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    Neurobiology
     Triggering receptor expressed on myeloid cell-2 modulating the polarization of mouse M2 microglia with oxygen-glucose deprivation/re-oxygenation model
    XU Hong-bei LUO Yong
    2021, 52 (3):  329-336.  doi: 10.16098/j.issn.0529-1356.2021.03.001
    Abstract ( )   PDF (6868KB) ( )  
    Objective  To  investigate the mechanism of TREM2 modulating the polarization of M2 microglia treated by oxygen-glucose deprivation/reoxygenation (OGD/R).   Methods  Mouse N9 microglial cells were cultured in vitro. N9 cells were transfected with lentivirus for TREM-2-overexpression (LV-TREM2), and LV-scramble acted as control group. OGD/R model was established. The OGD/R cells were randomly divided into OGD/R, OGD/R+LV-scramble and OGD/R+LV-TREM2 groups. Real-time PCR was used to detect the expression of TREM2 mRNA in OGD/R N9 cells within 72 hours after re-oxygenation. Immunofluorescence was applied to observe transfection of lentivirus LV-scramble and LV-TREM2 for normal N9 microglia, and Real-time PCR and Western blotting were used to verify the efficiency of lentivirus transfection. The mRNA and protein contents of M1 microglial markers tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS), M2 microglial markers arginase-1 (Arg-1) and interleukin-10 (IL-10) were detected by Real-time PCR and ELISA. The expressions of phosphorylated-phosphatidylinositol 3-kinases (p-PI3K), PI3K, phosphorylated protein kinase B (p-Akt), Akt, phosphorylated inhibitor of nuclear factor-κB (NF-κB)α (p-ⅠκBα) and ⅠκBα protein were detected by Western blotting. The distribution of NF-κB P65 (NF-κB P65) protein in N9 cells was analyzed by immunofluorescence method.    Results  TREM2 mRNA content in the OGD/R group cells increased significantly within 72 hours after re-oxygenation, and peaked at hour 24 and hour 48. Lentivirus LV-TREM2 effectively promoted the expression of TREM2 mRNA and protein of N9 cells in OGD/R model (P<0.001, P<0.01). Compared with the OGD/R group, the mRNA and protein content of TNF-α, IL-1β and iNOS decreased significantly, while Arg-1 and IL-10 in OGD/R+LV-TREM2 group increased  significantly(P<0.05). Besides, the ratios of p-PI3K/PI3K and p-Akt/Akt increased  obviously (P<0.05), the ratio of p-ⅠκBα/ⅠκBα  decreased significantly in OGD/R+LV-TREM2 group (P<0.001), and the nuclear translocation of NF-κB P65 protein was obviously weakened.   Conclusion TREM-2 overexpression exerts anti-inflammatory effect by modulating the polarization of microglia from M1 to M2 type, which is associated with PI3K/Akt and NF-κB signaling pathways regulated by TREM2 in N9 microglia with OGD/R model.
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     Acupoint catgut embedding inhibiting neuroinflammation in vascular dementia rats
    ZHU Shi-jie SUN Qiu-ying TANG Zhong-sheng LUO Ya-fei XIE Gao-yu WU Chun-peng KOU Yun-fang FAN Rui-juan
    2021, 52 (3):  337-343.  doi: 10.16098/j.issn.0529-1356.2021.03.002
    Abstract ( )   PDF (5236KB) ( )  
    Objective  To observe the effect of acupoint catgut embedding on the expression of inflammatory factor mRNA in cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) signal pathway of vascular dementia (VD) rats, and to explore the protective mechanism of acupoint catgut embedding on the brain inflammatory response of VD rats.    Methods  VD model was established by the modified Pulsinelli’s four vessel blocking method . Totally 148 male rats were randomly divided into VD model group, non acupoint catgut embedding group and acupoint catgut embedding group. On the 7th day after operation, catgut embedding at acupoints and catgut embedding at non acupoints were performed in the two treatment groups respectively, and materials were taken out 15 days later. Western blotting was used to detect the expression of COX-2 and PGE2, and real-time PCR was used to detect the mRNA expression of tumor necrosis factor α(TNF-α), intercellular cell adhesion molecule 1 (ICAM-1), interleukin(IL)-6, macrophage inflammatory protein 2 (MIP-2), IL-1β, and monocyte chemotactic protein 1 (MCP-1) in rat hippocampus.   Results Compared with the sham group, the expressions of COX-2, PGE2, TNF-α, ICAM-1, IL-6, MIP-2, IL-1β and MCP-1 in hippocampus of the other three groups were significantly higher (P<0.01). Compared with the model group, the expressions of COX-2, PGE2 protein and TNF-α, ICAM-1, IL-6, MIP-2, IL-1β, MCP-1 mRNA in the hippocampus of the acupoint catgut embedding group and the non acupoint catgut embedding group decreased significantly (P<0.01).   Conclusion  Acupoint catgut embedding can protect the brain from inflammatory injury by down-regulating the expression of related inflammatory factors in COX-2/PGE2 signaling pathway and reducing the inflammatory response induced by VD rats.
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     Intrauterine hypoxia slowing the development of Purkinje cells and reducing the expression of cerebellin in mice
    XU Ji-wei YANG Chang-qing CHEN Xiao-ping CHEN Xu-dong FAN Wen-juan
    2021, 52 (3):  344-351.  doi: 10.16098/j.issn.0529-1356.2021.03.003
    Abstract ( )   PDF (15889KB) ( )  
    Objective  To investigate the effects of the development of Purkinje cells and expression of cerebellin in postnatal mice with intrauterine  hypoxia.    Methods  Twenty healthyice were randomly divided into two groups: control group and hypoxia group, with 10 mice in each group. Mice in the hypoxia group were placed in the hypoxia chamber of the animal since the 14th day of gestation to make an animal model of intrauterine hypoxia. After the mother gave birth, the experimental animals were divided into hypoxia group and control group. There were 6 age groups including postnatal day  (P)0, P5, P9, P14, P21 and P30 in each group, and 5 mice in each age group. The cerebellum tissue was taken for vibrating sectioning. The developmental changes of calbindin-positive Purkinje cells were detected by immunofluorescence technique. The expression of cerebellin in Purkinje cell protuberances was detected by cerebellin (CBLN)1, CBLN4 and calbindin double labeling. Finally, Western blotting was used to semiquantitatively analyze the protein expression of cerebellar peptide in cerebellum at each time point.   Results  Compared with the control group of the same age, the number of cerebellar Purkinje cells in the hypoxic group decreased, the dendritic branches decreased, and the arrangement was disordered, and the expression of CBLN1 and CBLN4 in the cortex were significantly reduced.    Conclusion  Intrauterine hypoxia leads to abnormal development of the cerebellar Purkinje cells and synaptic changes.
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    Relationship between complete Freund’s adjuvant-induced chronic pain-induced negative emotions and morphological changes of hippocampal microglia
    LI Wei LIU Zhi-wen ZENG Jia-yu ZENG Xu-qing YUAN Jing ZHONG Xiao-lin WAN Wei
    2021, 52 (3):  352-357.  doi: 10.16098/j.issn.0529-1356.2021.03.004
    Abstract ( )   PDF (2183KB) ( )  
    Objective  To investigate the alteration of mood and hippocampal microglia morphology in a mouse model of chronic inflammatory pain induced by complete Freund’s adjuvant (CFA).    Methods  Thirty-two male ICR mice were randomly divided into two groups, including normal saline control group (NS) and CFA model group (CFA). The pain model was established by right hindpaw intraplantar CFA injection. The change of mechanical pain threshold after CFA injection was measured by von Frey fiber needle, the locomotor activity and anxiety-like behavior were determined by open field test (OFT), the depression-like behavior was determined by sucrose preference test (SPT) and forced swimming test (FST). The expression of microglia marker ionized calcium binding adaptor molecule-1 (IBA-1) in the hippocampus was determined by immunohistochemistry and its morphological change was analyzed by Sholl analysis.   Results  Compared with the NS group, the mechanical pain threshold of CFA group decreased significantly (P<0.01). The behavior result  showed that the CFA group showed remarkably reduced time in the inner area (P<0.01) compared with the NS group in the open field test;In the sucrose preference test, the percentage of sucrose preference (P<0.01) of CFA mice decreased significantly compared with the NS mice, while the immobility time of CFA mice (P<0.01) increased significantly in the forced swimming test compared with the NS mice. The immunohistochemistry showed that the number of microglia in the dentate gyrus (DG) of CFA mice increased significantly compared with the NS mice. The Sholl analysis result  showed that compared with the NS mice, the number of intersections of microglia in hippocampal DG decreased significantly in CFA mice.   Conclusion  Our finding  indicates that the negative emotions in CFA-induced chronic inflammatory pain may be related to the morphological changes of hippocampal microglia in the mice.
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    Expression of phosphatidylinositol 3-kinase/protein kinase B/FoxO1 signal pathway and interleukin-17 in experimental autoimmune encephalomyelitis in mice
    LI Qian GAO Jie HU Rong HAN Feng LI Hong SU Min
    2021, 52 (3):  358-364.  doi: 10.16098/j.issn.0529-1356.2021.03.005
    Abstract ( )   PDF (8136KB) ( )  
    Objective  To investigate the relevant mechanisms of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/FoxO1 and interleukin-17(IL-17) in the oneset of experimental autoimmune encephalomyelitis(EAE) mice.    Methods   Sixty C57BL/6 mice were randomly divided into control group and model group (EAE), 30 in each group. The EAE model was induced by myelin oligodendrocyte glycoprotein (MOG35-55) together with complete Freund’s adjuvant. The behavioral score of each group was observed. The spinal cord,spleen and peripheral blood were obtained when the behavioral score was 4. IL-17 and interferon-γ (IFN-γ) contents in mouse serum and supernatant of splenocytes stimulated with a-CD3 and MOG35-55,respectively,in vitro for 7 days were detected by ELISA. The percentage of CD4+IL-17+ cells in the mouse spleen was monitored via flow cytometry. The expressions of IL-17 and PI3K/Akt/FoxO1 in the spinal cord were detected by Western blotting.    Results Compared with the control group, the behavioral score of the EAE group was significantly higher than that of the control group. HE staining and Luxol fast blue staining indicated that EAE spinal cord increased prominently inflammatory infiltration and demyelination(P<0.05). IL-17 and IFN-γ contents of the EAE group in serum and supernatant of splenocytes cultured in vitro increased remarkably(P<0.05). The percentage of  CD4+IL-17+ T cells in splenocytes also increased markedly(P<0.05). The protein levels of IL-17 and phosphorylated Akt (p-Akt)in the spinal cord of the EAE group increased observably,while that of phosphorylated FoxO1 (p-FoxO1)decreased significantly(P<0.05).   Conclusion  The increased secretion of proinflammatory factor IL-17 in the spinal cord of the EAE group may be related to the activation of the PI3K/Akt/FoxO1 signaling pathway and T-cell function.
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    Effects of up-regulation of zinc finger and BTB domain containing 20 expression on learning and memory of APPswe/PSE9 transgenic Alzheimer disease mice
    WANG Yue-jing ZHANG Meng LANG Wei-ya ZHANG Hai-yan LIAN Jie FENG Hua-jie SUN Li-hui
    2021, 52 (3):  365-369.  doi: 10.16098/j.issn.0529-1356.2021.03.006
    Abstract ( )   PDF (3917KB) ( )  
    Objective  To investigate the effects of up-regulation of zinc finger and BTB domain containing 20(A20)expression on learning and memory and apoptosis of hippocampal neurons in APPswe/PSE9 transgenic Alzheimer disease(AD)mice.    Methods  Forty APPswe/PSE9 transgenic mice were divided into four groups: control group, AD group, small interfering RNA(siRNA)-AD group and A20-AD group. Adenoviruses carrying high expression of A20 were selected as interfering drugs and Morris water maze was used to observe spatial learning and memory. Western blotting was used to detect the expression of A20. Immunohistochemistry was used to observe the expression of P53 in the CA1 region.    Results  Compared with the AD group, the learning and memory function was mproved, the apoptosis rate of hippocampal CA1 neurons decreased significantly and the expression of P53 in the CA1 region decreased  significantly in A20-AD group mice.    Conclusion  Upregulation of A20 expression can improve the learning and memory ability of transgenic mice, reduce the expression of P53 in hippocampal CA1 region of the transgenic mice.
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    Edaravone intervening the proliferation and differentiation of  neural stem cells in rats with permanent cerebral ischemia
    LIANG Xiao-yan GU Yu ZHAO Meng DU Xin LIU Hong-wei ZHOU Yang ZHANG Tie-jun
    2021, 52 (3):  370-376.  doi: 10.16098/j.issn.0529-1356.2021.03.007
    Abstract ( )   PDF (6238KB) ( )  
    Objective  To observe the effect of edaravone on endogenous neural stem cells in rats with permanent cerebral ischemia.    Methods  The rat model of permanent cerebral ischemia was established by electrocoagulation. Thirty SD rats were randomly divided into three groups: sham operation group, brain injury group and edaravone group. Six hours after the establishment of the model, the edaravone group was intraperitoneally injected with 1.5 g/L edaravone (10 ml/kg) once a day. The sham operation group and the cerebral ischemia group were intraperitoneally injected with saline of equal volume for 7 days. 24 hours after the last administration, BrdU positive cells, Nestin/BrdU positive cells, neuronal class Ⅲ β-tubulin(Tuj1)/BrdU positive cells and glial fibrillary acidic protein (GFAP)/BrdU positive cells were observed by immunofluorescent staining, Tuj1 and GFAP protein expressions were detected by Western blotting.    Results  Compared with the cerebral ischemia group, the BrdU positive cells, Nestin/BrdU positive cells, Tuj1/BrdU positive cells and GFAP/BrdU positive cells increased significantly in the infraventricular area and the cortex area around the ischemia in the edaravone group (P<0.05).Compared with the cerebral ischemia group, the expression of Tuj1 and GFAP protein in the cerebral cortex of edaravone group increased (P<0.05).    Conclusion  Edaravone can promote the proliferation of endogenous neural stem cells and astrocytes in the subventricular area and the cortex around ischemia, and promote the differentiation of endogenous neural stem cells into neurons.
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    Medical Education
    Application of flipped classroom teaching mode supported by 3D printing model in embryology experiment teaching
    CAO Xin-yuan LIU Run-zhu ZHANG Han-lin CHEN Yong-mei XU Yuan-yuan QIAN Xiao-jing QIU Wen-ying
    2021, 52 (3):  479-484.  doi: 10.16098/j.issn.0529-1356.2021.03.023
    Abstract ( )   PDF (1550KB) ( )  
    Objective  To explore the teaching effect of flipped classroom teaching mode supported by the 3D printing model in the embryology experiment.    Methods  Totally 76 students from class 2016 were randomly divided into two groups: the experimental group previewed the embryo 3D models before class and taught other students in class, while the control group did not preview the embryo 3D models before class. An embryo knowledge test and a questionnaire survey were carried out before and after the experimental class to compare the differences of knowledge mastery and self-evaluation between the two groups.   Results  In the aspect of self-evaluation, the self-evaluation of the experimental group students in explaining the causes and explaining the process of development was significantly improved after the lecture (P<0.05), while the self-evaluation of the control group students in identifying the structure, understanding knowledge, telling the causes and explaining the process of development was significantly improved (P<0.05). In terms of knowledge test scores, the scores of students in the experimental group and the control group after teaching were higher than those before teaching (P<0.05). The improvement of the knowledge test in the control group was significantly higher than that in the experimental group (P<0.05). The experiment group’s grades of knowledge test were significantly higher than the control group in the second experiment. 70.27% of the students thought that they were still lack of knowledge in the introspection of the causes of wrong answers. The misunderstanding of English nouns (15.54%),  psychological factors (5.41%) and other factors (8.78%) also caused the students to lose points in the knowledge tests.    Conclusion  The combination of the 3D printing model and the flipped classroom teaching mode can effectively improve the teaching effect, which should be paid attention to in the reform of embryology experimental teaching.
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    Application of cases-based flipped class in hybrid teaching for neuroanatomy
    MENG Hai-wei DING Zhao-xi FENG Lei LIU Zhen TANG Yu-chun LIU Shu-wei
    2021, 52 (3):  485-488.  doi: 10.16098/j.issn.0529-1356.2021.03.024
    Abstract ( )   PDF (821KB) ( )  
    Objective  To explore the effects of cases-based flipped class in the hybrid teaching for neuroanatomy.    Methods  A comparative study was conducted among 102 students majored in clinical medicine of 2019 grade, one was the experiment group (n=51) and another was the control group (n=51). The traditional teaching method  was applied in control group, while the teaching during neuroanatomy based on cases-based flipped class was applied in experimental group. The teaching effects were evaluated by theory and experiment examination and investigated by the questionnaire of students’ satisfaction with the new teaching mode.    Results  Most students supported the cases-based flipped class teaching and thought it was helpful to improve the autonomous learning ability. The satisfaction rate of experimental group on the cases-based flipped class teaching effectiveness was 98.04%. The total scores, the scores of neuroanatomy and the student number who got 81-90 and 91-100 of experiment group were significantly higher than those of control group, and the difference was statistically significant (P<0.05).   Conclusion  Cases-based flipped class could effectively improve the quality of neuroanatomy teaching and students’ learning ability and effects for undergraduates.
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    Histology,Embryology and Developmental Biology
    Effects of angelica lactone combined with Xiaoke pill on regulation of vascular endothelial growth factor signal pathway and on renal protection in diabetic nephropathy rats
    GUO Yang-zhi DU Juan JIANG Min
    2021, 52 (3):  439-445.  doi: 10.16098/j.issn.0529-1356.2021.03.017
    Abstract ( )   PDF (10851KB) ( )  
    Objective  To investigate the effects of angelica lactone combined with Xiaoke pill on blood glucose regulation, renal protection and vascular endothelial growth factor signal pathway in diabetic nephropathy rats.     Methods  Totally 75 rats were randomly divided into control group, model group, Xiaoke pill group, angelica lactone group and angelica lactone combined with Xiaoke pill group, with 15 rats in each group. Except for the control group, the rest of the rats established diabetic nephropathy model. Rats in Xiaoke pill group, angelica lactone group and angelica lactone combined with Xiaoke pill group were given Xiaoke pill (0.8 g/kg), angelica lactone (20 mg/kg), Xiaoke pill (0.8 g/kg) combined with angelica lactone (20 mg/kg), respectively, once for 8 weeks. The levels of 24-hour urinary protein, fasting and postprandial blood glucose, glycosylated serum albumin and glycosylated hemoglobin, fasting insulin and insulin sensitivity index, serum creatinine (SCr) and urea nitrogen (BUN) were measured. The levels of insulin (INS) and glucose-6-phosphatase (G6Pase) mRNA in islet tissue were measured by Real-time PCR. Renal histopathological changes were detected by HE staining. The levels of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) in rat kidney were detected by Western blotting and immunohistochemistry.    Results  Compared with the model group, 24-hour urinary protein, fasting and postprandial blood glucose, glycosylated serum albumin and glycosylated hemoglobin, fasting insulin, SCr and BUN, VEGF and VEGFR2 protein levels and relative integral absorbance of kidney tissue in angelica lactone combined with Xiaoke pill group were significantly lower than those in model group(P<0.05). The insulin sensitivity index, the levels of INS and G6Pase mRNA in islet tissue were significantly increased(P<0.05), and the renal histopathology of diabetic nephropathy rats was significantly improved.    Conclusion  Angelica lactone combined with Xiaoke pill can reduce blood glucose level, increase insulin sensitivity index, and its mechanism may be related to the regulation of VEGF signal pathway.
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    Effect of excessive caffeine intake on fetal cartilage ossification in female rats during pregnancy and its mechanism
    LIU Kan WANG Qiu-ming SHI Xu-feng TAO Tao WANG Huan-ping WU Hai-ying
    2021, 52 (3):  453-459.  doi: 10.16098/j.issn.0529-1356.2021.03.019
    Abstract ( )   PDF (12530KB) ( )  
    Objective  To investigate the effect of excessive caffeine intake on fetal cartilage ossification in female rats during pregnancy and its mechanism.    Methods  From gestational day(GD) 9 to GD 20, the pregnant Wistar rats in caffeine exposure group were intragastrically administered 120 mg/kg day caffeine, and the control group was administered the same volume of distilled water. The pregnant mice were sacrificed at day 20, and the body length of the fetal mice was measured. The distal femur of fetal rats was isolated, the length of distal femur cartilage was measured, and primary chondrocytes were prepared. The cells were treated with caffeine (0.1, 1 and 10 μmol/L), insulin-like growth factor 1 (IGF-1, 100 μg/L) and extracellular regulated protein kinases(ERK)inhibitor (10 μmol/L), respectively. Then the cells were harvested for apoptosis, gene and protein analysis.    Results  Compared with the control group, the body length and femur length of the fetuses in the caffeine exposed group decreased significantly (P<0.05), and the serum corticosterone levels increased significantly (P<0.05). Immunohistochemical analysis showed that the expressions of IGF-1, proliferating cell nuclear antigen (PCNA) and sex determining region Y box protein 9(SOX9) in mast chondrocyte area of caffeine exposed group were significantly lower than those of control group (P<0.05). In vitro, caffeine treatment reduced the expression of IGF-1, PCNA, SOX9 mRNA and p-ERK protein in primary chondrocytes in a concentration-dependent manner, while exogenous IGF-1 could reversed these changes induced by caffeine, and the effect of exogenous IGF-1 was reduced by ERK inhibitors (all P<0.05).    Conclusion  Prenatal caffeine exposure leads to shortening of the long bones of the fetus and prolongation of the hypertrophy by inhibiting the proliferation of chondrocytes. The IGF-1/MAPK/ERK signaling pathway in chondrocytes may be partially involved in the adverse effects of caffeine on chondrocyte proliferation.
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    EEffect of urantide on the expression of osteopontin and α-smooth muscle actin in the heart of atherosclerotic rats
    LI Ying XIE Li-de WANG Tu ZHAO Juan
    2021, 52 (3):  446-452.  doi: 10.16098/j.issn.0529-1356.2021.03.018
    Abstract ( )   PDF (16382KB) ( )  
    Objective  To observe the effect of urantide on the expression of osteopontin (OPN) and α-smooth muscle actin (α-SMA) in the heart tissue of atherosclerosis (AS) rats, and to explore its mechanism of prevention and treatment of myocardial fibrosis injury in rats.    Methods  Totally 120 3-week-old healthy male Wistar rats in SPF grade were randomly divided into six groups: control group, model group, simvastatin group, urantide (3 days, 7 days, 14 days). HE and Masson trichrome staining were used to observe the morphology of rat heart and the expression of collagen fibers. Immunohistochemistry and Western blotting were used to detect the expression of OPN and α-SMA protein.    Results   In AS model group, cardiomyocyte hypertrophy or atrophy, a large number of inflammatory cell infiltration and a small amount of foam cells were observed in the heart tissue of rats. The increase of collagen fibers and the expression of OPN and α-SMA protein in cardiac tissue were significantly higher than those in the control group. Compared with the AS model group, after urantide treatment, cardiac injury was significantly improved, and the expression of collagen fiber, OPN and α-SMA protein was decreased.    Conclusion  Urantide can inhibit the expression of OPN and α-SMA protein in the heart tissue of AS rats to alleviate myocardial fibrosis and play a protective role in the heart tissue of AS rats.
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    Cell and Molecules Biology
    Targeted quantitative analysis of energy metabolites in the priming phase during rat liver regeneration
    YANG Hui XU Cun-shuan
    2021, 52 (3):  377-383.  doi: 10.16098/j.issn.0529-1356.2021.03.008
    Abstract ( )   PDF (2736KB) ( )  
    Objective  To investigate the regulation of liver regeneration (LR) by changes in energy metabolites in the initiation phase during rat liver regeneration.    Methods  Rats were randomly divided into 3 groups with 5 rats in each group, including two partial hepatectomy (PH) groups and one normal control group. Selective reaction monitoring/multiple reaction monitoring (SRM/MRM) was employed in the targeted metabolomics identification of 29 energy metabolites. Ingenuity Pathway Analysis (IPA) was applied for integration analysis, including canonical pathway and molecular interaction network.    Results  The levels of 3-phospho-D-glycerate, AMP, cyclic AMP, D-fructose 1, 6-bisphosphate, dihydroxyacetome phosphate(DHAP), guanosine monophosphate(GMP), guanosine triphosphate(GTP), nicotinamide adenine dinucleotide(NAD) and nicotinamide adenine dinueleotide phosphate(NADP) significantly increased. The levels of alpha-ketoglutarate, beta-D-fructose 6-phosphate, cis-aconitate, D-glucose 6-phosphate, lactate, NADPH, oxaloacetate and pyruvate dramatically reduced. Through hierarchical clustering analysis of energy metabolisms, these energy metabolisms can be grouped into four clusters. IPA showed that the biomolecular changes in the priming phase of liver regeneration are mainly related to carbohydrate metabolism, cellular growth and proliferation, and organismal development. During the priming phase of liver regeneration, adenosine 5’-monphosphate-activated protein kinase (AMPK), hypoxia-inducible factor 1α (HIF-1α), peroxisome proliferator-activated receptor (PPAR), protein kinase A (PKA) and phosphatid  linositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathways are involved in energy metabolism, and glycolysis may be the main mode of energy supply.    Conclusion  The result  suggests that the changes of energy matabolites during the initial stage of LR play a regulatory role in live regeneration.
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    Expression and localization of Copine-3-enhanced green fluorescent protein fusion protein
    LI You XU Li-ming ZHANG Qi-min YANG Su-fang LU Chun-hua
    2021, 52 (3):  384-390.  doi: 10.16098/j.issn.0529-1356.2021.03.009
    Abstract ( )   PDF (11725KB) ( )  
    Objective  To construct the eukaryotic expression vector plasmid enhanced green fluorescent protein(pEGFP)-N1-CPNE3, and identify the expression and localization of Copine-3 protein in cells.    Methods  The Copine-3 coding sequences (CPNE3) was amplified by RT-PCR from human bronchial epithelial (HBE) cells and inserted into eukaryotic expression vector pEGFP-N1. The recombinant plasmid pEGFP-N1-CPNE3 was confirmed by endonuclease digestion and sequencing before it was transfected into 293T and H1299 cells. Cellular localization of Copine-3-EGFP fusion protein was detected by con-focal laser scanning. Expression of Copine-3 in 293T and H1299 cells was detected by Western blotting analysis. Localization of Copine-3 in clinical samples of the lung adenocarcinoma patients was detected by immunohistochemistry.    Results  CPNE3 was successfully constructed into the eukaryotic expression vector pEGFP-N1 and expressed in 293T and H1299 cells. Furthermore, the location of Copine-3 protein in cytoplasm and nucleus was determined by immunofluorescence staining, immuno Western blotting and immunohistochemistry in those cells and clinical samples.    Conclusion  The eukaryotic expression vector pEGFP-N1-CPNE3 is constructed successfully, and Copine-3 protein is localized in cytoplasm and nucleus.
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    Screening and identification the hub genes of cardiac hypertrophy in mice
    SHI Zhuo LI Jia-hui GAO Jing DONG Yan-li REN Fu WEN Pu-shuai
    2021, 52 (3):  391-397.  doi: 10.16098/j.issn.0529-1356.2021.03.010
    Abstract ( )   PDF (6956KB) ( )  
    Objective  To screen and identify  the hub genes  closely related to cardiac hypertrophy by using bioinformaticsmethod  and biological experiments.   Methods  The chip data related to cardiac hypertrophy in mice were downloaded from the Gene Expression Omnibus(GEO) database, and the GEO2R online tool was adopted to screen for differentially expressed genes; DAVID 6.7, String 11.0 and Cytoscape 3.7.0 softwares were used to analyze differentially expressed genes; Kunming mice were randomly divided into a normal saline group (n=6) and an angiotensin Ⅱ (Ang Ⅱ) group (n=6) to establish a cardiac hypertrophy model, the expression of  hub genes in Kunming mouse model of cardiac hypertrophy induced by AngⅡ was detected by Real-time PCR method .    Results  A total of 202 common differentially expressed genes and 12 hub genes were selected; the Real-time PCR result  demonstrated that decorin(Dcn), HADHA and heat shock protein(HSP)90αA1 were significantly down-regulated in the AngⅡ group.    Conclusion  The selected hub genes can influence the development of cardiac hypertrophy in Kunming mice through extracellular matrix and transforming growth factor β(TGF-β).
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    Effect of neurogenin 3 overexpression on the differentiation of human umbilical cord mesenchymal stem cells into insulin-producing cells
    KONG Yin-hao XU Long-fei WANG Qi HAN Jing HONG Yan
    2021, 52 (3):  398-404.  doi: 10.16098/j.issn.0529-1356.2021.03.011
    Abstract ( )   PDF (11008KB) ( )  
    Objective  To investigate whether the overexpression of neurogenin 3(Ngn3)can promote the induced differentiation of human umbilical  cord mesenchymal stem cells(HUCMSCs)into insulinproducing cells(IPCs).    Methods  HUCMSCs were isolated and cultured, identified by flow cytometry, and the differentiation potential was identified by adipogenesis and osteogenesis induction. HUCMSCs were induced into IPCs in different stages, including a low glucose induction stage and a high glucose induction stage. The experiment was divided into two groups, the control group was induced by the above scheme, while the experimental group was additionally infected the lentivirus overexpression vector carrying the target gene Ngn3 on the 6th day of the same induction process. After induction, the changes of cell structure were observed by electron microscope; mRNA and protein was collected and Real-time PCR and Western blotting were used to compare the expressions of insulin and musculoaponeurotic fibrosarcoma oncogene homolog A Mafa in the two groups; medium supernatant was collected and C-peptide content was determined by ELISA.   Results  HUCMSCs were successfully isolated with positive expression of CD105 and CD90 and negative expression of CD34 and CD45, which had adipogenic and osteogenic differentiation ability. Then, HUCMSCs were induced into IPCs by stage induction, and the cells expressed Mafa and insulin positively. Ngn3 was overexpressed in the experimental group during the induction. After induction, electron microscopy showed that the cell structure was more mature in the experimental group. The expression levels of insulin and Mafa in the experimental group were significantly higher than those in the control group. During the induction process, the amount of C-peptide secreted by the experimental group was higher than that of the control group.    Conclusion  Lentivirus-mediated Ngn3 overexpression improves the differentiation efficiency of HUCMSCs into IPCs.
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    Review
    Distribution of Nogo in the heart and its role in cardiovascular disease
    ZHANG Su-xin SUN Yan-rong WANG Wen-juan SUN Hao-zhe HU Yao-wen QIN Lin-hua
    2021, 52 (3):  495-498.  doi: 10.16098/j.issn.0529-1356.2021.03.026
    Abstract ( )   PDF (836KB) ( )  
    As a member of the reticulin family, Nogo is mainly involved in processes such as tissue regeneration, apoptosis and tumor growth after tissue injury. Cardiovascular disease is one of the main diseases that threaten human health at present. In recent years, research on Nogo in the cardiovascular system has become increasingly extensive. Changes in the expression of Nogo during myocardial fibrosis, myocardial cell apoptosis and vascular remodeling suggest that it may play a certain role. This article reviews the distribution of Nogo in the heart and its role in cardiovascular disease, in order to reveal its possible role and mechanism in cardiovascular diseases.
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    Suppressing and activating effects of Nogo-B receptors in the pathogenesis of malignant solid tumors
    SU Hao REN Jie QIN Li-hua
    2021, 52 (3):  489-494.  doi: 10.16098/j.issn.0529-1356.2021.03.025
    Abstract ( )   PDF (1587KB) ( )  
    Nogo-B is a major family member of the reticulon protein family 4. It is widely expressed in the central nervous system and peripheral tissues. Studies have shown that Nogo-B binds to three different receptors: Nogo receptor-1 (NgR1), Nogo-B specific receptor(NgBR) and  paired immunoglobulin like receptor B(PirB). These receptors play a dual role of suppression and promotion in angiogenesis, proliferation and apoptosis, invasion and migration, which are important events in tumor development and progression, through various post-receptor signaling pathways, including RhoA/Rho-associated coiled-coil contaning protein kinase(RhoA/ROCK), phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt), adenosine 5-monophosphate-activated protein kinase α/liver X receptor α(AMPAα/LXRα), extracellular signal-regulated kinase(ERK), epithelial-mesenchymal transition(EMT), unfolded protein response(UPR) and so on. An in-depth understanding of the mechanisms by which Nogo-B receptors are involved in tumor pathogene is will provide new insights into the development of drugs. Here, we will summarize the up-to-date researches on the basic structure and expression of Nogo-B/Nogo-B receptors and the suppressing/activating effects of post-receptor signaling pathways in the pathogenesis of malignant solid tumors.
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    Anthropology
    Correlation between serum angiotensin converting enzyme and angiotensinogen levels and pregnancy-induced hypertension and analysis of risk factors
    WANG Ru ZHUANG Wen-ting WANG Xiang-lin LI Hong-rong LI Chang-xing KONG De-xia LI Jian-hua
    2021, 52 (3):  460-464.  doi: 10.16098/j.issn.0529-1356.2021.03.020
    Abstract ( )   PDF (870KB) ( )  
    Objective  To investigate the relationship between serum angiotensin converting enzyme (ACE) and angiotensinogen (AGT) and gestational hypertension syndrome (HDCP) and the risk factors of HDCP.    Methods  A total of 135 pregnant hypertensive patients (HDCP group) and 100 normal pregnant women as control check (CK) group were selected. Serum ACE and AGT levels were measured by ELISA, and correlation analysis was performed. The age and gestational age of the two groups, prepregnancy body mass index (BMI), parity, number of births, family history of hypertension, family history, education, and other general information, single factor analysis of risk factors for maternal HDCP, single factor regression analysis Statistically significant factors were all used for multivariate logistic regression analysis.    Results  The serum ACE level in the HDCP group (90.49±47.65)μg/L was significantly higher than that in the CK group (58.72±27.58)μg/L, P<0.05, the difference was statistically significant. The serum AGT level in the HDCP group was (64.57±19.71)μg/L was higher than CK group (58.22±18.64)μg/L, P>0.05, the difference was not statistically significant; single factor analysis showed: age, BMI, hypertension, family history of diabetes, ACE level was maternal (P<0.05), while gestational age, parity, number of births, and education were no significant differences in risk factors for HDCP  (P>0.05). Multivariate analysis showed: age, BMI, history of hypertension and ACE. It was a risk factor for pregnancy-induced hypertension.    Conclusion  ACE levels are associated with HDCP. AGT levels are not associated with HDCP. Patients with a high age, high BMI, and hypertension history have an increased risk of gestational hypertension syndrome.
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    Somatotype characteristics of Dong nationality adults in Hu’nan Province
    ZHANG Hui-juan WANG Yi-ming LIAO Wei-cheng CHEN Xi-wen QIN Da-bao HUANG Da-yuan
    2021, 52 (3):  465-472.  doi: 10.16098/j.issn.0529-1356.2021.03.021
    Abstract ( )   PDF (1172KB) ( )  
    Objective  To investigate the somatotype characteristics of Dong adults in rural areas of Hu’nan province.    Methods  The Heath-Carter somatotyping method  was used to study the somatotype of 749 adults (304 males and 445 females) of Dong in the rural areas of Hu’nan.    Results  The mean somatotype in males of Dong was endomorph-mesomorph category (4.83-4.95-1.80) and was mesomorphic endomorph category (6.09-4.68-1.37) in females. With their age increasing, the values of endomorphy decreases gradually in males, the values of endomorphy of females and mesomorphy of males and females increased at first and then decreased gradually, the values of ectomorphy of males and females decreased at first and then increased. The values of endomorphy of males were significantly lower than those of females with the same age groups, and the values of mesomorphy and the values of ectomorphy of males were significantly higher than those of females in some age groups. Compared with other ethnic groups, the values of endomorphy of Dong were larger, the values of mesomorphy were smaller, and the values of ectomorphy were in a middle level.    Conclusion  Adults of Dong in Hu’nan have thick subcutaneous fat, underdeveloped skeletal and muscular systems, and medium linearity. The somatotype of Dong adults in Hu’nan is close to that of Jiangsu Han, Fujian Han and Liaoning Han.
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    Relationship between exercise and bone strength, body composition, sex hormones in postmenopausal women
    YE Zhen-zhen YI Jian-feng PAN Jian-xi
    2021, 52 (3):  473-478.  doi: 10.16098/j.issn.0529-1356.2021.03.022
    Abstract ( )   PDF (884KB) ( )  
    Objective  To investigate the effects of exercise on bone strength, body composition, sex hormones and their relationship in postmenopausal women of Han nationality in Lanzhou.    Methods  From Jan. 2018 to Jun. 2019, 233 cases postmenopausal women of Han nationality in Lanzhou (110 cases in exercise group and 123 cases in non exercise group) were selected by stratified random sampling method , whose bone strength, body composition indexes and sex hormone were measured by ultrasonic bone mineral density meter, body composition analyzer and electrochemiluminescence automatic immune analyzer, respectively.    Results  There were lower body weight, body mass index and fat tissue composition of postmenopausal women of Lanzhou Han nationality (P<0.05), and there was higher bone strength, estradiol and muscle tissue composition in the exercise group (P<0.01). The prevalence of osteoporosis and obesity was lower in the exercise group (P<0.01). Pearson correlation analysis showed that estradiol and muscle tissue composition were positively correlated with the bone strength (P<0.05), and it was negatively correlated with fat tissue composition in postmenopausal women (P<0.01). Logistic regression analysis showed that limb muscle mass and estradiol were protective factors for bone, and visceral fat content was the risk factor of bone abnormality in postmenopausal non-exercise women. Estrogen was the protective factor of bone in postmenopausal exercise women.    Conclusion  The bone strength of postmenopausal women is determined by muscle and fat tissue, and the relationship between the both is affected by exercise. Exercise could effectively prevent and control osteoporosis in postmenopausal women of Lanzhou Han nationality by promoting estrogen production, increasing limb muscle and reducing visceral fat mass.
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    Cell and Molecules Biology
    Effects of microRNA-4286 on the growth, migration and invasion of gastric cancer cell line HGC-27 by regulating inositol polyphosphate-4-phosphatase type Ⅰ
    LIU Dong-tao YANG Zhi-juan DING Bo MA Jun-wen
    2021, 52 (3):  405-409.  doi: 10.16098/j.issn.0529-1356.2021.03.012
    Abstract ( )   PDF (3425KB) ( )  
    Objective  To investigate the effects of microRNA-4286 (miRNA-4286) on the growth, migration and invasion of gastric cancer cell line HGC-27 by regulating inositol polyphosphate-4-phosphatase type Ⅰ (INPP4A).    Methods  HGC-27 cells were divided into blank control group, negative control group and intervention group. The blank control group received no treatment, negative control group was transfected with pEGFP-N1 plasmid, and intervention group was transfected with pEGFP-N1-miRNA-4286 plasmid. Real-time PCR was used to detect the expression of miRNA-4286 in HGC-27 cells, and Western blotting was used to detect the expression of INPP4A protein in HGC-27 cells. The proliferation rate, invasion ability and migration ability of HGC-27 cells were detected by cholecystokinin octapeptide, cholecystokinin octapeptide(CCK8), Transwell chamber assay and scratch test, respectively.    Results  The miRNA-4286 expression level, proliferation rate, and number of transmembrane cells in intervention group were significantly higher than those in blank control group and negative control group (P<0.05). The expression level of INPP4A protein and the scratch healing ability of intervention group were significantly lower than those of blank control group and negative control group (P<0.05). The miRNA-4286 expression, INPP4A protein expression, proliferation rate, number of transmembrane cells, and the scratch healing ability of negative control group had no significant difference with those of blank control group (P>0.05).    Conclusion  miRNA-4286 may promote the growth, migration and invasion of gastric cancer cell line HGC-27 by down-regulating the expression of INPP4A.
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    Effect of small ubiquitin-like modifier 1 pseudogene 3 on the proliferation and apoptosis of non-small cell lung cancer cell line H1299 through protein kinase B signaling pathway
    XU Liang YANG Jian-ye YANG Fei-yan YANG Guo-biao
    2021, 52 (3):  410-417.  doi: 10.16098/j.issn.0529-1356.2021.03.013
    Abstract ( )   PDF (9132KB) ( )  
    Objective  To investigate the effect of long noncoding RNA (lncRNA) small ubiquitin-like modifier 1 pseudogene 3(SUMO1P3) on the proliferation and apoptosis of non-small cell lung cancer cell line 1299.   Methods  Determination of SUMO1P3 expression in non-small cell lung cancer cells by Real-time PCR. SUMO1P3 small interfering RNA(siRNA) was transfected into H1299 cells, the down regulation effect was determined by Real-time PCR.Cell proliferation was measured by MTT, 5-ethynyl-2’-deoxyuridine(EdU) method, the cell cycle was determined by PI single staining, apoptosis was detected by annexin Ⅴ-FITC/PI, detection of apoptosis by TUNEL,Western blotting was used to detect the expression of cleaved Caspase-3(c-Caspase-3), cyclin D1, P27, phosphorylated phospoinositide 3-kinase (p-PI3K)and phosphorylated protein kinase B(p-Akt). Akt signal activator treated H1299 cells transfected with SUMO1P3 siRNA, cell proliferation, apoptosis and cycle change were also measured by the above methods. The number of samples was 9.    Results  SUMO1P3 was up-regulated in non-small cell lung cancer cells. The expression of SUMO1P3 in H1299 cells decreased after transfection with SUMO1P3 siRNA, cell proliferation decreased, the ratio of G0/G1 phase increased, apoptosis rate increased, c-Caspase-3 and P27 protein in the cells increased, the protein levels of cyclin D1, p-PI3K and p-Akt decreased. Akt signal activator could reverse the inhibition of proliferation, cycle arrest and apoptosis of H1299 cells by SUMO1P3 siRNA.    Conclusion  Down-regulation of SUMO1P3 inhibits the proliferation of nonsmall cell lung cancer H1299 cells and induces apoptosis, the mechanism of action is related to the reduction of the activation level of the Akt signaling pathway.
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    Cancer Biology
    Expression and clinical significance of visfatin, hsamiR-199a and extracellular signal regulating protein kinase 1/2 in colorectal cancer
    LIU Ting YU Xiu-wen LIU De-shui XU Cheng LI Xue-song RONG Wei
    2021, 52 (3):  418-424.  doi: 10.16098/j.issn.0529-1356.2021.03.014
    Abstract ( )   PDF (5578KB) ( )  
    Objective  To investigate the expression and correlation of visfatin, hsa-miR-199a and extracellular regulated protein kinase 1/2 (ERK1/2) in colorectal cancer (CRC) tissues.    Methods  The expression levels of visfatin mRNA and hsa-miR-199a were detected by Real-time PCR in 64 primary cases of radical resection of colorectal cancer who underwent pathological clinical examination in the clinicopathological examination in our hospital center from January 2017 to December 2017, the protein expression levels of visfatin and ERK1/2 were detected by immunohistochemistry and Western blotting. The relative expression levels of hsa-miR-199a in each group were compared according to the expression levels of visfatin and ERK1/2.   Results  The levels of visfatin gene and protein in colorectal cancer tissue(observation group, 64 cases) were higher than those in normal colorectal cancer(control group, 64 cases), and the expression was consistent. The colorectal cancer tissue(observation group) expression level of hsa-miR-199a was lower than that of normal colorectal tissue(control group), and visfatin mRNA was significantly negatively correlated. The colorectal cancer tissue(observation group) expression of ERK1/2 was higher than normal colorectal tissue(control group) and visfatin was significantly positively correlated. The relative expression of hsa-miR-199a in the visfatin and ERK1/2 positive group was significantly different from that in the test variables.    Conclusion  The overexpression of visfatin in colorectal cancer affects the down-regulation of hsa-miR-199a and is related to the ERK signaling pathway.
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    Expression of SOX4 gene and its biological effects in endometrial carcinoma
    LIU Jia-shu FAN Bo HUANG Jin LIU Sheng-feng
    2021, 52 (3):  425-431.  doi: 10.16098/j.issn.0529-1356.2021.03.015
    Abstract ( )   PDF (6924KB) ( )  
    Objective  To investigate the expression of sex determining region Y box protein 4(SOX4) gene and its biological effects in endometrial carcinoma.    Methods  156 cases of endometrial carcinoma tissues, adjacent tissues of endometrial carcinoma and 156 cases of endometrial atypical hyperplasia were collected; Immunohistochemistry was used to detect the expression of SOX4 in endometrial cancer, endometrial dysplasia and normal endometrial tissue, and to analyze the relationship between SOX4 and the clinical characteristics of patients with endometrial cancer. After establishing the SOX4 overexpression/silencing Ishikawa cell strain using the lentivirus transfection technique, MTT, flow cytometry and Transwell chamber method  were used to detect the cell proliferation, apoptosis, migration and invasion ability, and Western blotting method  was used to detect SOX4, βcatenin and E-cadherin protein expression changes.     Results  The expression of SOX4 in endometrial cancer tissue was higher than that in normal endometrial tissue and endometrial atypical hyperplasia (P<0.05). SOX4 expression was correlated with invasion depth, International Federation of Obstetrics and Gynecoogy(FIGO) stage and lymph node metastasis (P<0.05). Compared with control group, SOX4 overexpressed Ishikawa cells have significantly increased proliferative ability, migration and invasion ability, significantly reduced apoptosis rate, significantly increased SOX4 and β-catenin protein expression, and significantly reduced E-cadherin protein expression (all P<0.05). while the proliferation ability, migration and invasion ability of Ishikawa cells interfered by SOX4 were significantly reduced, the apoptosis rate was significantly increased, the expression of SOX4 and β-catenin protein was significantly reduced, and the expression of E-cadherin protein was significantly increased (all P<0.05).     Conclusion  SOX4 gene is highly expressed in endometrial cancer tissues, which may promote the development of endometrial cancer by activating Wnt/β-catenin signaling pathway.
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    MicroRNA-98-5p targeting ribonucleotide reductase small subunit M2 regulating cisplatin resistance in cervical cancer cells
    ZHANG Xia JIANG Zhi-ming CHENG Xiao-yan XIANG Yan ZHAO Xiao HUANG Yu-ping YU Hao
    2021, 52 (3):  432-438.  doi: 10.16098/j.issn.0529-1356.2021.03.016
    Abstract ( )   PDF (4237KB) ( )  
    Objective  To investigate the regulation and mechanism of microRNA(miR)-98-5p on cisplatin sensitivity in cisplatinresistant cervical cancer cells.    Methods  The cisplatin(DDP)+miR-NC group (transfected miR-NC), DDP+miR-98-5p group (transfected miR-98-5p mimics), DDP+si-NC group (transfected si-NC), DDP+si-ribonucleotide reductase subunit M2(RRM2) group (transfected si-RRM2), DDP+miR98-5p+pcDNA group (co-transfected miR-98-5p mimics and pcDNA), DDP+miR-98-5p+pcDNA-RRM2 group (co-transfected miR-98-5p mimics and pcDNA-RRM2) were transfected into HeLa/DDP cells by liposome method . Real-tim PCR, Western blotting, CCK-8, Transwell chamber and dual luciferase reports gene detection assay were used to detect the expression of miR-98-5p, RRM2, cyclin D1, P21, matrix metalloproteinase(MMP)-2 and MMP-9 in cells, inhibition rate, half inhibitory concentration(IC50), migration and invasion and fluorescence activity.    Results  Compared with the HeLa group, the expression of miR-98-5p was significantly decreased in HeLa/DDP group, the expression of RRM2 was significantly increased, the IC50 value was significantly increased (P<0.05). Overexpression miR-98-5p or inhibition RRM2 could significantly inhibit the proliferation, migration and invasion of HeLa/DDP cells, down-regulated proteins expression of cyclin D1, MMP-2 and MMP-9, and up-regulated proteins expression of P21; miR-98-5p inhibited the fluorescence activity of wild-type RRM2 cells, and overexpress RRM2 could reverse the inhibitory effect of miR-98-5p on proliferation, migration and invasion of HeLa/DDP cells.    Conclusion  miR-98-5p can inhibit the proliferation, migration and invasion of cisplatin-resistant cervical cancer cells and enhance the sensitivity to cisplatin. The mechanism is related to the targeting of RRM2, which will provide directions for the treatment of cisplatin-resistant cervical cancer cells.
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