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Objective:To investigate the effect of high glucose on intracellular cholesterol content in rat glomerular mesangial cells and the underlying mechanism.Methods:Rat glomerular mesangial cells were cultured in vitro and divided into high glucose culture group (high glucose group, medium glucose concentration of 30 mmol/L) and normal glucose culture group (normal group, medium glucose concentration of 5.5 mmol/L). Lipid content was determined by oil red O staining and spectrophotometer colorimetry at 24, 36 and 48 h of culture. Intracellular protein imprinting was used to detect the expression of low density lipoprotein cholesterol receptor (LDLR) and adenosine triphosphate binding cassette A1 (ABCA1). Results:Oil red O staining showed that the intracellular lipid drops in the high glucose group were less than those in the normal group at 36 h, and there was no significant difference between the two groups at 24 h and 48 h of culture. The total cholesterol (TC) and cholesterol ester (CE) in glomerular mesangial cells of rats in the high glucose group were significantly lower than those in the normal group ( P=0.028, 0.029), while there was no significant difference in the free cholesterol (FC) between the two groups ( P=0.306). There was no significant difference in TC, CE and FC between the two groups at 24 and 48 h of culture (all P>0.05). The expression of LDLR in mesangial cells of rats in high glucose group was significantly lower than that in normal group at 24, 36 and 48 h of culture ( P=0.043, 0.004, 0.028), and the expression of ABCA1 was significantly higher than that in normal group at 24, 36 and 48 h of culture, ( P=0.050, 0.009, 0.006). Conclusions:High glucose may reduce intracellular cholesterol content in rat glomerular mesangial cells by reducing LDLR protein expression and increasing ABCA1 protein expression.
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Objective To analyze the transcription factor binding sites(TFBS)in the promoter region of E3 ubiquitin protein ligase 1(MIB1)gene in zebrafish,the types of MIB1 interacting genes and proteins and their roles in signaling pathways,and to investigate the regulatory mode and potential function of MIB1 gene.Methods Non-coding RNA(ncRNA)was predicted by National Genomics Data Center(NGDC).Alggen and AnimalTFDB online software were used to predict the TFBS types of MIB1 gene.GeneMANIA and STRING were used to analyze the interacting genes and proteins of MIB1.The related data were obtained through DA-VID website,and gene ontology(GO)visual analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)metabolic pathway analysis were performed.Results ncRNA could be transcribed from the promot-er region and 5'untranslated region of MIB1 gene.A total of 121 TFBS were obtained by prediction.P53 tran-scription factor could bind to the promoter region of MIB1 gene and interact with MIB1 protein.A total of 6 co-expressed genes of MIB1 were predicted online,and 20 interacting genes were screened.GO visual analysis showed that MIB1 and its interaction genes had functions in regulating the growth and differentiation of cells,tissues and organs and regulating the NOTCH signaling pathway in the biological process,and were mainly enriched in the cytoplasmic perinuclear region,cell membrane,postsynaptic dense area and so on.It had molec-ular functions such as binding NOTCH proteins and PDZ domain proteins.KEGG metabolic pathway analysis showed that MIB1 and its interacting genes were involved in 4 metabolic pathways.Conclusion MIB1 con-tains a variety of TFBS,and affects a variety of biological processes such as cell carcinogenesis and immune regulation by interacting with specific transcription factors.MIB1 may also play an important role in cell growth regulation,hematopoietic stem cell differentiation,embryonic development and neuronal information transmission through the mediation of its interacting genes and proteins.
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Objective To investigate the protective effect of retigabine (a M-type potassium channel opener) on brains and its mechanism in male mice after acute cerebral ischemia reperfusion(I/R)injury.Methods Seventy male C57BL/6J mice were randomly divided sham-operated group (n=10),middle cerebral artery occlusion (MCAO) group (n=10) and prevention group (n=50) according to the random number table method;mice in the prevention group were then divided into XE991 (a M-type potassium channel blocker) group,RTG-treatment 0 h group,RTG-treatment 1 h group,RTG-treatment 3 h group,and RTG-treatment 6 h group (n=10).The MCAO models were established by suture method,and reperfusion was performed 90 min after cerebral ischemia.In RTG-treatment groups,a single dose of 10.5 mg/kg RTG was injected at the designated varying time points (0,1,3 and 6 h after the reperfusion);in XE991 group,a single dose of 3.0 mg/kg XE991 was injected after the reperfusion;mice in the sham-operated group and MCAO group received the same volume of saline.Twenty-four h after model making,infarct size was measured by TTC staining.HE staining was used to observe the morphological changes of neurons in hippocampal CA1 regions.The apoptotic neurons level and membrane protein CD40L expression in the ischemic penumbra were detected by TUNEL staining and Western blotting.Results In the sham-operated group,brain tissues had no obvious change,no infarction was observed,there was no CD40L expression,and TUNEL staining positive neurons were hardly found.(1) Cerebral artery territory infarction was visible in the MCAO group and intervention group;however,the infarction volume of the RTG-treatment groups was significantly lower than that in the MCAO group (P＜0.05);the infarction volume of the RTG-treatment 6 h group was increased as compared with that of the RTG-treatment 0 h group,RTG-treatment 1 h group,and RTG-treatment 3 h group,without significant difference (P＞0.05).(2) HE staining showed that hippocampal neurons were obviously swollen and necrotic in the MCAO group and XE991 group,while the pathological damages such as brain edema and neuron necrosis were ameliorated significantly in the RTG-treatment groups.(3) As compared with those in the MCAO group,the number of TUNEL staining positive neurons in the RTG-treatment 0 h group,RTG-treatrnent 1 h group,and RTG-treatment 3 h group and CD40L number in the RTG-treatment 0 h group and RTG-treatment 3 h group were decreased significantly (P＜0.05);as compared with that in the MCAO group,the number of TUNEL staining positive neurons increased significantly in the XE991 group (P＜0.05).Conclusion RTG has protective effect on cerebral I/R,and its mechanism might relate to reducing cell excitability and inflammation,thereby inhibiting cell apoptosis;these protection would be less effective when RTG is used outside a defined critical period of time.