miR-126-5p Targets SP1 to Inhibit the Progression of Parkinson's Disease.

BACKGROUND: At present, symptomatic treatment may improve the life quality of Parkinson's disease (PD) patients to a certain extent but cannot completely cure PD. Therefore, it is urgent medical problem to be solved for improving the efficacy and safety of PD treatment. METHODS: SH-SY5Y and SK-N-SH cells were treated with 1-methyl-4-phenylpyridinium (MPP+) to establish PD model cells. miR-126-5p and specific protein-1 (SP1) expression levels were detected by quantitative Real-Time PCR (qRT-PCR). Western blot was applied to measure protein levels of SP1, Bax, and Bcl-2. The viabilities and apoptosis rates of treated cells were measured using cell counting kit-8 assay and flow cytometry analysis. Enzyme-linked immunosorbent assay was performed to measure TNF-α and IL-1ß releases. Interaction between miR-126-5p and SP1 was examined by dual-luciferase reporter assay. RESULTS: MPP+ treatment greatly downregulated miR-126-5p expression while upregulated SP1 expression in SH-SY5Y and SK-N-SH cells in a time- and does-dependent manner. Overexpression of miR-126-5p facilitated cell viability, while reduced cell apoptosis and inflammatory responses induced by MPP+ treatment. Moreover, SP1 was a target of miR-126-5p and could be negatively regulated by miR-126-5p. Overexpression of SP1 could reverse the effects of miR-126-5p on MPP+-administrated cells. CONCLUSION: Our results suggested that miR-126-5p attenuated the neurotoxicity induced by MPP+ in vitro through targeting SP1 (Graphical abstract), which further enhanced our understanding of the pathological mechanism of PD.

Correlation of diet, microbiota and metabolite networks in inflammatory bowel disease.

OBJECTIVES: Microbiota dysbiosis in inflammatory bowel disease (IBD) has been widely reported. The gut microbiota connect diet to the metabolism by producing small molecules via diverse metabolic pathways. In this study we aimed to investigate the dietary preferences of IBD patients, and to explore the interactions among gut microbiota composition, dietary components, and metabolites in relation to IBD. METHODS: Dietary preferences of IBD patients (including those with ulcerative colitis [UC] and Crohn's disease [CD]) and health controls were investigated, and their gut microbiota were analyzed using 16S rRNA gene sequencing and metagenomic analyses of fecal and biopsy samples. The metabolite profiles of the samples were then analyzed using gas and liquid chromatography-mass spectrometry analyses. RESULTS: The daily intake of folic acid, niacin, vitamins C and D, calcium, and selenium differed significantly between patients with IBD and healthy controls. A decrease in long-chain (such as arachidic, and oleic acid) and medium-chain fatty acids (sebacic acid and isocaproic acid) as well as bile acid was observed in patients with IBD. Compared with healthy controls, 22 microbial species (including Sulfolobus acidocaldarius, and Clostridium clostridioforme CAG132) in the UC group and 37 microbial species (such as Bacteroides fragilis and Fusobacterium nucleatum) in the CD group were found to be correlated to diet and metabolites. Bacteroides fragilis was enriched in patients with IBD and associated with multi-nutrients, and 21 metabolites including 25-hydroxyvitamin D3 and taurolithocholic acid. CONCLUSIONS: This study provides an interaction network to identify key micronutrients, microbiota components and metabolites that contribute to IBD.

Therapeutic effect of acupuncture combining standard swallowing training for post-stroke dysphagia: A prospective cohort study.

OBJECTIVE: To assess the therapeutic effect of acupuncture combining standard swallowing training for patients with dysphagia after stroke. METHODS: A total of 105 consecutively admitted patients with post-stroke dysphagia in the Affiliated Hospital of Gansu University of Chinese Medicine were included: 50 patients from the Department of Neurology and Rehabilitation received standard swallowing training and acupuncture treatment (acupuncture group); 55 patients from the Department of Neurology received standard swallowing training only (control group). Participants in both groups received 5-day therapy per week for a 4-week period. The primary outcome measures included the scores of Videofluoroscopic Swallow Study (VFSS) and the Standardized Swallowing Assessment (SSA); the secondary outcome measure was the Royal Brisbane Hospital Outcome Measure for Swallowing (RBHOMS), all of which were assessed before and after the 4-week treatment. RESULTS: A total of 98 subjects completed the study (45 in the acupuncture group and 53 in the control group). Significant differences were seen in VFSS, SSA and RBHOMS scores in each group after 4-week treatment as compared with before treatment (P<0.01). Comparison between the groups after 4-week treatment showed that the VFSS P=0.007) and SSA scores (P=0.000) were more significantly improved in the acupuncture group than the control group. However, there was no statistical difference (P=0.710) between the acupuncture and the control groups in RBHOMS scores. CONCLUSIONS: Acupuncture combined with the standard swallowing training was an effective therapy for post-stroke dysphagia, and acupuncture therapy is worth further investigation in the treatment of post-stroke dysphagia.

Maternal serum lipid levels during late pregnancy and neonatal body size.

OBJECTIVE: The aim of this study was to determine the predictive value of maternal serum lipid levels during late pregnancy for neonatal body size. METHODS: This study was conducted from January 1, 2011 to July 31, 2012 at a Maternal and Child Health Hospital. Fasting blood glucose, serum triglyceride, total cholesterol, HDL and LDL were estimated in maternal collected before delivery. Detailed anthropometry of the neonates was performed. RESULTS: Women who delivered a large for gestational age baby were older, taller, had a higher pre-pregnancy weight, higher pre-pregnancy BMI, and higher weight gain during pregnancy than women who delivered an appropriate for gestational age infant. After adjusting for maternal age, pre-pregnancy BMI, weight gain during pregnancy, parity, neonatal sex and gestational age at delivery, we found that only maternal HDL level was inverse associated with birth weight, length and head circumference (p<0.05). On logistic regression analysis, the significant metabolic predictors of large for gestational age was HDL (OR 0.57, 95%CI: 0.38-0.84, per 1 mmol/L increase) after adjusting for the confounders. CONCLUSIONS: Maternal serum HDL level determined in maternal blood taken close to delivery was independently associated with neonatal size and was the independent predictor for large for gestational age.

[Effects of decitabine on proliferation and apoptosis of NB4 and K562 cells].

This study was aimed to investigate the effects of decitabine (DAC) on proliferation and apoptosis of leukemia NB4 and K562 cells. The proliferation inhibition of DAC on NB4 and K562 cells was detected by Trypan blue staining. After treatment of DAC at different concentrations, the changes of cell cycle and CD11b expression was determined by flow cytometry. The cell morphological changes were observed by Wright's staining. The DNA ladder was used to detect cell apoptosis. The results indicated that DAC significantly inhibited the proliferation of NB4 and K562 cells in dose-and time-dependent manner. The median inhibitory concentration (IC50) of DAC-treated NB4 and K562 cells for 72 h was 0.113 µmol/L and 0.138 µmol/L, respectively. After treating these two cell lines with DAC at different concentration for 72 h, the cell ratio in G0/G1 phase significantly increased, while the cell ratio in S phase obviously decreased in 0.15 µmol/L DAC group (P < 0.05). The expression levels of myeloid differentiation antigen CD11b of both cell lines significantly increased in contrast to the control group (P < 0.05). The cell morphology detected by Wright's staining displayed partial differentiation and apoptosis after treating NB4 and K562 cells with DAC for 48 h. Typical apoptotic DNA ladder was observed in 0.15 µmol/L DAC group at 48 h. It is concluded that DAC can inhibit NB4 and K562 cell proliferation, induce cell differentiation and apoptosis, but more obviously for NB4 cells.

Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis.

Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43-53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood-brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite-host interactions.

[Construction of a eukaryotic expression vector harboring the small interfering RNA targeting HCMV-IE1 gene and its gene silencing efficiency].

OBJECTIVE: To investigate the effect of RNA interference targeting human cytomegalovirus immediate early gene 1 (HCMV- IE1) on the gene expression in vitro. METHODS: According to the sequence of HCMV-IE1 gene, the small interfering RNA (siRNA) sequences were designed and introduced into the eukaryotic expression vector containing the U6 promoter. After verification by sequence analysis, the recombinant eukaryotic plasmid (pHCMV-IE1i) was transfected into HEL HCMVAD169 cells. The effectiveness of HCMV-IE1 gene silencing was investigated by fluorescence microscopy, flow cytometry and RT-PCR. RESULTS: Sequence analysis confirmed successful construction of the recombinant eukaryotic expression plasmid pHCMV-IE1i. The expression of HCMV-IE1 was effectively suppressed by pHCMV-IE1i transfection in HEL cells as shown by fluorescence microscopy, flow cytometry (P < 0.05) and RT-PCR (P < 0.05). CONCLUSION: The expression of HCMV-IE1 can be effectively suppressed by RNA interference technique in vitro, which provides experimental data for prevention and treatment of HCMV infection.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis.

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

Matrix metalloproteinase-9 delays wound healing in a murine wound model.

BACKGROUND: Metalloproteinase-9 (MMP-9) is a type IV collagenase found at elevated levels in chronic wounds. As wounds heal, MMP-9 diminishes. In this study, we investigated whether MMP-9 directly contributes to chronic wound pathogenesis. METHODS: Recombinant proMMP-9 was prepared using immortalized keratinocytes transduced by a lentivirus. ProMMP-9 was purified from cell culture media and activated using 4-aminophenylmercuric acetate. Active MMP-9 was then suspended in xanthan gum to a concentration paralleling that found in human chronic wounds. Two parallel 6-mm punch biopsies were made on the backs of C57BL mice. Wounds were treated daily with MMP-9 or vehicle. Wound areas were measured and tissues examined by densitometry, real-time RT-PCR, histology, and immunohistochemistry at days 7, 10, and 12. RESULTS: Exogenous MMP-9, at the level found within chronic wounds, delayed wound healing in this animal model. By 7 days, wounds in the MMP-9-injected group were 12% larger than control wounds (P = .008). By day 12, wounds in the MMP-9-injected group were 25% larger than those of the control group (P = .03). Histologic examination shows that high levels of active MMP-9-impaired epithelial migrating tongues (P = .0008). Moreover, consistent with elevated MMP-9, the collagen IV in the leading edge of the epithelial tongue was diminished. CONCLUSION: MMP-9 appears to directly delay wound healing. Our data suggests that this may occur through interference with re-epithelialization. We propose that MMP-9 interferes with the basement membrane protein structure, which in turn impedes keratinocyte migration, attachment, and the reestablishment of the epidermis.

[Cryoablation for prostate cancer induces tumor-specific immune response].

OBJECTIVE: To assess the anti-tumor immune response to percutaneous cryoablation in patients with local prostate cancer. METHODS: We treated 10 patients with local prostate cancer by percutaneous cryoablation, collected the blood samples before and 2 weeks after the treatment and isolated peripheral blood mononuclear cells (PBMCs). Protein lysates were made by biopsy from autologous prostate cancer or non-cancer tissues. The levels of serum TNF-alpha, IFN-gamma, IL4 and IL-10 were determined by enzyme-linked immunosorbent assay (ELISA) and the Th1/Th2 ratio was calculated by the IFN-gamma/IL-4 ratio. The number of IFN-gamma + T cells under the stimulation of different protein lysates was counted by enzyme link immunol spot (ELISPOT). And the cytolytic activity of cytotoxic T lymphocytes (CTL) was detected by LDH assay. RESULTS: Compared with pre-treatment, the levels of TNF-alpha and IFN-gamma, the Th1/ Th2 ratio and the number of IFN-gamma + T cells induced by tumor protein lysates in PBMCs were increased significantly after cryosurgery (P < 0.01), while the levels of IL4 and IL-10 decreased slightly, and the non-tumor protein lysates induced no obvious changes in the number of IFN-gamma T cells. The cytolytic activity of cytotoxic T lymphocytes against human prostate cancer cells LNCaP was markedly increased, but not that against renal cancer cells GRC-1. One case of recurrence was found during the 3-6 months follow-up. CONCLUSION: Percutaneous cryoablation for prostate cancer could induce a tumor-specific immune response.

[Mechanism of oligochitosan-induced macrophage activation].

OBJECTIVE: To study the mechanism of oligochitosan-induced macrophage activation. METHODS: Oligochitosan was chemically modified with fluorophore 2-aminoacridone (2-AMAC). The cellular events of 2-AMAC-oligochitosan-macrophage interaction were analyzed with confocal laser microscopy and the fluorescence intensity of cells was analyzed by BD LSR flow cytometer. The mechanism of oligochitosan uptake by macrophages was studied by competitive inhibition test and the effect of calcium, trypsin and colchicine on oligochitosan recognition and internalization were also determined. RT-PCR was performed to investigate the level of TNF-alpha secretion. RESULT: Macrophage could bind and uptake oligochitosan, which was dependent on the temperature: the uptake proceeded rapidly at 37 degrees C and at 4 degrees C macrophage could only bind oligochitosan. EDTA decreased oligochitosan uptake. Trypsin treatment significantly reduced the internalization, and uptake was recovered by trypsin termination. Colchicine significantly inhibited the internalization process and was dose dependent. 0.1 mol/L mannose inhibited TNF-alpha expression induced by oligochitosan. CONCLUSION: Macrophage could uptake oligochitosan via mannose receptor mediated pinocytosis. Mannose receptor is crucial for the oligochitosan-induced macrophages activation.

Genetic variations in the pgm locus among natural isolates of Yersinia pestis.

A PCR-based screening method was used to study the genetic variations of the pgm locus among natural isolates of Yersinia pestis from China. Our results indicate that genetic variations in the pgm locus are well correlated with biovars of Y. pestis and plague foci, suggesting that the pgm locus plays a role in Y. pestis adaptation to its environment. The gene encoding two-component regulatory system sensor kinase became a pseudogene in all strains of biovar Orientalis due to a thymidine deletion, while it is intact in all the strains of the other biovars. Only strains from Foci H and L are the same as Yersinia pseudotuberculosis in that they have an intact transmembrane helix in the sensor kinase protein, which is lost in all the other strains because of the 18 bp in-frame deletion. The IS100 element that flanks the 39 terminus of the pgm locus was inserted into the chromosome during the within-species microevolution of Y. pestis, which is absent in strains from Foci G, H and L and also in Y. pseudotuberculosis. This fact indicates that the strains from these three foci are of an older lineage of Chinese Y. pestis. It is this IS100 element's absence that maintained high stability of the pgm locus in the Y. pestis strains from these three foci. The IS285 element insertion in the pigmentation segment and the IS100 element insertion in the downstream flanking region of the pgm locus are only present in strains from Foci H and L. The flanking region outside the 59 terminus of the upstream IS100 element is identical in the strains from these two foci, which is different in the other strains. All of these unique characteristics suggest that they are of a special lineage of Chinese Y. pestis.

[Analysis of contents of flavonoids in Mongolian medicine zhenbao pills II and III].

Both zhenbao pills II and III are Mongolian medicine of a kind. Zhenbao pill II was the artificial syntheses and zhenbao pill III was made from natural materials. In this paper, the flavonoids of Mongolian medicine zhenbao pills II and III were extracted by Soxhlet apparatus with methyl alcohol as extracting agent, the colorimetric method was applied to the determination of flavonoids, and the experimental procedure was studied with zhenbao pill II as the test sample. The result showed that the linear range of quantitative determination was 8.05-48.28 microg x mg(-1). The standard addition recovery (SAR) was 99.49%-100.50%. The RSD (n = 3) was 0.54%. The range of contents of flavonoids was 1.47-1.55 mg x g(-1) in zhenbao pill II and was 2.88-3.00 mg x g(-1) in zhenbao pill III. This method was simple and accurate with good reproducibility, and is suitable for the determination of flavonoids in all kinds of pills. The contents of flavonoids can be used to prove whether zhenbao pill is artificial syntheses or natural material.