[Identification of potential targets and synergistic mechanism of Kushen Decoction for the treatment of cryptosporidiosis].

OBJECTIVE: To explore the potential targets and synergistic mechanisms of Kushen Decoction for the treatment of cryptosporidiosis using network pharmacology and molecular docking methods. METHODS: The main active ingredients of Kushen Decoction were captured from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TC-MSP) and the Universal Protein Resource (UniProt) database, and the potential targets were predicted. In addition, the active ingredients of Kushen Decoction that were not included in the TCMSP database were retrieved in CNKI, WanFang Data, CBM, PubMed and Web of Science databases, and the target genes of all supplemented active ingredients were predicted using the online TargetNet database. Network construction and analysis were performed using the Cytoscape software, and cryptosporidiosis-related targets were retrieved in the Comparative Toxicogenomics Database and GeneCards database. The protein-protein interaction (PPI) network was created using the STRING database, and the DAVID database was used for GO enrichment and KEGG pathway analyses. The tissue distribution of key targets was investigated using the BioGPS database, and the AutoDockTools software was employed to verify the molecular docking results. RESULTS: A total of 38 active ingredients of Kushen Decoction were screened, and the core ingredients included quercetin, (+)-14α-hydroxymatrine and apigenin. A total of 831 targets of Kushen Decoction and 512 cryptosporidiosis-related targets were predicted, and PPI network analysis revealed 69 key targets, including AKT1, TNF and IL-6. There were 303 biological processes, 46 molecular functions and 29 cellular components involved in the treatment of cryptosporidiosis with Kushen Decoction, and 13 KEGG pathways played a therapeutic role in the synergistic mechanisms of multiple targets, such as Toll-like receptor (TLR), nuclear factor kappa B(NF)-κB, nucleotide binding oligomerization domain like receptor (NLR) signal pathways. The core targets were mainly distributed in the hematologic and immune systems. Molecular docking analysis showed that the binding energy between active ingredients and key targets were all less than 0 kJ/mol, indicating the strong binding of ligands to receptors. CONCLUSIONS: The active ingredients of Kushen Decoction, such as quercetin, (+)-14α-hydroxymatrine and apigenin, may act on targets like AKT1, TNF, IL-6 to modulate TLR, NLR and NF-κB signaling pathways to play a synergistic role in the treatment of cryptosporidiosis in the hematologic and immune system.

[Study of mumps immunity after administrating measles-mumps-rubella vaccine among children aged 2-7 years old in Jiangsu Province in 2015].

Objective: To investigate the immunity to mumps after administrating measles-mumps-rubella vaccine (MMR) among children aged 2-7 years old in Jiangsu province in 2015. Methods: A total of 4 190 healthy children aged 2-7 years old, living in local places for at least 3 months, and having been vaccinated at least 1 dose MMR were recruited to the study from Wujin district of Changzhou city, Gaogang district of Taizhou city and Ganyu district of Lianyungang city by using stratified cluster random sampling method between September and November, 2015. Those who did not accept MMR vaccination, who refused venous blood collection, who had affected mumps according to the memory of parents or teachers and who were diagnosed serious disease by clinical doctors were excluded from study. The self-designed questionnaire was used to collect the general information of the subjects and their MMR immunization history; and 0.5-2.0 ml of venous blood was collected from each subject. ELISA was used to detect the mumps antibody level in the serum of patients. Positive was defined as the antibody level ≥108 mU/ml, and negative as <108 mU/ml. χ(2) test was used to compare the difference in positive rates among subjects; and analysis of variance was used to compare the GMC changes in different time points after MMR vaccination. Results: Among 4 190 children, 2 280 were males (54.42%) and 1 910 were females(45.58%), and the positive rate of IgG antibody was 81.38% (3 344). There were 3 156 (95.18%) children vaccinated with one dose MMR, 187 (4.80%) children with two dose MMR, and 1 (0.02%) child with three dose MMR. The difference in positive rate of IgG antibody among different aged subjects showed statistical significance (χ(2)=58.61, P<0.001), the highest positive rate was in group of subjects aged 4-5 years old, at 89.43% (406/454), while the lowest positive rate was found among subjects aged 6-7 years old, at 75.63% (1 648/2 179). The positive rate after one dose of MMR vaccination was 79.14% (3 156/3 988), significantly less than it after two doses (93.03%, 187/201) (χ(2)=22.93, P<0.001). The GMC level at years<1, 1-<2, 2-<3, 3-<4, ≥4 following one dose MMR in the 3 988 children was 152.47, 227.78, 167.08, 126.91, 79.43 mU/ml, whose difference was statistically significant (F=51.29, P<0.001). Conclusion: The sero-prevalence of IgG antibody in the children aged 2-7 years old in Jiangsu province was high. The positive rate among who received two doses MMR was significantly higher than it among who received just one dose, and the GMC level waned with times.

Metabolic profiling reveals new serum biomarkers of lupus nephritis.

Metabolomics has been applied to explore altered metabolite profiles in disease and identify unique metabolic signatures specific to certain pathologies. The aim of the current study is to characterize the metabolic profile of patients diagnosed with lupus nephritis (LN) and explore new insights into underlying disease processes. A metabolomic approach using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) was developed in serum samples from 32 LN patients, 30 idiopathic nephrotic syndrome (INS) patients and 28 healthy controls (HCs). Potential biomarkers were screened from orthogonal projection to latent structures discriminate analysis (OPLS-DA) and further evaluated by receiver operating characteristic analysis (ROC). A total of 14 potential biomarkers were screened and tentatively identified for LN patients compared to HCs. Compared to HCs and INS patients, the LN patients had increased serum levels of sorbitol and glycocholic acid metabolites and decreased levels of cortisol, creatinine and L-aspartyl-L-phenylalanine. A panel of three metabolomics (theophylline, oxidized glutathione and capric acid) was identified as biomarkers of LN with a sensitivity of 87.50% and a specificity of 67.86% using ROC analysis. Our results suggest that UPLC-HRMS based quantification of circulating metabolites was a useful tool for identification of biomarkers with the ability to segregate LN patients from INS patients and HCs. The potential biomarkers indicated that the LN metabolic disturbance may be closely associated with inflammation injury, oxidative stress and phospholipid metabolism.

Association between NAD(P)H oxidase p22phox gene variants and acute myocardial infarction in a Han Chinese population.

BACKGROUND: We investigated the association between five selected single-nucleotide polymorphisms (rs12933505, rs3180279, rs3794264, rs4673, rs1049255) in the NAD(P)H p22phox gene and acute myocardial infarction (AMI) as well as severity of coronary artery stenosis in a Han Chinese population. PATIENTS AND METHODS: A total of 168 patients with AMI and 138 healthy controls were recruited. The TaqMan allelic discrimination assay was used to genotype five single-nucleotide polymorphisms. RESULTS: The frequency of the rs1049255 G allele was significantly lower in patients with AMI than in controls (p = 0.022). Compared with subjects with an AA genotype, subjects with a GG or AG genotype had a lower risk of AMI [multivariate-adjusted odds ratio (OR), 0.53; 95 % CI, 0.29-0.95; p = 0.031). Subjects with the GG and AG genotypes of rs1049255 showed a decreased susceptibility for triple-vessel disease (TVD) as compared with controls (multivariate-adjusted OR, 0.43; 95 % CI, 0.19-0.98; p = 0.042). Multiple logistic regression analysis revealed that the rs1049255G variant was an independent protective factor for AMI/TVD. CONCLUSION: The results suggest there is an association between the p22phox rs1049255 polymorphism with the prevalence of AMI and the severity of coronary artery stenosis in the Han Chinese population.

Probable longer incubation period for human infection with avian influenza A(H7N9) virus in Jiangsu Province, China, 2013.

Human infection with the emerging avian influenza A(H7N9) virus in China in 2013 has raised global concerns. We conducted a retrospective descriptive study of 27 confirmed human influenza A(H7N9) cases in Jiangsu Province, to elaborate poultry-related exposures and to provide a more precise estimate of the incubation periods of the illness. The median incubation period was 6 days (range 2-10 days) in cases with single known exposure and was 7·5 days (range 6·5-12·5 days) in cases with exposures on multiple days, difference between the two groups was not significant (Z = -1·895, P = 0·058). The overall median incubation period for all patients was estimated to be 7·5 days (range 2-12·5 days). Our findings further highlight the necessity for public health authorities to extend the period of medical surveillance from 7 days to 10 days.

Endothelial nitric oxide synthase and nicotinamide adenosine dinucleotide phosphate oxidase p22phox gene (C242T) polymorphisms and systemic lupus erythematosus in a Chinese Population.

Genetic polymorphisms in the endothelial nitric oxide synthase (eNOS) and nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase p22phox are linked with the expression and/or progression of vascular disease. We hypothesized that these polymorphisms may influence the development and/or progression of systemic lupus erythematosus (SLE), given their linkage with vascular disease. DNA from patients with SLE (n = 90) and their age- and sex-matched controls (n = 86) from The Second Xiangya Hospital of Central South University was assessed for eNOS and NADPH oxidase p22phox polymorphisms. These polymorphisms were examined by restriction fragment length polymorphism-polymerase chain reaction. The allele frequency of the NADPH oxidase p22phox gene C242T polymorphisms significantly varied between the SLE patients and the controls. We found no association of the eNOS polymorphism with the development of renal disease. These results indicated that the etiology of patients with SLE is associated with NADPH oxidase p22phox gene C242T polymorphisms. There was no significant increased risk of SLE associated with eNOS polymorphisms in the Chinese population.

Oxidative stress involvement in Physalis angulata-induced apoptosis in human oral cancer cells.

In this report, we investigated the role of oxidative stress in Physalis angulata-induced apoptosis of human oral cancer cells. P. angulata-induced apoptosis was characterized by nuclear morphological changes, membrane blebbing and activation of caspase-9. Exposure of HSC-3 cells to P. angulata caused production of reactive oxygen species and up-regulation of oxidative stress markers heme oxygenase-1 (HO-1), superoxide dismutase (SOD), heat shock protein 70 (HSP70) and caspase-4. Down-regulation of HO-1, SOD and HSP70 proteins expression by attenuation of oxidative stress, pretreatment with glutathione or N-acetylcysteine, significantly decreased P. angulata-triggered cell death. The present study also demonstrated that the mitochondria and the endoplasmic reticulum are the targets of P. angulata in HSC-3 cells. Our results revealed that: (1) reactive oxygen species may play a dominant role in this process, (2) P. angulata induces oxidative stress in HSC-3 cells, (3) P. angulata-initiated apoptosis is caused through oxidative stress-dependent induction of heme oxygenase-1, Cu/Zn SOD and HSP70 proteins expression and (4) antioxidants inhibited P. angulata-induced cell death through inhibition of the proteins expression of HO-1, Cu/Zn SOD and HSP70.

Folate restriction and methylenetetrahydrofolate reductase 677T polymorphism decreases adoMet synthesis via folate-dependent remethylation in human-transformed lymphoblasts.

The homozygous mutation (677TT) in the methylenetetrahydrofolate reductase (MTHFR) gene reduces enzyme activity and alters cellular folate composition. Previous epidemiological studies reported a potential protective effect of MTHFR677C --> T against acute lymphocytic leukemia and malignant lymphoma, but the mechanism remains to be determined. We investigated the biochemical impacts of MTHFR677C --> T on cellular S-adenosyl methionine (adoMet) synthesis, global DNA methylation, and de novo purine synthesis, all of which are potential regulatory pathways involved in tumorigenesis. Metabolic fluxes of homocysteine remethylation and de novo purine synthesis were compared between Epstein-Barr virus-transformed lymphoblasts expressing MTHFR 677C and MTHFR 677T using stable isotopic tracers and GCMS. MTHFR TT genotype significantly reduced folate-dependent remethylation under folate restriction, reflecting limited methylated folates under folate restriction. Data also suggested increased formylated folate pool and increased purine synthesis when folate is adequate. The impacts of MTHFR 677T polymorphism appeared closely related to folate status, and such alterations may modulate metabolic pathways involved in cancer onset/progression. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in hematological malignancies. These transformed cells are potential models for studying the consequences of human genetic variation and cancer pathogenesis.

Green tea catechins and vitamin E inhibit angiogenesis of human microvascular endothelial cells through suppression of IL-8 production.

Epidemiological and animal studies have indicated that consumption of green tea and high vitamin E intake are associated with a reduced risk of developing certain forms of cancer. However, the inhibitory mechanism of green tea catechins and vitamin E in angiogenesis, an important process in tumor growth, has not been well established. In the present study, alpha-tocopherol and several major catechins of green tea (catechin, epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate) were tested for their ability to inhibit tube formation in vitro using a model in which human microvascular endothelial cells were exposed to a constant rate of a physiologically low level of H2O2. In this model, the production of interleukin (IL)-8 by human microvascular endothelial cells at a low level of H2O2 was required for angiogenesis, as assessed by tube formation in three-dimensional gel in culture. Vitamin E (d-alpha-tocopherol, 40 microM) in the culture media significantly reduced IL-8 production and angiogenesis. Among the green tea catechins, epigallocatechin (0.5-1 microM) was the most effective in reducing IL-8 production and inhibiting angiogenesis. These results suggest that consumption of green tea catechins or supplemental intake of vitamin E may have preventive effects on tumor development, mediated, at least in part, through inhibition of angiogenesis via suppression of IL-8 production.

Effect of testosterone on gonadotropin response to DBcAMP, cAMP binding, and cAMP production in pituitary cultures.

The role of testosterone (T) in the modulation of pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) sensitivity to DBcAMP was examined in the pituitary monolayer cultures prepared from intact young female rats. Hormone contents in media and cell homogenates were determined by radioimmunoassays. Incubation with 8 and 4 mM DBcAMP for 4 h consistently induced a significant (P less than 0.05) increase in FSH and LH release, respectively. Pretreatment with 10 nM T for 4 days reduced the minimal dose of DBcAMP required to stimulate FSH release (2 vs. 8 mM) but had no effect on the DBcAMP-induced LH release. Data indicate that T treatment for 4 or 7 days stimulated total FSH contents (sum of hormone contents in medium and cells). Similarly, incubation with 10 mM DBcAMP for 4 h significantly increased total FSH content per dish in both the T-treated and non-T-treated cultures. Neither T nor DBcAMP had any effect on LH production under these conditions. Intracellular cAMP was significantly increased to three- to eightfold of control after T treatment for 3 or 6 h, respectively. Furthermore, cAMP-binding activities were significantly increased after T treatment for 1 or 4 days (174 or 422% of control). Our previous data indicate that estrogen increases LH production, cAMP binding, cAMP production, and LH sensitivity to DBcAMP, and these data indicate that T exerts stimulatory effects on FSH in a similar fashion. These results support the concept that the two gonadotropins are regulated independently via different gonadal steroids.

Effect of estrogen and progesterone on the development of endometrial hyperplasia in the Fischer rat.

The effect of changing endocrine status on the uterine endometrium of the female Fischer rat was observed in vivo after neonatal androgenization, after aging, after estrogen (E) implant in ovariectomized (OVX) rats, and in vitro after progesterone (P) addition to monolayer cultures of uterine tumor cells. Neonatal androgenization produced cystic ovarian follicles and persistent vaginal cornification for 10-14 mo, indicative of an anovulatory, persistent E status. The constant estrous (CE) state, induced by androgenization, uniformly produced focal glandular hyperplasia of the endometrium at 9 mo of age. Focal glandular hyperplasia, which was absent in the cycling 6-mo-old estrous endometrium, was also apparent in the 12-mo-old rats which had ceased cycling and entered natural CE. Subcutaneous estradiol-17 beta (E2) implants in 12-mo-old OVX rats induced early cystic and adenomatous changes in the foci of hyperplasia. Acyclic control rats, 21 mo old, in persistent diestrus (PD) exhibited a 3-fold elevation of serum P associated with glandular atrophy of the endometrium. Ten nM P also inhibited proliferation of endometrial cells in cultures prepared from tumors of 21-mo-old rats. On the other hand, adenomatous hyperplasia was observed in 29-mo-old PD rats, despite the presence of low serum E and elevated serum P. These results indicate that either induced or natural constant E status leads to focal glandular hyperplasia in the Fischer rat. E2 implants in the 12-mo-old OVX Fischer rat induced early cystic and adenomatous changes in the focal hyperplasia. Inhibition of these focal hyperplasias was associated with elevated serum P at 21 mo. The development of adenomatous hyperplasia in the aged endometrium, on the other hand, occurred despite elevated serum P at 29 mo.

Luteinizing hormone response to N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate in aged, young and androgenized rats: estradiol effect in vitro.

The studies in this report were designed to investigate whether the loss of pituitary luteinizing hormone (LH) responses to N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) in aged noncycling rats was the result of age, endocrine status associated with diestrus, or noncyclic low estrogen status. Pituitary monolayer cultures were prepared from female Fischer 344 rats. Aged (18-month-old) persistent diestrous (PD) rats, young diestrous (D) rats, or noncycling neonatally androgenized-constant estrous (AN-CE) rats were used. Enzymatically dispersed cells were maintained in the same batch of medium supplemented with dextran-coated charcoal adsorbed serums. Total LH contents were 1.75 +/- 0.04, 1.15 +/- 0.03, and 1.71 +/- 0.02 micrograms LH/dish in Day 5 cultures prepared from aged PD, young D, and AN-CE rats, respectively. Incubations with 5 mM DBcAMP for 4 h significantly (P less than 0.05) stimulated LH release in cultures prepared from young D and AN-CE rats but inhibited LH release in cultures prepared from aged PD rats even though a 4-h incubation with 10 nM LH releasing hormone (LHRH) stimulated LH release similarly in cultures of all three types of cells. The loss of DBcAMP-induced LH release in cultures prepared from aged PD rats was reversed by 17 beta-estradiol (E2). This treatment also reduced the basal LH release and increased the cellular LH content. These results indicate that the loss of DBcAMP-induced LH response in the aged rat is not an irreversible aging phenomenon but appears to be associated with the chronically low E2 status of aged PD rats but not young cycling D or noncycling AN-CE rats.

Estradiol stimulation of pituitary cAMP production and cAMP binding.

The role of 17 beta-estradiol (E2) in the modulation of N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-induced hormone release was examined in pituitary monolayer cultures prepared from intact and ovariectomized female rats. Incubations with 5 mM DBcAMP for 4 h significantly (P less than 0.05) stimulated both luteinizing hormone (LH) and prolactin (PRL) release in pituitary cultures prepared from rats at diestrus and from cycling rats at random stages of the estrous cycle. However, DBcAMP failed to stimulate the LH or PRL release in cultures prepared from ovariectomized rats in which the basal LH and PRL release was approximately three-fold and one-tenth of that in cycling rats, respectively. Pretreatment with 1 nM E2 augmented or restored the DBcAMP-induced LH release but not the DBcAMP-induced PRL release in cultures prepared from cycling or ovariectomized rats, respectively. Furthermore, E2 treatment alone of cultures prepared from cycling rats significantly increased intracellular cAMP concentrations and cAMP-binding activities by at least twofold over that of the non-E2-treated controls. The E2-induced rise in cellular cAMP concentration preceded the E2-induced rise in cAMP binding. These results indicate that the priming effect of E2 on pituitary LH responsiveness to DBcAMP is associated with increased cAMP production and cAMP binding.

cAMP-dependent protein kinase II interactions with nuclei derived from rat mammary tumor.

cAMP-dependent protein kinase from bovine heart exhibited significant interactions with nuclei from rat mammary tumors. These enzyme-nuclear interactions occurred when the intact holoenzyme was preincubated with 10 nM cAMP, 1 mM MgATP at 24 degrees C to produce high-affinity monophasic cAMP-dissociation kinetics. The enzyme-nuclear interactions are correlated with the loss of cAMP and PO4 from the purified enzyme. The data indicate that the high affinity cAMP-dependent protein kinase II exhibits significant nuclear interaction which may be related to cAMP function in rat mammary tumors.

LH responses to LHRH, DBcAMP, and 17 beta-estradiol in cultures derived from aged rats.

Effects of luteinizing hormone-releasing hormone (LHRH), N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAmP), and 17 beta-estradiol (E2) on LH responses were compared in 4-day-old pituitary cultures derived from female rats (Fischer 344) of 9 and 31 mo of age. Pituitary LH contents were similar in 5 X 10(5) freshly dispersed cells prepared from the two groups of rats. LH release in response to a 4-h incubation with 10 nM LHRH was virtually the same in cultures derived from 9- and 31-mo-old rats, 140 +/- 2 ng and 138 +/- 18 ng/4 h, respectively. In cultures derived from 9-mo-old rats, incubation with 8 mM DBcAMP for 4 h significantly stimulated LH release (286% of control level), and treatment with 10 nM E2 for 3 days significantly increased both basal LH release and LH accumulation (sum of LH contents in cells and medium) to 162 and 125% of control level, respectively. Neither DBcAMP nor E2 affected cultures derived from the 31-mo-old rats. These results indicate that there is a difference in the LH response to DBcAMP and E2 between cultures derived from 9- and 31-mo-old rats. The loss of pituitary responsiveness to DBcAMP and E2 and the loss of pituitary cyclicity in the aged female rat may be causally related.

Effect of 17 beta-estradiol on pituitary cAMP binding and responses to DBcAMP.

Effects of 17 beta-estradiol (E2) on adenosine 3',5'-cyclic monophosphate (cAMP) binding and luteinizing hormone (LH) and prolactin (PRL) responses to N6-O-2'-dibutyryl cAMP (DBcAMP) were examined in pituitary monolayer cultures prepared from female rats. Incubation with 8 mM DBcAMP for 4 h significantly (P less than 0.05) increased both LH and PRL release into medium by two- to threefold. E2 pretreatment augmented the DBcAMP-induced LH release but not PRL release to 160% of the non-E2-treated controls. However, the cellular and total accumulation of both LH and PRL were significantly increased in cultures pretreated with E2. The effect of E2 was time dependent, and the maximal effect was observed after 3 days of treatment. Furthermore, E2 treatment significantly increased cAMP-binding activities to 254% of the non-E2-treated controls. The time course of the E2 effect on cAMP-binding activities closely resembled the time course of the E2 effect on LH and PRL accumulation as well as the DBcAMP-induced LH release. These results suggest that the priming effect of E2 on pituitary LH and PRL responses to DBcAMP is associated with increased hormone synthesis and cAMP binding stimulated by E2 pretreatment.

Effect of MgATP on cAMP-dissociation kinetics of lactating rat mammary gland.

Mammary gland cytosols exhibit temperature-dependent interconversion of cAMP-dissociation rates from low to high affinity (k-1 = 0.14 min-1 at 0 degree C to k-1 = 0.02 min-1 at 24 degrees association). This interconversion corresponds to a change from a site 2 to a site 1 cAMP-dissociation rate for the type II cAMP-dependent protein kinase in mammary gland cytosols. This report presents data which indicates a requirement for MgATP in the temperature-dependent interconversion of cAMP-dissociation rates. The effect of MgATP on the generation of the high affinity state was observed at 24 degrees C but not 0 degree C association. The effect of MgATP was not mimicked by equimolar MgAMP-PNP, but did require an intact type II protein kinase holoenzyme which can undergo autophosphorylation of its regulatory subunit. The effect of MgATP was reproduced with partially purified preparations of beef heart type II protein kinase. These results suggest that MgATP may act through autophosphorylation of the type II holoenzyme. The data suggest a novel role of MgATP in the regulation of cAMP binding to cAMP-dependent protein kinase II.