Objective To develop a matrix metalloproteinase(MMP)-responsive hyaluronic acid(HA)-based controlled-release material for brain-derived neurotrophic factor(BDNF)to provide a novel therapeutic strategy for intervention and repair of traumatic brain injury(TBI).Methods HA was modified with amination,followed by condensation with Suflo-SMCC carboxyl group to form amide,and then linked with glutathione(GSH)to synthesize HA-GSH.The recombinant glutathione S-transferase(GST)-tissue inhibitor of metalloproteinase(TIMP)-BDNF(GST-TIMP-BDNF)expression plasmid was constructed using molecular cloning technique with double enzyme digestion by Bam H Ⅰ and Eco R Ⅰ.The recombinant GST-TIMP-BDNF protein was expressed in the Escherichia coli prokaryotic expression system,and purified by ion exchange chromatography,confirmed by Western blotting.MMP diluents were supplemented with PBS,MMP inhibitor marimastat,and varing concentrations(0.4,0.6,0.8 mg/ml)of GST-TIMP-BDNF or GST-BDNF.MMP-2 activity was analyzed using an MMP activity detection kit to evaluate the inhibitory effect of the recombinant protein on MMP.Primary rat neurons were extracted and cultured to establish an iron death model induced by RSL3.The effect of recombinant protein GST-TIMP-BDNF on neuronal injury was detected by immunofluorescence staining.Results MRI hydrogen spectrum identification confirmed the successful synthesis of HA-GSH.Western blotting results showed the successful expression of the recombinant protein GST-TIMP-BDNF containing the GST tag using the E.coli prokaryotic expression system.MMP activity detection results indicated that the recombinant protein GST-TIMP-BDNF had a superior inhibitory effect on MMP-2 activity compared to GST-BDNF(P<0.05).Immunofluorescence staining results showed a significant increase in fluorescence intensity in rat neurons treated with GST-TIMP-BDNF after RSL3 induction(P<0.05).Conclusion A MMP-responsive HA-based BDNF controlled-release material has been successfully developed,exhibiting a protective effect on neuron damage.

Objective:To investigate the pathogenic role and possible mechanism of NADPH oxidase 4 (Nox4) in type 1 diabetic keratopathy mouse models.Methods:Forty Nox4 knockout ( Nox4-/-) heterozygous male mice were selected and 120 age- and sex-matched wild-type C57BL/6 ( Nox4+ /+ ) mice were selected as controls. Nox4-/- and Nox4+ /+ mice were randomized into diabetic group (DM group) and non-DM group by random number method.Type 1 DM model was established in DM groups by intraperitoneal injection of streptozotocin.The DM and non-DM groups of Nox4+ /+ mice were randomized into regular feed group and Nox4 inhibitor GKT137831 (GKT) supplementary feed group by random number method.At 16 weeks after modeling, tear secretion of mice in different groups was measured by the phenol red thread test.Corneal epithelial integrity was evaluated by fluorescent staining.Changes in corneal never fiber density were observed by the in vivo laser scanning confocal microscopy.Reactive oxygen species (ROS) products in corneal epithelium were assayed by CellROX staining.The expressions of E-Cadherin and nuclear factor-κB (NF-κB) proteins were detected by immunofluorescence staining.Central corneal nerve fiber density was examined by flatmount staining with TUBB3 antibody.The use and care of laboratory animals complied with ARVO statement.The study protocol was approved by Laboratory Animal Care Committee of Xi'an Jiaotong University (No.XJTULAC201301). Results:In Nox4+ /+ mice, the tear secretion was (2.40±1.18)mm/minute in DM group, which was significantly less than (5.30±1.02)mm/minute in non-DM group ( P<0.01).The tear secretion was (4.19±0.63)mm/minute in DM group of Nox4-/- mice, which was significantly more than that in DM group of Nox4+ /+ mice ( P<0.05).Significant difference was found between (2.23±0.83)mm/minute of regular feed group and (4.02±0.71)mm/minute of GKT supplementary feed group ( P<0.01).In Nox4+ /+ mice, the DM group showed significantly increased corneal staining score, reduced corneal nerve fiber density, increased fluorescence intensity of ROS in corneal epithelium, weakened fluorescence intensity of E-Cadherin protein expression, and enhanced fluorescence of NF-κB protein expression compared with non-DM group.In Nox4-/- mice and mice fed with GKT supplementary feed, the increased fluorescence of ROS and decreased fluorescence of E-Cadherin protein expression were seen in the corneal epithelium of the DM groups compared with non-DM groups.In Nox4-/- mice and mice fed with GKT supplementary feed, NF-κB protein fluorescence was weak in corneal epithelial cells in DM groups, which was similar to that in non-DM groups.Immunofluorescence staining of corneal flatmount showed that the density of TUBB3-stained nerve fibers in DM group of Nox4+ /+ mice was significantly lower than that in non-DM group of Nox4+ /+ mice, and there was no significant reduction of nerve fibers in the corneal stromal layer in DM group of Nox4-/- mice or mice fed with GKT supplementary feed. Conclusions:Nox4 is involved in the pathogenic process of diabetic keratopathy, and its mechanism may be related to oxidative stress-induced aggregation of ROS products and activation of NF-κB-mediated inflammatory responses.

Human physiological indicators have become an important standard for assessing health in modern society.Traditional detection methods often require a separate laboratory,complex operation process and long detection time,so it is urgent to develop portable,fast and accurate on-site detection technologies for bioanalysis.Point-of-care testing(POCT),which differs from traditional laboratory testing,can realize the rapid in situ detection of biomarkers without the complicated analytical process of the laboratory.Smartphones,which are an essential tool in our daily life,not only have independent operating systems and built-in storage functions,but also have high-definition cameras,which have great application potential in POCT visualization.The combination of various biosensing technologies and smartphones has developed into a new direction in the field of POCT.This review mainly introduced the research progress of smartphone-based visual biosensors in POCT in recent years,including colorimetric sensors,fluorescence sensors,chemiluminescence sensors and electrochemiluminescence sensors.Finally,the problems faced by smart-phone-based visual biosensors in the application of POCT were summarized,and their future development was prospected.

Objective To explore the mechanism of'Wenyang Tongmai'(warming yang to unblock meridians)moxibustion in preventing and treating atherosclerosis.Methods Ten C57BL/6J mice fed with normal diet were set as blank group.Thirty ApoE-/-mice were fed with high-fat diet to establish atherosclerosis model,and were randomly divided into model group,Simvastatin group and moxibustion group,with 10 mice in each group.The intervention began on the first day of modeling.The mice in the moxibustion group were given moxibustion at Danzhong(RN17),Shenque(RN8),Neiguan(PC6),Xuehai(SP10)points,and the Simvastatin group was given Simvastatin distilled water suspension by gavage for 12 weeks.After administration,the pathological structure of thoracic aorta in mice was observed by hematoxylin-eosin(HE)staining method.The ultrastructure of thoracic aortic endothelial cells in mice was observed by transmission electron microscopy.The levels of serum tumor necrosis factor α(TNF-α),intercellular adhesion molecule 1(ICAM-1)and vascular cell adhesion molecule 1(VCAM-1)in mice were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA expression levels of silent information regulator 1(SIRT1)and forkhead box O3a(FOXO3a)in thoracic aorta were detected by real-time quantitative polymerase chain reaction(qRT-PCR).The protein expression levels of SIRT1 and FOXO3a in thoracic aorta were detected by Western Blot.Results Compared with the blank group,the pathological changes of thoracic aorta and vascular endothelial cells in the model group were obvious,the levels of serum inflammatory factor TNF-α,ICAM-1 and VCAM-1 were increased(P＜0.05 or P＜0.01),the mRNA and protein expressions of SIRT1 in thoracic aorta were decreased(P＜0.01),and the mRNA and protein expressions of FOXO3a had no significant difference(P＞0.05).Compared with the model group,the thoracic aorta and vascular endothelial cell structure of the mice in the Simvastatin group and the moxibustion group were obviously improved,the levels of serum TNF-α,ICAM-1 and VCAM-1 were decreased(P＜0.05),the mRNA and protein expressions of SIRT1 in the thoracic aorta were increased(P＜0.05 or P＜0.01),and the mRNA and protein expressions of FOXO3a had no significant difference(P＞0.05).There was no significant difference in the above indexes between the Simvastatin group and the moxibustion group(P＞0.05).Conclusion'Wenyang Tongmai'moxibustion can prevent and treat atherosclerotic in rats via regulating and controlling SIRT1/FOXO3a signaling pathway to reduce inflammatory response.

Objective:To explore the genotypes and frequency distribution of thalassemia in Lingui District,Guilin City,and provide reference for the prevention and control of thalassemia in this area. Methods:The results of genetic testing for thalassemia in 1501 suspected cases at the Second Affiliated Hospital of Guilin Medical University were analyzed retrospectively. The deletional mutations of α-thalassemia were detected by gap-PCR,the non-deletional mutations of α-thalassemia and β-thalassemia mutations were detected by PCR-reverse dot blot (PCR-RDB). Results:In 1501 samples,a total of 678 cases of thalassemia carriers were detected,with a detection rate of 45.17％. Among them,379 cases were α-thalassemia (including deletional α-thalassemia and non-deletional α-thalassemia),with a detection rate of 25.25％,the most common genotype was--SEA/αα (227 cases,15.12％),followed by-α3.7/αα (53 cases,3.53％). 270 cases of β-thalassemia were detected,with a detction rate of 17.99％,and βCD41-42/βN (144 cases,9.59％) was the main genotypes,followed by βCD17/βN (66 cases,4.40％) . In addition,there were 29 cases of αβ compound thalassemia,accounting for 1.93％,and the most common genotype was--SEA/αα complex βCD41-42/βN (5 cases,0.33％). Conclusion:Lingui District in Guilin City is a high-incidence area of thalassemia,and the genotypes of carriers are complex and diverse,with genetic heterogeneity. The results of this study provide a scientific basis for genetic counseling and prenatal diagnosis in this area.

Aim To explore the effects of Huannao Yicong decoction(HYD)on the learning and memory ability and brain iron metabolism in APP/PS1 mice and the correlation of HAMP knockout mice and APP/PS1 double transgenic model mice.Methods The ex-periment was divided into five groups,namely,HAMP-/-group(6-month HAMP gene knockout mice),APP/PS1 group(6-month APP/PS1-double-transgenic mice),HAMP-/-+HYD,APP/PS1+HYD,and negative control group(6-month C57BL/6J mice),with six mice in each group.The dose was ad-ministered(13.68 g·kg-1 weight),and the other groups received distilled water for gavage once a day for two months.After the administration of the drug,the mice in each group were tested for learning and memory in the Morris water maze;Biochemical detec-tion was performed to detect iron ion content in each mouse brain;Western blot and RT-qPCR were carried out to analyze hippocampal transferrin(TF),transfer-rin receptor1(TFR1),membrane iron transporter1(FPN1)divalent metal ion transporter 1(DMT1)and β-amyloid protein(Aβ)protein and mRNA expression levels in each group.Results Compared with the normal group,both HAMP-/-mice and APP/PS1 mice had reduced the learning and memory capacity,in-creased iron content in brain tissue,Aβ protein ex-pression increased in hippocampus of HAMP-/-group and APP/PS1 group mice(P＜0.01),the protein and mRNA expression of TF,TFR1 and DMT1 increased in hippocampal tissues of HAMP-/-and APP/PS1 groups(P＜0.01),and the FPN1 protein and mRNA expres-sion decreased(P＜0.01).Compared with the HAMP-and APP/PS1 groups,respectively,HAMP-/-+HYD group and APP/PS1+HYD group had improved learning and memory ability,decreased iron content,decreased Aβ protein expression(P＜0.01),decreased TF,TFR1,DMT1 protein and mR-NA expression(P＜0.01),and increased expression of FPN1 protein and mRNA(P＜0.01).Conclusions There is some association between HAMP-/-mice and APP/PS1 mice,HYD can improve the learning and memory ability of HAMP-/-and APP/PS1 mice and reduce the Aβ deposition.The mechanism may be related to the regulation of TF,TFR1,DMT1,FPN1 expression and improving brain iron overload.

Objective:To systematically screen and identify long noncoding RNA(lncRNA)associated with bone marrow adiposity changes in aplastic anemia(AA).Methods:The PPARγ and C/EBPα ChIP-Seq data in ChIPBase was analyzed by bioinformatics and the potential lncRNA co-transcriptionally regulated by PPARγ and C/EBPα was screened.The expression of candidate lncRNA was verified by qRT-PCR in the in vitro adipogenic differentiation model of BM-MSC,BM-MSC infected with lenti-shPPARγ and lenti-shC/EBPα as well as clinical BM-MSC samples derived from AA and controls.Results:PPARγ and C/EBPα were significantly highly expressed in AA BM-MSC,and knock-down of PPARγ and C/EBPα impaired the adipogenic capacity of AA BM-MSC.PPARγ and C/EBPα cotranscriptionally activate LINC01230 promoter activity in binding sites dependant manner.The LINC01230 was also aberrantly highly expressed in AA BM-MSC compared with controls.Conclusion:PPARγ and C/EBPα are aberrantly expressed in AA BM-MSC and may promote the adipogenic differentiation of AA BM-MSC,and to a certain extent mediate the bone marrow adiposity alteration by transcriptionally activating LINC01230 expression.

Aconitine,a common and main toxic component of Aconitum,is toxic to the central nervous system.However,the mechanism of aconitine neurotoxicity is not yet clear.In this work,we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine.HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system.Meanwhile,to confirm the metabolic mechanism of aconitine toxicity on HT22 cells,the levels of lactate dehydrogenase,intracellular Ca2+,reactive oxygen species,glutathione and superoxide dismutase,and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology.Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid,which was followed by the accumulation of lactic acid and reduction of glucose.The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca2+overload and oxidative stress,and eventually result in cell apoptosis.In general,we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.

ObjectiveTo investigate the effect of myosin heavy chain 7 gene-derived miRNA-208b-3p on the fibrotic phenotype of cardiac fibroblasts. MethodsmiRNA chip array was performed to detect the dysregulated miRNAs in the myocardium of diabetic db/db mice and db/m control mice. Neonatal mouse ventricular cardiomyocytes （NMVCs） and cardiac fibroblasts （CFs） were isolated from C57BL/6 mice and cultured. Real-time quantitative PCR （RT-qPCR） was conducted to determine the expression of miR-208b-3p in mouse CFs and NMVCs subjected to angiotensinⅡ（AngⅡ） and high glucose plus glucose oxidase （G/Go） treatment， respectively. Cell counting kit 8（CCk8） assay， flow cytometry and determination of fibrosis-related protein， including COL1A1， COL3A1and α-SMA， were performed in mCFs transfected with miR-208b-3p. Dual luciferase reporter assay was performed to confirm the interaction between miR-208b-3p and the 3'-UTR of metal response element binding transcription factor 2 （Mtf2） and progesterone receptor membrane component 1（Pgrmc1）， respectively. The expressions of Mtf2 and Pgrmc1 at the mRNA and protein levels in mCFs after miR-208b-3p mimic transfection were determined using RT-qPCR and Western blot assay， respectively. The small interfering RNA （siRNA） was used to inhibit Mtf2 and Pgrmc1 expression in mCFs， and the effects of Mtf2 siRNA， Pgrmc1 siRNA and miR-208b-3p on fibrosis-related protein expression in mCFs were investigated. ResultsResults of miRNA chip array and RT-qPCR assay showed that miR-208b-3p was up-regulated in the myocardium of the diabetic db/db mice. miR-208b precursor and the host gene of Myh7 were consistently increased in db/db mice. miR-208b-3p and Myh7 mRNA were expressed in mCFs and NMVCs， but the levels of miR-208b-3p and Myh7 mRNA in NMVCs were much higher than those in mCFs. miR-208b-3p was up-regulated in mCFs and NMVCs subjected to Ang Ⅱ and G/Go treatment， respectively. miR-208b-3p could significantly enhance fibrosis-related protein， including COL1A1， COL3A1 and α-SMA， in mCFs， without affecting the proliferation activity and cell cycle distribution of mCFs. Dual luciferase reporter assay revealed the interactions of miR-208b-3p with the 3'-UTR of Mtf2 and Pgrmc1. The results of RT-qPCR and Western blotting confirmed that miR-208b-3p inhibited Mtf2 and Pgrmc1 expression at the post- transcriptional level. Transfection with miR-208b-3p mimic， Mtf2 siRNA and Pgrmc1 siRNA could consistently enhance the fibrosis-related protein expression in the cardiac fibroblasts. ConclusionsmiR-208b-3p enhances fibrosis-related gene expression by targeting Mtf2 and Pgrmc1in mCFs.

MicroRNAs (miRNAs) are mediators of the aging process. The purpose of this work was to analyze the miRNA expression profiles of spermatozoa from men of different ages with normal fertility. Twenty-seven donors were divided into three groups by age (Group A, n = 8, age: 20-30 years; Group B, n = 10, age: 31-40 years; and Group C, n = 9, age: 41-55 years) for high-throughput sequencing analysis. Samples from 65 individuals (22, 22, and 21 in Groups A, B, and C, respectively) were used for validation by quantitative real-time polymerase chain reaction (qRT-PCR). A total of 2160 miRNAs were detected: 1223 were known, 937 were newly discovered and unnamed, of which 191 were expressed in all donors. A total of 7, 5, and 17 differentially expressed microRNAs (DEMs) were found in Group A vs B, Group B vs C, and Group A vs C comparisons, respectively. Twenty-two miRNAs were statistically correlated with age. Twelve miRNAs were identified as age-associated miRNAs, including hsa-miR-127-3p, mmu-miR-5100_L+2R-1, efu-miR-9226_L-2_1ss22GA, cgr-miR-1260_L+1, hsa-miR-652-3p_R+1, pal-miR-9993a-3p_L+2R-1, hsa-miR-7977_1ss6AG, hsa-miR-106b-3p_R-1, hsa-miR-186-5p, PC-3p-59611_111, hsa-miR-93-3p_R+1, and aeca-mir-8986a-p5_1ss1GA. There were 9165 target genes of age-associated miRNAs. Gene Ontology (GO) analysis of the target genes identified revealed enrichment of protein binding, membrane, cell cycle, and so on. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of age-related miRNAs for target genes revealed 139 enriched pathways, such as signaling pathways regulating stem cell pluripotency, metabolic pathways, and the Hippo signaling pathway. This suggests that miRNAs play a key role in male fertility changes with increasing age and provides new evidence for the study of the mechanism of age-related male fertility decline.
Humans ; Male ; Young Adult ; Adult ; Middle Aged ; MicroRNAs/genetics* ; Signal Transduction/genetics* ; Spermatozoa/metabolism* ; Gene Expression Profiling