[Mechanism of Mongolian drug Naru-3 in initiation of neuroinflammation of neuropathic pain from MMP9/IL-1ß signaling pathway].

Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1ß(IL-1ß). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1ß signaling pathway-mediated microglia p38/IL-1ß inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.

Long-term observation after transplantation of cultured human corneal endothelial cells for corneal endothelial dysfunction.

BACKGROUND: Corneal transplantation is the only way to treat serious corneal diseases caused by corneal endothelial dysfunction. However, the shortage of donor corneal tissues and human corneal endothelial cells (HCECs) remains a worldwide challenge. We cultivated HCECs by the use of a conditioned medium from orbital adipose-derived stem cells (OASC-CM) in vitro. Then the HCECs were used to treat animal corneal endothelial dysfunction models via cell transplantation. The purpose of this study was to conduct a long-term observation and evaluation after cell transplantation. METHODS: Orbital adipose-derived stem cells (OASCs) were isolated to prepare the conditioned medium (CM). HCECs were cultivated and expanded by the usage of the CM (CM-HCECs). Then, related corneal endothelial cell (CEC) markers were analyzed by immunofluorescence. The cell proliferation ability was also tested. CM-HCECs were then transplanted into monkey corneal endothelial dysfunction models by injection. We carried out a 24-month postoperative preclinical observation and verified the long-term effect by histological examination and transcriptome sequencing. RESULTS: CM-HCECs strongly expressed CEC-related markers and maintained polygonal cell morphology even after 10 passages. At 24 months after cell transplantation, there was a CEC density of more than 2400 cells per square millimeter (range, 2408-2685) in the experimental group. A corneal thickness (CT) of less than 550 µm (range, 490-510) was attained. Gene sequencing showed that the gene expression pattern of CM-HCECs was similar to that of transplanted cells and HCECs. CONCLUSIONS: Transplantation of CM-HCECs into monkey corneal endothelial dysfunction models resulted in a transparent cornea after 24 months. This research provided a promising prospect of cell-based therapy for corneal endothelial diseases.

[Effects of organic fertilizer application on crop yield and soil properties in rice-wheat rotation system: A meta-analysis]. / 稻麦轮作体系下有机肥施用对作物产量和土壤性质影响的整合分析.

To clarify the effects of organic fertilizer application on crop yield and soil properties in rice-wheat rotation system in China, we carried out a meta-analysis to quantitatively evaluate the effects of organic fertilizer types (ordinary organic fertilizer, biochar, and straw), fertilization regimes (organic fertilizer alone, organic fertilizer + partial chemical fertilizer, and organic fertilizer + full amount of chemical fertilizer), and experiment duration (short term, medium term, and long term) on soil properties and the yield of rice and wheat, as well as their responses to soil conditions (acid, neutral, basic). Results showed that the application of organic fertilizer had similar yield-increase effect on rice yield (3.1%) and wheat yield (3.0%) compared to chemical fertilizer application alone. The effect of organic fertilizer application on soil quality was more obvious, significantly reducing soil bulk density by 5.7%, and increasing the concentrations of soil organic matter, total nitrogen, total phosphorus, alkali-hydrolyzable nitrogen, available phosphorus, available potassium, microbial biomass carbon, and microbial biomass nitrogen by 11.7%-38.4%. Among different types of organic fertilizer, the effects of ordinary organic fertilizer and biochar on soil properties improvement were better than straw. Compared to the organic fertilizer application alone, the effects of organic fertilizer combined with chemical fertilizer on crop yield was better, but poorer on soil property improvement. With the increasing duration of organic fertilizer application, crop yield and soil fertility gradually increased. Under the condition of acid soil, the effect of organic fertilizer application on crop yield was the best. The annual yield of rice and wheat showed significant negative correlation with soil bulk density, but a significant positive correlation with the concentrations of soil total nitrogen, available phosphorus, available potassium, and microbial biomass nitrogen.

Bioinformatics analysis of key biomarkers for retinoblastoma.

OBJECTIVE: To identify key genes involved in occurrence and development of retinoblastoma. METHODS: The microarray dataset, GSE5222, was downloaded from the gene expression omnibus (GEO) database. Differentially expressed genes (DEGs) between unilateral and bilateral retinoblastoma were identified and functional enrichment analysis performed. The protein-protein interaction (PPI) network was constructed and analysed by STRING and Cytoscape. RESULTS: DEGs were mainly associated with activation of cysteine-type endopeptidase activity involved in apoptotic process and small molecule catabolic process. Seven genes (WAS, GNB3, PTGER1, TACR1, GPR143, NPFF and CDKN2A) were identified as HUB genes. CONCLUSION: Our research provides more understanding of the mechanisms of the disease at a molecular level and may help in the identification of novel biomarkers for retinoblastoma.

miR­15a­3p affects the proliferation, migration and apoptosis of lens epithelial cells.

The present study investigated the effect of microRNA (miR)­15a­3p on the proliferation, migration and apoptosis of lens epithelial cells and its potential mechanism, in order to further elucidate the pathogenesis of age­related cataracts (ARCs). The HLE­B3 human lens epithelial cell line was transfected with miR­15a­3p mimic. Expression of the miR­15a­3p mimic was measured by fluorescence­based reverse transcription­quantitative polymerase chain reaction analysis. Cell proliferation, apoptosis, invasion and migration were investigated using MTT and plate clone formation assays, terminal deoxynucleotidyl transferase dUTP nick end labeling and flow cytometry, and a wound healing assay and Transwell assay, respectively. The protein expression levels of B­cell lymphoma 2 (BCL2) and myeloid cell leukemia sequence 1 (MCL1) were also compared between transfected and wild­type HLE­B3 cells by western blot analysis. The results showed that transfection with the miR­15a­3p mimic significantly suppressed the proliferation of HLE­B3 cells, induced cell apoptosis and increased the proportion of early apoptotic cells. The migration of HLE­B3 cells was significantly inhibited following transfection with miR­15a­3p mimic (P<0.01), whereas cell invasion was unaffected (P>0.05). In addition, reduced protein levels of BCL2 and MCL1 were observed in the miR­15a­3p mimic­transfected HLE­B3 cells (P<0.01). In conclusion, miR­15a­3p may suppress cell proliferation and migration, and induce cell apoptosis in lens epithelial cells through inhibiting the expression of BCL2 and MCL1, which contributes to the onset of ARCs.

MicroRNA Profiling of Transgenic Mice with Myocardial Overexpression of Nucleolin.

BACKGROUND: Nucleolin (NCL) is the most abundant RNA-binding protein in the cell nucleolus and plays an important role in chromatin stability, ribosome assembly, ribosomal RNA maturation, ribosomal DNA transcription, nucleocytoplasmic transport, and regulation of RNA stability and translation efficiency. In addition to its anti-apoptotic properties, the underlying mechanisms associated with NCL-related roles in different cellular processes remain unclear. In this study, the effect of NCL on microRNA (miRNA) expression was evaluated by generating transgenic mice with myocardial overexpression of NCL and by analyzing microarrays of mature and precursor miRNAs from mice. METHODS: Using microinjection of alpha-MyHc clone 26-NCL plasmids, we generated transgenic mice with myocardial overexpression of NCL firstly, and then mature and precursor miRNAs expression profiles were analyzed in NCL transgenic mice (n = 3) and wild-type (WT) mice (n = 3) by miRNA microarrays. Statistical Package for the Social Sciences version 16.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform Student's t-test, and statistical significance was determined at P < 0.05. RESULTS: Several miRNAs were found to be differentially expressed, of which 11 were upregulated and 4 were downregulated in transgenic mice with myocardial overexpression of NCL compared to those in WT mice. Several differentially expressed miRNAs were subsequently confirmed and quantified by real-time quantitative reverse transcription-polymerase chain reaction. Bioinformatics analysis was used for the prediction of miRNA targets. Furthermore, in vitro experiments showed that NCL regulated miR-21 expression following hydrogen peroxide preconditioning. CONCLUSIONS: Myocardial-protection mechanisms exerted by NCL might be mediated by the miRNAs identified in this study.

[TCM Combined Western Medicine Treatment of Advanced NSCLC: a Preliminary Study of mIRNA Expression Profiles].

Objective To preliminarily observe miRNA gene profiles in benefit serum of advanced non-small cell lung cancer (NSCLC) treated by TCM combined Western medicine (WM) , and to seek for molecular markers for its efficacy monitoring and prediction. Methods Recruited were 5 advanced NSCLC patients who received TCM combined WM treatment and obtained efficacy benefit ( as the treatment group) , 3 advanced NSCLC patients who received early treatment ( as the lung cancer group) , and 3 healthy subjects (as the control group). Serum samples were collected and total RNA was extracted using Trizol method. Using microRNA PCR ARRAY chip technology (product of Exiqon Company) , differentially miRNA expression profiling in serum between the lung cancer group and the control group, and between the treatment group and the lung cancer group were detected. Benefit miRNA expression profiling was ob- tained based on cluster analysis and comparative analysis. Results After tested by miRNA PCR ARRAY and managed by data analysis, a total of 42 miRNAs with more than 2 folds difference were screened in the lung cancer group and the control group, including 29 up-regulated and 12 down-regulated miRNAs. Be- sides, miR-10b-5p, miR-21-5p, miR-182-5p, miR-361-3p, and miR-382-5p were statistically different (P < 0. 05). A total of 45 miRNAs with more than 2 folds difference were screened in the treatment group and the lung cancer group, including 12 up-regulated and 33 down-regulated miRNAs. Fifteen miRNAs were statistically different including miR-137-3p, miR-182-5p, miR-376a-3p, miR-382-5p, miR-409-3p, miR-10a-5p, miR-21-5p, miR-29a-3p, miR-141-3p, miR-150-5p, miR-200c-3p, miR-342-3p, miR-365a-3p, miR-375, miR- 502-3p (P<0.05). Totally 22 miRNAs were screened in the treatment group with more than 2 folds differ- ence as compared with the lung cancer group and with less than or equivalent to 2 folds difference as com- pared with the control group, including 7 up-regulated and 15 down-regulated miRNAs, of which, miR-127- 3p, miR-182-5p, miR-382-5p, miR-409-3p, miR-10a-5p, miR-21-5p, miR-141-3p, miR-342-3p were statistically different (P <0. 05). Conclusion miRNAs including miR-21-5p, miR-182-5p, miR-382-5p are promising to become molecular markers for efficacy monitoring and prediction of advanced NSCLC treated by TCM combined WM, which provides reference for individualized treating advanced NSCLC.

Oxidized low-density lipoprotein attenuated desmoglein 1 and desmocollin 2 expression via LOX-1/Ca(2+)/PKC-ß signal in human umbilical vein endothelial cells.

Numerous studies have reported the presence of oxidized LDL (ox-LDL) and expression of its lectin-like receptor, LOX-1, have been shown in atherosclerotic regions. The present study aims to investigate the effects of ox-LDL on expression of desmoglein 1 (DSG1) and desmocollin 2 (DSC2) in endothelial cells, and to explore the role of LOX-1 mediated signal in the permeability injury associated with DSG1 and DSC2 disruption induced by oxidized lipoprotein. RT-PCR and Western blotting were applied to determine the mRNA and protein expression levels of DSG1 and DSC2 in human umbilical vein endothelial cells (HUVECs) respectively. Immunoreactivities of DSG1 and DSC2 were detected by laser scanning confocal microscope (LSCM). HUVEC monolayers permeability was evaluated by FITC-labeled LDL in transwell assay system. The possible signal was assessed using in vitro blocking LOX-1 or Ca(2+) channel or PKC. The DSG1 and DSC2 expression were decreased by ox-LDL in concentration- and time-dependent manner. The effects of ox-LDL were mediated by its endothelial receptor, LOX-1. In parallel experiments, ox-LDL increased the influx of extracellular calcium, activation of protein kinase C (PKC) and permeability to LDL, which was inhibited by the LOX-1blocking antibody (10 µg/ml), Ca(2+) channel blocker (Diltiazem, 50 µmol/L) and PKC-ß inhibitor (hispidin, 4 µmol/L). These results suggested that ox-LDL-induced decrease in DSG1 and DSC2 expression and monolayer barrier injury via calcium uptake and PKC-ß activation following up-regulation of LOX-1 is one of the mechanisms of inducing greater permeability in HUVECs.

An involvement of SR-B1 mediated PI3K-Akt-eNOS signaling in HDL-induced cyclooxygenase 2 expression and prostacyclin production in endothelial cells.

It is well-known that sphingosine-1-phosphate (S1P), the phospholipid content of HDL, binding to S1P receptors can raise COX-2 expression and PGI(2) release through p38MAPK/CREB pathway. In the present study we assess the action of SR-B1 initiated PI3K-Akt-eNOS signaling in the regulation of COX-2 expression and PGI(2) production in response to HDL. We found that apoA1 could increase PGI(2) release and COX-2 expression in ECV 304 endothelial cells. Furthermore, SR-B1 was found to be involved in HDL induced up-regulation of COX-2 and PGI(2). Over-expressed SR-B1 did not significantly increase the expression of COX-2 and the PGI(2) levels, but knock-down of SR-B1 by siRNA could significantly attenuate COX-2 expression and PGI(2) release together with p38MAPK and CREB phosphorylation. Consistently, the declines of p-p38MAPK, p-CREB, COX-2 and PGI(2) were also observed after incubation with LY294002 (25µmol/L; PI3K special inhibitor) or L-NAME (50µmol/L; eNOS special inhibitor). In addition, we demonstrated the increases of PGI(2) release, COX-2 expression and p38MAPK phosphorylation, when nitric oxide level was raised through the incubation of L-arginine (10 or 20nmol/L) in endothelial cells. Taking together, our data support that SR-B1 mediated PI3K-Akt-eNOS signaling was involved in HDL-induced COX-2 expression and PGI(2) release in endothelial cells.

[Study on oligosaccharides from Morinda officinalis].

OBJECTIVE: To study the oligosaccharides from Morinda officinalis How. METHODS: Compounds were isolated by chromatography, and their structures were identified by spectral analysis and chemical evidences. RESULTS: Six compounds were isolated and identified as sucrose (I), inulin-type trisaccharide (II), inulin-type hexasaccharide (III), inulotriose (IV), inulotetraose (V), inulopentaose (VI). CONCLUSION: Compound IV, V and VI are isolated from Morinda officinalis for the first time.

[A clinical study on gestational transient thyrotoxicosis].

OBJECTIVE: To investigate the prevalence of gestational transient thyrotoxicosis (GTT) and analyze the cause of thyrotoxicosis encountered in this period. METHODS: An epidemiologic survey in ten hospitals in Shenyang was performed and 534 pregnant women during the first trimester of pregnancy filled questionnaire, received physical examination and had serum thyroid-stimulating hormone (TSH), free T(4) (FT(4)), free T(3) (FT(3)), thyroid peroxidase antibody (TPOAb), thyrotrophin receptor antibody (TRAb), and human chorionic gonadotrophin (hCG) tests. RESULTS: (1) The total prevalence of thyrotoxicosis was 9.75% (52/534) in the first trimester and the prevalence of GTT was 7.86%, which accounted for 80.77% of the thyrotoxicosis encountered in this period. A total of 88.89% of the overt GTT showed only elevated FT(3) level. (2) The level of serum hCG increased gradually in the first trimester. The medians of hCG were 25 300, 85 220 and 81 780 IU/L 6, 8 - 10 and 12 weeks after gestation, respectively (P = 0.000). The medians of serum TSH were 1.45, 1.10 and 0.84 mIU/L 6, 8 - 10 and 12 weeks after gestation, respectively (P < 0.01). (3) When serum hCG was more than 50 000 IU/L, the prevalence of GTT increased obviously. When serum hCG was between 80 000 IU/L and 110 000 IU/L, subclinical GTT increased significantly. When serum hCG was more than 110 000 IU/L, overt GTT increased significantly. Correlation analysis showed that serum hCG was related negatively with TSH (r = -0.402, P = 0.000) and positively with FT(3) (r = 0.165, P = 0.000), but not related with FT(4). CONCLUSIONS: The prevalence of GTT is 7.86% in the first trimester and it is the main cause of thyrotoxicosis found in the first trimester, accounting for 80.77% of all the causes. The serological characteristic of overt GTT is mainly the elevation of serum FT(3) level. Serum hCG level is related with the severity of GTT.