Objective ToexploretheMRIfeaturesofthepseudoaneurysmsandarteriovenousmalformationsinuterus,andtoimprove ourunderstandingonthemforhigherdiagnosticaccuracy.Methods TheMRIfeaturesofthe4casesofuterinepseudoaneurysmsand the7casesofuterinearteriovenousmalformationswhichdiagnosedandcuredbyourhospitalwereanalyzedretrospetively.Results TheMRIofuterinepseudoaneurysms(n＝4)shownodular(n＝3)oroval(n＝1)abnormalsignalinthemyometriumsofuterus,low signalintensityonT1WI,highsignalintensityonT2WI.Thelesionenhancementissimilartotheuterinearterycommunicatingwiththe lesion.TheMRIofuterinearteriovenousmalformations(n＝7)showuterinepatchyabnormalsignal,andtheirboundaryarenotclear. Thereareanumberoftortuousbloodvessels.Thelesionsareobviouslyheterogenouslyenhanced,andthethickeneddrainagevessels couldalsobeseenoncontrastGenhancedimages.Thelesionsofuterinepseudoaneurysmsandarteriovenousmalformationsdon’tshow highsignalintensityonDWI,suggsetingnolimiteddiffusion.Conclusion The MRIcharacteristicsoftheuterinepseudoaneurysms showthattheuterinelesionenhancementissimilartotheuterinearterycommunicatingwiththelesion.The MRIcharacteristicsof theuterinearteriovenous malformationsarethatthereareanumberoftortuousbloodvesselsanddrainagevesselsintheuterus. Therearen’tlimiteddiffusioninthesetwodiseasesonDWI.

Objective To investigate the effects of thyroid hormone on extremely severe traumatic brain injury (TBI).Methods A retrospective case-control study was conducted to analyze the treatment of 105 patients with extremely severe TBI admitted from July 2010 to April 2014.There were 79 males and 26 females,with an average age of 32.9 years.The patients were divided into conventional treatment group (Group A,35 cases),conventional treatment ± thyroxine treatment group (Group B,35 cases) and thyroxine treatment group after the condition that thyroxine level was low (Group C,35 cases) according to the random number table method.The incidence of low T3 and T4,incidence of hypotension,the dosage of vasoactive drugs,function evaluation of liver and kidney damage,Glasgow outcome scale (GOS),and acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) within 20 days after admission,and mortality rate within 30 days after admission were compared and analyzed.Results Within 20 days after admission,the rates of low thyroxine levels and hypotension of the Group B (22.9％,77.1％) were significantly lower than those in the other two groups (Group A:40％,100％;Group C:37％,100％) (all P ＜ 0.05).The doses of dopamine and norepinephrine in Group B was significantly lower than the other two groups and the combination rate of vasopressors in Group B was significantly lower than the other two groups (P ＜ 0.05),while there was no significant difference between Group A and Group C (P ＞ 0.05).The corresponding data in Group A and Group C had no statistically significant difference (P ＞ 0.05).The liver and renal dysfunction rates of Group B (29％,31％) were significantly lower than those of the other two groups (Group A:49％,51％;Group C:43％,51％) (all P ＜ 0.05).The corresponding data in Group A and Group C had no statistically significant difference (P ＞ 0.05).GOS in Group B [(4.8 ± 1.9) points] was significantly higher than that in Group A [(3.3 ± 0.2) points] (all P ＜ 0.05) within 30 days after admission and significantly higher than that of itself at the beginning [(3.6 ± 1.1) points] (P ＜ 0.05).The APACHE Ⅱ in Group A was significantly higher than those in other two groups as well as that in Group A at admission (P ＜ 0.05).Mortality rates in Group B (31％) and Group C (29％) were significantly lower than that in Group A (69％) within 30 days after admission (P ＜ 0.05).Conclusions Thyroxine can reduce the incidence of hypotension,liver and kidney injury rate in extremely severe TBI.Prevention is better than the supplementary treatment after severe TBI.Thyroxine can also reduce the mortality of extremely severe TBI within 30 days after admission.

Objective To investigate the effect of the overexpression of Krüppel-like factor 6 (KLF6)towards the apoptosis of human lens epithelial cells (HLECs) induced by ultraviolet B (UVB) radiation.Methods The eukaryotic expression plasmid pEGFP-C2-KLF6 which was successfully constructed were transfected into HLECs,followed by the detection of KLF6 level by using Western blot,and then companied by UVB stimulation.Cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay.The morphology of the cells was observed by using hematoxylin-eosin staining method.The cell damage was examined by Live/Dead staining.The apoptotic markers bax and bcl-2 were detected by Western blot.Quantitative apoptotic levels were measured with the apoptosis detection kit;the expression level of reactive oxygen species (ROS) was analyzed by DCFH-DA probe.Results The cell viability of the 0.5 μg transfection group and the 1.0 μg transfection group was significantly lower than that of the blank vector control group (both at P＜0.05).In high KLF6 expression group,the cells were sparse,long and narrow in size and shape,and the cytoplasm was concentrated.The cells in the normal control group were green living cells with stable morphology and even quantity.The number of red dead cells was increased significantly in the KLF6 highexpression group.After UVB irradiation,the apoptosis value,relative bax expression,bax/bcl-2 ratio and ROS expression of HLECs cells in the KLF6 high-expression group were all higher than those in the blank vector control group,with statistically significant differences between them (all at P＜0.05).Conclusions Overexpression of KLF6 can exacerbate apoptosis of HLECs caused by UVB,by regulating the expression of apoptosis-related proteins and promoting the accumulation of ROS in the endoplasmic reticulum.Down-regulation of KLF6 expression by biological tools may play a protective role on LECs to a certain extent.

Objective To observe the inhibitory effect of lentiviral vector miR-191 (LV-191) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).Methods Eighty healthy 7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, non-intervention group, normal saline (NS) group, LV-191 group and LV-green fluorescent protein (GFP) group, 16 mice in each group. The OIR model was established in the non-intervention group, NS group, LV-191 group and LV-GFP group. NS group, LV-191 group and LV-GFP group were given an intravitreal injection of 1 μl of NS, LV-191 and LV-GFP at the age of 12 days. No injection was performed in the non-intervention group. In normal group,newborn mouse were maintained in room air form P0 to P17, and no treatment was performed. Mice in all five groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR (RT-PCR) to detect miR -191 and P21 expression of retinal tissue.Results In the LV-191 group, the non-perfusion area were both significantly smaller than those in non-intervention group, NS group and LV-GFP group (F=127.20,P<0.001). The number of pre-retinal neovascular cell nuclei in retinas from LV-191 group were obviously lower than those in the retinas from non-intervention group, NS group and LV-GFP group (F=31.71,P<0.05). RT-PCR showed that the LV-191 and P21 level of LV-191 group increased significantly than other groups (F=10.95, 15.60;P<0.05).Conclusion Intravitreal injection of LV-191 inhibits RNV in mice model of OIR possibly through up-regulating p21.

Objective:To observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).Methods:A three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.Results:The LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group ( t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group ( t=11.30) and OIR + The LV-Vec group ( t=15.47), and the differences were statistically significant ( P<0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group ( t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups ( t=5.26, 5.46, 3.73), the differences were statistically significant ( P<0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours ( t=8.35, 13.84; P<0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant ( t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P<0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced ( t=9.33, 6.15; P<0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group ( t=15.23, 21.09; P<0.05), but no change in the mRNA expression of PSF ( t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased ( t=10.18, 13.10; P<0.05), but the PSF mRNA expression increased ( t=65.00, 85.79; P<0.05). Conclusion:PSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.

Objective:To investigate the protection and the corresponding molecular mechanisms of polypyramidine tract binding protein-associated splicing factor (PSF) overexpression on human retinal microvascular endothelial cells (hRMECs) induced by advanced glycation end-products (AGEs).Methods:The hRMECs were divided into the normal group, the vector group, PSF group, zinc protoporphyrin (ZnPP) group and PSF+ZnPP group for experiment. Cells in the normal group were cultured in a DMEM medium containing 10% fetal calf serum, penicillin/streptomycin, and placed in a closed constant temperature incubator at 37 °C, 95% air, and 5% CO 2. Cells in the vector group were infected with empty lentivirus. The cells in the PSF group were infected with overexpressing PSF lentivirus. Cells in the ZnPP group were treated with ZnPP (10 mol/L) for 2 h. The PSF+ZnPP group cells were infected with overexpressing PSF lentivirus, and then pretreated with ZnPP (10 mol/L) for 2 h. With the last four groups of cells stimulated with AGEs, HE, Hoechst33258 staining and flow cytometry were used to observe the protective effect of high expression of PSF on cell damage and the antagonistic effect of ZnPP on PSF. Western blot was used to detect the protein expression of heme oxygenase-1 (HO-1), phosphorylated (p) extracellular regulatory protein kinase (ERK), and Nrf2 in the cells. U0126, a specific antagonist of ERK pathway, was introduced, and Western blot verified the reversal effect of U0126 on the expression of HO-1 induced by PSF protein. Results:HE staining and Hoechst33258 staining showed that the number of nuclei of damaged cells of PSF group were significantly increased compared with control group, while decreased compared with PSF+ZnPP group ( F=27.5, 38.7; P<0.05). The results of flow cytometry showed that the ROS produced by cells in the PSF group was significantly increased compared to the normal group, and significantly decreased compared to the PSF+ZnPP group, the difference was statistically significant ( F=126.4, P<0.05). Western blot results showed that HO-1 expression of PSF group was significantly increased compared with control and the vector group ( F=70.1, P<0.05). AGEs inducement of 30, 60, 120 and 240 min could significantly improve pERK expression compared with 15 min ( F=474.0, P<0.05). The expression of HO-1 and Nrf2 proteins in the PSF+/U0126- group was significantly more than those in the PSF-/U0126-group, the expression of HO-1 and Nrf2 proteins in the PSF+/U0126+ group was significantly lower than that in the PSF+/U0126- group, and the differences were statistically significant ( F=30.2, 489.4; P<0.05). Conclusion:Over expression of PSF can promote the HO-1 expression by activating ERK pathway and promoting the Nrf2 to the nucleus, thus protect hRMECs against AGEs-induced oxidative damage.

Objective:To investigate the regulatory effect of transient receptor potential cation channel subfamily C member 3 (TRPC3) on the retina in oxygen-induced retinopathy (OIR) mice and biological behavior of human retinal vascular endothelial cells (HREC).Methods:A total of 32 healthy SPF grade 7-day-old C57BL/6 mice were selected and randomly divided into a control group and an OIR group by the random number table method, with 16 mice in each group.The control group received no special treatment, and the OIR model was established in the OIR group.On postnatal day 17 (PN17), the success of the model establishment was verified by immunofluorescence staining of the retinal patch.The in vitro cultured HREC were divided into a normal control group, a transfection reagent group, and a si-TRPC3 group.The normal control group received no special treatment, while the transfection reagent group and the si-TRPC3 group were transfected with transfection reagent or transfection reagent + si-TRPC3.The relative expression of TRPC3 mRNA was detected by real-time quantitative fluorescence PCR.The relative expressions of TRPC3, transcription factor NF-E2 related factor (Nrf2), and superoxide dismutase (SOD) proteins were determined by Western blot.HREC were further divided into a normal control group, a vascular endothelial growth factor (VEGF) group, a si-TRPC3 group, and a Pyr3 (TRPC3 channel inhibitor) group, which were cultured in complete medium, medium containing 20 ng/ml VEGF recombinant protein, medium containing 20 ng/ml VEGF recombinant protein (si-TRPC3 transfection for 72 hours), and medium containing 20 ng/ml VEGF recombinant protein+ 1 μmol/L Pyr3 for 48 hours, respectively.The proliferation ability of HREC was detected using cell counting kit 8 (CCK-8). The horizontal and vertical migration ability of cells were detected by cell scratch assay and transwell assay, respectively.This study followed the 3R principles of animal welfare and was approved by the Ethics Committee of Hebei Eye Hospital (No.2023LW04). Results:Pathological neovascular clusters with strong fluorescent staining appeared in the retina of OIR mice on PN17.The relative expressions of TRPC3 mRNA and protein in the retina of OIR mice were 2.057±0.244 and 1.517±0.290, respectively, significantly higher than 0.983±0.033 and 0.874±0.052 of control group ( t=6.165, 3.094; both at P<0.05). The relative expression levels of TRPC3 mRNA and protein were significantly lower, and the relative expression levels of Nrf2 and SOD proteins were higher in the si-TRPC3 group than in the normal control and transfection reagent groups, and the differences were statistically significant (all at P<0.05). The CCK-8 experiment results showed that the cell absorbance value was higher in the VEGF group than in the normal control group, and lower in the si-TRPC3 and Pyr3 groups than in the VEGF group, with statistically significant differences (all at P<0.05). The results of the cell scratch experiment showed that the lateral migration rate of VEGF group cells was higher than that of normal control group, while the lateral migration rate of si-TRPC3 group and Pyr3 group cells was lower than that of VEGF group, and the differences were statistically significant (all at P<0.05). The transwell experiment results showed that the number of stained cells in the VEGF group was higher than that in the normal control group, and the number of stained cells in the si-TRPC3 group and Pyr3 group was lower than that in the VEGF group, with statistically significant differences (all at P<0.05). Conclusions:Hypoxia induces increased TRPC3 expression in OIR mouse retina, and downregulation of TRPC3 inhibits HREC proliferation and migration.The mechanism is related to the activation of the Nrf2-related oxidative stress pathway.

Objective:To observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.Methods:hRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.Results:Different concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant ( t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant ( t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1 285-3p, hsa-miR-30d-3p, hsa-miR-3 170, hsa-miR-7 976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions. Conclusion:FA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.

According to different experimental methods of induced diabetic retinopathy (DR) and different characteristics of the observed retinopathy, DR animal models can be divided into drug or dietary-induced models, oxygen-induced retinopathy (OIR) models, spontaneous inheritance models, and transgenic models. At present, induced model is one of the most commonly used animal model for DR study, which has the advantages of short modeling cycle, low cost, simple experimental operation and good repeatability. However, the drugs have certain side effects on various organs of animals and the risk of animal death is higher. OIR model has good repeatability, good stability and relatively low cost. However, due to the lack of metabolic changes of hyperglycemia in OIR mice, this model cannot accurately reflect the effects of metabolism on retina under hyperglycemia. The pathological changes of the spontaneous model are relatively stable, however, the application of this model is limited because the genetic homogeneity of diabetes differs from that of human and the cost is high. Transgenic model has definite etiology, however, its application is limited owing to the high cost and the high requirements of technology, operation and equipment. Therefore, researchers should comprehensively consider characteristics and limitations of different models while choosing suitable DR model based on research objectives, observation indicators, experimental conditions, and funds. In addition, animal models that can more accurately simulate DR need to be developed to provide more effective tools for studying the mechanism of DR and developing feasible prevention and treatment approaches.

Objective:To observe the inhibitory effect of lentivirus (LV)-mediated miR-191 on the proliferation and angiogenesis of human retinal vascular endothelial cells (hREC) cultured in vitro.Methods:The hREC cell lines were cultured in vitro and divided into control group, hypoxia group, LV-empty vector (LV-vector) group, and LV-miR-191 (LV-191) group. The LV-vector group and LV-191 group were transferred to the corresponding lentiviral vector respectively. Flow cytometry was used to detect cell transfection efficiency. Cell Counting Kit-8 (CCK-8) test was used to detect cell proliferation ability. Scarification test and invasion chamber (Transwell) test were used to detect cell migration ability. Matrigel test was used to detect cell lumen formation ability. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the relative expression of miR-191 and relative mRNA expression of its downstream target genes p21, vascular endothelial growth factor (VEGF), cell division protein kinase (CDK) 6, cyclin-D1 (Cyclin D1). Independent sample t test was used for pairwise comparison. Results:The results of flow cytometry showed that the transfection efficiency of cells in the control group and the LV-191 group were 0.615% and 99.400%, respectively. The results of CCK-8, scarification, Transwell and Matrigel test showed that, compared with the control group, the number of cell proliferation ( t=6.130, 4.606), the cell mobility ( t=4.910, 6.702), the number of stained cells on the microporous membrane ( t=7.244, 6.724) and the lumen formation ability cells ( t=8.345, 9.859) were significantly increased in the hypoxia group and the LV-vector group ( P<0.01), while the LV-191 group showed completely opposite performance ( t=14.710, 6.245, 5.333, 5.892; P≤0.01). The qPCR test results showed that, compared with the control group and the LV-vector group, the relative expression of miR-191 mRNA in the cells of the LV-191 group was significantly up-regulated ( t=44.110, 42.680), the relative expression of Cyclin D1 mRNA ( t=29.940, 14.010) and CDK6 mRNA ( t=15.200, 7.645) decreased significantly, and the difference were statistically significant ( P<0.01); the relative expression of p21 mRNA increased, however, the difference was not statistically significant ( t=2.013, 2.755; P>0.05). There was no significant difference in the relative expression of VEGF mRNA in the 4 groups of cells ( F=0.966, P>0.05). Conclusions:LV-191 can inhibit the proliferation, migration and tubing of hREC by up-regulating p21 and down-regulating CDK6 and Cyclin D1.