injection of rAAV-HIF-1α resulted in a significant reduction of the apoptotic ratio in the AD rats ([12.07±2.06]%) (P<0.05).

CONCLUSIONIntracerebroventricular injection of rAAV-HIF-1α can inhibit hippocampal neuronal apoptosis in the rat model of AD.

Alzheimer Disease ; metabolism ; therapy ; Animals ; Apoptosis ; Disease Models, Animal ; Hippocampus ; cytology ; Hypoxia-Inducible Factor 1, alpha Subunit ; administration & dosage ; genetics ; metabolism ; Lateral Ventricles ; Male ; Neurons ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate and compare the histopathology effects and mechanisms of the methods of "invigorating spleen to remove phlem & promoting blood circulation to remove meridian obstruction" and "invigorate the kidney & promoting blood circulation to remove meridian obstruction" preventing hormone induced femoral head necrosis in hens.

METHODSSixty-four healthy hens were randomly divided into 4 groups: blank control group, model group, Jianpi group (with therapeutics of invigorating spleen to remove phlem), Bushen group (with the effect of warming kidney for duresis). All hens were injected intramuscularly with Medrat once a week for 16 weeks but normal saline in blank control group. Bilateral femoral heads were dissected on 8 weeks or 16 weeks. Paraffin tissue sections were prepared to detect histopathologic change with hematoxylin and eosin, or mason staining.

RESULTSHistological analysis showed that Huogu recipe I and Huogu recipe II can promote osteogenesis and repair osteonecrosis, increase blood circulation of bone marrow, and inhibit pimelosis of bone marrow. Compared with blank control group, the areas of adipose cells increased significantly (t = -12.9, P < 0.01), the area of immature collagen increased significantly (t = -2.0, P < 0.05) and the ratio of empty lacuna in medullary cavity (t = -3.7, P < 0.05). Compared with model group, both the area of adipose cells and the ratio of empty lacuna decreased in Jianpi group and Bushen group (F = 26.8, 13.5, P < 0.01), so it was with the Bushen group immature collagen (F = 4.6, P < 0.01).

CONCLUSIONBoth the methods of "invigorating spleen to remove phlem & promoting blood circulation to remove meridian obstruction" and "invigorate the kidney & promoting blood circulation to remove meridian obstruction" can prevent hormone induced femoral head necrosis. The time taking effect in the method of "invigorating spleen to remove phlem" was shorter.

Animals ; Chickens ; Drugs, Chinese Herbal ; Female ; Femur Head ; pathology ; Femur Head Necrosis ; chemically induced ; drug therapy ; pathology ; Glucocorticoids ; adverse effects ; Medicine, Chinese Traditional

OBJECTIVETo study the mechanism of Huogu I formula (I) in treating osteonecrosis of femoral head.

METHODSForty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 (BMP2), transforming growth factor beta1 (TGFβ(1)), Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand (OPG/RANKL) mRNA was detected by in situ hybridization.

RESULTSCompared with the control group, serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the model group rose significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant.

CONCLUSIONHuogu I formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ(1), Smads and OPG/RANKL of osteoclast in femoral head.

Animals ; Bone Morphogenetic Protein 2 ; metabolism ; Bone Regeneration ; drug effects ; physiology ; Chickens ; Chondrocytes ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Female ; Femur Head Necrosis ; chemically induced ; drug therapy ; metabolism ; Lipid Metabolism ; physiology ; Osteocytes ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Smad4 Protein ; metabolism ; Smad7 Protein ; metabolism ; Steroids ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo observe effects of blood circulation promoting compounds combined with medicinal guides on content of bone glaprotein (BGP), bone morphogenetic protein-2 (BPM-2) and expression of BMP-2 mRNA in rabbits with femoral head necrosis, and explore its mechanism.

METHODSNinety-eight healthy Spragur-Dawley male rabbits were collected and weighted 2.2 to 2.8 kg. Eighty-four rabbits were built femoral head necrosis model by freezing left femoral head in liquid nitrogen, then randomly divided into 6 groups, 14 in each group. The 6 groups included model group,promoting blood circulation to remove meridian obstruction group,promoting blood circulation to remove meridian obstruction combined with achyranthes bidentata group,radix angelicae pubescentis, asarum group, and platycodon grandiflorum group,other 14 rabbits were sham operation group. While drug groups were administrated corresponding Chinese herb after molding,model group and shamp operation group were given saline. Recombinant human granulocyte-colony stimulating factor ( 30 microg x kg(-1) x d(-1))were injected into all rabbits for 7 days. Samples were taken on the second and fourth week,the content of BGP and BMP-2 were detected by enzyme-linked immunosorbent assay (ELSA) and radioimmunoassay (RIA), histopathological changes of left femoral head were observed by Hematoxylin and Eeosin staining (HE), and expression of BMP-2 mRNA were tested by fluorescence in situ hybridization.

RESULTSCompared with sham operation group, the rate of empty lacunae femoral head were obviously increased in model group, and the content of BGP were increased on the second week, and BMP-2 and BMP-2 mRNA were decreased on the fourth week. Compared with model group, the content of BGP, BMP-2 and BMP-2 mRNA were higher both of the second and fourth week in promoting blood circulation to remove meridian obstruction group. The rate of empty la- cunae femoral head were lower in achyranthes bidentata group, BGP, BMP-2 and BMP-2 mRNA were higher on the fourth week. The rate of empty lacunae femoral head were lower in platycodon grandiflorum group, and BGP were decreased on the second and fourth week, BMP-2 were lower on the second week ,while BMP-2 mRNA were decreased on the fourth week; the content of BMP-2 and BMP-2 mRNA were increased in radix angelicae pubescentis group on the second week; while there was no change in asarum group.

CONCLUSIONRadix angelicae pubescentis can increase the content of BGP, BMP-2 and expression of BMP-2 mRNA ,which is an effective mechanism of preventing femoral head necrosis.

Animals ; Blood Circulation ; drug effects ; Bone Morphogenetic Protein 2 ; analysis ; genetics ; Femur Head Necrosis ; drug therapy ; pathology ; physiopathology ; Male ; Meridians ; Osteocalcin ; blood ; Osteogenesis ; drug effects ; Rabbits

OBJECTIVETo investigate the effect of Huogu II Formula (II) with medicinal guide Radix Achyranthis Bidentatae (Ach) on bone marrow stem cells (BMSCs) homing to necrosis area after osteonecrosis of the femoral head (ONFH) frozen by liquid nitrogen in rabbit as well as to explore the mechanism of prevention and treatment for ONFH.

METHODSThe animal model of ONFH was established by liquid nitrogen frozen on the rabbit left hind leg. Forty-eight Japanese White rabbits were randomly assigned to sham-operated group, model group, Huogu II group, and Huogu II plus Ach group, with 12 rabbits in each. During the course of ONFH animal model establishment, all rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor [rhG-CSF, 30 μg/(kg·day) for continuous 7 days]. Meanwhile, normal saline and decoction of the two formulae were administrated by gavage, respectively. White blood cells (WBC) were counted in peripheral blood before and after injection of rhG-CSF. Materials were drawn on the 2nd and 4th weeks after model built; bone glutamine protein (BGP) and bone morphogenetic protein 2 (BMP2) levels in serum were tested. Histopathologic changes were observed by hematoxylin and eosin (HE) staining. BMP2 mRNA levels were detected with in situ hybridization (ISH) staining. 5-Bromo-2'-deoxyuridine (BrdU) and stromal cell derived factor 1 (SDF-1) were measured by immunohistochemical assay in femoral head of the left hind leg.

RESULTSCompared with the shamoperated group, the ratio of empty lacuna, serum BGP, and SDF-1 level in the model group increased significantly, and BMP2 in both serum and femoral head decreased significantly. However, in comparison with the model group, the empty lacuna ratio of Huogu II group and Huogu II plus Ach group decreased obviously in addition to the levels of serum BGP and BMP2, and the expressions of BMP2 mRNA, BrdU, and SDF-1 increased significantly. Above changes were particularly obvious in Huogu II plus Ach group. BGP and SDF-1 on the 2nd week and empty lacuna rate and serum BMP2 level on the 4th week in Huogu II group significantly exceeded their counterparts. On the 2nd week, only in Huogu II plus Ach group that the BrdU counting rose significantly. On the 4th week, empty lacuna rate and serum BMP2 level in Huogu II plus Ach group exceeded those in Huogu II group distinctively.

CONCLUSIONSTo a certain extent, the medicinal guide Ach improves the preventive and therapeutic effects of Huogu II Formula on experimental ONFH model. The possible mechanism of this is related to its promoting effect on directional homing of BMSCs to the necrosis area.

Achyranthes ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Bone Morphogenetic Protein 2 ; blood ; genetics ; Bromodeoxyuridine ; metabolism ; Cell Movement ; Chemokine CXCL12 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Femur Head ; drug effects ; pathology ; Femur Head Necrosis ; blood ; genetics ; pathology ; therapy ; Gene Expression Regulation ; drug effects ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; pharmacology ; Humans ; Leukocyte Count ; Male ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Radioimmunoassay ; Stem Cell Transplantation ; Stem Cells ; cytology ; drug effects