Objective To investigate the effect of sufentanil preconditioning on the expression of Toll-like receptor 4 (TLR4) in myocardium during myocardial ischemia-reperfusion (I/R) in rats.Methods Thirty-six male Sprague-Dawley rats,weighing 250-300 g,were randomly divided into 3 groups (n =12 each):sham operation group (group S),I/R group and sufentanil preconditioning group (group SPC).Myocardial ischemia was induced by occlusion of anterior descending branch of left coronary artery for 30 min followed by 2 h of reperfusion.In group SPC,the rats were subjected to 3 consecutive cycles of 5 min sufentanil infusion at 0.2 μg· kg-1 ·min-1 via the femoral vein at 5 min interval before ischemia.In groups S and I/R,the rats were subjected to the equal volume of normal saline instead.HR and mean arterial pressure (MAP) were recorded at 30 min before ischemia (T0),immediately before ischemia (T1),at 30 min of ischemia (T2),and at 30 and 120 min of reperfusion (T3-4).At the end of reperfusion,blood samples were obtained to determine the serum concentration of TNF-α.The rats were then sacrificed and hearts were removed for measurement of myocardial infarct size and expression of TLR4 and NF-κB p65 in myocardium (using Western blot).Results There was no significant difference in HR among the three groups (P ＞ 0.05).Compared with group S,MAP was significantly decreased at T2-4,the serum concentration of TNF-α was increased,and the expression of TLR4 and NF-kB p65 protein in myocardium was upregulated in groups I/R and SPC (P ＜ 0.01).Compared with group I/R,no significant difference was found in MAP at all time points (P ＞ 0.05),and myocardial infarct size was significantly decreased,serum concentrations of TNF-α were decreased,and the expression of TLR4 and NF-κB p65 protein in myocardium was down-regulated in group SPC (P ＜ 0.01).Conclusion The mechanism by which sufentanil preconditioning alleviates myocardial I/R injury may be related to down-regulation of TLR4 expression in rat myocardium.

OBJECTIVETo study immunomodulating activity of Lonicera Japonica flavone by investigating immune enzymatic activity of serum and antoxidized activity of lymphoid organs in mice.

METHODSFifty KM mice were randomly divided into control group, model group, low dose group, middle dose group and high dose group(n = 10), respectively. And low dose group, middle dose group and high dose group were given Lonicera Japonica flavone with 100 mg/kg, 200 mg/kg and 400 mg/kg every day, respectively, while control group and model group were administered with NS. After continuously giving drug 7 weeks, other groups were injected with Dexamethasome (Dex: 25 mg /kg) for 3 days by subcutaneous injection, but the control group were treated with NS. And after giving Lonicera Japonica flavone 1 week simultaneously, organ indexes , the activity of acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LSZ) in serum , and the content of monoamine oxidase (MAO), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD) and malondialdehyde (MDA) in lymphoid organs in mice were tested, respectively.

RESULTSLonicera Japonica flavone could significantly improve the organ indexes, and significantly improve the activity of ACP, AKP and LSZ in serum, and significantly improve the contents of T-AOC and SOD, but reduce that of MAO and MDA in lymphoid organs in immunosuppressed mice.

CONCLUSIONIonicera Japonica flavone can significantly improve the activity of immune enzyme in serum and the antioxidized activity of lymphoid organs in mice. It suggests that Ionicera Japonica flavone has a good immunomodulatory effects.

Acid Phosphatase ; blood ; Alkaline Phosphatase ; blood ; Animals ; Antioxidants ; metabolism ; Flavones ; pharmacology ; Immunomodulation ; Lonicera ; chemistry ; Malondialdehyde ; metabolism ; Mice ; Monoamine Oxidase ; metabolism ; Muramidase ; blood ; Superoxide Dismutase ; metabolism

Objective To investigate the effects of small interfering RNA(siRNA)on the expression of CD147 on peripheral blood T-lymphocytes from patients with psoriasis vulgaris,and its effect on the proliferation and activation of these cells.Methods Peripheral blood monouclear cells(PBMC)were obtained from 10 patients with psoriasis vulgaris,and T lymphocytes were isolated.CD147 siRNA was chemically synthesized,then electroporated into the peripheral T-lymphocytes.Untransfected cells,blank-transfected cells and unspecifically transfected cells served as the control.After 24-,48-,72-and 96-hour incubation,RT-PCR was used to detect the mRNA expression of CD147 in these cells.MTT assay and flow cytometry were utilized to assess the proliferation of these cellas,and the expression Of CD25 at 24,48,and 72 hours after the transfection.Results Compared with untransfected cells,the mRNA expression of CD147 declined significantly in CD147 siRNA-transfected cells at 24 hours(P＜0.05),reached to the minimum at 48 hours (P＜0.01):there was no significant difierence in the expression of CD147 between the two groups of cells at 96 hours after the transfection(P＞0.05).There was a decrease of cell proliferation level by(44.5±3.13)%,(50.7±3.5)%and(53.98±4.15)%in CD147 siRNA-transfected cells 24,48 and 72 hours following the transfection,respectively;the corresponding decrease in blank-transfected cells was (37.28±3.56)%,(33.73±3.29)%,and(28.80±1.49)%,respectively,and that in unspecifically transfected cells,(31.29±2.46)%,(36.1±2.62)%and(32.08±2.78)%,respectively.A significant decrease was observed in the proliferation of CD147 siRNA-transfected cells compared with that of blank-transfected cells and unspecifically transfected cells at these three time points(P＜0.05,0.01,0.01 respectively).The expression rate of CD25 at 24,48 and 72 hours was(47.23±3.65)%,(31.50±4.22)%and(23.05±4.15)%,respectively,on CD147 siRNA-transfected cells,and,(80.2±4.8)%,(81.6±3.35)%and(83.5±4.1)%,respectively,on untransfeeted cells;the differences between the two groups at the three time points were statistically significant(all P＜0.01).Conclusion CD147 is correlated with cell proliferation and activation of peripheral T lymphoeytes from patients with psoriasis vulgaris,and may serve as a new treatment target for psoriasis.

To explore the relationship between serum thyroid stimulating hormone (TSH) level and obesity and nonalcoholic fatty liver disease (NAFLD) in euthyroid subjects, 1322 subjects were subjected to a questionnaire survey and physical examination. Fasting blood samples were collected to test serum TSH, plasma glucose and lipids. Fatty liver was diagnosed by type B ultrasonography. The relationship between serum TSH level and body mass index (BMI), percentage of body fat and NAFLD was analyzed. The results showed that serum TSH level was significantly higher in females than in males at the same group, and it was significantly higher in overweight group than in control group. Levels of body weight, BMI, waist circumference and percentage of body fat were increased in TSH >2.5 group compared to TSH ≤2.5 group in women. However, plasma lipids showed no significant differences. In males all the parameters showed no significant differences between two groups. Serum TSH was significantly correlated with body weight, BMI, waist circumference and percentage of body fat after adjustment for age in females. Multiple linear regression analysis revealed that percentage of body fat and BMI contributed significantly to the variance of TSH. Serum TSH level was significantly higher in nonalcoholic fatty liver group than in normal group in females. Multiple logistic regression analysis showed that TSH level was not the independent risk factor of NAFLD. Taken together the data suggest that serum TSH in normal range is significantly correlated with BMI and percentage of body fat in females. And the change of TSH level would not influence the prevalence of NAFLD.

Objective To explore the influence of fluorine on mRNA and protein expression of the insulin-like growth factor-1 (IGF-1) and its receptor of rat osteoblasts.Methods Osteoblasts were isolated from rat bone by enzyme digestion.Different fluorine concentration [0 (control),10-7,10-6,10-5,10-4,10-3 mol/L] was add to the second generation osteoblasts.The IGF-1 in the culture medium was determined by radioimmunoassay (RIA) at different fluorine concentration and different time (24,48 h).The expression of IGF-1 receptor was measured by the method of fluorescent quantitation PCR and the expression of protein IGF-1 receptor was measured by Western blotting.Results ①With increased dose of fluoride exposure,IGF-1 concentration in the osteoblastic culture medium increased first and then decreased at 24,48 h,respectively.Compared to the control group [(38.83 ± 3.48)ng/L],IGF-1 concentration of the 24 h 10-6 mol/L group[(65.45 ± 4.84)ng/L] was higher,and the difference was statistically significant(P ＜ 0.05).The same result was also shown in the 48 h 10-5 mol/L group [(59.14 ± 1.53)ng/L] to its corresponding control group [(33.79 ± 1.84)ng/L,P ＜ 0.05].②The mRNA expression of IGF-1 receptor of the 24,48 h 10-5 mol/L groups (0.0055 ± 0.0004,0.0262 ± 0.0040) was significantly higher than their corresponding control groups (0.0022 ± 0.0001,0.0073 ± 0.0008,all P ＜ 0.05).③With increased dose of fluoride exposure,the protein expression of IGF-1 receptor increased first and then decreased ;the expression of 24 h 10-5 mol/L group (1.39 ± 0.16) was compared with the corresponding control group (0.86 ±0.12),and the difference was statistically significant (P ＜ 0.05) ; the expression of 48 h every fluorine group was also compared with the corresponding control group,and the difference was not statistically significant(all P＞ 0.05).Conclusions Fluorine can affect the mRNA and protein expression of osteoblastic IGF-1 and its receptor.It indicates that IGFS signal transduction pathways play an important role in fluorine regulation of bone metabolism.

Cognitive impairment is a common complication of diabetes. Hippocampus plays an important role in cognitive function. In hyperglycemia, synaptophysin, a crucial synaptic vesicle membrane protein in hippocampus neuron is found to be down-regulated. Recent evidences have shown that angiotensin IV can facilitate memory acquisition and recovery. However, whether it can also improve cognitive functions of diabetic rats with cognitive disorder, and the possible mechanisms are uncertain. Hence, the objectives of this study. Forty fi ve Sprague Dawley male rats were randomly divided into three groups: Control, diabetic group and diabetes with angiotensin IV treatment group. The cognitive functions, mainly learning and memory of the rats were evaluated using Morris water maze task. The synapses ultrastructure, relative mRNA concentrations and protein expression levels of synaptophysin in hippocampus CA1 area were estimated using transmission electron microscope, RT-PCR, immunohistochemistry and western blotting, respectively. Our study showed that in the diabetic rats with angiotensin IV treatment, the cognitive impairment as measured by Morris water maze task improved, the ultrastructure of synapses in hippocampus reversed, the relative mRNA concentrations and protein levels of synaptophysin in hippocampus signifi cantly increased, when compared with diabetic rats. We conclude that angiotensin IV plays an important role in improving cognitive function of diabetic rats. The possible mechanisms are up-regulating the expression of synaptophysin and normalizing the ultrastructure of synapses in hippocampus.

Background Leber hereditary optic neuropathy (LHON)is a common inherited eye disease,which generally affects young adults with bilateral loss of central vision.Mutation frequency of Leber hereditary has not been fully clarified. Objective This study was to investigate the mutation frequency of mitochondrial NDI gene associated with LHON in Chinese population. Methods The proposal of the study was approved by Ethic Committee of Wenzhou Medical College.Written informed consent was obtained from each subject initial of this trial.Eight hundred and ninety-four LHON patients and 134 normal subjects were collected.Genomic DNA was extracted from peripheral blood leukocytes of the all participants.Polymerase chain reaction (PCR) was used to amplify and sequence analysis of the mitochondrial ND1 gene was performed and aligned with revised Cambridge Reference Sequence(rCRS) of mitochondrial DNA.Then mutated gene frequency was screened and analyzed. Results Mutational analysis of mitochondrial ND1 gene in 894 LHON patients revealed the presence of G3316A,T3394C,G3460A,C3497T,G3635A,G3733A,and T4216C.11.19％ LHON patients (100/894 ) were found to be associated with the gene mutations mentioned above,and 3.24％ patients (29/894) showed the co-occurrence of three primary mutations.Mutation frequencies in LHON patients were 2.57％,2.23％,1.45％,3.80％,0.67％,0.11％,0.34％,respectively,and G3316A,T3394C,C3497T and T4216C also were detected in 134 normal controls with the mutation frequencies of 4.48％,2.99％,4.48％ and 1.49％,respectively.Mutation frequency analysis showed an insignificant difference in the mutations of G3316A,T3394C,C3497T and T4216C between LHON patients and normal controls (x2 =0.926,P=0.336;x2 =0.052,P=0.820; x2 =0.142,P=0.707;P=0.129).G3376A,G3496T,G3700A,A4136G,T4160C and C4171A were absent in Chinese LHON patients. Conclusions Mitoehondrial ND1 gene in LHON is a mutational hotspot in Chinese population,11.19％ (100/894)associated with LHON was caused by ND1 gene mutation.G3635A,G3733A may be rare pathological mutation in Chinese population.However,G3316A,T3394C,C3497T and T4216C are insufficient to produce the clinical phenotype,but they may play a synergic role for penetrance and phenotypic manifestation in LHON.

Near infrared (NIR) spectroscopy as a kind of rapid process analysis technology has been successfully applied in Chinese medicine pharmaceutical process. In this research, the technology was adopted to establish the rapid quantitative analysis models of main indicators from the Lonicera japonica and Artemisia annua alcohol precipitation process of Reduning injection. On-line NIR spectra of 142 samples from alcohol precipitation process were collected and the content of main indicators for each sample were detected through off-line HPLC. With eliminating outliers, determination of spectra pretreatment method and selecting optimal band, the NIR quantitative calibration model for each indicator was established using partial least squares (PLS). These models were used to predict the unknown samples from precipitation process of Reduning injection to achieve the goal of rapid detection. The results showed that the models were ideal. The correlation coefficients of models for neochlorogenic acid, chlorogenic acid, 4-O-caffeoylquinic acid and secoxyloganin were 0.973 872, 0.985 449, 0.975 509 and 0.979 790, respectively and their relative standard errors of prediction (RSEP) were 2.922 49%, 2.341 37%, 2.930 40% and 2.184 60%, respectively. This study indicated that the NIR quantitative calibration model showed good stability and precision, and it can be used in rapid quantitative detection of main indicators of efficacy in order to on-line monitor the alcohol precipitation process of Reduning injection.
Chemical Precipitation ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Ethanol ; chemistry ; Lonicera ; chemistry ; Quality Control ; Spectroscopy, Near-Infrared ; methods

Forty batches of Lonicerae Japonica Fse i collected extensively and prepared as the test solution. Their chromatographic fingerprints and anti-influenza virus IC50 value (half maximal inhibitory concentration) were determined respectively. Then Unscrambler software was used, and spectrum-efficient correlation analysis was done for chromatographic fingerprints data and IC50 data by partial least squares regression method, to establish spectrum-efficient correlation model for anti-influenza virus of Lonicerae Japonicae Flos. Then the other 10 batches of Lonicerae Japonicae Flos were used to verify the model and explore the adaptability of this spectrum-efficient correlation model based on partial least squares regression method. The mathematical model obtained R2 of 0.969489 and RM-SEC of 0.070691 for calibration set; R2 of 0.959042 and RMSECV of 0.084005 for cross validation set. The verification experiment results showed that the relative error between the predicted values and measured values was within 10% in all 10 hatches, and within 5% in 80% of them. The results showed that the established spectrum-efficient correlation model could be used to evaluate the biological activity of anti-influenza virus of Lonicerae Japonicae Flos by determining its HPLC fingerprints.
Antiviral Agents ; analysis ; pharmacology ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Flowers ; chemistry ; Least-Squares Analysis ; Lonicera ; chemistry ; Molecular Structure ; Orthomyxoviridae ; drug effects

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.
Enzyme-Linked Immunosorbent Assay ; Genes, Synthetic ; genetics ; physiology ; Models, Theoretical ; Peptide Library ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Transcription Factors ; chemical synthesis ; chemistry ; metabolism ; Zinc Fingers ; genetics ; physiology