This paper reported a new home-made nonpoisonous substrate of horseradish peroxidase(HRP),3,3′,5,5′-tetramethylbenzidine sulphate(TMBS),used in ELISA with the aqueous soluble egg antigen(SEA)as antigen;proved patients'sera as antibo- dy and the antihuman IgG was replaced by protein A conjugate. The optimun condition obtained by orthogonal design was 1.0 millimolarity of TMBS,1.2 millimolarity of H_2O_2,and pH value at 5.0.The best time to stop reaction with H_2SO_4,was 30-60 min after adding substrate TMBS.At the same time,TMB and OPD were used as compared substrates.It showed that the reaction colour of both TMBS and TMB remained much more stable than that of OPD when being exposured in light and oxygen.After the solutions of TMBS and TMB were stored under 4℃ in refrigerator for 60 days,the coefficient of variation among different experiments was less than 10%. The results in detecting specific antibody in sera from 28 patients with proved schistosomiasis also showed that the sensitivity of TMBS was remarkably higher than OPD.It was found that TMBS used in enzyme immunoassay for demonstrating specific antibody was suitable not only for optical density(OD)read by spectrophotometer but also for estimation judged by naked-eye.During TMBS and TMB used in indirect immunoperoxidase method(IIP),the specific antibody in the tissue can be easily dete- cted and located as well.

0.05),and NK cells function(CD56),humoral immunologic function(IgG,IgM,IgA)were significantly elevated after TP-5 administration for 3 months(Pa

Objective To study the serum level of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) in patients with congestive heart failure (CHF) and assess the value of BNP. Methods The serum levels of BNP and ANP were determined by enzyme- linked immnoabsorbent assay.Cardial mdex(CI)and left ventricular election fraction(LVEF)in heart failure stage and period recouery of children with heart failare were determined by Doppler ultrasonography cardiogram. Results The BNP serum levels began to increase before heart failure (P

Objective To observe the immunologic function of critically ill newborn and the relative function of nuclear factor kappa B(NF-?B).Methods The critically ill group contained 50 cases,and 25 cases from healthy newborns were used as control group.Blood samples were collected in each case,levels of cytokine interleukin(IL)-4,interferon(IFN)-?,tumor necrosis factor(TNF)-? and NF-?B were detected.Result Compared with control group,NF-?B of the critically ill newborn activated and the cytokine were disorder,and IL-4 and TNF-? increased,but IFN-? decreased.Conclusions Critically ill newborn exist immune functional disorder.Furthermore,NF-?B activation may be involved in the process in infants with critically illness.

OBJECTIVETo observe the clinical efficacy of Qi-supplementing and blood-activating (QSBA) in treating diabetic peripheral nephropathy (DPN).

METHODSSixty-eight type 2 diabetes mellitus patients with Qi deficiency-blood stasis Syndrome were randomly divided into two groups, the neurotrophic agents were used in both groups, while QSBA herbs were used in the treated group additionally. The treatment course was 2 months. Blood glucose (BG), triglyceride (TG), total cholesterol (TC) and nerve conduction velocity (NCV) were detected before and after treatment.

RESULTSAfter treatment, the BG, blood lipid, NCV were improved significantly (P < 0.05) in both groups, but the improvement was more significant in the QSBA treated group than in the control group (P < 0.05).

CONCLUSIONQSBA, in treating DPN, can not only improve its symptoms, but also ameliorate the NCV.

Adult ; Aged ; Diabetic Neuropathies ; drug therapy ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Neural Conduction ; drug effects ; Phytotherapy ; Qi

In this study, we investigated the effect of Cigu Xiaozhi formula on HSC-T6 activity in hypoxic microenvironment based on network pharmacology and computer-aided drug design, and predicted and verified its possible targets and related signaling pathways. The potential active components and targets of Cigu Xiaozhi formula were screened by searching Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Encyclopaedia of Traditional Chinese Medicine (ETCM) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) databases, and the liver fibrosis related targets retrieved from Gene Cards and Pharm GK database were integrated to obtain the potential targets of Cigu Xiaozhi formula in the treatment of liver fibrosis. GO enrichment analysis and KEGG signaling pathway enrichment analysis were performed on Omic Share platform, and Cytoscape software was used to construct the "potential active ingredient-key target-pathway" network. The active components and target proteins were subjected to molecular docking analysis by Auto Dock software. According to the results of molecular dynamics simulation and binding free energy calculation, the top 5 active components with degree were scored. The active components stigmasterol and β-sitosterol were subjected to molecular docking. CoCl₂ was used to induce HSC-T6 cells to construct hypoxia model in vitro. The cell viability was detected by CCK-8 assay, and the optimal time and concentration of hypoxia model of HSC-T6 cells was determined to be 100 µmol·L^-1 CoCl₂ for 24 h. Under hypoxia condition, HSC-T6 cells were activated, the wound healing rate was significantly increased, and the fluorescence signal of activation marker protein α-smooth muscle actin (α-SMA) was significantly enhanced. However, 6% drug-containing serum could inhibit the activation of HSC-T6 cells, and the wound healing rate was significantly decreased, and the fluorescence signal of α-SMA was significantly weakened. Further studies showed that the expressions of hypoxia-inducible factor-1α (HIF-1α), α-SMA and key proteins of Hedgehog (Hh) signaling pathway in HSC-T6 cells were up-regulated under hypoxia, while the expressions of HIF-1α, α-SMA, Patched-1 (Ptch-1) and glioma related oncogene homology-1 (Gli-1) were down-regulated in 6% drug-containing serum group, the YC-1 group and the cyclopamine group. These results indicated that HIF-1α and Hh signaling pathways were involved in the activation of HSC-T6 cells, and the traditional Chinese medicine Cigu Xiaozhi formula could inhibit the activation of HSC-T6 cells, and the mechanism may be related to the inhibition of HIF-1α expression and the blocking of Hh signaling pathway. In conclusion, Cigu Xiaozhi formula can inhibit the activation of HSC-T6 cells by directly acting on HIF-1α and Hh signaling pathway, and exert an anti-hepatic fibrosis effect. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Gansu University of Chinese Medicine, in compliance with the Institutional Animal Care Guidelines.

Background Retinal retinoic acid (RA) plays an important role in the formation of the lensinduced myopia.However,it is not clear how RA transfer the myopic signal to choroid throughout the retinal pigment epithelium (RPE) barrier.Objective The aim of this study was to investigate the effect of all-trans retinoic acid (atRA) on the barrier of RPE in lens-induced myopic eye of guinea pig.Methods Thirty left eyes of 30 21-dayold clean guinea pigs were randomized into normal control group and the model group.The models of out of focus were induced by covering of-6.00 D concave lens on the left eyes for 15 days.Radius of corneal curvature was measured using corneal curvimeter,and diopeter of the guinea pig was examined by mydriatic optometry.The length of ocular axis was detected by A-sonography.The animals were sacrificed and the retinas of the left eyes were isolated for the culture and passage of RPE cells.The third generation of cells were incubated Millcell-PET microporous film,and atRA at the concentration of 1 × 10-6,1 × 10-7,1 × 10-8 and 1 × 10-9 mol/L was added to the micropore respectively for 12 hours,and the micropores with equal-solvent served as negative control group.Methyl thiazolyl tetrazolium (MTT)colorimetric method was used to detect the survival rate of the cells.Subsequently,the transepithelial electrical resistance (TER) of the monolayer cells was determined with CN10-EVOM2 resistance measuring meter.The vesicular transport change of RPE membrane in different groups was evaluated by FM1-43 fluorescence staining.Results The mean diopter was (-2.20±0.95) D in the models,and that in the controls was (+ 1.15 ±0.30) D,with a significant difference between them (t =14.57,P＜ 0.01).The axial length was (8.24 ± 0.09) mm in the models and it was significantly longer than (7.81±0.05) mm in the controls (t=17.20,P＜0.01).RPE cells grew well to form a monolayer in Millcell culture pool after one week.After 24 hours of the atRA treatment,the survival rate of RPE cells reduced gradually with the increase of atRA concentration with the highest rate in the 1 × 10-9 mol/L atRA group (93.3 ％) and followed by the 1 × 10-8 mol/L atRA group (88.2％).More than half of the cells dead in the 1 × 10-6mol/L and 1 × 10-7mol/L atRA groups (53.8％ and 47.1％).Significant differences in the TER value and fluorescence staining intensity of the cells were seen among the various groups (F =43.89,P =0.00 ; F =26.13,P =0.00),with the maximal values in the 1 × 10-8mol/L atRA group.The FM1-43 fluorescence located on the cellular membrane and cytoplasm.Conclusions AtRA can increase the functional state of tight junction and vesicular transport,which regulated the RPE cell barrier in the guinea pig.

OBJECTIVETo investigate the effect of hypoxia on the human pulmonary artery smooth muscle cells two pore domain potassium channels TASK-1 and the regulation of non-receptor tyrosine kinase c-Src in this process.

METHODSThe cultured human pulmonary artery smooth muscle cells (hPASMCs) were divided into: normal group, hypoxia 30 minute group, hypoxia 6 hours group and hypoxia 48 hour group, and hypoxia 48 hour + PP2 group, hypoxia 48 hour + PP3 group, hypoxia 48 hour + bpV group. Flow cytometry was used to analyze the cell cycle, RT-PCR and Western blot technique were carried out to detect the expression changes of TASK-1 mRNA and protein in different groups.

RESULTS(1) Cell Cycle Show: Compared with normal control group, with prolonged hypoxia, the percentages of hPASMCs in S phases of cell cycle were increased. While compared with hypoxia 48 hour group, the percentages of hypoxia 48 hour + PP2 group hPASMCs in S phases of cell cycle were decreased. The expression of TASK-1 mRNA on hPASMCs in acute hypoxia 6 hour group was increased, while the expression of TASK-1 protein on hPASMCs in the acute and chronic hypoxia group was decreased, and the expression of TASK-1 mRNA on hPASMCs in the chronic hypoxia group was decreased; After pre-incubation of a potent and selective inhibitor of the Src family of protein tyrosine kinases PP2, the expression of TASK-1 mRNA and protein in hypoxia 48 hour group was increased, however after pre-incubation of the inhibitor of the Src family of protein tyrosine phosphatase bpV, the expression of TASK-1 protein in hypoxia 48 hour group was decreased.

CONCLUSIONHypoxia promotes human pulmonary artery smooth muscle cell proliferation, and non-receptor tyrosine kinase c-Src may participate in the expression of two pore domain potassium channels TASK-1 regulated by hypoxia. Therefore, we hypothesized that TASK-1 channels and c-Src participatein the acute and chronic hypoxic human pulmonary vasoconstriction.

Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Humans ; Myocytes, Smooth Muscle ; cytology ; Nerve Tissue Proteins ; metabolism ; Potassium Channels, Tandem Pore Domain ; metabolism ; Pulmonary Artery ; cytology ; RNA, Messenger ; Vasoconstriction ; src-Family Kinases ; metabolism

OBJECTIVETo explore the effect of Xinfeng Capsule (XC) on lipoprotein metabolism of rheumatoid arthritis (RA) patients.

METHODSTotally 180 RA patients were assigned to the experimental group and the control group by random digit table, 90 in each group. Patients in the experimental group took XC (three pills each time, three times daily), while those in the control group took Methotrexate Tablet (four tablets each time, once per week). One month consisted of one therapeutic course and all patients were treated for two therapeutic courses. A healthy control group consisting of 60 patients was also set up. Changes of lipoprotein indices, clinical efficacy, lipid metabolism, joint symptoms and signs, activity indicators were observed, and correlation analyses were performed.

RESULTSCompared with the healthy control group, expression levels of prealbumin (PA), globulin (GLO), high-density lipoprotein (HDL), apolipoprotein Al (Apo-A1) were lowered in RA patients (P <0. 05, P <0. 01). Correlation analyses showed that PA was negatively correlated with joint tenderness, morning stiffness time, disease activity score (DAS-28), C-reactive protein (CRP), interleukin (IL)-6, respectively. Total protein (TP) was negatively correlated with joint tenderness. GLO was negatively correlated with joint tenderness and DAS-28. HDL was negatively correlated with erythrocyte sedimentation rate (ESR) and endothelin (ET)-1. Apo-Al was negatively correlated with joint pain; Apo-B was negatively correlated with CRP; LDL was negatively correlated with morning stiffness time (P <0. 05, P <0. 01). Compared with before treatment, expression levels of PA, HDL, Apo-A1 , Apo-B, and serum IL-10 contents increased, and expression levels of ESR, CRP, IL-6, ET-1 , joint pain, joint swelling, morning stiffness time, and DAS-28 decreased in the experimental group (P <0. 05, P <0. 01). PA increased more after treatment than before treatment in the control group (P <0. 01). There was statistical difference in joint symptoms (except joint tenderness) and activity indices (except ET-1) in the control group (P <0. 05, P <0. 01). Compared with the control group after treatment, PA and HDL increased, ET-1 and duration of morning stiffness decreased in the experimental group (all P <0. 05).

CONCLUSIONSLipoprotein metabolic disorder exists in RA patients, and it is associated with disease activity. XC could obviously improve lipoprotein metabolism and joint symptoms.

Arthritis, Rheumatoid ; drug therapy ; metabolism ; Blood Sedimentation ; C-Reactive Protein ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Humans ; Interleukin-10 ; Interleukin-6 ; Lipoproteins ; Lipoproteins, HDL ; metabolism ; Methotrexate