Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice,accompanied by the downregulation of DNA methyltransferases(DNMTs)in the brain.However,the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood.This study aimed to establish an in vitro model of hypoxic preconditioning,using a cultured mouse hippocampal neuronal cell line(HT22 cells),to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning.HT22 cells were divided into a control group,which received no exposure to hypoxia,a hypoxia group,which was exposed to hypoxia once,and a hypoxic preconditioning group,which was exposed to four cycles of hypoxia.To test the ability of hypoxic preadaptation to induce hypoxic tolerance,cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay.Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group.The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry.Compared with the hypoxia group,the expression levels of caspase-3 and spectrin,which are markers of early apoptosis and S-phase arrest,respectively,noticeably reduced in the hypoxic preconditioning group.Finally,enzyme-linked immunosorbent assay,real-time polymerase chain reaction,and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning.The results showed that compared with the control group,hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B.In conclusion,hypoxic preconditioning may exert anti-hypoxic neuroprotective effects,maintaining HT22 cell viability and inhibiting cell apoptosis.These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Reliability and validity of an indicator system for assessing the quality of ophthalmic nursing

Objective： The aim of this study was to examine the reliability and validity of an indicator system for assessing the quality of ophthalmic nursing. Methods： A questionnaire-based study was performed for 218 individuals in clinical nursing manage- ment using this indicator system. The resulting data were compiled and analyzed using Excel 2003 and SPSS 13.0, and tests of reliability and validity were conducted. Results： The indicator system yielded an overall Cronbach＇s o value of 0.948, a test-retest reliability of 0.793, 3-dimensional Cronbach＇s o values of 0.870-0.930, and a content validity of 0.92. Exploratory factor analysis was then performed to extract three common factors, which generated a cumulative contribution rate of 54.04%. The load range of each factor was 0.43-0.83. Conclusions： The indicator system for evaluating the quality of ophthalmic nursing has favorable reli- ability and validity, which means that it is a suitable clinical tool for assessing the quality of ophthalmic

dentification of Circular RNAs as a Novel Biomarker for Ovarian Endometriosis

Background： Endometriosis is a challenging disease with symptoms such as dysmenorrhea and infertility. However, its etiology is still vague and there is still no effective markers or treatment. This study aimed to profile the circular RNAs （circRNAs） expressed in eutopic endometrium from patients with ovarian endometriosis and explore potential clues to the pathogenesis of endometriosis, providing an evidence for clinical diagnosis and treatment. Methods： A total of 63 clinical samples, including control endometrium 01 = 22） and eutopic endoxnetrium （n = 41）, were collected from Peking Union Medical College Hospital between May 1,2016, and December 31,2016. Of them, four samples in each group were used for circRNA microarray. Then, tbur upregulated circRNAs were screened out for quantitative real-time polymerase chain reaction （qRT-PCR） validation. After that, bioinformatics analysis was pertbrmed to predict miRNAs targeted by validated circRNAs and investigate the circRNA-miRNA-mRNA interactions. Results： Among 88 differentially expressed circRNAs, 11 were upregulated and 77 were downregulated in eutopic endometrium of patients with endometriosis, qRT-PCR validation results for two upregulated circRNAs （circ0004712 and circ_O002198） matched the microarray results. The area under the receiver operating characteristic curve of circ_0002198 for distinguishing ovarian endometriosis was 0.846 （95% confidence interval [C1]： 0.752-0.939; P 〈 0.001 ） while that ofcitv_O004712 was 0.704 （95% CI： 0.571-0.837; P = 0.008）. On the basis of target prediction, we depicted the molecular interactions between the identified circRNAs and their dominant target miRNAs, as well as constructed a circRNA-miRNA-mRNA network. Conclusions： This study provides evidence that circRNAs are differentially expressed between eutopic and normal endometrium, which suggests that circRNAs are candidate factors in the activation ofendometriosis, circ_0002198 and circ0004712 may be potential novel biomarkers for the diagnosis of ovarian endometriosis.

Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-KB- related Inflammation during Traumatic Brain Injury in Mice

Background： In addition to neurons, all components of the neurovascular unit （NVU）, such as glial, endothelial, and basal membranes, are destroyed during traumatic brain injury （TBI）. Previous studies have shown that excessive stimulation ofcalpain is crucial for cerebral injury after traumatic insult. The objective of this study was to investigate whether calpain activation participated in NVU disruption and edema formation in a mouse model of controlled cortical impact （CCI）. Methods： One hundred and eight mice were divided into three groups： the sham group, the control group, and the MDL28170 group. MDL28170 （20 mg/kg）, an efficient calpain inhibitor, was administered intraperitoneally at 5 rain, 3 h, and 6 h after experimental CCI. We then measured neurobehavioral deficits, calpain activity, inflammatory mediator levels, blood-brain barrier （BBB） disruption, and NVU deficits using electron microscopy and histopathological analysis at 6 h and 24 h after CCI. Results： The MDL28170 treatment significantly reduced the extent of both cerebral contusion （MDL28170 vs. vehicle group, 16.90 ± 1.01 mm3 and 17.20±1.17 mm3 vs. 9.30 ± 1.05 mm^3 and 9.90 ± 1.17 mm3, both P 〈 0.001 ） and edema （M DL28170 vs. vehicle group, 80.76 ± 1.25% and 82.00 ± 1.84% vs. 82.55 ± 1.32% and 83.64 ± 1.25%, both P 〈 0.05）, improved neurological scores （MDL28170 vs. vehicle group, 7.50 ±0.45 and 6.33 ±0.38 vs. 12.33 ± 0.48 and 11.67±0.48, both P 〈 0.001）, and attenuated NVU damage resulting （including tight junction （TJ）, basement membrane, BBB, and neuron） from CCI at 6 h and 24 h. Moreover, MDL28170 markedly downregulated nuclear factor-κB-related inflammation （tumor necrosis factor-α [TNF-α]： MDL28170 vs. vehicle group, 1.15 ± 0.07 and 1.62± 0.08 vs. 1.59±0.10 and 2.18± 0.10, both P 〈 0.001 ： inducible nitric oxide synthase： M DL28170 vs. vehicle group, 4.51± 0.23 vs. 6.23± 0.12, P 〈 0.001 at 24 h; intracellular adhesion molecule- I ： MDL28170 vs. vehicle group, 1.45± 0.13 vs. 1.70 ± 0.12, P 〈 0.01 at 24 h） and lessened both myeloperoxidase activity （MDL28170 vs. vehicle group, 0.016± 0.001 and 0.016± 0.001 vs. 0.024± 0.001 and 0.023 ± 0.001, P 〈 0.001 and 0.01, respectively） and matrix metalloproteinase-9 （MMP-9） levels （MDL28170 vs. vehicle group, 0.87±0.13 and 1.10 ± 0.10 vs. 1.17 ± 0.13 and 1.25 ± 0.12, P 〈 0.001 and 0±05, respectively） at 6 h and 24 h after CCI. Conclusions： These findings demonstrate that MDL28170 can protect the structure of the NVU by inhibiting the inflammatory cascade, reducing the expression of MMP-9, and supporting the integrity of TJ during acute TBI.

Expression of Potassium Channels in Uterine Smooth Muscle Cells from Patients with Adenomyosis

Background： Adenomyosis （AM） has impaired contraction. This study aimed to explore the expression of potassium channels related to contraction in myometrial smooth muscle cells （MSMCs） of AM. Methods： Uterine tissue samples from 22 patients （cases） with histologically confirmed AM and 12 （controls） with cervical intraepithelial neoplasia were collected for both immunohistochemistry and real-time polymerase chain reaction to detect the expression of large conductance calciumand voltage-sensitive K＋ channel （BKCa）-a/β subunits, voltage-gated potassium channel （Kv） 4.2, and Kv4.3. Student＇s t-test was used to compare the expression. Results： The BKCa-a/β subunits, Kv4.2, and Kv4.3 were located in smooth muscle cells, glandular epithelium, and stromal cells. However, BKCa-β subunit expression in endometrial glands of the controls was weak, and Kv4.3 was almost undetectable in the controls. The expression of BKCa-a messenger RNA （mRNA） （0.62 ± 0.19-fold decrease, P 〈 0.05） and Kv4.3 mRNA （0.67 ±0.20-fold decrease, P 〈 0.05） decreased significantly in the MSMCs of the control group compared with the AM group. However, there were no significant differences in BKCa-β subunit mRNA or Kv4.2 mRNA. Conclusions： The BKCa-a mRNA and the Kv4.3 mRNA are expressed significantly higher in AM than those in the control group, that might cause the abnormal uterus smooth muscle contractility, change the microcirculation of uterus to accumulate the inflammatory factors, impair the endometrium further, and aggravate the pain.