Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose.

AIM: To determine whether the microRNA-27b-3p (miR-27b-3p)/NF-E2-related factor 2 (Nrf2) pathway plays a role in human retinal pigment epithelial (hRPE) cell response to high glucose, how miR-27b-3p and Nrf2 expression are regulated, and whether this pathway could be specifically targeted. METHODS: hRPE cells were cultured in normal glucose or high glucose for 1, 3, or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species (ROS) levels using a dihydroethidium kit. miR-27b-3p, Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunocytofluorescence (ICF), respectively. Western blot analyses were performed to determine nuclear and total Nrf2 protein levels. Nrf2, NQO1, and HO-1 expression levels by RT-qPCR, ICF, or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection. Finally, the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine. RESULTS: Persistent exposure to high glucose gradually suppressed hRPE Nrf2, NQO1, and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels. High glucose also promoted ROS release and inhibited cellular proliferation. Nrf2, NQO1, and HO-1 mRNA levels decreased after miR-27b-3p overexpression and, conversely, both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor. After treating hRPE cells exposed to high glucose with pyridoxamine, ROS levels tended to decreased, proliferation rate increased, Nrf2, NQO1, and HO-1 mRNA and protein levels were upregulated, and miR-27b-3p mRNA levels were suppressed. CONCLUSION: Nrf2 is a downstream target of miR-27b-3p. Furthermore, the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

Effectiveness and safety of acupoint application for chronic obstructive pulmonary disease: A protocol for updated systematic review and meta-analysis.

BACKGROUND: Acute exacerbation is a primary cause of repeated hospitalization and death in chronic obstructive pulmonary disease (COPD) patients. Therefore, how to control the symptoms of COPD at stable stage and reduce the number of acute exacerbation is a hot spot of medical research. Acupoint application (AA) is a significant part of external treatment of traditional Chinese medicine (TCM), Previous researches have reported that AA can be applied to the treatment of COPD. Nevertheless, its effectiveness is still inconclusive. This systematic review (SR) and meta-analysis is designed to appraise its effectiveness and safety for the treatment of patients with COPD. METHODS: Eight databases will be systematically retrieved from their inceptions to February 2021. Inclusion criteria are randomized control trials of AA combined with routine western medicine interventions in the treatment of COPD at stable stage. The primary outcomes we focus on comprise clinical effective rate, TCM symptom score, quality of life, dyspnea, exercise capacity, lung function, frequency of acute exacerbation, adverse events. The research screening, data extraction, and risk of bias assessment will be conducted by 2 individuals independently, and divergence will be adjudicated by a third senior investigator. The Stata 13.1 software will be used for meta-analysis. The confidence of evidence will be classified adopting grading of recommendations assessment, development and evaluation (GRADE) algorithm and methodological quality of this SR will be assessed using assessment of multiple systematic reviews-2 (AMSTAR-2) tool. RESULTS: This SR will provide evidence-based medical proof for the treatment of COPD at stable stage by AA combined with conventional western medicine interventions. The findings of this SR will be presented at relevant conferences and submitted for peer-review publication. CONCLUSIONS: The findings of this SR will provide up-todated summary proof for evaluating the effectiveness and safety of AA for COPD. REGISTRATION NUMBER: INPLASY 202140080.

[Determination of avermectin and ivermectin in swine muscular tissues using high performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To develop a method for simultaneous determination of avermectin (AVM) and ivermectin (IVM) in swine muscular tissues using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). METHODS: The AVM and IVM were extracted with acetonitrile, cleaned with basic alumina solid phase extraction cartridges, separated through C8 column, and then detected by mass spectrometry in positive ion mode with multiple reaction monitoring (MRM) using an electrospray ionization interface, with quantitative ion pair of 890.4/305.3 and 892.5/307.3 for AVM and IVM, respectively. RESULTS: The linear ranges for both AVM and IVM were between 5 microg/L and 100 microg/L, with a correlation coefficient of 0.9999. The detection limit was 2.0 microg/kg. The recoveries at the spiked concentration of 2 microg/kg, 10 microg/kg and 20 microg/kg ranged from 77.4% to 83.1% for AVM and 77.2% to 84.8% for IVM, with relative standard deviation (RSD) ranging from 8.7% to 11.7% for AVM and 6.3% to 10.3% for IVM, respectively. No AVM and IVM residues were detected in samples taken from Chengdu markets. CONCLUSION: A HPLC-MS/MS method was successfully developed for determining AVM and IVM in swine muscular tissues, which is simple, sensitive and precise and can meet both domestic and international requirements.

[Long term outcome of posterior lumbar pedicle screw fixation combined with isthmic bone graft fusion in the treatment of lumbar spondylolysis in young patients].

OBJECTIVE: To investigate the long-term effect of posterior lumbar pedicle screw fixation combined with isthmus bone grafting and fusion in young patients with spondylolysis. METHODS: A retrospective study was carried out, consisting of 16 young patients with lumbar spondylolysis without spondylolisthesis treated by lumbar posterior pedicle screw fixation combined with isthmic bone grafting fusion from January 2006 to July 2014. There were 11 males and 5 females, aged from 18 to 21 years old, with an average age of 19.3 years old, and the course of disease ranged from 12 to 26 months, with an average of 22 months. All the patients suffered from lumbar pain and difficulty in getting out of bed. Preoperative CT confirmed 12 cases of L5 isthmus fissure and 4 cases of L4 isthmus fissure. Bone graft fusion was confirmed and internal fixation was removed after operation. Lumbar spondylolysis was evaluated by lumbago visual analogue scoring method at preoperative and postoperative time points. Lumbar isthmic fusion was evaluated by lumbar CT, and degeneration of fixed and adjacent segments of lumbar intervertebral disc was evaluated by lumbar MRI. RESULTS: Of the 16 patients, 13 patients (26 sides) were followed up, with a mean duration of 96 months. The operation time ranged from 80 to 105 minutes, with an average of 95 minutes. The intraoperative bleeding volume ranged from 150 to 300 ml, with an average of 225 ml. All the patients were successfully operated without any complications related to the operation. VAS scores at each time point after operation were improved compared with those before operation(P<0.01). Postoperative CT scans of lumbar spine showed osseous fusion at 6 to 14 months, with an average of 12 months. There were no changes of adjacent segment degeneration, fixed segment disc degeneration and protrusion on lumbar spine MRI, and no symptomatic recurrence or recurrent spondylolysis in the long term. CONCLUSIONS: The posterior lumbar pedicle screw fixation combined with isthmic bone grafting and fusion is safe and effective in the treatment of young spondylolysis. The fusion rate is high and the interference of normal physiological range is reduced. The long-term effect is satisfactory.

Inhibition of potassium- and ischemia-evoked [3H] D-aspartate release from isolated bovine retina by cannabinoids.

We investigated the effect of cannabinoids on potassium chloride (K+)- and ischemia-induced [3H]D-aspartate release from isolated bovine retinae. The superfusion method was employed for studies of [3H]-neurotransmitter release. Cannabinoid receptor CB1 agonists, but not the CB2 agonist JWH 015, inhibited K+ -induced [3H]D-aspartate release from bovine retinae with the following rank order of activity: anandamide > ACEA > methanandamide > WIN 55,212-2. In the ischemic model, the rank order of activity was as follows: methanandamide > ACEA > WIN 55,212-2. The CB1 receptor antagonist AM 251 blocked inhibitory responses produced by cannabinoids in both experimental conditions. In conclusion, cannabinoids inhibit evoked [3H]D-aspartate release from isolated bovine retinae via an effect on CB1 receptors.

[Identification of two novel mutations of human blood coagulation factor V gene in a Chinese family with congenital factor V deficiency].

OBJECTIVE: To discover the mutations of human blood coagulation factor V (FV) gene in a Chinese family with congenital factor V deficiency, and to explore the molecular mechanism associated with the congenital factor V deficiency. METHODS: PCR and DNA sequencing were used to look for the FV gene mutations in the proband. And the novel mutation were testified by PCR restriction fragment length polymorphism technique or reverse DNA sequencing. One hundred healthy volunteers were chosen as controls at random. RESULTS: Two novel mutations were discovered in the FV gene of proband, which were the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16 (G-->T). The pedigree analysis showed that the two mutations inherited from his parents respectively: the A1763C came from his father, and the G-->T from his mother. The A1763C missense mutation in exon 11 was not found in each of 100 healthy volunteers. CONCLUSION: The congenital deficiency of FV in the proband might be caused by the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16, which jointly caused the proband to be a double heterozygote.

[Investigation of dietary fiber intakes and varies in 53 patients with diabetes].

OBJECTIVE: To investigate the intakes of total dietary fiber (TDF), soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) in Subjects With Type 2 Diabetes so as to provide the base for making the adequate intakes of dietary fiber. METHODS: The enzymatic-gravimetric methods for dietary fiber were established on basis of a collaborative study. Dietary intake was measured by means of 3-day food records through weighting and using food pictures. TDF, SDF and IDF were analyzed by enzymatic-gravimetric methods. RESULTS: The reproducibility relative standard deviations for DF ranged from 2.63% to 9.67%. Vegetable foods were the mainly sources of DF. The total dietary intakes, insoluble and soluble fibers were 26.5 +/- 9.8, 14.6 +/- 5.8, 10.4 +/- 4.4 (g/d) respectively. CONCLUSION: The dietary fiber intake of the diabetes remains in the range of intakes recommended by American Diabetes Association.

Neither the tumor necrosis factor alpha-308 A/G polymorphism nor the alpha2-macroglobulin polymorphism was associated with late-onset Alzheimer's disease in the Chinese population.

Late-onset Alzheimer's disease (LOAD) is the most common cause of dementia in the elderly. It is a complex and genetically heterogeneous disorder. Epidemiological studies demonstrated that nonsteroidal anti-inflammatory drugs could prevent or delay the onset of LOAD suggesting inflammation may be involved in AD. Tumor necrosis factor (TNF) is a potent immunomodulator and it might increase the production of amyloid beta(Abeta), which makes it an appropriate AD candidate gene. Alpha2 macroglobulin (A2M) is a serum protease inhibitor and a major low-density lipoprotein receptor-related protein (LRP) ligand. It can bind Abeta and mediate its clearance and degradation, suggesting it might be another AD candidate gene. In the present study, we analyzed the a 5 bp Ins/Del polymorphism of A2M gene (A2M-2), TNF alpha-308 A/G polymorphism and apolipoprotein E (APOE) polymorphisms of 67 sporadic late-onset AD patients and 142 normal elderly controls in the Chinese population. Our data showed that the APOE epsilon4 allele frequency in AD was significantly higher than that in the normal controls (chi2 = 11.66, P < 0.01) neither the frequencies of genotypes nor alleles of the TNF alpha-308 A/G and A2M polymorphisms were significantly different between AD and controls,suggesting the two polymorphisms were not risk factors to LOAD in Chinese.

[Expression and significance of transforming growth factor-beta1, matrix metalloproteinase-2 and its inhibitor in lens epithelial cells of diabetic cataract].

OBJECTIVE: To investigate the expression and significance of transforming growth factor-beta(1) (TGF-beta(1)), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in human Lens epithelial cells (LEC) of diabetic cataract. METHODS: Immunohistochemical staining was used to detect the expression of TGF-beta(1), MMP-2 and TIMP-2 in the diabetic LEC (23 cases) and the normal LEC (7 cases). RESULTS: There was significant difference of expression of MMP-2 and TGF-beta(1) between diabetic LEC and normal LEC (P < 0.01). But there was no statistic significance for TIMP-2 expression (P > 0.05). There was correlation between the expression of TGF-beta(1) and MMP-2. CONCLUSION: The imbalance induced by TGF-beta(1) between the expression of MMP-2 and TIMP-2 may play a critical role in the pathological fibrosis of anterior and posterior subcapsular during the development of diabetic cataract.

Association between APOC1 polymorphism and Alzheimer's disease: a case-control study and meta-analysis.

BACKGROUND: Previous association studies examining the relationship between the APOC1 polymorphism and susceptibility to Alzheimer's disease (AD) have shown conflicting results, and it is not clear if an APOC1 variant acts as a genetic risk factor in AD etiology across multiple populations. METHODS: To confirm the risk association between APOC1 and AD, we designed a case-control study and also performed a meta-analysis of previously published studies. RESULTS: Seventy-nine patients with AD and one hundred fifty-six unrelated controls were included in case-control study. No association was found between the variation of APOC1 and AD in stage 1 of our study. However, our meta-analysis pooled a total of 2092 AD patients and 2685 controls. The APOC1 rs11568822 polymorphism was associated with increased AD risk in Caucasians, Asians and Caribbean Hispanics, but not in African Americans. APOE ε4 carriers harboring the APOC1 insertion allele, were more prevalent in AD patients than controls (χ(2) = 119.46, OR = 2.79, 95% CI = 2.31-3.36, P<0.01). CONCLUSIONS: The APOC1 insertion allele, in combination with APOE ε4, likely serves as a potential risk factor for developing AD.

[Molecular mechanisms of Arg77Cys missense mutation and Arg174stop nonsense mutation of factor XIII A gene causing severe factor XIII deficiency].

The present study was aimed to investigate the molecular mechanisms responsible for the pathogenesis of severe factor XIII (FXIII) deficiency. Site-directed mutagenesis was conducted to obtain human FXIIIA expression plasmids bearing the mutations. Wild type FXIIIA recombinant plasmid (pcDNA3.1-FXIIIA-wt) and 2 mutant FXIIIA recombinant plasmids (pcDNA3.1/FXIIIA/77mut, pcDNA3.1/FXIIIA/174mut) were transfected into the cultured COS-7 cells using lipofectamine 2000 transfection reagent, respectively. FXIII activities were measured by the Berichrom(®) FXIII chromogenic assay. The expression levels of FXIIIA mRNA were detected by real-time RT-PCR. The recombinant FXIIIA mutants were determined by using Western blot and ELISA. The results showed that the normalized mRNA levels of 2 mutants in transfected COS-7 cells were 0.82 ± 0.21 and 0.76 ± 0.17, respectively. The relative levels of both mRNA transcripts were not significantly decreased as compared with the wild type (1.06 ± 0.51). FXIII activity and FXIIIA antigen levels in concentrated media of cell expressing the wild type protein were (24.0 ± 2.9)% and (13.2 ± 2.3)%, respectively. FXIII activity and FXIIIA antigen levels in cell lysates containing the wild type recombinant protein were (61.6 ± 30.4)% and (32.8 ± 14.5)%, respectively. However, the antigen levels and activity of 2 mutants were severely decreased as compared to the wild type. It is concluded that both mutations severely disturb the normal expression of FXIIIA protein. The reduction of expression levels and decreased activities of the 2 mutants provides a convincible explanation for the deficiency phenotype in the index case.

[Identification of Arg77Cys and Arg174stop double heterozygous mutation in a Chinese family with inherited FXIII deficiency].

OBJECTIVE: To identify the gene mutation type of an inherited coagulation factor XIII (FXIII) deficiency pedigree. METHODS: PCR and DNA sequencing were used to identify the mutations in the 15 exons and the flank sequence of FXIII gene in the proband. The identified mutations were validated by allele specific PCR, PCR restriction fragment length polymorphism technique or DNA sequencing in the family members and 100 healthy volunteers. RESULTS: Arg77Cys and Argl74stop double heterozygous mutations were discovered in the proband. The pedigree analysis showed that Arg77Cys missense mutation inherited from her father, and Arg174stop from her mother. The Arg77Cys missense mutation in exon 3 was not found in her husband and the other 100 healthy volunteers. CONCLUSION: A novel Arg174stop nonsense mutation was discovered in human FXIII gene. A simple DNA assay based on PCR for detection of this mutation was developed. The congenital FXIII deficiency in the proband might be caused by the coinheritance of the Arg77Cys missense mutation in exon 3 and the Arg174stop nonsense mutation in exon 4.

[Clinical study on the vertebral plasty for the treatment of osteoporotic vertebral compressed fractures in elderly].

OBJECTIVE: To study therapeutic effects of Sky-bone expander for the treatment of osteoporotic vertebral compressed fractures. METHODS: Fifteen patients (18 vertebrae) suffering from vertebral compression fractures were treated with Sky bone expander system which expanded and reconstructed the vertebral body with PMMA cement. The clinical effect was evaluated by observing the changing of visual analogue scale (VAS). The preoperative and postoperative mean VAS scores were compared by paired-sample t test. All the patients were followed up by telephone or clinic consulting after being discharged from our hospital. RESULTS: The procedure was performed successfully in 15 patients. The operation time ranged from 45 to 110 minutes (65 minutes per vertebra on average). The patients were followed up and the duration ranged from 6 to 12 months (8 months on average). The mean VAS score of the patients were improved significantly at the third postoperative day compared with those before the operation (2.5 +/- 1.3, vs 7.7 +/- 1.1, all P < 0.05). The mean VAS score at the end of the follow-up was 2.2 +/- 1.2. CONCLUSION: Sky bone expander system provides significant pain relief effect in the cases of osteoporotic vertebral compression fractures, shortens the duration of lying in bed, and its procedure is convenient with few complications.

Dual effect of isoprostanes on the release of [3H]D-aspartate from isolated bovine retinae: role of arachidonic acid metabolites.

The effect of 8-isoprostanes on potassium (K+)-depolarization-evoked release of [3H]D-aspartate from bovine isolated retinae was investigated. Isolated bovine retinae were prepared for studies of K(+)-evoked release of [3H]D-aspartate using the Superfusion Method. Low concentrations of 8-isoPGF(2alpha) (1-100 nM) inhibited whereas higher concentrations of this 8-isoprostane (100 nM-30 microM) enhanced K(+)-induced [3H]D-aspartate overflow. The excitatory effect of 8-isoPGF(2alpha) was mimicked by thromboxane receptor agonist, U-46619 and blocked by thromboxane receptor antagonist, SQ 29,548 (10 microM). Pretreatment of tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen unmasked an inhibitory effect of high concentrations of 8-isoPGF(2alpha) (1-30 microM) on [3H]D-aspartate release that was attenuated by AH 6809 (10 microM). In conclusion, 8-isoPGF(2alpha) exhibits a dual regulatory effect on K(+)-induced [3H]D-aspartate release in isolated bovine retinae. The inhibitory action caused by 8-isoPGF(2alpha) is due to the activation of EP1/EP2 receptors while the excitatory effects are due to the activation of thromboxane receptors.